Autism

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Library of Congress Cataloging-in-Publication Data
Autism : oxidative stress, inflammation, and immune abnormalities / edited by Abha

Chauhan, Ved Chauhan, and Ted Brown.
p. ; cm.
Includes bibliographical references and index.
ISBN 978-1-4200-6881-8 (hardcover : alk. paper)
1. Autism--Pathophysiology. 2. Oxidative stress. 3. Inflammation. 4.
Neuroimmunology. I. Chauhan, Abha, 1957- II. Chauhan, Ved. III. Brown, Ted, 1946-
[DNLM: 1. Autistic Disorder--etiology. 2. Inflammation. 3. Oxidative Stress. WM
203.5 A93805 2010]
RC553.A88A8726 2010
616.85’882--dc22 2009024353
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This book is dedicated to the children and adults who
have Autism spectrum disorders, to the family members
and health care professionals who provide care for them,
and to the scientists and sponsoring agencies devoted
to research on autism and related disorders.
Contents
Preface.......................................................................................................................xi
Editors ....................................................................................................................xvii
Contributors ............................................................................................................xix
Chapter 1 Type, Topography, and Sequelae of Neuropathological Changes

Shaping Clinical Phenotype of Autism ................................................1
Jerzy Wegiel, Thomas Wisniewski, Abha Chauhan,
Ved Chauhan, Izabela Kuchna, Krzysztof Nowicki, Humi Imaki,
Jarek Wegiel, Shuang Yong Ma, Teresa Wierzba Bobrowicz,
Ira L. Cohen, Eric London, and W. Ted Brown
Chapter 2 Evidence for Oxidative Damage in the Autistic Brain ....................... 35

Teresa A. Evans, George Perry, Mark A. Smith,
Robert G. Salomon, Woody R. McGinnis,
Elizabeth M. Sajdel-Sulkowska, and Xiongwei Zhu
Chapter 3 Oxidative Stress and Neurotrophin Signaling in Autism ................... 47

Elizabeth M. Sajdel-Sulkowska
Chapter 4 Genetics of Autism ............................................................................. 61

W. Ted Brown
Chapter 5 Phenotypic Expression of Autism, Maternal Depression,

and the Monoamine Oxidase-A Gene ................................................ 73
Ira L. Cohen
Chapter 6 Paraoxonase 1 Status, Environmental Exposures,

and Oxidative Stress in Autism Spectrum Disorders ......................... 91
Maria Dronca and Sergiu P. Paşca
Chapter 7 The Redox/Methylation Hypothesis of Autism: A Molecular

Mechanism for Heavy Metal-Induced Neurotoxicity ...................... 113
Richard C. Deth and Christina R. Muratore
vii
viii Contents
Chapter 8 Autism and Oxidative Stress: Evidence from an Animal Model ....... 131
Michelle A. Cheh, Alycia K. Halladay, Carrie L. Yochum,
Kenneth R. Reuhl, Marianne Polunas, Xue Ming,
and George C. Wagner
Chapter 9 Neurotoxic Brainstem Impairment as Proposed

Threshold Event in Autistic Regression ........................................... 153
Woody R. McGinnis, Veronica M. Miller, Tapan Audhya,
and Stephen M. Edelson
Chapter 10 Abnormalities in Membrane Lipids, Membrane-Associated

Proteins, and Signal Transduction in Autism ................................... 177
Ved Chauhan and Abha Chauhan
Chapter 11 Mitochondrial Component of Calcium Signaling

Abnormality in Autism ....................................................................207
J. Jay Gargus
Chapter 12 Inflammation and Neuroimmunity in the Pathogenesis

of Autism: Neural and Immune Network Interactions..................... 225
Carlos A. Pardo-Villamizar and Andrew W. Zimmerman
Chapter 13 Possible Impact of Innate Immunity on Autism .............................. 245

Harumi Jyonouchi
Chapter 14 Autism, Gastrointestinal Disturbance, and Immune

Dysfunction: What Is the Link ......................................................... 277
Paul Ashwood, Amanda Enstrom, and Judy Van de Water
Chapter 15 Possible Mechanism Involving Intestinal Oxytocin,

Oxidative Stress, and Signaling Pathways in a Subset
of Autism with Gut Symptoms ......................................................... 299
Martha G. Welch and Benjamin Y. Klein
Chapter 16 Cytokine Polymorphisms in Autism:

Their Role in Immune Alterations ................................................... 315
Fabián Crespo, Rafael Fernandez-Botran,
Christopher Tillquist, Lonnie Sears, Meghan Mott,
and Manuel Casanova
Contents ix
Chapter 17 Autism, Teratogenic Alleles, HLA-DR4, and Immune Function ..... 325
William G. Johnson, Steven Buyske, Edward S. Stenroos,
and George H. Lambert
Chapter 18 Autism: The Centrality of Active Pathophysiology and

the Shift from Static to Chronic Dynamic Encephalopathy ............ 343
Martha R. Herbert
Chapter 19 A Reevaluation of the State of Autism Treatment:

The Need for a Biopsychosocial Perspective ................................... 389
Eric London
Index ...................................................................................................................... 419
Preface
Autism spectrum disorders (ASD) are a group of behaviorally defined neuro-
developmental disorders characterized by deficits in social interaction; impairments
in verbal and nonverbal communication; and restricted, stereotyped patterns of
behaviors and interests. In 2007, the Centers for Disease Control and Prevention
issued an “autism alarm,” with 1 in 150 children estimated to be affected by ASD.
Epidemiological studies suggest that more than 1.5 million people in the United
States are affected with this disorder. Autism is the most severe disorder in the
broad spectrum of pervasive developmental disorders (PDDs), which also include
Asperger’s syndrome, Rett’s disorder, childhood disintegrative disorder, and PDD-not
otherwise specified. Within this spectrum, there are variations in the severity, level of
cognitive functioning, the presence or absence of associated medical conditions
such as seizures or other neurological disorders, and whether or not there is a history
of regression from apparent normal development. Currently, there is no biochemical
or genetic marker to support the behavioral diagnosis of autism.
Autism is a heterogeneous disorder, both etiologically and phenotypically. While
the cause of autism remains elusive, autism is considered a multifactorial disorder
that is influenced by genetic, epigenetic, and environmental factors. Accumulating
evidence from our studies and that of other groups suggests that oxidative stress may
be a common feature in autism linking the mechanism through which the environ-
mental factors exert their deleterious effects with the purported genetic alterations
in autism. The oxidative stress and intracellular redox imbalance can be induced
or triggered in autism by prenatal or postnatal exposure to certain environmental
factors such as heavy metals, viruses, bacterial infections, air pollutants, toxins, val-
proic acid, thalidomide, terbutaline, retinoic acid, and ethanol. Genetic factors can
also modulate the threshold for vulnerability to oxidative stress in autism. In addition
to behavior impairments, some individuals with autism may have a higher preva-
lence of gastrointestinal (GI) disturbances. Several studies suggest that inflamma-
tory phenomena, immune dysregulation, and certain autoimmune risk factors may
also contribute to the development and pathogenesis of autism. The chapters in this
book provide a comprehensive overview of neuropathological abnormalities, genet-
ics, oxidative stress, inflammation, immune dysfunction, aberrant cellular signaling,
gene–environment interactions, diagnostic tools, and treatment in autism.
In Chapter 1, Jerzy Wegiel and colleagues review neuropathological changes in
autism contributing to the clinical phenotype. These changes include (a) pathological
acceleration of brain growth in early childhood, (b) developmental heterochronicity
with different rates of growth for different brain regions/structures, (c) brain structure–
specific delay of neuronal growth in early childhood and partial correction of cell
size in late childhood/adulthood, and (d) regional cytoarchitectonic abnormalities
with consistent changes of structure of minicolumns, and variable topography and
severity of dysplastic changes and ectopias. These developmental abnormalities are
paralleled by signs of metabolic changes with modified expression and processing
xi
xii Preface
of β-amyloid precursor protein, enhanced turnover of cell organelle and pigment

accumulation, as well as oxidative stress.
Under normal conditions, a dynamic equilibrium exists between the production
of reactive oxygen species (ROS) and the antioxidant capacity of the cell. Oxidative
stress occurs when ROS levels exceed the antioxidant capacity of a cell. These ROS
are highly toxic and oxidize vital cellular components such as lipids, proteins, and
DNA, thus causing cellular damage and subsequent cell death via apoptosis or necrosis.
Oxidative stress is known to be associated with premature aging of cells and can
lead to inflammation, damaged cell membranes, autoimmunity, and cell death. In this
book, several chapters present evidence that support the concept of oxidative stress
in autism.
The brain is highly vulnerable to oxidative stress because of its limited antioxi-
dant capacity, higher energy requirement, and high amounts of unsaturated lipids
and iron. Based on immunocytochemical and biochemical studies, in Chapter 2,
Xiongwei Zhu and colleagues demonstrate lipid-derived oxidative protein modifica-
tions in postmortem brain samples from autistic subjects and suggest carboxyethyl
pyrrole (CEP) and iso[4]levuglandin E2 protein adducts as possible oxidative stress
markers for the autistic brain. From a structural perspective, their findings suggest
that axons of cholinergic neurons in the white matter are the primary site of oxidative
damage. At the molecular level, they have identified neurofilament heavy chain to
be the major target for CEP-modification. In Chapter 3, Elizabeth Sajdel-Sulkowska
reviews the evidence of oxidative stress–related protein and DNA modifications in
autism, and discusses data supporting altered neurotrophin signaling in autism and
the possible association between oxidative stress and altered neurotrophin expres-
sion. These findings not only support the notion that brain oxidative stress plays
an important role in autism but also warrant future in-depth mechanistic studies to
provide new targets for therapeutic intervention.
The genetics underlying autism is highly complex, with estimates of heritability at
greater than 90%. While no single gene has been found to be associated with autism,
multiple genes and interactions between genetic and environmental factors have been
postulated in autism. In Chapter 4, Ted Brown reviews this subject. The association
of single-gene disorders, such as fragile X syndrome with autism, and the role of copy
number variations, noncoding RNAs, parental age, and prenatal environmental stres-
sors, such as exposure to hurricanes in autism, are discussed in this chapter.
Reactive oxygen and nitrogen species are generated endogenously during oxi-
dative metabolism and energy production by mitochondria in the cell. While oxida-
tive phosphorylation in the mitochondria generates superoxide anions, the enzymatic
oxidation of biogenic amines by monoamine oxidase (MAO) in mitochondrial outer
membrane produces H2O2. A functional polymorphism in the monoamine oxidase
A (MAOA) promoter region has been reported to be associated with the severity
of autism. MAOA catalyzes the oxidation of amine-containing neurotransmitters,
such as serotonin and norepinephrine. Several studies suggest that serotonin function
is abnormal in autism. In Chapter 5, Ira Cohen reviews evidence on maternal depres-
sion and a polymorphism in the MAOA gene affecting the severity of autism using
a PDD behavior inventory (PDDBI). He also discusses the advantages of using the
PDDBI.
Preface xiii
In Chapter 6, Maria Dronca and Sergiu Paşca present evidence for the involvement
of paraoxonase 1 (PON1) in the pathogenesis of ASD. PON1, an esterase/lactonase
enzyme, plays an important role in hydrolyzing pesticides, protecting against oxida-
tive stress, and modulating the immune/inflammatory response. First, the authors
review the general biochemical properties of PON1, with the recent biochemical and
genetic studies indicating impaired PON1 status in ASD and the literature suggesting
a correlation between pesticide (mainly organophosphates) exposure and neurodeve-
lopmental delays or neuropsychiatric conditions. Second, the authors describe sev-
eral gene × environment models for autism, which include PON1: organophosphates
exposure × reelin × PON1 and organophosphates exposure × acetylcholine receptor
(AchR) × PON1. Finally, they illustrate how PON1 could contribute to the aberrant
immune response and abnormal redox status in autism. They also discuss how these
disturbances could affect the PON1 status and further increase the susceptibility to
various environmental neurotoxic agents during neurodevelopment.
Sulfur metabolism serves a number of critical roles, including the maintenance
of cellular redox status and support for several methylation reactions. Methylation
capacity is reduced during oxidative stress, affecting many processes such as epige-
netic regulation of gene expression, which is critical for development, as well as
dopamine-stimulated phospholipid methylation, which is involved in the synchroni-
zation of neuronal firing. The unique features of sulfur metabolism in the brain make
it highly vulnerable to heavy metals, which bind with high affinity to thiol and sele-
nocysteine oxidoreductases and interfere with redox regulation. Methionine synthase
(folate and vitamin B12–dependent enzyme) plays a key role as a monitor of cellular
redox status and as a regulator of the flux of homocysteine through transsulfuration to
glutathione synthesis. Levels of methionione synthase mRNA are significantly lower
in brain samples from autistic subjects, reflecting an adaptive response to neuroin-
flammation and oxidative stress. These factors allow the formulation of a “redox/
methylation hypothesis of autism,” described by Richard Deth in Chapter 7, which
outlines a molecular mechanism whereby heavy metals promote oxidative stress and
impaired methylation, leading to disrupted development and autism.
In Chapter 8, George Wagner and colleagues describe novel models of autism
in which mice are exposed either pre- or post-natally to toxicants such as valproic
acid and methylmercury. The early toxicant exposure results in neurodevelopmental
deficits in mice that are analogous to deficits observed in humans affected by autism.
Evidence that oxidative stress is involved in autism is provided by the ability of
pretreatment with antioxidants (trolox, a water-soluble vitamin E derivative) to fully
protect the developing mice against the neurobehavioral deficits induced by these
toxicants. These observations are discussed in the context of autism prevention.
In Chapter 9, Woody McGinnis and colleagues discuss an interesting hypothesis
for the unexplained phenomenon of regression in autism. In their model, visceral
dysfunction in autism occurs in conjunction with lost phonation and social function
because of selective toxicant effects on a relatively minute region of the brainstem,
which is known to remain permeable to a broad class of neurotoxins after the closure
of the blood–brain barrier elsewhere. By converging on the same site and sharing
oxidative modes of injury, these toxins may act independently, additively, or sequen-
tially to result in autistic regression.
xiv Preface
In Chapter 10, Ved Chauhan and I review evidence that ASD are associated
with abnormalities in lipid metabolism, membrane-associated proteins, and signal
transduction. Phospholipids and their lipid raft domain play important roles
in cellular signaling, and phosphoinositides are major signaling molecules in
G protein–coupled receptor signaling. We discuss our findings on altered levels
of amino-glycerophospholipids in the membrane, increased peroxidation of lipids,
decreased membrane fluidity, increased activity of phospholipase A 2 (lipid-
metabolizing enzyme), and altered activities of protein kinase C and protein kinase
A in autism, suggesting that membrane signaling may be affected in autism.
We also review the evidence of an association of the phosphatidylinositol 3-kinase
gene in autism, altered brain levels of Bcl2 and p53 (involved in apoptosis), altered
levels of cytokines and inflammation and mutational changes in the proteins
involved in cell signaling such as neuroligins, Pten, SHANK3, Wnt, reelin, and
voltage-dependent calcium channels, all suggesting impairment in signal transduc-
tion in autism. These abnormalities in the signal system may account for some of the
structural changes and cognitive deficits in the brains of individuals with autism.
Mitochondria play a major role in ROS generation and cytosolic calcium seques-
tration, and a primary defect in mitochondrial electron transport and oxidative phos-
phorylation impairs both processes. Conversely, abnormal calcium signaling will
secondarily perturb these mitochondrial functions, as the mitochondria have recently
been shown to participate with the endoplasmic reticulum in this important process
that governs a wide array of cellular functions. In Chapter 11, Jay Gargus presents a
genetic scheme as the basis for the elevated levels of ROS observed in autism, which
integrates earlier observations of genetic and functional mitochondrial dysfunction
in autism with newer observations of defects in calcium signaling in the disease.
Recently, Timothy syndrome, a rare monogenic form of autism, was shown to be
a channelopathy caused by a mutation in a calcium channel. In addition, diseases
comorbid with autism, such as migraine and seizures, share a channelopathy patho-
genesis, strengthening the notion that defects in calcium signaling may be a cardinal
aspect of the disorder that may represent a target for novel therapeutics.
Some parents of autistic children report frequent infection, prolonged illness, or
chronic sinopulmonary symptoms, which are suggestive of immune abnormalities
in autism. Emerging evidence from several independent research groups indicates
the role of the immune system and inflammation in the pathogenesis and pathophys-
iology of ASD. Increased oxidative damage and/or mitochondrial dysfunction can
also lead to inflammation because oxidative stress serves as a major upstream
component in the signaling cascade involved in the activation of redox-sensitive
transcription factors and pro-inflammatory gene expression resulting in an inflam-
matory response.
Recently, it has become evident that the neuroimmune network is crucial for
immune homeostasis and the function of the central nervous system (CNS). In
Chapter 12, Carlos Pardo-Villamizar and Andrew Zimmerman review the findings
on the activation of neuroglia and the neuroimmune system, as evidenced by neu-
roinflammation in brains, reactive astrogliosis, activated microglia, and cytokine
abnormalities. They also discuss the role of the maternal immune environment and
immunogenetic factors in autism.
Preface xv
Innate immunity plays a key role in the neuroimmune network. However, the role
of innate immunity in the onset and progression of ASD is not well understood. In
Chapter 13, Harumi Jyonouchi reviews immune abnormalities reported in children
with ASD, following an overview of innate immunity in the GI tract and the CNS.
Finally, the possible impact of innate immunity on neuroimmune interactions in
autistic children is discussed. In Chapter 14, Paul Ashwood and colleagues review
cell-mediated immune response, autoimmunity, cytokine abnormalities, gut inflam-
mation, and GI dysfunction and suggest a relationship between GI-related immune
dysfunction and autistic behaviors. Abnormal immune responses may predispose
individuals to frequent infections, adverse reactions to benign environmental factors,
and possibly autoimmune conditions, leading to increased oxidative stress.
Oxytocin (OT) is known to be dysregulated in some autistic children. In Chapter 15,
Martha Welch and Benjamin Klein offer the hypothesis that autism arises from
the dysregulation of a unified gut/brain system rather than originating in the brain
alone. They postulate that autism stems from physiological stress, including oxida-
tive stress, which, if unmodulated, triggers a cascade of adverse interrelated auto-
nomic, endocrinological, neurological, and immunological reactions. They review
evidence that dysregulated OT levels and signaling pathways downstream of the
oxytocin receptor combined with oxidative stress in the gut may dysregulate a uni-
fied gut/brain network and be involved in the pathogenesis of a subset of autism.
They also discuss a chain of possible cellular events in gut Paneth cells, involving
ROS, β-catenin, matrix metalloproteinase-7, prodefensin, and defensin, which could
impact various ion channels in enteric neurons and ultimately influence behavior.
A possible mechanism for dysregulation of gut/brain signaling under conditions of
abnormal OT levels during a time window critical for newborn development is dis-
cussed and compared with the same mechanism when modulated by adequate OT
levels in normal newborns. Finally, they discuss possible early therapeutic interven-
tions aimed at the OT-related mechanism postulated in this chapter.
Cytokines play an important role in the regulation of inflammatory responses and
are involved in the regulation of both innate and acquired immunities. They are often
encoded by highly polymorphic genes. Some of these polymorphisms are responsi-
ble for quantitative interindividual differences in cytokine production, thereby influ-
encing the relative strength of immune responses. In the last 10 years, evidence has
accumulated that increased levels of some pro-inflammatory cytokines are present in
the peripheral blood mononuclear cells of children with ASD. In Chapter 16, Fabián
Crespo and colleagues suggest that there are phenotypes of the immune system that
are predisposed to stronger or weaker inflammatory immune responses, and these
phenotypes can manifest from several different combinations of genotypes of differ-
ent cytokine genes with variable expressions. They propose that certain expression
polymorphisms in key cytokine genes may contribute to the etiology or the emergence
of autism by predisposing individuals carrying those genotypes (or their mothers)
to altered immune activation to certain antigens. The authors also suggest that the
maternal immunogenetic makeup may be associated with the fetal pathogenesis of
ASD since cytokines are able to cross the placenta.
In Chapter 17, William Johnson and colleagues discuss alleles of maternal genes
that act in the mother to contribute to the phenotype of their affected offspring. These
xvi Preface
alleles most likely act in the mother during pregnancy to modify the development of
the embryo or fetus, for example, brain development in the affected children. Of the
34 reports so far of these maternal alleles, nearly all were in neurodevelopmental
disorders, including autism. HLA-DR4 was originally suspected of being a maternally
acting gene allele for autism because its allele frequency was increased in individuals
with autism and their mothers, but not their fathers. This has now been confirmed by a
case–parent study design. HLA-DR4 may act in autism by affecting synapse develop-
ment, by a mechanism including oxidative stress, by a combination of these, or by an
as-yet-unknown mechanism.
Chapter 18 is a commentary by Martha Herbert. On the basis of active pathophysi-
ological processes, such as oxidative stress and inflammation in autism, she discusses
that the classical autism model, which frames ASD as a genetically determined devel-
opmental disorder of the brain whose main manifestation is behavioral alterations, does
not predict persistent pathophysiological disturbances in autism. Herbert describes a
pathophysiology-centered model of autism, in which it is argued that ASD is not only
developmental but also a chronic condition based on active pathophysiology; is not
only behavioral but also has somatic and systemic features; is not only genetic but
also environmental; and is not a static encephalopathy but is a dynamic, recalcitrant
encephalopathy.
The history of the treatment of autism has been dominated by a technical
approach mostly highlighted by applied behavior analysis and, to a lesser extent, by
psychopharmacology. In Chapter 19, Eric London proposes the utility of using the
biopsychosocial method elaborated by George Engel as a conceptual way to treat
autism. The implications of these concepts for both research and clinical works are
discussed.
I would like to express my gratitude to my coeditors, Drs. Ved Chauhan and Ted
Brown, for their help in the review process, and to all the contributors for their chap-
ters. My sincere thanks to CRC Press/Taylor & Francis Group, especially Barbara
Norwitz, Patricia Roberson and Jennifer Smith, for their support in compiling and
publishing this book. I hope that it will stimulate hypothesis-driven research and be
a valuable reference source not only to scientists in the laboratory but also to clinicians
and caregivers in the field of autism and related disorders.
Abha Chauhan, PhD

Head, Developmental Neuroscience Laboratory
New York State Institute for Basic Research in Developmental Disabilities
1050 Forest Hill Road
Staten Island, New York 10314, U.S.A.
Tel: 718-494-5258; Fax: 718-698-7916

Email: [email protected]
Editors
Abha Chauhan, PhD, is the head of the Developmental Neuroscience Laboratory
at the New York State Institute for Basic Research in Developmental Disabilities
(IBR), Staten Island, New York.
As a National Science Talent Scholar, Dr. Chauhan received her BS (chemistry
honors) in 1976 from the University of Delhi, her MS (biochemistry) in 1978 and her
PhD in 1982 from the Department of Biochemistry, Postgraduate Institute of Medical
Education and Research, Chandigarh, India. From 1983 to 1984, she worked as a
research associate in the Department of Biochemistry at the Mount Sinai School of
Medicine, New York. Dr. Chauhan then joined the Department of Neurochemistry
at IBR, where she has over 60 publications in the fields of membrane biochemistry,
signal transduction, Alzheimer’s disease, and autism.
Currently, Dr. Chauhan’s major interest is to investigate the biochemical and
immunological changes associated with autism in blood samples, lymphoblast cell
cultures, and postmortem brain samples, particularly as they relate to markers of
oxidative stress, inflammatory response, and the function of the immune system.
She is also studying the relationship, if any, between these abnormalities and low-
or high-functioning autism groups, as well as the severity of behavior deficits and
neuropathological abnormalities in autism. She has been awarded research grants
as a principal investigator from the Department of Defense, Autism Speaks, and the
Autism Research Institute for her work on autism.
In 2008, Dr. Chauhan served as the guest editor of the “Special Issue on Autism
Spectrum Disorders” of the American Journal of Biochemistry and Biotechnology
(April 2008). She also organized and chaired a colloquium on “Oxidative Stress
and Inflammation in Autism Spectrum Disorders” at the American Society for
Neurochemistry Meeting in 2009.
Ved Chauhan, PhD, is the head of the Cellular Neurochemistry Laboratory at the
New York State Institute for Basic Research in Developmental Disabilities (IBR),
Staten Island, New York.
Dr. Chauhan received his MS (biochemistry) in 1975 and his PhD (biochemis-
try) in 1980 from the Postgraduate Institute for Medical Education and Research,
Chandigarh, India. After working as a research associate for two years in the
Department of Biochemistry at the University of Southern California, Los Angeles,
he joined IBR as a research scientist in 1983.
Dr. Chauhan has published more than 70 research articles in peer-reviewed
journals. His work includes but is not limited to phospholipid methylation, calcium
traversal across bilayers, the role of phosphoinositides in the activation of protein
kinase C, lipid and amyloid β-protein interactions, hydrophobic domain formation
by fibrillar amyloid β-protein and its regulation by gelsolin, and membrane abnor-
malities and cellular signaling in autism.
xvii
xviii Editors
Dr. Chauhan is a member of the editorial board of the International Archives of

Medicine, an associate editor of the Journal of Alzheimer’s Disease, and an associate
editor of the “Special Issue on Autism Spectrum Disorders” of the American Journal
of Biochemistry and Biotechnology (April 2008). He has organized and chaired sev-
eral national and international symposia on autism.
W. Ted Brown, MD, PhD, is the director of the New York State Institute for Basic
Research in Developmental Disabilities (IBR), the chairperson of IBR’s Department
of Human Genetics, and the director of IBR’s George A. Jervis Clinic. He is a fellow
of the American College of Medical Genetics and an adjunct professor at the State
University of New York-Downstate Medical Center in Brooklyn.
Dr. Brown received his BA in 1967, his MA in 1969 and his PhD in biophysics in
1973 from The Johns Hopkins University, Baltimore, Maryland. He received his MD
from Harvard Medical School (cum laude) in 1974. He trained in internal medicine
in New York City, undertook a fellowship in clinical genetics, and was appointed
as an assistant professor of medicine at the New York Hospital-Cornell University
Medical Center in 1978. He began research into premature aging syndromes and
Down syndrome while on the Cornell Medical School Faculty, and was an attend-
ing physician at New York Hospital and a faculty member of Rockefeller University.
In 1981, he became the chairperson of the Department of Human Genetics at IBR.
In 1991, he was appointed the director of IBR’s Jervis Clinic, and in 2005, he became
the director of IBR.
Dr. Brown is the author of more than 300 publications. At IBR, his initial research
was on Down syndrome genes. He then focused his research on the fragile X syn-
drome, which was then newly recognized and is now considered the most common
inherited cause of mental retardation. At IBR, he established a DNA diagnostic and
molecular laboratory and developed a screening and prenatal testing program for
fragile X. He was the first to discover a relationship between autism and the fra-
gile X syndrome. His work on fragile X has ranged from clinical studies relating to
phenotype, to family inheritance studies, to mouse model development, and to basic
molecular research. His current research focuses on autism genetics and the fragile
X syndrome. Dr. Brown is also a recognized world authority on progeria, a rare and
tragic disease that afflicts young children with premature aging. He was instrumental
in the discovery of the genetic mutation that causes this disease.
Dr. Brown serves on the editorial board of the American Journal of Intellectual
and Developmental Disability. He has served on the scientific advisory board for
Cure Autism Now, the Progeria Research Foundation, and the National Fragile X
Foundation.
Contributors
Paul Ashwood, PhD Abha Chauhan, PhD
Department of Medical Microbiology Department of Neurochemistry
and Immunology New York State Institute for Basic
and Research in Developmental
Disabilities
M.I.N.D Institute Staten Island, New York
University of California at Davis
Davis, California Ved Chauhan, PhD
Department of Neurochemistry
Tapan Audhya, PhD
New York State Institute for Basic
Division of Endocrinology
Research in Developmental
Department of Medicine
Disabilities
New York University School
Staten Island, New York
of Medicine
New York, New York
Michelle A. Cheh, PhD
and Department of Neuroscience
Vitamin Diagnostics Laboratory Rutgers University
Cliffwood Beach, New Jersey New Brunswick, New Jersey
Teresa Wierzba Bobrowicz, MD, PhD Ira L. Cohen, PhD

Department of Neuropathology Department of Psychology
Institute of Psychiatry and Neurology New York State Institute for Basic
Warsaw, Poland Research in Developmental
Disabilities
W. Ted Brown, MD, PhD Staten Island, New York
Department of Human Genetics
New York State Institute for Basic Fabián Crespo, PhD
Research in Developmental Department of Anthropology
Disabilities
Staten Island, New York and
Steven Buyske, PhD Department of Psychiatry

Departments of Statistics and Genetics and Behavioral Sciences
University of Louisville
Rutgers University
New Brunswick, New Jersey Louisville, Kentucky
Manuel Casanova, MD Richard C. Deth, PhD

Department of Psychiatry Department of Pharmaceutical
and Behavioral Sciences Sciences
University of Louisville Northeastern University
Louisville, Kentucky Boston, Massachusetts
xix
xx Contributors
Maria Dronca, PhD Alycia K. Halladay, PhD

Department of Medical Biochemistry Department of Psychology
Iuliu Haţieganu University
of Medicine and Pharmacy and
Cluj-Napoca, Romania
Department of Pharmacology
and Toxicology
Stephen M. Edelson, PhD Rutgers University
Autism Research Institute New Brunswick, New Jersey
San Diego, California
Martha R. Herbert, MD, PhD

Amanda Enstrom, PhD Department of Pediatric
Department of Medical Microbiology Neurology
and Immunology Massachusetts General Hospital
and Harvard Medical School
Charlestown, Massachusetts
M.I.N.D Institute
Humi Imaki, PhD
Davis, California
Department of Developmental
Neurobiology
Teresa A. Evans, BS New York State Institute for Basic
Department of Pathology Research in Developmental
Case Western Reserve University Disabilities
Cleveland, Ohio Staten Island, New York
Rafael Fernandez-Botran, PhD William G. Johnson, MD

Department of Pathology Department of Neurology
and Laboratory Medicine
School of Medicine and
Louisville, Kentucky Center for Childhood Neurotoxicology
and Exposure Assessment
Robert Wood Johnson
J. Jay Gargus, MD, PhD Medical School
Department of Physiology and University of Medicine and
Biophysics Dentistry of New Jersey
Piscataway, New Jersey
and
Division of Human Genetics Harumi Jyonouchi, MD

Department of Pediatrics Department of Pediatrics
School of Medicine University of Medicine and
University of California, Irvine Dentistry of New Jersey
Irvine, California Newark, New Jersey
Contributors xxi
Benjamin Y. Klein, MD Woody R. McGinnis, MD

Division of Developmental Autism House of Auckland
Neuroscience Autism New Zealand, Inc.
Department of Psychiatry Auckland, New Zealand
and Veronica M. Miller, PhD
Department of Pathology and Cell Biology Wadsworth Center for Laboratories
Columbia University Medical Center and Research
New York, New York New York State Department of Health
Albany, New York
Izabela Kuchna, MD, PhD
Department of Developmental Xue Ming, MD, PhD
Neurobiology Department of Neuroscience
New York State Institute for Basic University of Medicine and
Research in Developmental Dentistry of New Jersey
Disabilities Newark, New Jersey
Meghan Mott, MS
George H. Lambert, MD Department of Psychiatry
Department of Pediatrics and Behavioral Sciences
School of Medicine
and University of Louisville
Louisville, Kentucky
Center for Childhood Neurotoxicology
and Exposure Assessment Christina R. Muratore, BS
Robert Wood Johnson Department of Pharmaceutical
Medical School Sciences
University of Medicine and Northeastern University
Dentistry of New Jersey Boston, Massachusetts
Piscataway, New Jersey
Krzysztof Nowicki, MD
Eric London, MD Department of Developmental
Department of Psychology Neurobiology
New York State Institute for Basic New York State Institute for Basic
Research in Developmental Research in Developmental
Disabilities Disabilities
Staten Island, New York Staten Island, New York
Shuang Yong Ma, MD, PhD Carlos A. Pardo-Villamizar, MD

Department of Developmental Division of Neuroimmunology
Neurobiology and Neuroinfectious Disorders
New York State Institute for Basic Department of Neurology
Research in Developmental Johns Hopkins University School
Disabilities of Medicine
Staten Island, New York Baltimore, Maryland
xxii Contributors
Sergiu P. Paşca, MD Mark A. Smith, PhD

Center for Cognitive and Neural Studies Department of Pathology
Cluj-Napoca, Romania Case Western Reserve University
Cleveland, Ohio
George Perry, PhD
College of Sciences Edward S. Stenroos, BS
University of Texas at San Antonio Department of Neurology
San Antonio, Texas
and
and
Center for Childhood Neurotoxicology
Department of Pathology and Exposure Assessment
Case Western Reserve University Robert Wood Johnson
Cleveland, Ohio Medical School
University of Medicine and
Marianne Polunas, MS Dentistry of New Jersey
Department of Pharmacology Piscataway, New Jersey
and Toxicology
Rutgers University Christopher Tillquist, PhD, MPH
New Brunswick, New Jersey Department of Anthropology
Kenneth R. Reuhl, PhD Louisville, Kentucky
Department of Pharmacology
and Toxicology George C. Wagner, PhD
Rutgers University Department of Psychology
New Brunswick, New Jersey Rutgers University
New Brunswick, New Jersey
Elizabeth M. Sajdel-Sulkowska, DSc
Department of Psychiatry Judy Van de Water, PhD
Harvard Medical School Division of Rheumatology, Allergy
and Clinical Immunology
and
Department of Internal Medicine
Department of Psychiatry
and
Brigham and Women’s Hospital
Boston, Massachusetts M.I.N.D Institute
Robert G. Salomon, PhD Davis, California
Department of Chemistry
Case Western Reserve University Jarek Wegiel, MS
Cleveland, Ohio Department of Developmental
Neurobiology
Lonnie Sears, PhD New York State Institute for Basic
Department of Pediatrics Research in Developmental
University of Louisville Disabilities
Louisville, Kentucky Staten Island, New York
Contributors xxiii
Jerzy Wegiel, VMD, PhD Carrie L. Yochum, MS

Department of Developmental Department of Psychology
Neurobiology Rutgers University
New York State Institute for Basic New Brunswick, New Jersey
Research in Developmental
Disabilities
Xiongwei Zhu, PhD
Department of Pathology
Martha G. Welch, MD Case Western Reserve University
Division of Developmental Cleveland, Ohio
Neuroscience
Department of Psychiatry Andrew W. Zimmerman, MD
and Department of Neurology
and Developmental Medicine
Department of Pathology and Cell Biology Kennedy Krieger Institute
Columbia University Medical Center Baltimore, Maryland
New York, New York
and
Thomas Wisniewski, MD, PhD
Department of Psychiatry Departments of Neurology and
New York University School Psychiatry and Behavioral
of Medicine Sciences
Silberstein Aging and Dementia Johns Hopkins University School
Research and Treatment Center of Medicine
New York, New York Baltimore, Maryland
1 Type, Topography,
and Sequelae
of Neuropathological
Changes Shaping
Clinical Phenotype
of Autism
Jerzy Wegiel,1,* Thomas Wisniewski,2 Abha
Chauhan,3 Ved Chauhan,3 Izabela Kuchna,1
Krzysztof Nowicki,1 Humi Imaki,1 Jarek Wegiel,1
Shuang Yong Ma,1 Teresa Wierzba Bobrowicz,4
Ira L. Cohen,5 Eric London,5 and W. Ted Brown6
Departments of 1Developmental Neurobiology,
3 Neurochemistry, 5 Psychology, and 6 Human Genetics,
New York State Institute for Basic Research in

Developmental Disabilities, Staten Island, NY 10314, USA
2Department of Psychiatry, New York University School
of Medicine, Silberstein Aging and Dementia Research
and Treatment Center, New York, NY 10016, USA
4Department of Neuropathology, Institute of
Psychiatry and Neurology, Warsaw, Poland
CONTENTS
1.1 Introduction .....................................................................................................2
1.2 Clinical, Etiological, and Neuropathological Diversity in Autism .................3
1.2.1 Clinic.................................................................................................. 3
1.2.2 Etiology ..............................................................................................4
1.2.3 Neuropathology..................................................................................5
* Corresponding author: Tel.: +1-718-494-5231; fax: +1-718-982-4856; e-mail: [email protected]
1
2 Autism: Oxidative Stress, Inflammation, and Immune Abnormalities
1.3 Deregulation of Brain Growth in Early Childhood ........................................6

1.3.1 Developmental Heterochronicity .......................................................7
1.3.2 Functional Consequences of Abnormal Brain Development............. 8
1.4 Cortical and Subcortical Neuropathology ......................................................8
1.4.1 Cortical Dysgenesis, Lamination Defects,
Migration Disturbances .....................................................................8
1.4.2 Brain Structure–Specific Delay of Neuronal Growth .......................9
1.4.3 Minicolumnar Abnormalities in Autism ......................................... 10
1.5 Neuronal Oxidative Stress and Metabolic Changes...................................... 10
1.5.1 Oxidative Stress in Autism .............................................................. 10
1.5.2 Lipofuscin in Autism ....................................................................... 11
1.5.3 β-Amyloid Precursor Protein and Intraneuronal
Amyloid β in Autism .......................................................................12
1.6 Clinicopathological Correlations .................................................................. 13
1.6.1 Speech, Language, and Verbal and Nonverbal
Communication................................................................................ 13
1.6.2 Face Perception ................................................................................ 13
1.6.3 Social Attachment—The Role of the Hypothalamus
in Behavioral Deficits ...................................................................... 14
1.6.4 Sensorimotor Deficits, and Repetitive
and Stereotyped Behaviors .............................................................. 15
1.6.5 Cognitive Deficits............................................................................. 17
1.6.6 Epilepsy-Associated Pathology ........................................................ 17
1.7 Mechanisms Affecting Brain Development .................................................. 18
1.7.1 BDNF and Neurotrophins in Autism ............................................... 18
1.7.2 Brain Stem and the Role of Serotonin in Brain
Development and Clinical Features of Autism ................................ 19
1.8 Closing Remarks.............................................................................................20
Acknowledgments....................................................................................................20
References ................................................................................................................ 21
1.1 INTRODUCTION
The aim of this chapter is to identify the type, topography, and sequelae of neuro-
pathological changes that contribute to the clinical phenotype of autism. Results
of recent magnetic resonance imaging (MRI) and postmortem neuropathological
and stereological studies of autism brain suggest a dynamic model of sequen-
tial subdivision of age- and brain-specific structural and functional changes.
Acceleration of brain growth in the first year of life and deceleration in the second
and third years appear to play a pivotal role in the onset of clinical signs of
autism (Courchesne and Pierce, 2005b; Courchesne et al., 2001, 2003; Dawson
et al., 2007; Dementieva et al., 2005; Gillberg and de Souza, 2002; Redcay and
Courchesne, 2005). The range of deviation from the normal trajectory of brain
growth may be a factor determining the severity of the disease (Courchesne et al.,
2003). Developmental heterochronicity (differential rates of growth of various
brain regions compared to controls), resulting in selective overgrowth of some brain
Type, Topography, and Sequelae of Neuropathological Changes 3
regions, appears to be a key factor determining topography and brain region–

specific type of cytoarchitectonic changes (Carper and Courchesne, 2005; Carper
et al., 2002; Courchesne et al., 2001; Hazlett et al., 2005; Sparks et al., 2002).
Topographic developmental heterochronicity may result in impairment of both
local and global connectivity, leading to local overconnectivity and impairment of
long-distance connectivity (Baron-Cohen, 2004; Casanova et al., 2006; Courchesne
and Pierce, 2005a). Stereological studies have revealed neuronal developmental het-
erochronicity in early childhood, resulting in selective developmental delay of the
growth of neurons in some subcortical structures and the cerebellum during the most
critical stage of development of social behaviors and communication skills (Wegiel
et al., 2008). Distortions of brain and neuronal development are reflected in abnormal
cortical minicolumn organization (Casanova et al., 2002, 2006), local dysgenesis, and
ectopias (Bauman and Kemper, 1985; Bauman et al., 1997; Kemper and Bauman,
1993, 1998). These complex developmental abnormalities appear to lay the foundation
for secondary and tertiary metabolic, structural, and functional changes, including
seizures and risk of sudden unexpected death; signs of oxidative stress, early and
enhanced accumulation of products of cell organelle degradation with lipofuscin
deposition; modified processing of β-amyloid precursor protein with accumulation of
truncated amyloid beta; and other as of yet unidentified changes. Secondary patho-
logic changes appear to be indicators of the susceptibility of abnormally developing
neurons to further modifications during cell maturation and aging. The pattern of
morphological changes emerging from these multidisciplinary studies appears to
represent a major trend. However, modifications of the course of disease and sub-
patterns of developmental changes result in a broad spectrum of morphological and
clinical interindividual differences.
1.2 CLINICAL, ETIOLOGICAL, AND NEUROPATHOLOGICAL

DIVERSITY IN AUTISM
Autism is the prototype of a pervasive developmental disorder (PDD) and is charac-
terized by (a) qualitative impairments in reciprocal social interactions, (b) qualitative
impairments in verbal and nonverbal communication, (c) restricted repetitive and
stereotyped patterns of behavior, interests, and activities, and (d) onset prior to the
age of 3 years. PDD also includes childhood disintegrative disorder, Asperger’s dis-
order, Rett syndrome, and pervasive developmental disorder—not otherwise specified
(PDD-NOS). The common features of all these disorders are qualitative deficits in
social behavior and communication (American Psychiatric Association, 2000).
1.2.1 CLINIC
In most cases (90%–95%), it is not presently possible to detect a known or specific
etiology. These cases are referred to as idiopathic or nonsyndromic autism (Boddaert
et al., 2009; Gillberg and Coleman, 1996). In 6% (Fombonne, 2003), 5% (Tuchman et al.,
1991), or 10% (Rutter et al., 1994) of cases, autism was diagnosed in association with
other disorders. About 30% of children with idiopathic autism have complex autism,
defined by the presence of dysmorphic features, microcephaly and/or a structural brain
malformation (Miles et al., 2005). About 70% of children with autism have essential
autism, defined by the absence of physical abnormalities. For most children, the onset
of autism is gradual. However, a multisite study revealed significant regression at ages
of 18 to 33 months (regressive autism) in about 13.8% (Colorado) to 31.6% (Utah) of
autistic subjects (Department of Health and Human Services, 2007). Moreover, the
manifestations of autism vary greatly, depending on developmental level and chrono-
logical age of the affected individual. The majority of patients exhibit serious social
and communicative impairments throughout life but some improve enough to be able
to live relatively independently as adults. In 44.6% of children, autism is associated
with cognitive impairment (defined as having intelligence quotient scores of <70;
Department of Health and Human Services, 2007). Expressive language function in
individuals with autism may vary from mutism to verbal fluency (Rapin, 1996;
Stone et al., 1997; Wetherby et al., 1998). Sensorimotor deficits also show significant
interindividual differences, with more frequent and severe impairments of gross and
fine motor function (motor stereotypes, hypotonia, limbic apraxia) in subjects with
lower IQ (Rogers et al., 1996). Hand mannerisms and body rocking are reported in
37% to 95% of individuals with autism (Lord and Rutter, 1995; Rapin, 1996; Rogers
et al., 1996), whereas preoccupation with sensory features of objects, abnormal
responsiveness to environmental stimuli, or paradoxical responses to sensory stimuli
are seen in 42% to 88% of people with autism (Kientz and Dunn, 1997). Epilepsy is a
comorbid complication, occurring in up to 33% of individuals with autism (Tuchman
and Rapin, 2002).
1.2.2 ETIOLOGY
The clinical diversity of autism reflects the etiologic heterogeneity of this disorder.
Genetic factors; pre-, peri-, and postnatal pathological factors; and concurrent dis-
eases may contribute to autism (Muhle et al., 2004; Newschaffer et al., 2002; Rutter
et al., 1994). About 5% to 10% of cases are associated with several distinct genetic
conditions including fragile X syndrome, tuberous sclerosis, phenylketonuria, Rett
syndrome, and chromosomal anomalies such as Down syndrome (DS) (Folstein
and Rosen-Scheidley, 2001; Fombonne, 2003; Smalley et al., 1988; Yonan et al.,
2003). Autism spectrum disorders (ASDs) in people with DS have been described
in several reports (Ghaziuddin et al., 1992; Howlin et al., 1995; Prasher and Clarke,
1996; Wakabayashi, 1979), and the prevalence of autism in boys with DS was esti-
mated as at least 7% (Kent et al., 1999). The prevalence of autism in the fragile X
syndrome is estimated as 15%–28% (Hagerman, 2002). Cytogenetic abnormalities
(partial duplications, deletions, inversions) in the 15q11-q13 region account for 1% to
4% of autism cases (Cook, 1998; Gillberg, 1998). Several potential candidate genes
have been identified in both autosomes and X chromosomes, including the tuberous
sclerosis gene on chromosomes 9 and 16; serotonin transporter on chromosome 17;
gamma-aminobutyric acid receptor-beta 3 on chromosome 15; neuroligins on the
X chromosome (see Vorstman et al., 2006); and possibly PTEN on chromosome 10
(Butler et al., 2005). Modifications in the tryptophan hydroxylase gene may play a
modest role in autism susceptibility (Coon et al., 2005).
1.2.3 NEUROPATHOLOGY
While knowledge of the clinical and genetic factors in autism is based on examination of
thousands of patients, postmortem neuropathological studies are based on reports
of a very small number of brains. A review by Palmen et al. (2004) revealed that
between 1980 and 2003, only 58 brains of individuals with autism have been exam-
ined, and results of only a few neuropathological and stereological studies were pub-
lished. Usually, neuropathological reports and morphometric reports were based on
evaluation of one or several brains. Due to the broad age spectrum and the etiological
and clinical diversity in autism, the pattern of neuropathological changes reported is
incomplete and often inconsistent. As a result, the morphological markers and neu-
ropathological diagnostic criteria of autism have not yet been established (Lord et al.,
2000; Pickett and London, 2005). In the past, the contribution of postmortem studies
to the detection and characterization of neuropathological changes and mechanisms
leading to structural and functional manifestations of autism was limited because
of (a) the deficit of autism brains, resulting in a lack of statistical power, (b) the lack of
efficient mechanisms for sharing the limited tissue resources, (c) the lack of complex
studies of the dynamic of changes during the life span, (d) the infrequent application
of unbiased morphometric methods to detect quantitative differences, and (e) the averag-
ing of results from subjects with different clinical and morphological manifestations
of autism. Heterogeneity within the autism spectrum is the major obstacle to autism
research at all levels (Newschaffer et al., 2002), including neuropathological stud-
ies and attempts at detection of clinicopathological correlations. Recent evidence of
genetic fractionation of social impairment, communication difficulties, and rigid and
repetitive behaviors indicates that heterogeneity in ASD could be an unavoidable con-
sequence of the contribution of nonoverlapping genes. If different features of autism
are caused by different genes associated with different brain regions and related to dif-
ferent core cognitive impairments (Happe et al., 2006), it seems likely that many brain
networks are involved in the pathology of autism. The diversity of neuropathological
findings and the commonly reported inconsistencies in regional findings correspond
to developmental impairments in many interacting brain networks and to expansion
from “local” abnormalities to “nonlocal” effects of the emerging cognitive system.
In spite of these limitations, “localizing” models are still the main approach to the
identification of pathological changes as a component of the structural and functional
abnormalities of the networks (Müller, 2007).
The possibility that autism is associated with neuropathological changes was
explored in the first studies reported between 1980 and 1989 (Bauman and Kemper,
1985; Courchesne et al., 1987, 1988; Damasio et al., 1980; Gaffney et al., 1987;
Hashimoto et al., 1989, 1993; Murakami et al., 1989; Ritvo et al., 1986). Expansion
of these studies through examination of larger cohorts and application of stereology,
functional and structural MRI, and biochemistry resulted in the identification of
several major forms of pathology contributing to the clinical phenotype including
abnormal acceleration of brain growth in early childhood (Redcay and Courchesne,
2005), delay of neuronal growth (Wegiel et al., 2008), changes in brain cytoarchitec-
ture (Bailey et al., 1998; Bauman and Kemper, 1985; Casanova et al., 2002, 2006),
metabolic modifications with abnormal amyloid precursor protein (APP) processing

(Bailey et al., 2008; Brown et al., 2008; Sokol et al., 2006), enhanced oxidative stress
(reviewed in Chauhan and Chauhan, 2006), and turnover of cell organelle with pigment
accumulation and glial activation (Lopez-Hurtado and Prieto, 2008).
1.3 DEREGULATION OF BRAIN GROWTH

IN EARLY CHILDHOOD
The major measures of age-related changes are head circumference, MRI-based
volumetry of the brain and brain structures, and postmortem brain weight and volume
of brain subdivisions. Between 1990 and 2000, several groups reported increased
head circumference (Bailey et al., 1995; Bolton et al., 1995; Davidovitch et al., 1996;
Fidler et al., 2000; Fombonne et al., 1999; Lainhart et al., 1997; Miles et al., 2000;
Steg and Rapoport, 1975; Stevenson et al., 1997), whereas MRI-based studies revealed
increased brain volume (Piven et al., 1995, 1996). According to Fombonne et al.
(1999), the prevalence of macrocephaly in autism is about 20%. In a report by Bailey
et al. (1998), four of six subjects with autism 4 to 24 years of age had macrocephaly.
Increased brain weight was reported in postmortem studies by Bailey et al. (1993)
and Kemper and Bauman (1998). Increase in the volume was regional and not gen-
eralized, with the greatest enlargement in the occipital and parietal lobes (Filipek,
1996; Filipek et al., 1999; Piven et al., 1995, 1996). However, in several studies, an
increase in brain size was not detected (Garber and Ritvo, 1992; Haznedar et al.,
2000). Inconsistency in detection of abnormal head and brain size can be associated
with interindividual differences, the age of examined individuals, and the methods
applied. Courchesne et al. (2003) integrated their work and that of other researchers
into the concept of four phases of modified brain growth, described below.
At birth, the head circumference of neonates later diagnosed with autism is
normal or slightly less than that observed in normally developing children (Courchesne
et al., 2003; Dawson et al., 2007; Dementieva et al., 2005; Dissanayake et al., 2006;
Gillberg and de Souza, 2002; Hazlett et al., 2005; Lainhart et al., 1997; Mason-Brothers
et al., 1990; Stevenson et al., 1997). Slight undergrowth is independent of body growth
and may be a reflection of prenatal neural developmental defects corresponding to
pathology detected in postmortem studies of the brains of autistic adults (Bailey
et al., 1998; Casanova et al., 2002; Courchesne et al., 2003; Kemper and Bauman,
1998). In only 5% of neonates diagnosed later as autistic was the head circumference
more than that in normally developing infants (Courchesne et al., 2003; Dementieva
et al., 2005).
In the second phase, by 1 or 2 years of age, a rapid and large increase in head
circumference distinguished children diagnosed later with autism from normally
developing children (Courchesne et al., 2003; Dawson et al., 2007; Dementieva
et al., 2005; Dissanayake et al., 2006; Hazlett et al., 2005). Ninety percent of 2- and
3-year-old children with autism had brain volumes larger than those of control
children (Courchesne at al., 2001). According to Dawson et al. (2007), a period of
exceptionally rapid head growth is limited to the first year of life, and head growth
decelerates after 12 months of age. Acceleration of growth in head circumference
appears to begin at about 4 months (Courchesne and Pierce, 2005a; Gillberg and
de Souza, 2002; Redcay and Courchesne, 2005). Using meta-analysis based on eval-
uation of head circumference converted to brain volume, brain volume measured
from MRI, and brain weight from postmortem studies, Redcay and Courchesne
(2005) revealed that brain size increases from 13% smaller than in control subjects
at birth to 10% larger than in control infants at 1 year, but only 2% greater by ado-
lescence. The greater growth rate of head circumference in the first year, and its
return to normal rates thereafter, is not accounted for by an overall growth in stature.
Studies of behavioral development in infants later diagnosed with autism suggest
that the period of acceleration of head growth precedes and overlaps with the onset
of behavioral changes, and that the period of deceleration coincides with a period of
behavioral decline or worsening of symptoms in the second year of life (Dawson
et al., 2007). Coincidence of acceleration of brain growth rate with onset and
worsening of clinical symptoms may indicate that structural developmental changes
critical for a lifelong phenotype occur in early infancy. Acceleration of brain growth
in the first year and deceleration in the second year of life suggest that failure of the
mechanism controlling brain growth in the first year of life plays an essential role in
the onset of clinical features of autism. Identification of these mechanisms may lead
to conceptualization of early preventive treatments.
In the third phase, of 2 to 4 years, the overall rate of brain growth slows but is still
10% more than in normally developing children (Carper et al., 2002; Courchesne
et al., 2001; Hazlett et al., 2005; Sparks et al., 2002). In 4- to 5-year-old autistic
children, MRI-based estimated brain volume is 1350 mL, whereas in normally devel-
oping children, a comparable volume (1360 mL) is reached about 8 years later. In post-
mortem studies, the brain weight of 3- to 5-year-old autistic males was 15% higher
(1451 g) than in control males of this age (1259 g) (Redcay and Courchesne, 2005).
In the fourth phase, the volume of the brain decreases, and this trend extends from
middle/late childhood through adulthood. Head (Aylward et al., 2002) or brain enlarge-
ment (Bailey et al., 1998; Hardan et al., 2001; Lainhart et al., 1997; Piven et al., 1995,
1996) has also been observed in studies of older populations of autistic individuals.
However, by adolescence and adulthood, the average size of the brain is only 1% to 3%
greater in autistic than in control cohorts (Redcay and Courchesne, 2005).
Moreover, the pattern of brain growth reflects the severity of clinical manifestation
of autism (Courchesne et al., 2003). Among infants who have the more severe form of
autism, 71% showed increases during their first year of more than 1.5 S.D., with 59%
showing increases between 2.0 and 4.3 S.D. In children with a less severe form of
autism, PDD-NOS, acceleration of brain growth is observed later, and the increase
is less pronounced. Later onset and slower rate of progression of autism appear to be
associated with a better outcome.
1.3.1 DEVELOPMENTAL HETEROCHRONICITY

Developmental heterochronicity studies indicate that autism is a disorder involving a
transient period of pathological acceleration of brain growth. Developmental het-
erochronicity, with different rates of growth for different brain regions/structures,
appears to be the second major factor contributing to the clinical phenotype. MRI
studies showed that overgrowth of the frontal and temporal lobes and amygdala, brain
regions that are involved in cognitive, social, and emotional functions as well as lan-
guage development, is synchronized with brain overgrowth in 2- to 4-year-old autistic
children in contrast to a different rate of growth of the occipital cortex (Carper and
Courchesne, 2005; Carper et al., 2002; Courchesne et al., 2001; Hazlett et al., 2005;
Sparks et al., 2002). The reduced size of the body and posterior subregions of the
corpus callosum noted in subjects with autism may indicate disproportions in brain
subregions development (Piven et al., 1997b). The cellular and molecular basis for
transient acceleration of brain growth and enhanced growth of some brain regions
is not known, but Courchesne et al. (2003) proposed that the observed pattern is
associated with an excessive number of neurons, enhanced rate of growth of size of
neurons, and increased number of minicolumns as well as excessive and premature
expansion of the dendritic tree.
1.3.2 FUNCTIONAL CONSEQUENCES OF ABNORMAL BRAIN DEVELOPMENT

Using a computational model analogue of autism, Cohen (2007) has argued that an
interaction between stochastic and above-average or “excessive” numbers of neural
connection factors has implications for understanding the disorder. In particular, a
relative excess of connections could lead to enhanced recognition of complex patterns
in the environment. In Cohen’s chapter, it was noted that if large and complex brains
are in part familial (Courchesne et al., 2003; Fidler et al., 2000), and brain size is her-
itable (Pfefferbaum et al., 2000) and positively correlated with IQ (Pennington et al.,
2000), then behavioral outcomes both within and across generations of family members
could result in (a) individuals who may be unusually gifted in their ability to handle
complex nonlinear problems such as mathematics or computer science, (b) individuals
with autism, or (c) individuals with a combination of autism or autistic-like behavior
and giftedness (many typical Asperger’s cases). These trends are detected among
relatives of subjects with autism (Folstein and Rutter, 1988).
The effect of an abnormal trajectory of brain development observed in autism are
well-validated characteristics of the learning style of children with autism, includ-
ing (a) greater attention to idiosyncratic than socially relevant stimuli, (b) stimulus
overselectivity or a lack of drive for central coherence, (c) problems with acquiring
fuzzy concepts, (d) development of savant skills, (e) problems with generalization
of previously acquired skills, (f) rigidity and resistance to change, (g) social and
communication deficits, and (h) difficulty in learning complex higher-order concepts
(Cohen, 2007).
1.4 CORTICAL AND SUBCORTICAL NEUROPATHOLOGY

1.4.1 CORTICAL DYSGENESIS, LAMINATION DEFECTS, MIGRATION DISTURBANCES
The fundamental characteristics of the neuropathological changes described by
Kemper and Bauman (1993, 1998), Bauman and Kemper (1985, 1996), and Bauman
et al. (1997) suggest three major neuropathologies in the brain of people with autism:
(a) curtailed development of neurons in the structures that are substrates for memory
and emotions—the entorhinal cortex, hippocampus, subiculum, anterior cingulate
gyrus and mamillary body; (b) a congenital decrease in the number of Purkinje
cells in the cerebellum; and (c) age-related differences in cell size and number of neu-
rons in the cerebellar nuclei and in the inferior olivary nucleus. Microdysgenesis is
represented by increased neuronal density in the cortical layer, clustering of cortical
neurons, disorganization of cortical layers, neuron cytomegaly, ectopic neurons, and
nodular heterotopias. A detailed study of serial sections from the brain of a 29-year-old
man with autism revealed reduced neuronal size and increased cell-packing density
(Bauman and Kemper, 1985), both features of an immature brain (Friede, 1975).
Cell-packing density was increased by 66% in the hypothalamus and mamillary
body, and by 54% in the medial septal nucleus, with smaller nerve cells. The reduced
size of neurons and the selective increase in cell-packing density were seen in central
(40%), medial (28%), and cortical nuclei (35%). Atrophy of the neocerebellar cortex,
with marked loss of Purkinje cells and, to a lesser extent, of granule cells, was pres-
ent in gracile, tonsil, and inferior semilunar lobules. Changes were not detected in
the anterior lobe or the vermis. Reduced numbers of cells were noticed in fastiglial,
globose, and emboliform nuclei, and cells were small and pale. The dentate nucleus
was distorted. Retrograde neuronal loss in the inferior olive related to neuronal loss
in cerebellar cortex was not found, but olivary neurons were small and pale. Brain
cytoarchitecture abnormalities were not associated with gliosis. In a 21-year-old
female with autism, Rodier et al. (1996) found that the brain was smaller than a con-
trol brain, and the length of facial nerve nucleus was less than 500 μm as compared
to 2610 μm in the control subject.
1.4.2 BRAIN STRUCTURE–SPECIFIC DELAY OF NEURONAL GROWTH

The reduced size of neurons and their nuclei in the cortex of autistic subjects reported
by Casanova et al. (2006) could be an indicator of reduced or impaired functional
connectivity between distant cortical regions (Casanova et al., 2006; Just et al., 2004;
Koshino et al., 2005). Our ongoing studies of series of brains from age-matched autis-
tic and control subjects (Wegiel et al., 2008) indicate that reduced size of neurons
is a brain structure–specific marker. In 4- to 7-year-old autistic children, Purkinje
cells were smaller by 38%. Neurons in the dentate nucleus were reduced by 26%; in
the amygdala, by 24%; in the nucleus accumbens, by 41%; in caudate, by 20%; and
in the putamen, by 27%. Neurons in the nucleus of the facial nerve and the nucleus
olivaris did not show a significant difference from controls. The second significant
feature of the pattern of neuronal size abnormalities is the partial or complete correc-
tion of the size of neurons (for example, in the nucleus accumbens) observed in late
childhood or adulthood. This study indicates that the delay of growth of neurons is
the most consistent pathology detected in the brains of examined people with autism.
Pathology is brain structure–specific. Changes may range from no delay to severe
developmental delay. The youngest examined children (4 to 7 years old) show the
most severe deficit in the volume of the neuronal body and nucleus. Partial correc-
tion of cell volume is observed in late childhood and adulthood, which indicates that
brain structure and function undergo modifications during the life span. The study
of basal ganglia and cerebellum supports the hypothesis that clinical manifestations of
autism are the result of regional neuronal maldevelopment.
One may assume that mechanisms regulating growth of the neuron in early child-
hood are the target of factors that are the cause of autism. The result of deregulation
of these mechanisms could be (a) significantly delayed growth of neuronal body,
nucleus, dendritic tree, spines, and reduced number of synapses and (b) functional
deficits corresponding to these structural developmental delays. These abnormalities
of very early childhood might be the major contributor to clinical deficits that are the
basis for the clinical diagnosis of autism at the age of 3 years.
1.4.3 MINICOLUMNAR ABNORMALITIES IN AUTISM

The next significant contribution to detection of neocortical developmental pathol-
ogy is the result of studies of minicolumns by Casanova’s group (Buxhoeveden and
Casanova, 2002; Casanova et al., 2002, 2006). Malformations of cortical develop-
ment are observed in heterogeneous disorders caused by abnormalities of cell pro-
liferation, apoptosis, cell migration, cortical organization, and axon pathfinding
(Hevner, 2007). Clinically malformations of cortical development are significant
causes of mental retardation, seizures, cerebral palsy, and neuropsychiatric disor-
ders (Barkovich et al., 2005; Guerrini and Marini, 2006; Sarnat and Flores-Sarnat,
2004). Minicolumns are considered a basic architectonic and functional unit of
the human neocortex (Buxhoeveden and Casanova, 2002; Casanova et al., 2002).
Increased neuron density by 23%, reduced size of neurons in minicolumns, and a
concomitant increase in the total number of minicolumns appears to illustrate the
bias of local rather than global information processing (Casanova et al., 2002, 2006),
resulting in a “hyper-specific brain” (McClelland, 2000). Synchronization of inter-
actions requiring the involvement of distant brain regions is impaired in autism as a
result of developmental connectivity deficits (underconnectivity) of smaller neurons
(Just et al., 2004; Koshino et al., 2005; Zilbovicius et al., 1995). Structural imaging
studies also suggest the overrepresentation of short association fibers in autism, with
a regional increase in the volume of white matter (Herbert et al., 2004) favoring the
local information processing observed in autistic subjects (Happe, 1999).
1.5 NEURONAL OXIDATIVE STRESS AND METABOLIC CHANGES

An increasing body of evidence suggests that the abnormal rate of development of
neurons and neuronal networks in early infancy is followed by metabolic changes,
with signs of oxidative stress, enhanced autophagocytosis, and lipofuscin accumulation,
leading to early selective neuronal structural and functional changes.
1.5.1 OXIDATIVE STRESS IN AUTISM

Oxidative stress is known to be associated with premature aging of cells and can lead
to inflammation, damaged cell membranes, autoimmunity, and cell death. The brain
is highly vulnerable to oxidative stress due to its limited antioxidant capacity, higher
energy requirement, and high amounts of unsaturated lipids and iron (Juurlink and
Peterson, 1998). The brain makes up about 2% of body mass but consumes 20% of
metabolic oxygen. The vast majority of energy is used by the neurons (Shulman
et al., 2004). Glutathione (GSH) is the most important antioxidant for detoxification
and elimination of environmental toxins. Due to the lack of glutathione-producing

capacity by neurons, the brain has a limited capacity to detoxify reactive oxygen
species (ROS). Therefore, neurons are the first cells to be affected by the increase in
ROS and shortage of antioxidants and, as a result, they are most susceptible to oxida-
tive stress. Antioxidants are required for neuronal survival during the early critical
period (Perry et al., 2004). Children are more vulnerable than adults to oxidative
stress because of their naturally low glutathione levels from conception through
infancy (Erden-Inal et al., 2002; Ono et al., 2001). The risk created by this natural
deficit in detoxification capacity in infants is increased by the fact that some envi-
ronmental factors that induce oxidative stress are found at higher concentrations in
developing infants than in their mothers, and accumulate in the placenta.
Accumulating evidence from our and other groups suggests increased oxidative
stress in autism (reviewed in Chauhan and Chauhan, 2006). Lipid peroxidation is a
chain reaction between polyunsaturated fatty acids and ROS, producing lipid perox-
ides and hydrocarbon polymers that are both highly toxic to the cell. We have reported
that levels of malondialdehyde (MDA), a marker of lipid peroxidation, are increased in
the plasma from children with autism (Chauhan et al., 2004). Other studies on erythro-
cytes (Zoroglu et al., 2004) and urine samples (Ming et al., 2005) have also indicated
increased levels of lipid peroxidation markers in autism, thus confirming an increased
oxidative stress in autism. Recent studies with the postmortem brain samples from
autism and control subjects have provided further evidence on increased oxidative
stress in autism. Increased levels of lipid-derived oxidative protein modifications, i.e.,
carboxyethylpyrrole and iso[4]levuglandin E2–protein adducts, and heme-oxygenase-1
(an inducible antioxidant enzyme) have been reported in the autistic brain, primarily
in the white matter (Evans et al., 2008). Sajdel-Sulkowska et al. (2008) have reported
elevated levels of 3-nitrotyrosine (a specific marker for oxidative damage to proteins)
in the cerebella of subjects with autism. In addition, we have observed increased lipid
peroxidation in cerebellum and temporal cortex of brain in autism (Chauhan et al.,
2009). MDA levels were significantly increased by 124% in the cerebellum, and by
256% in the temporal cortex in autism as compared to control subjects.
1.5.2 LIPOFUSCIN IN AUTISM

Lopez-Hurtado and Prieto (2008) revealed a significant increase in the number of
lipofuscin-containing cells in the brain of 7- to 14-year-old autistic subjects (by 69% in
area 22, 149% in area 39, and 45% in area 44). The increase in the number of lipofuscin-
containing cells was paralleled by neuronal loss and glial proliferation. Lipofuscin
accumulation is a component of aging (Brunk and Terman 2002a,b; Brunk et al., 1992;
Szweda et al., 2003), the neurodegeneration observed in Alzheimer’s (Stojanovic
et al., 1994) and Parkinson’s diseases (Tórsdóttir et al., 1999), developmental syndromes
such as Rett syndrome (Jellinger et al., 1988), and autism (Lopez-Hurtado and Prieto,
2008), and such psychiatric disorders as bipolar affective disorder (Yanik et al., 2004)
and schizophrenia (Akyol et al., 2002; Herken et al., 2001).
Lipofuscin is an intralysosomal deposit of products of autophagocytosis and
degradation of cytoplasmic components, including mitochondria, which cannot be
degraded further or exocytosed. Oxidative stress is considered the factor contribut-
ing to lipid and protein damage and degradation, resulting in lipofuscin production
and accumulation (Brunk et al., 1992; Sohal and Brunk, 1989). The presence of
oxidatively modified proteins and lipids in lipofuscin supports the causative link
between enhanced oxidative stress, autophagocytosis, and deposition of products of
degradation in the lysosomal pathway and lipofuscin (Brunk and Terman, 2002a,b;
Szweda et al., 2003; Terman and Brunk, 2004) and suggests that in autism, abnormal
development is associated with early signs of oxidative stress and enhanced degradation
and, possibly, turnover of cytoplasmic components.
1.5.3 b-AMYLOID PRECURSOR PROTEIN AND INTRANEURONAL

AMYLOID b IN AUTISM
Sokol et al. (2006) detected signs of overexpression of APP in about 40% of autistic
subjects. The levels of secreted APP in plasma in children with severe autistic behav-
ior and aggression were two or more times the levels in children without autism,
and up to fourfold more than in children with mild autism. The trend observed
in autistic children, with higher levels of secreted β-APP and nonamyloidogenic
secreted β-APP, and lower levels of Aβ 1–40 compared to controls, suggests an
increased α-secretase pathway in autism (anabolic nonamyloidogenic APP process-
ing). Enzyme-linked immunosorbent assay (ELISA) study of blood plasma from 25
autistic children 2–4 years of age and 25 age-matched control children revealed sig-
nificantly increased level of secreted amyloid precursor protein alpha (sAPP-α) in
60% of autistic children (Bailey et al., 2008). Western blotting analysis confirmed
higher levels of sAPP-α in autistic children.
Amino-terminally truncated intraneuronal amyloid (Aβ) is present in the neu-
rons of control subjects, and the amount of intraneuronal Aβ increases with age
(Wegiel et al., 2007). This study of 10 brains of autistic people revealed enhanced
intraneuronal accumulation of amino-terminally truncated Aβ in 50% of autistic
subjects, including in one 5-year-old child and four adults 20, 23, 52, and 62 years of
age. A similar pattern was also found in four examined brains of people with autism
and isodicentric chromosome 15 (idic15) (Brown et al., 2008). In idic15, excessive
accumulation of intraneuronal Aβ might be related to an extra copy of one of the
amyloid precursor protein-binding protein (APBA-2) genes localized on chromo-
some 15. In many brain regions, Aβ is accumulated in large cytoplasmic gran-
ules corresponding to deposits of lipofuscin. Numerous large lipofuscin deposits
with very strong Aβ immunoreactivity in the neurons of several children and adults
with autism appear to reflect severe metabolic stress affecting all of the neurons
in the amygdala, all large neurons in the caudate/putamen, a majority of Purkinje
cells, and the neurons in the dentate nucleus and nucleus olivaris, but only about
30%–40% of cortical pyramidal neurons. Accumulation of truncated Aβ appears
to be a by-product of enhanced degradation of transmembrane APP. The aggregated
intracellular Aβ induces the production of ROS and lipid peroxidation products and
ultimately results in leakage of the lysosomal membrane (Glabe, 2001). This pro-
cess appears to affect many neuronal populations, not only in young and old adults,
but also in children diagnosed with autism. A metabolic shift with Aβ accumulation
in neurons in these brain areas that are involved in the expression of emotions, ste-
reotypic behaviors, and social deficits, such as the amygdala, hippocampus, some
striatal subdivisions, and cerebellum, may contribute to cellular dysfunction and the
clinical expression of autism.
1.6 CLINICOPATHOLOGICAL CORRELATIONS

Studies of clinicopathological correlations cover several domains of functional deficits
in people with autism, including (a) speech, language, and verbal and nonverbal
communication, (b) social deficits and face perception, (c) sensorimotor deficits, and
(d) cognitive deficits.
1.6.1 SPEECH, LANGUAGE, AND VERBAL AND NONVERBAL COMMUNICATION

Expressive language function of individuals with autism ranges from complete mutism
to verbal fluency. Verbal abilities are often accompanied by errors in word meaning
(semantics) or language and communicative deficits in social context (social pragmatics)
(Rapin, 1996; Stone et al., 1997; Wetherby et al., 1998). Studies of the language-related
neocortex, including Wernicke’s area (BA 22, speech recognition), Broca’s area (BA
44, speech production) and the gyrus angularis (BA 39, reading) of 7- to 44-year-old
autistic and 8- to 56-year-old control individuals, revealed reduced numerical density
of neurons by 38% in area 22, and by 24% in area 39 in autistic subjects, as well as an
increased numerical density of lipofuscin-containing neurons by 50% in BA 22, and
44% in BA 44. These neuronal changes were paralleled by an increase of numerical
density of glial cells in all three examined regions. Lopez-Hurtado and Prieto (2008)
hypothesized that structural alteration in one or more of these cortical areas may
contribute to the communication impairment observed in autism.
1.6.2 FACE PERCEPTION

All subjects with ASDs have disturbance of social behavior, including abnormalities in
social reciprocity and difficulties in use of eye contact, facial expression, and social
motivation. Social functioning includes eye contact, processing of faces, identifica-
tion of individuals, and monitoring of face expression (Baron-Cohen et al., 1994).
Patients with autism reveal deficits in face-processing (Grelotti et al., 2001), perception
(Schultz, 2005), and recognition (Joseph and Tanaka, 2003).
The face-processing network includes the visual cortex (BA17), which projects
via the inferior occipital gyrus to the fusiform gyrus. Fibers from the fusiform gyrus
project to the amygdala, and inferior frontal gyrus and orbital cortex (Fairhall and
Ishai, 2007; van Kooten et al., 2008). Functional magnetic resonance imaging (fMRI)
identified the fusiform gyrus and other cortical regions as supporting face-processing
in control subjects, and hypoactivity of the fusiform gyrus in autistic patients (Bolte
et al., 2006; Kanwisher et al., 1999; Pierce et al., 2004). Hypoactivation of the fusi-
form gyrus is believed to be associated with the failure to make direct eye contact in
autism (Dalton et al., 2005). Results of imaging-based fusiform volume estimation
are inconsistent. Increased (Waiter et al., 2004) and unchanged (Pierce et al., 2001)
volume in both hemispheres and increased fusiform gyrus in the left hemisphere
(Herbert et al., 2002) were reported. Morphometric studies of the brain of 7 autistic
and 10 control subjects revealed a reduced number of neurons in layers III, V, and
VI, and reduced volume of neuronal soma in layers V and VI in the fusiform gyrus. No
alterations in Brodman area 17 in these autistic individuals suggest that the input from
the visual cortex to the fusiform gyrus is intact. These results indicate the underdevel-
opment of connections in the fusiform gyrus that may contribute to abnormal face
perception in autism (van Kooten et al., 2008).
Bailey et al. (1998) noted abnormalities in cytoarchitectonic organization and
neuronal density in the superior frontal cortex and superior temporal gyrus in
autism. Neurons in the superior temporal sulcus are sensitive to the angle of gaze
(Perrett et al., 1985). Neurons that are attuned to particular facial expressions were
found in the inferior and superior temporal lobes (Hasselmo et al., 1989). Cortical
areas responsive to faces, facial expressions, and angle of gaze send direct projec-
tions to the amygdala (Stefanacci and Amaral, 2000). Pathological changes in the
amygdala may play a central role in the dysfunction seen in autism, including
disturbed components of social cognition such as attention to and interpretation of
facial expressions. fMRI studies show that judging from the expression of another
person’s eyes what the other person might be thinking or feeling is associated with
activation in the superior temporal gyrus, frontal cortex, and amygdala, whereas in
subjects with autism, activation appears in the temporal and frontal cortex but not
in the amygdala (Baron-Cohen et al., 1999).
1.6.3 SOCIAL ATTACHMENT—THE ROLE OF THE HYPOTHALAMUS

INBEHAVIORAL DEFICITS
Experimental studies revealed that the hypothalamic nucleus paraventricularis
(NPV) and the nucleus supraopticus (NSO), producing oxytocin (OT) and vasopressin
(VAS), regulate emotional responses, social attachment, cognitive functions, sleep,
and appetite (Barden, 2004; Ehlert et al., 2001; Manaye et al., 2005). OT and VAS
are relayed from the human brain into the bloodstream via the posterior pituitary.
The presence of receptors for both peptides throughout the forebrain, limbic system,
thalamus, brain stem, and spinal cord (Raggenbass, 2001) indicates that hypotha-
lamic neuropeptides modulate the function of many brain regions. Developmental
changes in the distribution and expression of receptors suggest that the hypothalamic
peptides play a significant role both in brain development and function (Shapiro and
Insel, 1989). OT is required for the development of social memory. In OT knockout
mice, the loss of social memory could be rescued by OT treatment (Ferguson et al.,
2000). VAS is necessary for the formation of social memory and OT for retention of
newly formed social memories (Popik and Van Ree, 1992; Popik et al., 1992). OT
facilitates the learning of social interactions and the formation of associations that
are specifically related to the mother (Nelson and Panksepp, 1996).
The initial product of oxytocin mRNA is a polypeptide containing both nanopeptide
OT and neurophysin I, separated by tripeptide glycine-lysine-arginine. The result of
enzymatic cleavage are intermediate forms containing 10, 11, or 12 amino acids, col-
lectively referred to as carboxy-extended forms of OT (OT-X), and oxytocin (Gabreels
et al., 1998; Gainer et al., 1995; Mitchell et al., 1998; Rao et al., 1992). In 5.8- to
11.5-year-old autistic individuals, reduced plasma OT level, deficits in OT prohormone
processing (increase in OT-X), and an increase in the ratio of C-terminal extended

forms to OT were found. In control children, nearly all OT-X is metabolized to OT,
whereas in autistic children, the immature OT forms serve as the primary circulating
molecule in the absence of or in compensation for OT (Green et al., 2001). However,
experimental studies show that OT-X is not an effective agonist at OT-sensitive sites
(Mitchell et al., 1998). Deficient conversion of OT-X to OT in autism could be the result
of alterations in the level of prohormone convertases associated with genetic defects
(Cook, 1998; Szatmari et al., 1998). The identification of four single nucleotide poly-
morphisms located within the OT receptor gene of 195 Chinese autistic subjects indi-
cates that abnormal modulation of the OT receptor results in autism (Wu et al., 2005).
OT and VAS are known to play a role in repetitive behaviors. Patients with ASDs show
a significant reduction in repetitive behaviors following OT infusion (Hollander et al.,
2003). In about 60% of subjects with autism, abnormal sleep patterns are observed.
VAS is involved in the control of circadian rhythmicity (Swaab, 1997). VAS enhances
aggressiveness, anxiety, stress levels, and the consolidation of fear memory (Bielsky
et al., 2004; Griebel et al., 2002; Landgraf and Neumann, 2004). OT decreases anxiety
and stress; facilitates social encounters, maternal care, and the extinction of condi-
tioned avoidance behavior (Bale et al., 2001; Champagne et al., 2001; Windle et al.,
1997); reduces activation of the amygdala and modulates fear processing (Kirsch et al.,
2005). The presence of abnormal levels of hypothalamic neuropeptides in patients
with autism provides strong evidence that an abnormality in OT, VAS and other hypo-
thalamic neuropeptides may have a significant contribution to the behavioral features
of autism. However, the morphology and biochemistry of the hypothalamus of autistic
subjects remains unknown. The only study of the hypothalamic mammillary body of
a 26-year-old autistic man revealed that the cell-packing density was increased by 66%
(Bauman and Kemper, 1985).
1.6.4 SENSORIMOTOR DEFICITS, AND REPETITIVE AND STEREOTYPED BEHAVIORS

In individuals with autism, impairments of gross and fine motor function recognized as
hypotonia, limbic apraxia, and motor stereotypes are common findings and are more
severe in subjects with lower IQ (Rogers et al., 1996). Hand mannerisms, body rocking,
or unusual posturing are reported in 37% to 95% of individuals (Lord, 1995; Rapin, 1996;
Rogers et al., 1996). In 42% to 88% of subjects with autism, aberrant sensory processing
results in a preoccupation with sensory features of objects, over- or underresponsive-
ness to environmental stimuli or paradoxical responses to sensory stimuli (Kientz and
Dunn, 1997). Sensorimotor deficits may by associated with pathological changes in both
the nigrostriatal system (basal ganglia) and the cerebellum (Bailey et al., 1998; Kemper
and Bauman, 1998; Ritvo et al., 1986; Saitoh and Courchesne, 1998; Sears et al., 1999).
Cerebellar abnormality with a deficit/loss of Purkinje cells (Bailey et al., 1998; Kemper
and Bauman, 1993, 1998; Ritvo et al., 1986) has been a common finding. Individuals
with autism have been classified as affected by cerebellar hyper- or hypoplasia (Saitoh
and Courchesne, 1998). A reduced number of Purkinje cells without significant glial
activation and a reduced size of Purkinje cells were noticed in the majority of cerebellar
reports (Bailey et al., 1998; Fehlow et al., 1993; Kemper and Bauman, 1993; Lee et al.,
2002; Ritvo et al., 1986) in 21 of 29 examined cases (Palmen et al., 2004).
Results of evaluation of the size of the cerebellum using MRI are inconsistent.
In several MRI studies, smaller cerebellar hemispheres (Gaffney et al., 1987;
Murakami et al., 1989) and vermis (Ciesielski et al., 1997; Courchesne et al., 1988;
Hashimoto et al., 1995) were reported. In a study by Piven et al. (1997a), the total
cerebellar volume was found to be greater in subjects with autism than in the control
group, and the increase was proportional to the increased total brain volume. In the
cerebellum, boys with autism had less gray matter, a smaller ratio of gray to white
matter, and smaller lobules VI and VII than did controls. Despite the inconsistency of
reports characterizing topographic autism-associated vermian hypoplasia (Hashimoto
et al., 1993; Kaufmann et al., 2003; Levitt et al., 1999; Piven et al., 1997a; Schaefer et al.,
1996), several reports show associations between the size of the vermis and deficits in
attention-orienting (Harris et al., 1999; Townsend et al., 1999), stereotypic behavior,
and reduced exploration in autism (Pierce and Courchesne, 2001).
The reduced size of the pons, midbrain, and medulla in autism reported by
Hashimoto et al. (1992, 1993, 1995) was not confirmed in other studies (Hsu et al.,
1991; Piven et al., 1992).
Changes in neurons in the deep cerebellar nuclei were noticed by some authors
(Kemper and Bauman, 1998) but not by others (Bailey et al., 1998). Structural
MRI shows variable patterns of changes. Volumetry of the cerebellum may show
no change, hypoplasia, or hyperplasia. Courchesne et al. (1988) reported selective
hypertrophy of lobules VI and VII, but these results were not confirmed in other
subjects. In part, the pattern may correspond to the functional status of subjects. In
highly functioning subjects with autism, hypoplasia of the cerebellum has not been
detected (Holttum et al., 1992).
A decrease in the number of GABAergic Purkinje cells is considered the most
consistent neuropathological finding in autism, as it was detected in at least 50% of
examined cases (Arin et al., 1991; Bailey et al., 1998). Recent studies indicate that
preserved Purkinje cells reveal a 40% decrease in the expression of glutamic acid
decarboxylase 67 (GAD67) mRNA in autistic subjects relative to control patients
(Yip et al., 2007). Moreover, in autism, the basket cells likely provide increased
GABAergic feed-forward inhibition to Purkinje cells. The result may include dis-
ruption in the timing of Purkinje cell firing and altered inhibition of the cerebellar
nuclei, which could directly affect cerebellocortical output, leading to changes in
motor behavior and cognition (Yip et al., 2008).
Repetitive and stereotyped behaviors defined as recurring, nonfunctional activi-
ties, or interests that occur regularly and interfere with daily functioning are a defining
signs of autism. These behaviors include lower-order repetitive motor behavior,
intense circumscribed patterns of interests, and higher-order rituals and compul-
sions (Gabriels et al., 2005). Several studies implicated the role of basal ganglia and
frontostriatal circuitry in the pathophysiology of autism, especially in repetitive and
stereotyped behaviors. Increased volume of the basal ganglia was reported in sev-
eral MRI studies (Herbert et al., 2003; Hollander et al., 2005; Langen et al., 2007;
Sears et al., 1999). Sears et al. (1999) and Hollander et al. (2005) observed a positive
correlation between caudate volumes and repetitive behavior scores. A significant
increase in caudate nucleus volume, disproportional to brain volume, was detected
in MRI studies in two independent samples of medication-naive subjects with autism
(21 high-functioning children and adolescents, and 21 typically developing subjects;

21 high-functioning adolescents and young adults, and 21 healthy subjects) (Langen
et al., 2007). Our studies showing a significantly smaller size of neurons in the cau-
date, putamen, and nucleus accumbens, especially in the brains of children 4–7 years
of age suggest a developmental delay in the growth of neurons in the basal ganglia
of autistic subjects, which may contribute to basal ganglia dysfunction (Wegiel et al.,
2008). MRI and postmortem morphometric studies support the hypothesis that
developmental abnormalities in frontostriatal circuitry contribute to repetitive and
stereotyped behaviors, which are one of three defining symptoms of autism.
1.6.5 COGNITIVE DEFICITS

Many individuals with autism demonstrate a particular pattern on intellectual tests
that is characteristic of autism. Performance IQ is usually higher than verbal IQ, and
block design is the highest subtest, whereas comprehension is usually the lowest (Siegel
et al., 1996). Individuals with autism have poorer adaptive function than would be
predicted by IQ alone (Volkmar et al., 1993).
Cognitive deficits might be related in part to the memory system and limbic region
abnormalities. Reduced volume of both the hippocampal formation and amygdala
were noticed in subjects examined by Aylward et al. (1999), but not in populations
examined by other researchers (Piven et al., 1998). Neurons in the hippocampus
have reduced complexity of dendritic arbors. They are smaller and more densely
packed in various portions of the hippocampal formation, entorhinal cortex, medial
nuclei of the amygdala, medial septal nucleus, mammillary nuclei, and anterior cin-
gulate gyrus (Bauman and Kemper, 1985; Kemper and Bauman, 1993). Haznedar
et al. (1997) observed reduced volume of the anterior cingulate gyrus and decreased
positron emission tomography (PET) activity in subjects with autism. Because the
cerebellum is involved in a variety of cognitive and affective processes, abnormalities
of both the limbic system and the cerebellum may be linked to the core social and
communicative deficits in autism.
The caudate nucleus is an integral component of the frontostriatal network
involved in cognitive functions (Chow and Cummings, 1999; Voelbel et al., 2006),
including learning (Poldrack et al., 1999), short- and long-term memory (Fuh and
Wang, 1995), and planning and problem-solving (Mendez et al., 1989; Schmidtke
et al., 2002). The increased volume of the caudate observed in autistic children may
be indicative of impaired neuronal pruning, contributing to a decrease in executive
function (Voelbel et al., 2006).
1.6.6 EPILEPSY-ASSOCIATED PATHOLOGY

The 1% prevalence of epilepsy in the general population increases to 8% in DS,
10% in AD (Menendez, 2005; Risse et al., 1990; Velez and Selwa, 2003), and 33%
in autism (Tuchman and Rapin, 2002). The interpretation of developmental changes
in autism has been challenged by the need to differentiate among lesions that are
not associated with epilepsy, that cause epilepsy, and that are produced by epilepsy
(Sutula and Pitkanen, 2001). Recent studies support the hypothesis that epilepsy
induces brain alterations that contribute to changes in circuitry, which potentiates the
seizure-genic focus (Armstrong, 2005).
Studies of nonautistic subjects indicate that epilepsy-associated pathology
includes patchy or laminar neuronal loss and gliosis in the cerebral cortex in one or
both hemispheres. In temporal epilepsy, abnormalities were reported in 75% of the
specimens examined, and hippocampal sclerosis was found in 50% (Bruton, 1988).
Loss of hippocampal neurons correlates with the frequency of tonic-cloning seizures
and the total duration of epilepsy (Dam, 1980; Tasch et al., 1999). Loss is accentu-
ated in the CA4 sector and is observed in the granule cell layer in the dentate gyrus.
Dispersion of dentate gyrus granular neurons might be a result of seizure-related,
disturbed migration of neurons (Bengzon et al., 1997), or epilepsy-enhanced neuro-
genesis (Ericksson et al., 1998). Ammon horn sclerosis is a progressive lesion that
can be induced and propagated by seizures (Armstrong, 2005).
In nearly all cases with hippocampal pathology, changes are also observed in
other brain regions. In about 25%, the amygdala, thalamus and mammillary body
are affected with neuronal loss. More severe neuronal loss and gliosis in the hip-
pocampus is paralleled by severe neuronal loss and gliosis in the lateral nucleus
in the amygdala (Bruton, 1988; Hudson et al., 1993; Thom et al., 1999). Ectopias
with more than 8 neurons per 2 sq. mm of white matter occurred in 43% of epileptic
patients but in none of the controls (Hardiman et al., 1988). In 45% of severely affected
epileptics, significant neuronal loss and astrocytosis spreading out into the overlying
molecular layer is observed in the cerebellar cortex. The severity of the cerebellar
damage may range from gross atrophy of most or many folia to the restricted neu-
ronal loss in some folia, especially at their basal portion (Gessaga and Urich, 1985).
Central apnea, asphyxia, and pulmonary edema occurring during a seizure
(Nashef et al., 1996) as well as life-threatening cardiac arrhythmias during seizures
(Earnest et al., 1992; Jallon, 1997; Nashef et al., 1996; Reeves et al., 1996; Saussu
et al., 1998) have been suggested as possible causes of sudden unexpected death in
epilepsy (Thom et al., 1999).
Enhanced electric activity of neurons and/or increased cell synaptic transmis-
sion with enhanced vesicle exocytosis, both in normal and in disease-affected
brains are a common cause of modifications of APP processing and Aβ levels.
Epilepsy is associated with an elevation of APP expression (Sheng et al., 1994) and
occurs in 10 of 11 examined subjects with diffuse nonfibrillar Aβ plaque forma-
tion (mean age 47.9 ± 8.8 years of age) (Mackenzie and Miller, 1994; Mackenzie
et al., 1996).
1.7 MECHANISMS AFFECTING BRAIN DEVELOPMENT

1.7.1 BDNF AND NEUROTROPHINS IN AUTISM
The neurotrophins, a related family of growth factors, including nerve growth factor
(NGF), brain-derived neurotrophic factor (BDNF), and neurotrophins (NT) NT-3
and NT-4/5, have a major role in the survival, growth, and differentiation of neu-
rons (Conner et al., 1997). During typical brain development, only neurons making
the appropriate connections survive and form synapses, whereas neurons that fail
to obtain adequate neurotrophins die (Oppenheim, 1991). BDNF is broadly distrib-
uted throughout the human central nervous system (CNS) and provides neurotrophic
support for many neuronal populations in the cortex, amygdala, hippocampus, and
striatum (Murer et al., 2001; Schmidt-Kastner et al., 1996; Tapia-Arancibia et al.,
2004). The hypothalamus is the brain structure that contains the highest BDNF pro-
tein levels (Katoh-Semba et al., 1997; Nawa et al., 1995; Yan et al., 1997) and BDNF
mRNA (Castren et al., 1995; Kawamoto et al., 1996; Yan et al., 1997). In the cerebel-
lum, immunoreactivity was observed in Purkinje cells and the olivary complex of
the nuclei (Kawamoto et al., 1996; Murer et al., 2001).
In the basal forebrain of autistic individuals, the level of BDNF was three times
higher than in controls (Perry et al., 2001). Miyazaki et al. (2004) observed a higher
level of BDNF in the blood samples of young children with autism than in adult
control subjects. The mean BDNF levels in sera of children diagnosed with autism
and childhood disintegrative disorder were about four times higher than in control
children (Connolly et al., 2006). Children with autism and childhood disintegrative
disorder have both elevated BDNF levels and levels of autoantibodies against BDNF.
Serum BDNF has been shown to be increased after seizures (Binder et al., 2001;
Chavko et al., 2002).
1.7.2 BRAIN STEM AND THE ROLE OF SEROTONIN IN BRAIN

DEVELOPMENT AND CLINICAL FEATURES OF AUTISM
Because 5-hydroxytryptamine (5-HT; serotonin) serves as both a neurotransmitter
and an important developmental signal in the brain, dysregulation of the 5-HT sys-
tem during development may be responsible for many of the abnormalities seen in
autism (Whitaker-Azmitia, 2005). In fact, all known chemical inducers of autism
including cocaine, thalidomide, valproate, and alcohol modulate 5-HT levels in the
brain (Harris et al., 1995; Kramer et al., 1994; Narita et al., 2002; Rathbun and Druse,
1985; Stromland et al., 1994; Williams et al., 2001). A high proportion of children
with autism exhibit elevated blood 5-HT levels (hyperserotonemia) and specific alter-
ations in 5-HT biosynthesis. The severity of hyperserotonemia is correlated with the
severity of autistic behaviors (Chandana et al., 2005; Chugani et al., 1999; Kuperman
et al., 1987). A causal role for serotonergic abnormalities in the etiology of autism is
also suggested by studies indicating autism-specific genetic polymorphisms in 5-HT
metabolizing enzyme, transporter, or receptor genes (Cohen et al., 2003; Sutcliffe
et al., 2005). Also, gender-specific differences in serotonergic regulation during
development (Chandana et al., 2005; Chugani et al., 1999), combined with a 52%
higher rate of 5-HT biosynthesis in the male than female brain (Nishizawa et al.,
1997), and the increased susceptibility of males to early insults imposed by elevated
levels of 5-HT (Johns et al., 2002), may contribute to the fourfold higher propensity
of males to develop autism compared to females.
As a result of the regulatory role of serotonin affecting the size of neurons, the
size of dendritic tree and the number of synapses in innervated cortical and sub-
cortical structures and cerebellum, developmental abnormalities in the serotonergic
system may contribute to structural and functional changes in target brain regions
and structures. Virtually all regions of the brain receive serotonergic afferents from
raphe system neurons. The rostral raphe nuclei form ascending pathways of axons
mainly to the forebrain. The caudal raphe system innervates the lower brain stem
and the spinal cord (Aitken and Törk, 1988; Lidov and Molliver, 1982). The func-
tions of serotonin are mediated by 14 subtypes of 5-HT receptors in the nervous
system (Hoyer et al., 1994; Martin and Humphrey, 1994; Saudou and Hen, 1994a,b).
The serotonin2A (5-HT2A) receptor is known to be one of the major subtypes and is
associated with psychological and mental events (Roth, 1994). The 5-HT2A receptor
plays a role in facilitating the formation and maintenance of synapses (Niitsu et al.,
1995). Staining for 5-HT2A shows the entire somata and dendritic tree of Purkinje
cells in a rat cerebellum (Maeshima et al., 1998). In vitro studies have shown that
that 5-HT inhibits the growth and arborization of Purkinje cell dendrites through
5-HT2A receptors and stimulates them through the 5-HT1A receptor (Kondoh et al.,
2004). 5-HT promotes the formation of synapses in developing and mature brain
and spinal cord (Chen et al., 1997; Niitsu et al., 1995; Okado et al., 1993), and this
process is mediated by the 5-HT2A receptor in the spinal cord (Niitsu et al., 1995).
Biochemical studies support the hypothesis that developmental defects of the raphe
nuclei may make a major contribution to the structural and functional defects of
cortical and subcortical structures. However, raphe nuclei have not yet been examined
in autistic subjects.
1.8 CLOSING REMARKS

The detected brain structure–specific patterns of structural aberrations in a major-
ity of examined anatomic subdivisions in autism brain may contribute to deficits
in expression of emotions, processing of social stimuli, learning of social behav-
iors, verbal and nonverbal communication, and stereotypic behaviors. Pathological
acceleration of brain growth and immaturity of neurons and neuronal networks
in early childhood indicate that (a) a significant portion of structural/functional
defects starts in early infancy and (b) causative factors dysregulate the mechanisms
controlling brain/neuron development. The deceleration of brain growth in the
second year of life and the increase of neuronal size in late childhood/adulthood
suggests delayed activation of correcting mechanisms. However, the delayed cor-
rection of brain and neuronal size does not result in functional recovery. Analysis
of the detected pattern of abnormal brain development in autism indicates that early
diagnosis and early treatment may prevent or reduce developmental delay, reduce/
eliminate secondary structural and functional changes, and improve clinical status
throughout the life span.
ACKNOWLEDGMENTS
This study was supported in part by funds from the New York State Office of
Mental Retardation and Developmental Disabilities and grants from Autism Speaks
and the Department of Defense Autism Spectrum Disorders Research Program
(AS073234).
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2 Evidence for Oxidative
Damage in the
Autistic Brain
Teresa A. Evans,1 George Perry,1,3 Mark
A. Smith,1 Robert G. Salomon,2 Woody
R. McGinnis,4 Elizabeth M. Sajdel-
Sulkowska,5 and Xiongwei Zhu1,*
Departments of 1Pathology and 2Chemistry, Case
Western Reserve University, Cleveland, OH 44106, USA
3
College of Sciences, University of Texas at San
Antonio, San Antonio, TX 78249, USA
4Autism House of Auckland, Autism New
Zealand, Inc., Auckland, New Zealand
5
Department of Psychiatry, Brigham and
Women’s Hospital, Boston, MA 02115, USA
CONTENTS
2.1 Oxidative Stress Is a Common Feature during
Neurodegeneration in the Brain .................................................................... 36
2.2 Peripheral Oxidative Stress in Autism .......................................................... 37
2.3 Oxidative Stress in the Autistic Brain ........................................................... 38
2.4 Axon Is the Primary Site for Oxidative Damage in the Autistic Brain:
Possible Mechanisms Underlying Functional Underconnectivity ................40
2.5 Formation of CEP–Adduct Is Not a Postmortem Artifact ............................ 41
2.6 Conclusion ..................................................................................................... 43
Acknowledgments....................................................................................................44
References ................................................................................................................44
35
2.1 OXIDATIVE STRESS IS A COMMON FEATURE DURING

NEURODEGENERATION IN THE BRAIN
The production of reactive oxygen species (ROS) takes place in all biological sys-
tems, however, under normal physiological conditions, redox homeostasis is main-
tained through antioxidant defense systems including antioxidants and antioxidant
enzymes (Zhu et al., 2005). Oxidative stress entails breaching antioxidant defense to
an extent that is sufficient to lead to damage to cellular components. Increased ROS
and oxidative stress markers such as oxidative modifications to lipids, proteins, and
nucleic acids are all considered evidence of oxidative stress and these changes are
frequently accompanied by changes in antioxidants and antioxidant enzymes (Zhu
et al., 2005). Neurons of the central nervous system are subject to a number of unique
conditions that make them particularly vulnerable to oxidative stress, including a
high energy and oxygen consumption rate, a high unsaturated lipid content of neu-
ronal membranes, high levels of transition metals, a relative scarcity of antioxidant
defense systems compared with other organs, and the postmitotic nature of neu-
ronal populations. Not surprisingly, extensive evidence suggests that oxidative stress
occurs in the brain of various neurodegenerative diseases including Alzheimer’s dis-
ease, Parkinson’s disease, amyotrophic lateral sclerosis, Huntington’s disease, Prion
disease, and stroke (Smith et al., 1994; Takeda et al., 2000). Although it is debatable
whether oxidative stress is the cause or consequence of the disease, multiple lines
of evidence suggest that oxidative stress is one of the earliest and most prominent
changes suggesting a more causative role in disease pathogenesis (Nunomura et al.,
2001, 2004). The pathology in these cases is often considered to be the result of
cellular damage from ROS or cellular response to elevated ROS (Lee et al., 2005).
Furthermore, oxidative stress is not always linked to the brain of age-related neu-
rodegenerative disease patients in their later stages of life since it is also found in
younger patients like those with Down syndrome where it is also believed to play an
essential role in disease pathogenesis (Nunomura et al., 2001).
It is well known that autism patients suffer extensive neuronal loss in the cerebellum
(Courchesne et al., 1994). Recently, unbiased stereological studies also demonstrated
significant neuronal loss in the amygdala area, especially at the lateral nucleus sub-
division (Schumann and Amaral, 2005). How these neurons become lost is of debate
in the field. Although it is widely believed that the neuronal loss in these brain areas
is due to the neurodevelopmental abnormalities, emerging evidence suggest that neu-
rodegeneration may also be involved. For example, younger autistic patients have
larger amygdala, which becomes normal later in life where neuronal loss is identi-
fied. One important piece of support for the neurodevelopmental hypothesis is the
lack of gliosis in the autistic brain, but at least in a subset of autism patients, gliosis
is identified suggesting neuronal insults later in life (Ahlsen et al., 1993; Bailey et al.,
1998; Laurence and Fatemi, 2005). Given the prominent role of oxidative stress in
neurodegeneration, we hypothesized that oxidative stress occurs in the autistic brain
and results in neurodegeneration that in turn contributes to the autistic symptoms.
Indeed, the evidence that oxidative stress is present in children with autism contin-
ues to accumulate and, given the sharp increased incidence of autism, defining the
mechanisms that result in clinical symptoms is urgently needed.
Evidence for Oxidative Damage in the Autistic Brain 37
2.2 PERIPHERAL OXIDATIVE STRESS IN AUTISM

A number of factors have been implicated in the pathogenesis of autism, including
genetic, environmental, immunological, and neurological. Nonetheless, the precise
molecular mechanisms involved in the pathogenesis of autism remain unknown. Some
similarities in groups of children with autism have been recognized. Chromosomal
abnormalities have been found in 5%–10% of those with an autism spectrum disor-
der (ASD) and genetic susceptibility loci have also been identified in even greater
numbers of those with ASD (Grice and Buxbaum, 2006). High concordance rates
are present in siblings of autistic probands as well as in monozygotic twins (Bailey
et al., 1995). From this and other evidence, it is generally accepted that autism has
both genetic and environmental influences.
The presence of oxidative stress in peripheral tissues in children with autism
has been well established (Keller and Persico, 2003; Chauhan and Chauhan,
2006). Children with autism often present with abnormal immune and digestive
systems. Digestive pathology such as inflammation of the bowel and changes in
digestive enzymes are common, and children often suffer from bowel problems
and constipation and, more occasionally, liver problems (Horvath and Perman,
2002). The cause of these systemic changes is not entirely known but may be
related to an increase in ROS. Increased nitric oxide (NO) levels in red blood
cells and higher antioxidant enzyme activity as well as zinc deficiency and cop-
per excess in plasma (Sogut et al., 2003; Chauhan et al., 2004; Sweeten et al.,
2004) and elevated urinary isoprostanes levels, a lipid peroxidation product, are
all reported in autism patients (Ming et al., 2005; Yao et al., 2006). Additionally,
mitochondrial abnormalities (Filipek et al., 2003), with elevated levels of lactate,
pyruvate, and ammonia, and lower levels of carnitine are reported in autistic case
studies (McGinnis, 2004). However, how these oxidative products in the body in
children with autism are related to the changes seen in the brain and the symp-
toms of autism is not known. Treatment with vitamin C, carnosine, or vitamin
B6 leads to significant improvement in autistic children compared with placebo
(Kleijnen and Knipschild, 1991; Dolske et al., 1993; Chez et al., 2002), and this
may be a result of protection against oxidative injury, perhaps caused by mito-
chondrial dysfunction.
The cause of the increase in oxidative species in the periphery is not known,
but could be brought about by exposure to a number of environmental toxins such
as mercury or infection that leads to increased production of ROS or decreased
activity of superoxide dismutase (SOD), catalase, or glutathione peroxidase
enzymes responsible for removing the ROS that are produced during normal
metabolism in the cell. SOD catalyzes the conversion of the superoxide anion
into hydrogen peroxide, and catalase and glutathione peroxidase act to convert
this hydrogen peroxide into water. SOD, through the Haber–Weiss reaction, is
also capable of mediating the formation of a hydroxide radical, a potent ROS,
when free copper is present and other conditions are met. Related to this, signifi-
cantly higher levels of ZnCuSOD, which could lead to an increase in the forma-
tion of hydroxide radicals, have been found in erythrocytes and in platelets of
autistic individuals compared with controls (Zoroglu et al., 2004). Low levels of
enzymes responsible for the removal of oxidative species are also seen in autism.
Plasma concentrations of reduced glutathione (GSH) are lower (4.1 ± 0.5 μmol/L)
for autistics compared with healthy controls (7.6 ± 1.4 μmol/L), p < 0.001 (James
et al., 2004, 2006). Glutathione peroxidase, an antioxidant enzyme, requires
GSH as a cofactor to break down oxidative species and lower levels of glutathione
peroxidase (−44.4%) are found in erythrocytes of autistic individuals compared
with controls (Golse et al., 1978; Yorbik et al., 2002). These lower levels of glu-
tathione peroxidase can lead to a decrease in the ability of the cells to remove
H 2O2 formed by the action of ZnCuSOD. In autistic patients, the combination of
an increase in the formation of oxidative species by SOD and a reduction in the
removal of these species by glutathione peroxidase might result in an overall
increase in oxidative stress.
Along with apparent genetic connections and the presence of systemic oxida-
tive stress indicators, families with a high rate of autoimmune diseases and immune
abnormalities also have higher rates of autism, for example, autoantibodies IgG, IgM,
IgE, and IgA against brain proteins are present in some ASD patients (Jyonouchi
et al., 2001) and proinflammatory cytokines can be produced in excessive amounts
by peripheral blood mononuclear cells from autistic children (Jyonouchi et al., 2001).
Inflammation including autoimmune conditions can cause an increase in oxida-
tive stress since macrophage activity increases during inflammation and causes an
increase in the production of ROS by the macrophages. Autoantibodies can be used
as a marker for an increase in immune responses, and these are found at higher
levels in patients with autism. For example, serum IgG antibrain autoantibodies are
present in 35% of children with ASD compared with 5% from healthy children and
IgM autoantibodies are present in 50% of children with ASD compared with 10%
of controls (Vojdani et al., 2002). In another similar study, sera from 40 healthy
subjects and 40 autistic children was analyzed for the presence of IgG, IgM, and
IgA antibodies against nine neuron-specific antigens and three encephalitogenic and
cross-reactive proteins. For one of these antigens, namely neurofilament, only up to
10% of controls had IgA, IgG, or IgM antibodies against this antigen compared to up
to 57.5% of subjects with autism (Gu et al., 2003).
Our previous studies demonstrated significantly elevated levels of carboxyethyl
pyrrole (CEP) epitopes and the corresponding autoantibodies in the blood of age-
related macular degeneration (AMD) patients compared with healthy age- and
sex-matched controls (Miyagi et al., 2002). However, while we measured levels
of CEP and iso[4]levuglandin E2 [iso[4]LGE2]–protein adducts and levels of CEP
and iso[4]LGE2 –protein autoantibodies in plasma from patients with documented
ASD with age-matched healthy controls, we found no significant difference (Evans
et al., 2008).
2.3 OXIDATIVE STRESS IN THE AUTISTIC BRAIN

The autistic brain is much younger than is typically seen in neurodegenerative diseases
and may be at an increased risk for oxidative damage because the young brain has
immature antioxidant systems that may be unable to protect against any increase in
ROS (Meagher and FitzGerald, 2000). Oxidative species mediate the formation of
lipid peroxides and other products that contribute to loss of membrane function and
cellular damage. Lipids are likely very important in the physiology and function
of the brain, and a decrease in their function and integrity likely plays a role in the
etiology of neurodegenerative disorders. Membrane damage due to lipid peroxida-
tion could lead to a decrease in functional neurons or a decrease in the conductive
ability of axons in the white matter and a decrease in conductivity between different
portions of the brain.
Lipid hydroperoxides and peroxidation products of common cellular lipids are
produced in tissue after death, are unstable and short lived, making analysis of the
levels of these substances in autopsy tissue difficult (Gu et al., 2003). Therefore, to
avoid problems with directly measuring oxidative species, it is possible to identify
other substances that are modified by these oxidative processes. We have recently
developed and characterized a method of detecting CEP adducts using a specific
antibody (Evans et al., 2008). CEP adduct is derived exclusively from free radical–
induced oxidative cleavage of docosahexaenoates, e.g., the docohexaenoic acid
(DHA) ester of 2-lysophosphatidylcholine (DHA-PC), to afford 4-hydroxy-
7-oxohept-5-enoic acid (HOHA) ester of 2-lysophosphatidylcholine (HOHA-PC)
that then reacts with protein to generate CEP modifications of the ε-amino groups
of lysyl residues (Figure 2.1) (Gu et al., 2003). DHA is the most oxidizable fatty acid
in humans, and while a minor lipid in most tissues, it is rich in specific regions of
the brain and retina (Smith et al., 1994; Takeda et al., 2000). Since DHA is present
in the brain at such high levels, CEP adducts can be used as a reliable and sensitive
dosimeter for local oxidative damage (Skinner et al., 1993; Alvarez et al., 1994) and
it provides a reasonable way for detecting oxidative damage in brain tissue in autism.
We also used antibodies against iso[4]LGE2 –protein adducts that arise exclusively
through free radical–induced cyclooxygenation of arachidonates, e.g., the arachi-
donic acid (AA) ester AA-PC, and subsequent adduction of an intermediate iso[4]
LGE2-PC with protein (Figure 2.1). Iso[4]LGE2–protein adducts identify cumulative
damage to cells from oxidative injury, such as that associated with inflammation
(Money et al., 1971; Comi et al., 1999; Poliakov et al., 2003; Sweeten et al., 2003).
In addition to oxidative modification, oxidative stress in biological systems usually
elicits an antioxidant response and therefore some antioxidant enzymes such as heme
oxygenase-1 (HO-1) that are induced by oxidative stress are also widely used as an
acceptable oxidative stress marker in neurodegeneration (Chung et al., 2004).
Using immunocytochemistry, CEP was found localized primarily in the white
matter, often extending well into the gray matter of axons in every case of autism
examined, and was not found in any age-matched or even older control cases that
were analyzed (Evans et al., 2008). Clearly, this pattern of staining is a hallmark of
the autistic brain. The white matter is composed mostly of axons, and staining in
Protein
OH
O2 OHC N (CH2)2COOH
C2H5 4 (CH2)2COOH (CH2)2COOH
Docosahexaenoic acid (DHA) HOHA
CEP
FIGURE 2.1 Generation of CEP.

this region may be indicative of a decreased functionality of the axons. Oxidative

damage in the white matter in the autistic brain involves more than DHA-containing
lipid since there are also elevated levels of iso[4]LGE2–protein adducts, an oxida-
tive protein modification derived from arachidonate-containing lipids. More impor-
tantly, the notion of elevated oxidative stress in autistic brain is further supported
by the increased expression of HO-1, an inducible antioxidant enzyme. The induc-
tion of HO-1 is likely a response to elevated oxidative stress, which indicates that the
elevated oxidative damage as evidenced by increased CEP and iso[4]LGE2–protein
adducts elicit functional consequences, e.g., by inducing an antioxidant response.
The increase in HO-1 indicates that the antioxidant response is still intact and the
body is attempting to retain homeostasis in the brain. In this regard, it is worthy of
noting that brain-derived neurotrophic factor (BDNF), which can act as an antioxi-
dant factor in brain development, is also increased in the autistic brain (Joosten and
Houweling, 2004). However, given the presence of oxidative damage as evidenced
by the CEP– and iso[4]LGE2–protein adducts, it is likely that the total antioxidant
response may be compromised and insufficient (Miyazaki et al., 2004; Connolly
et al., 2006). Overall, our data represents the first direct experimental evidence of
elevated oxidative stress in the autistic brain. The increase in ROS results in oxida-
tive modifications, not limited to lipid, and recent studies also indicate an increased
protein and DNA modification as evidenced by increased 3-nitrotyrosine (3-NT) lev-
els (Sajdel-Sulkowska et al., 2008) and 8-OH-dG (Sajdel-Sulkowska et al., in press)
in the autistic brain. The cause of the increased oxidative stress in the autistic brain
is not known, but could be due to the exposure to environmental insults that lead to
an increase in the production of oxidative species or a decrease in the ability of the
body to process oxidative species that are normally produced in metabolism. It is
now of great importance to understand the mechanisms underlying enhanced brain
oxidative stress, whether they correlate with neuronal loss, and how they may lead to
or compound the phenotype of autism patients.
2.4 AXON IS THE PRIMARY SITE FOR OXIDATIVE DAMAGE

IN THE AUTISTIC BRAIN: POSSIBLE MECHANISMS
UNDERLYING FUNCTIONAL UNDERCONNECTIVITY
One of the most astonishing aspects of our observation is that all three oxidative
stress markers we used in our study demonstrated similar localizations within
processes in the white matter in the autistic brain (Evans et al., 2008). The striking
threadlike pattern in the white matter appears to be a hallmark of the autistic brain,
suggesting that the axon may be the primary site of oxidative damage. Most of the
axons connecting different sections of the brain are found in the white matter, and
disruption of these connections may lead to a decrease in functional connectivity
in the brain. This connection of parts of the brain has been shown to be respon-
sible for many higher-order cognitive tasks such as reasoning, language, and social
judgment (Halliwell et al., 1999). Likely not coincidentally, such tasks appear to be
central in the symptoms seen in autism and it is suggested that underconnectivity
in the brain may be responsible for the appearance of symptoms. Although more
(A) (B)
FIGURE 2.2 In autism, cellular processes contain choline acetyltransferase (A), in a pattern
similar to that seen for CEP modifications (B).
research is warranted on the direct connection between white matter and con-
nectivity in autism, our findings suggested a possible mechanism regarding how
oxidative damage may be involved in underconnectivity and thereby contribute to
the development of disease.
More recently, to characterize the identity of the neurons affected by oxidative
modification, we have explored the association of CEP with various abnormal systems
(i.e., GABAergic, glutamatergic, cholinergic, and serotoninergic systems) in autism
and found that the immunostaining pattern of choline acetyltransferase, a marker
of cholinergic neurons, was very similar to the pattern seen with the CEP antibody
(Figure 2.2), suggesting that cholinergic neurons may be more vulnerable to CEP
modifications in autism.
Consistent with the immunocytochemical detection of CEP primarily in axons,
our further biochemical study suggested that neurofilament heavy (NFH) chain may
be the primary target for CEP modification. Western blot analysis of CEP antibody
revealed a band around 200 kDa, similar to the molecular size of NFH and, coimmu-
noprecipitation experiments using autism brain homogenates confi rmed that NFH is
modified by CEP. Indeed, NFH also demonstrated an immunostaining pattern very
similar to that of CEP in autism tissues and a coimmunostaining assay confirmed the
colocalization of CEP and NFH within the same processes (not shown). This is the
first study implicating a specific protein in autism, which has the potential to advance
our understanding of the mechanisms underlying functional underconnectivity.
2.5 FORMATION OF CEP–ADDUCT

IS NOT A POSTMORTEM ARTIFACT
The most obvious concern of any immunocytochemical detection of oxidative modi-
fications in postmortem human tissues is whether the positive signal is simply due to
postmortem artifact. To test for the effect of postmortem interval (PMI) on the accumu-
lation of these modifications within tissue structures, we examined CEP in a rat model
of PMI. Initial experiments found no CEP immunoreactivity in the brain in any of the
samples up to 24 h postmortem delay using WKY rat strain. However, use of an addi-
tional spontaneous hypertensive rat strain (SHR), proved to be very informative. Using
(A) (B)
(C) (D)
FIGURE 2.3 In a rat model, CEP is localized to cellular processes within the brain
strikingly similar to the CEP localization in autism. To test for any postmortem changes
in CEP modifications, rat pup brains were fi xed following either 0 h (A), 4 h (B), or 8 h (C)
postmortem delay. All rats displayed similar levels of CEP and following image analysis
showed no significant differences (not shown). Paralleling the fi ndings in human autism
cases, CEP localization overlaps with neurofilament protein accumulation on adjacent sec-
tions (C, CEP and D, neurofi lament protein). Scale bar = 50 μm.
this model, rat pups were kept at room temperature for different interval (0–8 h) prior to
brain dissection to simulate increased PMI. All rat pups from all dams tested accumu-
lated CEP in areas of the brain that overlapped significantly with neurofilaments accu-
mulation as seen using monoclonal antibody SMI-31 (Covance) (Figure 2.3). Image
analysis was performed to measure both the percent area stained and the intensity of
labeling for both CEP and neurofilaments. No correlation was found between either the
amount or intensity of CEP accumulation and PMI. These data strongly argue for neu-
rofilament protein being a specific target for CEP modification (Evans et al., 2008).
To further correlate the rat model data with human disease, the levels of CEP
modifications were compared in autism and control brain sections, which had
varying and disparate PMI. There was no significant correlation between CEP and
PMI, with times ranging from 13 to 39 h (Figure 2.4). Even in the control case with
the longest PMI (36 h), there was no recognizable immunoreactivity to CEP (data
not shown). Densitometric analysis of the staining in these autistic cases clearly
did not show any significant correlation with differences in PMI (Figure 2.4).
Taken together, the human and rat models provide strong evidence that CEP
modifications do not accumulate after death, and therefore may be a direct result
(A) (B)
35
Relative CEP intensity

30
25
20
15
10
5
0
0 10 20 30 40 50
(C) (D) PMI (h)
FIGURE 2.4 In autism, CEP is localized specifically to processes within the brain.
Morphologically, no differences are apparent between cases collected following a relatively
short PMI of 13 h (A), or 24 h (B), or even after a long PMI of 39 h (C). Computer-assisted
image analysis of the intensity of CEP accumulation in nine cases of autism found no signifi-
cant correlation when compared with PMI (D). Scale bar = 50 μm.
of oxidative damage in the living brain. Although this excludes a direct correlation
between postmortem time and staining, this does not account for any differences in
the presence of these substances in the rat brain.
2.6 CONCLUSION
There is ample evidence suggesting systemic oxidative stress in autism patients
and evidence for brain oxidative stress is now beginning to accumulate. Our
immunocytochemical and biochemical studies provide the fi rst experimental evi-
dence demonstrating lipid modification in autistic brain and suggests CEP– and
iso[4]LGE2 –protein adducts, products of lipid peroxidation, as possible hallmark
oxidative stress markers for the autistic brain. Supplementary animal experiments
show that CEP formation is unlikely due to postmortem artifact, supporting CEP
as a specific oxidative stress marker. From a structural perspective, our findings
suggest that axons of cholinergic neurons in the white matter are the primary site
of oxidative damage. At the molecular level, we have identified NFH to be the
major target for CEP modification. Our findings not only support the notion that
brain oxidative stress plays an important role in autism but now warrant future
in-depth mechanistic studies, which have the potential to provide new targets for
therapeutic efforts.
ACKNOWLEDGMENTS
This research was supported by Autism Research Institute. Human tissue was
obtained from the NICHD Brain and Tissue Bank for Developmental Disorders at
the University of Maryland under contracts N01-HD-4-3368 and N01-HD-4-3383.
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3 Oxidative Stress
and Neurotrophin
Signaling in Autism
Elizabeth M. Sajdel-Sulkowska1,2,*
1Department of Psychiatry, Harvard Medical School
and 2Department of Psychiatry, Brigham and
Women’s Hospital, Boston, MA 02115, USA
CONTENTS
3.1 Further Evidence for Involvement of Oxidative Stress in Autism ................ 48
3.2 Neurotrophins and Brain Development: Could the Imbalance
in Brain Neurotrophin Expression Lead to the Abnormalities
Observed in Autism? ..................................................................................... 50
3.2.1 Evidence for Altered Neurotrophin Expression in Autism .............. 51
3.2.2 Possible Mechanisms Involved in Altered Neurotrophin
Signaling in Autism: An Environmental Impact ............................. 52
3.2.3 Neurotrophin Signaling in Autism: Genetic Component ................ 53
3.2.4 Dual Role of Neurotrophins............................................................. 53
3.2.5 Neurotrophin Signaling and Gender ................................................ 54
3.2.6 Possible Role of Neurotrophins as Targets of Future Therapies ...... 55
3.3 Conclusions ................................................................................................... 55
Acknowledgments.................................................................................................... 55
References ................................................................................................................ 55
Autism is a neurodevelopmental disorder characterized by social and language

deficits, ritualistic-repetitive behaviors, and disturbance in motor functions. As con-
cordance in monozygotic twins is less than 100% and the phenotypic expression is
variable, both genetic predispositions and environmental triggers, many of which
induce oxidative stress, are likely to be involved in the etiology of autism, but the
cause(s) remain elusive. Data from brain imaging and head-circumference studies,
as well as Purkinje cell analysis showing early brain overgrowth followed by slow
brain growth and a decrease in Purkinje cell number in a subset of autistic cases sug-
gest that regulatory mechanisms in brain growth and differentiation are abnormal
* Corresponding author: Tel.: +1-617-732-5859; fax: +1-617-713-3078; e-mail: [email protected].

harvard.edu
47
in autism. Cell proliferation, differentiation, and the elimination of an excess number

of neurons produced during the course of normal brain development are regulated
by neurotrophins. We have recently reported an altered expression of brain neu-
rotrophins, and specifically an increased level of neurotrophin 3 (NT-3) in the autis-
tic cerebellum. Neurotrophin imbalance is likely to result in the initial overgrowth of
brain tissue followed by an arrested growth resulting in abnormal brain connectiv-
ity. Here, we review the evidence for increased oxidative stress in autism, the role of
neurotrophins in brain development, data supporting altered neurotrophin signaling
in autism, as well as animal data suggesting both the dual role of neurotrophins
in the developing brain and the possible association between oxidative stress and
altered neurotrophin expression. We also address the therapeutic potential of neu-
rotrophins in autism.
3.1 FURTHER EVIDENCE FOR INVOLVEMENT

OF OXIDATIVE STRESS IN AUTISM
Autistic pathology results most likely from the interplay between genetic predisposi-
tion and the action of environmental triggers. Several environmental toxins, such as
heavy metals (Bokara et al., 2008) including mercury (Palmer et al., 2009; Windham
et al., 2006) and pesticides (D’Amelio et al., 2005), polychlorinated biphenyls (PCBs)
(Kimura-Kuroda et al., 2007), as well as maternal infection (Brown et al., 2008) have
been implicated in autism; these in turn share the ability to increase oxidative stress.
Oxidative stress occurs in the course of normal physiological processes. The dam-
aging action of reactive oxygen species (ROS) targets key cellular macromolecules,
including lipids, carbohydrates, proteins, DNA, and RNA. This action is kept in check
by the body’s natural antioxidant defense system that includes metal-binding metallo-
proteins and other antioxidants. In response to exposure to environmental toxins and in
a number of disease conditions, this defense system appears to be overburdened, lead-
ing to unchecked oxidative stress damage. Specifically, oxidative damage to proteins is
mediated by peroxynitrate formed by the reaction of superoxide with nitric oxide (NO),
reacting with tyrosine residues (Beckman and Koppenol, 1996), and resulting in the
formation of 3-nitrotyrosine (3-NT, a specific marker of oxidative damage to proteins).
Modification of tyrosine residues in proteins can alter their conformation and function,
which in turn has a profound effect on the developing brain.
There is growing evidence supporting the role of oxidative stress and specifically
modification of proteins, as evidenced by increased 3-nitrotyrosine in over 50 pathol-
ogies including Alzheimer disease (AD), Parkinson disease (PD), and cancer (Beal,
2002; Neumann et al., 2008). Increased oxidative stress is also being implicated in
autistic pathology. We have recently reported an increase in 3-nitrotyrosine in autis-
tic cerebella (Sajdel-Sulkowska et al., 2008). Furthermore, it appears that tyrosine
residues likely to be modified under increased oxidative stress are specifically related
to the particular pathology and are genetically determined (Neumann et al., 2008).
A proteonomic approach has identified specific target proteins of nitration in the
Alzheimer’s hippocampus (Sultana et al., 2006); others have shown that more than
half of the modified proteins in AD and PD were related to the respective pathologies
Oxidative Stress and Neurotrophin Signaling in Autism 49
(Sacksteder et al., 2006). It is possible that the genetic predisposition implicated in

autism involves genes involved in oxidative defense systems that render the individu-
als more sensitive to oxidative stress triggers. Such a possibility is very intriguing
since early screening could identify individuals “allergic” to environmental toxins.
Once identified, the families may be able to implement dietary and other measures to
reduce the risk of autism and/or control the extent of symptoms.
Indeed, there is evidence for the disruption of antioxidant defense mechanisms in
autism manifested by lower than control levels of glutathione peroxidase (GSPHx)
(Yorbik et al., 2002), by lower levels of plasma glutathione levels and higher ratios
of oxidized glutathione to reduced glutathione (James et al., 2004, 2006) and by
lower levels of two major serum antioxidant metalloproteins ceruloplasmin (copper-
binding protein) and transferrin (iron-binding protein (Chauhan and Chauhan, 2006;
Chauhan et al., 2004).
Concomitant with a decrease in the oxidative defense in autism, there is an
increased oxidative stress in autism indicated by observations of increased lipid
oxidation markers in blood (Chauhan et al., 2004; Zoroglu et al., 2004) and in urine
(Ming et al., 2005; Yao et al., 2006), increased levels of NO in red blood cells (Sogut
et al., 2003), and plasma levels of NO metabolites (Sweeten et al., 2004).
We have previously reported a 65% increase in cerebellar 3-nitrotyrosine (Figure 3.1;
Sajdel-Sulkowska et al., 2008). More recently, we have observed an increase in the
DNA oxidation marker, 8-OH-dG, in the autistic cerebellum (Sajdel-Sulkowska
et al., 2009). Our study showed an overall 63.4% increase in the level of 8-OH-dG
in the autistic cerebellum with four out of five autistic samples higher than mean
control level. However, one of the autistic cases fell within the control values, sug-
gesting that there may be a subset of autistic cases with little or no DNA damage.
A similar conclusion was recently arrived at with respect to decrease in Purkinje
cell number in a subset of autistic cases (Whitney et al., 2008); it would be of inter-
est to compare directly Purkinje cell number and the levels of 8-OH-dG directly in
the same cerebellar samples. A trend toward increased urinary 8-OH-dG levels in
70
60
50
3-NT (pmol/g)
40
30
20
10
0
Control Autism
FIGURE 3.1 Increased 3-nitrotyrosine levels in autistic cerebella. 3-NT, 3-nitrotyrosine.

(From Sajdel-Sulkowska, E. M. et al., Am. J. Biochem. Biotechnol. (Special Issue on Autism
Spectrum Disorders), 4, 73, 2008. With permission.)
children has been reported in autism (Ming et al., 2005). Increased 8-OH-dG levels
have been also observed in lymphocytes (Mecocci et al., 2002), the cerebrospinal
fluid (CSF)-DNA (Lovell et al., 1999), and in the urine of patients with AD (Lee
et al., 2007) and PD (Sato et al., 2005). The children with brain damage showed
increased urinary 8-OH-dG levels (Fukuda et al., 2008). An immunohistochemi-
cal approach showed 10 times higher brain levels of 8-OH-dG in schizophrenia
(Nishioka and Arnold, 2004).
The oxidative stress in autism could result from (1) the exposure to high levels of
environmental pro-oxidants such as pesticides (Abdollahi et al., 2004; D’Amelio et al.,
2005) and mercury (Hg) (Mutter et al., 2005a,b; Palmer et al., 2008; Windham
et al., 2006); (2) inability to metabolize and clear the toxicant, such as heavy metals,
from the system (Bradstreet et al., 2003; McGinnis 2004; Serajee et al., 2004); (3) the
decreased internal antioxidant defense mechanisms (James et al., 2004; Yorbik
et al., 2002, 2006; Zoroglu et al., 2004); or (4) increased sensitivity to oxidative
stress (Buyske et al., 2006; Yang et al., 2008). It is possible that all four mechanisms
may be involved in precipitating autistic pathology.
3.2 NEUROTROPHINS AND BRAIN DEVELOPMENT: COULD

THE IMBALANCE IN BRAIN NEUROTROPHIN EXPRESSION
LEAD TO THE ABNORMALITIES OBSERVED IN AUTISM?
Neurotrophins play a critical role in the regulation of growth, development, survival,
differentiation, and function of neurons in both central and peripheral nervous
system and synaptic development and maintenance, thus they are likely players in
autistic pathology. Each of the four major mammalian neurotrophins, brain-derived
neurotrophic factor (BDNF), nerve growth factor (NGF), neurotrophin 3 (NT-3),
and NT-4, binds to its receptors, tropomysin-related kinase (TrkA, TrkB, TrkC),
and neurotrophin receptor (75NTR), and activates signaling pathways affecting
transcription. Neurotrophins are synthesized and secreted by the target organs, bind
to the receptors, and act locally to regulate target innervations and nerve function.
The neurotrophin–receptor complexes are also internalized and transported to cell
bodies where they promote neuronal survival and differentiation (Reichardt, 2006).
Thus the activity of neurotrophins can be regulated by factors involved in the tran-
scriptional and posttranscriptional steps, receptor binding, and transport.
During development, neurotrophins are involved in the regulation of neuronal
density. Developmental expression of neurotrophins has been examined in the rat
hippocampus, neocortex, and cerebellum (Das et al., 2001). The results of this study
indicate brain region–specific neurotrophin expression and independent regulation
of neurotrophin mRNAs and proteins.
The involvement of neurotrophins in neuronal plasticity is suggested by the observa-
tions of altered brain levels of neurotrophins in response to social and physical stimula-
tion in mice (Zhu et al., 2006) and learning in rats (Klinstova et al., 2004). Furthermore,
exposure to environmental toxins such as lead (Bokara et al., 2008) or chlorpyrifos
(Betancourt et al., 2007), resulting in increased oxidative stress, is associated with an
increase in hippocampal BDNF (Chao et al., 2007), and maternal infection during
pregnancy results in altered levels of brain NGF and BDNF (Marx et al., 1999).
3.2.1 EVIDENCE FOR ALTERED NEUROTROPHIN EXPRESSION IN AUTISM

Analysis of archived neonatal blood samples by recycling immunoaffinity chroma-
tography showed an increase in BDNF and NT-4 in newborns that subsequently
developed autism and mental retardation (Nelson et al., 2001). However, subsequent
enzyme-linked immunosorbent assay (ELISA) analysis of these samples did not
confirm the increase (Nelson et al., 2006). Yet serum BDNF levels were elevated
severalfold in older children with autism (Connolly et al., 2006). Similarly, increased
levels of plasma BDNF were observed in children with early-onset autism (Enstrom
et al., 2008). In the archived neonatal samples, the NT-3 levels were significantly
lower than in controls (Nelson et al., 2006). More recently, serum BDNF has been
found to increase with age, while that of NT-3 and NT-4/5 decreased with age
(Nelson et al., 2006). Serum BDNF levels were found to increase during the first
several years, which then decreased slightly in adults. They were significantly lower
in autism cases aged 0–9 years compared to age-matched controls (Katoh-Semba
et al., 2007). In adult (25 years of age) autistic cases, serum levels of BDNF were
lower than in controls (Hashimoto et al., 2006). An increase in serum BDNF and
NT-4 levels in autism has been reported (Miyazaki et al., 2004). Serum levels of
BDNF were lower in adult male patients with autism than controls (Hashimoto et al.,
2006). BDNF hyperactivity may be associated with early brain overgrowth and
increased prevalence of seizures in autism. These differences in the results of indi-
vidual studies may reflect an expected spectrum of changes in neurotrophin expres-
sion in autism. However, irrespective of the direction, altered brain neurotrophin
levels are likely to be associated with impaired brain structure and function.
Despite evidence pointing to the role of neurotrophins in the pathophysiology
of autism discussed above, data on the levels of brain neurotrophins in autism
have been limited to two studies, and NT-3 between control and autistic cases have
not been directly compared. The only direct assay of brain neurotrophin levels was
reported by Perry et al. (2001). Using an ELISA, they reported a threefold higher
level of BDNF in the basal forebrain in autism with no changes in NGF in teen-
age cases. NT-3 levels were not measured in this particular study. They interpreted
this increase as an intrinsic component of the autism disease process. The results
of the only other study of human brain neurotrophins in control subjects using an
immunohistochemical approach indicated that all four neurotrophins (NGF, BDNF,
NT-3, and NT-4) are expressed in the human hippocampus at all ages, with the
higher number of immunoreactive perikarya present in pre- and perinatal ages than
in adults (Quartu et al., 1999).
Our recent report (Sajdel-Sulkowska et al., 2009) is the first to compare the
levels of NT-3 between autistic and control brains. We examined the possible link
between increased oxidative stress and brain neurotrophin levels in autism using
cerebellar samples from the subset of autistic and control cases used in our previ-
ous study on brain 3-nitrotyrosine levels (Sajdel-Sulkowska et al., 2008). We have
selected the cerebellum because of its intimate involvement in autism indicated by
increased cerebellar volume, decreased ratio of gray to white matter (Courchesne
et al., 2001, 2003), and decrease in Purkinje cell number (Courchesne, 1991;
Kemper and Bauman, 1993; Rivto et al., 1986; Whitney et al., 2008). NT-3 was
900
800
700
NT-3 (pg/g) 600
500
400
300
200
100
0
Control Autism
FIGURE 3.2 Increased NT-3 levels in autistic cerebella. (From Sajdel-Sulkowska, E.M.,
Ming, X., and Koibuchi, N. (2009), Cerebellum [Epub PMID 19357934] With permission.)
selected because of its high expression in the developing rat cerebellum (Das et al.,
2001) and its critical role in migration and survival of cerebellar granule (Li et al.,
2004) and Purkinje cells (Kawakami et al., 2000). The data presented in Figure
3.2 shows a statistically significant NT-3 increase (40.3%) in the autistic cerebel-
lum and support a concept of altered brain neurotrophin expression in autism. As
stated above, NT-3 levels in human brain have not been previously quantified; our
data is thus the fi rst on the brain levels of NT-3 in autism. It is intriguing that the
expression of this particular neurotrophin expression appears to decrease with age
in the rodent brain (Das et al., 2001). Consequently, if a similar developmental
pattern is present in human cerebellum, then prolonged and persistent elevation
of NT-3 in autism could upset the balance of neurotrophic factors and affect brain
growth and development. Specifically, overexpression of NT-3 could contribute
to the cerebellar overgrowth observed in some autistic cases (Courchesne et al.,
2001). Furthermore, persistent elevation of NT-3, specifically involved in neuronal
differentiation (Ghosh and Greenberg, 1995), neurite fasciculation (Segal et al.,
1995), and axonal targeting, may have other profound effects such as on synapse
formation.
We also observed a positive correlation between cerebellar NT-3 and cerebellar
3-nitrotyrosine, which suggests an association between increased oxidative stress
and altered brain neurotrophin levels in autism. This association is not perfect
(r = .83), suggesting the presence of a subset of autistic cases with normal neurotro-
phin levels. It is possible that subsets of autistic cases with unique clinical symptoms
are related to specific molecular changes.
3.2.2 POSSIBLE MECHANISMS INVOLVED IN ALTERED NEUROTROPHIN

SIGNALING IN AUTISM: AN ENVIRONMENTAL IMPACT
The discussion presented above thus seems to support the notion that abnormalities
in neurotrophin signaling pathway are involved in autistic pathology. Evidence dis-
cussed below suggests that changes in neurotrophin expression could be brought about
by the environmental triggers of oxidative stress as well as a genetic component.
Neonatal exposure to lead induces an increase in hippocampal BDNF (Chao

et al., 2007), and the levels of NGF and BDNF are lower in women with evidence of
infection during pregnancy (Marx et al., 1999). Our data revealing the correlation
between NT-3 and oxidative stress seems to support environmental cause of neu-
rotrophin imbalance in the autistic brain and increased oxidative stress as a causal
factor in autism.
3.2.3 NEUROTROPHIN SIGNALING IN AUTISM: GENETIC COMPONENT

It is quite likely that mutations affecting neurotrophin signaling pathway are also
involved in autism. While the polymorphism in BDNF has been linked to impaired
hippocampal function (Egan et al., 2003), a significant association has been reported
between autism and SNP 16 in lymphocyte BDNF DNA (Nishimura et al., 2007).
Such association is also found with obsessive compulsive disorder, attention defi-
cit disorders, anxiety disorders, and anxiety-related personality traits (Nishimura
et al., 2007). Abnormal, alternatively spliced forms of protein, Ca2+-dependent acti-
vator protein (CAPS2/CADPS2), involved in release of neurotrophins, have been
reported in some of the autistic cases (Sadakata et al., 2007). Because of the close
association between BDNF and Trk-B, a polymorphism in the receptor could affect
BDNF trafficking. Hyperactivity of BDNF-TrkB signaling, associated with epilepsy
(Binder et al., 2001), has also been found in autism (Tsai, 2005). With respect to
NT-3, one would expect to find mutations in Sp3/4 (Tabuchi, 2008). Finally, in the
context of abnormal neurotrophin signaling in autism, one should not ignore pos-
sible mutations in the BDNF transcriptional repressor (MeCP2) in Rett syndrome
(Chen et al., 2003). It is possible that polymorphic forms of proteins involved in the
neurotrophin signaling cascade in autism express an increased number of tyrosine
residues in the positions that render them more accessible to oxidative modification
and formation of 3-nitrotyrosine. A model incorporating this concept is presented
in Figure 3.3.
3.2.4 DUAL ROLE OF NEUROTROPHINS

This discussion would be incomplete without pointing out the dual role of neurotro-
phins. In addition to the trophic function, NT-3 may also under certain conditions
exacerbate oxidative stress (Bates et al., 2002) as part of a response to hypoxia
(Pasarica et al., 2005). NT-3 is also known to reduce survival of Purkinje cells in
vitro (Morrison and Mason, 1998).
In that respect, NT-3 behaves like NO that plays an important role in neuronal
differentiation by nitrative modification of specific proteins (Cappelletti et al., 2003).
However, overproduction of nitric oxide leads to peroxynitrite formation and is a
source of neurotoxicity (Vargas et al., 2004). Increase in peroxynitrite results in 3-NT
protein modification, which is observed in a number of human pathologies. It has
been suggested that the negative action of NT-3 may be related to altered glutamate
receptors (Behrens et al., 1999). In this respect, it is of interest that glutaminergic
system abnormalities (Yip et al., 2007) have been implicated in a selective Purkinje
cell decrease in autism (Rout and Dhossche, 2008).
Normal oxidative
defense
3-NT
T
T T Normal brain
T T Proteins of development
neurotrophin
signaling cascade
Heavy metals NT-3

Toxins
Maternal infection BDNF
T Abnormal brain
T development;
T 3-NT 3-NT NGF
T 3-NT decrease in Purkinje
T cell number?
Oxidative
stress
Behavior
Genetic abnormalities
abnormalities in
oxidative defense
Autism?
FIGURE 3.3 Proposed model of gene–environment interaction and altered neurotrophin

signaling cascade in autism. 3-NT, 3-nitrotyrosine; NT-3, neurotrophin 3.
3.2.5 NEUROTROPHIN SIGNALING AND GENDER

Autism disproportionately affects boys more than girls, with a male-to-female ratio
of 4:1 for autism (Baird et al., 2000; Scott et al., 2002). Thus, it is of interest that
sexual dimorphism can be observed in response to environmental toxins, oxidative
stress, neurotrophin expression, brain structure, and behavior.
A number of environmental or pharmacological toxins, whose action involves
oxidative stress, have been observed to evoke different responses in males and in
females. These include exposure to chlorpyrifos, organophosphate pesticides (Dam
et al., 2000), ethanol (Rintala et al, 2001), methylazoxymethanol acetate (MAM)
(Ferguson et al., 1996), glucocorticoids (Vicedomini et al., 1986), and naltrexone use
in ethanol detoxification (de Cabo and Paz Viveros, 1997). Rats exposed prenatally
to cocaine had impaired motor coordination, with males being more affected than
females (Markowski et al., 1998).
Gender appears to affect the extent of hypertension-associated oxidative stress,
lipid peroxidation, and protein damage in heart and brain tissue (Ren, 2007). In male
rats, infection just before birth results in increase in protein carboxylation and reduced
ratio of reduced/oxidized forms of glutathione in the hippocampus. The effect appears
to be sex-dependent because it does not occur in the female offspring (Lante et al.,
2007). Furthermore, other studies suggest that females are more efficient in dealing
with oxidative stress (Ballerio et al., 2007).
Brain neurotrophins also exhibit a sexually dimorphic profile of expression
(Gilmore et al., 2003), and gender differences in hippocampal BDNF expression have
been suggested (Das et al., 2001) but not directly studied.
3.2.6 POSSIBLE ROLE OF NEUROTROPHINS AS TARGETS OF FUTURE THERAPIES

Neurotrophin expression appears to be plastic; it can be affected by various dis-
ease states, but also by pharmacological means. Decreased BDNF activity is
found in Rett syndrome (Chen et al., 2003), a severe neurodevelopmental disorder
that is caused by mutations in the gene encoding methyl-CpG-binding protein 2
(MeCP2), which is thought to act as a transcriptional repressor of BDNF. MeCP2
null mice, which develop a Rett-like phenotype, exhibit progressive deficits in
BDNF expression (Ogier et al., 2007). Ampakine (a regulator of glutamate recep-
tors) treatment of MeCP2 null mice increases BDNF and results in functional
improvement (Ogier et al., 2007). Rare mutations in MeCP2 as well as polymor-
phic markers have also been identified in autistic individuals (Loat et al., 2008).
Semax, a heptapeptide analog of adrenocorticotropic hormone, stimulates mem-
ory and attention in rats and stimulates BDNF synthesis (Tsai, 2007). Further
studies are needed to profi le signaling cascades of other neurotrophins in autism.
However, the studies discussed above suggest a potential for regulation of neu-
rotrophin expression and possible therapeutic means of improving neurotrophin
imbalance in autism.
3.3 CONCLUSIONS
The data discussed here support the key role of the following abnormalities in
autism: (1) increased oxidative stress targeting both cerebellar protein and DNA;
(2) genetic predisposition to oxidative stress triggers; (3) altered expression of brain
neurotrophins; and (4) interaction between oxidative stress and neurotrophins. In
particular, developmental elevation in cerebellar NT-3 expression could contribute to
the initial cerebellar overgrowth and a subsequent reduction on cerebellar Purkinje
cell number and/or result in abnormal brain connectivity in a subset of autistic cases.
Chronic elevation in NT-3 in autism could further predispose the autistic individuals
to oxidative damage.
ACKNOWLEDGMENTS
We thank Autism Research Institute for continuing support. We thank NICHD Brain
and Tissue Bank for Developmental Disorders at the University of Maryland for
providing us with human postmortem brain specimens.
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4 Genetics of Autism
W. Ted Brown*
Department of Human Genetics, New York State
Institute for Basic Research in Developmental
Disabilities, Staten Island, NY 10314, USA
CONTENTS
4.1 Introduction ................................................................................................... 61
4.2 Epidemiology of Autism ............................................................................... 62
4.3 Heritability of Autism ................................................................................... 63
4.4 Integrative Genetics ...................................................................................... 63
4.5 Single-Gene Mutations Associated with Autism ..........................................64
4.6 Copy Number Variations in Autism.............................................................. 65
4.7 Parental Age and Autism ..............................................................................66
4.8 Prenatal Maternal Stress and Autism............................................................ 67
4.9 Potential Role of Noncoding RNAs in Autism ............................................. 67
4.10 Summary and Conclusions ........................................................................... 68
References ................................................................................................................ 68
4.1 INTRODUCTION
Autism has a strong genetic component. It is highly heritable. Heritability is a genetic
term that provides an estimate of the proportion of phenotypic variation in a popula-
tion that is due to genetic variation. Studies comparing the ratio of concordance of
autism in monozygotic twin pairs (70%–90%) to dizygotic twin pairs (0%–10%) have
provided heritability estimates for autism of >90% (Folstein and Rosen-Sheidley,
2001; Freitag, 2007). The autism heritability estimate of >90% suggests that the
environmental component of autism is <10%, but not zero. The identical twin dis-
cordances may be due to epigenetic changes (Cheung et al., 2008; Kaminsky et al.,
2009), somatic mutations (Bruder et al., 2008), and chorionic environmental influences
(Bohm and Stewart, 2009). The inheritance of autism is not simple: it is rather com-
plex (Abrahams and Geschwind, 2008). Autism is a heterogeneous condition of variable
severity, ranging from severe cognitive impairment with seizures and lack of speech
to the milder Asperger syndrome (AS) with social communication deficits but normal
intelligence. It is now commonly regarded as an autism spectrum disorder (ASD), a
classification that includes autism, pervasive developmental disability disorder—not
otherwise specified (PDD-NOS), and AS (Freitag, 2007).
61
It is likely that there are subtypes of ASDs. For example, regression is seen in
a certain proportion of cases. One recent study reported that regression was seen
in 41% of a sample of 333 subjects with autism; 26% lost either language or social
skills, while 15% lost both (Hansen et al., 2008). Other domains in which subtypes
are likely include the presence or absence of behavioral inflexibility, macrocephaly,
seizures, and syndromic appearance.
4.2 EPIDEMIOLOGY OF AUTISM

A large epidemiological study overseen by the Centers for Disease Control and
Prevention (CDC) concluded that in 14 large areas in the United States, among
over 400,000 eight-year-old children, the mean prevalence of ASD was 6.6/1,000
(one of every 152 children). The male-to-female ratio overall was found to be
approximately 4 to 1. With the altered sex ratio, approximately 1 in 81 boys and 1
in 325 girls were found to have an ASD. Among all sites, 42% of males and 58%
of females with ASD had IQ scores of ≤70. Since the males in general had higher
IQ scores, it would be expected that the sex ratio would be higher for those with
IQ scores >70. Compared to findings of a CDC study done 2 years earlier, for the
majority of sites, there was no variation in prevalence over 2 years (CDC, 2007).
The prevalence figures of 6.6/1000 are similar to those in other recent epidemio-
logical studies (Chakrabarti and Fombonne, 2001, 2005; Fombonne et al., 2006;
Williams et al., 2008).
AS is a fairly new diagnosis and is a subcategory of ASD, representing a high-
functioning subgroup of ASD. Whether it is a genetically separate subtype of
ASD is an open question. It does occur within families in which there is also
autism (Ghaziuddin, 2005). The main clinical features are markedly impaired
social interactions and repetitive patterns of behavior, but no delay in language
and cognitive development (American Psychiatric Association, 1994). Witwer and
Lecavalier (2008) reviewed 22 studies on ASD subtypes and concluded that dif-
ferences between autism and AS were largely unsupported on the basis of current
criteria. Currently universal consensus for the essential clinical features does not
exist, and several different sets of criteria are used. Kopra et al. (2008) compared
four widely used criteria (ICD-10, DSM-IV, Gillberg and Gillberg, 1989; Szatmari
et al., 1989), and found the highest agreement between the ICD-10 and the DSM-IV
criteria. Mattila et al. (2007) reviewed the literature and conducted an epidemio-
logical study of AS among 5484 eight-year-olds in Finland. These researchers
reported that previous prevalence surveys for AS had widely ranged from 0.03
to 6 per 1000 due to varying criteria and age of diagnosis. Their study found
prevalence rates per 1000 of 2.5, according to DSM-IV (American Psychiatric
Association, 1994); 2.9 as per ICD-10 (World Health Organization, 1992); 2.7,
to Gillberg and Gillberg (1989); and 1.6 as per Szatmari criteria (Szatmari et al.,
1989), emphasizing the need to carefully reconsider the diagnostic criteria in epi-
demiological studies. When all children who meet any of the clinical criteria for
AS were included, the male-to-female ratio was relatively low (1.7:1). Thus, the
true prevalence and the actual sex ratio of AS, if different from those of ASD in
general, are not known.
Genetics of Autism 63
4.3 HERITABILITY OF AUTISM

In autism, as in many other complex genetic conditions, we are dealing with a lack of
success in identifying the underlying genetic components. For example, the heritabil-
ity of height is estimated to be 80%–90%. However, large genome-wide association
studies (GWASs) have identified genetic variants that account for little more than 5%
of the heritability of height (Maher, 2008). The same is true for large-scale studies of
schizophrenia, attention-deficit hyperactivity disorder (ADHD), obesity, diabetes, and
heart disease. A large-scale GWAS of general cognitive ability (IQ) recently reported
only six potential genetic loci, with the strongest candidate having an effect size of
only 0.4% (Butcher et al., 2008; Zimmer, 2008).
Where is the missing heritability? One possibility is that the assumptions about the
underlying genetic basis of these conditions may be wrong. The most popular model
employs the common disease common variant (CDCV) model, which assumes the
existence of genetic variants that are common and confers relative risks on the order
of 1.1–1.5. Most GWASs use genetic variants (single-nucleotide polymorphisms
[SNPs]) when the polymorphisms under study are relatively common, with allele fre-
quencies of 5%–50%. An alternative assumption is the common disease multiple rare
variant (CDMRV) model, which postulates that complex genetic conditions result
from many different mutations, each of which is relatively or exceedingly rare but
has very strong effects when, for example, the relative risk conferred is greater than
10-fold (Psychiatric GWAS Consortium Steering Committee, 2009). If these condi-
tions or disorders are caused by a large number of rare mutations, then larger sample
sizes, and possibly the sequencing of each individual, may be required, which is not
currently economically feasible, although it may be so in the near future. A second
possibility is that due to the heterogeneous nature of ASD, it will be necessary to
categorize families for linkage analysis by underlying phenotypes. An initial applica-
tion of this approach, for the domains of social interaction and repetitive behaviors,
produced modestly higher linkage scores for several loci (Liu et al., 2008).
4.4 INTEGRATIVE GENETICS

An alternative perspective to autism heritability is that it may be necessary to
take an integrative genetics approach and identify multiple interacting networks
that may be involved. With this approach, variations in hundreds of thousands of
DNA markers are correlated to expression levels of many multiples of genes as
measured by microarrays, which in turn are related to quantitative phenotypes
or disease traits (Schadt et al., 2005). Data derived by this approach are used to
develop models of how (1) DNA variations, (2) variations in gene expression, and
(3) variations in disease trait are related, often by complex interacting networks.
This approach has been applied with success to the study of gene expression
related to human obesity (Emilsson et al., 2008) and to the study of human liver,
a metabolically active tissue important in a number of common human diseases,
such as diabetes and atherosclerosis (Schadt et al., 2008). Analysis of such mul-
tiple interacting networks requires intense computing power, but already has pro-
vided a number of new pharmaceutical compounds that are in various phases of
clinical trials (Nelson, 2008; Zhu et al., 2008). Applications of this approach to
neurodevelopmental disorders such as autism will be a challenge because of dif-
ficulties in measuring gene expression levels in the target tissue. Perhaps initially
these difficulties will be addressed with mouse models. A similar approach has
been proposed that employs a probabilistic framework to combine genetic linkage
information with whole-genome molecular-interaction data to predict networks of
interacting genes that contribute to common disorders (Feldman et al., 2008; Goh
et al., 2007; Lossifov et al., 2008). This approach was applied to three disorders—
autism, bipolar disorder, and schizophrenia—and it yielded a number of candidate
genes and predicted gene targets that are likely to be shared among the disorders
(Lossifov et al., 2008).
4.5 SINGLE-GENE MUTATIONS ASSOCIATED WITH AUTISM

A wide variety of chromosomal abnormalities, submicroscopic copy number
variations (CNVs), single-gene disorders, and rare point mutations have been
associated with ASDs (Abrahams and Geschwind, 2008). A list of some 25 ASD
candidate genes can be compiled, each of which may be found in mutated form
in various ASD subjects (Table 4.1). As a whole, the various identified abnormali-
ties probably now account for up to 20% of ASDs (Abrahams and Geschwind,
2008; Marshall et al., 2008). Among single-gene disorders, fragile X syndrome
was originally found in as many as 18% of autistic subjects (Brown et al., 1982).
As the criteria for ASDs broadened to include more high-functioning individuals,
and molecular testing for fragile X eliminated some subjects (originally consid-
ered to be cytogenetically positive for fragile X at low frequencies of <2%), the
number of autism subjects with fragile X syndrome decreased, and now about 3%
of males with ASD test positive for fragile X (Szatmari et al., 2007). Chromosome
15q11–13 duplications are found in 1%–2% of ASD subjects (Abrahams and
Geschwind, 2008). Tuberous sclerosis (mutations of TSC1 or TSC2) is found in
about 1% of ASD subjects. Down syndrome (DS) has been associated with ASD
in 7%–10% of cases (Lowenthal et al., 2007), but because it is generally recog-
nized at birth, it is probably not included in most studies of ASDs (alternatively,
if 0.1% of the population has DS, then by chance 0.1% of ASD subjects would
have DS). Other syndromes associated with ASD, each accounting for about 1%,
include Chr16p11 del, 22q13 del (SHANK3 gene), and Rett syndrome (Abrahams
and Geschwind, 2008).
The overlapping phenotypes of fragile X syndrome and autism have been of con-
siderable interest for some time because approximately 30% of males with fragile X
syndrome meet the full criteria for autism, and an additional 30% have PDD-NOS
(Harris et al., 2008). Based on promising animal studies, trials of metabotropic glu-
tamate receptor 5 antagonists as well as several other drugs are beginning with indi-
viduals with fragile X syndrome, with exciting preliminary results (Berry-Kravis
et al., 2009; Bilousova et al., 2009; Hagerman et al., 2009). If fragile X and autism
have some underlying pathophysiological genetic networks in common, as appears
likely (Belmonte and Bourgeron, 2006), then these new drugs may have promising
therapeutic roles for ASDs as well.
TABLE 4.1
ASA Gene Candidates
Gene Name Gene Description
RELN Reelin
UBE3A Ubiquitin protein ligase E3A
MECP2 Methyl CpG binding protein 2
GABRB3 Gamma-aminobutyric acid (GABA) A receptor beta 3
TSC1 Tuberous sclerosis 1
TSC2 Tuberous sclerosis 2
PTEN Phosphatase and tensin homolog
NLGN3 Neuroligin 3
NLGN4 Neuroligin 4
FMR1 Fragile X mental retardation 1
DHCR7 7-Dehydrocholesterol reductase
CADPS2 Ca2+-dependent activator protein for secretion 2
SLC6A4 Solute carrier family 6 (tr-serotonin) member 4
SHANK3 SH3 and multiple ankyrin repeat domains 3
OXTR Oxytocin receptor
MET Met protooncogene
CNTNAP2 Contactin-associated protein-like 2
CACNA1C Calcium channel voltage-dependent L type alpha 1C subunit
SLC25A12 Solute carrier family 25 (mitochondrial, Aralar) member 12
NRXN1 Neurexin 1
GRIK2 Glutamate receptor ionotropic kainate 2 precursor
EN2 Engrailed homeobox 2
AHI1 Abelson helper integration site 1
ITGB3 Integrin beta 3
Source: After Abrahams, B.S. and Geschwind, D.H., Nat. Rev. Genet., 9, 341, 2008; for
original references, see OMIM, Online Mendelian Inheritance in Man.
Note: These genes are found mutated or strongly associated in some families with
autism.
4.6 COPY NUMBER VARIATIONS IN AUTISM

CNVs include insertion, deletions, and duplications of DNA and can range from less
than a kilobase to several million bases. To determine the role of CNVs in ASDs, a
number of studies using differing approaches (oligonucleotides, SNPs, and clones)
have come to reasonably similar results (reviewed in Cook and Scherer, 2008).
Resolution of the minimum size of the CNV is an important variable, because the
frequency of detected CNVs should increase as the resolution increases. De novo or
noninherited CNVs are found in 7%–10% of ASD samples from simplex families
(having only one child affected, the majority), in 2%–3% from multiplex families,
and in ∼1% in non-ASD controls; ∼10% of ASD subjects with de novo CNVs carry
two or more (Christian et al., 2008; Marshall et al., 2008; Sebat et al., 2007; Szatmari
et al., 2007). In these studies, a relatively increased frequency of CNVs involving

neuronal synaptic complex genes, such as SHANK3, NLGN4, and NRXN1, has been
found. In about 1% of ASD subjects, a duplication or deletion of a 600 kb region at
chromosome 16p11.2 that contains some 25 genes has also been found (Weiss et al.,
2008). In subjects with syndromic ASD (having an unusual or dysmorphic appear-
ance), up to 27% may have de novo CNVs (Jacquemont et al., 2006).
Recent studies on CNVs in schizophrenia have reported similar findings to those
on CNVs in ASDs (Walsh et al., 2008; Xu et al., 2008). For example, in the Xu
study, de novo CNVs were found in 10% of sporadic cases and in 1.3% of unaffected
controls (Xu et al., 2008). It is anticipated that further research will be forthcoming
regarding the role of de novo CNVs in other neuropsychiatric conditions such as
bipolar disorder, obsessive-compulsive disorder, Tourette’s syndrome, and ADHD
(Cook and Scherer, 2008).
Inherited CNVs reportedly are found in up to 50% of ASD subjects for whom one
of the presumably normal parents also has the duplication/deletion. These familial
CNVs could include candidate genes relevant to ASD if they are rare in the normal
population, but further analysis of this possibility is needed. As regards to what is
normal, a recent analysis of 2500 controls found that 5%–10% of individuals carry
individual CNVs larger than 500 kb in size, and 1%–2% of controls carry CNVs
greater than 1 Mb. Resolution of the minimum size of the CNV is an important vari-
able, and when size was assayable down to 10 kb, it was found that an average of 3–7
CNVs were present per control subject, with a global average of 540 kb of CNV DNA
per person (Itsara et al., 2009).
Zhao et al. (2007) have proposed a unified theory for the genetic basis of autism
based on the observed high frequency of CNVs found in ASD. They propose that
most families with ASD fall into two types. The large majority are families in which
male offspring have a low risk of having a de novo mutation such as a CNV. A small
minority of families includes those in which the risk of ASD in male offspring is
close to 50%, essentially a dominant model with reduced penetrance in female
offspring and carrier mothers. To explain the 4:1 male-to-female ratio, Zhao et al.
hypothesize, that for unknown reasons that are yet to be determined, females have a
greatly reduced risk of expressing the mutation, such as a CNV. This model nicely
fits with the prevalence data for ASD, but the sex-dependent penetrance assumption
is not satisfying: why should random CNVs be four times more penetrant in males?
4.7 PARENTAL AGE AND AUTISM

Advanced parental ages have been associated with increased risk of ASDs. A CDC-
sponsored study of a 1994 birth cohort of ∼250,000 study-site births, including
1250 ASD cases with complete parental age information, concluded that the odds
ratio for maternal age ≥35 versus 25–29 years was 1.3, and the odds ratio for pater-
nal age ≥40 versus 25–29 years was 1.4 (Durkin et al., 2008). This study came to
essentially the same conclusions as an earlier study of 593 children with ASD, in
which the risk of ASDs increased significantly with each 10 year increase in mater-
nal age (RR of 1.31) and paternal age (RR of 1.28), and generally confirmed several
earlier studies (Croen et al., 2007). The observed paternal age effect could suggest
the role of accumulating new mutations because spermatogonia constantly divide

in the male. The independent effect of maternal age could suggest environmental
exposures during pregnancy, complications of pregnancy, or age-related chromo-
somal changes.
4.8 PRENATAL MATERNAL STRESS AND AUTISM

Do hurricanes cause autism? Kinney et al. (2008a) reviewed weather service data to
identify hurricanes and severe weather storms in Louisiana from 1980 to 1995 and
correlated these data with autism prevalences in the various localities. They found
that the prevalence of autism increased in a dose–response fashion with severity
of prenatal storm exposure. This effect was most significant near the middle and
near the end of gestation. Children who had been exposed to storms during prena-
tal gestation months 5–6 or in the last month of pregnancy had a 3.8 times greater
risk of having an ASD than did children exposed during other months of gestation
(p < 0.001). Kinney et al. (2008b) suggest a number of potential mechanisms by
which prenatal stress could disrupt normal brain development in the fetus, includ-
ing neuroinflammatory effects; reduced fetal blood circulation; stimulated release of
maternal stress hormones that could cross the placenta, causing altered development
of the hypothalamic-pituitary-adrenal axis; complications of pregnancy; and epige-
netic effects on expression of genes involved in the stress response. An interesting
hypothesis is that prenatal stress could cause elevations of maternal stress hormones
including testosterone (Cruess et al., 2000) and that risk for ASD may be increased
by fetal exposure to high levels of testosterone during critical periods of pregnancy,
which would support the “extreme male brain” theory of ASD (Baron-Cohen, 2002;
Baron-Cohen et al., 2005; Knickmeyer and Baron-Cohen, 2006). The “extreme male
brain” theory proposes that the behaviors seen in autism are an exaggeration of typi-
cal sex differences and that exposure to high levels of prenatal testosterone might be
an added risk factor. This theory offers a potential explanation for the 4:1 male-
to-female ratio for ASDs.
4.9 POTENTIAL ROLE OF NONCODING RNAs IN AUTISM

The human genome is more complex than was originally thought. Although only
about 1.5% of the genome codes for proteins, about 5% is evolutionarily highly con-
served, but even more than 5% may encode important functional information and, in
fact, at least 70% of the human genome is transcribed (Pheasant and Mattick, 2007).
A number of classes of functional nonprotein-coding RNAs (ncRNAs) are being
identified, including microRNAs (miRNAs), small interfering RNAs (siRNAs), Piwi-
interacting RNAs (piRNAs), and long noncoding RNAs (lincRNAs) (Ghildiyal and
Zamore, 2009). There are over 1000 miRNAs, more than 1000 lincRNAs and pos-
sibly, 200,000 different piRNAs in the human genome (Betel et al., 2007; Guttman
et al., 2009). The functions of these various ncRNAs are being intensively investi-
gated. They very likely have important roles in regulating the expression of protein-
coding RNAs. They may even be enriched in regions that are involved with CNVs in
autism (Armengol et al., 2007).
A search for regions of the genome that are highly evolutionarily conserved
among mammals but have rapidly diverged from our last common ancestor, chim-
panzees, led to the discovery of human accelerated region 1 (HAR1) (Pollard et al.,
2006). This 118 bp sequence had accumulated 18 base pair changes, when zero or
one would have been expected to occur by chance. The expression of HAR1 in the
brain overlaps nearly identically with that of the reelin gene, which is a protein that
helps regulate processes of neuronal migration as well as neuronal positioning in the
cortex and the hippocampus, and modulates synaptic plasticity (Niu et al., 2008).
Thus, a rapidly evolving ncRNA gene appears to play an important role in human
brain development. Further, other ncRNAs appear to play key roles in dendritic spine
development (Schratt et al., 2006) and in long-term memory formation (Mercer et al.,
2008). Further research in this exciting new area could help us to better understand
the genetics of autism.
4.10 SUMMARY AND CONCLUSIONS

ASDs have a strong genetic component, but they have a complex pattern of inheri-
tance and are highly heterogeneous. GWASs have not defined strong gene candi-
dates, leading to the conclusion that multiple rare variants may be involved. Genetic
network and integrative genetic approaches are leading to deeper insights into the
underlying genetic problems. A number of single-gene disorders, such as fragile X,
have been associated with ASDs and may lead the way for the development of new
drug therapeutic approaches. The recent discovery of the significant role that CNVs
play in ASD and other neurodevelopmental disorders also may help us understand
and explain the underlying common genetic networks that are involved. Parental
age effects in ASD suggest genetic mechanisms. Prenatal exposure to hurricanes
has been associated with autism, pointing to the potential role of environmental
stressors. Finally, the exciting role that the ncRNAs may play in regulating brain
development and functioning are just beginning to help explain the complex genet-
ics of autism.
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5 Phenotypic
Expression of Autism,
Maternal Depression,
and the Monoamine
Oxidase-A Gene
Ira L. Cohen*
Department of Psychology, New York State
CONTENTS
5.1 Introduction ................................................................................................... 73
5.2 Assessment Issues and the PDD Behavior Inventory ................................... 74
5.3 Maternal Depression and Autism Severity ................................................... 76
5.4 Monoamine Oxidase-A Gene as a DEP/AUT Gene ..................................... 78
5.5 Association of an MAOA-uVNTR Polymorphism
with Behavior in Autism ............................................................................... 81
5.6 MAOA Gene and Behavior Profiles in Autism, a Maternal Effect............... 82
5.7 Summary and Conclusions ...........................................................................84
References ................................................................................................................ 86
5.1 INTRODUCTION
Autism and the other pervasive developmental disorders (PDD), including childhood
disintegrative disorder, Rett’s disorder, Asperger’s disorder, and PDD—not other-
wise specified, comprise a set of disorders which share a “triad” of impairments
in socialization and communication along with repetitive and ritualistic behaviors
(American Psychiatric Association, 2000). Etiologically, autism is a disorder with
a strong genetic basis (Bailey et al., 1995) but research has yet to identify a single
gene or even a set of genes that account for the vast majority of the identified cases
73
although progress is being made (Abrahams and Geschwind, 2008). Indeed, Happe
and Ronald (2008) have noted that the triad of impairments characteristic of autism
fractionate when examined in large-scale twin populations and are associated with
nonoverlapping genes, suggesting that it may be fruitless to assume a single cause
is responsible for the disorder. To complicate the matter further, there is strong
heterogeneity not only across diagnostic classes in PDD but within autism itself.
For example, Miles et al. (2005) have suggested two groupings: essential versus
complex autism, with the latter categorized by evidence of significant dysmorphol-
ogy or microcephaly. Such individuals were also more likely to have an identifiable
syndrome. Yet the notion that autism is a syndrome with many different “organic”
etiologies is not new (Coleman, 1976).
These observations suggest that when attempting to understand the puzzle of
autism, it may be more beneficial to consider examining how behavioral compo-
nents of the disorder (rather than the syndrome as a whole) are individually linked
to specific, theoretically relevant, biobehavioral variables. The advantages of this
approach include identifying possible etiologies for behaviors associated with
autism as well with as other overlapping disorders, identifying targets for interven-
tion, and for explaining the wide variation in the severity of autism. The latter fac-
tor impacts development over time, as well as stress within the family. This tactic
has been the focus of our research on autism over the past 25 years, starting with
our initial studies on behaviors that differ in children with autism who have fragile
X syndrome relative to those who do not (Cohen, 1992, 1995, 1996; Cohen et al.,
1989, 1991, 1996).
5.2 ASSESSMENT ISSUES AND THE PDD BEHAVIOR INVENTORY

Of course, in order for this approach to be efficacious, it is critical to have relevant
measures of autism that are reliable, valid, and continuous or “dimensional” in nature.
Categorical classifications of behaviors into “normal” or “abnormal” are often not
very productive from a research perspective (e.g., statistical power), are difficult to
interpret, and do not readily map onto the way behaviors occur in the natural
environment, i.e., in a continuously varied manner. For example, impairments in
social interaction can manifest themselves as varying degrees of shyness, aloofness,
avoidance, aggressiveness, or a combination of these behaviors. This varying nature
of behavior is often better captured by dimensional rating scales rather than by cat-
egorical diagnostic classification systems.
Tools for assessing autism, such as the Autism Diagnostic Interview-Revised
(ADI-R; Lord et al., 1994), the Autism Screening Questionnaire (ASQ; Berument
et al., 1999), now known as the Social Communication Questionnaire (SCQ), and
the Autism Diagnostic Observation Schedule (ADOS; Lord et al., 1999), do yield
dimensional measures of the severity of components of the disorder. However,
these measures provide “raw scores” and not “standardized scores.” That is, there
is no reference against which the meaningfulness of the size of the scores can
be interpreted. For example, IQ scores are easily understood because they are
standardized against the “typical” population. Therefore, if two people receive IQ
scores of 95 and 100, it tells us that they are actually quite similar to each other
Autism, Maternal Depression and the MAOA Gene 75
as well as to most people in the population (since most IQ tests are standardized
with a mean of 100 and an SD of 15). However, raw scores do not provide this type
of information. There is no way to know if raw scores of 10 and 15 in two differ-
ent people on an autism measure such as the ADI-R represent “normal” variation
or an actual meaningful difference in severity. Similar criticism applies to other
autism scales such as the Childhood Autism Rating Scale (CARS) (Schopler et al.,
1980). Related to this, such assessment tools are not age-standardized. This is a
critical issue since children with autism at 2 years of age do not behaviorally look
the same as children with autism at age 12.
Finally, most assessment tools for autism exclusively assess problem behaviors.
They do not measure the person’s abilities in socialization, communication, and
play. There are several problems with this approach. First, there is no way to mea-
sure actual improvement or worsening in skills over time, only a change in problem
behaviors can be assessed. Second, there is no way to differentiate subgroups within
the autism spectrum that differ mainly in their relative assets (e.g., Asperger’s cases
that have intact language but poor social skills). Third, there is no way of identifying
cases that differ on the basis of a relative lack of appropriate skills, and not the pres-
ence of deviant behaviors (e.g., language impaired children).
For the above reasons, we developed the PDD Behavior Inventory (PDDBI)
(Cohen, 2003; Cohen et al., 2003b; Cohen and Sudhalter, 2005), a reliable and
valid informant-based assessment tool standardized on a large, well-diagnosed
sample of children, aged 2−12.5 years with PDD (autism in 86%; PDD-NOS in
12%, and other PDD 2%). Three hundred sixty-nine parents and 277 teachers com-
prised the informant sample. Filling out the PDDBI is relatively straightforward
(items are between the fifth and eighth grade reading level) and can be completed
by most informants in about 30−45 min.
The PDDBI provides age- and norm-referenced (T-scores: mean = 50; SD = 10)
measures of behaviors typically seen in children with autism across two primary
dimensions designated “Approach/Withdrawal Problems” and “Receptive/Expressive
Social Communication Abilities.” The Approach/Withdrawal Problems dimension is
comprised of seven behavioral domains covering a variety of the problem behaviors
seen in children with autism while the Receptive/Expressive Social Communication
Abilities dimension is comprised of three domains capturing social, language, and
memory skills that may be deficient in some children with autism. Domain T-scores
are also used to compute an “Autism Composite” score designed to reflect overall
severity. Each of these domain and composite T-scores are normally distributed
within the reference sample. T-scores from the PDDBI have proven to be sensitive to
relevant independent variables (Chauhan et al., 2004; Cohen et al., 2003a,b; Cohen
and Tsiouris, 2006) and correlate well with diagnosis (Cohen, 2003; Cohen et al.,
2003a,b; Cohen and Sudhalter, 2005).
In this chapter, I will summarize what we have learned, using the PDDBI, about
two related factors influencing the severity of autism: maternal depression and a
polymorphism in the monoamine oxidase A (MAOA) gene. Overall, our data indicate
that maternal depression influences the behavioral profile of autism in a somewhat
paradoxical manner and this effect is remarkably similar to what we see with this
MAOA polymorphism, suggesting a link between mood disorders and autism.
5.3 MATERNAL DEPRESSION AND AUTISM SEVERITY

One of the most unusual factors influencing the severity of autism is a family history
of anxiety and depression. Familial mood and anxiety disorders and autism have
been shown to be associated in a number of studies (DeLong and Dwyer, 1988;
Piven et al., 1991; DeLong, 1994; DeLong and Nohria, 1994; Smalley et al., 1995;
Piven and Palmer, 1999). Smalley et al. (1995) reported a lifetime prevalence of DSM
(Diagnostic and Statistical Manual of Mental Disorders-III-Revised) (American
Psychiatric Association, 1987) defined major mood disorders in 40.3% of parents of
children with autism, relative to 19.2% of parents of children with tuberous sclerosis,
or of children with an unspecified seizure disorder. The majority of these parents
(64%) had their first onset of depression prior to the birth of their child with autism.
Further, 32.4% of siblings (mean age = 17 years) were found to have developed major
mood disorders. Similar results were reported by Piven et al. (1999). Smalley et al.
(1995) also reported that 20% of parents and 20.6% of siblings of children with
autism had social phobia. Although depression occurs at higher rates in families
with autism, the two disorders are not directly related. That is, in families, autism
does not cause depression and depression does not cause autism (Bolton et al., 1998;
Piven and Palmer, 1999).
Given the fact that one of the primary characteristics of depression is withdrawal
from the environment and that social phobia is characterized by intense social
withdrawal, one would expect that caregivers who suffer from these conditions
would have children with autism whose outcome would be less than optimal. This
expectation would be wrong. Smalley et al. (1995) found that a history of social
phobia was more likely in families where the child with autism had an IQ greater
than or equal to 70. Similar observations have been noted by DeLong (2004) with
respect to a family history or mood disorders. We reported (Cohen and Tsiouris,
2006), based on a large sample of 122 families of young children (mean (SD)
age = 5.4 (1.4) years) with an autism spectrum disorder, that a maternal lifetime
history of recurrent major depression (there were no significant paternal effects or
maternal anxiety disorder effects) is associated with significantly higher IQs
(differences as great as 20 IQ points), higher adaptive skills and higher T-scores on
the Receptive/Expressive Social Communication Abilities dimension of the PDDBI
(p < 0.025) in these children.
Associations between recurrent major depression in the mothers and the Approach/
Withdrawal domain scores on the PDDBI were complicated but suggestive of
increased anxiety and more social withdrawal in these young children. Specifically,
teachers, but not parents, rated the children whose mothers had a history of recurrent
depression as having more problems with sensory behaviors, rituals, and fears, and
fewer problems with overarousal (i.e., relatively less hyperactivity), a more fearful
and inhibited (internalizing) behavior style. These types of problems are suggestive
of early-onset anxiety or internalizing problems. The fact that these effects were
evident in the teacher but not the parent reports could reflect separation anxiety or
social phobia problems in this young age group. In the non-PDD population, social
phobia and major depression have been noted to be more common among young
children (mean age of 6.7 years) whose parents have a history of major depression
(Biederman et al., 2001). Thus, the children of mothers with recurrent depression
in our study resembled, to some extent, their non-PDD cohorts. Unlike these chil-
dren, however, their internalizing problems manifested as an increased severity of
certain “autistic” behaviors.
These observations are indeed puzzling. Why would high functioning autism
be linked to a lifetime history of mood disorders in their families, especially their
mothers? In our study, we could not attribute the relatively increased IQ association
to psychosocial factors, selection bias, or other treatments such as applied behavior
analysis (ABA). While it is conceivable that those mothers with a depression
history may be more intelligent and, therefore, their children with autism would be
more likely to be higher functioning, IQs of the children in our study were unre-
lated to maternal educational level, and type of maternal depression was unrelated to
maternal educational level. Further, studies have concluded that IQs of children with
autism do not correlate significantly with IQs of their parents (Szatmari and Jones,
1991; Fombonne et al., 1997).
Genetic factors could be a possible explanation. Smalley et al. (1995) speculated
that the association of familial mood/anxiety disorders with high functioning autism
is due to greater etiological heterogeneity in children with autism who also have intel-
lectual disabilities. That is, in such children, genetic and/or environmental factors
unrelated to mood disorder likely play a role in contributing to causation. DeLong
(2004) has made a similar argument. However, there is no a priori reason for why genes
associated with intellectual disability should not also be involved in depression or
social phobia. Intellectual disability is a common feature in males with fragile X
syndrome. Yet, social anxiety is prominent in these males at all levels of intelligence
(Cohen et al., 1988) and major depression has been reported to be common in their
mothers (Thompson et al., 1996).
In our paper (Cohen and Tsiouris, 2006), we hypothesized a “genetic modifier”
model (cf. Slavotinek and Biesecker, 2003) as an explanation. In this model, “autism
genes” and “depression genes” share common alleles, which interact with one
another (epistatic interactions) to modify the expression of each disorder. In the case
of autism, this would include a relatively “protective” effect of recurrent depression
alleles on IQ in children with autism.
This modifier model is displayed in the Venn diagram (Figure 5.1). Here, the
phenotypes of autism and depression are depicted as separate entities arising from
gene sets that are largely independent. However, the two disorders share some
risk alleles (denoted here as DEP/AUT alleles). When these DEP/AUT alleles are
present, they interact with each other to modify the expression of each disorder
(the shaded regions). Therefore, this model predicts that DEP/AUT alleles should
result in individuals more likely to have a specific autism or depression pheno-
type, depending upon the number and type of these alleles, and that these DEP/
AUT alleles have a protective effect on the deleterious effects on language and
IQ caused by other autism genes, accounting for the increased cognitive ability in
these affected children. If this hypothesis were true, it would have clear implica-
tions for prognosis and treatment. Is there any evidence that genes associated with
mood and anxiety disorders have a protective effect on language and IQ in children
with autism? The answer is yes.
Autism
Autism phenotype
genes
DEP/AUT
Depression
genes Depression
phenotype
FIGURE 5.1 This figure presents a Venn diagram of the hypothesized overlap of risk alleles
(DEP/AUT alleles) that are common to both autism and recurrent mood disorders. The dashed
arrows emerging from the DEP/AUT overlap represent this modifier gene effect. The shaded
regions represent DEP/AUT modified subgroups of autism and depression. (From Cohen, I.L.
and Tsiouris, J.A., J. Aut. Dev. Disord., 36, 1077, 2006. With permission.)
5.4 MONOAMINE OXIDASE-A GENE AS A DEP/AUT GENE

In searching for genes that would have an impact on both autism and depression,
genes involved with the catecholamine and serotonin systems are reasonable can-
didates. There are two reasons for this. First, the catecholamines and serotonin, as
neurotransmitters, are involved with mood, arousal, reward, and neural development
(Azmitia, 2001). Second, drugs that affect these neurotransmitters improve some of
the symptoms of autism, especially repetitive behaviors (Campbell et al., 1978, 1988;
Cohen et al., 1980; Anderson et al., 1989; Hollander et al., 2005; McDougle et al.,
2005) as well as depression.
Serotonin plays an important role in neurogenesis in utero, along with its
well-characterized neurotransmitter role (Gillberg and Coleman, 2000; Azmitia,
2001). As such, it has theoretical relevance for autism, as well as for mood dis-
orders. There is evidence that serotonin function is abnormal in autism. One of
the earliest and most consistent findings is that serotonin blood concentration is
elevated in a subset of cases with autism (Anderson, 1987). However, the meaning
of these elevated blood serotonin levels is unclear. Elevated blood serotonin levels
are not specific to autism (Gillberg and Coleman, 2000) and are not necessarily
indicative of elevated brain serotonin levels. For example, Coleman et al. (1977)
reported the case of a boy with infantile spasms who had consistently elevated
levels of blood serotonin. This child died at 21 months of age and, on autopsy,
gray matter levels of serotonin were lower than a control case. Nevertheless, ele-
vated blood serotonin levels may indicate a dysfunction in the serotonin system
that is indirectly related to the function of the central nervous system (CNS). For
example, in some studies, blood serotonin levels have been found to correlate
inversely with verbal IQ (Cook et al., 1990) and to correlate positively with severity
of autism (Kuperman et al., 1987). Blood serotonin levels have also been found
to be related to mood and anxiety disorders. Cook et al. (1994) reported that
parents of children with autism who had elevated blood serotonin levels also had
increased levels of symptoms of depression and obsessive–compulsive disorder,
relative to parents of children with autism who had normal serotonin levels. More
evidence for a serotonin dysfunction is found in the fact that increased binding
of paroxetine to the serotonin transporter in blood platelets has been observed
in autism (Marazziti et al., 2000), suggesting that a similar mechanism may be
present centrally. Indeed, at the CNS level, Chugani et al. (1999) reported, in a
positron emission tomography (PET) scan study, that brain serotonin synthesis
declines with age in typically developing children; but in children with autism,
brain serotonin synthesis fails to decrease with age.
Many factors affect overall CNS serotonin levels as well as the availability of serotonin
in the synapse. Metabolically, serotonin is synthesized from 5-hydroxytryptophan
(5-HTP) by l-amino acid decarboxylase (l-AADC). 5-HTP is synthesized from tryp-
tophan by tryptophan hydroxylase (TPH), the rate-limiting enzyme in serotonin
synthesis. When released into the synapse, serotonin interacts with a multitude of
pre- and postsynaptic receptor sites. These postsynaptic receptors can induce gene
expression by activating CREB1 (cAMP-responsive element-binding protein), a gene
that has been implicated in recurrent depression in women (Zubenko et al., 2003).
An inhibitory presynaptic autoreceptor (HTR1B/HTR1DB) modulates the release of
serotonin into the synapse. The serotonin reuptake transporter (SERT/SLC6A4/5-HTT)
brings serotonin back into the presynaptic neuron where it is eventually metabolized
by MAO in the mitochondria. Thus, the extent to which serotonin can effect changes
on the postsynaptic neuron is influenced by availability of precursors, enzyme activ-
ity, presynaptic receptor sensitivity and function, genes involved with the cAMP
signaling pathway, and excitatory or inhibitory influences of other neuronal systems.
The density of serotonergic synapses will also influence overall brain serotonin levels.
Understanding how the serotonin system affects behavior in autism will ultimately
require examining the contribution of many of these factors, both singly and in com-
bination. For reasons enumerated below, the gene that codes for MAOA is a reasonable
starting point.
Two forms of MAO exist (A and B). The genes that encode these isoenzymes map
to Xp11.23 ∼ 11.4. MAOA is of interest in this discussion because it preferentially
deaminates serotonin and norepinephrine while MAOB acts on the phenethylamines
and benzylamine (Sabol et al., 1998). Both MAOA and MAOB are present in various
regions of the body including the brain and fibroblasts. Some tissues preferentially
express only one of these isoenzymes. For example, platelets only express MAOB
(Sabol et al., 1998).
A number of different studies indicate that MAOA levels and MAOA gene dif-
ferences are associated with neurotransmitter and behavioral changes. For example,
MAOA knockout mice show large increases in brain serotonin levels and disturbed
CNS development (Cases et al., 1998). Thus, absence of MAOA has marked effects
on brain development and serotonin function. Abnormal behaviors (aggression),

borderline mental retardation, and marked decreases in MAOA activity have been
reported in a family with a point mutation in the MAOA gene (Brunner et al., 1993).
A blind male with Norrie disease, who also had a microdeletion in both MAOA and
MAOB genes, had profound retardation and stereotypy habit disorder (Collins et al.,
1992). Psychiatric problems were noted in his mother. This obligate carrier had been
diagnosed with “chronic hypomania and schizotypal features,” suggesting that MAO
plays a role in mood and thought disorders. A recent PET scan study in fact has indi-
cated that MAOA levels are uniformly increased in the brain during major depres-
sion (Meyer et al., 2006). A study of females with Turner syndrome has implicated
the MAO gene (MAOB and, possibly, MAOA) in behaviors associated with autism
(such as deficits in recognition of fear expression in faces) and in neuroanatomical
regions associated with processing of emotion such as the amygdala and orbitofron-
tal cortex (Good et al., 2003).
An upstream variable number tandem repeat (uVNTR) polymorphism within
the MAOA promoter region has been identified by Sabol et al. (1998). It consists
of a 30-bp repeat sequence consisting of 3, 3.5, 4, or 5 copies. Expression studies
indicate that the number of repeats is related to transcriptional efficiency of the
gene. The 3-repeat allele (i.e., the one with 3 copies of the 30 bp repeat sequence)
is associated with reduced transcription (Denney et al., 1999) relative to the other
alleles (Sabol et al., 1998), leading to less MAOA activity. These same MAOA-
uVNTR alleles have been shown to affect central serotonin functioning, as well as
behavior. The low activity alleles have been found to be associated with increased
serotonergic responsivity (prolactin response to fenfluramine challenge) (Manuck
et al., 2000). Williams et al. (2003) reported that males (N = 48) carrying the
3.5 and 4 repeat alleles have higher 5-hydroxyindoleacetic acid (5-HIAA) levels
in cerebrospinal fluid (CSF) than those with 3 or 5 repeats (N = 36). This effect
was independent of racial background. These effects were, however, not present
in women.
Behaviorally, Caspi et al. (2002) reported that the low activity 3-repeat MAOA
allele was associated with antisocial behavior (an “externalizing” behavior) only
in those males who had been subjected to abuse as children, suggesting a gene–
environmental stress interaction effect. More germane to our model, alleles with
more than 3 copies of the repeat sequence were found to be associated with recurrent
major depression in females in at least two studies (Schulze et al., 2000; Yu et al.,
2005). Thus, the 3-repeat allele seems to be involved with “externalizing”-type
behaviors while the longer repeats may be associated with more “internalizing”
(withdrawal) behaviors.
Given that (a) MAOA affects serotonin levels; (b) serotonin function may be
abnormal in autism; (c) serotonin reuptake inhibitors are potent antidepressants;
(d) MAOA polymorphisms associated with high activity of the gene are associated
with depression; (e) MAOA polymorphisms associated with low transcriptional
activity of the gene are associated with externalizing behavior (perhaps in reaction
to stress); (e) MAOA plays an important role in brain development; and (f) MAO
inhibitors are antidepressants, the MAOA gene is a reasonable DEP/AUT candidate
gene for modulating the pattern of expression of autism.
5.5 ASSOCIATION OF AN MAOA-uVNTR

POLYMORPHISM WITH BEHAVIOR IN AUTISM
We have reported (Cohen et al., 2003a,b), in a study of 41 males hemizygous for low
or high activity MAOA alleles, that these alleles are associated with unique behavior
patterns as measured by the PDDBI, IQ, and Adaptive Behavior assessments. In order
to control for age-related effects and variability in assessment procedures, all of the
males were less than 12.5 years of age and all received the same behavioral tests.
We found the high activity (4-repeat) allele in boys was associated with both higher
IQ and lower Autism Composite scores (as measured by the parent and teacher forms
of the PDDBI). There were no significant informant effects. That is, results were
the same from both the parent and teacher PDDBIs. Analysis to determine which
domains were specifically affected in the Approach/Withdrawal Problems dimen-
sion indicated that the SENSORY (a measure of repetitive interest in sensory-type
behaviors such as hand flapping, staring at objects, repetitive play with objects, etc.)
(p < 0.002) and AROUSAL (a measure of hyperactivity and lack of responsiveness)
(p < 0.02) domains were highest in the 3-repeat group. These externalizing-type
behavior effects replicate the above studies performed in other populations. Within
the Receptive/Expressive Social Communication Abilities dimension, domains
assessing social skills (p < 0.005), and measures of language and memory (p < 0.02)
were highest in the 4-repeat group. These effects are shown in Figure 5.2. Thus, the
4-repeat behavior pattern resembled that of children having mothers with a history
of recurrent depression.
The results of our study differed from that of Jones et al. (2004). They reported
an effect of these MAOA alleles on Leiter performance IQ, but their data indicated
that the effect was only seen when the mother’s genotype was examined, i.e., a
maternal effect. Mothers who were homozygous for the 3-repeat allele (N = 9) had
children with much lower IQs (mean (SD) = 51.1 (32.7)) than mothers who were
heterozygous (i.e., with both the 3-repeat and 4-repeat alleles; N = 37; mean (SD) =
78.5(25.5)) or homozygous for the 4-repeat allele (N = 31; mean (SD) = 72.5 (26.8)).
The explanation may relate, in part, to the nature of the two samples. Our sample
consisted of boys less than 12.5 years of age whereas age was undefined in the
study by Jones et al. (2004). Further, the MAOA allelic distribution of their affected
males was atypical. The distribution of 3-repeat and 4-repeat alleles in affected
males in the Jones et al. (2004) sample significantly differed from our study sample
(Yates corrected χ2 (1) = 7.4, p < 0.01; numbers provided by M.B. Jones, personal
communication), suggesting population stratification effects. Fifty-nine percent of
their sample had the 3-repeat allele whereas only 36% of our sample had this same
allele, a percentage identical to that of the general male population (Sabol et al.,
1998). As well, effects may only be on verbal IQ and not performance IQ, although
we did not see this in our study. More importantly, in order to disentangle maternal
and child MAOA associations, boys should be stratified in groups according to
their and their mothers’ genotypes. An interaction between the alleles of the moth-
ers and their sons, for example, could have wiped out any child effect. We have
since examined this question in a larger sample. There is indeed a maternal effect
for most behaviors.
Informant × Subscale × MAOA

70
65
60
Mean (±95% Cl)
55
50
45
40
35
Soc Appr
LMRL
Sem/Prag Abil
Autism Score
Soc Appr
LMRL
Sem/Prag Abil
Autism Score
Stereotypies
Stereotypies
Parent Teacher
3-repeat 4-repeat
FIGURE 5.2 This figure shows the similarity in parent and teacher profiles for the signifi-
cant PDDBI domain T-scores (mean ± 95% confidence interval) across 3-repeat and 4-repeat
MAOA-uVNTR genotypes (see text). Note that the domain names differ slightly from the
current version of the PDDBI (Cohen and Sudhalter, 2005) and these changes are noted
below. Vertical lines separate the SENSORY (stereotypies) domain (higher scores indicate
increased severity) from the Receptive-Expressive Social Communication Abilities domains
(where higher scores indicate increased ability) and from the overall Autism Score (higher
scores indicate increased severity). Soc Appr, Social Approach domain; LMRL, Learning,
Memory and Receptive Language domain; Sem/Prag Abil is a component of the Expressive
Language domain. (From Cohen, I.L. et al., Clin. Genet., 64, 190, 2003. With permission.)
5.6 MAOA GENE AND BEHAVIOR PROFILES

IN AUTISM, A MATERNAL EFFECT
Since our paper was published, my collaborators and I have recruited additional
boys on whom we have both PDDBI and MAOA-uVNTR data. Some of this infor-
mation has been reported elsewhere (Holden et al., 2006). We currently have data
on 115 simplex and multiplex families who have sons who are positive for autism
or autism spectrum disorder on the ADI-R or ADOS-G, have autism scores on
the PDDBI greater than 36 (a cutoff that agrees well with the above measures as
well as with clinical diagnosis of autism), have no other medical explanation for
their disorder, who were born full-term, and who were in the correct age range
for the PDDBI (18 months to 12.5 years of age). For multiplex families, only the
earliest born affected male was selected for analysis. Of the 115 mothers, 4 were
homozygous for the 3-repeat allele, 63 were heterozygous for the 3- and 4-repeat
alleles (34 boys with the 3-repeat allele and 29 with the 4-repeat allele), and 48 were
homozygous for the 4-repeat allele. Due to the small sample size of the homozygous
3-repeat mothers, their data were dropped from the rest of the analyses yielding
three groups: (1) 3_34, i.e., 3-repeat boys from heterozygous mothers; (2) 4_34,
or 4-repeat boys from heterozygous mothers; and (3) 4_44 or 4-repeat boys from
homozygous 4-repeat mothers. If the associations with the MAO-A were maternal
in nature, we would predict that groups 1 and 2 would be similar and both would
differ from group 3. If the associations were strictly due to the alleles inherited by
the boys, then group 1 would differ from groups 2 and 3, with the latter groups
similar to one another. If the boys’ and the mothers’ alleles interacted, then all three
groups would differ from one another.
Data were analyzed with multivariate analyses of variance (MANOVAs) with
domains within each dimension of the PDDBI serving as the dependent variables.
Analysis of variance (ANOVA) was used to analyze the overall Autism Composite
score. Family status (simplex or multiplex) also served as a predictor to control for
the effects of living with a sibling having autism. Post-hoc analyses were used to
compare groups.
Within the Receptive/Expressive Social Communication Abilities dimension,
higher levels of ability in association with the 4-repeat allele were predicted based
on our previous data. This was confirmed for the Social Approach Behaviors domain
(SOCAPP; a measure of nonvocal social skills such as eye contact, gesture, play, and
empathy), Expressive Language domain (EXPRESS; a measure of phonological,
semantic and pragmatic verbal ability), and the Learning, Memory and Receptive
Language domain (LMRL; a measure of memory and receptive skills) except that
these were indeed maternal in nature. The 3_34, 4_34, and 4_44 groups had mean
SOCAPP (SE) T-scores of 44.9 (2.1), 47.2 (2.3), and 51.1 (1.6). Thus, there was about
a 0.5 SD difference between the 4_44 group and the other two groups (p < 0.022)
who did not differ from each other. Similar effects were present for the other two
domains. Interestingly, the LMRL domain has the highest correlation with IQ
(r (74) = 0.77; Cohen and Sudhalter, 2005) and the difference across groups for this
domain was the largest: the 3_34, 4_34, and 4_44 groups had mean (SE) T-scores
of 47.5 (2.2), 47.3 (2.4), and 54.6 (1.7). Thus, there was almost a 1 SD difference
between the 4_44 group and the other two groups (p < 0.014). These results confirm
our earlier findings but indicate that these differences are maternal in nature.
Within the Approach/Withdrawal Problems dimension of the PDDBI, signifi-
cant differences (p < 0.05) were evident across the three groups for three domains:
Sensory/Perceptual Approach Behaviors (SENSORY), replicating our original
observation; Ritualisms/Resistance to Change (RITUAL; a measure of engaging
in rituals or resisting changes in routines or in the environment); and Specific
Fears (FEARS; a measure of overall anxiety, sensitivity to noises, separation prob-
lems, etc.). The SENSORY effect was not maternal with the 3_34 group showing
higher T-scores (mean (SE) = 52.7 (1.7)) than the 4_34 or 4_44 groups (mean =
(SE) 48.1 (1.8) and 48.4 (1.4), respectively, p < 0.05). All of these scores were in
the autism range but the means differed by about 0.5 SD. However, the RITUAL
and FEARS effects were maternal in nature. The 3_34, 4_34, and 4_44 groups
had mean (SE) RITUAL T-scores of 50.8 (1.5), 47.7 (1.7), and 53.6 (1.3) and mean
(SE) FEARS T-scores of 50.2 (1.6), 48.0 (1.8), and 54.6 (1.4). Thus, again, there
was about a 0.5 SD difference between the 4_44 group and the other two groups
(p < 0.005). Conceptually, both fears and ritualistic behaviors are related to one
another as “internalizing” behaviors. Indeed, obsessive–compulsive disorder is
characterized in the DSM as an anxiety disorder.
A nonmaternal effect was present, however. Boys with the 3-repeat allele had
higher SENSORY scores, as noted, and higher Autism Composite Scores, a mea-
sure of overall severity, as predicted based on our previous data. The Autism
Composite was worse in boys with the 3-repeat allele by about 0.5 SD (p < 0.028).
The 3_34, 4_34, and 4_44 groups had mean (SE) T-scores of 55.2 (1.6), 49.6 (1.7),
and 50.9 (1.4).

These results are consistent with the maternal depression effects cited above. This
particular MAOA polymorphism (or a closely linked site) is associated with anxiety-
like behaviors in children with autism and with adaptive abilities in socialization
and language, and these effects are maternal in nature. Recurrent major depression
in the mothers of these children is also associated with the same behavior pattern.
Thus, the “maternal environment” seems to play a role in modulating internalizing
behaviors and social and language skills in these children. Mothers homozygous
for the higher-activity 4-repeat MAOA allele have children on the autism spectrum
who appear to be relatively better off compared to children with autism who have
heterozygous mothers, irrespective of their MAO genotype in that they are “higher
functioning,” a good prognosticator for future development.
It should be noted that our results were specific to only certain behaviors; there
was no “global” association of these alleles with the full spectrum of behaviors
characteristic of autism. Thus, we did not find evidence for any associations of
these MAOA alleles with PDDBI domains assessing social pragmatic problems
and repetitive language (or aggression, at this age). The association with the global
autism score was quantitative in nature and unlikely to be associated with an actual
difference in diagnostic category. The 3-repeat cases were simply “more autistic,”
by 0.5 SD, than the other two groups whose scores were at the expected mean for
children with autism. Therefore the notion that genes affect only certain behaviors
linked to autism is supported by our data.
Recent data indicate that this MAOA-uVNTR polymorphism is also associ-
ated with brain size differences in autism. Davis et al. (2008) reported in an MRI
study that cerebral gray and white matter volume was increased in children with
autism having the low-activity 3-repeat allele (n = 12) versus those with the high
activity 4-repeat allele (n = 17). Thus, there is an association of this polymorphism
with brain growth in autism. These researchers did not determine if their effects
were maternal in nature or not but they did note that MAOA is expressed early in
development.
Taken together, these studies are consistent with the idea that alleles associated
with depression are protective for cognitive functioning in children on the autism
spectrum but enhance internalizing problems. These effects are maternal in nature,
and, given the MRI data, likely prenatal in origin. Indeed, as noted, the primary
substrate for MAO is serotonin and Cote et al. (2007) noted that this molecule plays
a strong role in development prior to its role as a neurotransmitter. In mice, these

researchers reported that this morphogen effect is maternal, i.e., that embryonic
development is dependent on serotonin that is of maternal, and not fetal, origin. They
also noted (Cote et al., 2007, p. 333), with respect to autism, that “it is conceivable
that variations in maternal serotonin levels exert subtle effects on brain development
during early ontogeny irrespective of the proper levels of peripheral serotonin in the
affected child.” Our data are consistent with this notion. Perhaps genes like MAOA
that may affect maternal serotonin levels during early gestation mitigate some of
the deleterious effect of other autism genes (or autism predisposing prenatal stress
or epigenetic factors) on cognitive development but enhance the child’s disposition
to react with intense anxiety to the environment. This behavior pattern is typically
seen in Asperger’s type individuals. These findings suggest it may be beneficial
to explore the use of maternal and child genotyping for these alleles in order to
predict the therapeutic efficacy of medications affecting the serotonin system or
MAOA activity.
It should also be noted that the notion that prenatal factors are involved in autism has
been of increasing interest to the research community. Studies have noted increased
risk for development of autism in association with a variety of birth complications
(Kolevzon et al., 2007; Durkin et al., 2008; Kinney et al., 2008; Limperopoulos et al.,
2008; Pinelli and Zwaigenbaum, 2008; Schendel, 2008; Schendel and Bhasin, 2008;
Tsuchiya et al., 2008; Williams et al., 2008).
My colleagues and I (Karmel et al., 2008) have reported on factors associated with
the later diagnosis of autism in neonatal intensive care unit (NICU) infants. Such
infants were more likely than the rest of the cohort to be of low birth weight, to have
shorter gestations, to show evidence for greater CNS involvement, to be male, and
to have more educated mothers. We also noted that, by 4 months of age, the group
that went on to develop autism had a greater preference for high frequency visual
stimulation than a matched cohort, suggesting unique problems with the attention-
arousal system in these infants. In fact, this preference was strongly correlated with
a variety of domains on the PDDBI in a subgroup of these children (n = 12) who were
seen at an average age of 4 years. When compared with other autistic children seen
by me who were not in a NICU, the NICU children, at 4 years of age, showed higher
RITUAL and AGGRESS (aggressiveness and irritability) domain scores, suggesting
long-term influences of these birth complications on certain aspects of their behav-
iors. It would be interesting to analyze the MAOA genotypes of these children and
their mothers to see if there is an association with the severity of their disorder, the
specific type of birth complication or their interaction.
In summary, our data indicate that, by assuming that autism is a syndrome com-
prised of several different behavioral categories that can be independently influenced
by many different factors, and by having appropriate assessment tools, it is possible
to determine which variables modulate their severity and the magnitude of these
effects. Further, to the extent that these variables yield similar effects, it may be
possible to determine what they have in common. Our data indicate that a maternal
lifetime history of recurrent depression and the high activity alleles of the MAOA-
uVNTR polymorphism yield similar PDDBI profiles, and both may theoretically be
linked through a common effect on prenatal serotonin levels.
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6 Paraoxonase 1
Status, Environmental
Exposures, and Oxidative
Stress in Autism
Spectrum Disorders
Maria Dronca1,* and Sergiu P. Paşca2
1Department of Medical Biochemistry,
Iuliu Haţieganu University of Medicine
and Pharmacy, Cluj-Napoca, Romania
2Center for Cognitive and Neural
Studies, Cluj-Napoca, Romania
CONTENTS
6.1 Introduction ................................................................................................... 91
6.2 Human Serum Paraoxonase 1 .......................................................................92
6.3 PON1 in Autism Spectrum Disorders ...........................................................96
6.4 Environmental Exposures ............................................................................. 98
6.5 Oxidative Stress .......................................................................................... 101
6.6 Inflammation ............................................................................................... 102
6.7 Conclusions ................................................................................................. 104
Acknowledgments.................................................................................................. 104
References .............................................................................................................. 104
6.1 INTRODUCTION
Autism spectrum disorders (ASDs) are a group of clinically and etiologically
heterogeneous disorders, affecting the core domains of communication and social
development and involving abnormal repetitive and restrictive behaviors (Rapin and
Tuchman, 2008). To date, ASDs are considered to be genetically influenced disorders,
* Corresponding author: Tel.: +40-766-530-042; fax: +40-264-597-257 e-mail: [email protected]
91
which are also subject to considerable environmental contributions (Abrahams and

Geschwind, 2008; Herbert et al., 2006; Persico and Bourgeron, 2006). It is becoming
increasingly recognized that no single explanation can account for all types of autism,
and that multiple factors (genetic, epigenetic, and environmental) are involved in shap-
ing the systemic phenotype. Exposures to environmentally toxic agents (e.g., pesti-
cides), disturbances in the redox homeostasis (i.e., decrease in antioxidant defenses
plus an increase in reactive oxygen species [ROS] production), as well as perturbed
immune balance, have all been proposed, to a certain degree, as contributing fac-
tors to the pathophysiology of autism. Within this context, evidence has emerged in
recent years to suggest the possible involvement in ASD of Paraoxonase 1 (PON1),
an enzyme that travels in blood attached to high-density lipoprotein (HDL), which is
involved in the degradation of neurotoxic pesticides, protects against oxidative stress,
and is also a negative acute-phase protein. In this chapter, after briefly reviewing the
basic structural and functional properties of PON1, we present the current evidence
for an impaired PON1 status in ASD, as well as its relation to organophosphates
(OP) exposure, oxidative stress, and immune/inflammatory modulation. In the end,
we suggest future directions for research that could substantiate the knowledge on
PON1’s role in neurodevelopmental disorders.
6.2 HUMAN SERUM PARAOXONASE 1

Human paraoxonase 1 (PON1) is a member of a family of esterases/lactonases that
also includes PON2 and PON3; the genes encoding for these enzymes are clustered
in tandem on the long arm of chromosome 7 (7q21.3–q22.1), and appear to be
approximately 27–28 kb long (Clendenning et al., 1996; Harel et al., 2004; Primo-
Parmo et al., 1996). Their order was found to be PON1, PON3 and PON2, with
PON1 being the most centromeric (Figure 6.1a) (Mochizuki et al., 1998). These
genes share approximately 65% similarity at the amino acid level, and approxi-
mately 70% similarity at the nucleotide level, suggesting that they have arisen
by gene duplication. Phylogenetic analysis demonstrated that PON2 is the oldest
member of the family from which PON3 and afterwards PON1 arose (Draganov
and La Du, 2004). In humans, PON1 and PON3 are expressed mainly in the liver
and are then transferred from the hepatocyte membrane to HDL, while these HDL
particle are transiently associated with the scavenger receptor SR-B1 (Deakin et al.,
2002; Gaidukov and Tawfik, 2005; Reddy et al., 2001). PON1 can be competitively
removed from HDL by phospholipids, thus being able to move between HDL and
other phospholipid-rich areas, such as cell membranes and sites of lipid damage
(Deakin et al., 2002; Sorenson et al., 1999). PON1 may also stimulate cellular
cholesterol efflux, the first step in reverse cholesterol transport. In contrast, PON2
remains in the intracellular milieu, being expressed in a number of tissues, including
the liver, kidney, heart, brain, and placenta, but also in the cells of the arterial wall
(i.e., endothelial cells, smooth muscle cells, and macrophages) (Ng et al., 2001,
2005; Primo-Parmo et al., 1996).
Of the three PON enzymes, PON1 (paraoxonase activity, EC 3.1.8.1; arylesterase
activity, EC 3.1.1.2) is the most studied and best understood, and has been repeat-
edly associated with cardiovascular diseases (Durrington, Mackness, and Mackness,
Paraoxonase 1 Status, Environmental Exposures, and Oxidative Stress 93
Centromere
7q21–q22
PON2 PON3 PON1
(a) 140 kb
L55M Q192R
C-909G A-832G A-162G C-126G C-108T Start
T11714A A20352G
(b)
XRE like
XRE
SRE like
NF-Y
NF-1
Sp1
Sp1
Sp1
(c) A-162G C-108T
FIGURE 6.1 Structure of paraoxonases’ genes. (a) PON gene family and location of PON1,
PON2, and PON3 on human chromosome 7, bands q21–q22. (b) The locations of polymor-
phisms in the promoter and coding region of the PON1 gene; substitutions T11714A and
A20352G in the exons 3 and 6 of the coding sequence correspond to L55M and Q192R
variation in the protein sequence respectively. (c) The –162 and –108 polymorphisms and
potential transcription factor binding sites in the 200 bp region of the promoter. SRE, sterol
regulatory element; Sp1, transcription factor; XRE, xenobiotic responsive elements; NF-Y,
nuclear factor-Y.
2001; Mackness, Arrol, and Durrington, 1991; Ng et al., 2005; Rozenberg, Shih, and
Aviram, 2005). PON1 received its name because of the ability to hydrolyze paraoxon,
the microsome-activated form of the insecticide parathion. Besides paraoxon, PON1
has the ability to hydrolyze other OP toxins, arylesters (e.g., phenylacetate), cyclic
carbonates, and lactones (Draganov and La Du, 2004). Recent comprehensive struc-
ture–activity studies with PON1 and PON1 variants generated through directed
evolution have demonstrated that PON1’s native activity is that of a lactonase, while
arylesterase and phosphotriesterase are promiscuous activities (Khersonsky and
Tawfik, 2006).
From a structural perspective, PON1 consists of 354 amino acids residues (355 with
N-terminal methionine) and has a molecular weight that can range from 38 to 45 kDa,
depending on its glycosylation state (Clendenning et al., 1996; Gan et al., 1991). PON1
is unusual in retaining the N-terminal signal sequence, which provides a hydrophobic
anchor for attachment to the HDL particle (Sorenson et al., 1999). Both activity and
stability of PON1 are dramatically dependent on the HDL components (phospholipids
and/or apolipoproteins) (Cabana et al., 2003; Rochu et al., 2007). At present, the exact
structure and catalytic mechanism of the PON1 enzyme are still unknown, although
a gene-shuffled bacterially expressed chimerical PON1 variant suggests that human
PON1 is a six-bladed β-propeller (each blade consisting of four β-pleated sheets) with
a “velcro” closure sealed by a disulfide bond between Cys42 and Cys353 (see Figure 6.2
for more details).
N
1
D 6 H1
D H2
C
C
2 B
B
H3
A A C
5
C
N
(a) 4 (b)
FIGURE 6.2 (See color insert following page 200.) Overall structure of PON1. (a) View
of the six-bladed-propeller from above. Shown are the N- and C-termini, and the two calcium
atoms in the central tunnel of the propeller (the “catalytic calcium” or Ca1, green (left); the
“structural calcium” or Ca2, red (right)). (b) A side view of the propeller. At the top of the
propeller, there are three helices H1–H3 which determine the PONs’ cell distribution, trans-
location and secretion (H1), and protein–lipid and protein–protein interactions (H2 and H3).
(Reproduced from Harel, M. et al., Nat. Struct. Mol. Biol., 11, 412, 2004. With permission.)
From a functional perspective, PON1 displays several activities:
1. Phosphotriesterase activity: PON1 hydrolyzes the oxygen analogs of com-

monly used organophosphorus insecticides such as paraoxon, chlorpyrifos-
oxon, diazinon, as well as nerve agents such as sarin, soman, tabun
(Draganov and La Du, 2004).
2. Arylesterase activity: PON1 hydrolyzes phenylacetate and other aromatic ary-
lesters, but it could also play a role in drug metabolism (i.e., the release of sali-
cylate, the more active form of aspirin) (Santanam and Parthasarathy, 2007).
3. Phopholipase A2-like activity: PON1 has the ability to hydrolyze the platelet-
activating factor (Rodrigo et al., 2001), and biologically active oxidized
phospholipids (Ahmed et al., 2002).
4. Peroxidase-like activity: PON1 is implicated in the decomposition of lipid
peroxides and H2O2 (Aviram et al., 2000; Ferretti et al., 2004), although
there are some controversies in this regard (Kriska et al., 2007).
5. Lactonase and lactonizing activities: PON1 can alter the biological activity
and/or distribution of endogenous (homocysteine thiolactone, δ-valerolactone
derivatives) and exogenous lactones (e.g., antibacterial agents, glucocorticoid
γ-lactones) (Draganov and Teiber, 2008; Draganov et al., 2005; Jakubowski,
2000).
At the moment, the physiological role of PON1 in vivo is uncertain. Experimental

data support the hypothesis that PON1’s antiatherosclerotic activity is due to its
implication in HDL-mediated cholesterol efflux from macrophages (Rosenblat et al.,

2005), protection of LDL and HDL against oxidation (Aviram et al., 1998; Mackness
et al., 1993), and prevention of proteins’ modification by homocysteine thiolactone
(Jakubowski, 2000).
To a considerable degree, PON1 level and/or catalytic efficiency are determined
by genetic variants (polymorphisms) in the coding and regulatory regions (Furlong
et al., 2008; Roest et al., 2007). With respect to regulation of PON1 expression,
it appears that only about 30% of the variability of PON1 plasma levels can be
explained by known single-nucleotide polymorphisms (SNPs). Sequencing of
promoter region of the PON1 gene led to the discovery of at least five SNPs with
varying degrees of influence over gene expression: −909/907 (C or G), −832/824
(A or G), −162 (A or G), −126 (C or G), and −108/−107 (C or T) (Figure 6.1b). These
SNPs are frequent in the population and, at least those at positions −108, −162,
and −909, have an impact on promoter’s activity of up to twofold difference in gene
expression (Brophy et al., 2001). The −108 polymorphism, lying within a consensus
binding site for transcription factors Sp1 and Sp3 (Figure 6.1c), has the greatest effect
on PON1 levels in serum (23%–24% of the total variation), with −108C providing
higher levels of plasma PON1 (Costa et al., 2003; Roest et al., 2007).
In the coding regions of PON1, two missense mutations were observed (T11714A
and A20352G), resulting in a Leu(L)/Met(M) and Gln(Q)Arg(R) substitutions at
codons 55 and 192, respectively (Humbert et al., 1993) (Figure 6.2b). Gene frequen-
cies of PON1L55M range from 0.57 in Caucasians populations to 0.99 in Oji-Cree
population, while the gene frequencies of PON1Q192R range from 0.75 in Caucasians
of Northern European origin to 0.3 for some Asian populations (Brophy, Jarvik,
and Furlong, 2002). The L55M substitution has not been found to affect catalytic
efficiency (Adkins et al., 1993; Davies et al., 1996; Humbert et al., 1993), but
the PON1M55 alloenzyme has been associated with a lower plasma concentration
(Mackness et al., 1998). In addition, due to the location in the N-terminal side of
PON1, the L55M polymorphism influences the binding of PON1 to HDL (Humbert
et al., 1993). Deakin et al. (2003) suggested that C-108T is the major genetic deter-
minant of PON1 bioavailability while the effect of the L55M genotype on PON1
concentration may be the reflection of linkage between two loci. For a “nonpoly-
morphic” substrate such as phenylacetate, the amount of the enzyme is important,
but for a “polymorphic” substrate such as paraoxon, both alleles present and the
amount of protein are important. On the other hand, the Q192R polymorphism is
responsible for a striking substrate specific difference in the hydrolytic activity of
the enzyme (Davies et al., 1996; Humbert et al., 1993). The Gln/Arg polymorphism
leads to two allozymes that clearly differ in paraoxon-hydrolyzing ability: the Arg
allozyme has a relatively higher activity and shows a greater degree of stimula-
tion by NaCl than does the Gln allozyme (Humbert et al., 1993). Further stud-
ies suggested that the effects of this polymorphism may be substrate-dependent,
as the PONQ192 isoform hydrolyzes diazoxon, sarin and soman more rapidly than
PON1R192 with in vitro assays (Davies et al., 1996). It is noteworthy to mention that
Q192R and L55M alleles are in strong linkage disequilibrium, which favors the
simultaneous presence of those alleles associated with “high paraoxonase activity”
(Garin et al., 1997).
In a given population, plasma PON1 activity can vary up to 40-fold (Davies et al.,
1996; Richter and Furlong, 1999), and PON1 protein levels, within a given genotype
class, can vary up to 13-fold (Jarvik et al., 2000). Although genetic determinants play
an important role in determining the individual PON1 status, the contribution of other
factors in modulating PON1 activity may also be important. Age, gender, ethnicity,
diet (polyphenols, vitamin C and E, unsaturated fats), environmental chemicals
(OP exposure, heavy metals), drugs (statins, fibrates), as well as certain disease con-
ditions (e.g., liver disease, diabetes, inflammatory states), have been found to influence
PON1 activity (Costa et al., 2005; Deakin and James, 2004; Rainwater et al., 2009).
6.3 PON1 IN AUTISM SPECTRUM DISORDERS

A relation between ASD and PON1 was first investigated by Serajee et al. (2004).
These authors studied possible association between polymorphisms in xenobiotic
metabolism genes (MTF1, ABCC1, SLC11A3, SLC11A2, PON1, and GSTP1) and
autism. By genotyping the T/A polymorphism in exon 3 in the PON1 gene (L55M),
they found that the marker showed deviation from Hardy–Weinberg equilibrium,
while the transmission disequilibrium test did not demonstrate any association with
autism in the 196 trios taken from the AGRE database (p = 0.12).
In 2005, D’Amelio et al. (2005) conducted a more extensive study by investigat-
ing three functional SNPs (C-108T, L55M, and Q192R) in the PON1 gene in 312
autistic patients and 676 first-degree relatives, belonging to 177 Italian families and
107 Caucasian-American families. Interestingly, they found that North American
but not Italian families displayed a significant association between autism and a less
active PON1 isoform on diazinon (L55/R192); patients also exhibited a nonsignifi-
cant trend toward increased allele frequencies and transmission rates of the −108T
allele. Following these results, our group reported in a small cohort from Romania
that the arylesterase activity of PON1 is reduced in children with ASD as compared
to healthy control subjects, whereas PON1 paraoxonase activity was found to be
similar (Paşca et al., 2006). The fact that the salt-stimulated paraoxonase activity
was not found to be impaired in autistics could be explained by the higher variability
in this catalytic activity or the modest sample of subjects in our study. More recently,
we evaluated in 50 children with ASD and 30 age- and sex-matched controls from
Romania whether the measurement of arylesterase and NaCl-stimulated paraoxo-
nase enzymatic activities of PON1, together with the assessment of PON1Q192R and
PON1L55M polymorphisms might yield more information than either genotype or
activity alone in a cohort of patients with ASD (Paşca et al., 2009b). We found that
both PON1 arylesterase (i.e., bioavailability) and PON1 paraoxonase (i.e., catalytic)
activities were decreased in autistic patients (p < 0.001, p < 0.05, respectively), but no
association with less active variants of the PON1 gene was found. Given that in a
recent study investigating one-carbon metabolism, we found preliminary evidence
for metabolic and genetic differences between clinical subtypes of ASD (Paşca
et al., 2009a), we also checked for potential differences in the PON1 status between
typical autism, pervasive developmental disorder—not otherwise specified (PDD-NOS),
and Asperger syndrome, but we failed to locate any statistical dissimilarities
(unpublished observations). The reduction in PON1 activities (arylesterase and
diazoxonase) in children with autism has also been confirmed recently in a bigger
sample size comprising children from Europe and North America (A.M. Persico
et al., unpublished results).
Most studies investigating the association of PON1 with various diseases have
examined only three SNPs: C-108T, L55M, Q192R. However, even if an individual
was genotyped for all known PON1 polymorphisms, this analysis would not
provide the level of plasma PON1 activity or the phase of polymorphisms (which
polymorphisms are on each of an individual’s two chromosomes). A functional
genomic analysis provides a much more informative approach. This is accomplished
through the use of a high-throughput enzyme assay involving two substrates:
paraoxon and phenyl acetate (Eckerson et al., 1983), diazoxon and paraoxon (Richter
and Furlong, 1999), and more recently the non-OP substrates: phenyl acetate and
4-(chloromethyl)-phenyl acetate (Richter et al., 2009). The two-dimensional enzyme
analysis, referred to as the determination of “PON1 status,” is much more useful and
informative than PCR-based genotypes alone for epidemiological studies examining
the role of PON1 in OP or lipid metabolism. Plotting rates of hydrolysis of one sub-
strate against a second substrate provided a clear resolution of the individuals with
low activities (phenotype AA) from individuals with intermediate and high activities
(phenotype AB and BB, respectively). We examined the PON1 phenotype (using
arylesterase and salt-stimulated paraoxonase activities) and found a similar distribution
in the ASD group and the control group (Paşca et al., 2009b). The similar phenotypic
distribution and the fact that there was no difference in the activities’ ratio between
groups suggest that the relationship between the two activities is generally main-
tained in autistics, regardless of a reduction in the hydrolytic protection. On the other
hand, while in the ASD group, the PON1 catalytic activity/Q192R dependency was
more pronounced than in controls, the dependency of the bioavailability of PON1
on the L55M polymorphism was absent in the ASD patients, whereas it reached a
borderline small level in healthy participants. Interestingly, in children with ASD
and first-degree relatives, the PON1R192 allele has been previously associated with a
reduction in PON1 arylesterase activity (Gaita and Persico, 2006). In a recent report,
we also showed, although in a small sample, that the distribution of the PON1 C-108T
genotypes was analogous in autistics and healthy controls (Kaucsár et al., 2009).
Nevertheless, we observed in this study that the PON1 promoter polymorphism had
a lower influence on the arylesterase activity’s variance in autistic patients than controls,
while the paraoxonase activity was not affected.
The intracellular status of PON2 still waits to be investigated in ASD, although
there is circumstantial evidence for a disturbed gene expression in hypothalamic
neurons in a rodent model for Rett syndrome (fold change in MeCP2-Tg versus WT
was 0.327, p = 0.0089) (Chahrour et al., 2008). Although it is expected to find pertur-
bations in PON2 in cells from autistic patients considering the previously reported
alterations in intracellular redox status, the exact contribution of PON2 is worth
exploring in future studies.
It is also worth mentioning that PON1 has a potentially imprinted expression in
mouse placenta (Okita et al., 2003). If confirmed in humans, the imprinted status of
PONs’ genes could have implications for the imprinting hypothesis for the develop-
ment of autism (Badcock and Crespi, 2006).
6.4 ENVIRONMENTAL EXPOSURES

In recent years, great concern has been raised about the negative impact of chronic
OP (and other pesticides) exposure on the normal development of the nervous system
(for a more comprehensive resource, see Eskenazi et al., 2008; Rosas and Eskenazi,
2008). Both animal studies and clinical reports of OP exposure in children have con-
firmed the deleterious effects of these agents on neurological functioning. Pesticides
cross both the blood–brain barrier and placenta, and the fetus is generally more
susceptible to neurotoxic agents intervening during development. As a matter of fact,
significant decreases in birth weight and head circumference have been reported after
OP exposure (Berkowitz et al., 2004; Whyatt et al., 2004). The CHAMACOS and
Mt. Sinai studies revealed a dose-related number of impaired neonatal reflexes with
higher in utero exposure to OP (Engel et al., 2007; Young et al., 2005). Noteworthy is
that when an abnormal maternal PON1 status (i.e., lower first and second tertile) was
taken into account, a higher risk for abnormal reflexes was detected (Engel et al., 2007).
On the other hand, a study from Ecuador found only a decrease in the Stanford-Binet
copying test with higher prenatal pesticide exposure in their cohort of older children
(Grandjean et al., 2006).
Developmental disorders, including atypical autism, have been described among
farming families who normally use OP (Worth, 2002). Currently, there are two studies
reporting a statistical association between OP exposure during pregnancy and the
risk for developing ASD. In a longitudinal birth cohort study in New York City, Rauh
et al. (2006) reported that children exposed to higher levels of chlorpyrifos (i.e.,
levels of >6.17 pg/g cord plasma) were more likely to display mental and psychomotor
developmental delays, attention problems, attention-deficit/hyperactivity disorder,
and pervasive developmental disorder (PDD) at 36 months of age. Eskenazi et al.
(2007) investigated the relationship between prenatal and postnatal biomarkers of
OP exposure (as measured by six nonspecific dialkylphosphate [DAP] metabolites)
and children’s neurodevelopment in the CHAMACOS birth cohort living in an agri-
cultural community in California. Children with higher prenatal and postnatal total
DAPs were at significantly higher risk of being diagnosed with a PDD, displaying
approximately twofold increase in the risk for each 10-fold increase in metabolites
(prenatal DAPs: OR = 2.3; CI95% = [1.0–5.2], p = 0.05; 24 month DAPs: OR = 1.7;
CI95% = [1.0–2.9], p = 0.04). Not only OP, but also other pesticides widely dispersed
in human environment have been associated with a risk for neurological impairment
or autism. For example, an association between residential proximity to organochlo-
rine pesticide applications during gestation and ASD among children was reported
(OR = 6.1, CI95% = [2.4–15.3]) (Roberts et al., 2007). In addition, noncoplanar poly-
chlorinated biphenyls (PCBs) cause abnormal development of the auditory cortex in
rats, including a perturbed balance of neuronal inhibition to excitation (Kenet et al.,
2007), while use of household pesticides from pet shampoos (containing pyrethrins)
were associated with an increased risk for autism (adjusted OR = 2.0, CI95% = [1.2–3.6];
strongest effect during the second trimester) (Hertz-Picciotto et al., 2008).
Chronic, occupational exposure to OP toxins has been shown to reduce PON1
activity (Costa et al., 2005), while temporary inhibition of PON1 activity has been
reported after acute poisoning with OP pesticides (Sozmen et al., 2002). In smaller
children, this effect could be enhanced due to a physiologically precarious PON1 status.
Studies in humans have shown that serum PON1 activity in newborns is fourfold
lower than the mothers’ PON1 levels, and it increases over time to reach a plateau
between 15 and 25 months of age (Cole et al., 2003; Furlong et al., 2005). Moreover,
there is an increased influence of genetic variation on PON1 activity in neonates
(Chen et al., 2003), while low PON1 activity was also reported to be associated
with smaller neonatal head circumference (Berkowitz et al., 2004). Interestingly,
birth outcomes (e.g., birth length, head circumference) have been connected with
PON1 status in mothers (Wolff et al., 2007). In premature babies (33–36 weeks of
gestation), PON1 activity measured using phenyl acetate as a substrate is 24% lower
compared to full-term babies (Ecobichon and Stephens, 1973).
Therefore, the highly reactive intermediates of pesticides might account for the
decreased paraoxonase activity of PON1 in ASD, as these compounds can inactivate
the enzyme (Hernandez et al., 2004). An upregulation of PON1 following chronic
OP exposure was described only in carriers of the PON1192R allele (Browne et al.,
2006), but this allele has been associated with a reduction in PON1 arylesterase
activity in children with ASD and first-degree relatives (Gaita and Persico, 2006).
In addition, it was also reported that carriers of the PON1192R allele, as compared
to individuals with the QQ genotype, exhibited lower acetylcholinesterase activity
(Hernandez et al., 2004). Notably, neonatal exposure to parathion in rats (at doses
straddling the threshold for cholinesterases inhibition) compromises the indices of
acetylcholine synaptic function in adolescence and adulthood (Slotkin, Levin, and
Seidler, 2009). Therefore, an altered PON1 status could also be related, at least con-
ceptually for now, with the previously described alterations in the cholinergic system
in autism (Perry et al., 2001). Differences in the regional expression (e.g., parietal
cortex, cerebellum, and thalamus) of nicotinic acetylcholine receptors’ subunits have
been documented in postmortem brains from autistics and these results are sugges-
tive of a potential enhanced susceptibility to OP exposure (Court et al., 2000; Lee
et al., 2002). Consequently, an impaired catalytic efficacy of PON1 to degrade OP
during an environmental exposure could have far more deleterious consequences in
children displaying subtle alterations in the central cholinergic transmission (Pessah
et al., 2008). This gene (PON1, AchR subunits) × environment (OP exposure, inhibi-
tors of PON1 activity) interaction should be further explored in future prospective
studies in relation to the autism risk.
The data indicating OP exposure as a risk factor for ASD (Eskenazi et al., 2007;
Rauh et al., 2006) and the fact that overall these patients display lower PON1 activi-
ties (Paşca et al., 2006; Paşca et al., 2009b) are congruent with another recently
proposed gene × environment interaction model, which includes PON1, a history
of OP exposure, and a genetic or epigenetic based impairment in Reelin (Persico
and Bourgeron, 2006) (Figure 6.3). Reelin is an extracellular serine protease that
plays a pivotal role in the central nervous system by regulating the processes of neu-
ronal migration and positioning in the developing brain and by modulating synaptic
plasticity in the adult cerebral cortex. Reelin, secreted by Cajal–Retzius cells in the
cortex and external granular layer and cerebellar nuclear neurons in the cerebellum,
is crucial for the configuration of the mature cortical architecture, and exerts its
functions by binding to a variety of receptors (Vldlr, ApoER2, a3b1 integrins) and
High PON1 activity Low PON1 activity
Reelin protease activity
= 7–10 GGC = Exposure to organophosphates (OP)

= ≥ 12 GGC = Threshold for correct neuronal migration
FIGURE 6.3 (See color insert following page 200.) The gene × environment model for
autism. The model involves the Reelin (RELN) and Paraoxonase 1 (PON1) genes, and pre-
natal exposure to organophosphates (OP). RELN variants carrying either “normal” (7−10
repeats) or “long” (≥12 repeats) GGC alleles genetically determine whether levels of reelin
are normal or reduced, respectively. In principle, both conditions are compatible with normal
neurodevelopment. However, prenatal exposure to OP can transiently inhibit the proteolytic
activity of reelin, which might then fall below the threshold required for correct neuronal
migration, also depending on baseline levels of RELN gene expression determined geneti-
cally and epigenetically. In addition, exposure to identical doses of OP can affect reelin to
a different extent depending on the amount and affinity spectrum of the OP- inactivating
enzyme paraoxonase produced by the PON1 alleles of each individual. (Reproduced from
Persico, A.M. and Bourgeron, T., Trends Neurosci., 29, 349, 2006. With permission.)
by means of a proteolytic activity on extracellular proteins (Huang and D’Arcangelo,

2008). Several lines of evidence suggested that Reelin is associated to autism (Lintas
and Persico, 2008). Case–control and family-based studies pointed out that longer
triplet repeats (≥11 GGC) in the in 5′ untranslated region of the RELN gene confer
vulnerability to autistic disorder (Persico et al., 2001), while in vivo and in vitro
experiments suggested that the “long” RELN allele significantly reduces the expres-
sion of the gene. Moreover, postmortem studies have confirmed reductions in Reelin
protein and mRNA in brains from ASD patients (Fatemi et al., 2005). Although a
malfunctioning RELN gene variant may contribute to the pathogenesis of ASD,
it is credible to say that the “long” allele is not sufficient to cause autism (Lintas and
Persico, 2008). Following these observations, Persico and Bourgeron (2006) pro-
posed that an autistic phenotype could arise in genetically or epigenetically vulnerable
individuals producing lower amounts of RELN (i.e., carriers of the “long” allele)
that are exposed to OP during critical pre/postnatal neurodevelopmental periods and
in which neuronal migration, corticogenesis, and synaptogenesis would be affected
to a different extent depending on the enzymatic efficacy of PON1. An interaction
between prenatal chlorpyrifos exposure and reeler mutation has been uncovered,
although these interactions were more complex than expected (Laviola et al., 2006).
While the model was initially conceived as a gene × gene × environment interaction
(i.e., less active PON1 variants × “long” allele of RELN × pre/postnatal OP exposure),
results from our studies indicated that PON1 activities may be impaired in ASD
patients in the absence of an association with less active PON1 gene variants (i.e.,
Q192R, L55M, C-108T) (Kaucsár et al., 2009; Paşca et al., 2009b). Future studies
should better delineate the effects of RELN dosage in the context of a pesticides
exposure and reduced PON1-mediated degradation of toxic agents.
Besides OP, exposure to several other environmental chemicals can have a sub-
stantial impact on serum PON1 activity, and could therefore, be relevant to autism.
For instance, heavy metals cations (cadmium, iron, zinc, and mercury) have been
shown to be, at least in vitro, potent inhibitors of PON1R192 activity, while PON1Q192
appear to be less sensitive to metal inhibition, excepting lead (Cole et al., 2002).
6.5 OXIDATIVE STRESS

The brain is highly vulnerable to oxidative stress, particularly during the early part
of development, due to its naturally low levels of antioxidants, higher energy require-
ment, and higher content of fat and iron (Juurlink and Paterson, 1998). Due to a defi-
cient glutathione-producing capacity, neurons are the first cells to be affected by an
increase in ROS and/or a shortage in antioxidants (Perry et al., 2004). Especially
in genetically susceptible individuals (e.g., carrying polymorphic oxidative genes),
free radicals resulting from ingested or inhaled environmental toxins, food or food
additives, inflammation or infection, may seriously disrupt neurodevelopmental
milestones and lead to neuropsychiatric impairments.
Currently, it is generally recognized that autism is associated with a perturbed
redox homeostasis (Chauhan and Chauhan, 2006). Increased oxidative stress in
autism may be due to environmental pro-oxidants exposure, intensive production of
endogenous pro-oxidants (i.e., nitric oxide [NO], xanthine oxidase, homocysteine),
and/or decreased level/activity of antioxidants (i.e., ceruloplasmin, transferrin, super-
oxide dismutase [SOD]), glutathione peroxidase (GPx), catalase (CAT), PON1, and
reduced glutathione (GSH). In fact, reduced levels of GPx (Yorbik et al., 2002), CAT
(Zoroglu et al., 2004), and GSH (GPx cosubstrate) (James et al., 2004, 2006; Paşca
et al., 2009a) are found in erythrocytes/whole blood of autistic individuals compared
with controls. On the contrary, higher erythrocyte’s SOD was observed in autism by
Zoroglu et al. (2004). Oxidative stress in ASD is associated with increased plasma
levels of malonyldialdehyde, a lipid peroxidation biomarker (Chauhan et al., 2004).
Recently, carboxyethyl pyrrole and iso[4]levuglandin–protein adducts, markers of
oxidative damage resulting from lipid peroxidation, were detected in postmortem
brain tissue from autistic subjects (Evans et al., 2008).
ASDs have also been associated with one-carbon metabolism perturbations,
as reported in studies investigating plasma metabolites (Adams et al., 2007; Geier
and Geier, 2006; Geier et al., 2009; James et al., 2004, 2006; Paşca et al., 2006,
2009a) or immune cells (Suh et al., 2008). For instance, our group recently
reported differences in the severity of the metabolite profiles across the spec-
trum of autistic disorders, with typical autistic patients exhibiting disturbances in
both the methionine cycle and the transsulfuration pathway concomitant with a
slightly more prevalent T allele frequency for the MTHFR C677T polymorphism,
while patients with the less severe form of the disease (i.e., PDD-NOS) pre-
sented only with disturbances in the methionine cycle; in addition, since the lev-
els of both folate and vitamin B12 were within the normal range, we excluded
vitamin deficiencies as a cause for these impairments (Paşca et al., 2009a).
A disturbed one-carbon metabolism, although (apparently) nonspecific to autism,
could lead to a diminished capacity for methylation, with consequences on gene
expression, neurotransmitter synthesis, and, potentially, on neuronal synchroni-
zation. It remains to be further investigated how much the metabolic profiles in
plasma echo the actual intracellular metabolic dynamics, and whether corrections
of these impairments will lead to genuine clinical improvements.
PON1 was found to efficiently hydrolyze not only lipoprotein-associated perox-
ides (including cholesteryl linoleate hydroperoxides), but also hydrogen peroxide
(H2O2), and may thus play an important role in eliminating potent oxidants involved
in atherogenesis (Aviram et al., 1998). Conversely, PON1 also serves as a target for
lipid peroxidation products, resulting in an inhibition of PON1 activities (arylesterase
and lactonase) and a reduction in PON1 gene expression (Aviram et al., 1999; Van
Lenten et al., 2001). PON1 induces the lysophosphatidylcholine (LPC) formation,
which might act in both direction as pro-oxidant (based on the upregulated effects
on adhesive molecules) and antioxidant (by increasing the expression of extracellular
SOD in monocyte-macrophages) (Rosenblat, Oren, and Aviram, 2006). Moreover,
PON1 can be inactivated by S-glutathionylation, resulting from a reaction between
its free sulfhydryl group at Cys284 and oxidized glutathione (GSSG) (Rozenberg and
Aviram, 2006). Considering these facts, one may assume that in ASD, an altered
PON1 could contribute to the perturbed redox homeostasis, while an enhanced oxi-
dative stress could easily disturb the PON1 status.
6.6 INFLAMMATION
Experimental and clinical evidence accumulated in the last decades have led to
the idea that, at least in a subgroup of patients with autism, immune imbalance or
even autoimmune processes may play a considerable role in the pathophysiology
of the autistic disorder (Ashwood, Wills, and Van de Water, 2006; Pardo, Vargas,
and Zimmerman, 2005). Previous studies have already reported a wide plethora of
immune abnormalities, including T-cell, B-cell, and NK-cell dysfunction; autoanti-
body production; and increased proinflammatory cytokines.
Studies published more than a decade ago indicated that HDL protects against bac-
terial lipopolysaccharide (LPS), the endotoxin found in the outer membrane of Gram-
negative bacteria (Levine et al., 1993). Animal experiments have later showed that
purified PON1 (type Q) is able to protect cells from LPS and prevent or greatly reduce
the release of cytokines (La Du et al., 1999), suggesting a potential role for PON1 in
modulating immune responses. Interestingly, Jyonouchi and coworkers showed that
peripheral blood mononuclear cells (PBMCs) from ASD patients exposed to LPS pro-
duced excessive amounts of tumor necrosis factor-α (TNF-α), interleukin 1 (IL-1),
and/or interleukin 6 (IL-6) compared to controls, indicative of an aberrant innate
immune response in a considerable percentage of children with ASD (Jyonouchi,
Sun, and Le, 2001). Moreover, Sweeten, Posey, and McDougle, (2003) showed that
the mean absolute monocyte count was significantly higher in the autistic children,
which was accompanied by increased levels of neopterin; these findings were inter-
preted as heightened cell-mediated immunity in ASD. In the context of the maternal
immune activation (MIA) model for developmental disorders, it is worth mentioning
that maternal injections of LPS in rodents result in increased anxiety, deficits in social
interaction in the adult offspring; this suggests that short systemic maternal inflam-
mation has long-lasting consequences on the adult mouse stress and social behavior
(Hava et al., 2006). On the other hand, the cytokine IL-6 was already identified
as a key intermediary contributing to MIA in rodents (Smith et al., 2007). Vargas
et al. (2005) demonstrated in postmortem autistic brain, excessive significantly higher
level of subsets of cytokines (IL-6, IL-10, MCP-3, etc.) in brain regions that have been
previously associated with autism (i.e., anterior cingulated gyrus). In a small sample
of patients with autism displaying regressive features, Chez et al. (2007) reported
elevated levels of TNF-α in cerebrospinal fluid. These and other studies suggest that
at least some autistic children display a subclinical inflammatory state.
During inflammation, particularly during the acute-phase response, there is a
reduction in several proteins important in reverse cholesterol transport and inhibit-
ing oxidation. These include cholesterol ester transfer protein, lecithin cholesterol
acyltransferase, hepatic lipase, apoA-I, and PON1. It is thought that reduction in
these proteins, accompanied by an increase in proteins such as apoJ and serum
amyloid A (SAA), changes HDL from an anti-inflammatory to a proinflammatory
particle (Van Lenten et al., 2006). Several studies provide evidence that hepatic
PON1 mRNA is downregulated in response to inflammatory cytokines (Deakin
and James, 2008). Treatment with LPS and oxidized LDL, known to induce an
immune response, reduced PON1 mRNA in human hepatocyte cells line (Feingold
et al., 1998). A similar effect was observed with administration of TNF-α, IL-1,
and IL-6 (Feingold et al., 1998; Kumon et al., 2002). Therefore, HDL loses its anti-
inflammatory properties during acute infection, and PON1 activity and concentration
are regulated by inflammatory cytokines and endotoxin administration. In this
context, it could be concluded that PON1 is a negative acute-phase protein whose
mRNA is rapidly downregulated (Feingold et al., 1998). In an ongoing study, we did
not identify any statistical correlation between arylesterase or paraoxonase activi-
ties of PON1 and plasma C-reactive protein (CRP) levels in a population of children
with typical autism (unpublished observation).
NO is a unique biological messenger molecule involved in neurodevelopment, but
also a versatile player in the immune system. Recent studies have shown that NO
may also play a role in the pathophysiology of autism. Apparently, autistic children
display increased NO levels in red blood cells (Sogut et al., 2003) and increased levels
of plasma NO metabolites (Sweeten et al., 2004; Zoroglu et al., 2003). A recent arti-
cle reported a nitrite-mediated inactivation of PON1, in a dose- and time-dependent
manner; this inactivation is probably generated through nitration of enzyme phenyl
residues, while tryptophan could be of important value in minimizing this effect
(Abd-Allah and Mariee, 2008). Future research should elucidate in more details the
role of PON1 in modulation of immune responses and, consequently, how is the
altered PON1 status in autism contributing to the aberrant inflammatory responses
in autism.
6.7 CONCLUSIONS
The biochemical and genetics studies indicate that the PON1 status is impaired in
children with ASD. The current evidence points toward differences in the frequency
of PON1 gene variants in autistic patients between North America and Europe.
Currently, the reasons behind the PON1 status alteration in autism are not known,
nor are all its consequences. Besides the SNPs investigated (i.e., C192R, L55M,
C-108T) to date, other SNPs in the PON1 gene or at distant loci could be associated
with ASD. Chronic OP exposure, increased and prolonged oxidative stress, an altered
hepatic status, environmental heavy metals, or a silent subclinical inflammatory
state are all factors that could contribute to lessening the PON1 bioavailability and/
or catalytic activity. Whatever are the causes, an altered PON1 status would increase
the susceptibility to neurotoxic pesticides (especially in Reelin-deficient subjects
during critical periods) and lead to deficiencies in corticogenesis and cholinergic
neurotransmission, as well as contribute to the perturbation in the redox homeostasis
and immune dysfunctions; and all these despite the relative nonspecificity of the
PON1 alterations in autism.
We suggest that future studies should be performed on bigger, prospec-
tive cohorts of ASD patients, and that they should address the following issues:
(a) investigate for a possible relationship between PON1 and the severity of the
clinical phenotype; (b) elucidate the effect of HDL, inflammation, and oxidative
biomarkers on PON1 in autism; (c) replicate and verify for causality the asso-
ciation between OP exposure and autism; (d) measure the putative physiological
activity of PON1, i.e., lactonase activity; and (e) investigate the effect of other
SNPs or distant genetic loci.
ACKNOWLEDGMENTS
The authors would like to thank Drs. Anca M. Paşca, Raul C. Muresan, and Carmen
Gherasim for fruitful discussions and pertinent comments on previous versions of
this manuscript.
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7 The Redox/Methylation
Hypothesis of Autism:
A Molecular Mechanism
for Heavy Metal-Induced
Neurotoxicity
Richard C. Deth* and Christina R. Muratore
Department of Pharmaceutical Sciences,
Northeastern University, Boston, MA 02115, USA
CONTENTS
7.1 Introduction ................................................................................................. 113
7.2 Redox: An Evolutionary Perspective .......................................................... 114
7.3 Redox Metabolism in Human Brain ........................................................... 115
7.4 Redox and Methylation ............................................................................... 117
7.5 Heavy Metals and Redox Status ................................................................. 118
7.6 Oxidative Stress and Neuroinflammation in Autism .................................. 120
7.7 A Molecular Mechanism for Heavy Metal-Induced Autism ...................... 122
7.7.1 Preexisting Genetic Risk Factors ................................................... 122
7.7.2 Heavy Metal Exposure................................................................... 123
7.7.3 Molecular Targets .......................................................................... 124
7.7.4 Cellular Consequences................................................................... 125
7.8 Summary ..................................................................................................... 126
References .............................................................................................................. 127
7.1 INTRODUCTION
The rapid rise of autism rates over the past 25 years suggests a potential toxic effect
from one or more environmentally encountered substances on the normal trajectory
of neurological development, although increased diagnosis may also contribute. Two
major autism categories can be identified, based upon whether or not individuals
did or did not initially exhibit normal cognitive function and language acquisition.
113
(1) Classical autism, described prior to 1980, corresponds to the latter pattern where
infants fail to achieve standard developmental milestones from birth. (2) Regressive
autism, which is more typical of the contemporary condition, reflects a loss of pre-
viously acquired abilities that can occur over a short period of time (i.e., days to
weeks). Anecdotal reports from some parents link regression to the time period after
vaccination, raising the possibility that vaccine components, including mercury and/
or aluminum and the accompanying inflammatory activation they promote, might
contribute to autism. While this proposal is both frightening and controversial, it
requires serious consideration and investigation. Autistic children exhibit signs of
oxidative stress and neuroinflammation, which may be attributed in part to heavy
metal toxicity. This chapter reviews clinical observations and relevant molecular
mechanisms, which bear on this critical public health question.
7.2 REDOX: AN EVOLUTIONARY PERSPECTIVE

Many biochemical reactions involve reduction or oxidation, the gain or loss of elec-
trons, respectively. This is especially true in aerobic organisms, such as humans,
which utilize oxygen as a primary source of energy, creating a constant source
of oxidative risk that must be counterbalanced by an effective antioxidant redox-
buffering system (Benzie, 2000). Since the origin of life, molecules containing
reduced sulfur (e.g., thiols) have served as primary antioxidants, based upon the
ease with which they release their hydrogen atom, making them excellent reducing
agents. Indeed, the simplest thiol, hydrogen sulfide, a gas released from under-
water volcanic vents, may have been essential for earliest life forms (Clark et al.,
1998). Thiols within proteins (e.g., enzymes) can be modified by reactive oxygen
or nitrogen species, and by formation of mixed disulfides, resulting in a change of
protein function. Via these mechanisms, a vast number of cellular processes come
under the influence of cellular redox status. Under oxidative stress conditions,
these thiol-based changes in protein function can serve to restore redox balance,
representing a major example of homeostatic control.
It has been proposed that metabolic adaptation to increasing levels of oxygen
has been an important driving force during the 2–3 billions of years over which
life on earth has evolved (Falkowski, 2006; Raymond and Segrè, 2006). At the
ocean depths where geothermal conditions allowed synthesis of biomolecules
(e.g., amino acids) and primitive life forms, oxygen exists almost exclusively in its
fully reduced state, i.e., water (H2O). Accordingly, early organisms depended upon
anaerobic metabolism, including the abstraction of reducing equivalents (hydro-
gen atoms) from hydrogen sulfide (H2S). Photosynthesis by algae and plants began
converting H2O to O2, a reaction that liberates both electrons and reducing equiva-
lents, which can react with carbon dioxide (CO2) to form organic materials (e.g.,
carbohydrates). Based upon the utility of these reactions, plants emerged from the
ocean and became a dominant species, accelerating oxygenation of the atmosphere.
As damage from accumulating oxygen became more problematic, organisms with
novel antioxidant metabolic strategies emerged and evolved, illustrating the power
of negative selection pressure. Along this evolutionary path, the selective advantage
of combining cell types with different metabolic networks was realized, resulting
The Redox/Methylation Hypothesis of Autism 115
in complex plant and animal organisms. As we reflect on the extreme complexity of

humans, with multiple organ systems and a resident population of microorganisms
in our intestine, we must appreciate that the primitive elements of our metabolic
origins are still operative and the ability to sustain redox balance is the heart of
metabolism for every cell type.
7.3 REDOX METABOLISM IN HUMAN BRAIN

Autism is primarily a neurodevelopmental disorder, and systemic oxidative stress
has been documented in autistic children (Chauhan and Chauhan, 2006; James et al.,
2004, 2006). This strongly suggests that the brain and neuronal function are more
sensitive to disturbances involving redox metabolism than other tissue and cell types.
This conclusion is consistent with the notion that the brain, indeed the human brain,
reflects the highest degree of evolution with unique metabolic features that are nec-
essary for its normal function. Reciprocally, this perspective implies that the human
brain function is uniquely vulnerable to the toxic effects of agents such as heavy
metals, which interfere with redox metabolism.
The tripeptide glutathione, in its reduced (GSH) and oxidized (GSSG) forms,
is the primary determinant of redox status in all human cells, reflected by its high
intracellular concentration (∼10 mM) and the multiplicity of redox reactions in
which it participates (Schafer and Buettner, 2001). The cysteine residue of GSH,
flanked by glutamate and glycine, is the basis of its redox activity, reflecting its
hydrogen sulfide origins. Prevailing levels of GSH depend upon its de novo synthe-
sis, for which the concentration of cysteine is rate-limiting, as well as its dynamic
utilization in multiple reactions, many of which are reversible, allowing regeneration
of GSH. Most important among the latter is reduction of oxidized GSSG to GSH,
which can be directly accomplished by glutathione reductase or indirectly via
involvement of protein thiols in conjunction with the thioredoxin system. In both
cases, reducing equivalents are provided by NADPH, whose level is maintained by
glucose metabolism through the successive actions of hexokinase and glucose-6-
phosphate dehydrogenase.
De novo synthesis of GSH is supported by cysteine made available from the
breakdown of proteins, through cellular uptake, or via transsulfuration of homo-
cysteine, which is elaborated during the methionine cycle of methylation (Figure 7.1).
Various cell surface amino acid transporters bring either cysteine or oxidized cystine
into cells. Cell type–specific differences in transporter expression can lead to varia-
tions in redox regulation and can also influence local glutamate levels. Astrocytes,
for example, express a transporter which exchanges cystine for glutamate, EAAT1
(Excitatory Amino Acid Transporter 1), and within the cell cystine is reduced to pro-
vide cysteine for GSH synthesis (Banner et al., 2002). Under normal circumstances,
astrocytes synthesize excess GSH, and the excess is released to the extracellular
space where it is converted to cysteine by the action of two proteases. Neurons take
up astrocyte-derived cysteine via EAAT3, which can alternatively transport gluta-
mate or aspartate, the predominant transporter in mature neurons (Aoyama et al.,
2006; Gegelashvili and Schousboe, 1998). EAAT3 activity is regulated by protein
kinase C and phosphatidylinositol 3 kinase (Do et al., 2002; Huang and Zuo, 2005),
Cysteine Cysteinylglycine GSH Astrocyte

Glutamate
EAAT3
GSSG GSH GSCbl
Glutamate OHCbl SAM
γ-Glutamylcysteine
MeCbl
Cysteine H2S
Partial block in human brain
Cystathionine
Adenosine Adenosine
D4SAH D4HCY HCY SAH
MethylTHF (–)
MethylTHF >150
Phospholipid Methionine
methylation synthase Methylation
THF THF reactons
D4SAM D4MET MET SAM
PP+Pi ATP ATP PP+Pi
Dopamine
Neuron
FIGURE 7.1 Pathways of thiol metabolism in human neuronal cells. In human neurons,
transsulfuration is impaired and cysteine for synthesis of GSH is provided primarily by
uptake via EAAT3, which is dependent upon GSH released from astrocytes. GSH is required
for synthesis of methylcobalamin (MeCbl) and for activity of methionine synthase. During
oxidative stress, low levels of GSH and MeCbl cause a decrease in a large number of SAM-
dependent methylation reactions, and a decrease in dopamine-stimulated, D4 dopamine
receptor-mediated phospholipid methylation.
linking it to receptor signaling pathways, and implying that neurotransmitters and

neurotrophic factors may regulate redox status in neurons.
Transsulfuration of homocysteine (HCY) provides a major source of cysteine
in most cells. However, in the brain, activity of cystathionine-gamma-lyase is
low, resulting in accumulation of cystathionine and decreased cysteine availabil-
ity (Finkelstein, 1990). Early studies concluded that transsulfuration did not func-
tion in brain, but more recent studies confi rm partial activity, since inhibition
of cystathionine-gamma-lyase leads to a significant decrease in GSH (Vitvitsky
et al., 2006). Interestingly, levels of cystathionine are 40-fold higher in human
brain versus rat or mouse, with intermediate levels in nonhuman primates, although
levels in other human tissues are low (Tallan et al., 1958). This pattern reflects a
progressive decrease in transsulfuration with evolution among these mammals,
accompanied by a progressive increase in the risk of oxidative stress. It is also
of interest that the transsulfuration pathway operates in the opposite direction in
plants, allowing synthesis of HCY (and methionine) from cysteine, reflecting a
lower requirement for GSH in association with the release of oxygen (Giovanelli and
Mudd, 1967).
Restricted transsulfuration in human brain has several implications: (1) Capacity
for GSH synthesis is limited. (2) EAAT3-dependent uptake of cysteine is more critical
for maintaining cellular redox status. (3) Agents affecting EAAT3 activity will exert
a more powerful influence on redox status. (4) The limited available pool of GSH
may be more dynamically utilized. (5) Human brain function is more vulnerable to
toxic substances, which impair redox metabolism.
7.4 REDOX AND METHYLATION

Methylation involves the addition of a carbon atom to a molecule, usually causing
a change in the function of the methylated molecule. S-adenosylmethionine (SAM),
the ATP-activated form of the essential amino acid methionine, is the methyl donor
for more than 150 methyltransferase-dependent methylation reactions, which regu-
late a large number of cellular functions (Katz et al., 2003). Donation of a methyl
group by SAM results in formation of S-adenosylhomocysteine (SAH), which inhib-
its methyltransferase activity by competing with SAM, and is reversibly converted
to HCY (Figure 7.1). Ongoing activity of the methionine cycle of methylation is
sustained by the cobalamin (vitamin B12)-dependent enzyme methionine synthase,
which utilizes 5-methyltetrahydrofolate (methylfolate) as the methyl donor for con-
version of HCY to methionine. Since HCY formation from SAH is reversible, any
decrease in methionine synthase activity will be reflected as an increase in both
HCY and SAH, resulting in inhibition of SAM-dependent methylation reactions.
Clearly methionine synthase exerts a powerful influence over cell function via its
control over methylation.
As illustrated in Figure 7.1, methionine synthase is positioned at the inter-
section between transsulfuration and methylation pathways. As a consequence,
its level of activity exerts control over cellular redox status, since it determines
the proportion of HCY that will be diverted toward cysteine and GSH synthesis.
Methionine synthase activity is exceptionally sensitive to inhibition during oxida-
tive stress, primarily because its cobalamin cofactor is easily oxidized (Liptak
and Brunold, 2006). This allows methionine synthase to serve as a redox sensor,
lowering its activity whenever the level of oxidation increases, until increased
GSH synthesis brings the system back into balance. Electrophilic compounds,
such as oxygen-containing xenobiotic metabolites, also react with cobalamin,
inactivating the enzyme and increasing diversion of HCY toward GSH synthesis
(Watson et al., 2004). Thus methionine synthase is a sensor of both redox and
xenobiotic status.
One of the most important roles of methylation is epigenetic regulation of gene
expression through DNA methylation. Cytidine residues preceding guanine residues
(i.e., CpG sites) frequently occur in promoter regions and their methylation facilitates
binding of a series of proteins that favor histone binding and inhibits transcription of
adjacent genes (Miranda and Jones, 2007). Significant changes in patterns of DNA
methylation occur during development, as genes are differentially turned off or on,
and disruption of methylation by oxidative stress and impaired methionine synthase
activity will therefore adversely affect development. Indeed, several neurodevelop-
mental disorders including Rett, Prader-Willi, and Angelman syndromes, as well as
fragile X syndrome, have been linked to genetic defects involving DNA methylation
(McConkie-Rosell et al., 1993; Thatcher et al., 2005; Wan et al., 1999). Oxidative
stress associated with environmental exposure to xenotoxins may therefore mimic

certain aspects of these disorders. Moreover, xenotoxin exposure can amplify the
impact of methylation-related genetic risk factors, increasing the likelihood of devel-
opmental disorders.
7.5 HEAVY METALS AND REDOX STATUS

It is widely recognized that heavy metals exert many of their toxic effects via binding
to thiols. Moreover, thiol-containing compounds, especially GSH, are critical for
heavy metal detoxification and elimination. This is particularly true for mercury,
and the term mercaptan is a synonym for thiol-containing compounds, which “cap-
ture mercury.” Since thiols play a central role in maintaining cellular redox status, it
is not surprising that mercury and other heavy metals would disrupt redox status.
Moreover, cell type-specific differences in thiol metabolism, as described above, can
lead to differences in vulnerability to heavy metals.
Mercury exists in elemental (Hg•), inorganic (Hg2+), or organic (e.g., methyl-
mercury or ethylmercury) states. Hg• is liquid at room temperature and has a high
vapor pressure, which allows it to readily enter the gas phase. These unique physical
properties increase the spread of mercury throughout the earth and its atmosphere
and also facilitate its use in industrial products such as liquid electrical switches and
light bulbs. Methylmercury from maternal seafood ingestion and dental amalgams
is the primary source of in utero exposure for infants, while ethylmercury from the
vaccine preservative thimerosal can be a postnatal source of exposure. Vaccination-
associated mercury exposure significantly increased starting in the late 1980s, but
was dramatically reduced following a 1999 FDA report (Centers for Disease Control
and Prevention, 1999), which led to the availability of nominally thimerosal-free
infant vaccines starting in 2001. Removal of thimerosal was not, however, associ-
ated with a decrease in rising autism rates (Schechter and Grether, 2008), casting
doubt on a causative role for thimerosal (Fonbonne, 2008). Nonetheless, since epi-
genetic effects of toxic exposures can be transmitted across generations, significant
concerns remain about the impact of mercury exposure during the two previous
decades. In addition, aluminum, which shares many of the effects of mercury on
thiol metabolism and redox status (Sharma and Mishra, 2006; Verstraeten et al.,
2008), remains as an adjuvant additive in a number of vaccines at levels much higher
than previous levels of mercury. Lead exposure, from paint, dust, contaminated soil,
or other sources is an additional, potentially important contributor to neurotoxicity
and autism via its effects on redox status (Quig, 1998; Verstraeten et al., 2008).
Organomercurials have greater access to the brain than inorganic mercury, since
methyl and ethyl groups increase hydrophobic character and facilitate diffusion across
the blood–brain barrier. However, the methyl and ethyl groups dissociate from mer-
cury, leaving inorganic mercury trapped within the brain compartment, where it can
remain for years. Studies in nonhuman primates showed that a greater proportion of
inorganic mercury remained in the brain from thimerosal than from methylmercury,
consistent with the weaker chemical bond of mercury to the ethyl group (Burbacher
et al., 2005). Lacking methyl or ethyl groups, aluminum has an intrinsically lower
ability to cross the blood–brain barrier than organomercurials, but significant levels
in brain can be detected after vaccination (Flarend et al., 1997).
Within the brain compartment, mercury and other metals affect thiol metabolism
in different cell types, including pluripotent stem cells, neurons, astrocytes, micro-
glia, and oligodendrocytes. Under mild oxidative stress conditions, an increased
proportion of pluripotent stem cells become astrocytes, whereas mild reducing con-
ditions increase the proportion of neuronal cells (Prozorovski et al., 2008). Since
astrocytes serve as reservoirs of GSH and provide cysteine for neurons (Figure
7.1), this mode of regulation appears to adjust cell fate in response to prevailing
redox conditions, and heavy metal-induced oxidative stress would reduce neuronal
development. Neuronal stem cells are particularly sensitive to mercury, and low
nanomolar concentrations of methylmercury activate caspase-dependent apopto-
sis (Tamm et al., 2006). Heavy metal-induced oxidative stress in oligodendrocytes
can lead to impaired myelination (Crang and Jacobson, 1982), while it can lead to
activation and the release of proinflammatory cytokines in microglia (Kim and de
Vellis, 2005). Importantly, each of these conditions has been observed in the brain
of children with autism.
In 2004, our group first described the potent inhibitory effects of mercury,
thimerosal, aluminum, and lead on methylation and methionine synthase activity
in SH-SY5Y human neuronal cells (Waly et al., 2004). Subsequently, we determined
that inhibition reflected the ability of these heavy metals to lower GSH levels
(M. Waly et al., unpublished observation), resulting in decreased synthesis of meth-
ylcobalamin (methylB12), which is required for methionine synthase activity in
these neuronal cells, as illustrated in Figure 7.1. These heavy metals potently inhibit
EAAT3-mediated uptake of cysteine, which accounts for their ability to decrease
GSH, methylB12, and methionine synthase activity. Together, these studies illustrate
the critical role of EAAT3 in regulating redox status and methylation activity in
human neuronal cells, as well as their vulnerability to heavy metals.
An important breakthrough in understanding the molecular mechanism of mer-
cury toxicity was provided from studies carried out by Holmgren and colleagues
at the Karolinska Institute in Sweden (Carvalho et al., 2008). They compared the
potency of inorganic mercury and methylmercury to inhibit several enzymes, each
of which promote a reduced intracellular redox state, including thioredoxin, thiore-
doxin reductase, glutathione reductase, and glutaredoxin. Among these, thioredoxin
and thioredoxin reductase showed exceptionally high sensitivity to both mercury
compounds, strongly suggesting that they are primary targets for mercury-induced
neurotoxicity. Thioredoxin has multiple activities, including the ability to release
GSH from glutathionylated proteins (i.e., proteins with a thiol-bound GSH), while
thioredoxin reductase, a selenoprotein, serves to reactivate thioredoxin after it has
carried out deglutathionylation (Figure 7.2). The extent of protein glutathionylation
reflects the level of cellular oxidative stress, and mercury inhibition of the thiore-
doxin system will promote the accumulation of glutathionylated proteins, producing
and sustaining a state of high oxidative stress. The ultrahigh affinity of mercury for
selenoproteins has long been recognized, and selenium supplementation has been
suggested as a treatment for mercury toxicity.
Oxidation GSH
GSH GSSG HS- Protein
Thioredoxin
GSH or
Hg2+
glutaredoxin
GSS- Protein Thioredoxin

reductase
Change in function NADPH
FIGURE 7.2 Mercury inhibits deglutathionylation of proteins. Mixed disulfide exchange

allows glutathionylation of proteins by oxidized glutathione (GSSG), which can result
in an alteration of protein function associated with oxidative stress. Thioredoxin and
glutaredoxin remove glutathione utilizing NADPH-derived reducing equivalents pro-
vided by thioredoxin reductase. Mercury potently inhibits all three proteins involved in
deglutathionylation.
7.6 OXIDATIVE STRESS AND NEUROINFLAMMATION IN AUTISM

As detailed in this book, a substantial and growing body of evidence indicates that
oxidative stress and neuroinflammation are closely associated with autism and are
likely to be critical factors in causing the disorder (for reviews see: Chauhan and
Chauhan, 2006; Deth et al., 2008; Kern and Jones, 2006; McGinnis, 2004). Plasma
levels of glutathione, as well as methionine cycle and transsulfuration metabolites
are abnormal in autistic individuals (Geier and Geier, 2006; James et al., 2004,
2006). Adenosine and SAH levels are increased while HCY, methionine and SAM
levels are low, consistent with decreased methionine synthase activity and increased
cystathionine-beta-synthase (CBS) activity, while the SAM/SAH ratio is signifi-
cantly reduced, indicating impaired methylation capacity. Cystathionine, cysteine,
and GSH levels are decreased, along with the GSH/GSSG ratio, reflecting increased
oxidative stress. Elevated HCY levels have also been reported in autism (Pasca et al.,
2006). Supplementation with a combination of betaine (trimethylglycine) and folinic
acid (5-formylTHF) normalized methionine cycle metabolites, but transsulfuration
metabolites remained abnormal (James et al., 2004). Upon further addition of meth-
ylcobalamin, levels of all metabolites, as well as SAM/SAH and GSH/GSSG ratios
returned to normal. If these abnormal metabolic profiles are confirmed by others,
they will represent a critically important clue to the origins of autism.
Oxidative stress in autism is associated with increased plasma levels of mal-
onyldialdehyde, urinary levels of fatty acid and lipid peroxidation biomarkers
(Chauhan et al., 2004; Ming et al., 2005; Yao et al., 2006; Zoroglu et al., 2004).
Elevated levels of inflammatory cytokines and evidence of microglial activation
are observed in postmortem brain sections, indicating the presence of neuroin-
flammation (Vargas et al., 2005). Microglial cells monitor the local environment
and provide a macrophage-like function in the brain, releasing proinflammatory
substances upon activation. In addition, microglia take up organic mercury and

convert it to the more toxic inorganic mercury (Charleston et al., 1995), and in pri-
mate cortex, chronic methylmercury exposure leads to a large increase in activated
microglia (Charleston et al., 1994). Heavy metals can therefore cause oxidative
stress in neurons not only by their direct influence on sulfur metabolism, but also
by promoting microglia-based neuroinflammation.
Oxidation of cobalamin during oxidative insults provides a short-term mechanism
to augment transsulfuration and GSH synthesis, but chronic neuroinflammation and
prolonged oxidative stress can activate additional, longer-term adaptive responses to
restrict methionine synthase activity. These could include decreased transcription of
the methionine synthase gene, decreased translation of its mRNA, increased deg-
radation of the protein, and/or decreased cellular uptake of cobalamin or folic acid
cofactors. To evaluate brain methionine synthase status in autism, we carried out
quantitative reverse transcriptase polymerase chain reaction (RT-PCR) using cortex
RNA samples from autistic subjects and age-matched neurotypical control subjects.
The tissues from which these RNA samples were derived included those in which
Vargas et al. (2005) described the presence of neuroinflammation. We utilized primers
directed against both cobalamin-binding and cap domains of methionine synthase
and in both cases, the level of mRNA was significantly lower in autism samples,
amounting to a decrease of two- to threefold (Figure 7.3; unpublished observation).
As outlined above, lower activity of methionine synthase increases HCY diversion
to transsulfuration and GSH synthesis, thus we interpret the reduction in methionine
synthase mRNA as an adaptive response to oxidative stress and neuroinflamma-
tion. This finding confirms impaired methylation in the brain during autism, and in
particular, it indicates that the supply of methyl groups for dopamine-stimulated
phospholipid methylation (PLM) activity will be reduced.
2.0
Control
Autistic
1.5
Methionine synthase
mRNA (fold change)
1.0
**
0.5
*
0.0
CAP COB
FIGURE 7.3 Methionine synthase mRNA levels in human cortex are reduced in autism.
RNA samples from autistic and nonautistic subjects were probed using qRT-PCR with spe-
cific primers to the CAP and COB domains of methionine synthase. n = 11 for each group
(∗ = p < 0.05 compared to control for the CAP primer set; ∗∗ = p < 0.05 compared to control
for the COB primer set).
Genes play a major role in autism, as reflected by high concordance rates

between monozygotic twins (Smalley et al., 1988), but this does not necessarily
imply that genetic defects are the cause of autism. A study of single-nucleotide
polymorphisms (SNPs) in genes affecting redox and methylation found that autis-
tic subjects had a significantly higher prevalence of risk-inducing SNPs (James
et al., 2006). These genes included transcobalamin II and the reduced folate car-
rier, which transport cobalamin and folate into cells, as well as methionine syn-
thase reductase, which is responsible for reduction of oxidized cobalamin, and
catecholamine-O-methyltransferase (COMT), which inactivates dopamine and
norepinephrine. Importantly, these SNPs are normal features of human genes, con-
tributing to individual variability in the metabolic processes they control. When
combinations of SNPs were evaluated, an odds ratio up to sevenfold was found,
providing a clear example of how genetic risk factors might contribute to autism,
but at the same time not necessarily represent the critical causative factor.
7.7 A MOLECULAR MECHANISM FOR HEAVY

METAL-INDUCED AUTISM
The above-described observations provide a framework for specifying a molecular
hypothesis of how exposure to heavy metals may cause autism in genetically vulner-
able individuals. The hypothesis presented here uses organic mercury provided by
thimerosal as a prototypical example, but other sources of exposure and other heavy
metals can be expected to substantially act via the same mechanism. The possible
involvement of thimerosal-derived mercury in autism is a highly controversial subject,
which requires further research and investigation. The aim of providing a specific
molecular mechanism is to bring clarity to the proposed link between mercury expo-
sure and autism, and to guide further studies, which will ultimately refute or sustain
the hypothesis. If sustained, the hypothesis should lead to measures that will reduce
heavy metal exposure of infants in the future with the expectation that autism rates
will cease to increase and may even decrease. In addition, a useful hypothesis should
also lead to effective treatments for those who are already affected, a goal which
is partially realized by current biomedical treatments. The hypothesis is directed
toward “contemporary autism,” whose rising prevalence suggests a critical role for
environmental factors. It does not apply to “classical autism,” which is more reflec-
tive of profound genetic abnormalities. If the prevalence of contemporary autism
is taken as 10-fold higher than that for classical autism, this hypothesis is directed
toward 90% of current cases.
7.7.1 PREEXISTING GENETIC RISK FACTORS

Risk of heavy metal-induced autism can arise from normally occurring genetic
variants, which directly or indirectly affect the capacity for glutathione synthesis,
the activity of methionine synthase, CBS, the methylation of DNA or histones,
or the level of catecholamines and/or their receptor signaling activity. Additional
genetic risk arises from variants affecting the formation of neural networks
(e.g., neurogenesis, neural differentiation, synapse formation), particularly those
networks supporting gamma frequency synchronized oscillations during aware-

ness, attention, and cognition. Notably, these genetic risk factors would be latent in
the absence of environmental exposures, giving rise to differences in neurological
and cognitive function that would be considered to be normal individual variations
within the population.
7.7.2 HEAVY METAL EXPOSURE

The opportunities for infants to be exposed to heavy metals are limited. Moreover,
there are very few opportunities for exposure of sufficiently large number of infants
in the United States to cause rates of autism to increase across the entire country.
Prenatal exposure from maternal fish consumption is one possible source of heavy
metal exposure with sufficiently broad influence to potentially contribute to the cur-
rent “autism epidemic.” Vaccine-associated ethylmercury and aluminum represent
another potential source of society-wide heavy metal exposure.
Prenatal heavy metal exposure can occur through, for example, maternal consump-
tion of fish containing significant quantities of methylmercury. Since fish provide an
important source of ω-3 fatty acids and other nutrients important for brain develop-
ment, contamination of this vital food category represents a major threat to human
well-being. The Environmental Protection Agency (EPA) established a benchmark
range for methylmercury in cord blood of 46–79 μg/L (213–367 nM), based upon
neurocognitive testing of exposed populations, which they consider as a threshold
value for significant “developmental neuropsychological impairment” (U.S. EPA,
2001). However, methylmercury levels above 6 μg/L (27 nM) are associated with lower
IQ (Kjellstrom et al., 1986, 1989; Lederman et al., 2008), suggesting that neuronal
function is impaired at low nanomolar concentrations of mercury. Neurological
assessment of neonates revealed a correlation between blood mercury levels and
developmental status, with a 10-fold higher mercury level being equivalent to a loss
of 3 weeks in gestational development (Steuerwald et al., 2000). Since development
substantially reflects epigenetic influences over gene expression, decreased methyla-
tion of DNA and histones is likely to be an important aspect of this mercury-induced
developmental delay.
Vaccination with the ethylmercury-containing preservative thimerosal represented
a significant source of mercury exposure for infants in the United States prior to its
voluntary removal from mandated childhood vaccines starting in 2001. The preser-
vative action of thimerosal reflects the ability of mercury to potently interfere with
thiol metabolism in all organisms so as to limit their replication and survival. After
administration of a thimerosal-containing vaccine, peak blood mercury levels reach
5.0, 3.6, and 2.8 μg/L in newborn, 2-month-old, and 6-month-old infants, respectively,
and blood levels decrease with a t1/2 of 3.7 days (Pichichero et al., 2008). However,
for autism, the relevant issue is the level of mercury in brain tissue, particularly the
level of inorganic mercury, which is retained for long periods of time. A study in
nonhuman primate infants showed that total brain mercury reached approximately
40 ng/g immediately following four thimerosal-containing vaccinations, which were
timed to replicate the schedule for human infants, and the level of inorganic mercury
reached 16 ng/g of brain tissue (Burbacher et al., 2005). This tissue level of inorganic
mercury would be equivalent to a concentration of 80 nM if the mercury was in solu-

tion, but the vast majority of mercury is undoubtedly bound to thiols and thiol- or
selenium-containing proteins, where it exerts its neurotoxic effects (Ralston et al.,
2007). Consistent with high-affinity and essentially irreversible binding, the level of
inorganic mercury in brain failed to show any significant decline during 28 days
of washout, although organic mercury decreased with a t1/2 of 14 days.
The above studies indicate that vaccination with thimerosal-containing vaccines
creates a steady-state level of inorganic mercury in the brain that can bind to thiol
and selenium-containing proteins in the nanomolar concentration range. At the
same time, dose–response studies in cultured human neuronal cells show that both
thimerosal and inorganic mercury significantly lower GSH levels, inhibit methion-
ine synthase, and reduce methylation activity at subnanomolar concentrations (Waly
et al., 2004; M. Waly et al., unpublished observation). Thus vaccine-derived mercury
exposure can produce brain levels of mercury sufficient to interfere with methylation
of DNA and histone in a manner, which would lead to developmental disorders.
Aluminum is added to many vaccines, at concentrations 10-fold or higher than
mercury, as an adjuvant to boost immunity by promoting Th2 cell-mediated antibody
response (Lindblad, 2004), reflecting its ability to promote inflammation and induce
oxidative stress (Oteiza et al., 2004). Aluminum lowers brain levels of GSH and
disrupts neurodevelopment in rats (Domingo, 1995; Sharma and Mishra, 2006), and
has been implicated as a factor in neurodegenerative disorders such as Alzheimer’s
disease (Campbell, 2006). In studies with cultured human neuronal cells, we found
that aluminum inhibits methylation activity and lowers GSH in human neuronal
cells at nanomolar concentrations, similar to mercury (Waly et al., 2004; M. Waly
et al., unpublished observation). Thus although it has received less attention than
mercury, vaccine-derived aluminum is a significant source of heavy metal exposure
for infants, and may contribute to causing autism.
7.7.3 MOLECULAR TARGETS

As outlined above, mercury binds strongly to thiols, even stronger to selenocysteine,
and this binding forms the basis for its neurodevelopmental and neurotoxic effects.
Organic mercury binds to lower molecular weight thiols (e.g., GSH, cysteine, HCY),
which are present in the extracellular fluids at micromolar concentrations, via one
bond, while inorganic mercury can bind two thiols. When thiol-bound mercury
is excreted, this binding represents a mode of detoxification. Specialized proteins
containing multiple cysteines (e.g., metallothionein) or selenocysteines (e.g., sele-
noprotein P) provide addition protection. When the mercury-thiol bond does break,
mercury is free to redistribute to other partners, and over time, it migrates to sites of
higher affinity. High-affinity binding of inorganic mercury binding is provided by
proteins, in which it is able to simultaneously bind to two cysteine or selenocysteine
residues. In such dual binding locations, the occasional breakage of one bond does
not release mercury, resulting in ultrahigh-affinity binding, which can last for the
lifetime of the protein. Accordingly, the activity of such proteins can be inhibited in
an essentially irreversible manner.
Intramolecular disulfide bonds provide potential binding sites for mercury, par-
ticularly if they undergo cycles of oxidation and reduction. Vicinal (i.e., consecutive)
cysteine residues provide a special case, and the oxidized and reduced proteins have
significantly different conformations and activities. In such cases, the disulfide bond
serves as a redox sensor, and the binding of mercury stabilizes a conformation,
which mimics the oxidized condition. Another structural motif with high mercury
binding affinity is the thioredoxin fold, named after the ubiquitous protein in which
it was first characterized. In the thioredoxin fold, two cysteines are separated by two
residues (e.g., glycine and proline), which allow the formation of a redox-sensitive
disulfide bond that binds inorganic mercury with high affinity. Since thioredoxin
catalyzes removal of thiols such as GSH or cysteine from proteins, mercury inhi-
bition results in the accumulation of these thiol-modified proteins. Under normal
circumstances, thiolation of proteins is increased when GSSG and cystine levels are
high, so the net effect of mercury is to shift cells to an oxidative stress condition.
Mercury binds potently to thiols in their unprotonated thiolate state, but the
prevalence of this state is much higher for selenocysteine, resulting in a millionfold
higher affinity for mercury, as compared to cysteine, with a binding constant of 1045
(Dyrssen and Wedborg, 1991). This property is the basis for the unique redox capa-
bilities of selenoproteins, but also makes them highly vulnerable to mercury inhibition.
Moreover, redistribution of mercury away from thiols of lower affinity results in its
increasing association with selenoproteins. Thus the targets of mercury, which are
most likely to contribute to autism are selenoproteins, which include thioredoxin
reductases, glutathione peroxidases, thyroid hormone deiodinases, selenoprotein P,
and other proteins whose function remains to be established. Selenoproteins are par-
ticularly important in brain, and under deficiency conditions, other tissues release
their selenium, which is taken up by the brain (Behne and Kyriakopoulos, 2001).
Since both thioredoxin and thioredoxin reductase possess molecular features,
which render them highly sensitive to inorganic mercury, their combined inhibition will
result in a highly amplified accumulation of thiol-modified proteins (Figure 7.2).
When combined with the unique pattern of sulfur metabolism in human neuronal
cells (Figure 7.1), these actions may account for the extremely potent reduction in
GSH levels caused by mercury. Importantly, autistic subjects have a significantly
lower level of selenium, which may increase their sensitivity to mercury (Jory and
McGinnis, 2008).
7.7.4 CELLULAR CONSEQUENCES

A reduction in cellular GSH levels causes a decrease in the synthesis of methyl-
cobalamin, which is absolutely required for the activity of methionine synthase in
human neuronal cells (M. Waly et al., unpublished observation). Lower methionine
synthase activity leads to a decrease in the SAM to SAH ratio, resulting in impaired
methylation of DNA and histones, with adverse consequences for the highly orches-
trated expression of genes during development. Differentiation of neuronal stem
cells is particularly sensitive to mercury (Tamm et al., 2006). Lower methionine
synthase activity interferes with D4 dopamine receptor-mediated PLM, which has
been proposed to play a central role in gamma frequency synchronization of neural

networks during attention (Kuznetsova and Deth, 2008).
Mitochondrial dysfunction is a recognized risk factor for autism (Oliveira et al.,
2005; Poling et al., 2006). Complex I of the mitochondrial electron transport chain is
subject to glutathionylation at several cysteine residues during oxidative stress, which
is correlated with impaired activity (Hurd et al., 2008). Mercury-induced inhibition
of thioredoxin activity will increase complex I glutathionylation, thereby providing a
molecular link to the occurrence of mitochondrial dysfunction.
7.8 SUMMARY
The ability to counteract oxidation is a critical role for sulfur metabolism in all cells,
but human neuronal cells are particularly vulnerable to oxidative stress. As illustrated
in Figure 7.4, mercury and other heavy metals impair GSH synthesis and inhibit
methylation activity by binding with high affinity to thiols and selenoproteins. Studies
indicate that the concentration of inorganic mercury delivered to the brain following
vaccination is sufficient to inhibit the activity of selenoproteins such as thioredoxin
reductase. This inhibition creates a state of oxidative stress, which disrupts methy-
lation activities including epigenetic regulation of gene expression and dopamine-
stimulated PLM. Together, these observations provide a coherent and compelling
molecular mechanism by which mercury and other heavy metals can contribute to
the etiology of autism. Additional studies are needed to confi rm specific aspects
of this molecular hypothesis.
Heavy metals
(mercury)
Protein thiols Selenoproteins

(e.g., thioredoxin) (e.g., thioredoxin reductase)
Δ Protein thiol status Δ Protein activity

Genetic (e.g., glutathionylation) (e.g., mitochondrial dysfunction)
risk
factors
Oxidative stress + ROS
Impaired methylation
DNA methylation Dopamine-stimulated

Δ Gene expression phospholipid methylation
Developmental delay Impaired attention
FIGURE 7.4 Molecular hypothesis for heavy metal-induced autism. Heavy metals such as
mercury bind with high affinity to protein thiols and to selenoproteins, disrupting their role in
redox regulation. The resulting oxidative stress inhibits methionine synthase activity, causing
a decrease in DNA methylation and a decrease in D4 dopamine receptor-mediated phos-
pholipid methylation. In a genetically vulnerable subpopulation, these neurotoxic effects are
significant factors in the etiology of autism.
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8 Autism and Oxidative
Stress: Evidence from
an Animal Model
Michelle A. Cheh,1 Alycia K. Halladay,2,3 Carrie
L. Yochum,2 Kenneth R. Reuhl,3 Marianne
Polunas,3 Xue Ming,4 and George C. Wagner2,*
Departments of 1Neuroscience, 2Psychology,
and 3Pharmacology and Toxicology, Rutgers
University, New Brunswick, NJ 08854, USA
4Department of Neuroscience, University of Medicine
and Dentistry of New Jersey, Newark, NJ 07103, USA
CONTENTS
8.1 Introduction: An Overview of Autism ........................................................ 132
8.2 Etiology of Autism ...................................................................................... 133
8.3 Animal Model of Autism ............................................................................ 135
8.4 Oxidative Stress and Autism ....................................................................... 136
8.5 Valproic Acid, Autism, and Oxidative Stress.............................................. 137
8.6 Mercury Exposure and Autism ................................................................... 138
8.7 Trolox Protects Mice against Early Exposure to MeHg ............................. 139
8.7.1 Body Weight .................................................................................. 140
8.7.2 Reflex Development ....................................................................... 140
8.7.2.1 Midair Righting .............................................................. 140
8.7.2.2 Negative Geotaxis .......................................................... 140
8.7.2.3 Hanging Wire ................................................................. 140
8.7.3 Cognitive Development .................................................................. 141
8.7.3.1 Hidden Platform Water Maze......................................... 141
8.7.3.2 Reversal Learning .......................................................... 143
8.7.3.3 Visible Platform.............................................................. 144
8.7.4 Social Development ....................................................................... 144
8.7.4.1 Sniffing ........................................................................... 144
8.7.4.2 Attacks............................................................................ 145
131
8.8 Conclusions ................................................................................................. 145

References .............................................................................................................. 147
8.1 INTRODUCTION: AN OVERVIEW OF AUTISM

Autism is a neurodevelopmental disorder with core symptoms of impaired social
interactions, deficits in verbal and nonverbal communication, and the appearance of
stereotypic and sometimes self-injurious behaviors. These symptoms appear early and
persist throughout the life of the individual (Rapin, 1997). The intensity of symptom
manifestation varies along a continuum with mild but pervasive developmental dis-
turbances at one end of the spectrum to severely retarded and compromised indi-
viduals at the other end. Patients with Asperger syndrome are higher functioning
autistic individuals with impaired social skills but less severe language deficits.
Some autistic individuals manifest exceptional skills for restricted topics, but these
savant skills are embedded in an otherwise restricted behavioral repertoire that only
serves to heighten their occurrence by contrast.
That autism is a neurobiological disorder has been apparent since its earliest
description. In an editorial comment accompanying his initial report, Kanner (1943a)
characterized autism as an “…innate inability to form affective contact with people
in the ordinary way to which the human species is biologically disposed.” He then
provided 11 case histories of children; for each, the hallmark feature was a failure
of the child to engage in proper social interactions. Of interest, of the 10 cases for
whom the case history were reasonably complete, Kanner (1943b) noted that five had
an unusually large head circumference. In addition, Kanner (1943b) also noted that
six of these 10 cases had difficulty feeding, showing sensitivity to different diets, and
five showed frequent illness during development. Most important, in one case (case 3),
the child exhibited an adverse reaction to smallpox vaccination administered at the age
of 12 months. Two years later, the mother reported: “It seems that he has gone back-
wards mentally gradually for the last two years.” Kanner’s case 3 appears to be the first
association of autistic regression following an adverse reaction to a vaccination.
Several of the important biological features of autism include: (1) a male:female
ratio of about 4:1 in the prevalence of autism (Rapin, 1997; Fombonne et al., 1999);
(2) approximately 75% of those diagnosed with autism are also mentally retarded
(Rutter, 1979); (3) about 30% of all individuals with autism exhibit an increase
in plasma serotonin (Schain and Freedman, 1961); (4) about 40% of all autistic
individuals have a seizure disorder (Volkmar and Nelson, 1990; Ballaban-Gil
and Tuchman, 2000); (5) approximately 70% of those with autism engage in self-
injurious behavior sometime during their life (Bartak and Rutter, 1976); and (6)
there is a strong heritability of autism with about a 60% concordance rate between
monozygotic twins as compared to about a 1% concordance rate for dizygotic twins
(Bailey et al., 1996). Finally, one of the more dramatic features of autism is that
about 35%–40% of those destined to be diagnosed with the disorder have appar-
ent normal cognitive-social development through about the age of 18–30 months
but then experience a “regression” where acquired skills are lost (Tuchman and
Rapin, 1997). The lost skills are sometimes regained, at least partially, but only
Autism and Oxidative Stress: Evidence from an Animal Model 133
after considerable developmental delay. Collectively, these features reveal autism to

have a neurobiological basis with early onset.
Although it is clear that autism has a neurobiological basis, its neuropathology
has not been well characterized. Following histopathological analysis, Bauman and
Kemper (1985) reported limbic system and cerebellar abnormalities in the brain of
a 29-year-old mentally retarded individual with autism who had regressed at the age
of 30 months and exhibited a seizure disorder thereafter. This individual engaged in
stereotypic and self-injurious behavior and was medicated and institutionalized until
the time of his death. The major drawback to the conclusion that the described brain
pathology was attributable to autism was that there were no control cases for mental
retardation, protracted institutionalization with medication, or seizure activity, in
the absence of autism. In a later review summarizing histopathological observations
of the brains of nine autistic individuals, Kemper and Bauman (1998) reported a
consistent reduction in cerebellar Purkinje cells. Comparing these nine brains to the
controls, the authors noted variability in cerebellar lobe size. They also note that two
other cerebella from individuals with autism but normal intelligence both were similar
to the control. Since Soto-Ares et al. (2003) observed cerebella atrophy in 27 of 30
individuals with mental retardation, evidence suggests that the cerebellar pathology is
not specific to autism but better reflects the more common mental retardation.
Ritvo et al. (1986) also reported a reduction in cerebellar Purkinje cells in the brains
of four individuals with autism. Once again, three of their subjects were severely
retarded while the fourth (subject 2) had a normal IQ. The Purkinje cell counts of this
individual were nearly identical to those of the controls. Collectively, these data led to
the conclusion that cerebellar and limbic damage is associated with mental retardation,
protracted institutionalization with medication, seizure disorders, and/or autism.
In well-controlled magnetic resonance imaging studies, an increase in cerebral gray
and white matter was observed in autistic children as compared to normally develop-
ing children (Courchesne et al., 2001; Aylward et al., 2002; Sparks et al., 2002;
Hazlett et al., 2005). These differences, which appear very early in life, slowly dis-
appear between the ages of 5 and 12 (Courchesne et al., 2001; Aylward et al., 2002).
Unfortunately, when individual regions are compared, such as the amygdala and the
hippocampus, the results are less conclusive. Howard et al. (2000) reported increases in
the volume of the amygdala while Aylward et al. (2002) reported decreases. Likewise,
Saitoh et al. (1995) and Piven et al. (1995) reported no differences in the volume of the
hippocampus but Aylward et al. (2002) noted decreases in the volume of this structure.
Thus, one must use caution in concluding that neuropathological changes in limbic
structures and the cerebellum are associated specifically with autism.
8.2 ETIOLOGY OF AUTISM

In an important paper, Wakefield et al. (1998) revealed an apparent link between
gastrointestinal (GI) distress (including food intolerance, diarrhea, and/or abdomi-
nal pain) in children (12 case reports) and an autistic-like regression with loss of
acquired skills (notably social and communication) and, less frequently, the appear-
ance of stereotypic and self-injurious behaviors. This autistic regression followed the
GI distress either immediately or gradually over a period of a few weeks.
Unfortunately, the Wakefield et al. (1998) paper became a source of controversy.

In eight of the 12 cases, the authors noted that the GI distress and subsequent autis-
tic regression followed delivery of the measles-mumps-rubella (MMR) vaccination.
Specifically, in these eight cases, the GI distress along with other symptoms includ-
ing fever, delirium, and/or convulsions were part of an adverse reaction to the MMR
vaccination. The autistic regression appeared an average of 6.3 days after the onset
of the reaction. The authors clearly state “We did not prove an association between
measles, mumps, and rubella vaccine and the syndrome described” but despite this
caution, the report triggered a debate about the possible link between MMR and
autism. This debate soon expanded to include a possible association between one of
the constituents of the MMR vaccine, organic mercury, and autism.
The observations of Wakefield et al. (1998) are of interest because they replicate
aspects of Kanner’s (1943b) original case descriptions of autism associated with fre-
quent illness, food intolerance, and adverse reactions to a vaccination. There have
been attempts to refute the basic observations of Wakefield et al. (1998), whose report
focused on autistic regression and not the overall prevalence of autism. For example,
Dales et al. (2001) compared the percentage of children immunized with MMR on a
year-by-year basis in California over the years 1980 to 2000 with the number of diag-
nosed cases of autism. They observed that the percentage of children being immunized
remained very stable while, during these same years, the absolute number of cases of
autism increased dramatically. Although no correlation coefficient or any other statisti-
cal analysis was provided, they concluded that there was no relationship between the
two and that the MMR vaccination was not associated with autism. Interestingly, there
was an increase in the population of California over that 20-year period and, therefore,
a comparable increase in the absolute number of MMR vaccinations delivered. This
seems to indicate that the Dales et al. (2001) data reflect that the increased numbers
of autism cases were associated with increased administration of MMR. However, the
simplest conclusion is that the increase in the number of cases of autism was associ-
ated with an increase in the population of California. In any case, the link between the
MMR vaccination and autistic regression was not addressed by Dales et al. (2001).
Taylor et al. (1999) reviewed autism cases in the United Kingdom born between
1979 and 1998, comparing the incidence before and after October 1988, when the
MMR vaccine was introduced. They report that there was a dramatic increase in
the incidence in autism in their population starting 3 years after the introduction
of the MMR vaccination but concluded that there was no association between autism
and the MMR vaccination because, by 1992, the vaccine coverage had already
stabilized (while the autism rate was still climbing).
Madsen et al. (2002) assessed a large population of children born in Denmark
between 1991 and 1998. They divided their population into those children who
received the MMR vaccination and those who did not. They then looked for a diag-
nosis of autism or autism spectrum disorder in these two groups and concluded that
there was strong evidence against the hypothesis that the MMR vaccination causes
autism. Of interest, however, is that there were about 27% more cases of autism
diagnosed over the age of 3 in the vaccinated group as compared to the unvaccinated
group. More important, they failed to specifically examine autistic regression, pre-
ferring instead to combine all cases of autism. This controversy continues with most
recent reports continuing to emphasize the safety of the MMR vaccine (Demicheli
et al., 2005; Honig et al., 2008). Nonetheless, one consequence of the MMR contro-
versy is that there has been increased awareness that early exposure to environmental
toxicants may place certain individuals at risk for autism.
As noted, about one-third of those diagnosed with autism experience autistic
regression, having apparently normal development interrupted at some point by a
dramatic setback with a loss of acquired skills. Yet even in these cases, as well as
for the remaining two-thirds of the cases where autism appears to be the result
of a neurodevelopmental defect present at birth (Kanner, 1943b), it remains true that
the etiology of autism remains unknown. Evidence for a genetic contribution for the
disorder is clear, but no single gene has been identified; rather, a constellation of gene
polymorphisms appears to increase the risk for individuals. The controversy surround-
ing the link between autism and the MMR vaccination suggested the involvement of
an environmental toxicant exposure as a second factor, at least for autistic regression.
Other studies have linked autism to prenatal exposure to toxicants such as thalidomide
or valproic acid (VPA) (Rodier et al., 1997). Based upon these observations, together
with the fact that autism fails to reach 100% concordance rates in monozygotic
twins, a hypothesis has been developed that the etiology of autism may arise as the
result of a gene by environment interaction with a gene polymorphism(s) enhancing
the sensitivity of individuals to the deleterious effects of environmental toxicants
(London and Etzel, 2000). Based upon our observations in humans (Ming et al.,
2005, 2008a) and mice (Wagner et al., 2006; Ming et al., 2008b), we have advanced
this hypothesis with the proposal that autism is the result of a gene by environ-
ment interaction where the environmental toxicant triggers oxidative stress while the
genetic deficiency affects the ability of the individual to respond effectively to the
deleterious effects of oxidative stress (Ming et al., 2008b). Toward this end, we have
developed an animal model of autism and have used this model to assess the effects
of gene alterations (Cheh et al., 2006) as well as early exposure to toxicants such as
VPA (Wagner et al., 2006; Yochum et al., 2008) and mercury (Wagner et al., 2007)
on neurobehavioral development.
8.3 ANIMAL MODEL OF AUTISM

The symptoms of autism have proven difficult to model in other species, though there
has been some success, especially with respect to the stereotypic and self-injurious
behaviors (e.g., Wagner et al., 2004). We have initiated work on a novel strategy to
model the behavioral phenotype of autism in mice. In this model, the normal develop-
ment of key behaviors is carefully monitored from birth through adolescence. Once the
maturation of these key behaviors is understood (i.e., in terms of the postnatal day(s)
of life in which subjects are able to successfully perform the task or engage in the
behavior), the performance of mice with early toxicant exposure and/or genetic modi-
fication can be assessed. The model does not claim to produce autistic mice; rather,
developmental deficits can be identified against the vehicle-treated, wild-type controls.
Likewise, the model does not claim to be exclusive for autism. Rather, it provides a new
strategy for classifying alterations in the normal maturation of individual mice conse-
quent to toxicant exposure, genetic manipulation, or the interaction of the two.
The model strategy begins by characterizing behavioral manifestations of devel-

opmental disorders as retardations (i.e., a behavior fails to develop during a critical
period), regressions (i.e., a behavior develops at about the right time but then is lost
with later development, especially following toxicant exposure), or intrusions (i.e., the
appearance of behaviors aberrant in form or frequency which mask normal develop-
ment). Most developmental disorders (e.g., autism, attention deficit disorder, mental
retardation) include some combination of these conditions. In this framework, the
hypothesis that environmental toxicants are causally involved in developmental dis-
orders can be readily tested. That is, acute or repeated exposure to a toxicant should
disrupt neurobehavioral development, causing behavioral retardation, regression, or
intrusions. These conditions can be identified only if normal developmental patterns
are thoroughly understood.
For each of these three classes of developmental deficits (i.e., retardations, regres-
sions, and intrusions), neurodevelopmental tests may be employed to target the cognitive,
motor, emotional, and/or social maturation of the subject. Genetic manipulation or
toxicant exposure may, for example, impact the social maturation of the subjects, caus-
ing a retardation or regression in its development or by obscuring its manifestation by
inducing intrusive stereotypic or self-injurious behaviors.
Further refinement of this model enhances its relevance for autism. This may be
accomplished by making genetic alterations known to be relevant to autism (e.g.,
Cheh et al., 2006) and by using toxicants that have been associated with autism
(e.g., Wagner et al., 2006, 2007; Yochum et al., 2008). Likewise, the neurodevelopmental
tasks that are employed may assess the maturation of the cerebellum or hippocampus,
brain areas thought to be involved in autism and, likewise, known to be targets of
the selected toxicants. Most recently, we have used this model strategy to further
assess the hypothesis that oxidative stress may be a critical factor in the etiology
of autism.
8.4 OXIDATIVE STRESS AND AUTISM

The involvement of enhanced oxidative stress in autism has been derived from several
lines of evidence (reviewed in Chauhan and Chauhan, 2006). First, an increased excre-
tion of oxidative stress biomarkers has been reported. Specifically, nitric oxide, a free
radical that can block energy production, was increased in erythrocytes of patients with
autism as compared to age- and sex-matched controls (Sogut et al., 2003). In addition,
elevated nitrite concentrations along with thiobarbituric acid–reactive substances and
xanthine oxidase activity were reported in the plasma and red blood cells of autistic
individuals (Zoroglu et al., 2003, 2004; Chauhan et al., 2004). The elevation of these
substances indicates excess generation of free radicals in individuals with autism com-
pared to controls. Consistent with the increased oxidative stress biomarkers, children
with autism were found to have increased body burdens of environmental toxicants
that may generate oxidative stress (Edelson and Cantor, 1998, 2000).
A second line of evidence that oxidative stress may play a role in autism is
suggested by a reduced endogenous antioxidant capacity. Specifically, altered glu-
tathione peroxidase (Yorbik et al., 2002; Sogut et al., 2003), superoxide oxidase
(Yorbik et al., 2002), and catalase (Zoroglu et al., 2004) activities as well as total
glutathione, and GSH/GSSG and cysteine levels (James et al., 2004) were found in
autistic individuals compared to controls. Likewise, levels of exogenous antioxi-
dants were also found to be reduced in autism, including vitamin C, vitamin E, and
vitamin A in plasma, and zinc and selenium in erythrocytes (James et al., 2004).
Finally, evidence of altered oxidative stress in autism is derived from evidence of
impaired energy metabolism. Magnetic resonance spectroscopic study of the brains
of individuals with autism showed reduced synthesis of ATP (Minshew et al., 1993).
In addition, higher lactate (Coleman and Blass, 1985; Chugani et al., 1999) and pyru-
vate (Moreno et al., 1992) levels in autism may suggest mitochondrial dysfunction in
autism (Filipek et al., 2003); one major cause of mitochondria dysfunction is a result
of oxidative injury (Packer, 1984). Taken together, these lines of evidence support the
hypothesis that at least some children with autism exhibit enhanced oxidative stress.
As noted, we have used our animal model to examine the role of oxidative stress in
causing the neurobehavioral deficits following early exposure to VPA (Ming et al.,
2008b) and mercury.
8.5 VALPROIC ACID, AUTISM, AND OXIDATIVE STRESS

Following its administration, VPA elicits an oxidative stress response when it is
metabolized by a cytochrome P450-dependent hydroxylation, leading to a decrease
in cellular glutathione and suppressing the activities of hepatic glutathione reductase,
glutathione peroxidase, and glucose-6 phosphate dehydrogenase (Simon et al., 1994;
Jurima-Romet et al., 1996). Thus, it appears that the metabolism-dependent VPA-
induced cytotoxicity is the result of generation of hydrogen peroxide and subsequent
interaction with iron to produce the highly reactive hydroxyl free radicals. In this
regard, a series of antioxidants and iron chelators protected against metabolism-
dependent VPA-induced in vitro cytotoxicity (Jurima-Romet et al., 1996; Tabatabaei
and Abbott, 1999).
In adults, VPA is used as an anticonvulsant with few side effects. However, prena-
tal exposure to VPA causes behavioral and neuroanatomical abnormalities in children
that are strikingly similar to those found in autism. This fetal valproate syndrome
is characterized by deficits in language and communication, stereotypic behavior,
and hyperexcitability (Ardinger et al., 1988; Williams et al., 2001). In rodents, early
VPA exposure results in a reduction in Purkinje cell number and cerebellar cell
volume similar to what is observed in patients with autism (Rodier and Hyman,
1998; Ingram et al., 2000; Sobaniec-Lotowweska, 2001). Schneider and Przewlocki
(2005) found that prenatally. VPA administration to rats resulted in the appearance
of repetitive behaviors together with decreased social interaction.
In our previous study, we demonstrated that mice injected with sodium valproate
at crucial developmental time points displayed a retardation and/or regression of
certain critical behaviors such as surface righting, midair righting, and negative
geotaxis (Wagner et al., 2006). In addition, we showed that similarly treated mice
showed deficits in their later cognitive performance and social interactions (Wagner
et al., 2006; Yochum et al., 2008) and a 30-fold increase in the number of cells stain-
ing for apoptosis in the cerebellum together with a 10-fold increase in cells staining
for apoptosis in the hippocampus (Yochum et al., 2008).
In the context of our present chapter, we sought to determine if antioxidant pre-

treatment could protect against the behavioral deficits caused by VPA. Accordingly,
male BALB/c mice were treated with vitamin E or its vehicle on day P14. Pups then
received a second injection of either saline or 400 mg/kg of VPA for a total of four
groups. As in our initial studies, the ability to right in midair was evaluated on P13–19
by holding the pups upside-down, 45 cm above a padded surface and dropped for
three trials each test day. Their ability to right in midair was determined if the pup
landed with all four paws on the surface.
Consistent with our previous studies, we found that midair righting first appears
about postnatal day 14 of life. The VPA treatment caused a pronounced regression
in this response; pups that could perform the task on P14 completely lost the ability
to midair right after receiving the VPA. This regression-like loss of a motor skill
did gradually return over about a 1-week period. Of importance, pretreatment with
the antioxidant, vitamin E, completely protected the pups against the VPA-induced
loss of midair righting (Ming et al., 2008b). As noted, VPA has been associated with
human autism and its mechanism of action is thought to involve oxidative stress.
Therefore, this first set of data suggests that antioxidants administered at the time of
toxicant exposure might afford some protection against an autistic-like regression.
We then sought to extend these observations to a second toxicant that has also, at
least indirectly, been linked to autism.
8.6 MERCURY EXPOSURE AND AUTISM

It is well established that exposure to methylmercury (MeHg) can induce detrimental
effects in humans (for review, see Chang and Annau, 1984; Mendola et al., 2002).
The effects are most severe when exposure to high doses of MeHg occurs during
neural development and are manifest as severe developmental disabilities often asso-
ciated with brain malformations. MeHg is an environmental toxicant that bioaccu-
mulates in large predatory fish and, thus, human exposure comes almost exclusively
through fish consumption (Clarkson et al., 2003). A number of studies have examined
the effects of low-level MeHg exposure in fish-eating populations. Unfortunately, the
results of these studies are conflicting (reviewed in Davidson et al., 2004) with some
reporting that increased blood concentrations of MeHg correlate with poor develop-
mental outcomes while others observing no adverse effects. Nonetheless, it has been
postulated that an increase in low-level exposure to MeHg could be involved in some
neurodevelopmental disorders (Davidson et al., 2004).
The brain is the primary target for MeHg (Nagashima, 1997; Costa et al., 2004).
Following exposure, MeHg initiates an increase in the generation of reactive oxy-
gen species (ROS) and, simultaneously, decreases endogenous antioxidant activity
(Andersen and Andersen, 1993; Yee and Choi, 1994, 1996; Shanker and Aschner,
2003; Vicente et al., 2004). Neurons are particularly vulnerable to MeHg toxicity
because their endogenous antioxidant capacity is low compared to other cell types.
Therefore, the brain is particularly sensitivity to oxidative stress-induced injury con-
sequent to MeHg exposure (Usuki et al., 2001; Costa et al., 2004). The cerebellum
is one of the principle brain regions disrupted by MeHg exposure, with cerebellar
granule cells being a primary target in the adult human (Castoldi et al., 2000).
MeHg exposure during neural development leads to more severe toxicity than
comparable exposure in adults. Specifically, early exposure results in a more dif-
fuse neuropathology and can affect the organization of the cerebellum, cerebral
cortex, and striatum (Chang et al., 1977; Chang and Annau, 1984; Sager et al.,
1984; Sakamoto et al., 2002). Commonly reported neurobehavioral deficits in
rodents following early MeHg exposure include deficits in reflex development
and locomotor activity and coordination as well as cognitive deficits in learning
and memory (Olson and Boush, 1975; Chang and Annau, 1984; Vorhees, 1985;
Doré et al., 2001; Sakamoto et al., 2002; Stringari et al., 2006). In general, the
severity of the behavioral deficits following MeHg increases with dose and earlier
exposure times.
Antioxidants have been shown to confer significant protection against MeHg tox-
icity in vitro and in vivo, reducing the levels of ROS thereby attenuating the neuro-
pathological damage (Chang et al., 1978; Andersen and Andersen, 1993; Yee and
Choi, 1994; Usuki et al., 2001; Shanker and Aschner, 2003; Beyrouty and Chan,
2006). One recent study reported that pretreatment with vitamin E attenuated MeHg-
induced behavioral deficits in the acoustic startle response of rats (Beyrouty and
Chan, 2006). However, the protective effect of antioxidants on persistent MeHg-
induced neurobehavioral disruption is not well studied. Using our animal model
of autism, we have previously shown that early exposure to MeHg sensitizes the
developing mouse to the later appearance of stereotypic and self-injurious intrusive
behavior (Wagner et al., 2007).
8.7 TROLOX PROTECTS MICE AGAINST EARLY

EXPOSURE TO MeHg
We now report that, as with VPA, antioxidant pretreatment protects mice against the
neurobehavioral deficits induced by early exposure to MeHg. Mice were exposed to
MeHg in the presence or absence of antioxidants during the first 2 weeks of post-
natal life (see Wagner et al., 2007 for details). The developmental period selected
for MeHg exposure corresponds to the third trimester in human gestation, a time,
of rapid brain growth (Costa et al., 2004). Trolox (6-hydroxy-2,5,7,8-tetramethyl-
chroman-2-carboxylic acid), a water-soluble vitamin E derivative, was used as the
antioxidant pretreatment. Trolox has been reported to provide significant protection
against MeHg toxicity both in cell culture and in neuropathological studies (Usuki
et al., 2001; Shanker and Aschner, 2003).
Pregnant BALB/c mice (Taconic, Germantown, New York) were housed in plastic
cages in a temperature- and humidity-regulated colony room with a 12 h light/12 h
dark cycle, and given free access to food and water. Day of birth was recorded as
postnatal day (PND) 0; litters were culled to all male pups prior to treatment and
were weaned at PND 20.
Mice were treated with 4.0 mg/kg MeHg Cl (ICN, Costa Mesa) or phosphate-
buffered saline (PBS) vehicle subcutaneously on alternate days between PND 3–15.
Additional groups of pups were dosed with the same MeHg regimen, but given
high-dose (2.5 mg/kg) or low-dose (1.0 mg/kg) Trolox subcutaneously 1 hour prior
to each MeHg injection. We evaluated body weight during postnatal development
and reflexes including midair righting, negative geotaxis, and hanging wire strength.
Adolescent mice were then evaluated in the water maze. Finally, pairs of similarly
treated adult mice were tested in a resident-intruder paradigm. Details of the behav-
ioral test procedures may be found in Wagner et al. (2006, 2007).
We observed that MeHg-induced deficits in reflex development and spatial learning
and memory as well as in adult social interactions, and that Trolox pretreatment pro-
tected the mice against these MeHg-induced neurodevelopmental deficits. Specific
observations were as follows.
8.7.1 BODY WEIGHT

Body weight was monitored daily from PND 3–15. Across postnatal development, mice
of all treatment groups gained weight (F(6,450) = 1099.1, p < 0.0001). There was a
significant effect of treatment on body weight gain (F(4,75) = 4.5, p = 0.003) as well as
a significant treatment by day interaction (F(24,450) = 2.5, p < 0.0001). Specifically,
MeHg produced a significant decrease in body weight gain compared to controls from
PND 5–15. In addition, pretreatment with both high-dose and low-dose Trolox attenu-
ated the MeHg-induced reduction in weight gain, with high-dose Trolox reaching
significance on PND 7–15 and low-dose Trolox reaching significance on PND 5–11.
8.7.2 REFLEX DEVELOPMENT

8.7.2.1 Midair Righting
Mice were assessed on three daily trials for their ability to successfully right in mid-
air on PND 13–19. Control mice displayed significantly better performance in midair
righting than MeHg-exposed mice. As shown in Figure 8.1a, significant impairment
was observed in MeHg-exposed mice on PND 14–18 (day 14, H = 11.1, p = 0.01; day 15,
H = 9.8, p = 0.2; day 16, H = 19.7, p < 0.001; day 17, H = 10.2, p < 0.001; day 18, H = 17.8,
p < 0.001). Complete protection against this behavioral disruption was observed fol-
lowing pretreatment with high-dose Trolox on all test days, while pretreatment with
low-dose Trolox resulted in a significant attenuation of this deficit on PND 17–18.
8.7.2.2 Negative Geotaxis

Mice were monitored for their performance of the negative geotaxis response on days
13–19. All mice showed an improvement in performance of this reflexive response
across testing (F(6,192) = 7.3, p < 0.001). As shown in Figure 8.1b, MeHg-treated ani-
mals showed the longest latencies to perform the geotactic response, although this
difference did not reach statistical significance.
8.7.2.3 Hanging Wire

Mice were evaluated for their latency to fall when suspended from a wire on days
13–19. All mice showed an improvement in their ability to hang from the wire across
testing (F(6,156) = 30.4, p < 0.001). There was no significant effect of treatment on
hanging wire performance (data not shown).
Neonatal reflex development
Number of successful landings
2 * *
*
*
1
*
* *
0
13 14 15 16 17 18 19
(a) Postnatal day
30
28
26
24
22
Second to turn 180°
20
18
16
14
12
10
8
6
4
2
0
13 14 15 16 17 18 19
(b) Postnatal day
PBS MeHg4 Trolox 1.0 + MeHg Trolox 2.5 + MeHg
FIGURE 8.1 (a) The number of times out of three that mice were successful at midair righting
across postnatal development is illustrated. * Indicates significantly different from control using a
distribution free nonparametric post-hoc test. (b) Latency to perform the negative geotaxis response
across postnatal development is depicted. Error bars indicate SEM.
8.7.3 COGNITIVE DEVELOPMENT

8.7.3.1 Hidden Platform Water Maze
Mice were tested in the hidden-platform water maze for 16 trials across two days (8 trials
a day for two consecutive days). All treatment groups showed a significant reduction
in escape latency across the 16 trials of water maze testing (F(15,630) = 2.5, p < 0.001).
As shown in Figure 8.2a and b, MeHg-exposed mice showed poorer hidden-platform
water maze performance than controls (F(4,42) = 5.9, p = 0.001). Post-hoc analysis
revealed that the escape latency for MeHg-exposed mice was significantly greater
than controls for the average escape latency on day 1, as well as on trials 9, 10, 13, 14,
15 on day 2. This deficit was attenuated by high-dose of Trolox on day 2 of testing,
but not by the lower dose. As an additional measure, escape latency was converted
to the proportion of time spent in the quadrant where the platform was located. This
Hidden-platform water maze

70
60
Average escape latency (s)
50
*
40
30
20
10
0
PBS MeHg 4 Tro 2.5 + Tro 1.0 +
MeHg MeHg
(a) Average escape latency, day 1
60 *
* * *
50 + * *
+ *
40 +
+ +
+
30
20
10
0
9 10 11 12 13 14 15 16
(b) Escape latency by trial, day 2
PBS Trolox 2.5 mg/kg + MeHg
MeHg 4 mg/kg Trolox 1.0 mg/kg + MeHg
FIGURE 8.2 (a) Escape latency averaged for eight trials of hidden-platform water maze test-
ing on day one and (b) escape latency across eight trials of hidden-platform water maze testing
on day two is shown.
Reversal learning
60
*
50
Escape latency (s)
*+ * *
40 *
30 +
20
10
0
0 1 2 3 4 5 6 7 8
(c)
Proportion of escape latency
Proportion of time in former escape quadrant

0.8
0.6
0.4 *
* *
0.2
0.0
0 1 2 3 4 5 6 7 8
(d) Trial number
FIGURE 8.2 (continued) (c) Latency to escape to the hidden-platform when moved to the
opposite quadrant across eight trials, and (d) the proportion of time spent in the quadrant
where the platform was previously located across eight trials is depicted. * Indicates MeHg
significantly different from control. + Indicates significantly different than MeHg-treated.
Error bars indicate SEM.
proportion of time spent in the escape quadrant increased across trials for all mice
(F(15,630) = 2, p = 0.013). MeHg-treated mice showed a smaller proportion of time in
the escape quadrant compared to controls (F(4,42) = 3.5, p = 0.015), further indicating
that they did not learn as well. Trolox pretreatment partially improved this measure,
with both doses showing similar efficacy during the later trials (data not shown). Finally,
in separate group of mice, high-dose Trolox was administered in the absence of MeHg
and no effect on escape latency was observed (data not shown), indicating that Trolox
itself does not affect spatial learning, but is effective at attenuating the MeHg-induced
deficit in hidden-platform water maze performance.
8.7.3.2 Reversal Learning

One week after the initial spatial learning trials, the position of the hidden platform
was changed to the opposite quadrant of the maze. For this reversal learning task,
mice showed a significant reduction in escape latency across trials, demonstrating that
they were able to find the position of the platform in its new location (F(7,252) = 4.3,
p < 0.0001). There was also a significant effect of treatment on performance in this
reversal learning task (F(4,36) = 3.1, p = 0.026). As shown in Figure 8.2c, MeHg-exposed
mice did not learn the task as well as controls, displaying longer escape latencies
across testing. Post-hoc analysis revealed significant differences on trials 2, 5, and 7.
Pretreatment with high-dose Trolox significantly attenuated the MeHg-induced
impairment in reversal learning performance, which reached significance on trials 2

and 7. However, pretreatment with low-dose Trolox did not significantly improve the
MeHg-induced impairment. Again, high-dose Trolox by itself did not alter performance
in this task (data not shown). This indicates that Trolox alone does not alter reversal
learning, but is able to attenuate the MeHg-induced deficit in reversal learning.
During the reversal learning task, the time spent in the quadrant where the plat-
form was previously located was also assessed. Control mice spent a larger propor-
tion of time in this quadrant during early trials, which significantly decreased across
repeated testing (F(7,252) = 3.9, p = 0.001). This same pattern was not observed in
MeHg- or Trolox-pretreated mice. Correspondingly, there was a treatment effect on
the proportion of time spent in the previous platform location (F(4,36) = 4.2, p = 0.006)
and a significant interaction between trial and treatment (F(28,252) = 1.5, p = 0.04).
As shown in Figure 8.2d, MeHg-treated mice spent significantly less time in the for-
mer escape quadrant on trials 1, 2, and 5. This suggests that control mice developed
a spatial memory for the former escape location and were able to adapt their strategy
when they did not find the escape platform. Pretreatment with high-dose Trolox did
not shorten the proportion of time MeHg-exposed mice spent in the previous platform
location, even though it reduced the deficit in escape latency in both the hidden-
platform and reversal learning paradigms. This suggests that Trolox confers partial
protection against the MeHg-induced impairment in learning and memory.
8.7.3.3 Visible Platform

To determine whether motor skills may have influenced results of the water maze
tasks, another group of treatment and age-matched mice were tested using a cued-
version of the task. No significant difference was observed between groups in the
latency to escape to a visible platform. This demonstrated that mice of all treatment
groups were equally able to swim and escape to the visible platform. Therefore,
a motor deficit likely did not contribute to the poorer performance observed by
MeHg-exposed mice.
8.7.4 SOCIAL DEVELOPMENT

The social behavior of adult mice treated as neonates was examined using the resident–
intruder paradigm. The number of resident-to-intruder sniffing episodes and the number
of attacks were recorded for two sessions run on consecutive days in treatment-matched
pairs. Only mice pretreated with high-dose Trolox were assessed in this task.
8.7.4.1 Sniffing
There was a significant effect of day on the number of times resident mice sniffed
an intruder (F(1,26) = 5.9, p < 0.001). In addition, a significant effect of treatment
(F(1,26) = 6.68, p < 0.004) and a significant treatment-by-day interaction were found
(F(2,26) = 9.0, p < 0.001). As shown in Figure 8.3a, MeHg-exposed resident mice
made significantly fewer sniffs of the intruder on day 1 compared to control mice.
Pretreatment with Trolox resulted in complete reversal of the deficit in sniffing observed
in MeHg-treated mice. Furthermore, both control and Trolox-pretreated mice engaged in
a greater number of sniffing episodes on day one than on day 2. MeHg-exposed mice
Resident-intruder paradigm
35 10
30 *
8 *
Number of attacks
Sniffing episodes
25
+ 6
20
+
15
*
4
10
2
5
0 0
(a) Day 1 Day 2 (b) Day 1 Day 2
PBS MeHg 4 mg/kg MeHg 4 mg/kg + Trolox 2.5 mg/kg
FIGURE 8.3 (a) The number of times a pair of mice sniffed one another and (b) the number
of attacks resident mice made to the intruder in the resident–intruder paradigm on two consecu-
tive days is illustrated. * Indicates significantly different than control. + Indicates significantly
different than on day one of testing. Error bars indicate SEM.
engaged in a similar level of sniffing on both test days that was reduced compared to
the control and Trolox-pretreated mice.
8.7.4.2 Attacks
A significant effect of treatment on the number of attacks was also observed in the
resident–intruder paradigm (F(1,26) = 6.68, p < 0.004). MeHg-exposed resident mice
made many more attacks than controls. This increased aggression was completely
absent in Trolox-pretreated mice, such that none of the resident mice pretreated
with Trolox attacked the intruder (see Figure 8.3b). All three groups maintained a
similar level of attack behavior on the first and second day of testing. Results of the
resident–intruder paradigm indicate that less social investigation and greatly enhanced
aggressive behavior occur in adult mice that were exposed to MeHg during postnatal
development. This behavioral pattern was completely protected by high-dose Trolox
pretreatment.
8.8 CONCLUSIONS
Methylmercury is a developmental neurotoxicant that causes damage, in part, through
the generation of ROS. A number of studies have reported that MeHg exposure dur-
ing development leads to impaired cognitive and motor behavior. The postnatal
MeHg exposure regimen used here resulted in behavioral impairments consistent
with those reported by others including impaired reflex development and deficits in
a spatial learning task. In addition, the present study also revealed that early MeHg
exposure results in abnormal social and aggressive interactions behavior much later
in life. Pretreatment with Trolox, a water-soluble vitamin E derivative, resulted in a
robust protective effect against these MeHg-induced behavioral deficits. Trolox was
particularly effective at abolishing the MeHg-induced deficits in midair righting and
the increased aggressive behaviors of the adults. These data suggest that free-radical
scavenging antioxidants might be an effective means of reducing the potential health
risks of early MeHg exposure.
Dramatically increased aggressive behavior together with reduced social investiga-
tion was observed in MeHg-treated mice compared to controls. When control mice are
paired in a small chamber with a target intruder mouse for the first time, they quickly
develop a “social” memory (File and Seth, 2003). That is, they frequently approach and
investigate the intruder on the first test day but this approach behavior decreases when
they are exposed to the same intruder on subsequent days. MeHg-treated mice, how-
ever, approached the intruder significantly fewer times on the first test day, but main-
tained a similar level of investigation upon the second exposure to the same intruder.
Moreover, MeHg-exposed mice attacked the intruder much more frequently than con-
trols on both test days. This indicates that developmental MeHg exposure altered the
social behavior expressed in adulthood, leading to increased aggression with reduced
social investigation. We recently reported that mice treated with the same MeHg regi-
men exhibited significant increases in amphetamine-induced self-injurious behaviors
as adults (Wagner et al., 2007). In the present study, Trolox pretreatment protected
mice against the MeHg-induced alterations in social behavior observed in the resident–
intruder paradigm, completely preventing increase in intrusive attack behavior.
Learning and memory deficits were observed in MeHg-treated mice tested in the
water maze during adolescence. We found an increase in their escape latency in
the hidden platform water maze, as well as in the reversal-learning task. Sensorimotor
problems did not appear to contribute significantly to the MeHg-induced deficits,
since no difference in escape latency was observed between groups in the visible
platform version of the task. Trolox elicited a partial, dose-related protective effect
on hidden-platform and reversal-learning performance.
The proportion of time that mice spent in the previous escape quadrant was
recorded during the reversal-learning task. This measure functioned as a probe trial
to assess memory for the former escape quadrant. The control mice spent more time
in the former escape location on early trials. This decreased after just a few trials
and the controls were able to quickly adapt their strategy to learn the new plat-
form location. In contrast, mice exposed to MeHg showed a decreased proportion of
time spent in the former escape location, reflecting their overall poor performance
at finding the hidden-platform. However, Trolox pretreatment did not improve this
measure. The lack of protection on this measure suggests that subtle abnormalities
of MeHg exposure may still be present. Future studies determining the effect of
Trolox in other learning and memory tasks may lead to a clearer understanding of the
degree of protection conferred on MeHg-induced learning deficits.
Finally, the maturation of the mid-air righting reflex over the first 3 weeks of post-
natal life is mediated, in part, by continuing cerebellar maturation. MeHg exposure
resulted in a significant disruption in the maturation of the midair righting reflex and,
as above, Trolox pretreatment effectively attenuated this deficit.
It appears likely that pretreatment with the free radical scavenging antioxidant,
Trolox, protected mice against the behavioral deficits by decreasing the amount of
oxidative damage to neurons induced by the MeHg. Gutierrez et al. (2006) recently
reported that rats exposed to mercuric chloride displayed reduced levels of total
radical trapping antioxidant potential following MeHg, suggesting that oxidative

stress may be induced as intracellular stores of glutathione and other antioxidants
become depleted. Shanker et al. (2005) showed that intracellular glutathione and
antioxidants play an important role in protecting against MeHg-induced oxidative
stress in the brain. Usuki et al. (2001) reported that high-dose Trolox resulted in
significant protection against MeHg-induced toxicity in adult rats, reducing the num-
ber of apoptotic cells in the cerebellum as well as the degree of hind-limb crossing
(a behavioral manifestation of MeHg toxicity in the adult). Likewise, Shanker and
Aschner (2003) reported that Trolox was able to completely block the increase in
ROS following MeHg exposure in isolated PND1 cerebral astrocytes. Thus, it is con-
cluded that early exposure to MeHg leads to motor, cognitive, and social deficiencies
that resemble human developmental disorders such as autism and that pretreatment
with the antioxidant, Trolox, affords protection against the neurobehavioral deficits.
In summary, it appears that autism may be the result of early toxicant exposure acting
upon individuals sensitive to their damaging effects because of a constellation of gene
polymorphisms. While no single toxicant has been identified, a number of compounds
have been associated indirectly with autism and these appear to share a common mech-
anism of generating ROS. Likewise, no single gene has been identified but a number of
the polymorphisms that have been associated with autism directly or indirectly affect
the response to ROS. We (and others) have previously linked the occurrence of autism
to the proximity of toxic landfills (Ming et al., 2008a) and have demonstrated increased
excretion of biomarkers of ROS in a cohort of autism cases compared to age-matched
controls (Ming et al., 2005). Finally, using our animal model, we have shown that dele-
tion of genes in mice that have been associated with human autism polymorphisms
results in neurobehavioral deficits during development (Cheh et al., 2006), that early
administration of toxicants that have been linked to human autism produces a spectrum
of behavioral deficits quite similar to the gene deletions (Wagner et al., 2006, 2007;
Yochum et al., 2008), and most recently, that pretreatment with antioxidants protects
mice against the deleterious effects of these toxicants (Ming et al., 2008b; present chap-
ter). The protective effects afforded by antioxidant pretreatment against the neurobehav-
ioral deficits induced by VPA and MeHg are consistent with the interpretation that these
compounds exert their damaging effects, at least in part, through the generation of ROS.
The clinical ramifications of these observations await further study.
ACKNOWLEDGMENTS
Supported by: NIEHS grants ES05022, ES07148, ES11256, as well as the NJ Governor’s
Council on Autism (GCW) and Autism Speaks (GCW).
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9 Neurotoxic Brainstem
Impairment as Proposed
Threshold Event in
Autistic Regression
Woody R. McGinnis,1,* Veronica M. Miller,2
Tapan Audhya,3,4 and Stephen M. Edelson5
1
Autism House of Auckland, Autism New Zealand, Inc.,
Auckland, New Zealand
2
Wadsworth Center for Laboratories and Research,
New York State Department of Health, Albany,
NY 12201, USA
3
Vitamin Diagnostics Laboratory,
Cliffwood Beach, NJ 07735, USA
4
Division of Endocrinology, Department of Medicine,
New York University School of Medicine,
New York, NY 10016, USA
5 Autism Research Institute, San Diego, CA 92116, USA
CONTENTS
9.1 Introduction ................................................................................................. 154
9.2 Somatobehavioral Regression in Autism .................................................... 154
9.3 Toxicants as Potential Triggers for Regression in Autism .......................... 155
9.4 Toxicant Accumulation and Injury in CVO ................................................ 157
9.5 Findings Consistent with Brainstem Pathology in Autism ......................... 160
9.5.1 Clinical and Electrophysiological Brainstem
Findings in Autism......................................................................... 160
9.5.2 Abnormal Brainstem Morphology ................................................. 161
9.5.3 Biochemical Abnormalities in Autism .......................................... 161
9.5.4 Features of Regression in Autism .................................................. 162
153
9.5.5 Oxidative Mechanisms .................................................................. 164

9.5.6 Inflammatory Mechanisms in Autism ........................................... 166
9.6 Future Research .......................................................................................... 168
References .............................................................................................................. 168
9.1 INTRODUCTION
In this chapter, we propose a model for autistic regression resulting from passage
of toxicants into regions of brain unprotected by blood–brain barrier (BBB). These
portals surround the primitive brainstem, a reportedly abnormal brain region in
autistic subjects. We propose that toxicant-induced impairment of a specific struc-
ture, the dorsal vagal complex (DVC) of the medulla, sufficiently accounts for the
primary neurophysiological changes of autistic regression. Toxicant effects on other
unprotected sites may explain associated abnormalities in autism, including altered
melatonin and oxytocin production. In autistic regression, it is possible that brain-
stem is primary target for toxicant-induced inflammation, which ramifies to higher
structures in a pattern resembling the known stages of Parkinson disease (PD).
9.2 SOMATOBEHAVIORAL REGRESSION IN AUTISM

The earliest accounts of autism included descriptions of regression (Kanner, 1943), an
intriguing phenomenon, which has been well documented as a true loss of acquired
function in children later diagnosed with autism (Goldberg et al., 2003; Hansen
et al., 2008; Lord et al., 2004; Werner and Dawson, 2005). The reported incidence of
regression varies with methodology and approaches 50% in some studies (Goldberg
et al., 2003). As reviewed by Goldberg, regression is well documented as a second-year
phenomenon, usually occurring at 18–24 months (Goldberg et al., 2003). Behavioral
changes are well-characterized in regression, and fall broadly into two domains which
are congruent with classical diagnostic criteria for autism: (1) lost vocalization—
acquired words, or babbling in preverbal children and (2) lost social skills.
In a study distinguished by a relatively brief period between regression and
parental interview, Lord found that 29% of children with the diagnosis of autism lost
meaningful words and another 9% lost nonword vocalizations (Lord et al., 2004).
Word loss was found in 62% of autistic children with regression, and occurred over
days or weeks (Goldberg et al., 2003). Loss of spoken words nearly always associates
with loss in social behavior (Lord et al., 2004), but some children appear to suffer
isolated social loss (Goldberg et al., 2003). Concomitant motor losses are very rare
(Davidovitch et al., 2000; Tuchman and Rapin, 1997).
While regression commonly is viewed by parents as the first sign of abnormality
in children who eventuate the diagnosis of autism, studies suggest that some children
with discernible regression in the second year displayed prior developmental delay
(Torrente et al., 2002), or difficulties in regulatory behavior (Werner and Dawson,
2005). Multiple distinct regressions are documented in some cases (Weissman et al.,
2008). On the basis of parental questionnaires collected by the Autism Research
Institute (ARI) over five decades, Rimland proposed a proportionate increase in
regressive autism (Rimland and McGinnis, 2002).
Neurotoxic Brainstem Impairment as Proposed Threshold Event 155
Over the past decade, it has become clear that a variety of gastrointestinal
problems—constipation, diarrhea, abdominal pain, and distension—commonly
associate with autistic behavior. Over half of a mixed cohort of autistic children
had radiographic fecal loading or megacolon (Afzal et al., 2003), as did 100% of a
regressive cohort, with or without history of intercurrent diarrhea (Torrente et al.,
2002). Endoscopy of regressed cohorts demonstrated very high incidence of entero-
colitis (Wakefield et al., 1998, 2002) and reflux esophagitis (Horvath et al., 1999).
Case reports suggest that gastrointestinal symptoms and behavioral regression
occur at about the same time (Madsen and Vestergaard, 2004). On the basis of hun-
dreds of clinical interviews oriented to somatic as well as behavioral changes, we
(McGinnis, Audhya, and Edelson) concur with Goldberg’s published observation:
“Parents often report their children as having gastrointestinal symptoms that started
about the same time autistic symptoms appeared” (Goldberg, 2004). Refined data
for onset of gastrointestinal symptoms are not sufficiently evolved, but the collective
clinical knowledge strongly suggests a triad of changes in autistic regression: (1) lost
vocalization, (2) lost social skills, AND (3) gastrointestinal impairment.
9.3 TOXICANTS AS POTENTIAL TRIGGERS

FOR REGRESSION IN AUTISM
It has been postulated that regression results from brain damage after birth by
environmental toxicants, particularly in light of evolving evidence of gliosis and
neuronal loss in autism (Kern and Jones, 2006). The increasing incidence of autism
parallels progressive contamination of the environment (Grandjean and Landrigan,
2006; Lathe, 2008), and autism rates correlate with presence of toxic landfill sites
(Ming et al., 2008). A preliminary epidemiological study linked the incidence of
autism most strongly to the estimated environmental concentrations of cadmium
and mercury (Windham et al., 2006), and another suggested that proximity to point
sources of environmental mercury associates with autism (Palmer et al., 2009).
Greater cumulative exposure to mercury in autism is suggested by elevated dental
concentrations of mercury (Adams et al., 2007), and urinary porphyrin abnormality,
which correlates with autistic symptoms (Geier et al., 2008). A possible etiological
role for mercury in autism (Bernard et al., 2001) is unproven (Institute of Medicine,
2004; Nelson and Bauman, 2003).
The very timing of regression in the second year is potentially significant, because
the BBB, which shields brain from blood-born toxicants, is mature in humans by
about 1 year of age. Select regions of brain known as the circumventricular organs
(CVO) fail to develop BBB, so they are potential portals for toxicants otherwise
impeded by BBB after 1 year. The CVO are located conspicuously within or around
the subcortical brainstem, defined here broadly to include medulla, pons, midbrain,
and diencephalon (Figure 9.1). An extensive neural network interconnects multiple
CVO and receives input from the viscera.
A CVO of particular interest, the area postrema (AP), is the so-called “emesis
center” of the medulla. AP is one of the most highly vascularized regions of brain
(Wislocki and Putnam, 1920), and blood flowing through its capillaries has very long
residence time in comparison to other regions of brain (Gross et al., 1990). The AP is
Subfornical
organ
Organum
vasculosum
Pineal
Median
eminence
Posterior
Area pituitary
postrema
FIGURE 9.1 The CVO, in proximity to primitive brainstem (shaded), remain unprotected
by BBB after its maturation in humans by about 12 months of age.
a central site of action for circulating angiotensin II (Kim et al., 2008). A rich network
of axons emerges from the AP, including serotonergic connections to the pons.
AP-lesioned animals consume remarkable amounts of water (Curtis et al., 1996)
or concentrated salt water (Johnson and Edwards, 1991)—an interesting observation
in light of published reports of increased water consumption in autistic children
(Terai et al., 1999) and an 8% (2,090/25,637) incidence of salt-craving in autistic
children surveyed by ARI. Food aversions and cravings for carbohydrates and bland
diets result from ablation of AP (Edwards et al., 1997), and although inadequately
quantified in autism, are commonly reported by parents. Flavor aversion after cad-
mium exposure is reversed by dimercaptosuccinic acid (Peele et al., 1988), a chelating
agent which does not cross the BBB, but which reportedly improved behavior in a
number of children with autism (Geier and Geier, 2006).
AP is proximate to the dorsal motor nucleus of the vagus (DMV) and the nucleus
tractus solitarius (NTS). The three structures comprise the DVC, which mediates
autonomic function of the cervical, thoracic, and abdominal viscera (Figure 9.2).
Ablation of the DMV blocks viscerosecretory as well as visceromotor function.
The NTS receives abundant viscerosensory input via cranial nerves VII, IX, and X,
integrates peripheral and central signals, and sends both sympathetic and parasym
pathetic efferents to the viscera. Conceivably, reported rapid improvement in
children with autism after secretin infusion (Horvath et al., 1998) relates to NTS,
where binding of intravenous secretin is most prominent (Yang et al., 2004).
DMV is the consistent site of initial pathology in PD, which ascends in stages
to outlying brainstem and distal structures including neocortex (Figure 9.3). Not
surprisingly, digestive symptoms are frequent in PD and occasionally dominate
the clinical picture (Spellman and Warner, 1977). Disorders of gastrointestinal
motility are prominent in PD (Cersosimo and Benarroch, 2008). The gastroe-
sophageal sphincter is lax, and 61% of patients with PD complained of esophageal
AP
IV DMV
NTS
FIGURE 9.2 Cross section of luxol-blue stained medulla at level of area postrema (AP),
which adjoins the dorsal motor nucleus of the vagus (DMV) and the NTS in the floor of the
fourth ventricle (IV). AP lacks tight BBB and tight CSF barrier and is envisioned as key por-
tal for neurotoxicants in autistic regression.
symptoms (Bassotti et al., 1998). Many environmental risk factors have been
identified for PD (Onyango, 2008).
Years after autistic regression, reported physical changes in brain are evident in
widespread areas not limited to brainstem. Plausible mechanisms exist within our
model for ramifying changes after initial brainstem injury. Brainstem injury itself
may disturb development of higher structures (Geva and Feldman, 2008; Tanguay and
Edwards, 1982). The spread of pathology from brainstem CVO conceivably involves
diffusion of toxicants or reactive inflammatory cytokines. Elevated cytokine levels
in cerebrospinal fluid (CSF) of subjects with autism (Vargas et al., 2005) plausibly
result from toxicant-induced production in CVO, two of which—AP and median
eminence (ME)—lack tight junctions with CSF (Broadwell et al., 1983).
Experimentally, inflammatory cytokine diffuses from lateral ventricle along
white matter nerve bundles of the corpus callosum, external capsule, and striatum
all the way to amygdala (Vitkovic et al., 2000). Cytokine injected in striatum exac-
erbates excitoxicity in cortex (Lawrence et al., 1998). The pattern of inflammatory
cytokine diffusion along nerve bundles suggests an anisotropic diffusion pathway in
small channels located outside myelinated axons (Agnati et al., 1995).
9.4 TOXICANT ACCUMULATION AND INJURY IN CVO

As will be shown, cadmium, monosodium glutamate (MSG), and paraquat exem-
plify a class of neurotoxicants, which do not readily cross the BBB and which
preferentially accumulate in areas of brain unprotected by BBB. The data we will
discuss shows that mercury in its inorganic form poorly crosses BBB and therefore
Neocortex,
Presymptomatic Symptomatic primary,
phase phase secondary
Neocortex,
high order
association
Mesocortex,
thalamus
Threshold Substantia
nigra,
amygdala
Gain setting
nuclei
Dorsal
motor ×
nucleus
1 2 3 4 5 6 Stages of the
PD-related
(A) path, process
(B)
FIGURE 9.3 (See color insert following page 200.) The ascending pathology of Parkinson’s
disease occurs in six recognizable stages, beginning in the DMV of the medulla. Progressive
shading in table (A) corresponds to the like-shaded anatomic regions represented in diagram (B).
(From Braak, H. et al., Cell. Tissue Res., 31, 121, 2004. With permission.)
concentrates in CVO, and also that administration of mercury in the organic form—
which is commonly understood to traverse the BBB—results in accumulation of
inorganic mercury in CVO, with long-term residence after remote conversion and
redistribution via blood. A review of the literature will make it clear that oxidative
mechanisms of cytotoxicity are prominent for each of the aforementioned toxicants.
Cadmium injected intravenously into adult rats accumulated only in regions out-
side the BBB, including AP and pineal, but did not appear elsewhere in the brain
(Arvidson, 1986; Arvidson and Tjalve, 1986). Cadmium induces oxidative stress,
histopathological damage, and increased lipid peroxidation in brain tissue (Mendez-

Armenta et al., 2003; Santos et al., 2005). In rats, abnormal auditory brainstem
response, cytokine elevation, and apoptosis from cadmium exposure are blocked by
administration of antioxidants (Kim et al., 2008). The oxidative effect of cadmium
appears to be due to inhibition of complexes II and III of the mitochondrial electron
transport chain (Wang et al., 2004).
Paraquat administered subcutaneously to adult rats accumulated only in areas
that lie outside the BBB, including AP and pineal (Naylor et al., 1995). Paraquat
neurotoxicity, which involves increased tumor necrosis factor (TNF) alpha and
increased superoxide production by microglia, is mediated by oxidative stress
(Wu et al., 2005). In hepatocytes, herbicides uncouple oxidative phosphorylation,
inhibit complexes II and IV, deplete glutathione (GSH), and cause mammalian cell
death by inducing lipid peroxidation (Palmeira et al., 1995). Paraquat is suggested
as a risk factor for PD (Wu et al., 2005; Peng et al., 2007).
MSG added to food is a common source of concentrated free glutamate (Walker
and Lupien, 2000), which in excess is excitotoxic. Glutamic acid decarboxylase
levels in brain are noted to be much lower in autism (Fatemi et al., 2002). MSG
administered to neonatal rats resulted in widespread neuronal necrosis in the arcuate
nucleus (Rascher and Mestres, 1980). Administration of MSG after 1 month of age
resulted in injury to the ME—a CVO unprotected by BBB—but arcuate neurons
were spared, consistent with closure of the BBB (Peruzzo et al., 2000).
Subcutaneous injections of relatively small amounts of MSG (0.1–0.5 mg/g body
weight) in 7 day old mice reduced glutamic acid decarboxylase activity in hippocam-
pus and cerebellum at 60 days (Urena-Guerrero et al., 2003). But greatest MSG
toxicity was found in areas outside the BBB, particularly the ME, which receives
axon terminals from the nearby arcuate nucleus and other hypothalamic secretory
neurons (Meister et al., 1989).
A single subcutaneous dose of MSG to adult rats induced severe ultrastructural
alterations in acetylcholine-positive AP neurons, which transport acetylcholinest-
erase to the NTS (Karcsu et al., 1985). Intraperitoneal administration of MSG to rats
resulted in oxidative changes in CVO, including lipid peroxides, which persisted for
long periods after exposure (Bawari et al., 1995; Singh et al., 2003).
As reviewed by McGinnis (2001), worrisome levels of inorganic mercury exist
in domestic water supplies, carbon-burning emissions, and municipal sludge used
as fertilizer—and individual inorganic mercury ingestion can be much greater than
expected. Mercury cell chlor-alkali plants are used to produce many food products,
including high fructose corn syrup, which may contain mercury (Dufault et al.,
2009). Ingested inorganic mercury avidly binds intestine, causing inflammation at
low nanomolar concentrations; fractional systemic absorption of ingested inorganic
mercury is evidenced by Pink disease, a profound neurological disease in young
children who ingested inorganic mercury found in teething powders (McGinnis,
2001). Autopsy reports for Pink disease include reference to vagal degeneration and
“small cell” (microglial?) infiltration in the brainstem (Wyllie and Stern, 1931).
By intramuscular injection, inorganic mercury accumulated largely in AP and
brainstem motor nuclei (Arvidson, 1992), and was prominent in AP 16 days after
injection (Nordberg and Sereniu, 1969). Inorganic mercury added to drinking water
of rats concentrated in the motor nuclei of brainstem and deep nuclei of cerebellum
(Moller-Madsen and Danscher, 1986).
Organic mercury, as found in fish and as preservative in some vaccines (Poling
2008), passes the BBB readily. Most organic mercury is converted to inorganic mer-
cury for excretion in feces, but some recirculates to increase inorganic mercury con-
centration in blood (Havarinasab et al., 2007). Once in the brain, inorganic mercury
persists for years (Vahter et al., 1994). It is the inorganic form which associates
with immune stimulation (Havarinasab et al., 2007) and increased microglia in brain
(Geier and Geier, 2007). Notably, amalgam removal decreased plasma and red-cell
inorganic mercury levels by 73% (Halbach et al., 2008).
The absence of BBB increases accumulation of inorganic mercury during and
after chronic methyl mercury (organic) exposure. Methyl mercury was administered
orally for 18 months to adult female primates, and mercury levels were measured in
six regions of brain, including pituitary, a CVO lacking BBB. Inorganic mercury
concentrations increased on average in the six areas 30-fold at 6 months and 60-fold
at 18 months, but by far the highest concentrations of mercury—largely inorganic—
were achieved in pituitary. In animals in which methyl mercury was discontinued
at 6 months, inorganic levels continued to climb in the pituitary—doubling between
6 and 12 months—but not in regions with BBB. Mercury in control animals was
undetectable in most regions, but appeared higher in pituitary. One or two primates
in each exposure group had distinctly higher or lower fractions of inorganic mercury
across brain regions, assumed secondary to individual variations in demethylation
(Vahter et al., 1994).
9.5 FINDINGS CONSISTENT WITH BRAINSTEM

PATHOLOGY IN AUTISM
On the basis of clinical observations consistent with pathology of the reticular
formation, Rimland proposed brainstem—in spite of its phylogenic antiquity—as
site of primary pathology in autism (Rimland, 1964). It has been postulated that
inherent developmental flexibility of the serotonergic system, which richly endows
the brainstem, might help explain the varied clinical presentation of autism (Cordes,
2005). A detailed psychobiological model has evolved to explain dysregulated social
behavior as a consequence of disturbed vagal messenging (Porges et al., 1996).
Electroencephalographic studies suggested centrencephalic rather than circum-
scribed telencephalic damage in autism (Gilberg and Schaumann, 1983). Against
this conceptual background, we present findings in autism that are consistent with
brainstem pathology, including abnormality of specific CVO.
9.5.1 CLINICAL AND ELECTROPHYSIOLOGICAL BRAINSTEM FINDINGS IN AUTISM

Decreased or absent postrotational nystagmus, and lack of dizziness or nausea
after spinning suggested brainstem dysfunction in autism (Ornitz, 1983). Half
(15,306/30,676) of the autistic subjects in the ARI survey often or sometimes engaged
in “whirling like a top.” Abnormal auditory brainstem response in autism also sug-
gests brainstem dysfunction (Klin, 1993; Kwon et al., 2007).
Increased heart-rate variability, failure to habituate to respiratory response, and

enhancement of vascular reaction to visual input were interpreted by early autism
investigators as evidence of altered function of the reticular formation (Bonvallet and
Allen, 1963), which is distributed extensively within the brainstem. More recently,
abnormal heart rate, blood pressure, and respiratory activity during task performance
were ascribed to abnormal vagal tone (Althaus et al., 2004). Impaired cardiac barore-
flex and depressed vagal tone more specifically implicates medullary structures (Ming
et al., 2005). Baroreflex is mediated by afferents from the glossopharyngeal nerve IX,
which synapse in the NTS, from which excitatory signals to the DMV are relayed via
the vagus to slow heart rate.
9.5.2 ABNORMAL BRAINSTEM MORPHOLOGY

Magnetic resonance imaging (MRI) demonstrated hypoplastic brainstem
(Courchesne, 1997), smaller midbrain and medulla (Hashimoto et al., 1993), and
lesser brainstem gray-matter volume (Jou et al., 2008) in autism. In a classical 1985
survey of the entire brain of an autistic adult, Bauman and Kemper (1985) reported
unusual packing density and arborization of neurons of the inferior olive, imme-
diately adjacent to DVC. A follow-up series of six brains revealed enlargement of
inferior olivary neurons in the younger subjects, and unusually diminished size in
adults. Peripheral clustering of the inferior olivary neurons was observed in five of
six subjects (Kemper and Bauman, 1993).
Bailey reported variable developmental abnormalities in brainstems of six autistic
subjects. Description of a 4 year old male from this cohort is illustrative:
… medulla oblongata was large but the pyramids were relatively small. There was mild
widening of the sulci of the superior cerebellar vermis…apparent reduplication of the
medial accessory olive, and multiple small bilateral groups of ectopic neurons lay lateral
to the olives in the inferior cerebellar peduncles. An aberrant tract was present in both
sides of the pontine tegmentum…midbrain was unusually small and the periaqueductal
grey matter and raphe nuclei appeared disproportionately large… (Bailey et al., 1998).
Two subjects with autistic behaviors had numerous swollen axon terminals
(“spheroids”) in various motor and sensory nuclei and reticular formation of the
medulla, as well as hypothalamus, dorsomedial thalamus, hippocampus, and cere-
bral cortex (Weidenheim et al., 2001). Abnormal morphology was evident in neurons
of the superior olivary nucleus of the caudal pons in five autistic subjects (Kulesza
and Mangunay, 2008).
9.5.3 BIOCHEMICAL ABNORMALITIES IN AUTISM

Melatonin is produced only by a CVO (the pineal). Blood levels of melatonin
(Kulman et al., 2000; Nir et al., 1995) and its metabolite (Tordjman et al., 2005) are
depressed in autism, and none of 24 autistic subjects examined in one study demon-
strated normal circadian variations in melatonin (Kulman et al., 2000). Abnormal
melatonin production potentially disrupts neurodevelopment by affecting neuroplas-
ticity (Baydas et al., 2002; Yun et al., 2004).
Melatonin is an efficient antioxidant (Allegra et al., 2003), which directly scavenges

free radicals (Reiter et al., 2002), stimulates production of antioxidant enzymes (Kotler
et al., 1998), and increases activity of antioxidant enzymes (Reiter et al., 1998). Unlike
other endogenous antioxidants, melatonin easily crosses BBB (Gupta et al., 2003).
Melatonin administration reduced neural lipid peroxidation and levels of degraded
glial fibrillar acidic protein in hippocampus, cortex, and cerebellum in experimentally
stressed rats (Baydas et al., 2002). In models of oxidative stress—including exposure to
cadmium, paraquat, and kainic acid—melatonin blocks lipid peroxidation (Reiter
et al., 1998). Melatonin protects against gut ulceration in stressed animals by scavenging
free radicals and reducing oxidative damage (Bandyopadhyay et al., 2002).
Groups of autistic children had significantly lower plasma levels of oxytocin
(Green et al, 2001; Modahl et al., 1998), failure to demonstrate normal longitudinal
increases in oxytocin blood levels (Modahl et al., 1998), and predominance of the
nonamidated, physiologically inactive form of oxytocin (Green et al., 2001). Oxytocin
synthesis and storage are potentially sensitive to toxicants because oxytocin from
synthetic hypothalamic nuclei is transported for storage in the posterior pituitary
(no BBB) via axons, which pass through the ME (no BBB) (Larsen et al., 1991).
Patterns of social behavior, communications, and rituals in autism may attribute
to altered oxytocin (Carter et al., 1992; Insel, 1997; Insel and Hulihan, 1995; Insel
et al., 1999). Deficits in social investigation and memory in oxytocin–knockout mice
were rescued by intraventricular administration of oxytocin (Ferguson et al., 2000).
Oxytocin infusion also reduced repetitive behaviors in autism (Hollander et al.,
2003). Oxytocin is a potent antioxidant at nanomolar concentrations and prevents
peroxidation of brain membranes (Moosmann and Behl, 2002).
9.5.4 FEATURES OF REGRESSION IN AUTISM

The three primary somatobehavioral features of regression are explicable on the
basis of neurotoxicant-induced impairment of the DVC, and variation in clinical
presentation of these features may result from differential degrees of impairment in
these minute brainstem structures.
It is widely appreciated that parasympathetic visceromotor fibers of the vagus
that originate in the DMV stimulate normal peristalsis, gastroesophogeal sphincter
contraction, and secretory function in the gut. It follows that objective signs of con-
stipation and reflux in most regressed subjects, as presented earlier, are consistent
with impaired visceromotor signals from the brainstem.
Duodenal biopsies from regressed subjects frequently demonstrated enlarged
Paneth’s cells full of secretory granules (Horvath et al., 1999) (Figure 9.4). Since
section of the vagus nerve blocks release of Paneth’s cell granulations (Eletskii
et al., 1984), the finding in autism is consistent with impaired visceromotor signals
from brainstem. Interestingly, exposure of primates to mercury results in enlarged
Paneth’s cells full of secretory granules (Chen et al., 1983).
It should be noted that diarrhea—including onset of diarrhea in the regressive
period—is prominent in some autistic children. Plausible explanations exist within
the model. Overflow diarrhea from stasis proximal to impacted stool is not uncom-
monly reported by clinicians. Poor vagal tone potentially results in a net softening
(A) (B)
FIGURE 9.4 (See color insert following page 200.) Enlargement of Paneth’s cells, as
indicated by dark staining of secretory granules, was a frequent finding in a series of duodenal
biopsies from children with regressed autism (A), as compared to non-autistic controls
(B). (Photographs courtesy of Karoly Horvath MD, PhD, Professor of Pediatrics, Thomas
Jefferson University, Philadelphia, PA.)
effect on stool if reduction in secretory function results in microbial overgrowth

or malabsorption. Alternatively, hypermotility could result from toxicant-induced
disinhibition of sympathetic efferents from the NTS.
Impaired vagal signaling is considered sufficient basis for loss of vocalizations
during the early stage of speech development. The complex process of phonation—
word or nonword—is mediated by vagal motor efferents from DMV to the intrinsic
and extrinsic muscles of the larynx, posterior tongue, and tensor palati (the latter is
noted to affect opening of the eustachian tube). Importantly, vagal afferents to NTS
coordinate the position and shape of the vocal folds and muscles of phonation. “Tone
of voice” differences in autism (Paul et al., 2005) are consistent with vagal alteration,
which associates with altered pitch (Shaffer et al., 2005).
The model essentially states that altered mechanics of vocalization—dysarthria—
can underlie loss of vocalization in autistic regression. The existence of complex
cognitive impairments in older autistic subjects does not contradict brainstem-
mediated loss of vocalizations, because cognitive impairments in older subjects
could result from later changes in brain. The proposed vagal dysarthria should not
be confused with classical “motor apraxia of speech,” which includes additional
glossal and facial signs not seen in autism. The observation of marked improve-
ment in communication with an electronic speech-generating device (Thunberg
et al., 2007) is more consistent with brainstem-mediated vocalization deficit than
higher cognitive incapacity in some older autistic children. Parents in the ARI sur-
vey report that at age 3–5 years, 20% of children with autism (5,880/31,109) were
significantly better at correctly pointing to named objects than in saying the names
of the same objects.
Frank dysarthria was reported in 12% (3/25) of a cohort of autistic children
with identifiable mitochondrial abnormalities (Weissman et al., 2008). Specific
data from the ARI parent survey also are consistent with altered mechanical
vocalization: of children whose onset of autistic symptoms occurred after 1 year of

age, 17% (4,141/23,685) reportedly started talking normally, then had talk replaced
by whispering for at least 1 week. Of this subgroup, 1771 children deteriorated to
loss of all whispering as well as loss of talking, and another 679 children continued
whispering long-term.
Altered function of NTS is a plausible mechanism for loss of social function,
because NTS projects secondarily to limbic and cortical areas known to mediate
social behavior, arousal, mood, emotion, anxiety, seizure activity, and pain per-
ception (Marvel et al., 2004; Nemeroff et al., 2006). Vagal nerve stimulation for
depression is thought to act via innervation of the NTS (Nemeroff et al., 2006),
and vagal nerve stimulation for seizure control has been reported to improve sig-
nificantly varied autistic behaviors (Murphy et al., 2000). As discussed earlier,
concurrent CVO-mediated changes in oxytocin may contribute to loss of social
skills as well.
It is observed that: (1) social regression often precedes language regression
(Goldberg et al., 2003), (2) social regression may be unaccompanied by language
regression (Goldberg et al., 2003; Lord et al., 2004), and (3) language regression
nearly always is accompanied by social regression (Goldberg et al., 2003; Lord et al.,
2004). A cogent explanation for these variations exists within the detailed anatomy
of the DVC. NTS predictably is more vulnerable to blood-born toxicants than DMV,
because in its caudal portion, the dorsal aspect of NTS is unprotected by BBB (Gross
et al., 1990). Although the distances are not great, toxicants must enter the DMV by
diffusion from AP (and NTS) entry points.
The model’s site specificity allows different toxicants independently to provoke
regression by one-time or cumulative insult. Likewise, different toxicants may act in
combination or sequentially on CVO to exceed the neurophysiological threshold for
regression. The model does not imply entirely normal biochemistry or development
of the brainstem or other parts of the brain prior to regression, and in fact encour-
ages consideration of preexisting conditions, which potentially affect the regressive
threshold.
In this context, it is interesting that the vulnerable period for thalidomide exposure
as risk factor for autism (20–24 days) corresponds to the timing of formation of the
medullary motor nuclei (Rodier et al., 1997). Intriguing too is decreased autonomic
activation of heart rate (and brainstem auditory evoked potentials) in Faroes Island
children as a function of cord-blood levels of mercury (Grandjean et al., 2004). Earlier
weakening or maldevelopment of brainstem structures—including influences, which
are not modulated directly by BBB—conceivably potentiate regression triggered by
toxicant effects on brainstem.
A summary of objective findings in autism, which are consistent with brainstem
abnormality or abnormality of brainstem-proximate CVO, is presented in Table 9.1.
9.5.5 OXIDATIVE MECHANISMS

The mechanisms of relevant toxicants are substantially oxidative, so it is appropriate
to consider oxidative stress in the context of the proposed model. Greater oxida-
tive stress in blood and urine (Chauhan and Chauhan, 2006; Chauhan et al., 2008;
TABLE 9.1
Reported Findings in Autism, Which Are Consistent with Brainstem
Abnormality or Consistent with Abnormality of CVO
Found within or Proximate to the Brainstem
Studies References
Clinical observations
Excessive thirst Terai et al. (1999)
Salt-craving and flavor-aversion ARI parent survey
Tone-of-voice alterations Paul et al. (2005)
Talk → whisper ARI parent survey
Electrical speech generation Thunber et al. (2007)
Secretin effect Horvath et al. (1998)
Altered postrotatory response Ornitz (1983)
Cardiac baroreflex Ming et al. (2005)
Heart rate, respiratory and vascular Althaus et al. (2004), Bonvallet
and Allen (1963)
Electrical studies
Auditory brainstem response Klin (1993), Kwon et al. (2007)
Centrencephalic electroencephalograms Gilberg and Schaumann (1983)
Decreased cardiac parasympathetic tone Ming et al. (2005)
Vagal nerve stimulation Murphy et al. (2000)
Magnetic resonance imaging

Smaller brainstem Courchesne (1997)
Smaller medulla and midbrain Hashimoto et al. (1993)
Decreased brainstem gray matter Jou et al. (2008)
Radiographic studies
Fecal loading in 100% of regressed cohort Torrente et al. (2002)
Fecal loading in >50% of mixed cohort Afzal et al. (2003)
Endoscopic studies
Esophageal reflux 67% of regressed cohort Horvath et al. (1999)
Microscopic studies
Neuronal morphology/clustering inf. olives Kemper and Bauman (1993)
Neuronal morphology superior olives Kulesza and Mangunay (2008)
Ectopic neurons and aberrant tracts Bailey et al. (1998)
Swollen axon terminals in medullary nuclei Weidenheim et al. (2001)
Paneth’s cells enlarged with granules Horvath et al. (1999)
Biochemical studies
Abnormal oxytocin production Green et al. (2001), Modahl et al.
(1998)
Abnormal melatonin production Kulman et al. (2000), Nir et al. (1995),
Tordjman et al. (2005)
McGinnis, 2004; Suh et al., 2008; Yao et al., 2006) and greater oxidative modification
of hippocampus (Evans et al., 2008), cerebral cortex (Evans et al., 2008; Lopez-
Hurtado and Prieto, 2008), and cerebellum (Sajdel-Sulkowska et al., 2008) exists in
children already diagnosed with autism. Gastrointestinal inflammation, emotional
stress associated with noncommunication, sleep disturbance, and lower production
of melatonin and oxytocin may contribute variably to greater oxidative stress after
regression.
There is some support—elevated brain-derived neurotrophic factor (BDNF) in
cord blood, greater maternal stress during gestation, more perinatal complications—
for the suggestion that oxidative stress is heightened prior to regression (McGinnis,
2004, 2007). If oxidative stress were higher in brain prior to regression, then cor-
respondingly lower antioxidant protection in brain would be expected to influence
thresholds for brainstem injury by toxicants. Greater peripheral oxidative stress alone
plausibly lowers the threshold for CVO-mediated regression, because the cholinergic—
especially, muscarinic—nervous system is sensitive to oxidative stress (McGinnis,
2004), and extracranial vagal parasympathetics are exclusively cholinergic.
Cholinergic receptors are inhibited preferentially by oxidants (De Sarno and
Pope, 1998), and numbers of cholinergic receptors are reduced under conditions of
oxidative stress (Gajewski et al., 1988). Cholinergic receptor activity measured in
basal forebrain and parietal cortex was lower in autism (Perry et al., 2001), and it
has been suggested that cerebral hypoperfusion in autism results from muscarinic
inhibition (McGinnis, 2004). The cholinergic system is essential for brain develop-
ment, acting as modulator of neuronal proliferation, migration, and differentiation
(Roda et al., 2008).
We retrospectively compared acetylcholine (ACh) levels in plasma and CSF from
children aged 3–15 who were identifiable as autistic or nonautistic. Mean plasma
ACh was much lower in autistic children (192 ± 36 ng/L; n = 94) than in nonautistic
children (464 ± 85 ng/L; n = 66). Mean ACh in CSF also was lower in an autistic
cohort (941 ± 202 ng/L; n = 16) than a nonautistic cohort (1558 ± 510 ng/L; n = 8).
It is not known whether these levels reflect the state of neurological transmission
in autism. Chronic exposure of animals to cadmium or methyl mercury is noted to
lower ACh in brain (Hrdina et al., 1976), but existence of any such relationship in
autism is speculative.
9.5.6 INFLAMMATORY MECHANISMS IN AUTISM

Microglia are resident immune cells in the brain, which normally influence late
embryonic and postnatal brain development by exerting an important “pruning
effect.” Microglia also release potentially cytotoxic inflammatory cytokines and oxi-
dative factors in response to toxicants or infections. Microglial activation is assumed
to relate to the pathogenesis of PD and is associated with neuronal cell loss in many
toxic exposures (Mangano and Hayley, 2009). An increase in microglia is seen after
exposure to inorganic mercury (Charleston et al., 1994), and in association with inor-
ganic mercury accumulation after organic mercury exposure (Vahter et al., 1994).
Microglial activation in autism is evidenced by greater inflammatory cytokines in
CSF and microscopic changes in examined areas of brain (cerebral cortex, cingulate
gyrus, cerebellum) (Vargas et al., 2005). As yet, there is no clear understanding of

when neuroinflammation begins in autism, how neuroinflammation varies longitu-
dinally, or how variable patterns might reflect etiologies.
Many xenobiotics, including bacterial lipopolysaccharide (LPS), stimulate
peripheral mononuclear cells and brain microglia to increase production of TNF,
which plays a central role in the cascade of potentially cytotoxic factors in the
immune response. In cell cultures, ACh blocks TNF-induced cell activation
(Saeed et al., 2005). In PD, TNF is increased in CSF and brain (McCoy et al.,
2006). TNF in CSF was also elevated in two autistic cohorts, one exclusively com-
prised of regressed subjects (Chez et al., 2007); the other with 10 of 12 regressed
(Zimmerman et al., 2005). High CSF/blood ratios of TNF, which poorly transits
the BBB, implies increased TNF production in the brain, at least in the regressive
subgroup (Chez et al., 2007).
LPS poorly transits the BBB. Systemic administration of LPS to adult animals
results in immediate and robust TNF production in brain, detectable in neuronal cell
bodies, but only at CVO or immediately adjacent structures, and most intensely at
the AP and ME. LPS-induced TNF expression in the NTS was modest at 1.5 h, and
marked by 18 h. At the same intervals, TNF was absent initially in DMV, then pres-
ent at 18 h. TNF reached other nuclei, such as hypoglossal IX, by 18 h (Breder et al.,
1994). Another study administered LPS and initially found gene encoding for TNF
only in CVO, followed by a migratory pattern of TNF-positive cells around CVO at
three hours, and finally a ubiquitous presence of TNF-positive cells throughout the
brain at 6 h (Nadeau and Rivest, 1999).
Whether TNF plays a significant role in the pathogenesis of autism—as suspected
in PD on the basis of animal models (McCoy et al., 2006)—is unproven. Within our
model, it is possible that toxicant-induced TNF production by CVO (especially AP
and ME, which lack tight barriers to CSF) at time of regression is sufficient basis for
large increases in TNF and other inflammatory cytokines in CSF. The toxicants used
as examples in our model certainly stimulate TNF production.
Cadmium has been regarded as one of the “inflammation-related xenobiotics”
because low concentrations potently stimulate inflammatory cytokines, including
TNF, in hepatocytes (Souza et al., 2004). Exposure of neonatal rats provides clear
evidence that MSG increases TNF in brain unprotected by BBB, and that the increase
is associated with neuronal death, possibly via apoptosis (Chaparro-Huerta et al.,
2002). Paraquat-induced TNF has been implicated in pathogenesis of lung disease,
and production of TNF in response to LPS by peripheral monocytes is increased by
paraquat up to 18-fold (Erroi et al., 1992). An increase of the same order of magni-
tude in TNF upregulation in response to LPS was reported in peripheral monocytes
of a subgroup of autistic children (Jyonouchi et al., 2001), but the finding has not
been examined in the context of toxicant exposure.
TNF-alpha actions on AP and NTS substantially disrupt autonomic control of the
gut. For instance, by modulating release of glutamate by primary vagal afferents to the
NTS, TNF-alpha in animal experiments produced extreme gastric stasis (Hermann
and Rogers, 2008). Interestingly, work with experimental colitis suggests that intestinal
inflammation may affect adversely survival and function of DMV neurons. Exposure
of cultured DMV neurons to TNF diminished cell proliferation, increased apoptosis,
and altered calcium response to glutamate (Ammori et al., 2008). Besides endogenous
inflammatory factors, viruses also easily may enter at CVO (Banks, 2000).
9.6 FUTURE RESEARCH

Within the current technology, it may be possible to identify brainstem lesions, which
can account for regression and other deficits observed in autism. Special attention to
thin, unmyelinated “C-fibers” is warranted, since these comprise afferents to NTS
and efferents from the DMV, which may be highly sensitive to toxicant exposure.
Direct examination of the vagus nerve is needed to rule out extracranial neuropathy
in autism, and afford inference about status of the DMV and NTS. When possible,
identification of regressed subgroups may afford a useful methodology.
Physical detection of toxicants in brain—especially CVO and brainstem—of sub-
jects with autism is indicated. While tissue half-lives and combined toxicity might
obscure etiology in tissue-concentration studies, significant findings in even a subset
of the autistic population could be enormously important.
Oxidative residuals of neurotoxicity may be detectable in relevant areas of brain when
toxins are not. Lipofuscin, a relatively stable matrix of oxidized lipid and cross-linked
protein, was elevated in cerebral cortex in autism (Lopez-Hurtado and Prieto, 2008),
suggesting a need for complete lipofuscin mapping. Examination of lipofuscin itself for
accumulation of toxicants is an interesting possibility (Opitz et al., 1996). Carboxyethyl
pyrrole (CEP) is a promising marker for axonal oxidation in autism (Evans et al., 2008).
Distribution of oxidative markers in animal brain after exposures to toxicants may pro-
vide a valuable context for interpretation of findings in brain of autistic subjects.
Regional differences in distribution of receptors, inflammatory markers, and
oxidative modification are interpretable from the toxicological perspective. For
instance, while mercury levels in medulla from whales were considered only mildly
elevated in relation to estimated lowest-observable-adverse-effect levels, a significant
correlation existed between total mercury concentration and N-methyl-d-aspartate
(NMDA) receptor levels (Basu et al., 2009). TNF levels in brainstem structures
potentially reflect effects of toxicants. A preliminary study found a significant
correlation between concentrations of mercury and 3-nitrotyrosine in cerebellum
from subjects with autism (Sajdel-Sulkowska et al., 2008). Suspicion of toxicants as
triggers in autism will stimulate a host of creative research methodologies.
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10 Abnormalities in
Membrane Lipids,
Membrane-Associated
Proteins, and Signal
Transduction in Autism
Ved Chauhan* and Abha Chauhan
Department of Neurochemistry, New York State
CONTENTS
10.1 Introduction ................................................................................................. 178
10.2 Abnormalities in Membrane Amino-Glycerophospholipids
in Autism ..................................................................................................... 179
10.3 Membrane Fluidity and Role of Fatty Acids in Autism .............................. 179
10.4 Phospholipase A2 and Autism ..................................................................... 181
10.5 Polyphosphoinositides and Autism ............................................................. 182
10.6 Protein Kinases and Autism........................................................................ 183
10.7 Lipoprotein, Cholesterol, and Autism ......................................................... 183
10.8 Calcium and Calcium Ion Channels in Autism........................................... 184
10.9 Role of Membrane-Associated Proteins Such as Pten, Neuroligins,
SHANK3, Reelin, and Serotonin Receptors in Autism .............................. 185
10.9.1 Potential Role of Pten and PI3K/Akt Pathway in Autism .............. 188
10.9.2 Neuroligins and Autism ................................................................. 189
10.9.3 SHANK3 and Autism .................................................................... 189
10.9.4 Reelin and Autism.......................................................................... 190
10.9.5 Serotogenic Receptors and Autism ................................................ 190
10.9.6 Evidence of Altered Levels of Bcl2 and p53
(Markers of Apoptosis) in the Brains of Autism Subjects ............. 191
177
10.10 Oxidative Stress, Inflammation, and Cell Signaling................................... 192

10.10.1 Oxidative Stress in Autism ............................................................ 192
10.10.2 Cytokines, Inflammation, and Autism ........................................... 194
10.11 Lipid Rafts and Disorders of the Brain ....................................................... 195
10.12 Conclusions ................................................................................................. 195
References .............................................................................................................. 196
Membrane lipids play an important role in the control of cellular functions. In this
chapter, we present evidence that autism spectrum disorders (ASD) are associated
with abnormalities in lipid metabolism, membrane-associated proteins, and signal
transduction. Altered levels of amino-glycerophospholipids (AGP) in the membrane,
increased peroxidation of lipids, and decreased membrane fluidity in autism suggest
that membrane signaling may be affected in autism. Increased phospholipase A2
(PLA2) activity in the blood and lymphoblasts of autistic subjects suggests that this
lipid-metabolizing enzyme may be affected in autism. If membrane lipids abnormal-
ities occur in autism, then lipid rafts, membrane domains with a strong affinity for
signaling molecules, may also be involved in the etiology of autism. An association
of phosphatidylinositol 3-kinase (PI3K) gene in autism; decreased activity of protein
kinase C (PKC); increased activity of protein kinase A (PKA) in the lymphoblasts
from autistic subjects; altered brain levels of Bcl2 and p53 involved in apoptosis;
inflammation and altered levels of cytokines; and mutational changes in the pro-
teins involved in cell signaling such as neuroligins, Pten, SHANK3, Wnt, reelin, and
voltage-dependent calcium channels suggest impairment in signal transduction in
autism. These abnormalities in the signal system may account for some of the struc-
tural changes and cognitive deficits in the brains of individuals with autism.
10.1 INTRODUCTION
Autism spectrum disorders (ASD), also known as pervasive developmental disor-
ders (PDDs), cause severe and pervasive impairment in language, cognition, and
socialization. These disorders include autism (severe form), pervasive develop-
mental disorder—not otherwise specified (PDD-NOS), and a much milder form,
Asperger syndrome. They also include two rare disorders: Rett syndrome and
childhood disintegrative disorder. Autism is a heterogeneous disorder, both etio-
logically and phenotypically. Within this autism spectrum, there are variations in
the severity of the disorder, the level of cognitive functioning, the presence or
absence of associated medical conditions such as seizures or other neurological
disorders, and whether or not there is a history of regression from apparent normal
development. Etiologically, autism is generally considered to be a polygenetic dis-
order resulting from interactions of alleles at several loci (Sutcliffe, 2008).
The genetic predisposition of autism may be obvious, but there is limited knowl-
edge of causative or secondary abnormalities in the biochemical pathways in autism.
The concept of membrane abnormalities as a cause of neurodevelopmental disorders
was suggested by Horrobin (1999). Phospholipids make up the bulk of all internal
and external neuronal membranes. Alteration in membrane lipids can result in defec-
tive membrane functions and therefore may have a wide impact on learning and
Membrane and Signal Transduction Abnormalities 179
behavior. Some of the important membrane functions associated with phospholipids

that are essential for the proper functioning of the cells include generation of intra-
cellular second messengers, i.e., inositoltriphosphate (IP3) and diacylglycerol (DG),
by receptor-coupled cleavage of phosphoinositides, and maintenance of fluid envi-
ronment by unsaturated fatty acids. In addition, phosphoinositides are cofactors of
protein-phosphorylating enzymes such as Akt/protein kinase B (Kisseleva, Cao, and
Majerus, 2002) and endocytosis (Ikonomov et al., 2002). Sphingomyelin generates
sphingosine, an intracellular messenger (van Blitterswijk et al., 2003). Most neuronal
membrane proteins such as receptors and receptor-coupled enzymes are embedded
in or attached to phospholipid membranes. The quaternary structure of protein and,
therefore, the function of protein depend on the precise composition of the immedi-
ate phospholipid environment. Alterations in membrane lipid metabolism, such as
composition of fatty acids, oxidation of lipids, and/or altered levels of phospholipids
in the membrane, will affect the functions of proteins that are involved in signal
transduction. It is pertinent to mention that the fluid mosaic model of membrane,
which described membrane as a lipid bilayer in which proteins are diffused freely,
has been modified substantially. Membrane compartmentalization occurs by lipid–
lipid, lipid–protein, and membrane–cytoskeletal interactions (Nicolau et al., 2006),
and alterations in membrane composition will affect all of these interactions, and
thus signal transduction. In this chapter, we present evidence that suggests a role of
abnormalities in membrane biochemistry and signal transduction in autism.
10.2 ABNORMALITIES IN MEMBRANE AMINO-

GLYCEROPHOSPHOLIPIDS IN AUTISM
We previously reported that the levels of phosphatidylethanolamine (PE) were decreased
while phosphatidylserine (PS) were increased in the erythrocyte membranes from chil-
dren with autism as compared to typically developing siblings (Chauhan et al., 2004b).
PE and PS belong to a class of phospholipids known as amino-glycerophospholipids
(AGP). In addition to the erythrocyte membrane, AGP were also affected in the blood
plasma in autism. As shown in Figure 10.1, a significant increase in plasma levels of AGP
was observed in autism subjects (mean ± SE = 0.2022 ± 0.021) as compared to typically
developing siblings (mean ± SE = 0.1575 ± 0.017) (Chauhan et al., 2004b). The acidic lip-
ids are localized mainly on the cytoplasmic side of the membrane. During apoptosis
and also in oxidative stress, normal symmetry of the membrane is lost, and PE and PS
are externalized (Jain, 1984; Matsura et al., 2005). Externalization of PS also plays an
important role in the aggregation of platelets (Bucki et al., 2001). It remains to be studied
whether orientation of these lipids in the membrane is affected in autism. Alterations in
brain energy and phospholipid metabolism in autism have also been suggested to cor-
relate with the neuropsychologic and language deficits (Minshew et al., 1993).
10.3 MEMBRANE FLUIDITY AND ROLE OF FATTY

ACIDS IN AUTISM
Membrane fluidity refers to the viscosity of the lipid bilayer of a cell membrane.
The phospholipids of membrane contain fatty acids of varying lengths and saturation.
Shorter-chain fatty acids and ones with greater unsaturation are less stiff, less viscous,
0.35
0.30
Absorbance at 410 nm 0.25
0.20
0.15
0.10
0.05
0.00
Autism Siblings
FIGURE 10.1 Increased AGP content in the plasma of autism subjects as compared to
typically developing sibling controls. Total lipids extracted from the plasma samples of
14 children with autism (䊏) and their 14 nonautistic siblings (Δ) were labeled with trinitroben-
zene sulfonic acid (TNBS), and absorbance of the samples read at 410 nm. (Reproduced from
Chauhan, V. et al., Life Sci., 74, 1635, 2004b. With permission.)
and have lower melting points. We have observed that fluidity of the erythrocyte
membrane from children with autism is significantly lower than that of normal sib-
lings (Chauhan et al, 2005). These results suggest that membranes are more rigid
in autism. Biomembranes are generally in the fluid liquid-crystalline phase and
maintenance of optimal membrane fluidity is critical to biological functions. It has a
marked effect on membrane properties, modulating the activity of membrane-bound
enzymes and other membrane-associated molecules such as ion channels and recep-
tors (Houslay and Stanley, 1982). The activities of integral membrane proteins are
markedly affected by the physical state of the lipids in which they are embedded.
For example, fluid membranes have a greater number of insulin receptors and more
responsive receptors, resulting in heightened sensitivity to insulin (Neufeld and
Corbo, 1984). Rigid membrane adversely affects the function of tissues throughout
the body, including the brain. In autistic subjects with developmental regression,
Bu et al. (2006) observed increased levels of ω-9 fatty acids, i.e., eicosenoic acid
(20:1n9) and erucic acid (22:1n9) when compared with typically developing controls.
In addition, an increase in eicosadienoic acid (20:2n6) and a decrease in palmi-
etaidic acid (trans,16:1n7t) were observed in autistic children with clinical regression
compared to those with early onset autism. The above evidence on decreased fluid-
ity of the erythrocyte membrane, and alterations in the fatty acid composition in
autism suggest that abnormalities in fatty acid metabolism may be involved in this
disorder.
The composition of fatty acids in the membrane phospholipids can be influenced
by the dietary intake of polyunsaturated fatty acids, the status of oxidative stress,
and/or the action of phospholipase A2 (PLA2), an enzyme that removes the sn-2 fatty
acids of phospholipids. It is known that dietary ω-3 fatty acids affect osmotic fragility
and membrane fluidity (Hagve, Lie, and Gronn, 1993). Such effects may be respon-
sible for the decreased blood viscosity and improved microcirculation observed after
feeding ω-3 fatty acids to animals (Young and Conquer, 2005).
Long-chain ω-3 fatty acids such as eicosapentaenoic acid (EPA) and docosa-
hexaenoic acid (DHA) have important structural roles as components of cellular
membranes. Both DHA and EPA are linked with many aspects of neural function
including neurotransmission, membrane fluidity, ion channel, enzyme regulation,
and gene expression (Young and Conquer, 2005). If unsaturated fatty acids are more
in our diet, the cell membrane becomes more fluid. DHA is the most unsaturated of
all fatty acids, and it helps in fluid cell membranes. The incorporation of EPA and
DHA into membrane phospholipids increases after ingestion of large amounts of ω-3
fatty acids.
Epidemiological studies suggest that dietary consumption of EPA and DHA,
commonly found in fish or fish oil, may decrease the risk for certain neuropsychiatric
disorders. Decreased blood levels of ω-3 fatty acids have been reported in several
neuropsychiatric conditions, including attention deficit (hyperactivity) disorder,
Alzheimer’s disease, schizophrenia, and depression (Young and Conquer, 2005).
Supplementation studies, using individual or a combination of ω-3 fatty acids, sug-
gest that certain symptoms associated with some of these disorders may improve
with ω-3 fatty acid supplementation (Young and Conquer, 2005). Recently, Amminger
et al. (2007) reported an advantage of dietary supplementation of ω-3 fatty acids for
the hyperactivity and stereotypy of individuals with behavioral disorders.
10.4 PHOSPHOLIPASE A2 AND AUTISM

PLA2 hydrolyzes the sn-2 fatty acids of phospholipids, giving rise to polyun-
saturated fatty acids and lysophospholipids. The released fatty acid, i.e., arachi-
donic acid is a major source of the production of prostaglandins and leukotrienes.
Chromosomal linkage studies with genetic markers have provided possible genetic
links to autism. Multiple genes have been postulated to be involved in the etiology
of ASD (reviewed in Abrahams and Geschwind, 2008; Lamb et al., 2000). Several
studies have suggested that loci D1S1675 (Risch et al., 1999), D6S283-D6S261
(Philippe et al., 1999), D7S530-D7S684 (International Molecular Genetic Study of
Autism Consortium, 1998), and D13S800 (Barrett et al., 1999) are potential gene
sites for autism. While these studies point to the involvement of different loci in
autism, overlapping loci on chromosome 7 have been detected in all these studies.
Therefore, PLA2 (7q31-located at D7S530-D7S684 loci on chromosome 7) may be
involved in the etiology of autism.
We have observed that PLA2 activity is higher in lymphoblasts from autism

subjects than from control subjects (Chauhan and Chauhan, 2008; Chauhan et al.,
2007). Ward (2000) also observed significantly higher concentrations of PLA2 in
the erythrocytes from patients with regressive autism and classical autism/Asperger
disorder than from control subjects. Interestingly, supplementation with EPA in
patients with classical autism or Asperger disorder resulted in significantly reduced
PLA2 concentrations compared to nontreated individuals.
The role of PLA2 in the etiology of several other neurodevelopmental disorders
has also been documented previously (Bell et al., 2004). It has been suggested that
genetic inheritance may cause phospholipid abnormalities in dyslexia that are some-
what similar to those found in schizophrenia (Horrobin and Bennett, 1999). PLA2
levels were reported to be significantly higher in dyslexic-type adults and schizo-
phrenics than in control subjects (MacDonell et al., 2000).
Two functional candidate genes (phospholipase genes) have been identified: phos-
pholipase Cβ2 (PLCβ2) and PLA2, group IVB (cytosolic; PLA2G4B). Molecular
genetic studies have suggested a reading disability (RD, dyslexia) susceptibility
locus on chromosome 15q (Morris et al., 2004). The locus was mapped to the region
surrounding D15S994 and D15S944, which is located within PLCβ2 and is 1.6 Mb
from PLA2G4B, thus suggesting a role of phospholipases in neurodevelopmental
disorders.
10.5 POLYPHOSPHOINOSITIDES AND AUTISM

Phosphatidylinositol (PI), PI 4-phosphate (PI 4P), PI 4,5-P2, PI 3P, PI 3,4-P2, and
PI 3,4,5-P3 belong to a group of lipids collectively known as phosphoinositides.
The amount of these lipids is generally less than 1% of total lipids, but they are
essential lipids in signal transduction. Phospholipase C–mediated cleavage of PI
4,5-P2 produces two intracellular messengers: DG, an activator of protein kinase C
(PKC), and IP3, a calcium mobilizer (Chauhan et al., 1990). Phosphatidylinositol
3-kinase (PI3K) phosphorylates PI, PI 4P, and PI 4,5-P2 at the 3-OH group of inosi-
tol ring, giving rise to PI 3P, PI 3,4-P2, and PI 3,4,5-P3. Extensive evidence from
our group and other groups has shown that PIP2 and PIP3 are potent activators of
PKC (Chauhan and Brockerhoff, 1988; Chauhan, Chauhan, and Brockerhoff, 1991;
Chauhan et al., 1989, 1991; Derman et al., 1997; Lee and Bell, 1991; Singh et al.,
1993; Toker et al., 1994). The 3-OH-phosphorylated phosphoinositides also have
an important function in the activation of protein kinase B/Akt (enzyme involved in
apoptosis), and also in membrane trafficking. An association of inositol polyphos-
phate-1-phosphatase (INPP1), phosphoinositide-3-kinase catalytic gamma polypep-
tide (PIK3CG), and tuberous sclerosis complex 2 (TSC2) gene variants with autistic
disorder and its implications for phosphatidylinositol signaling in autism were sug-
gested by Serajee et al. (2003). In addition to lipid moiety of phosphoinositides,
its water-soluble part, i.e., myo-inositol was also altered in autism (Serajee et al.,
2003). A decrease in concentration of myo-inositol in autism compared to typi-
cally developing controls was reported. The additional role of phosphoinositides in
autism is discussed in Section 10.9.
10.6 PROTEIN KINASES AND AUTISM

Intracellular signaling is carried out by the phosphorylation of proteins by protein
kinases. These protein kinases have specific requirement for activation, e.g., PKC
requires calcium, PS and DG; protein kinase A requires cAMP; and protein kinase B
requires PIP3. We studied the activities of Ca2+ /Mg2+ -ATPase (which regulates intra-
cellular calcium concentration) in the erythrocytes, and of protein-phosphorylating
enzymes, namely, PKC and PKA, in lymphoblasts from autism and controls subjects
(Chauhan et al., 2007). The activity of membrane Ca2+ /Mg2+ -ATPase was higher
in autism subjects than in controls. The results suggested that intracellular calcium
levels might be increased in autism. Interestingly, basal intracellular calcium levels
were observed to be higher in the lymphoblasts from autism subjects than in lym-
phoblasts from control subjects. Since imbalance in calcium homeostasis can affect
the activity of PKC in autism, we also measured the PKC activity in lymphoblasts
from autism and control subjects. PKC activity was decreased in both cytosolic and
membrane fractions in autism as compared to controls. On the other hand, the activ-
ity of cAMP-dependent PKA was increased in autistic lymphoblasts as compared to
control lymphoblasts. Recently, Lintas et al. (2008) reported an association between
autism and PRKCB1 gene variants, pointing towards the role of PKCβ in altered
epithelial permeability, and demonstrating a significant downregulation of brain
PRKCB1 gene expression in autism. Our data on levels of intracellular calcium,
activities of Ca2+ /Mg2+ -ATPase, PKC, and PKA strongly support that signal trans-
duction may be abnormal in autism (Chauhan et al., 2007).
10.7 LIPOPROTEIN, CHOLESTEROL, AND AUTISM

The family of low-density lipoprotein (LDL) receptors represents a collection of
membrane receptors that have been remarkably conserved throughout evolution.
These multifunctional receptors known to regulate cholesterol transport are gaining
attention in neuroscience due to their ability to transduce a diversity of extracellular
signals across the membrane. Their roles in modulating synaptic plasticity and in
hippocampus-specific learning and memory have recently come to light. In addition,
genetic, biochemical, and behavioral studies have implicated these signaling systems
in a number of neurodegenerative and neuropsychiatric disorders involving loss of
cognitive ability, such as Alzheimer’s disease, schizophrenia, and autism (reviewed
in Qiu, Korwek, and Weeber, 2006).
The results of several genomic screens suggest the presence of an autism sus-
ceptibility locus on chromosome 19p13.2–q13.4. The apolipoprotein E (apoE) gene
on chromosome 19 encodes for a protein, apoE, whose different isoforms (E2, E3,
E4) influence neuronal growth. ApoE participates in lipid transport and metabolism,
repair, growth, and maintenance of axons and myelin during neuronal develop-
ment. Persico et al. (2004) reported a linkage/association between reelin (RELN)
gene polymorphisms and autistic disorder. ApoE participates in the reelin signaling
pathway by competitively antagonizing reelin binding to ApoE receptor 2 and to
very-low-density lipoprotein receptors (VLDL). ApoE2 displays the lowest recep-
tor binding affinity compared with ApoE3 and ApoE4. The linkage/association was
assessed between primary autism and ApoE alleles in 223 complete trios, from 119
simplex Italian families, and 44 simplex/29 multiplex Caucasian-American families.
Statistically significant disequilibrium favored the transmission of epsilon2 alleles
to autistic offspring, over epsilon3 and epsilon4 (Persico et al., 2004). However, the
study by Raiford et al. (2004) did not find an association between apoE and autism.
In blood, lipids are transported by lipoproteins. Corbett et al. (2007) reported
a potential role for dyslipidemia in the pathogenesis of some forms of ASD. Using
two-dimensional electrophoresis and analyzing 6348 peptide components of serum,
apolipoprotein (apo) B-100 had an effect size >0.99, with a p < 0.05 and a Mascot
identification score of 30 or greater, for autism compared to controls. In addition, apo
B-100 and apo A-IV were higher in children with high-functioning autism compared
to low-functioning autism.
Some alterations of lipids in autism may also be linked to secondary abnormalities.
For example, Smith-Lemli-Opitz syndrome (SLOS), a genetic condition of impaired
cholesterol biosynthesis, is associated with autism (Tierney et al., 2001). It has been
suggested that in addition to SLOS, there may be other disorders of sterol metabolism
or homeostasis associated with ASD (Tierney et al., 2006). However, the incidence
of SLOS and other sterol disorders among individuals with ASD is not known.
10.8 CALCIUM AND CALCIUM ION CHANNELS IN AUTISM

Calcium is a vital intracellular signaling molecule. The concentration of intracellu-
lar calcium is low in the resting cell; however, its levels rise from low nanomolar to
micromolar levels after receptor-coupled stimulation of the cell. The receptor-coupled
increase in intracellular levels of calcium is important for neuronal survival, differen-
tiation, migration, and synaptogenesis (Aamodt and Constantine-Paton, 1999; Cline,
2001; Komuro and Rakic, 1998; Moody and Bosma, 2005; Represa and Ben Ari, 2005;
Spitzer, Root, and Borodinsky, 2004). Defects in these developmental processes can
lead to some of the neuroanatomical abnormalities, such as increases in cell-packing
density, decreases in neuron size and arborization, and alterations in connectivity,
that are associated with ASD patients (Courchesne et al., 2005; DiCicco-Bloom
et al., 2006). Voltage-gated calcium channels mediate calcium influx in response to
membrane depolarization and regulate intracellular processes such as contraction,
secretion, neurotransmission, and gene expression. Their activity is essential for cou-
pling electrical signals in the cell surface to physiological events in cells.
Functional mutations in genes encoding voltage-gated Ca2+ channels have been
suggested as a possible cause of ASD (Hemara-Wahanui et al., 2005; Splawski et al.,
2004, 2006). Point mutations in the gene encoding the L-type voltage-gated Ca2+
channel CaV1.2 (CACNA1C) cause Timothy syndrome, a multisystem disorder that
includes cardiac abnormalities and autism (Splawski et al., 2004, 2005). CaV1.2
plays an important role in the activation of transcription factors, such as cAMP-
response-element-binding protein (CREB) and myocyte enhancer factor 2 (MEF2),
involving neuronal survival and dendritic arborization (West et al., 2001). The muta-
tions associated with Timothy syndrome prevent voltage-dependent inactivation of
CaV1.2, which causes the channels to have longer open periods and to carry more
Ca2+ than wild-type channels (Splawski et al., 2004, 2005). Similar evidence of
calcium involvement in autism comes from a mutation in CACNA1F, which encodes

the L-type voltage-gated Ca2+ channel CaV1.4. It was reported to cause autistic
symptoms in a New Zealand family of patients who have stationary night blindness
(Hemara-Wahanui et al., 2005; Hope et al., 2005).
ASD-associated mutations have been identified not only in genes encoding Ca2+
channels themselves but also in genes encoding ion channels whose activity is
directly modulated by Ca2+. Several point mutations in SCN1A and SCN2A genes,
which encode the voltage-activated Na+ channels NaV1.1 and NaV1.2, respectively,
are associated with childhood epilepsy and autism (Weiss et al., 2003). Mutations
in a C-terminal region of other voltage-gated Na+ channels reduce the amount
of channel inactivation, raising the possibility that excessive ion channel activity
leads to ASD (Glaaser et al., 2006; Kim et al., 2004). Another report suggests that
there is also an association between a Ca2+ -activated K+ channel (BKCa) and ASD
(Laumonnier et al., 2006). Disruption of the BKCa gene KCNMA1 led to haplo-
insufficiency and reduced BKCa activity. The reported decrease in BKCa channel
activity, together with the reduced inactivation of voltage-gated Ca2+ channels in
individuals with autism, suggests that some forms of autism are due to abnormally
sustained increases in intracellular Ca2+ levels.
Glutamate receptors are ligand-gated Ca2+ channels that are important in many
activity-dependent developmental processes. Polymorphisms in GRIN2A, which
encodes a NMDA receptor subunit, have also been associated with ASD (Barnby
et al., 2005). ASD-associated polymorphisms were reported in the gene encoding
subunit 2 of the kainate ionotropic glutamate receptor (GRIK2) and in metabotropic
glutamate receptor genes (Jamain et al., 2002; Serajee et al., 2003). The transcription
factor ARX is expressed in several cell types throughout the forebrain and is thought
to regulate the development of gamma-aminobutyric acid (GABA)-ergic neurons
in the basal ganglia and cortex (Friocourt et al., 2006). Mutations of ARX can lead
to epilepsy, movement disorders, cortical malformations, mental retardation, and in
some cases, autism (Stromme et al., 2002; Turner et al., 2002).
Wingless-type mouse memory tumor virus (MMTV) integration site family mem-
ber (Wnt) proteins form a family of highly conserved secreted signaling molecules
that regulate cell-to-cell interactions during embryogenesis. The role of WNT2 has
also been implicated in ASDs. Two families with mutations in WNT2 have been
identified, and a polymorphism in an upstream region of WNT2 has been associ-
ated with families defined by severe language abnormalities (Wassink et al., 2001).
Increases in Ca2+ concentrations have recently been shown to enhance the synthesis
and release of Wnt through activity of the Ca2+ -regulated transcription factor CREB
(Wayman et al., 2006). Considering the pivotal role of calcium in cellular signaling,
it is possible that calcium may have a wide role in the etiology of ASD.
10.9 ROLE OF MEMBRANE-ASSOCIATED PROTEINS

SUCH AS PTEN, NEUROLIGINS, SHANK3, REELIN,
AND SEROTONIN RECEPTORS IN AUTISM
Figure 10.2 represents the mechanism by which membrane-associated proteins par-
ticipate in the cellular signaling. Growth factor binds to its receptor and activates PI3K,
Growth factor Reelin

Ca2+ Ca2+
Voltage-gated
Growth factor receptor calcium channels
VLDLR ApoER2
Cellular
stress
PI
PI 4,5 - P2 PI3K PKB/Akt Dab-1
3, 4, 5 – P3
Ca2+
Pten mTOR
p53
GSK-3β
Bcl2
Tau
Neuronal migration Proliferation Apoptosis
FIGURE 10.2 (See color insert following page 200.) Participation of growth factor
receptors, reelin, voltage-gated calcium channels, p53, and Bcl2 in neuronal migration,
proliferation, and apoptosis. Growth factor binds to its receptor and activated phosphati-
dylinositol 3-kinase (PI3K), which converts phosphatidylinositol 4,5-bisphosphate (PI 4,
5-P2) to phopsphatidylinositol 3, 4, 5-trisphosphate (PI 3, 4, 5-P3). PI 3, 4, 5-P3 is an activa-
tor of protein kinase B (also known as Akt) that triggers proliferation pathways by activating
the mammalian target of rapamycin (mTOR). Akt also activates phosphatase and tensin
homolog on chromosome 10 (Pten), which dephosphorylates PI 3, 4, 5-P 3 to PI 4, 5-P2. Pten
activates glycogen synthase kinase-3β (GSK-3β), which phosphorylates tau protein involved
in neuronal migration. Reelin binds to very low-density lipoprotein receptor (VLDLR) and
apolipoprotein E receptor 2 (ApoER2), which activate Disabled-1 protein (Dab-1). Dab-1
activates the Akt involved in neuronal migration and proliferation. Cellular stress, on the
other hand, affects the activities of p53 and Bcl2, proteins involved in apoptosis.
which converts PI 4,5-P2 (PIP2) to PI 3,4,5-P3 (PIP3). PIP3 is an activator of PKB (also
known as Akt) that triggers proliferation of the cell. Akt also activates phosphatase
and tensin homolog on chromosome 10 (Pten), which dephosphorylates PIP3 to PIP2 and
may initiate apoptotic signaling. Pten activates glycogen synthase kinase-3β (GSK-3β),
which phosphorylates tau protein, a cytoskeletal protein. Phosphorylation of tau protein
is important for neuronal migration. Reelin binds to VLDL receptor (VLDLR) and
apolipoprotein E receptor 2 (apoER2), and it activates Disabled-1 protein (Dab1) that
also activates PKB. Calcium channels are important for controlled and fast requirement
of intracellular calcium, which is vital for the biological activities of numerous proteins.
Cellular stress initiates pathways leading to apoptosis by decreasing the levels of p53 (a
proapoptotic protein) and decreasing the levels of Bcl2 (an antiapoptotic protein).
In Figure 10.3, the role of neuroligins (Nlgn), neurexins (Nrxns), as well as SH3
and multiple ankyrin repeat domains 3 (SHANK3) proteins in cellular signaling are
Presynaptic vesicle
Neurexin
Ca2+ Ca2+
channel channel
Neuroligin
SHANK3/ProSAP
PSD-95 PSD-95
Foderin Cortactin
Actin filament
Postsynaptic vesicle
FIGURE 10.3 (See color insert following page 200.) Interaction of neurexin and
neuroligins. Neurexins participate in synaptic transmission by affecting calcium channels.
They also interact with neuroligins of postsynaptic vesicles. Neuroligin controls the actin
assembly by interacting with postsynaptic density protein 95 (PSD-95), which further inter-
acts with SHANK3. SHANK3/PSD-95 interact with foderin and cortactin, which bind
to actin filament.
described. Neurexins in presynaptic vesicles interact with neuroligins at the post-

synaptic vesicles, and neuroligins further interact with shank3 through postsynaptic
density protein 95 (PSD95). SHANK3 also interacts with actin filaments with the
help of foderin and cortactin.
Serotonin (5-hydroxytryptamine, i.e., 5HT) is a calming neurotransmitter that
is produced from the amino acid tryptophan in nerve cells. Its reuptake transporters
and receptors are shown in Figure 10.4. Imbalance of serotonin metabolism affects
mood and behavior. Selective serotonin reuptake inhibitors (SSRIs) such as fluox-
etine (Prozac) have been widely used for mood and behavioral problems. They block
serotonin from entering the neuron and keep it active for longer periods of time in
the synaptic gaps.
The potential role of these proteins in autism is discussed below.
Serotonin
Serotonin reuptake
transporter Synaptic gap
Serotonin receptors
FIGURE 10.4 (See color insert following page 200.) Serotonin and its receptors, and
serotonin reuptake transporter. Serotonin molecules from the presynaptic vesicles are released
into the synaptic gap, where they are internalized by serotonin receptors into the postsynaptic
vesicles. Serotonin molecules are also retaken from synaptic gaps to the presynaptic vesicle
by the serotonin reuptake transporters. An imbalance of the uptake and reuptake system in
serotonin causes brain dysfunctions.
10.9.1 POTENTIAL ROLE OF PTEN AND PI3K/AKT PATHWAY IN AUTISM

Pten is involved in mechanisms that control development of neuronal and synaptic
structures and, subsequently, synaptic function (Fraser et al., 2008). Pten mutations
have been reported in autistic individuals with macrocephaly (Butler et al., 2005;
Goffin et al., 2001; Zori et al., 1998). Several reports have suggested that germline
mutations in Pten can lead to multiple hamartoma syndromes and syndromes
clinically characterized by autism associated with macrocephaly (Buxbaum et al.,
2007; Greer and Wynshaw-Boris, 2006; Herman et al., 2007; Kwon et al., 2006). In
addition, mutations in Pten have been linked to other brain disorders such as macro-
cephaly, seizure, Lhermitte-Duclos disease, and mental retardation (Waite and Eng,
2002). Pten seems to have a wide range of implications in autism, although further
studies are warranted.
While Pten hydrolyzes the 3′ phosphate group of PIP3 (Maehama and Dixon,
1998), PI3K catalyzes the phosphorylation of the 3′ hydroxyl group of phosphoinosit-
ides, resulting in Akt activation. Upon activation, Akt phosphorylates proteins that
are involved in signal transduction such as TSC 2 gene product tuberin, GSK3β, and
the proapoptotic protein BAD (Luo, Manning, and Cantley, 2003). Alterations in
the PI3K/Akt pathway have been linked to many brain disorders. Decreased levels
of Akt are associated with schizophrenia (Emamian et al., 2004), and in individuals
with TSC mutations exhibiting central nervous system disorders, including autism
(Wiznitzer, 2004). The PI3K/Akt pathway has also been linked to fear conditioning
in rats (Lin et al., 2001). Bcl2, an antiapoptotic protein, is one of the downstream tar-
gets modulated by Akt. Activated Akt stimulates changes in Bcl2 expression, result-
ing in antiapoptotic effects in hippocampal neurons (Matsuzaki et al., 1999).
10.9.2 NEUROLIGINS AND AUTISM

Neuroligins are transmembrane proteins expressed on the postsynaptic cell that
bind to neurexins, which are presynaptic transmembrane proteins, and they appear
to play a crucial role in maintaining the functionality of the brain’s synaptic circuitry
(Figure 10.3). Synapses provide essential connections between nerve cells in the
brain that enable signals to be transmitted. Neural connections require precise orga-
nization of the presynaptic and postsynaptic neurons. Nrxns and Nlgns perform
important functions in synaptic transmission (Missler et al., 2003; Varoqueaux et al.,
2006) and differentiation of synaptic contacts (Graf et al., 2004; Scheiffele et al., 2000),
and both Nrxns and Nlgns have been identified as candidate genes for autism
(Jamain et al., 2003; Szatmari et al., 2007). The family of human neuroligins con-
sists of five genes (NLGN1 at chromosome 3q26, NLGN2 at 17p13, NLGN3 at
Xq13, NLGN4 at Xp22, and NLGN4Y at Yq11). Recent studies reported poly-
morphisms in neuroligin-3 (NLGN3) and neuroligin-4 (NLGN4) genes in ASD
(Talebizadeh et al., 2006; Ylisaukko-oja et al., 2005).
The role of genetic variants of NLGN has been controversial. One missense
variant, p.I679V in NLGN4Y gene, was identified in an individual with autism, as
well as his father, who had learning disabilities (Yan et al., 2008). Talebizadeh et al.
(2006) observed that splice variants of NLGN3 and NLGN4 genes might lead to
ASD. Yamakawa et al. (2007) reported that syntrophin-gamma2 (SNTG2) binds
to NLGN 4X and NLGN 4Y, and that the binding is affected by the autism-related
mutations. On the other hand, Wermter et al. (2008) reported that there is no evi-
dence for the involvement of NLGN3 and NLGN4X genetic variants with high-
functioning autistic individuals. Further research is warranted to clarify the role of
neuroligins in autism.
10.9.3 SHANK3 AND AUTISM

The SHANK3 gene is located on chromosome 22. SHANK3 (also known as ProSAP2)
regulates the structural organization of dendritic spines and is a binding partner of
neuroligins. Jeffries et al. (2005) reported that SHANK3 is a candidate gene for
autism and abnormal brain development. Durand et al. (2007) reported that a muta-
tion of a single copy of SHANK3 on chromosome 22q13 could result in language and/
or social communication disorders. A recent report suggests that SHANK1 (SH3 and
multiple ankyrin repeat domains 1) mutant mice exhibit altered postsynaptic density
(PSD) protein composition; reduced size of dendritic spines; smaller, and thinner
PSDs; and weaker basal synaptic transmission (Hung et al., 2008). Behaviorally,
mutant mice had increased anxiety-related behavior and impaired contextual fear
memory, suggesting the importance of SHANK1 for synapse structure and function
in vivo, and a role for SHANK1 in specific cognitive processes, a feature that may be
relevant to ASD in humans. Considering the involvement of SHANK3 in dendritic
spine formation and its binding to Nlgn, it is possible that SHANK3 may play an
important role in the etiology of autism.
10.9.4 REELIN AND AUTISM

Several genome-wide screens have indicated the presence of an autism susceptibility
locus within the distal long arm of chromosome 7 (7q). Mapping at 7q22 within this
region showed a candidate gene reelin (RELN). RELN encodes a signaling pro-
tein that plays a pivotal role in the migration of several neuronal cell types and
in the development of neural connections. Reelin modulates synaptic plasticity by
enhancing the induction and maintenance of long-term potentiation (D’Arcangelo,
2005; Weeber et al., 2002). It also stimulates dendrite (Niu et al., 2004) and dendritic
spine (Niu, Yabut, and D’Arcangelo, 2008) development and regulates the continuing
migration of neuroblasts generated in adult neurogenesis sites such as subventricular
and subgranular zones.
Several studies have suggested impairments in reelin signaling in autism. Fatemi
et al. (2001) reported that dysregulation of reelin and Bcl-2 may be responsible for
some of the brain structural changes and behavioral abnormalities observed in
autism. Fatemi, Stary, and Egan (2002) also reported reduced levels of reelin in the
blood of autistic subjects. Serajee, Zhong, and Huq (2006) studied 34 single nucle-
otide polymorphisms (SNPs) in the RELN gene, with an average spacing between
the SNPs of 15 kb, for evidence of an association with autism, and observed signifi-
cant differences in the transmission of the alleles of exon 22 and intron 59 SNP to
autistic subjects, suggesting a role for the RELN gene in susceptibility to autism.
In addition, Fatemi et al. (2005) reported reductions in reelin protein and mRNA
as well as Disabled 1 (Dab 1, a gene encoding a key regulator of reelin signaling)
mRNA and elevations in reelin receptor, i.e., VLDLR mRNA. Family-based associ-
ation analyses showed a significant association for the 5′-UTR repeat (PDT p-value =
0.002), suggesting the potential of RELN as an important contributor to genetic risk
in autism (Skaar et al., 2005). While most of the studies suggest a role of reelin in the
etiology of autism, a few studies did not find any link between reelin and the etiology
of autism. Krebs et al. (2002) suggested that GGC polymorphism of the RELN gene
is unlikely to be a major susceptibility factor in autism and/or genetic heterogeneity.
In another study, case–control and affected sib–pair findings did not support role
for RELN in susceptibility to ASD, but the more powerful family-based association
study demonstrated that RELN alleles with larger numbers of CGG repeats may play
a role in the etiology of some cases of ASD, especially in children without delayed
phrase speech (Zhang et al., 2002). Further research is warranted to assess the pre-
cise role of RELN in autism.
10.9.5 SEROTOGENIC RECEPTORS AND AUTISM

In the central nervous system, serotonin, i.e., 5HT plays an important role as a
neurotransmitter in the modulation of anger, aggression, mood, sleep, appetite,
and metabolism. Serotonin has broad activities in the brain, and genetic varia-
tions in serotonin receptors and the serotonin transporter, which facilitates
reuptake of serotonin into presynapses, have been implicated in neurological dis-
eases. Increased platelet serotonin levels have been consistently found in a cohort
of autistic subjects. Suggested mechanisms for hyperserotonemia in autism are
increased synthesis of serotonin by tryptophan hydroxylase, increased uptake
into platelets through 5HT transporter (5HTt), diminished release from platelets
through 5HT2A receptor (5HT2Ar), and decreased metabolism by monoamine
oxidase (MAOA) (Hranilovic et al., 2008). Recent studies suggest that sero-
tonin transporter (SERT)–binding capacity measured by single-photon emission
computed tomography (SPECT) is disturbed in autism (Makkonen et al., 2008;
McDougle, 2008).
The serotogenic system is also affected in prenatal viral infection, which also
has an association with neurodevelopmental disorders such as schizophrenia and
autism. Winter et al. (2008) reported disruption of serotonergic system after prenatal
viral infection that may be attributed to potential modeling disruptions occurring
in patients with schizophrenia and autism. Hranilovic et al. (2007) have reported a
significant negative relationship between platelets serotonin levels and speech devel-
opment, indicating the relationship between the peripheral 5HT concentrations and
verbal abilities in autistic subjects. However, no association of polymorphic variants
of the serotonin transporter (5-HTT) gene-linked polymorphic region (5-HTTLPR)
with ASD was observed (Zhong et al., 1999). Further research is warranted to con-
firm the role of serotonin in the etiology of autism.
10.9.6 EVIDENCE OF ALTERED LEVELS OF Bcl2 AND p53

(MARKERS OF APOPTOSIS) IN THE BRAINS OF AUTISM SUBJECTS
Bcl2 derives its name from B-cell lymphoma 2. Bcl2 is membrane-bound anti-
apoptotic protein with a neuroprotective role in the central nervous system (Lin
et al., 1996). The p53 tumor suppressor protein (tumor protein 53) plays a cen-
tral role in controlling cell growth and apoptotic cell death in response to cellular
stress and DNA damage (Araki et al., 2000). In autism, many areas of the brain
show abnormalities in autism, including loss of pyramidal neurons and granule
cells in the hippocampus as well as significant loss and atrophy of Purkinje cells
in the cerebellum (Bauman and Kemper, 2005; Fatemi and Halt, 2001; Ritvo et al.,
1986). Emerging evidence suggests that apoptotic mechanisms may be responsi-
ble for causing neuropsychiatric disorders such as schizophrenia (Jarskog et al.,
2000) and autism (Araghi-Niknam and Fatemi, 2003; Fatemi and Halt, 2001; Fatemi
et al., 2001). In particular, significant reduction in the levels of Bcl2 (a membrane-
bound antiapoptotic protein) and a concomitant increase in p53 levels have been
reported in cerebellum (Araghi-Niknam and Fatemi, 2003; Fatemi and Halt, 2001;
Fatemi et al., 2001), parietal cortex (Fatemi and Halt, 2001; Fatemi et al., 2001), and
superior frontal cortex of autism subjects (Araghi-Niknam and Fatemi, 2003). It is
not known whether altered levels of Bcl2 and p53 in autism are due to previous
prenatal or postnatal environmental insults or alternatively because of ongoing
neurodegeneration. The above studies indicate that apoptosis may be involved in

the pathogenesis of autism.
10.10 OXIDATIVE STRESS, INFLAMMATION,

AND CELL SIGNALING
During oxidative stress, free radicals, i.e., reactive oxygen species (ROS) and reac-
tive nitrogen species (RNS), react with proteins, lipids, and nucleic acids and affect
their functions, leading to cell death. ROS plays crucial role in both normal and
pathological cell signaling processes such as calcium homeostasis, kinase activ-
ity, and gene regulation (Esposito et al., 2004). Oxidative stress controls the activi-
ties of receptor and nonreceptor types of protein tyrosine kinases (Hardwick and
Sefton, 1995; Kharbanda et al., 1995; Pu et al., 1996), ras (Lander et al., 1996),
protein tyrosine phosphatases (Garcia-Morales et al., 1990; George and Parker,
1990), protein kinase C (Gopalakrishna, Chen, and Gundimeda, 1993), and tran-
scription factors such as NF-AT (Abate et al., 1990) and NF-kB (Hayashi, Ueno, and
Okamoto, 1993; Staal et al., 1990). Among these factors, transcriptional factors are
most important because of their role in different phases of brain development and
wiring. Transcriptional factors are central in cross-talk among cells and signaling
molecules. Oxidative stress leads to an increase in Fos expression, an event linked to
apoptosis and reduced synaptic plasticity. On the other hand, oxidative stress leads
to decreased levels of Jun D, a protein that upregulates antioxidant response element
(ARE)–mediated expression and induces the detoxifying enzymes in response to
oxidative insults. ROS can also increase the expression of p53 and Fas (Mao et al.,
2006). p53 and Fas can, in turn, lead to increased ROS production, indicating a feed-
forward mechanism to ensure the death of cells committed to die. Therefore, oxida-
tive damage of membrane lipids/proteins may result in abnormal cellular signaling.
10.10.1 OXIDATIVE STRESS IN AUTISM

Oxidative stress–induced production of lipid peroxides and their by-products is
known to result in the loss of membrane functions and integrity (Jain, 1984). Lipid
peroxidation is a chain reaction between polyunsaturated fatty acids and ROS,
producing lipid peroxides and hydrocarbon polymers that are both highly toxic to the
cell (Horton and Fairhurst, 1987; Jain, 1984; Tappel, 1973). In a recent review article,
we have gathered evidence of oxidative stress in autism (Chauhan and Chauhan,
2006). We previously reported that lipid peroxidation is increased in the plasma of
children with autism as compared to their developmentally normal siblings (Chauhan
et al., 2004a). Malonyldialdehyde (MDA) is an end product of peroxidation of poly-
unsaturated fatty acids and related esters, and therefore, is used as a marker of lipid
peroxidation (Jain, 1984). The plasma MDA contents measured by reaction with
thiobarbituric acid (TBA) were higher in 87% of autism subjects (Chauhan et al.,
2004a). Recently, we also observed increased lipid peroxidation in cerebellum and
temporal cortex of brain in autism (Chauhan et al., 2009). MDA levels were signifi-
cantly increased by 124% in the cerebellum and by 256% in the temporal cortex in
autism as compared to control subjects.
Other studies also indicated increased levels of other lipid peroxidation and
protein oxidation markers in autism, thus confirming increased oxidative stress in
autism. Zoroglu et al. (2004) reported increased TBA-reactive substances in the
erythrocytes of autism subjects as compared to normal controls. Ming et al. (2005)
reported increased excretion of 8-isoprostane-F2alpha in the urine of children with
autism. Isoprostanes are produced from the free radical oxidation of arachidonic
acid through nonenzymatic oxidation of cell membrane lipids. Evans et al. (2008)
reported increased levels of lipid-derived oxidative protein modification, i.e., car-
boxyethyl pyrrole and iso[4]levuglandin E2–protein adducts, in the autistic brain,
primarily in the white matter. Sajdel-Sulkowska et al. (2008) reported increased
levels of 3-nitrotyrosine (a specific marker for oxidative damage of protein) in the
cerebella of autistic subjects. Lipofuscin, a matrix of oxidized lipid and cross-linked
protein, forms as a result of oxidative injury in the tissues. Density of lipofuscin was
observed to be greater in cortical brain areas concerned with social behavior and
communication in autism (Lopez-Hurtado and Prieto, 2008).
Several studies have suggested alterations in the enzymes that play a vital role
in the defense mechanism against damage by ROS in autism. In autism, decreased
activity of glutathione peroxidase (GPx) in plasma (Yorbik et al., 2002) and in eryth-
rocytes (Pasca et al., 2006; Yorbik et al., 2002), reduced levels of total glutathione
and lower redox ratio of reduced glutathione (GSH) to oxidized glutathione (GSSG)
in plasma (James et al., 2004), decreased catalase activity in red blood cells (Zoroglu
et al., 2004), and decreased superoxide dismutase (SOD) activity in plasma, all point
toward oxidative stress. On the contrary, Sogut et al. (2003) reported unchanged
plasma SOD activity and increased GPx activity in autism.
Ceruloplasmin (a copper-transporting protein) and transferrin (an iron-transporting
protein) are major antioxidant proteins that are synthesized in several tissues includ-
ing brain (Arnaud, Gianazza, and Miribel, 1988; Loeffler et al., 1995). Ceruloplasmin
inhibits the peroxidation of membrane lipids catalyzed by metal ions such as Fe and
Cu (Gutteridge, Richmond, and Halliwell, 1979). It also acts as ferroxidase and SOD,
and it protects polyunsaturated fatty acids in red blood cell membranes from active
oxygen radicals (Arnaud, Gianazza, and Miribel, 1988). We reported reduced levels
of serum ceruloplasmin and transferrin in children with autism as compared to their
unaffected siblings (Chauhan et al., 2004a). The transferrin levels were observed to
be lower in 16 of 19 (84%) children with autism as compared to their unaffected sib-
lings, while ceruloplasmin levels were lower in 13 of 19 (68%) children with autism
as compared to their developmentally normal siblings (Chauhan et al., 2004a). It
was of particular interest to observe that the levels of ceruloplasmin and transferrin
were reduced more markedly in children with autism who had lost acquired lan-
guage skills (Chauhan et al., 2004a). Children who had not lost language skills had
levels similar to that seen in the typically developing siblings. These results suggest
that there is an altered regulation of transferrin and ceruloplasmin in children with
regressive autism. Such alterations may lead to abnormal iron and copper metabo-
lism in autism, which may have a pathological role in autism. Some preliminary
studies have suggested altered serum Cu/Zn ratios in autism (McGinnis, 2004).
We recently reported that there might be a link between decreased levels of PE
in the membrane and copper-mediated oxidation of lipids (Chauhan, Sheikh, and
Chauhan, 2008). We studied the effect of copper on the oxidation of liposomes
composed of mouse brain lipids, and also on the lymphoblasts from autism and
control subjects. Among the various metal cations (copper, iron, calcium, cadmium,
and zinc), only copper was found to oxidize PE, while having no effect on other
phospholipids. The action of copper on PE oxidation was time-dependent. Copper
oxidized PE in a concentration-dependent manner. No difference was observed
between copper-mediated oxidation of diacyl-PE and alkenyl-PE (plasmalogen).
Copper-mediated oxidation of PE in autistic lymphoblasts was higher than in con-
trol lymphoblasts. Taken together, our studies suggest that ceruloplasmin and cop-
per may be involved in the oxidative stress, and in reducing the levels of membrane
PE in autism.
10.10.2 CYTOKINES, INFLAMMATION, AND AUTISM

A number of studies have implicated oxidative stress as a major upstream com-
ponent in the signaling cascade involved in activation of redox-sensitive tran-
scription factors and proinflammatory gene expression, leading to inflammatory
response (Parola et al., 1999; Uchida et al., 1999) Cytokines are glycoproteins that
are involved in cell signaling and are used extensively in cellular communication.
Cytokines are critical to the development and functioning of both the innate and
adaptive immune response. They are often secreted by immune cells that have
encountered a pathogen, thereby activating and recruiting further immune cells
to increase the system’s response to the pathogen. Cytokines are also involved
in several developmental processes during embryogenesis. Several investiga-
tors have reported peripheral immune abnormalities in autism (Ashwood and
Wakefield, 2006; Jyonouchi et al., 2005; Korvatska et al., 2002; Pardo, Vargas,
and Zimmerman, 2005; Singh et al., 1991; Vojdani et al., 2008; Warren et al.,
1986; Yonk et al., 1990; Zimmerman et al., 2005). It is also known that cytok-
ines and chemokines play roles as mediators of inflammatory reactions in the cen-
tral nervous system and in the processes of neuronal–neuroglia interactions that
modulate the neuroimmune system. In a recent study, we examined the activation
levels of cytokines including the proinflammatory cytokines (IL-6, IL-1β, TNF-α,
and GM-CSF), Th1 cytokines (IL-2 and IFN-γ), Th2 cytokines (IL-4, IL-5, and
IL-10), and the chemokine IL-8 in the brains of ASD patients and compared with
age- and gender-matched normal subjects (Li et al., 2009). Our results showed that
TNF-α, IL-6, GM-CSF, IFN-γ, and IL-8 were significantly increased in the brains
of ASD patients compared with the control subjects. However, the Th2 cytokines
(Il-4, IL-5, and IL-10) showed no significant difference. Th1/Th2 ratio was also
significantly elevated in ASD patients. In addition, we found that regulatory IL-10
was not increased in compensatory response to the increased Th1 cytokines in
ASD patients (Li et al., 2009). Other groups have also reported that there are
abnormalities in immune responses in the central nervous system of individuals
with autism (Pardo, Vargas, and Zimmerman, 2005b; Vargas et al., 2005; Vojdani
et al., 2008), suggesting that neuroinflammation may have implications in the
pathogenesis of autism.
10.11 LIPID RAFTS AND DISORDERS OF THE BRAIN

A new and fairly rapid development in the field of lipid rafts is changing the gen-
eral view on how membrane complexity takes place. Lipid rafts are microdomains
within the membrane that are defined by their cholesterol and sphingomyelin con-
tent. Although research on lipid raft and its association with ASD is lacking, this
chapter would not be complete without postulating a role of lipid raft in neurode-
velopmental disorders. A recent review describes the role of the raft microdomain
in neurodevelopment (Allen, Halverson-Tamboli, and Rasenick, 2007). Drugs for
treating mood disorders (unipolar depression and bipolar disorder) and schizophre-
nia tend to accumulate in lipid raft (Eisensamer et al., 2005), and affect the signal-
ing of raft-associated proteins. Although chronic antidepressant treatment does not
alter membrane cholesterol content, treatment with chemically distinct antidepres-
sants results in the movement of Gαs (but not other G proteins) out of lipid rafts
and into closer association with adenyl cyclase (Donati and Rasenick, 2005; Menkes
et al., 1983; Toki, Donati, and Rasenick, 1999). This may contribute to the increased
cAMP and synaptic changes that are observed after chronic antidepressant treatment
(Donati and Rasenick, 2003).
Lipid raft has been reported to be involved in several neurodegenerative diseases.
Lipid rafts may function as platforms for the production of neurotoxic proteins, such
as amyloid beta-protein in Alzheimer’s disease (Ehehalt et al., 2003) and prion protein
in transmissible spongiform encephalopathies (Taylor and Hooper, 2006), which may
then be poised to modulate raft-associated signaling cascade. There is enough infor-
mation in the literature on the role of lipid rafts in neurotransmitter signaling. Allen,
Halverson-Tamboli, and Rasenick (2007) have proposed that lipid rafts of the mem-
brane are vehicles for neurotransmitter signaling, through a clustering of receptors
and components of receptor-activated signaling cascades. Ionotropic neurotransmitter
receptors (ligand-gated ion channels) are localized in lipid rafts (Allen, Halverson-
Tamboli, and Rasenick, 2007). The binding of receptors and neurotransmitters takes
place in lipid rafts (Burger, Gimpl, and Fahrenholz, 2000; Sooksawate and Simmonds,
2001). Lipid rafts also contribute in the trafficking of ionotropic receptors (Hering, Lin,
and Sheng, 2003; Pediconi et al., 2004). G-protein-coupled neurotransmitter receptors
(GPCR) signaling complexes are present in lipid rafts (Neubig, 1994), and lipid rafts
are involved in GPCR trafficking. In addition, lipid rafts have been shown to regulate
the functions of G-proteins, thereby affecting the function of phospholipase and cal-
cium signaling, a process of pathway for neurotansmitter-mediated cellular signaling
(reviewed in Allen, Halverson-Tamboli, and Rasenick, 2007). Considering the impor-
tant role that is played by raft microdomains in signal transduction, it is postulated
that these lipid rafts may also be involved in abnormal signaling in autism.
10.12 CONCLUSIONS
In this chapter, we have gathered evidence on how membrane lipids and proteins
may be involved in the development of autism. Genetic and environmental factors
are the most important factors in the etiology of autism. If we look into the down
stream target of these factors, i.e., membrane and proteins function, it becomes
apparent that membrane is the intermediate link between genetic/environmental
factors and the functions of proteins. Other signaling molecules that are directly or
indirectly connected to lipids such as reelin, SHANK3, Wnt, neuroligins, Bcl2 and
Pten can also contribute significantly to the etiology of autism.
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11 Mitochondrial
Component
of Calcium Signaling
Abnormality in Autism
J. Jay Gargus1,2,*
1Department of Physiology and Biophysics and
2Division of Human Genetics, Department of
Pediatrics, School of Medicine, University of
California, Irvine, Irvine, CA 92697, USA
CONTENTS
11.1 Introduction .................................................................................................208
11.2 Mitochondrial DNA Mutations in Autism ..................................................209
11.3 Functional Mitochondrial Defects in Autism ............................................. 210
11.4 Mitochondria are Central Participants in Calcium Homeostasis................ 211
11.5 Timothy Syndrome Causes Autism via a Defect in Calcium
Channel Function ........................................................................................ 212
11.6 Other Defects in the Calcium Signaling Pathway in Autism ...................... 214
11.7 Calcium Signaling Dysfunction Causes Defects in Neurosecretion ........... 215
11.8 Family of Autism-Related Diseases with Defective Neuronal
Calcium Signaling ....................................................................................... 216
11.9 Pharmacogenetic Calcium Signaling Abnormalities .................................. 218
11.10 Conclusion ................................................................................................... 218
Acknowledgment ................................................................................................... 219
References .............................................................................................................. 219
There are several suggestions in the literature that oxidative stress and mitochondrial
function are abnormal in autism. However, these defects produce such global
perturbations of cellular homeostasis that it is difficult to discern the critical
pathway leading to the disease phenotype. Resolving this pathway is important
207
since it provides the most promising target for novel drug development. Since
most reactive oxygen species (ROS) arise as by-products of electron transport
and oxidative phosphorylation, primary mitochondrial defects are a likely source
of ROS. Certainly, genetic defects in mitochondrial function do impose a mas-
sive oxidative stress. In the same fashion in which there have been many hints of
abnormal ROS levels in autism, many disparate clues have recently come to sug-
gest that abnormal neuronal calcium signaling also plays a role in autism. This is
a process recently recognized to be under mitochondrial regulation and capable
of disrupting neuronal synaptic function and hence behavior. Therefore, a body
of evidence now suggests that calcium signaling abnormalities are a fundamental
pathway perturbed in autism, with many lesions arising from primary defects in
mitochondrial function, but with other lesions primarily perturbing other com-
ponents of the calcium signaling pathway and only secondarily impairing mito-
chondrial function.
11.1 INTRODUCTION
The autistic spectrum disorders (ASD) all share the same characteristic core
deficits in social interaction, communication, and behavior; however, they remain
a group of developmental disorders that are only behaviorally, not yet pathophysi-
ologically, defined. The high heritability of ASD is assurance that genes and the
biochemical pathways they subserve underlie the phenotype, but to date these
pathways remain elusive. There are several suggestions in the literature that oxi-
dative stress and mitochondrial dysfunction are abnormal in autism, likely in
turn impacting a variety of downstream processes (Chauhan and Chauhan, 2006;
Gargus and Imtiaz, 2008; James et al., 2006). But since these impacts on cellular
homeostasis are so broad, it remains difficult to discern those pathways most
directly connected to the critical phenotypes of autism, and therefore those that
would appear the most promising targets for novel drug development. Oxidative
stress produces oxidative damage in a cell, tissue, or organ caused by the reac-
tive oxygen species (ROS), such as free radicals and peroxide, interacting with
an array of critical endogenous biomolecules. Is this ROS damage etiologically
important in the neurological phenotype, or does it merely serve the role of a use-
ful correlative biomarker? Since most ROS arise as by-products from essential
metabolic reactions, with electron transport and oxidative phosphorylation of the
mitochondria accounting for the vast majority, primary mitochondrial defects are
a likely source. Certainly genetic defects in mitochondrial function do impose
a huge endogenous oxidative stress (Esposito et al., 1999; Subramaniam et al.,
2008). But while they cause an increase in ROS, they also impose a dramatic
deficiency in the cell’s essential ATP energy supply and in the other essential cel-
lular processes subserved by the mitochondria, such as maintenance of the mem-
brane potential and calcium homeostasis. Just as there have been many hints of
abnormal ROS levels in autism, many genetic and biophysical clues have recently
come to suggest that abnormal neuronal calcium signaling may also play a role
in autism (Gargus, 2009). While calcium homeostasis and signaling are intensely
studied biophysical phenomena that are well recognized to play a central role
Mitochondrial Component of Calcium Signaling Abnormality in Autism 209
in many aspects of excitable cell biology and synaptic physiology (Flavell and
Greenberg, 2008), their role in disease processes is only beginning to emerge
(Bezprozvanny and Gargus, 2008). Likewise the coordinating role mitochon-
dria play in these processes has only recently been recognized (Szabadkai and
Duchen, 2008). In this chapter, evidence will be presented that calcium signaling
abnormalities are a critical and fundamental pathway perturbed in autism, with
many lesions arising from primary defects in mitochondrial function, but other
lesions perturbing this calcium signaling being independent of a direct impact on
the mitochondria.
11.2 MITOCHONDRIAL DNA MUTATIONS IN AUTISM

Arguably the most specific way to show the primacy of a mitochondrial defect in
disease is to show a heritable functional mutation in the small segment of the genome
carried on the mitochondrial chromosome, the mtDNA, since its encoded proteins
only contribute to the mitochondrial membranes. But only a few isolated case reports
of mitochondrial DNA mutations in autism have been reported. One patient had the
G8363A mutation in the mtDNA tRNA Lys gene documented in blood and skeletal
muscle (Graf et al., 2000), and five patients had large mtDNA deletions (Fillano
et al., 2002). But these were not typical autism patients as the predominant component
of their more severe phenotype was seizures. However, another study has shown
that it is very difficult to identify mtDNA mutations in standard, nonsyndromic
forms of ASD (Pons et al., 2004). In this study, the investigators explicitly docu-
mented the A3243G mtDNA mutation in families with autism. This mutation was
originally recognized to cause the MELAS phenotype, an acronym for “mitochon-
drial encephalomyopathy, lactic acidosis and strokes.” Pons et al. (2004) showed
that the mothers of four patients with autism had detectable levels of the mutant
mtDNA. This mutation was shown to be heteroplasmic, meaning that there were
two populations of mtDNA, the wild-type (WT) normal copy, and the mutant copy.
Since all cells have hundreds of mitochondria and each mitochondrion has mul-
tiple mtDNA molecules, mixed heteroplasmic mitochondrial populations can sup-
port cell survival even when the mutation causes a very severe functional defect.
However, at low levels of heteroplasmy, it becomes extremely difficult to molecu-
larly detect the mutation against a much higher abundance of the WT molecule.
Therefore, while these investigators could detect the mother’s mtDNA mutation
in two affected sons as well as in their mothers, no mutation could be detected,
even using five different tissues in the analysis, in the two remaining children with
autism whose mothers carried a heteroplasmic mutation. This may well reflect cur-
rent technical limitations for detecting low levels of heteroplasmy. This problem
is being addressed by emerging techniques that have already broadened the spec-
trum of recognizable mitochondrial disease (Bannwarth et al., 2008). By providing
a more complete picture of phenotypes produced by low levels of heteroplasmic
mutations that underlie modest defects in mitochondrial function, such techniques
hold the potential to assess the full impact of mtDNA mutations in autism. At this
point the data suggest, however, that we likely have only a significant underesti-
mate of this impact.
11.3 FUNCTIONAL MITOCHONDRIAL DEFECTS IN AUTISM

The mtDNA encodes only 13 of the thousands of proteins required to produce
functional mitochondria. The remainder of the mitochondrial proteins are encoded
by genes on the nuclear chromosomes (nDNA). The role of modest mitochondrial
defects in autism has been shown most commonly by demonstration of altered mito-
chondrial function in subsets of children with autism and a syndrome of energy
deficiency, some associated with specific nDNA lesions (Gargus and Imtiaz, 2008).
In a large retrospective study of cases of typical nonsyndromic autism, a biochem-
ical profile of mitochondrial energy deficiency was observed for the cohort with
autism (Filipek et al., 2004). The signature observed in this group of typical patients
with autism included a reduced plasma total and free carnitine (P < 0.001), moderate
hyperammonemia (P < 0.001), and chronic lactic acidosis. The lactic acidosis was
associated with a plasma lactate that itself was only modestly abnormal, but there
was an elevated plasma lactate/pyruvate ratio that was predominated by the abnor-
mal pyruvate values. This lactate/pyruvate ratio was confirmed to be a chronic lactic
acidosis since it was accompanied by an elevated plasma alanine level (P < 0.001).
Importantly, individual patients were not sufficiently abnormal to be resolved with
statistical significance, but rather a large cohort was required to provide the analyti-
cal power to discern these group differences (Filipek et al., 2004). Subsequently, a
comparably sized Portuguese population study revealed remarkably similar findings:
most patients with autism had an increased lactate/pyruvate ratio, but only 20% had
a statistically significant elevated lactate, even fewer (<7%) had a functional mito-
chondrial defect ascertained at muscle biopsy, and none had an identifiable mtDNA
mutation (Oliveira et al., 2005). Further epidemiological evaluation of the Portuguese
study revealed functional mitochondrial disorders to be the second most common
autism-associated disorder, accounting for 5% of cases (Oliveira et al., 2007).
Rare chromosomal rearrangements have the well established ability to link com-
plex phenotypes to specific chromosomal loci, and the most common chromosomal
rearrangement seen in autism is an inverted duplication of chromosome 15q11-q13
(Cook et al., 1997). The extra chromosomal material is generally maternal, and
carried as an extra marker chromosome, implicating imprinted genes at this locus
(Schanen, 2006), but the specific dosage-sensitive gene(s) causing the syndrome
remains to be discovered. Filipek and coworkers reported two such children with
autism and a maternal 15q-inverted duplication marker chromosome as well as a
panel of biochemical signs of mild mitochondrial dysfunction, including elevated
serum lactate, pyruvate, ammonia, and alanine, an elevated urinary lactic acid, a
secondary carnitine deficiency, and a biopsy-proven partial deficiency of mitochon-
drial respiratory complex III (Filipek et al., 2003). A similar pattern of biochemical
signs of modest mitochondrial energy deficiency have been observed in the syndro-
mic form of ASD caused by mutations in MECP2 that produce Rett syndrome. Rett
syndrome patients were shown to have modest lactic acidosis and hyperammonemia
(Eeg-Olofsson et al., 1990) and the mouse model shown to have functional defects in
mitochondrial respiratory complex III (Kriaucionis et al., 2006). Likewise, another
important syndromic form of ASD is tuberous sclerosis, caused by dominant muta-
tions in either TSC1 or TSC2. The protein products of these two genes heteromulti-
merize to regulate TOR, a key calcium-sensitive regulator of mitochondrial function
whose name arises from the fact that it is the target of rapamycin (Chen et al., 2008;
Liu and Butow, 2006). While magnetic resonance spectroscopy (MRS) has been
shown to detect elevated central nervous system (CNS) lactate in this disease (Yapici
et al., 2007), peripheral biochemical markers of mitochondrial energy deficiency
have yet to be reported. In addition, linkage and association studies have revealed
autism susceptibility loci at 1p, 2q, 3q, 5p, 7q, 9q, 11p, 15q, 16p, 17q, and Xq; how-
ever, none makes a major contribution to the common disease risk, each accounting
for <1%, and while many hypothetical candidates have been proposed, no specific
susceptibility genes in the loci have been proven (Abrahams and Geschwind, 2008).
Recently, linkage and association studies in nonsyndromic autism have focused
attention on mitochondrial dysfunction caused by variation in SLC25A12. This gene
on 2q24 encodes the brain-specific isoform of the mitochondrial calcium-regulated
aspartate/glutamate carrier. In a large study, nine candidate genes in this region
were scanned for autism-associated single nucleotide polymorphisms (SNPs) and
two SNPs, located in introns 3 and 16 of SLC25A12, were found associated with the
disease (Ramoz et al., 2004). The same risk haplotype at these two SNPs was then
confirmed to be linked and associated with the disease in 197 families (Ramoz et al.,
2004). Subsequent studies confirmed that autism was associated with other SNPs
within the locus, although none appeared to be functional (Segurado et al., 2005).
More recently, a study of postmortem brain tissue from six patients with autism and
matched controls showed significantly increased transport activity by the product of
the SLC25A12 gene in autism. However, no mutations or polymorphisms were found
associated with the disease (Palmieri et al., 2008). Furthermore, all of the excess
enzyme activity found in brain samples from patients with autism was calcium-
dependent and was found to be associated with elevated cytosolic calcium levels in
tissue from subjects with autism (Palmieri et al., 2008). They found that controlling
for the calcium levels, transport activity was identical in isolated mitochondria from
patients and controls. They therefore concluded that the critical link to this altered
mitochondrial metabolism observed in the brains of patients with autism was in fact
caused by altered calcium homeostasis, although it was never directly studied.
11.4 MITOCHONDRIA ARE CENTRAL PARTICIPANTS

IN CALCIUM HOMEOSTASIS
As discussed above, calcium plays a central role in mitochondrial function.
Mitochondria produce a transmembrane proton electrochemical potential (ΔμH+),
via electron transport along the inner membrane’s chain of respiratory complexes.
Therefore, ΔμH+ is the primary energy currency of the mitochondria. It is used to
either synthesize ATP or to carry out the coupled transport of other chemical species
including calcium. The accumulation of calcium is an important mitochondrial
function, and quantitatively calcium transported into the mitochondria represents
the major cytosolic reserve. Therefore, a major demand placed on mitochondrial
calcium transport collapses ΔμH+ and has the same net effects on the mitochondrial
currency available for ATP synthesis as a primary defect in mitochondrial electron
transport or oxidative phosphorylation.
Until recently, the endoplasmic reticulum (ER) had been thought to contain
the only dynamic pool of ionized calcium that participates in a host of cellular
signaling functions. This intracellular store of calcium could be rapidly released

via intrinsic ER channels, the inositol 1,4,5-triphosphate receptors (IP3R) and the
ryanodine receptors (RyR). Once released, this calcium would activate a host of
kinases, ion channels, and transcription factors, and then be resequestered via the
ER’s calcium ATPase (SERCA). The importance of this pathway in health and
disease is best shown in the pharmacogenetic malignant hyperthermia syndrome
(MHS) (Gargus, 2008a, 2009), and there are suggestions of a variant of this dis-
order in a rare endophenotype of autism (Gargus and Imtiaz, 2008). In genetically
susceptible individuals with MHS, the cytosolic calcium pool is hypersensitive.
Therefore, general anesthesia triggers a massive release of cytosolic calcium. This
in turn produces the hallmarks of the syndrome—muscle rigidity directly from
calcium activation of the contractile proteins through a process referred to as
excitation–contraction coupling, and then lactic acidosis, hypercapnea, hypox-
emia, and hyperthermia, as the mitochondria struggle to resequester the calcium
load. Three genes are known to carry MHS susceptibility alleles, but additional
MHS loci remain to be defi ned. The known MHS genes encode: the sarcoplas-
mic reticulum ryanodine receptor calcium release channel (RYR1) causing MHS
type 1, the sarcolemmal sodium channel (SCN4A) causing MHS type 2, and the
sarcolemmal calcium channel (CACNA1S) causing MHS type 5. In neurons,
these same channels and paralogous channels participate in the related process of
excitation–secretion coupling that drives neurotransmission, a process in which
defects are implicated in autism (see below).
While mitochondria have long been known to sequester the vast majority of intra-
cellular calcium, only recently, the dynamic nature of this mitochondrial calcium
pool has been recognized (Spat et al., 2008; Szabadkai and Duchen, 2008). The
mitochondria are now known to actively communicate with the ER calcium signal-
ing apparatus in the generation of rapid calcium signals, forming a bidirectional
link between energy metabolism and cellular signals transmitted via changes in the
cytosolic-free calcium ion concentration (Danial et al., 2003; Hayashi and Su, 2007;
Patterson et al., 2005). It is therefore likely that the modest abnormalities of mito-
chondrial energetics observed in autism will result in and/or be caused by abnormal
calcium signaling (Gargus, 2009). In fact, we have observed a presentation of the
constellation of MHS in two siblings with functional mitochondrial defects, con-
genital hypotonia, and autism. They do not have a RYR1 allele that accounts for
most MHS, and one child has a muscle biopsy-proven partial respiratory complex 3
deficiency, while the other’s muscle biopsy reveals only mitochondrial hyperprolif-
eration, a nonspecific indication of weak mitochondrial dysfunction. Their primary
genetic lesion remains to be discovered (Gargus and Imtiaz, 2008).
11.5 TIMOTHY SYNDROME CAUSES AUTISM VIA

A DEFECT IN CALCIUM CHANNEL FUNCTION
The calcium ion is one of the most ancient, universal, and versatile biological
signaling molecules, known to regulate physiological systems at every level from
membrane potential and ion transporters to kinases and transcription factors
(Berridge et al., 2000). Disruptions of intracellular calcium homeostasis underlie a
host of emerging diseases, the calciumopathies (Bezprozvanny and Gargus, 2008;

Stutzmann et al., 2006). Cytosolic calcium signals originate either as extracellu-
lar calcium enters a cell through plasma membrane ion channels, or, as discussed
above, from the release of an intracellular calcium store in the ER via IP3R and
RyR channels. Therefore to a large extent, calciumopathies represent a subset of
the ion channel diseases, the channelopathies (Gargus, 2003, 2005). They include
the mitochondria, in addition, as the major intracellular calcium repository that
dynamically participates with the ER stores in calcium signaling.
The importance of calcium signaling abnormalities in autism is most saliently
brought into focus through the lens of Timothy syndrome (TS). TS is predominated
by the lethal cardiac arrhythmia syndrome referred to as long QT (LQT) because of
its characteristic electrocardiographic (EKG) findings (Splawski et al., 2004, 2005).
LQT, a channelopathy disease, has been extensively reviewed and has now been
shown to be caused by mutations in all of the cardiac ion channels that contribute to
the ventricular action potential (Gargus, 2005, 2008a; Priori and Napolitano, 2006).
The pathogenic alleles in these eight ion channel loci all prolong the repolarization
of the working myocardium, prolonging the QT interval and setting the stage for a
fatal arrhythmia. Like most of the other LQT mutations, TS (also called LQT8) is
a simple monogenic dominant channelopathy. It additionally produces extracardiac
symptoms such as an invariant syndactyly. Other organ systems are often affected as
well, so that immune deficiency, intermittent hypoglycemia, and seizures occur in a
significant portion of cases as well. Surprisingly, over 80% of the patients also have
autism (Splawski et al., 2004, 2005). The same specific allele of CACNA1C, a gene
that encodes the “cardiac-expressed” voltage-gated calcium channel, was found to
cause TS in 12 de novo unrelated cases (Splawski et al., 2004). This channel is a close
paralog of the MHS5 channel discussed above, so lessons learned from that disease
should be informative. The specific recurrent de novo mutation, G406R, is located
in the minor alternatively spliced exon 8A of the gene. Two other alleles in this locus
cause a very similar syndrome but without the syndactyly. These are found in exon
8, not 8A, suggesting cutaneous expression of only the minor transcript (Splawski
et al., 2005). The two exons are mutually exclusive, with the vast majority of the
mRNA containing exon 8, and both exons encoding the same protein domain. One
of the exon 8 alleles produces exactly the same G406R missense as the classic TS
mutation, but causes a severe early lethal disease, likely because of the higher abun-
dance of this transcript isoform. The other allele in this exon was only found in a
mosaic individual, suggesting that most mutations in this gene are not compatible
with viability (Gargus, 2003).
Since so much is understood about the pathogenesis of LQT and the biophysics
of the ion channels involved, and it is so clear that one specific “mild” mutation in
a calcium channel causes this syndrome and autism, TS might well be the finely
honed scalpel that has been long sought to reveal the pathophysiology underlying
the enigma of autism. The TS mutant channel expressed in the heart is expressed
in the neurons of the brain, and it must cause the symptoms in both organs since
TS is a simple monogenic disease of both phenotypes. Since the TS channel con-
ducts a major component of the inward calcium current underlying the depolar-
ized QT interval, a lengthening of the QT to produce the LQT characteristic of the
syndrome suggests that excess current is conducted by the mutant channel. This is
supported by the finding that a loss-of-function allele at this locus causes the short
QT Brugada syndrome (Antzelevitch et al., 2007) and by pharmacology, since the
channel blocker verapamil is used to treat TS, and the channel opener Bay K 8644
can mimic the TS arrhythmia (Jacobs et al., 2006; Sicouri et al., 2007). The mutant
and normal wild-type (WT) versions of the channel have been expressed in vitro
and kinetic analysis has been applied to dissecting the molecular defect biophysi-
cally (Antzelevitch et al., 2007; Barrett and Tsein, 2008). It is clear that the major
effect of the TS mutation is to alter the speed with which the opened conducting
channel returns to a nonconducting conformation, a process called channel inacti-
vation. The channel inactivation arising from changes in the membrane potential
are slowed, as would be predicted from the cardiac findings, but a separate mecha-
nism of the inactivation regulated by the calcium signal itself is greatly accelerated.
The net result of the mutant is a very rapid inactivation of 50% of the current, and
then a very slow inactivation of the remainder (Barrett and Tsein, 2008). Therefore,
it remains to be determined exactly which aspect is key to neuronal dysfunction and
how downstream signaling is perturbed by this altered neuronal mechanism to pro-
duce the characteristic phenotype of autism. Nonetheless, these biophysical findings
greatly extend the pathophysiology of autism and begin to render it a neurobiologi-
cal rather than strictly behavioral phenotype. This brightens the prospect that new
molecular targets can be discovered against which new generations of drugs can be
developed in this disease.
11.6 OTHER DEFECTS IN THE CALCIUM SIGNALING

PATHWAY IN AUTISM
While the TS mutation is highly informative, clearly it does not account for even
a tiny fraction of the cases with classical autism. There are, however, additional
suggestions that the calcium signaling pathway illuminated by the TS mutation
is germane to autism. As discussed above, such a pathway clearly has the abil-
ity to integrate the mitochondrial lesions of autism into a consensus signaling
pathophysiology. Furthermore, mutations in CACNA1H, a paralog of the TS/ LQT8
and MHS5 channels, have been found in familial autism (Splawski et al., 2006).
These rare mutations behave more like those contributing to a multigenic disease,
such as most cases of autism appear to be, since they do not neatly segregate with
the disease as would be the case for a monogenic factor. So while they appear
to contribute susceptibility to autism pathogenesis, they are insufficient to cause
monogenic disease.
As a further suggestion that the pathophysiology of classical calcium signaling
diseases such as MHS might be informative in autism, mutations in a paralog of
the MHS2 and LQT3 sodium channel genes, SCN1A have been found in rare cases
of familial autism (Weiss et al., 2003). This neuronal sodium channel gene had
previously been shown to contribute pathogenic alleles to the seizure syndromes
generalized epilepsy with febrile seizures plus (GEFS+) and severe myoclonic
epilepsy of infancy (SMEI) (Ma and Gargus, 2007) as well as to the migraine
syndrome familial hemiplegic migraine (FHM3) (Dichgans et al., 2005; Gargus
and Tournay, 2007). It is particularly intriguing that the autism-associated alleles

are quite different from the seizure alleles, which produce a more severe lesion
in the channel protein, but that they are very similar to the mutations found in
the FHM3 families (Gargus and Tournay, 2007). Both FHM3 and autism alleles
perturb the same regions of the channel protein, and they are those intracellular
regions that interact with calmodulin, a bound protein subunit of the channel that
confers calcium sensitivity to its regulation (Gargus, 2009).
Recently in a large survey of consanguineous Middle Eastern families with
autism, the technique of microarray homozygosity mapping identified one family
that segregated a homozygous deletion of SCN7A (Morrow et al., 2008). This gene
lies adjacent to SCN1A within the sodium channel gene cluster on chromosome 2
and it is rapidly evolving, having arisen from SCN1A by endoduplication (Plummer
and Meisler, 1999). Its mRNA is neuronally expressed; however, no function of the
putative ion channel it encodes has yet been observed (Saleh et al., 2005). It therefore
becomes a promising candidate gene that reinforces a neuronal excitation–secretion
coupling MHS-like calcium signaling pathogenesis in autism.
11.7 CALCIUM SIGNALING DYSFUNCTION CAUSES

DEFECTS IN NEUROSECRETION
As discussed above, neuronal calcium signaling culminates in the release of neu-
rotransmitter vesicles into the synaptic cleft via the process of excitation–secretion
coupling. Also discussed above was the syndromic form of autism found in Rett
syndrome, shown to be caused by mutations in MECP2. Such mutations in mutant
mouse models reveal behavioral changes reminiscent of autism (Moretti et al., 2005).
In addition, neuronal function studied in the mouse MECP2 knock-out Rett model
revealed that excitatory, but not inhibitory, synapses showed less spontaneous activity
than control. This observation suggests a defect in the calcium-dependent processes
of excitatory neurosecretion and synaptic vesicle trafficking (Nelson et al., 2006).
In a consistent fashion, mutations in neuroligins, postsynaptic cell-adhesion mol-
ecules that participate in synaptic function, have been found in rare patients with
autism and have been implicated in the disease (Jamain et al., 2003; Sudhof, 2008).
The studies of the neuroligin NGLN3 mutant (R451C allele) mouse model also
showed impaired social interactions, enhanced spatial learning abilities, but in this
case inhibitory synaptic transmission was increased with no apparent effect on excit-
atory synapses (Tabuchi et al., 2007). Deletion of NGLN3, in contrast, did not cause
such changes, indicating that the pathogenic allele is a gain-of-function mutation.
Therefore, both the Rett and the NGLN3 mouse models reveal an increased ratio of
inhibitory/excitatory synaptic transmission and serve to suggest that such a neuronal
defect contributes to autism. Mutations have also been found in patients with autism
in the genes that encode proteins that interact with the neuroligins. These genes
include NRXN1 (Yan et al., 2008), SHANK3 (Durand et al., 2007), and CNTNAP2
(Arking et al., 2008). Together with the mitochondrial and ion channel defects, these
lesions begin to build a case that autism pathogenesis involves calcium signaling
defects that culminate in defective excitation–secretion coupling, synaptic vesicle
trafficking, and neurosecretion.
11.8 FAMILY OF AUTISM-RELATED DISEASES WITH

DEFECTIVE NEURONAL CALCIUM SIGNALING
A number of common highly heritable diseases have long been recognized to be
comorbid with autism—seizures, migraine, and bipolar disease (BPD) being the
most prominent (Gargus, 2009; McElroy, 2004; Pellock, 2004). While there are
many competing theories for such familial clustering of comorbid conditions, an
important consideration is that these superficially distinct common diseases share
some fundamental heritable components of vulnerability, likely arising from shared
subsets of susceptibility-conferring loci. On the other hand, while these common
diseases, like autism, are overwhelmingly multigenic, the strongest clues to under-
standing these phenotypes are still provided by analysis of rare monogenic forms
of these diseases. This is particularly clear for the seizure and migraine phenotypes
(Dodick and Gargus, 2008; Gargus, 2006; Gargus and Shih, 2007). The monogenic
seizure and migraine diseases are all caused by ion channel mutations and have
a common channelopathy pathogenesis. The mutations all produce constitutionally
hyperexcitable neurons that are susceptible to periodic decompensations, much like
the LQT heart or the MHS muscle (Gargus, 2006, 2008a). For example, the gene
families (FHM1/CACNA1A, FHM2/ATP1A2, and FHM3/SCN1A), the mutational
lesions and the integrated pathophysiology underlying familial hemiplegic migraine,
are all strikingly homologous to those in LQT and MHS, and provide a robust plat-
form for understanding the pathogenesis of the calcium channel autism syndrome,
TS/LQT8. The FHM3 and FHM1 genes are paralogs of the sodium and calcium
channel genes that contribute to MHS (MHS2 and MHS5, respectively) and to LQT
(LQT3 and LQT8, respectively). The close relationship of the FHM3 and autism
alleles of FHM3/SCN1A has been discussed above. FHM1 encodes the neuronal P/Q
calcium channel that participates in excitatory neurosecretion. In vitro expression
of FHM1 alleles of CACNA1A in murine pain fiber neurons showed that the mutant
alleles cause a dominant-negative loss of the P/Q calcium channels. Additionally, this
loss allows other calcium channel types to be overexpressed in their place, further
perturbing nerve function (Barett et al., 2005; Cao et al., 2004, 2005; Tao and Cao,
2008). This finding, however, remains controversial since other investigators found
conflicting results in their FHM1 models (Kaja et al., 2005; Tottene et al., 2002).
FHM2 is a channelopathy caused by mutations that functionally alter an ion pump,
not a channel (Segall et al., 2004, 2005). The FHM2 gene encodes the α2 subunit of
the Na, K- ATPase, a plasmalemmal enzyme that consumes over 30% of the cell’s
ATP energy to create the transmembrane ion gradients that allow the ion channels to
function. As a major consumer of mitochondrial ATP energy and a sustainer of chan-
nel function, this mechanism sits in an intriguing junction of multiple pathways. The
enzyme transports sodium and potassium; it does not transport calcium, however,
preliminary studies on in vitro expression of FHM2 alleles have shown that these
dominant ATP1A2 ion pump mutants primarily produce alterations in neuronal cal-
cium signaling and do so by suppressing calcium release from ER stores via IP3R
(Gargus, 2008b; Smith et al., 2009). Therefore, all three FHM loci, like the three
known MHS loci, encode membrane proteins that participate in calcium signaling.
It is of note while the FHM2 and MHS1 loci encode quite distinct proteins; both
serve as links to the release of ER calcium stores. Therefore, each syndrome involves
a sodium and calcium channel plus a link to release of the ER calcium stores. Finally,
this pathway seems to extend to the mitochondria since recent studies have directly
shown that for murine CACNA1A seizure alleles in the FHM1 locus, pathogenesis is
through a pathway that involves disruptions in calcium signaling leading to induced
mitochondrial dysfunction that is ultimately capable of causing apoptosis (Bawa and
Abbott, 2008).
There are no simple monogenic forms of BPD, however, an illustration of how
calcium signaling pathogenesis extends into behavioral neuropsychiatric syndromes
is revealed by the recent discovery that the same CACNA1C locus mutated in TS/
LQT8 was found to be the sole replicated significant association found in two large
genome-wide association studies (GWAS) of BPD (Sklar et al., 2008). The SNP
markers that were found to be associated with BPD were all in intron 3 of the gene,
but their effect has yet to be functionally characterized. Similarly, SNP variants in
BCL2, an autosomal gene encoding an antiapoptotic integral mitochondrial mem-
brane protein that controls calcium signaling and contributes to the modulation of
many cellular functions including gene expression and synaptic plasticity, was found
to be associated with BPD and additionally to cause functional defects in calcium
signaling in vitro and in mouse models (Du et al., 2008; Einat et al., 2005). The
BCL2 variant associated with increased risk for BPD decreased BCL2 protein levels,
increased baseline cytosolic calcium levels, and elevated IP3R agonist-stimulated
cytosolic calcium release and apoptosis.
Finally, calcium signaling abnormalities have been revealed in the monogenic
mitochondrial migraine syndromes, monogenic neurodegenerative diseases, such
as Huntington disease, and in multigenic neurodegenerative diseases such as
Alzheimer and Parkinson disease. Migraine is a common symptom of mitochondrial
encephalomyopathy (Finsterer, 2006) and these mitochondrial migraine syndromes
are the only migraine syndromes recognized to be caused by major-effect loci other
than FHM. These mitochondrial syndromes include disease caused by the classic
mtDNA mutations, such as the point mutations underlying mitochondrial encepha-
lomyopathy, lactic acidosis, and strokes (MELAS), or the more recently resolved
autosomal nDNA mutations, such as those causing cerebral autosomal dominant
arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) or in
POLG, the autosomal gene encoding mitochondrial DNA polymerase γ (Finsterer,
2006; Gladstone and Dodick, 2005; Hudson and Chinnery, 2006; Porter et al., 2005).
Huntington disease is caused by dominant polyglutamine-expansion mutations in
HTT, encoding huntingtin. Evaluation of striatal GABAergic neurons from mouse
models expressing full-length human huntingtin revealed that the toxic polyglu-
tamine-expanded huntingtin increased mitochondrial depolarizations in response
to N-methyl-d-aspartate (NMDA) receptor calcium signals, leading to apoptosis
(Fernandes et al., 2007; Zhang et al., 2008). An analysis of calcium signaling abnor-
malities in brain slices of Alzheimer disease transgenic mouse models highlight the
critical roles of calcium signaling in the neuronal pathophysiology, with some stud-
ies pointing to direct presenilin-induced ER calcium release (Nelson et al., 2007;
Tu et al., 2006), others to presenilin-linked disruptions in RyR calcium signaling
(Stutzmann et al., 2007), and still others to presenilin-linked disruptions in IP3R
calcium signaling (Cheung et al., 2008). The presenilin mutations are rare monogenic
causes of the disease, however, mitochondrial dysfunction secondary to oxidative
stress seems to be an important contributor to common forms of the disease as well
as to Parkinson disease (Yang et al., 2008). In the case of Parkinson disease, the
importance of the TS-related calcium channel CACNA1D in pacemaker activity of
the substantia nigra dopaminergic neurons that are vulnerable in this disease seems
critical. The demands the calcium signals driven by this pacemaker channel place
on mitochondria create the age-dependent vulnerability characteristic of the dis-
ease, and blockers of this channel spare the mitochondria and are neuroprotective
in a murine model (Surmeier, 2007).
11.9 PHARMACOGENETIC CALCIUM

SIGNALING ABNORMALITIES
Despite the presence of constitutively defective ion channels in most of the simple
monogenic channelopathy diseases such as those causing seizures, MHS, FHM, or
LQT, healthful homeostasis predominates the majority of the time. Periodic decom-
pensations occur, and a number of monogenic calcium signaling diseases are recog-
nized to be vulnerable to environmental triggers. Classically such an interaction is
referred to as a pharmacogenetic syndrome. Clearly MHS fits the classical definition
of such a disease, since the anesthesia trigger is required to manifest the disease in
the genetically susceptible individuals. LQT is essentially the same, since the fatal
arrhythmia is generally triggered by a massive adrenergic release that occurs as part
of the “fight or flight” response. Here the offending trigger “drug” is endogenously
synthesized epinephrine, so while not classically pharmacogenetic, it is transpar-
ently related. From this perspective, it is reasonable to note that a classic technique to
trigger calcium signaling through IP3R is the use of thimerosal (Kaplin et al., 1994),
a sulfhydryl oxidizing reagent that is known as a preservative once used in vaccines.
Likewise several drugs, but most notably valproate (DeVivo et al., 1998), cause a
carnitine deficiency syndrome similar to that seen in the energy-deficient endophe-
notype of autism (Filipek et al., 2004; Gargus and Imtiaz, 2008), and this drug is one
of the few drugs that produces a pharmacogenetic syndrome in human and rodents of
autism (Arndt et al., 2005; Schneider and Przewlocki, 2005; Wagner et al., 2006).
11.10 CONCLUSION
Just as clues began to accumulate several years ago that mitochondrial function and
ROS production are abnormal in autism, many new disparate clues have come to
suggest that abnormal neuronal calcium signaling may serve as the scaffold that
unites these and newly recognized genetic lesions into a consensus pathophysiology
of autism with a more mechanistic neurobiological underpinning. This evidence now
suggests that calcium signaling abnormalities are a fundamental pathway per-
turbed in autism, with the multigenic disease architecture including primary defects
in mitochondrial function, but also other lesions perturbing this calcium signaling
pathway independent of a direct impact on the mitochondria. Aspects of this same
pathway are beginning to be recognized in other complex neurobehavioral diseases,

and hopefully extensions of the work though metabolomics, physiomics, and work
in model systems will provide new windows to extend this work to the promotion of
new targets for novel drug discovery in autism (Gargus and Imtiaz, 2007).
ACKNOWLEDGMENT
Supported in part by grants to J.J.G. from the National Institutes of Health, the Doris
Duke Charitable Foundation and National Alliance for Autism Research/Autism
Speaks. The author has no conflicting financial interests.
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12 Inflammation and
Neuroimmunity in the
Pathogenesis of Autism:
Neural and Immune
Network Interactions
Carlos A. Pardo-Villamizar1,* and
Andrew W. Zimmerman2,3,4
1Division of Neuroimmunology and Neuroinfectious
Disorders, Department of Neurology and
Departments of 2Neurology and 3Psychiatry and
Behavioral Sciences, Johns Hopkins University
School of Medicine, Baltimore, MD 21287, USA
4Department of Neurology and Developmental Medicine,
Kennedy Krieger Institute, Baltimore, MD 21205, USA
CONTENTS
12.1 Introduction ............................................................................................... 226
12.2 Concept of Inflammation .......................................................................... 227
12.2.1 Immunological Responses in the CNS....................................... 228
12.2.2 What Is Neuroinflammation? ..................................................... 228
12.3 How Do Immune Factors and Inflammation Influence ASD? .................. 229
12.3.1 Influence of the Immune System on Brain Development
and the Pathogenesis of ASD ..................................................... 229
12.3.2 Cytokines and Chemokines in Brain Development ................... 230
12.3.3 Maternal Environment and Effects of Immune Responses
on Neurodevelopment in the Pathogenesis of ASD ................... 231
12.3.4 Neuroglia and Neuroinflammatory Responses
during Adulthood ....................................................................... 233
12.3.5 Neuroglia, Neuroinflammation, and Synaptic Plasticity ........... 234
12.3.6 Chronic Neuroimmune Reactions in ASD ................................. 235
225
12.4 Inflammation and Immunogenetics in ASD ............................................. 235

12.4.1 Genetic Susceptibility and Immune Responses
in Neurological Disease ............................................................. 235
12.4.2 Role of MHC on Susceptibility in ASD ..................................... 236
12.5 Conclusion ................................................................................................. 237
References .............................................................................................................. 237
The role of the immune system and inflammation in the pathogenesis and
pathophysiology of autism spectrum disorders (ASDs) is controversial. It is very
clear now that the interaction of the immune system with the central nervous sys-
tem (CNS) is critical for normal neurological and behavioral functions. Recent
studies support the view that immune responses are involved in the modeling of
the CNS during prenatal and postnatal stages, and that neuroimmune activity may
disrupt normal neurodevelopment and contribute to the neuropathological abnor-
malities found in ASDs. This review focuses on the most recent research that links
immunological factors, inflammation, and neuroimmune responses with autism.
The findings include maternal autoantibodies against fetal neural epitopes, the
activation of neuroglia and neuroimmune pathways and abnormalities in systemic
immune responses in children with autism. These immunological factors influence
two important stages of the CNS function: early brain development and neuronal
organization, and later neuronal and synaptic physiology. A better understanding
of the role of immunity and neuroinflammation in the pathogenesis of autism may
have important clinical and therapeutic implications. Future studies should focus
on the actions of neuroimmune factors during brain development in the pathogen-
esis of autism.
12.1 INTRODUCTION
Autism spectrum disorders (ASD) are neurodevelopmental disorders with neurobe-
havioral manifestations that involve communication, social interaction, and
behavioral abnormalities (Rapin and Tuchman, 2008). It is increasingly clear that
immunological factors and immune dysregulation are important in the pathogenesis
and persistence of neurobehavioral abnormalities in ASD (Ashwood, Wills, and Van
de, 2006; Pardo, Vargas, and Zimmerman, 2005). In addition, evidence from the
effects of maternal viral infections during pregnancy (Chess, Fernandez, and Korn,
1978), an excess of autoimmune disorders in mothers of subjects with ASD or their
families (Comi et al., 1999), and the effects of environmental factors on the immune
system supports the view that various types of disturbances of immune function
are important in the pathogenesis of ASD and in the perpetuation of their associ-
ated behavioral and neurological abnormalities. Growing evidence of immunologi-
cal abnormalities in patients with ASD (Ashwood, Wills, and Van de, 2006) also
indicates that, in addition to the spectrum of neurological and behavioral problems
exhibited by patients with ASD, other systems, including the immune system and
gastrointestinal tract, are also affected. The causes and effects of these immune system
Inflammation and Neuroimmunity in the Pathogenesis of Autism 227
abnormalities in autism are unknown but could be critical for maintaining, if not
also initiating, some of the abnormalities in central nervous system (CNS) function.
These abnormalities likely have both polygenic and environmental bases that will
have important clinical and therapeutic implications. Current evidence suggests that
neurobiological abnormalities in ASD are associated with changes in cytoarchitec-
tural and neuronal organization that may be determined by genetic, environmental,
immunological, and toxic factors (DiCicco-Bloom et al., 2006; Pardo and Eberhart,
2007). Since neuroimmune pathways and CNS cell populations, such as the neuroglia
(astroglia and microglia), play central roles during brain development, in cortical
organization, neuronal function, and the modulation of immune responses, it is quite
possible that these factors, acting in concert with host immunogenetic factors, con-
tribute to the pathogenesis of ASD.
12.2 CONCEPT OF INFLAMMATION

Inflammatory responses represent the main mechanism by which the immune sys-
tem reacts to challenges by either exogenous or intrinsic factors that disturb homeo-
stasis in the human body. Inflammation comprises a complex network of interacting
cells of the immune system and chemical mediators that act locally in all areas of
the body in response to those challenges. Inflammation has been viewed tradition-
ally as a defense mechanism to resist or prevent infection, react to tissue injury, or
limit the development of cellular or organ dysfunction. This concept has been enlarged
recently by new evidence that supports critical roles for inflammation for maintaining
cellular viability, initiating pathways of tissue repair and growth, and the sculpting
of new organs and functional systems during development (Christiaens et al., 2008;
Griffiths, Neal, and Gasque, 2007; Hagberg and Mallard, 2005; Medzhitov, 2008).
Although the traditional concept of inflammation has focused on the cellular reac-
tions mediated by leukocytes in areas of tissue damage, this simplistic approach
is no longer sufficient as increasing evidence suggests that the main mechanisms
of inflammatory responses by the immune system consist of a complex network of
interactions between the innate and adaptive immune responses that exert well-
coordinated control in development, homeostasis, and reaction to the dysfunc-
tion of specific organ systems and the internal environment (Medzhitov, 2008).
Although the functional dichotomy of the innate and adaptive systems is a way to
understand the immune response, it is also clear that there is a continuous overlap
of these two mechanisms of immune action, with the innate responses being less
specific but more consistent in maintaining homeostasis and function, and the adap-
tive response being more specific and focused on reactions to particular deleterious
challenges. Thus, inflammation is not necessarily limited to the cellular reaction to
injury mediated by leukocytes, but rather it consists of well-coordinated responses by
cellular elements as well as several types of chemical mediators such as cytokines,
chemokines, and others that maintain tissue homeostasis and function (Barton, 2008).
Although inflammation is mostly homeostatic and beneficial, it is clear that devia-
tions of immune system responses may also generate dysfunction and damage such
as those that occur in autoimmune disorders (e.g., systemic lupus erythematous
or multiple sclerosis), exaggerated responses to infection (e.g., acute disseminated
encephalomyelitis), or exogenous challenges (e.g., postvaccinal reactions) (Blanco

et al., 2008; Medzhitov, 2008).
12.2.1 IMMUNOLOGICAL RESPONSES IN THE CNS

Although the CNS had been considered an immune-privileged structure, several
studies have recently demonstrated that such a concept is no longer valid (Zipp
and Aktas, 2006). Recent studies support the view that the immune and CNSs
have constant interactions between them that preserve their homeostasis and func-
tion (Griffiths, Neal, and Gasque, 2007; Ransohoff, 2002). Both systems are highly
hierarchical in structure and maintain their functions based on complex interac-
tions among specialized cells and chemical mediators. While the central role of
the immune response in the CNS is to react to tissue injury and maintain an active
process of surveillance for the detection of infection and noxious factors, there is
growing evidence that the immune system in the brain also contributes to fundamental
processes of brain modeling and plasticity (Bauer, Rauschka, and Lassmann, 2001).
Because of the diversity in the microenvironments and cellular processes involved
in immune and nervous system activity, the elements and mechanisms involved in
neuroimmune responses in the CNS are different than those that operate in non-CNS
tissues. Some of these mechanisms are modulated by the interplay of chemical medi-
ators such as cytokines and chemokines as well as immune cells (e.g., leukocytes and
monocytes), together with elements of the CNS such as neurons, neurotransmitters,
neuroglia, and blood vessels (Ransohoff, 2002). In the CNS environment, neuroglial
cell populations and the blood–brain barrier (BBB) function as a neurovascular-
immune unit that facilitates the continuous functional association of the CNS with
the immune system and constitutes the main modulator of neuroimmune interactions
(Abbott, Ronnback and Hansson, 2006; Kim et al., 2006).
12.2.2 WHAT IS NEUROINFLAMMATION?

Inflammation in the CNS (neuroinflammation) is a host mechanism that involves
elements of the immune and nervous systems in response to the injury, infection,
or dysfunction of the CNS and its components. Neuroinflammation involves the
interplay of elements of innate and adaptive immunity such as cellular responses
by monocytes, B or T cells; mediators such as cytokines, chemokines, and their
receptors; complement and other chemical modulators, along with elements of the
CNS such as neuroglia, BBB, and neurochemical mediators (e.g., neurotransmitters
and metalloproteinases) (Griffiths, Neal, and Gasque, 2007; Infante-Duarte et al.,
2008; Tilleux and Hermans, 2007). Similar to immune activation in other organs or
tissues, neuroinflammation may involve both innate and adaptive types of immune
responses. Most of the adaptive neuroimmune responses are triggered by infectious
processes (e.g., viral encephalitis) or external injury to the CNS (e.g., head trauma)
and involve the disruption of the BBB, the infiltration of T cells into the brain and
spinal cord structures, or the production of specific antibodies that target CNS anti-
gens or other proteins involved in disease processes. Innate neuroimmune responses
involve mostly intrinsic responses by neuroglia, such as microglial and astroglial
activation, along with increased expression by CNS cells, neurons, neuroglia, and
elements of the BBB; and cytokines, chemokines, and other neuroimmune mediators
(e.g., metalloproteinases, Toll-like receptors [TLR], and microRNA) (Baltimore et al.,
2008; Cardona et al., 2008; Charo and Ransohoff, 2006; Page-McCaw, Ewald, and
Werb, 2007; Ransohoff, Liu, and Cardona, 2007). Innate neuroimmune responses
occur in neurodegenerative diseases with abnormalities of CNS homeostasis such
as those that occur in metabolic and seizure disorders. Frequently, both innate and
adaptive neuroimmune responses occur concomitantly; however, they also may have
distinctive pathogenic roles at different stages of neurological dysfunction. There is
a growing controversy about whether the roles of neuroinflammatory responses are
deleterious or protective in the setting of neurological dysfunction or injury, and it
is now evident from different experimental approaches that neuroinflammation may
play dual roles, both in providing neuroprotection as well as producing injury in the
CNS (Griffiths, Neal, and Gasque, 2007; Skaper, 2007; Tilleux and Hermans, 2007).
12.3 HOW DO IMMUNE FACTORS AND

INFLAMMATION INFLUENCE ASD?
Although ASDs are not recognized as classical immune mediated disorders, there
is increasing interest in examining the role of the immune system and inflamma-
tion in the development and persistence of the complex neurological and behavioral
abnormalities seen in these disorders. From early brain development to adulthood,
immune responses are involved in mechanisms of neurodevelopmental plasticity,
neuronal and cortical organization, synaptic function and adaptive synaptic plastic-
ity, and critical stages of brain function that determine neurological and behavioral
activity (Pardo, 2008; Pardo, Vargas, and Zimmerman, 2005). There is also recent
evidence in humans that maternal environmental factors such as autoantibodies
directed to the fetal brain may influence immune responses during fetal brain devel-
opment (Braunschweig et al., 2007; Singer et al., 2008; Zimmerman et al., 2007) and
lead, in experimental animals, to the development of neuropathological abnormali-
ties and behaviors similar to those present in ASD (Patterson, 2002).
12.3.1 INFLUENCE OF THE IMMUNE SYSTEM ON BRAIN

DEVELOPMENT AND THE PATHOGENESIS OF ASD
Brain development is a complex and dynamic process in which different molecular
pathways overlap with each other and change over time to produce a well-organized
and compartmentalized structure. The development of the cerebral cortex, for exam-
ple, involves highly complex and coordinated phases of cellular organization that
includes four overlapping stages: neurogenesis, fate determination, migration, and
differentiation, which finally result in the highly organized architecture and circuitry
that characterize the mammalian neocortex. The role of neuroimmune factors is
critical for all processes of neuronal migration, axonal growth, neuronal positioning,
cortical lamination, as well as dendritic and synaptic formation. All elements of the
neuroimmune network—including astrocytes and microglia; immune mediators such
as cytokines, chemokines, and soluble factors produced by neuroglia (e.g., growth
factors and neurotrophins); as well as other classical immune pathways such as those
associated with the major histocompatibility complex (e.g., MHC class I) and com-
plement—participate in mechanisms of neurodevelopment during intrauterine and
postnatal stages. Therefore, a dual role, developmental and immunological, is now a
well-recognized feature of all elements of neuroimmune responses (Boulanger and
Shatz, 2004; Tonelli, Postolache, and Sternberg, 2005).
Because the disorganization of cortical neurons, abnormalities in mini-columnar
organization and subcortical white matter, as well as brain growth abnormalities
are the dominant features of the neuropathology of ASD (Bauman and Kemper,
2005; Casanova, 2007), it is likely that the critical period of pathogenesis in autism
occurs during fetal brain development and the first year of life (Pardo, 2008; Pardo
and Eberhart, 2007). During this critical pathogenic period, neurodevelopmental
processes such as neuronal migration, cortical lamination, synaptic and dendritic
modeling, and the establishment of neuronal and cortical networks are influenced
not only by genetic factors, but also by important neuroimmune mechanisms that
involve astrocytes and microglia; interactions of cytokines, chemokines, and their
receptors; the developmental expression of TLRs; and complement activation (Bauer,
Kerr, and Patterson, 2007; Lagercrantz and Ringstedt, 2001; Stevens et al., 2007).
These neurobiological processes, along with the influence of the neuroimmune sys-
tem, are crucial for the development of neurological and behavioral trajectories, such
as social cognition, language and communication, and motor development (Pardo
and Eberhart, 2007). Disturbances of specific neurobiological trajectories triggered
by abnormalities in the maternal environment or by genetic influences may eventually
translate into abnormal patterns of neurodevelopment and behaviors that characterize
ASD. In addition to their actions during the period of pathogenesis, neuroimmune
responses also may be activated during post-pathogenic stages during which neuro-
logical regression, abnormal neuronal activity (e.g., seizures), and aberrant neural
networks may trigger such responses as the part of deleterious responses or even as
the part of neuroprotective pathways. It is unclear at present, whether neuroimmune
responses or neuroinflammation are common occurrences in the brain of patients
with ASD during postnatal stages or adulthood; however, our neuroimmunopatho-
logical studies suggest that a chronic stage of immune activation or neuroinflamma-
tion occurs at least in subsets of patients with ASD. The presence of these changes is
not determined by the age or duration of the disorder, and in the postmortem brain
tissues we examined, it appears to be an ongoing, long-term neuropathological
process (Vargas et al., 2005).
12.3.2 CYTOKINES AND CHEMOKINES IN BRAIN DEVELOPMENT

Along with the neuroglia, another important element of the neuroimmune response
that contributes to brain development is a complex network of cytokines, chemok-
ines, and their receptors. Both cytokines and chemokines are well-known media-
tors of immunological activity, maturation, and the trafficking of immune cells and
responses to injury (Engelhardt and Ransohoff, 2005; Ransohoff, Liu, and Cardona,
2007). Although few human studies are available, neuropathological studies have
demonstrated that subsets of chemokines are present in the CNS and exert their
modulatory and developmental functions on neurons, neuroglia, and BBB by inter-

acting with membrane receptors, even at early stages of brain development (Rostene,
Kitabgi, and Parsadaniantz, 2007). Some chemokines are critical for neuroimmune
function and are involved early in neurodevelopment. For example, CCL2 (also
known as macrophage chemoattractant protein-1, MCP-1) and CCL5 (also known as
RANTES) are important cues for processes of microglial migration and the coloni-
zation of the CNS (Rezaie, Cairns and Male, 1997). Experimental models in rodents
have demonstrated the crucial function of CXCL12 (also known as stromal derived
factor-1, SDF-1) and its receptor, and CXCR4 in mechanisms of cerebellar develop-
ment, neuronal migration, and axonal pathfinding (Lazarini et al., 2003; Ma et al.,
1998; Stumm and Hollt, 2007; Zou et al., 1998). CXCL12 appears to have an impor-
tant function in mechanisms that control migration of Cajal–Retzius cells, important
sources of the glycoprotein reelin and other factors that support radial glia in the
critical process of cortical lamination (Paredes et al., 2006). Functional disturbances
in reelin and Cajal–Retzius cells result in abnormalities of neuronal positioning,
cortical lamination, and the columnar organization of the cortical neurons, puta-
tive factors that may contribute to the neuropathological abnormalities in autism
(Fatemi et al., 2005). Similarly, cytokines have been recognized as important fac-
tors during brain development (Pousset, 1994; Pousset, Fournier, and Keane, 1997).
Cytokines traditionally recognized as “pro-inflammatory,” such IL-6, TNF-α, and
IL-1β, as well as “anti-inflammatory” cytokines, such as TGF-β, are involved in
pathways that contribute to CNS development (Bauer, Kerr, and Patterson, 2007;
Munoz-Fernandez and Fresno, 1998; Nakashima and Taga, 2002; Taga and Fukuda,
2005). The TGF-β cytokine family, for example, is involved in essential nervous
tissue remodeling, including cell-cycle control, regulation of early development and
differentiation, neuron survival, and astrocyte differentiation (Gomes, Sousa, and
Romao, 2005). Cytokines such as IL-6 appear to mediate long-lasting changes in the
transcription and behavior of adult offspring following maternal immune activation
during gestation (Smith et al., 2007).
12.3.3 MATERNAL ENVIRONMENT AND EFFECTS OF IMMUNE RESPONSES

ON NEURODEVELOPMENT IN THE PATHOGENESIS OF ASD
There is growing evidence that the maternal immune environment affects the devel-
oping fetal CNS and determines specific patterns of inflammation-mediated brain
and behavioral pathology (Hagberg and Mallard, 2005; Meyer, Yee, and Feldon,
2007; Meyer et al., 2006). The most exciting line of research that links maternal
immunological factors in the pathogenesis of ASD comes from the demonstration
of maternal autoantibodies in the serum of some mothers of patients with autism
(Braunschweig et al., 2007; Singer et al., 2008; Zimmerman et al., 2007) and their
presence correlates with a history of early developmental regression in their off-
spring (Braunschweig et al., 2007). Two different groups of researchers have dem-
onstrated the presence of antibodies that cross-react with fetal, but not adult, neural
antigens. These findings suggest that, at least in a subset of patients with autism, the
potential passive transfer of maternal antibodies during fetal life may have played a
pathogenic role in the presence of the disorder. It is unclear, however, what the specific
targets of these autoantibodies are or what triggers their production. Following an

earlier study by Dalton et al. (2003), both groups have successfully transferred the
recently described serum anti-fetal brain antibodies from mothers of children with
autism into animal models during gestation, and demonstrated behavioral changes
suggesting features of autism in the offspring of mice (Morris et al., 2008) and monkeys
(Martin et al., 2008).
Another recent topic of increasing interest focuses on the potential role of immu-
notoxicants and nutritional factors that activate maternal immune responses and
which in turn may affect early stages of brain development. There is growing evi-
dence for effects of environmental toxins such as herbicides and other environmental
chemicals on either maternal or fetal immune activation that may produce develop-
mental brain disarrangements associated with the neurological and behavioral prob-
lems observed in ASD (Dietert and Dietert, 2008; Roman, 2007). We have observed
neuroinflammation following the experimental neonatal exposure of rats (analogous
to early third trimester in humans) to terbutaline, a β-2 adrenergic receptor agonist
and drug for asthma that is commonly used for tocolysis (slowing premature labor)
(Zerrate et al., 2007), as well as in the brain of murine offspring following the ges-
tational transfer of human maternal anti-fetal brain antibodies (Morris et al., 2008).
These observations support the relevance of maternal immune states and are consis-
tent with descriptions of an increased frequency of autoimmune disorders in mothers
and families of subjects with autism (Comi et al., 1999; Croen et al., 2005; Molloy
et al., 2006; Mouridsen et al., 2007; Sweeten et al., 2003). Immunogenetic suscep-
tibility factors, particularly in maternal human leukocyte antigens (HLA), will be
discussed in Section 12.4.
In addition to the epidemiological and immune evidence from studies in human
subjects, experimental models of maternal infections and immune challenges have
demonstrated the effects of immune responses on the development of neurobiological
and behavioral abnormalities consistent with neuropsychiatric disorders such as autism
and schizophrenia (Patterson, 2002, 2007). Although different gestational periods of
vulnerability may determine specific outcomes, it appears that infection-associated
immunological events in early stages of fetal life may have a greater impact than later
stages on the occurrence and progression of neurodevelopmental abnormalities (Bach
et al., 2008; Meyer, Yee, and Feldon, 2007). In animal models, the disruption of nor-
mal cytokine and chemokine developmental pathways, triggered by either infections
or immune challenges, appears to modify the expression patterns of these immune
mediators in the fetal brain. In turn, these altered patterns determine the develop-
ment of neuropathological changes and the various forms of behavioral dysfunction
that emerge later in life (Meyer et al., 2006; Shi et al., 2003; Smith et al., 2007).
Interleukin-6, a well-known pro-inflammatory cytokine during adulthood, appears to
be a key factor in the development of pathogenic effects from maternal immune chal-
lenges (Smith et al., 2007). Other immune regulators such as bone morphogenetic
proteins and TGF-β also appear to play important roles during stages of maternal
immune reactions that challenge the developing fetal brain (Jonakait, 2007). These
experimental observations point out that future studies on the pathogenesis of ASD
should focus on a more detailed analysis of maternal infections as potential risk factors
associated with the development of these disorders.
12.3.4 NEUROGLIA AND NEUROINFLAMMATORY RESPONSES DURING ADULTHOOD

In the CNS, neuroglial cells such as astrocytes and microglia, along with perivascular
macrophages and endothelial cells, are important for maintaining normal neuronal
function and homeostasis (Aloisi, 2001; Bauer, Rauschka, and Lassmann, 2001;
Bessis et al., 2007; Dong and Benveniste, 2001; Neumann, 2001; Prat et al., 2001;
Trapp et al., 2007; Williams and Hickey, 2002). As such, these CNS elements are
also involved in immune function and the interaction of the immune and nervous
systems. Neuroglial cells also contribute in a number of ways to the regulation of
immune responses and neuronal activity in the CNS. Both microglia and astro-
cytes are involved in crucial neurobiological functions and contribute extensively
to processes of cortical organization, neuroaxonal guidance, and synaptic plastic-
ity (Fields and Stevens-Graham, 2002; Murai and Van Meyel, 2007). Astrocytes,
for example, play an important role in the detoxification of excess excitatory amino
acids (Nedergaard, Takano, and Hansen, 2002), the maintenance of the integrity
of the BBB (Prat et al., 2001), the production of neurotrophic factors (Bauer et al.,
2001), and the metabolism of glutamate (Nedergaard, Takano, and Hansen, 2002;
Tilleux and Hermans, 2007). Under normal homeostatic conditions, astrocytes
facilitate neuronal survival by producing growth factors and mediating the uptake
and removal of excitotoxic neurotransmitters, such as glutamate, from the synaptic
microenvironment (Nedergaard, Takano, and Hansen, 2002; Ransom et al., 2003).
Both astroglia and microglia are involved in pathogenic inflammatory mechanisms
that are common to diverse disorders of the CNS and are important factors in the
neuroimmune response that occurs in response to the disruption of homeostasis in
the CNS. During neuroglial activation secondary to injury or neuronal dysfunc-
tion, astrocytes and microglia produce several factors that modulate inflammatory
responses. For example, they secrete pro-inflammatory cytokines, chemokines, and
metalloproteinases that can magnify and accelerate immune reactions within the
CNS (Bauer, Rauschka, and Lassmann, 2001; Benn, Halfpenny, and Scolding, 2001;
Rosenberg, 2002). Microglial cells, for example, are involved in synaptic stripping,
cortical plasticity, as well as immune surveillance (Aloisi, 2001; Ambrosini and
Aloisi, 2004; Graeber, Bise, and Mehraein, 1993; Trapp et al., 2007). Perivascular
astrocytes and microglia modulate BBB activity and secrete factors that regulate the
trafficking of immune cells into the CNS.
In several degenerative and immune-mediated disorders of the CNS, such as
Alzheimer’s disease (AD), HIV dementia, epilepsy, schizophrenia, and multiple
sclerosis, astroglia and microglia associated with innate neuroimmune responses are
central to the pathogenic mechanisms of neurodegeneration and appear to mediate
important processes that lead to neuronal dysfunction (Aloisi, 2001; Golde, 2002).
For example, in HIV dementia, microglial activation and infiltration by macrophages
contribute to the neuronal damage responsible for the dementia (Kaul, Garden, and
Lipton, 2001). In other disorders such as epilepsy, and particularly in Rasmussen’s
syndrome, a rare pediatric epileptic disorder, both astroglial and microglial reac-
tions occur in parallel to adaptive neuroimmune responses mediated by the T-cell
infiltration of the cerebral cortex (Pardo et al., 2004). In neurodegenerative disorders
such as Parkinson’s disease (PD) and AD, the role of the neuroimmune response has
been exposed as a critical part of the cascade of molecular and cellular events lead-
ing to either the dysfunction of specific neuronal populations, such as nigral cells
in PD, or the processing and degradation of amyloid in AD (Blasko and Grubeck-
Loebenstein, 2003; McGeer and McGeer, 2002; Nagatsu and Sawada, 2005). Several
studies have shown that microglia are activated and increased in number in schizo-
phrenia, both in postmortem tissue and on positron emission tomography, which
may result from several possible underlying metabolic disturbances (Radewicz et al.,
2000; van Berckel et al., 2008; Wierzba-Bobrowicz et al., 2005). Clinical similarities
with schizophrenia raise the possibility of using analogous approaches to diagnostic
imaging in autism. Because of the central importance of neuroinflammatory path-
ways and neuroglia in response to neuronal dysfunction and their pathogenic roles in
diverse neurological disorders, we have hypothesized that neuroimmune responses
and neuroinflammation are associated with the pathogenic mechanisms involved in
cortical and neuronal dysfunction observed in ASD. Neuroinflammation in autism
may lend itself to treatment trials using anti-inflammatory drugs, which have shown
some benefit in PD and AD, as well as schizophrenia (Muller et al., 2002; Vlad
et al., 2008; Wahner et al., 2007).
12.3.5 NEUROGLIA, NEUROINFLAMMATION, AND SYNAPTIC PLASTICITY

Neuroglia are fundamental components of communication in neuronal networks by
the virtue of their contribution to the modulation of synaptic and dendritic func-
tion and by the generation of responses to neuronal activity. These functions are
facilitated by complex neuroglia–neuronal interactions in which both microglia and
astroglia maintain a dynamic structural and functional association with synapses
and dendrites (Volterra and Meldolesi, 2005). Neuronal–neuroglia interactions are
regionally specific and occur in all regions of the CNS. Astroglia exhibit extensive
and elaborate interplay with both presynaptic and postsynaptic structures in a highly
organized way, as individual astrocytes are associated with specific microdomains
and local circuits. In the hippocampus, for example, individual astrocytes maintain a
functional association with a specific neuronal territory that modulates the physiology
of a well-defined subset of synapses. It has been calculated that in the hippocam-
pus, an individual astrocyte domain may modulate the function of 140,000 synapses
(Bushong et al., 2002; Kirov and Harris, 1999). Similarly, Bergmann glia in the cere-
bellum have specific local interactions with individual synapses in discrete microdo-
mains (Grosche et al., 1999). Recent morphological and neurophysiological studies
have also demonstrated that individual astroglia in the cerebral cortex interact with
specific islands of functional synapses and that a single astrocyte may enwrap from
4 to 8 neurons and establish contacts with 300 to 600 dendrites (Halassa et al., 2007).
Astroglial responses to neuronal activity are facilitated by calcium signaling, trig-
gered by factors that include glutamate and ATP, and also involve purinergic recep-
tors and gap-junctional signaling (Scemes and Giaume, 2006). The demonstration
of the functional role of calcium signaling in the gliotransmitter environment has
exposed the critical role of astrocytes in modulating synaptic function (Nedergaard,
Ransom, and Goldman, 2003; Volterra and Meldolesi, 2005). The secretion of astro-
glial factors with neuromodulatory function is central to the regulation of synaptic
efficacy and long-term synaptic plasticity (Montana et al., 2006; Panatier et al.,
2006; Pascual et al., 2005). Among these factors, cytokines such as TNF-α appear
to be involved in mechanisms of glia-mediated homeostasis during the activity-
dependent remodeling of the developing and established neuronal circuits that
follow brain injury (Beattie et al., 2002; Stellwagen and Malenka, 2006). These
observations support the important role of the gliotransmitter environment in the
modulation of neuronal function.
12.3.6 CHRONIC NEUROIMMUNE REACTIONS IN ASD

Although most of the focus of the role of neuroinflammation is directed to the criti-
cal periods of prenatal and postnatal development, there is growing evidence that
neuroimmune reactions are also involved in neuropathological and behavioral distur-
bances during postnatal stages and into the adulthood of patients with autism. Our
neuropathological studies of postmortem brain tissues from patients with autism
demonstrate an active and ongoing neuroinflammatory process in the cerebral cortex
and white matter, characterized by astroglial and neuroglial activation. These find-
ings support a role for neuroimmune responses in the pathogenesis and persistence
of abnormalities in ASD (Vargas et al., 2005). Since both astroglia and microglia
are involved in pathogenic inflammatory mechanisms common to many different
disorders of the CNS, it is possible that different factors (e.g., genetic susceptibility,
maternal factors, and prenatal environmental exposures) may trigger the development
of these neuroglial reactions. Furthermore, more detailed studies of immune factors
performed by protein array techniques have demonstrated that cytokines/chemokines
such as MCP-1, IL-6, and TGFβ1, which are mainly derived from activated neuro-
glia, are the most prevalent cytokines in brain tissues (Vargas et al., 2005). Similar
observations are also seen in the cerebrospinal fluid from patients with autism. These
findings strongly support the hypothesis that neuroimmune reactions are part of
the neuropathological processes in ASD and are among the factors that contribute
to CNS dysfunction in ASD. However, the significance of the neuroinflammatory
response to the specific neuropathologies and behavioral disruptions in ASD and its
relevance to the etiology of ASD requires further exploration.
12.4 INFLAMMATION AND IMMUNOGENETICS IN ASD

12.4.1 GENETIC SUSCEPTIBILITY AND IMMUNE RESPONSES
IN NEUROLOGICAL DISEASE
A critical issue in the natural history of a disease is the influence that the immune
system and immunogenetic host factors may have on its pathogenesis. The patho-
genic mechanisms of many neurological diseases, including neurodegenerative and
neuroimmunological disorders, are influenced by the spectrum and functions of the
proteins associated with immunological pathways. The expression of proteins such
as MHC or its HLA, cytokines, chemokines, and integrins is closely associated with
the function of these immunological pathways and may determine the patterns of sus-
ceptibility and severity of disease. Allelic variations in regulatory regions of cytokine
genes, point mutations, or single nucleotide substitutions have been shown to affect
gene transcription and levels of cytokine expression and to produce interindividual
variation in cytokine production (Meenagh et al., 2002). Single nucleotide polymor-
phisms (SNPs) and haplotypes of cytokine and chemokine genes produce genetic
differences in cytokine expression that predispose to disease or confer resistance in
immune-mediated disorders by influencing the strength and duration of the immune
response (Bidwell et al., 1999, 2001; Haukim et al., 2002). The clinical outcomes of
autoimmune, inflammatory, and neurodegenerative disorders appear to be influenced
by the balance between pro-inflammatory and anti-inflammatory pathways, modu-
lated by cytokines and chemokines. This relationship has been supported by numer-
ous reports of the association of some cytokine alleles and expression phenotypes
with immune-mediated or autoimmune disorders (Bidwell et al., 1999, 2001; Haukim
et al., 2002). The association of SNPs of genes associated with inflammation has been
investigated in inflammatory and neurodegenerative disorders such as AD and PD, as
well as neuroinflammatory disorders such as multiple sclerosis and HIV-associated
dementia (Crusius et al., 1995; Epplen et al., 1997; Gonzalez et al., 2002; Hakansson
et al., 2005a,b; Mycko et al., 1998). These polymorphisms may modify the natural
history of the disease by producing increased or decreased susceptibility to immune-
mediated responses or other pathogenic factors. For example, one polymorphism of
the MCP-1 gene (A-2518G) is associated with an increased risk of developing HIV
dementia (Gonzalez et al., 2002), early onset of PD (Nishimura et al., 2003), increased
resistance to antipsychotic therapy in schizophrenic patients (Mundo et al., 2005), and
increased levels of MCP-1 in serum in AD patients (Fenoglio et al., 2004; Pola et al.,
2004). In other disorders, SNPs of cytokine genes have been shown to be protective
against the onset or increased severity of disease. For example, a polymorphism in
position 1082 of the IL-10 gene promoter has been shown to be protective against
severe forms of multiple sclerosis, with the effect increasing over the years (Luomala
et al., 2003). More recently, the potential association of a SNP of IL-6 has been
associated with the development of PD (Hakansson et al., 2005a,b).
12.4.2 ROLE OF MHC ON SUSCEPTIBILITY IN ASD

At present the clearest evidence for the importance of immunogenetic factors in
autism is derived from studies of HLA typing in families. Increased frequencies of
HLA DR4, DR13, A2, and B44 have been reported in children with autism (Daniels
et al., 1995; Lee et al., 2006; Torres et al., 2002, 2006). Studies of HLA in simplex
families indicated that mothers and their sons in a geographically defined group had
a significantly higher frequency of DR4 than normal control subjects, when com-
pared to a nationally distributed multiplex sample (Lee et al., 2006). Furthermore,
no HLA linkage was found in a series of multiplex families, which may have other
genetic etiologies for autism (Rogers et al., 1999). Since HLA DR4 is frequently
associated with autoimmune disorders such as rheumatoid arthritis, it may signify
increased susceptibility in mothers to form immune responses in response to fetal
antigens, a situation that may be analogous to Rh isoimmunization in hemolytic
disease of the newborn. There may also be regional susceptibility due to environ-
mental factors, since autoimmune disorders reported in mothers have varied among
studies from different areas (Comi et al., 1999; Molloy et al., 2006; Mouridsen et al.,
2007; Sweeten et al., 2003). Recent studies of HLA in sets of parents, grandparents,
and children with autism have demonstrated the selective transmission of HLA DR4
from maternal grandparents to the mother, with greater frequency than from the
mother to her child with autism (Johnson et al., 2008). This enlarges our concept of
a specific type of susceptibility in which the mother becomes the “genetic patient”
in an immune interaction with her fetus. This is one possible immunogenetic mecha-
nism for autism that requires further investigation. Future studies of other potential
immunogenetic factors should also focus on defining the presence of specific SNPs
and haplotypes in cytokines, chemokines, their receptors, and other immune factors,
such as complement, integrins, and matrix metalloproteinases.
12.5 CONCLUSION
Inflammation and immune factors may influence the pathogenesis and perpetuation
of pathophysiological events that lead to ASD by their effect on brain development
as well as CNS function during adulthood. Because specific immune challenges
may occur during neurodevelopment, immunological and neuroimmune responses
that follow such challenges, whether environmental, maternal, or neurogenetic,
may constitute critical pathogenic factors in the development of ASD. The role
of immune factors and neuroinflammation are still uncertain but could be critical
in maintaining, if not also in initiating, some of the abnormalities present in the
CNS in ASD. A better understanding of the role of the immune system and neu-
roimmune reactions in the pathogenesis of ASD may have important clinical and
therapeutic implications.
ACKNOWLEDGMENTS
Dr. Pardo is supported by the Peter Emch Fund for Autism Research, the Bart A.
McLean Fund for Neuroimmunology Research, Cure Autism Now, and NIH-NIDA
(K08-DA16160). Dr. Zimmerman is supported by the Hussman Foundation.
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13 Possible Impact of Innate
Immunity on Autism
Harumi Jyonouchi*
Department of Pediatrics, University of Medicine
and Dentistry of New Jersey, Newark, NJ 07028, USA
CONTENTS
13.1 Introduction ...............................................................................................246
13.2 Innate Immunity: An Overview ................................................................246
13.2.1 Innate Immunity in the Gut Mucosa .......................................... 247
13.2.1.1 The Epithelium .......................................................... 247
13.2.1.2 GALT .........................................................................248
13.2.1.3 Effector T-Cell Subsets..............................................248
13.2.1.4 Treg Cells................................................................... 249
13.2.1.5 Effects of Innate Immunity ....................................... 250
13.2.2 Innate Immunity in the CNS ...................................................... 251
13.2.2.1 Innate Immune Cells in the CNS .............................. 252
13.2.2.2 Role of Treg Cells in the CNS Homeostasis.............. 253
13.2.2.3 Neuro-Immune Network ........................................... 253
13.3 Evidence of Innate Immune Abnormalities in Autism ............................. 254
13.3.1 GI System ................................................................................... 254
13.3.1.1 Intestinal Permeability .............................................. 255
13.3.1.2 Dysbiosis.................................................................... 255
13.3.1.3 Autism Colitis ............................................................ 255
13.3.1.4 Food Allergy.............................................................. 256
13.3.2 CNS ............................................................................................ 258
13.3.2.1 CNS Inflammation..................................................... 258
13.3.2.2 Mechanisms of Chronic CNS Inflammation ............. 259
13.4 Possible Impact of Innate Immunity on Neuro-Immune
Interactions in Autism ............................................................................... 259
13.4.1 Impaired Signaling of Neurotransmitters ..................................260
13.4.1.1 Cholinergic Neurotransmission .................................260
13.4.1.2 GABA Neurotransmission.........................................260
13.4.1.3 Ca2+ Signaling............................................................ 261
13.4.1.4 β2 Adrenergic Receptor............................................. 261
245
13.4.2 A Redox/Methylation Hypothesis .............................................. 262

13.4.3 Effects of Immune Insult............................................................ 262
13.4.3.1 Insult In Utero............................................................ 262
13.4.3.2 Immune Insult in Postnatal Period ............................ 263
13.5 Conclusions ............................................................................................... 265
Acknowledgment ................................................................................................... 265
Abbreviations ......................................................................................................... 265
References ..............................................................................................................266
13.1 INTRODUCTION
Autism spectrum disorders (ASD) are complex developmental disorders with largely
unknown etiology. Although recent progress in genetics have defined various disor-
ders that manifest autistic features, this may account for up to 10%–15% of ASD,
usually manifested as severe classical autism. For the remaining ASD patients, the
diagnosis of ASD is solely based on subjective behavioral symptoms, which can
vary markedly time to time during development. The presence of comorbidities also
affects their behavioral symptoms. Gastrointestinal (GI) inflammation, one of the
most common comorbidities in ASD, likely affects mood, irritability, and concentra-
tion simply because of abdominal discomfort. Recent studies indicate the presence
of more complex mechanisms affecting the brain via gut neuro-immune network,
as detailed later.
Apart from GI symptoms, a subset of ASD children presents with frequent child-
hood infections including otitis media, rhinosinusitis, and upper respiratory-tract
infection. However, conventional immune workup is often unrevealing in such ASD
children. Subtle immune abnormalities have been described in ASD children, although
the effects of such abnormalities are unclear. Nevertheless, recent studies may indi-
cate the possible impact of innate immunity on the onset and/or progress of ASD in
some but not all ASD children.
In this chapter, the overview of innate immunity in the GI tract and in the central
nervous system (CNS) is discussed first, and then immune abnormalities reported in
the GI tract and in the CNS in autism are discussed. Lastly, the possible impact of
innate immunity on neuro-immune interactions in autism is discussed.
13.2 INNATE IMMUNITY: AN OVERVIEW

In addition to the obvious host immune defense that eliminates foreign pathogens,
the immune system exerts surveillance against aberrant cell growth and tissue injury
and facilitates tissue repair. In other words, the immune system orchestrates to main-
tain homeostasis and the integrity of our body. To carry out such a complex task,
the immune system is composed by various components, which may overlap in their
functions. Immune responses can be grossly divided into two components: innate
immunity and adaptive immunity. The innate immunity acts as the first line of immune
defense sensing the deviation of immune homeostasis such as pathogen invasion.
Innate immune cells sense pathogen-derived molecular patterns (PAMPs) via specific
receptors called pattern recognition receptors (PRRs). Toll-like receptors (TLRs) are
Possible Impact of Innate Immunity on Autism 247
the first family of PRRs characterized (Zedler and Faist, 2006). PRRs can also sense
molecular patterns derived from injured tissue (the so-called danger-signals) and
can initiate immune reaction, a process which appears to be vital for wound healing
(Zedler and Faist, 2006). It is now well recognized that the innate immunity bridges
subsequent adaptive immune responses that then reciprocally affect innate immune
responses. Namely, PRR-mediated signaling activates antigen (Ag)-presenting cells
(APCs) by up-regulating the expression of major histocompatibility complex (MHC)/
co-stimulatory molecules, augmenting the Ag processing, and facilitating the migra-
tion of APCs to the lymphoid organs (Teitelbaum and Allan Walker, 2005).
The innate immunity is exerted in an Ag-independent manner and components
of innate immunity are genetically predetermined. Thus, the innate immunity can
function at birth and may play more significant roles in the first years of life before
Ag-dependent adaptive immunity fully develops. However, such characteristics of
innate immunity may be accompanied by a possibility of being more vulnerable to
subtle genetic variation than adaptive immunity.
13.2.1 INNATE IMMUNITY IN THE GUT MUCOSA

The structures essential for composing the mucosal immune system include the
epithelial cell monolayer and gut-associated lymphoid tissue (GALT), primarily
composed of Peyer’s patches (PP) and lamina propria (LP).
13.2.1.1 The Epithelium

The gut epithelium plays a crucial role in innate immune defense in the gut mucosa.
However, the intestinal epithelial barrier is functionally immature in newborns as
evidenced in higher intestinal permeability than in adults. Molecules expressed by
epithelial cells are also important for the barrier function. The glycosylation patterns
of oligosaccharides expressed on epithelial cells are reported to change after birth
(Dai et al., 2000; Teitelbaum and Allan Walker, 2005), which make the intestinal
epithelial cells less susceptible to complement-mediated cell lyses and also appear to
affect colonization patterns of microbes. Impaired intestinal epithelial barrier func-
tion in patients with protein-losing enteropathy was implicated in the specific loss
of heparan sulfate proteoglycans from the basolateral surface of intestinal epithelial
cells (Murch et al., 1996; Westphal et al., 2000). The importance of heparan sulfate
proteoglycans was further supported in rodent models deficient of these molecules
(Bode et al., 2008).
In addition to their barrier functions, epithelial cells play a major role in gut
innate defense partly by producing active mediators. Anti-microbial peptides, which
are mainly produced by Paneth cells clustered at the bottom of the intestinal crypts,
serve as broad-spectrum antibiotics killing both gram-positive and gram-negative
bacteria (Porter et al., 2002). This is also true for respiratory epithelial cells but they
also produce interferon-β (IFN-β), a critical factor for innate defense against viral
pathogens (Wark et al., 2005). In contrast, colonic epithelial cells are also thought to
contribute to the maintenance of homeostasis in the colon where microbes cohabit
at high concentrations. The colonic epithelium was shown to express TLR9 on the
cell surface in rodents (Lee et al., 2007, 2008). The TLR9 expressed apically in
the epithelium is reported to compromise on the inflammatory cascade induced by

several TLRs expressed basolaterally (Lee et al., 2007, 2008). The TLR9-deficient
mice exhibited more severe epithelial ulceration in colitis induced by dextran sodium
sulfate (Lee et al., 2008).
13.2.1.2 GALT
The LP located beneath the intestinal epithelium serves as a meshwork of connective
tissue containing plasma blasts, T cells, and various innate immune cells including
dendritic cells (DCs), mast cells, macrophages, and granulocytes (neutrophils and
eosinophils). The presence of abundant mast cells in the LP during infancy and early
childhood is thought to be important for mucosal immune defense against extracel-
lular parasites. In addition, mast cells are also indicated to have a role in tolerance
induction against macronutrients (Lu et al., 2006). However, the excessive mast-
cell activation can cause various abdominal symptoms by the release of mediators
including histamine.
PPs are lymphoid aggregates composed of B-cell follicles, surrounding inter-
follicular CD4+ and CD8+ T cells and Ag-presenting innate immune cells (DCs,
macrophages, etc.) intervening beneath the follicle-associated epithelium. DCs
composed of various subsets are abundant in the subepithelial dome (Wershil and
Furuta, 2008). Activated DCs can migrate to mesenteric lymph nodes or LPs and can
exert stimulatory and modulatory actions, bridging innate immunity and adaptive
immunity (Coombes et al., 2007). The production of tumor necrosis factor (TNF)-α
and the expression of TNF-α receptors are important for the development of PPs
(Fu and Chaplin, 1999).
The presentation of luminal Ags is affected by the dose of Ag and the presence
or absence of adjuvant effects. Pathogenic bacteria produce microbial byproducts,
which can stimulate innate immune cells including gut APCs and serve as the major
source of adjuvant (Abreu et al., 2005; Franchi et al., 2006). In contrast, a commensal
flora often down-regulate APC activation (Smith and Nagler-Anderson, 2005).
13.2.1.3 Effector T-Cell Subsets

To maintain intestinal homeostasis, multiple subsets of T cells are present in the
intestinal mucosa including effector subsets of T-helper (Th) cells including Th1,
Th2, and Th17; regulatory T (Treg)-cell subsets; and CD8+ T cells. The functions of
effector T cells are suboptimal in neonates and most of them are naive T cells that
are sensitized with exposure to gut luminal Ags and their responses to naive Ags are
under the influence of innate immune responses in the gut mucosa. Th1 and Th2
cells are well established with distinct cytokine production patterns and their roles
in immune defense; Th1 cells produce IFN-γ and activate macrophages in defense
against intracellular organisms, while Th2 cells promote immunoglobulin E (IgE)-
mediated immune defense producing Th2 cytokines (IL [interleukin]-4, IL-5, and
IL-13). Th17 cells are characterized by the production of distinct cytokines (IL-17A,
IL-17F, and IL-22) and appear important for defense against fungal and certain
bacterial pathogens (Bettelli et al., 2007; Harrington et al., 2005; Schmidt-Weber
et al., 2007). It has been shown that IL-17A not only augments T-cell priming
but also activates innate immune cells (fibroblasts, macrophages, endothelial cells,
and epithelial cells), resulting in the production of potent inflammatory mediators

(IL-1, IL-6, TNF-α, iNOS, chemokines, etc.) and subsequent neutrophilic inflam-
mation (Bettelli et al., 2007; Schmidt-Weber et al., 2007). The key differentiation
factors for Th17 cells are IL-6 and IL-1β in humans (Acosta-Rodriguez et al.,
2007; McGeachy and Cua, 2008) and recent studies also indicate a role of IL-23
and TGF-β (Manel et al., 2008; McGeachy and Cua, 2008). IL-23 also serves as a
survival factor for Th17 cells, while IL-27 suppresses the development of Th17 cells
(Batten et al., 2006; Stumhofer et al., 2006). Th17 cells with their potent proinflam-
matory natures have been implicated in autoimmune conditions including multiple
sclerosis, rheumatoid arthritis, and inflammatory bowel disease (IBD) (Bettelli et al.,
2007; Schmidt-Weber et al., 2007). Recently decreased plasma levels of IL-23 but
not IL-17 were reported in children with autism in comparison with age-matched
normal controls in studies employing 40 autistic children and 20 controls (Enstrom
et al., 2008). However, it is unclear how this finding is associated with a potential role
of Th17 cells in autism. The enumeration of Th17 cells and ideally the assessment of
Th17 cell functions will be informative for further addressing a potential role of Th17
cell in autism. The differentiation of effector T-cell subsets is profoundly affected by
innate immune responses, especially cytokine concentrations in the microenviron-
ment, and is discussed in Sections 13.2.1.4 and 13.2.1.5.
13.2.1.4 Treg Cells

The intestinal immune homeostasis is partly maintained by active immune suppres-
sion. The major cells involved in this process are Treg cells. Thymus-derived, natu-
rally occurring Treg cells are known to play a role in maintaining self-tolerance in the
periphery (Torgerson and Ochs, 2007; Wildin et al., 2001). However, in the gut mucosa,
other types of regulatory T cells called induced or adaptive Treg (aTreg) cells appear
to play a crucial role in the induction and maintenance of immune tolerance. They are
thought to be derived from naive T cells, following Ag exposure in the presence of IL-2
and TGF-β (Chatila et al., 2008). Treg cells are characterized by the expression of CD4,
CD25high, Foxp3, cytotoxic T-lymphocyte associated Ag 4, and glucocorticoid-induced
TNF receptor-related protein. Milk protein–specific aTreg cells have been reported
to be present in children who have outgrown non-IgE-mediated food allergy (NFA)
against cow’s milk protein (Karlsson et al., 2004). The Foxp3− IL-10+ T regulatory type
1 cells are also thought to have derived from naive T-cell upon Ag exposure (Chatila,
2005). They were initially recognized in patients with severe combined immunodefi-
ciency following hematopoietic stem cell (Bacchetta et al., 1994), and also are known
to be abundant in the intestine (Chatila, 2005).
It is of note that suppressive Treg cells can reciprocally differentiate into proin-
flammatory Th17 cells and vice versa in both rodents and humans depending on
microenvironment. The high concentration of IL-6 overrides differentiation signal
by TGF-β preferring Th17 differentiation, while retinoic acid, a metabolite of vita-
min A, inhibits IL-6-driven Th17 cell differentiation and greatly augments Treg cell
differentiation in the presence of TGF-β (Bettelli et al., 2007; Denning et al., 2007;
Mucida et al., 2007). These findings indicate the importance of cytokines produced
by innate immune cells. This may be especially true in the first few years of life
when shaping T-cell responses in the later life.
13.2.1.5 Effects of Innate Immunity

Innate immune responses in the gut can augment and also suppress subsequent
adaptive immunity. Lipopolysaccharide (LPS), an endotoxin, is produced by both
pathogenic microbes and commensal floras and sensed by TLR4, a well-known
PRR. In the small intestine where bacterial density is low, intestinal epithelial cells
respond to LPS by producing proinflammatory cytokines (Hornef et al., 2002,
2003). In the colon where bacteria exist at high density, colonic epithelial cells
are much less responsive to LPS rendering the state of endotoxin tolerance (Abreu
et al., 2005; Smith and Nagler-Anderson, 2005). They also become less respon-
sive to other TLR-mediated signaling (cross-tolerance). The TLR expression on
macrophages and DCs in the small intestine is also down-regulated under normal
conditions, preventing excessive inflammatory responses (Hausmann et al., 2002;
Smith et al., 2001). Apically expressed TLR9 in the colonic epithelium also medi-
ates inhibitory signaling (Lee et al., 2007, 2008).
The above-described innate immune defense is genetically predetermined and
full-term babies are fully capable of mounting innate immune defense. However,
Ag-specific, adaptive immune responses are slow to develop. During the first few
months of life, the rapid expansion of Ag-specific T and B cells occurs (de Vries
et al., 2000). During this period, the subtle changes in innate immune responses can
significantly affect the development of GALT and may make certain individuals
more prone to aberrant responses to immune insults.
13.2.1.5.1 Genetic Factors

As noted above, the genetically predetermined innate immune defense renders
genetic variation (polymorphism) more influential in clinical manifestations as
shown in IBD patients. Nucleotide oligomerizing domain 2 (Nod2), a member of
PRRs called a Nod-like receptor family, recognizes a component of proteoglycans
(PGN), muranyl dipeptide common to most bacterial PGN (Abreu et al., 2005;
Franchi et al., 2006; Meylan et al., 2006). Intracellular Nod2 is highly up-regulated
in monocytes and Paneth cells and can also be induced on intestinal epithelial cells
by TNF-α and IFN-γ (Lala et al., 2003; Rosenstiel et al., 2003). Genetic varia-
tion in Nod2 is associated with increased susceptibility to various inflammatory
conditions, most notably Crohn’s disease (CD), at least in Caucasians (Abreu et al.,
2005; Franchi et al., 2006; Rodriguez-Bores et al., 2007). Human CD-associated
Nod2 variants exhibit reduced or loss of activity in cell lines, indicating that a defi-
cit in sensing bacterial presence likely triggers abnormal inflammatory responses
(Inohara et al., 2003; Ogura et al., 2001). Other multiple genetic factors involved in
innate immunity are also included in candidate genes in the development of IBD
(Rodriguez-Bores et al., 2007).
13.2.1.5.2 Environmental Factors

13.2.1.5.2.1 Commensal Flora The lack of the commensal flora or the TLR4 sig-
naling via endotoxin produced by the commensal flora is reported to augment immune
responses to food Ags in both animal models and humans (Bashir et al., 2004).
The restoration of oral tolerance in germ-free mice by feeding the mice LPS, a TLR4
agonist, has also been reported (Wannemuehler et al., 1982). This is partly attributed to
the fact that LPS enhances the suppressive activity of TLR4 +CD4+CD45RBlowCD25+
Treg cells (Caramalho et al., 2003). A prolonged exposure to LPS or TLR2 stimulants
also leads to tolerance and cross-tolerance to other PAMPs in intestinal epithelial cell
lines (Otte et al., 2004). Likewise, other TLR agonists derived from the commensal
flora also appear to be important in maintaining proper communication between the
gut mucosal immune system and the commensal intestinal flora (Abreu et al., 2005;
Kelly et al., 2005; Lee et al., 2007, 2008).
In addition to anti-inflammatory effects mediated by the host-immune system, the
commensal flora can exert direct anti-inflammatory actions. Bacteroides thetaiotao-
micron was shown to restrict the signaling induced by flagellin, a TLR5 agonist, and
flagellated pathogens. These microbes block downstream signaling associated with
NF-κB activation by promoting the nuclear export of transcriptionally active RelA
(Kelly et al., 2004). This effect of B. thetaiotanomicron may partly explain why
the gut tolerates large amounts of flagellated, potentially inflammatory commensal
bacteria. In addition, other commensal bacteria were shown to block the activation
of NF-κB by inhibiting IκB-α ubiquitination (Neish et al., 2000).
13.2.1.5.2.2 Micronutrients As described in Section 13.2.1.4, a novel role for

retinoic acid, the metabolite of vitamin A, in the gut mucosal immunity has been
shown. Unlike spleen DCs, LP DCs augment de novo generation of Foxp3+ Treg
cells, but their actions are dependent on TGF-β and retinoic acid (Denning et al.,
2007; Mucida et al., 2007; Sun et al., 2007) and inhibited by IL-6. Reciprocally,
retinoic acid antagonizes IL-6 in Th17 differentiation (Denning et al., 2007; Mucida
et al., 2007; Sun et al., 2007). Retinoic acid was also known to imprint gut-homing
specificity on T cells (Iwata et al., 2004) and it was reported that retinoic acid induces
Foxp3+ Treg cells expressing gut-homing receptors at high levels in both humans and
mice (Kang et al., 2007).
Another emerging important regulator in mucosal immunity is adenosine that is
likely derived from injured cells/tissues and can be also generated from extracel-
lular nucleotides by ectoenzymes CD39 and CD73 (Sitkovsky et al., 2008). Treg
cells were shown to express CD39 and CD73 and adenosine renders Treg cells to
produce suppressive cytokines (IL-10 and TGF-β). These findings indicate that Treg
cells play a role in restoring homeostasis in sterile tissue injury (Deaglio et al., 2007;
Sitkovsky et al., 2008). Adenosine is also reported to exert anti-inflammatory effects
independent of Treg cells (Sitkovsky et al., 2008). These findings indicate the impor-
tance of micronutrients in the gut mucosal homeostasis.
13.2.2 INNATE IMMUNITY IN THE CNS

As opposed to the gut, the brain is tightly regulated to maintain homeostasis by
limiting the influence of external stimuli to protect mostly nonrenewable neuronal
cells. The CNS immune system is also quite opposite to the gut immune system in
terms of much less exposure to Ag stimuli largely due to the presence of the blood
brain barrier (BBB), scant lymphoid tissue without lymphatic drainage, and limited
numbers of APCs. The CNS is poorly equipped to mount the immune responses, but
recent studies indicate the importance of innate immune cells in the CNS.
13.2.2.1 Innate Immune Cells in the CNS

Microglial cells are thought to be the resident macrophages in the brain and exert the
first line of defense in the CNS parenchyma as a sensor of the CNS microenviron-
ment. However, they also play a physiological role in the brain development/functions
such as cortical plasticity and synaptic stripping (Davoust et al., 2008; Marin-Teva
et al., 2004; Roumier et al., 2004). The quiescent microglial cells are distinguished
from other macrophage lineage cells in their morphology extending microglial pro-
cesses into the parenchyma (Davoust et al., 2008). This may allow them to detect
changes in the microenvironment. The quiescent microglial cells are also distin-
guished from other macrophages with the low expression of cell surface markers
including CD45, MHC II, and Fc receptor, rendering them poorly capable of Ag pre-
sentation and phagocytosis (Davoust et al., 2008). Upon activation, the microglial
cells change into the similar morphology of other macrophages with the retraction of
ramifications and start exerting functions of macrophages including phagocytosis, the
presentation of Ag, and the production of inflammatory mediators. They can also pro-
liferate, causing microgliosis in various pathological conditions (Streit et al., 1999).
In addition to the microglial cells, other types of macrophages are also present in
the CNS–CNS associated macrophages. These include perivascular macrophages,
meningeal macrophages, and choroid plexus macrophages. They express CD45high
and MHCII+. In the inflamed CNS, peripheral blood monocytes and/or myeloid pro-
genitors may be recruited to compose bone marrow–derived microglial (BMDM)
cells (Davoust et al., 2008; Djukic et al., 2006; Rodriguez et al., 2007). In the rodent
model of Alzheimer’s disease, BMDM cells are reported to be more effective in
clearing amyloid plaques (Simard et al., 2006).
Astrocytes, oligodenrocytes, and neuronal cells can also participate in innate
immune responses in addition to their physiological roles. Astrocytes and brain
endothelial cells interact to regulate water and electrolyte metabolism in the brain
(Abbott et al., 2006). Astrocytes produce neurotropic factors such as glutamate
in response to neurotransmitters or other neuronal signaling (Vesce et al., 2007;
Zlokovic, 2008). They also acts as a “salt sensor” affecting local neuronal activity in
response to changes in local lactate levels (Shimizu et al., 2007).
These innate immune cells are sensing PAMPs and danger signals via PRRs.
Among PRRs, TLRs are most extensively studied in the brain. Multiple stud-
ies revealed mRNA and/or protein expression of TLR1–TLR8 in microglial cells
(Crack and Bray, 2007; Olson and Miller, 2004). Some TLRs are also reported to be
expressed on astrocytes (Crack and Bray, 2007). Perivascular and choroids plexus
macrophages constitutively express CD14 and TLR4, which render them to sense
circulating LPS in the absence of BBB (Laflamme and Rivest, 2001); TLR4 ligands
to LPS with CD14 as a cofactor. In rodent models of endotoxemia, it was shown that
the initial detection of LPS by these macrophages trigger the rapid up-regulation
of CD14 in parenchymal microglial cells (Glezer et al., 2007). The transcription of
multiple genes associated with innate immune responses takes place upon signaling
that is mainly mediated by the NF-κB pathway (Glezer et al., 2007; Gosselin and
Rivest, 2007). In this model, TLR2 is also involved in the production of the second
wave of TNF-α and IL-12. TNF-α appears important for priming microglial cells
(Glezer et al., 2007). Such innate inflammatory responses need to be tightly regulated
considering largely nonrenewable neuronal tissues. These regulatory mechanisms

include the production of counter-regulatory cytokines (IL-10, TGF-β, etc.) and the
release of glucocorticoids (GCs) (Glezer et al., 2007; Haddad, 2008). Acute innate
inflammatory responses in the brain may be protected by rapidly resolving and phago-
cytosing the excitotoxic damage in acute brain injury models (Gosselin and Rivest,
2007). The protective action of acute innate responses in the brain is also supported
by the absence of neuronal damage with the single injection of LPS in the endotox-
emia model (Gosselin and Rivest, 2007) and also by the augmented repair of myelin
induced by LPS (Glezer et al., 2006).
However, the chronic activation of innate immunity is likely neurotoxic as indi-
cated in various neurodegenerative diseases causing the production of reactive
oxygen and nitrogen species, the recruitment of peripheral blood leukocytes and
lymphocytes, the impairment of BBB, or other damages. BMDM cells are thought
to be preferentially recruited at the site of neuronal insults and BMDM cells can
penetrate even intact BBB and expand (Davoust et al., 2008), but their role in neuro-
degenerative diseases are not well understood.
13.2.2.2 Role of Treg Cells in the CNS Homeostasis

In addition to the above-described regulatory mechanisms in the innate immunity,
Treg cells also known to play a role in controlling actions of effector stage T and B
cells recruited to the site of neuronal insult/inflammatory in the brain (O’Connor and
Anderton, 2008). The effector cells may not only be specific for pathogens invaded
to the CNS, but may also be auto-reactive to neuronal tissues. In rodent models of
multiple sclerosis (MS), the accumulation of Treg cells has been reported in the recov-
ery stage (Kohm et al., 2006; Korn et al., 2007; Liu et al., 2006). These CNS Treg
cells are distinguished from other Treg cells by their strong ability to proliferate,
and they may be recruited in an Ag-independent manner by the CNS (O’Connor and
Anderton, 2008; O’Connor et al., 2007). It is reported that Treg cells in MS patients
is deficient in their function to suppress the proliferation of IFN-γ-producing Th cells,
although normal numbers of Treg cells are present in the CNS (Astier and Hafler,
2007; O’Connor and Anderton, 2008; Venken et al., 2008). Treg cells differentiation
and proliferation are dependent on cytokines produced by innate immune cells such
as TGF-β in the microenvironment. Thus, innate immune cells may be important for
supporting regulatory actions of Treg cells in the CNS as shown in the GI mucosa.
13.2.2.3 Neuro-Immune Network

An intricate neuro-immune interaction persists throughout one’s life beginning in
the stage of embryogenesis. This interaction is thought to be especially critical in the
first few years of life. The disruption of this intricate network can lead to pathological
condition in the CNS with the developing brain possibly being the most vulnerable.
13.2.2.3.1 Cytokines
In addition to PAMPs, numerous studies report the effects of cytokines on the CNS.
Cytokine produced outside the brain can affect the CNS; neuronal tissues are one
of the target organs. They may directly affect the brain at the level of brain paren-
chyma crossing the BBB or entering the brain area lacking the BBB (Goncharova
and Tarakanov, 2007). They may also indirectly affect the brain via peripheral nervous
systems and also via secondary messengers induced by cytokines (Goncharova and
Tarakanov, 2007). For example, IL-2 can penetrate the intact BBB and affect cell
growth and survival, the release of multiple mediators, and neuronal activities via
IL-2 receptor expressed on neuronal and glial cells (Goncharova and Tarakanov,
2007). Proinflammatory cytokines released by innate immune responses (IL-1β,
IL-6, and TNF-α) are known to cause “sickness behaviors” affecting multiple
aspects of the CNS functions (Goncharova and Tarakanov, 2007; Gosselin and
Rivest, 2007). A part of their actions may be attributed to their regulation of the
activity of the serotonin transporter via p38 mitogen-activated protein-kinase path-
way (Zhu et al., 2006). IL-1 is also implicated in long-term memory (Goeb et al.,
2006; Pickering and O’Connor, 2007). Type 1 IFNs are also known to affect multiple
CNS functions. It is well known that a high dose of exogenous type I IFNs can cause
depression (Goeb et al., 2006).
These cytokines are also produced by the brain-resident innate immune cells as
described in Section 13.2.2.1. TNF-α and IL-1β have been implicated in neurotoxic
effects observed in neuronal damages (Gosselin and Rivest, 2007). IL-1β induces
the production of reactive oxygen/nitrogen species, modulate activities of metallo-
proteinases, and increase the synaptic release of nitric oxide. TNF-α triggers the
recruitment of neutrophils and monocytes to the site of inflammation and augments
their phagocytic functions to eliminate pathogens and tissue debris. Such effects can
not only be neuroprotective but also be neurotoxic.
Reciprocally, the CNS affects cytokine network by releasing various mediators such
as GCs. For example, acute stress responses via hypophysis-pituitary-adrenal (HPA)
axis limit excessive immune responses and restore immune homeostasis. However,
chronic stress responses lead to the chronic production of GCs and catecholamines
(CAs), which can dysregulate immune functions. Evidence indicates that both GCs and
CAs render Th2-skewed responses by suppressing the production of IL-12 by APC;
IL-12 is the key cytokine for Th1 differentiation (Blotta et al., 1997; Elenkov, 2008).
GCs are also reported to up-regulate the production of IL-4, IL-10, and IL-13 by Th2
cells (Blotta et al., 1997; Elenkov, 2008). Both GCs and CAs affect the production of
proinflammatory cytokines by macrophage-monocyte lineage cells (Elenkov, 2008).
The cholinergic signaling provides anti-inflammatory action to leukocytes via cholin-
ergic receptors (Franco et al., 2007). Evidence indicates that anti-inflammatory signal-
ing via HPA axis and cholinergic nervous system is dysregulated in the IBD patients
as evidenced by markedly reduced 5-hydroxyindoleacetic acid (5-HT) production
(Franco et al., 2007).
13.3 EVIDENCE OF INNATE IMMUNE ABNORMALITIES IN AUTISM

13.3.1 GI SYSTEM
In addition to the behavioral symptoms, certain medical conditions are present
in many but not all the ASD children. One of the most common comorbidities in
ASD children is GI symptoms (Ashwood and Wakefield, 2006; Ashwood et al.,
2004; Horvath et al., 1999; White, 2003). This led to the speculation of GI pathology
in the onset and progress of autism. Proposed abnormalities may be more or less asso-
ciated with “presumed,” impaired gut mucosal innate immunity in autistic children.
13.3.1.1 Intestinal Permeability

As discussed in Section 13.2.1, gut epithelial cells are thought to play multiple roles
in the gut mucosal innate immunity not only serving as an epithelial barrier but also
producing multiple mediators. In the view of the high frequency of GI symptoms, the
dysfunction of intestinal epithelial barrier has been hypothesized in ASD children—
“leaky gut hypothesis” (White, 2003). This hypothesis postulated that impaired gut
permeability in autistic children permits the entry of macromolecules like milk pro-
tein, causing the sensitization of gut mucosal immune system and subsequent food
allergy (FA). It is also postulated that such macromolecules entered to the blood
stream affect the CNS directly. However, it is unclear whether impaired gut perme-
ability is present prior to the onset of GI symptoms or altered permeability is a result
of gut mucosal inflammation. It should be noted that children with NFA have been
reported to exhibit impaired intestinal permeability that resolves after the implemen-
tation of the restricted diet avoiding offending food (Dupont and Heyman, 2000).
Recent study in 14 autistic children with current or previous GI complaints found no
evidence of changes in intestinal permeability (Robertson et al., 2008).
13.3.1.2 Dysbiosis
Intestinal commensal flora also plays a role in maintaining intestinal homeostasis
as discussed in Section 13.2.1.5.2. There have been anecdotal reports of the onset of
autism following the administration of antibiotics and the subsequent appearance of
GI symptoms. It was hypothesized that antimicrobial administration led to the dis-
ruption of commensal flora and the colonization of bacteria producing neurotoxin. In
open-label trial, the administration of oral vancomycin in 10 children with regressive
autism resulted in short-term improvement in their behavioral symptoms (Sandler
et al., 2000). Two studies that examined the constitution of gut microflora in ASD
children reported differences from normal controls. One study examined 7 children
with regression autism and documented GI symptoms as well as 4 control children
and their results revealed significant difference in the upper and the lower intestinal
floras between autistic and control children (Finegold et al., 2002). Another study
examined 58 ASD children, 15 normal siblings, and 10 unrelated healthy children.
The authors reported a higher incidence of Clostridium histolyticum group in ASD
children; most of ASD children had the history of multiple antibiosis and significant
GI symptoms (Parracho et al., 2005). In these studies, it is possible that other factors
such as previous frequent antibiosis and the restricted diet on which many ASD chil-
dren were already placed at the time of the study entry may have affected the results.
The careful selection of the study subjects with appropriate case controls may shed a
light whether dysbiosis has any role in the pathogenesis of autism.
13.3.1.3 Autism Colitis

Secondary to the high prevalence of GI symptoms in ASD children, abnormalities
of GI mucosa has been implicated in the development of autism. Macroscopic (ileo-
colonic lymphoid nodular hyperplasia [LNH]) and histological findings indicating
mild GI mucosal inflammation have been reported in ASD children (Furlano et al.,
2001; Wakefield et al., 2000, 2005). Immunohistochemical studies of biopsy speci-
men from ASD children revealed higher numbers of CD3+CD8+ T cells in the epi-
thelium as well as higher numbers of CD3+ T cells and CD19+ B cells in the LP as
compared to those from normal controls (Ashwood et al., 2003). In children with
regressive autism, Torrente et al. (2002) reported lymphocytic colitis that is char-
acterized with epithelial IgG and complement deposition. LNH can be observed in
normal children and the significance of “autism colitis” appears still controversial.
However, one study indicated significantly higher prevalence of LNH in the ileum
and colon in ASD children than controls; LNH appears to be not affected by the diet
or age at the time of colonoscopy (Wakefield et al., 2005).
In subsequent studies, these authors report the up-regulation of proinflammatory
cytokines in the intestinal mucosa. It was reported that LP CD3+ T cells in the duo-
denum express higher IL-2, TNF-α, and IFN-γ, but less IL-10 and also the higher
expression of TNF-α and IFN-γ by LP CD3+ T cells in the colon in ASD children
with GI symptoms (Ashwood and Wakefield, 2006; Ashwood et al., 2004). In 18
ASD children with GI symptoms, authors reported increased expression of TNF-α
and IFN-γ and less expression of IL-10 by CD3+ T cells in both intestinal mucosa and
peripheral blood as compared to normal controls (Ashwood and Wakefield, 2006).
On the other hand, other studies failed to reveal any difference between ASD
children and normal controls in the concentration of proinflammatory cytokines
(IL-6, IL-8, and IL-1β) in the GI mucosa (DeFelice et al., 2003) or in the stool
concentration of calprotectin or rectal nitric oxide: these are nonspecific markers
of GI inflammation. Such conflicting results may be partly attributed to the small
numbers of study subjects, although most of the studies focus on ASD children
with GI symptoms. It still remains to be seen whether “autism colitis” is present or
such GI inflammation may be associated with other immune abnormalities such as FA.
Unfortunately, in all these studies, the presence or absence of IgE-mediated FA or
NFA has not been addressed.
How is the GI inflammation documented in ASD children associated with
innate immune abnormalities? Innate immunity is crucial for maintaining immune
homeostasis. If there exists aberrant innate immune responses in the gut mucosa
as indicated in the peripheral blood of a subset of ASD children (Jyonouchi et al.,
2002, 2005b), these ASD children may be vulnerable to GI inflammation triggered
by immune insults, developing chronic inflammation as postulated in patients with
Crohn’s disease.
13.3.1.4 Food Allergy

As detailed in the previous section, young children are more vulnerable to sensitiza-
tion to common food proteins due to the immature gut mucosal immune system.
Secondary to the high frequency of GI symptoms in ASD children, the presence of
FA has been suspected. Previously reported immunological abnormalities such as
increase in the higher production of Th2 cytokines without stimuli and the higher fre-
quency of Th cells expressing Th2 cytokines suggested the higher prevalence of atopy
in ASD children (Gupta et al., 1998; Molloy et al., 2006). However, to my knowledge,
there has been no report documenting the high prevalence of atopy in ASD children.
TABLE 13.1
Prevalence of Atopic Disorders
Study Group Atopic Disorders AR + AC AD Atopic Asthma
ASD children 34/123 (27.6%) 34/123 (27.6%) 10/123 (8.1%) 6/123 (4.9%)
a
ROM/CRS 3/25 (12.0%) 3/25 (12.0%) 0/25 (0%) 3/25 (12.0%)2
FA 6/27 (22.2%) 4/27 (14.8%) 4/27 (14.8%) 3/27 (11.1%)
Control 10/46 (21.7%) 10/46 (21.7%) 3/46 (6.5%) 7/46 (15.2%)2
Note: AC, allergic conjunctivitis; AD, allergic dermatitis; AR, allergic rhinitis.
a Among ROM/CRS patients and controls, 16 and 2 children were diagnosed with non-atopic asthma,
respectively.
One study examining 30 autistic children reports the higher frequency of skin prick
test reactivity in their study population, but it is unclear whether these children dem-
onstrated corresponding clinical features; authors report normal IgE levels and no
asthma symptoms in these autistic children (Bakkaloglu, 2007). In 123 ASD children
evaluated in the Pediatric Allergy/Immunology clinic at our institution, we found
no evidence of the high prevalence of atopy (Table 13.1). These ASD children were
evaluated by standard diagnostic measures for atopic disorders including the mea-
surement of allergen-specific IgE and skin prick testing. Likewise, we did not find the
high frequency of IgE-mediated FA in ASD children evaluated in our clinic.
Various dietary intervention measures have been tried on the basis of anec-
dotal reports despite the lack of evidence of IgE-mediated FA. Among such dietary
intervention measures, a casein-free, gluten-free (cf/gf) diet appears most popular
with frequent beneficial effects per parental reports. However, prospective studies
addressing the effects of the cf/gf diet revealed conflicting results (Elder et al., 2006;
Knivsberg et al., 2002). This may be partly attributed to the random selection of the
study subject without proper workup for FA. These studies may have been formu-
lated to test the “leaky gut hypothesis.”
In our previous studies, we hypothesized that GI symptoms frequently seen in
ASD children can be partly explained by NFA and immune reactivity to food pro-
teins can be detected by measuring the production of TNF-α and other inflammatory
cytokines by peripheral blood mononuclear cells (PBMCs) as reported in NFA chil-
dren (Benlounes et al., 1999). Our results revealed the presence of cellular immune
reactivity to common dietary proteins (mainly milk protein) in young ASD children
with GI symptoms (Jyonouchi et al., 2005a). We also found that such immune reac-
tivity to food protein was correlated with aberrant responses to LPS (TLR4 agonist)
by PBMCs (Jyonouchi et al., 2005b). However, NFA may be playing a lesser role
in GI symptoms in older ASD children, since most children likely outgrow NFA
condition with the maturation of the gut immune system and the establishment of
oral tolerance. We also observed the less prevalence of NFA in older (>6 years) ASD
children (unpublished observation).
Given our findings, it is possible that aberrant innate immune responses may
provoke undesired immune reactivity against commensal flora in ASD children as
observed in IBD patients. In the studies of children who underwent the elimination
diet, we found the decline of the cellular reactivity to milk proteins but persistent
reactivity to candida Ag, a representative microbial luminal Ag (Jyonouchi et al.,
2007). Taken together, our data indicate a role of NFA in GI symptoms in some ASD
children in association with aberrant innate immune responses.
In summary, there exist convincing data supporting the presence of chronic GI
inflammation in ASD children, but the etiology of GI inflammation, which is likely
affected by multiple genetic and environmental factors, is still not well understood.
NFA can partly explain the GI symptoms and beneficial effects of dietary interven-
tions in some ASD children. However, aberrant innate immune responses, if present
as suggested by our data, likely affect immune reactivity not only to food protein but
also to commensal flora. Apparent effects of oral vancomycin and altered commensal
flora reported in ASD children may be explained partly by aberrant innate immune
responses. Further studies will be required to understand a role of innate immunity in
the GI symptoms observed in ASD children.
13.3.2 CNS
Autism has been considered as a heterogeneous neuro-developmental disorder
defined by behavioral symptoms and in a majority of patients, the development of
autism is likely affected by multiple genetic and possibly environmental factors
starting from fetal life. A role of the immune system in the brain pathology has been
controversial secondary to the lack of clear evidence of immune reactions unlike
other neurodegenerative diseases. However, recent studies indicate the evidence of
chronic mild inflammation in the CNS in autism patients.
13.3.2.1 CNS Inflammation

Ongoing inflammation in the CNS in autism has been suggested by several investiga-
tors with findings of elevated inflammatory markers in cerebrospinal fluid (CSF).
In 12 children with autism, decreased quinolinic acid and neopterin but increased
biopterin were reported along with the increase in soluble tumor necrosis factor
receptor II (Zimmerman et al., 2005). Others also reported increased levels of TNF-α
in both CSF and serum in 10 children with regressive autism (Chez et al., 2007).
Elevated cerebellar 3-nitrotyrosine, a marker of oxidative stress, was also reported
in 9 autistic children (Sajdel-Sulkowska et al., 2008). Such findings are consistent
with active neuro-inflammatory process evidenced by the activation of microglial
cells and astrocytes in the cerebral cortex, white matter, and cerebellum in autopsied
brains in 11 autistic patients (Vargas et al., 2005). In their studies, they also reported
the up-regulation of protein levels of macrophage chemoattractant protein (MCP)-1
and TGF-β1 in the brain tissue along with elevated levels of MCP-1 in CSF. Authors
report that the major source of inflammatory cytokines is likely activated astro-
cytes. Since the numbers of patients in these studies are relatively limited and these
study subjects appear to have overt clinical symptoms indicating inflammation, it is
uncertain whether such inflammatory processes can be observed in all the autistic
children. Nevertheless, it is likely that a subset of autistic children can reveal the
evidence of ongoing inflammation involving innate immune cells in the CNS.
13.3.2.2 Mechanisms of Chronic CNS Inflammation

As a cause of “presumed” CNS inflammation, several possibilities have been postu-
lated. These include impaired responses to oxidative stresses and auto-inflammatory/
autoimmune conditions. If such mechanisms are present, it may be reasonable to assume
that such changes can be detected in the periphery. Several authors have reported
changes in serum parameters either indicating oxidative stress (Chauhan and Chauhan,
2006; Corbett et al., 2008; Deth et al., 2008; James et al., 2006) or dysregulated
adaptive and innate immune responses (Ashwood et al., 2006, 2008; Sweeten et al.,
2003b, 2004). Effects of multiple environmental factors on genetically susceptible
individuals (xenobiotics) have been implicated in the development of autism as detailed
in Section 13.4.2. A role of mitochondrial dysfunction has been suspected in association
with the evidence of increase in oxidative stress in autistic children (Oliveira et al., 2007;
Poling et al., 2006). However, it is unclear whether impaired oxidative stress can solely
explain reported inflammatory processes in the brain.
The presence of autoantibodies against brain tissues has also been reported as
reviewed by Ashwood et al. (Ashwood and Wakefield, 2006; Ashwood et al., 2006).
Interestingly, one study reports the higher presence of anti-brain antibodies in autis-
tic children as well as their unaffected siblings (Singer et al., 2006). Epidemiological
studies indicate the higher prevalence of autoimmune diseases in parents of children
with autism (Mouridsen et al., 2007; Sweeten et al., 2003a). These findings led to the
hypothesis that maternally derived anti-brain antibodies have a role in the develop-
ment of autism. Several authors report the presence of antibodies against fetal brain
in sera of mothers with autistic children including antibodies against myelin basic
protein and glial acidic fibrillary protein (Croen et al., 2008; Singer et al., 2008;
Zimmerman et al., 2007). Others reported that maternal mid-pregnancy autoantibodies
to fetal brain may be an early marker for autism (Croen et al., 2008). Then how do these
maternal autoantibodies affect fetal and infantile brain? Most of these autoantibodies
are IgG in isotype and how these autoantibodies can access to the CNS is unclear in
the presence of BBB. It is also unclear how the presence of maternal autoantibodies is
associated with frequent comorbidities such as GI symptoms in ASD children.
In summary, evidence is slowly accumulating indicating the presence of ongo-
ing inflammation in the CNS in some, if not all, autistic children involving innate
immune cells. However, mechanisms leading to ongoing inflammation are unclear.
13.4 POSSIBLE IMPACT OF INNATE IMMUNITY

ON NEURO-IMMUNE INTERACTIONS IN AUTISM
Autism was once thought to be a static encephalopathy caused by structural and
genetic abnormalities. However, clinical features in autistic children indicate oth-
erwise. That is, a high frequency of comorbidities such as GI symptoms and sleep
disturbance indicates chronic inflammatory condition involving other organ systems.
Various studies reported the evidence of ongoing inflammation and chronic oxida-
tive stress not only in the CNS but also in the periphery in autistic children (Ashwood
and Wakefield, 2006; Ashwood et al., 2008; Chauhan et al., 2004; Deth et al., 2008;
James et al., 2006; Pardo and Eberhart, 2007; Pardo et al., 2005; Vargas et al., 2005;
Zimmerman et al., 2005). Autism without clearly defined metabolic/genetic causes

is better to be considered as a chronic multisystem disease(s) under the influence of
multiple genetic and environmental factors.
It is generally agreed that there are at least two types of ASD in terms of the dis-
ease development: ASD children whose abnormal cognitive development is evident
from birth (classical autism) and those who reveal developmental regression gener-
ally between 18 and 24 months of age following an apparent span of normal devel-
opment (regressive autism) (Lainhart et al., 2002). The diagnosis of autism may not
be definite, and some ASD children drop the diagnosis of autism, which may occur
spontaneously or possibly in response to therapeutic measures (Fein et al., 2005;
Kelley et al., 2006; Kleinman et al., 2008; Sutera et al., 2007). What kind of biologi-
cal abnormalities can lead to the above described clinical features of ASD affecting
the multiple organ systems? In this section, postulated mechanisms that may explain
many aspects of complex clinical features of ASD are discussed focusing on potential
roles of innate immunity.
13.4.1 IMPAIRED SIGNALING OF NEUROTRANSMITTERS

13.4.1.1 Cholinergic Neurotransmission
Cholinergic signaling plays a vital role in many aspects of brain functions and its
dysregulation has been implicated in various neurological diseases. The decreased
protein expression of α3, α4, and β2 nicotinic acetylcholine receptor (nAChR) has
been reported in cerebral cortex and the cerebellum of autopsied brains from adult
autistic patients (Martin-Ruiz et al., 2004). As for the immune system, the cholin-
ergic neurotransmission plays an important physiological role in down-regulating
inflammatory responses (de Jonge and Ulloa, 2007). In rodent models of sepsis,
the vagal nerve stimulation attenuates inhibitory responses partly through down-
regulating the production of proinflammatory cytokines via α7nAChR (de Jonge
and Ulloa, 2007). In the brain, both neuronal and glial cells express nAChR. On the
other hand, it was reported that in rat embryonic brain cells, TLR agonist and CD40
ligand (CD154) promote excess cholinergic differentiation from undifferentiated pro-
genitors (Ni et al., 2007); as stated previously, both neuronal and glial cells express
TLRs. Nicotinic agonists have been investigated for their potential therapeutic effects
on neurodegenerative diseases, since nicotine itself has significant toxicity (de Jonge
and Ulloa, 2007). The cholinergic neurotransmission is inhibited by various chemi-
cals, including organic phosphates and nicotinoids, that one may be exposed in daily
life. It was proposed that low but chronic exposure to such chemicals may be asso-
ciated with impaired cholinergic neurotransmission in some autistic children with
genetic vulnerability. However, the role of exposure to such toxic chemicals in the
development of autism is not well understood.
13.4.1.2 GABA Neurotransmission

The gamma-aminobutyric acid (GABA) receptors (GABR) transmit excitatory
responses in adult brains, but they function differently during the development of
neuronal maturation. Impaired GABAergic circuits have been implicated in many
neuro-developmental disorders including autism (Di Cristo, 2007). Polymorphisms
of GABRγ1 and other genes located on chromosome 15q11–13 are reported to be risk
factors for autism (Ashley-Koch et al., 2006; Vincent et al., 2006). On the other hand,
Tochigi et al. (2007) reported no association between GABR genes in 15q11–13 and
autism in a Japanese population. In the periphery, GABA also serves as a neurotrans-
mitter as well as a hormone like factor in both peripheral nervous system and endocrine
organs. For example, GABA increases insulin secretion (Gladkevich et al., 2006).
In type 1 diabetes, a T-cell-mediated organ-specific autoimmune disease, GABA
expression is down-regulated with the presence of autoantibodies against GABA and
glutamic acid decarboxylate (GAD) 65 that catalyzes glutamate to GABA and carbon
dioxide by decarboxylation (Gladkevich et al., 2006). GAD65 autoantibody is also
known to be present in patients with stiff person’s syndrome (SPS), a rare autoimmune
neurological disorder. In SPS patients, others report the presence of autoantibody against
GABAA-receptor (Raju et al., 2006). Innate immune cells do also express GABAA
receptors and selective GABAA receptor agonist was shown to activate macrophages
independent of TLRs in vitro and in vivo (Lubick et al., 2007). Environmental expo-
sures to chemicals that inhibit GABA neurotransmission can thus potentially cause
various symptoms affecting nervous, endocrine, and immune systems in genetically
vulnerable individuals. However, it is not known how such environmental exposure has
a role in the development of autism.
13.4.1.3 Ca2+ Signaling

Ca2+ signaling is fundamental in cell biology and the impaired Ca2+ signaling causes
a chronic illness affecting multiple organs. Timothy syndrome arises from a single
nucleotide change that leads to a mutation in the pore-forming subunit of the L-type
Ca2+ channel Ca(V)1.2 (Splawski et al., 2004). Their clinical features are charac-
terized by autistic features, severe cardiac arrhythmia, and developmental abnor-
malities (Splawski et al., 2004). The immune system is also profoundly dependent on
Ca2+ signaling along with neuronal cells on its function and development. However,
little is known regarding any evidence of impaired Ca2+ signaling in autism without
defined etiology. In the Allergy/Immunology Clinic, we have evaluated over 200
ASD children and did not find any patient indicating impaired Ca2+ signaling in the
immune system. However, subtle changes in the Ca2+ signaling may not be detect-
able in conventional immune workup. It remains to be seen how potentially impaired
Ca2+ signaling affects the development of autism.
13.4.1.4 b2 Adrenergic Receptor

The prenatal stimulation of the β2 adrenergic receptor with the use of terbutaline,
a β2 agonist and a tocolytic agent, has also been implicated in the increased risk of
autism in genetically susceptible individuals (Cheslack-Postava et al., 2007). When
terbutaline was administered to rats equivalent to ages of the second and third trimes-
ters of humans, microglial activation was observed, supporting possible effects of a
tocolytic agent in autism in susceptible individuals (Zerrate et al., 2007). However,
β2 agonists have been used by pregnant women suffering from asthma, without any
known adverse effects on offspring. This may be due to a minute amount of β2 ago-
nist entering the blood stream when inhaled as opposed to a large dose of β2 agonist
administered intravenously when used as a tocolytic agent.
13.4.2 A REDOX/METHYLATION HYPOTHESIS

As described in the previous sections, the evidence of chronic oxidative stress has
been described in ASD children (Deth et al., 2008). It has been hypothesized that
altered methionine synthase, which is an effective defense mechanism against oxida-
tive stress in the acute stage but not in chronic condition by affecting DNA methyla-
tion and dopamine receptor phospholipid methylation, may be associated with the
development of autism along with impaired responses to oxidative stress (Deth et al.,
2008). Individuals with genetic susceptibility to xenobiotics will be affected in such
conditions. The risk of autism is implicated in environmental exposure to heavy
metals and xenobiotics in this hypothesis. However, epidemiological studies failed
to reveal a causal association between early exposure to mercury from thimerosal-
containing vaccines and immunoglobulins and deficits in neuropsychological func-
tioning at the age of 7–10 years (Thompson et al., 2007). It is possible that such
risk may only be objectively found in individuals with defined genetic susceptibility.
Likewise, xenobiotics can affect the immune system and alter gene expression by
inhibiting DNA methylation, but how the low dose of chronic environmental exposure
to xenobiotics can impact the immune system is not well understood.
13.4.3 EFFECTS OF IMMUNE INSULT

13.4.3.1 Insult In Utero
As indicated in the previous section, the immune system can reciprocally affect neu-
rotransmitter signaling. Thus, immune insults mediated by cytokines and other
mediators can profoundly affect the development of brain and other organ systems in
vulnerable period. It has been known that maternal infection during pregnancy is a
risk factor in neuro-developmental disorders including autism (Arndt et al., 2005). The
effects of maternal infection in utero have been studied in animal models, often using
TLR agonists such as LPS (endotoxin), mimicking infection-induced innate immune
responses without using live pathogens. By using TLR3 agonist mimicking viral infec-
tion in rodents, others reported multiple neuro-pathological symptom clusters in adult-
hood (Meyer et al., 2008a,b; Pessah et al., 2008). Authors reported that abnormalities
found depended on the precise timing of prenatal immune insult (Meyer et al., 2008b).
Such effects of innate immune activation in this model are likely affected by
genetic susceptibility. This was indicated by the attenuated effects of TLR3 ago-
nist in their model in transgenic mice over-expressing IL-10, a counter-regulatory
cytokine (Meyer et al., 2008b). Others also revealed the similar effects of nonpatho-
genic immune insult in utero in rodents using cytokines as a source of immune
insults (Smith et al., 2007). During pregnancy, maternal infection does not neces-
sarily induce fetal infection, but inflammatory mediators induced by innate immune
responses can affect fetal brain via inflammatory mediators such as LPS or other
means. These findings indicate that maternal innate immune responses, if excessive,
can affect lasting effects in the CNS of offspring.
In animal models with maternal viral infection (influenza), deleterious effects on
brain structure and functions have also been reported when mice were infected in
the late second trimester (Fatemi et al., 2008). As shown in models using a TLR3
agonist, infection with influenza in the late second trimester revealed significant
changes in the expression of genes implicated with autism and schizophrenia in the
brain in addition to brain atrophy (Fatemi et al., 2008). Interestingly, authors also
reported altered levels of serotonin, 5-HT, and taurine in this model; they report
decreased levels of serotonin in the cerebella of offspring at postnatal day 14 but not
at day 56 (Winter et al., 2008). Elevated levels of serum serotonin has been reported
in a subset of autistic children (Burgess et al., 2006), while low plasma serotonin lev-
els in mothers of autistic children was indicated as a risk factor for autism (Connors
et al., 2006). These results indicate that altered serotonin levels may be associated
with prenatal immune insult.
The pitfalls of the above-described rodent models are that the growth pattern of
the brain differs in humans and rodents. The timing of in utero exposure in these
animal models is considered to be still in the early stage of gestation in humans even
if in utero challenge occurs in the late pregnancy in rodents. It will be necessary to
collect data in pregnant women and outcomes of neuropsychiatric functions in off-
spring prospectively.
13.4.3.2 Immune Insult in Postnatal Period

It is not well understood whether postnatal immune insult further affects the devel-
opment/progress of autism. Immune insult can certainly cause oxidative stresses,
which may contribute in disease progress in vulnerable individuals after birth as
discussed in the previous sections. GI inflammation caused by FA and dysbiosis can
also affect the CNS via cytokines, neurotransmitters, and other means. For example,
Shultz et al. (2008) reported impaired social behaviors in the rat with the intrac-
erebroventricular injection of propionic acid (PPA), an enteric bacterial metabolic
end-product. The brain of the animals injected with PPA revealed astrogliosis—
the evidence of neuroinflammation via the activation of CNS innate immune cells
(Shultz et al., 2008). The PPA production is likely to increase with the disruption
of commensal flora triggered by prolonged antibiosis, severe FA, and viral gastro-
enteritis. The high frequency of GI symptoms and apparent therapeutic effects of
probiotics, vancomycin (Ashwood et al., 2004), and dietary intervention in ASD
children (Levy and Hyman, 2005) may support a potential role of PPA in their behav-
ioral symptoms.
In patients with regressive autism, parents often attribute the onset of regression to
apparent systemic immune insult such as viral infection and adverse drug reactions.
Common viral infection such as influenza causes the systemic activation of innate
immunity and the CNS responds to such insults by sensing PAMPS, proinflamma-
tory cytokines, and other mediators as detailed in Section 13.1. In autistic subjects
with genetic susceptibility, the activation of the CNS innate immune system could
lead to undesirable neuroinflammation and brain dysfunction. Apart from genetic
susceptibility associated with neurotransmitter signaling and oxidative stresses as
discussed before, are there any primary immune abnormalities or genetic poly-
morphisms that affect CNS responses to immune insults in ASD children? Given
the CNS immune system, such abnormalities, if present, may be likely associated
with innate immunity and its key inflammatory mediators. However, so far, no such
abnormalities are clearly described in autistic children.
In studying the gene expression of whole peripheral blood from children with
autism or ASD, the up-regulation of genes associated with innate immunity has
been reported, albeit up-regulation is modest (about twofold) (Gregg et al., 2008).
These genes are those expressed by natural killer (NK) cells and many of them
belong to the NK cytotoxicity pathways (Gregg et al., 2008). NK cells are one of the
major effector cells in innate immunity at the time of viral syndrome. Such findings
may support a role of innate immunity in the development of autism. However, such
changes were found in both ASD and autism patients (Gregg et al., 2008) and it is
unclear whether such changes are more significantly observed in a subset of ASD
children with apparent susceptibility to recurrent infection or regression triggered
by microbial infection.
Previously, we retrospectively reviewed ASD subjects seen in our clinic for the past
3 years with regard to infection-induced exacerbation. We defined such exacerbation
as significant exacerbation in behavioral symptoms as well as the loss of once-acquired
cognitive skills documented by caretakers/teachers/therapists independent of parents,
excluding initial regression. With such definition, up to 10% of ASD children falls
into this category and this does not seem to be associated with atopy or autism sub-
types (autism, PDD-NOS, or ASD) (Table 13.2). The diagnosis of primary immunode-
ficiency such as specific polysaccharide antibody deficiency is not associated with this
subset of ASD children. Our findings indicate a more potent role of innate immunity in
TABLE 13.2
Prevalence of Microbial Infection–Induced
Exacerbationb in ASD Children Evaluated
in the Pediatric Allergy/Immunology Clinic
Infection-Induced Exacerbation
ASD without atopya
Autisma 9/107 (8.4%)
PDD-NOS 6/84 (7.1%)
ASD 2/32 (6.3%)
ASD with atopy
Autism 4/44 (9.1%)
PDD-NOS 1/43 (2.3%)
ASD 1/11 (9.1%)
a Atopy was defined as the presence of atopic disorders (atopic

asthma, allergic rhinoconjunctivitis, and atopic dermatitis) with
positive skin-test reactivity and/or positive allergen-specific IgE
antibodies.
b Specific polysaccharide antibody deficiency was diagnosed in a
total of 4 ASD children and 2 of them revealed infection-induced
exacerbation. No other primary immunodeficiency was diagnosed
in this study population.
ASD children with the above-described clinical features and in such children, genetic
susceptibility may be defined by altered innate immune responses.
It is also of note that when we tested the effects of the elimination diet in ASD
children with NFA (mainly to milk protein), we observed the decline of cellular
reactivity to offending food protein, but persistent altered responses to LPS, a TLR4
agonist, in ASD/NFA children but not in non-ASD/NFA children. Such findings may
also indicate the presence of aberrant innate immune responses affecting responses
to immune insult after birth.
13.5 CONCLUSIONS
No definite evidence exits that the innate immunity affects the development of autism.
However, the clinical and laboratory findings described in this chapter appear suf-
ficient to support a role of innate immunity in some autistic children. Further studies
in a well-defined subset of ASD children indicative of innate immune abnormali-
ties will help in defining the role of innate immunity in the development/progress
of autism.
ACKNOWLEDGMENT
The author is thankful to Dr. L. Huguenin for critically reviewing this chapter.
ABBREVIATIONS
5-HT 5-hydroxyindoleacetic acid
ASD autism spectrum disorders
Ag antigen
APC Ag-presenting cells
BBB blood brain barrier
BMDM bone marrow–derived microglial
CAs catecholamines
CD Crohn’s disease
cf/gf casein-free, gluten-free
CNS central nervous system
CSF cerebrospinal fluid
DCs dendritic cells
FA food allergy
GABA gamma-aminobutyric acid
GABR GABA receptors
GAD glutamic acid decarboxylate
GALT gut-associated lymphoid tissue
GCs glucocorticoids
GI gastrointestinal
HPA hypophysis-pituitary-adrenal
IBD inflammatory bowel disease
Ig immunoglobulin
IFN interferon
IL interleukin
LNH lymphoid nodular hyperplasia
LP lamina propria
LPS lipopolysaccharide
MCP-1 macrophage chemoattractant protein-1
MHC major histocompatibility complex
MS multiple sclerosis
nAChR nicotinic acetylcholine receptor
NFA non-IgE-mediated food allergy
NK natural killer
Nod2 nucleotide oligomerizing domain 2
PAMPs pathogen associated molecular patterns
PBMCs peripheral blood mononuclear cells
PGN proteoglycans
PP Peyer’s patches
PRRs pattern recognition receptors
SPS stiff person’s syndrome
TGF transforming growth factor
Th T-helper
TLR Toll-like receptors
TNF tumor necrosis factor
Treg regulatory T cells
aTreg adaptive Treg
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14 Autism, Gastrointestinal
Disturbance, and
Immune Dysfunction:
What Is the Link?
Paul Ashwood,1,3,* Amanda Enstrom,1,3
and Judy Van de Water2,3
1Department of Medical Microbiology and
Immunology, 2Division of Rheumatology, Allergy
and Clinical Immunology, Department of Internal
Medicine, and 3M.I.N.D. Institute, University of
California at Davis, Davis, CA 95616, USA
CONTENTS
14.1 Introduction ............................................................................................... 278
14.2 Autism and Immune Dysfunction ............................................................. 279
14.2.1 Autoimmunity and Autism ......................................................... 279
14.2.2 Cell-Mediated Immune Response in Autism ............................. 281
14.2.2.1 Cytokines in Autism .................................................. 282
14.3 Gastrointestinal Dysfunction in Autism ................................................... 282
14.4 Discussion: Possible Links between Immune Dysfunction,
Gastrointestinal Dysfunction and Autism ................................................. 285
References .............................................................................................................. 291
Autism spectrum disorders (ASD) are complex debilitating neurodevelopmen-

tal disorders. Despite diagnostic advances and a broader awareness, its causes and
underlying pathology remain largely a mystery. Studies have suggested both genetic
and environmental components in its etiology, and much attention has been drawn
toward a possible role for immune dysfunction. Indications of an increased incidence
of chronic gastrointestinal (GI) disturbance in some children with ASD have also
generated interest in GI dysfunction as a contributing factor to certain aspects or
symptoms and even behavioral characteristics in ASD. Endoscopic, histologic, and
* Corresponding author: Tel.: +1-916-703-0405; fax: +1-916-703-0367 e-mail: [email protected]
277
immunohistochemical analyses of the GI tract in children with ASD who present with
GI symptoms have revealed chronic inflammatory changes throughout the gut, includ-
ing extensive immune cell infiltration of the gut wall and increased pro-inflammatory
responses in mucosal T-lymphocyte cells. In addition, some of the changes observed
suggest the potential involvement of an autoimmune response that is directed toward
the epithelial barrier surrounding the gut lumen. Here, we review evidence supporting
a potential role of GI dysfunction in some cases of ASD, the potential link between
immune dysfunction and gut inflammation, and the hypotheses regarding the rela-
tionship between autistic behaviors and GI-related immune dysfunction.
14.1 INTRODUCTION
Autism, or autistic disorder, is now recognized in the DSM-IV-TR as a heterogeneous
syndrome, belonging to a group of neurodevelopmental disorders known as the
autism spectrum disorders (ASD) (APA, 2000) that include other specific diagnostic
subtypes such as Asperger’s disorder and Rett’s disorder.
The etiology of autism is largely unknown, although ASD are highly heritable.
The evidence for a genetic link came initially from twin studies, where the concor-
dance rate for autism was 60%–90% in monozygotic twins compared with 3%–5%
in dizygotic twins (Bailey et al., 1995; Folstein and Rutter, 1977). More recent stud-
ies have also produced evidence that twinning is a risk factor for developing autism
(Taniai et al., 2008). Betancur, Leboyer, and Gillberg (2002) reported the proportion
of twins with autistic sibling pairs was 10%, compared to the expected twinning
rate based on the normal population of 2.4%. There is also an indication of fourfold
increased autism prevalence among dizygotic twins (Greenberg et al., 2001). Autism
has also been associated with single-gene defects such as tuberous sclerosis com-
plex, 15 q duplications, and fragile X syndrome, but these account for only a small
percentage of cases (Muhle, Trentacoste, and Rapin, 2004). While these studies
yield evidence that there are genes that strongly impact the likelihood of developing
autism, no definitive pattern of genes has been identified and a multitude of different
and varied candidate genes have been implicated in autism (Muhle, Trentacoste, and
Rapin, 2004). Moreover, the replication of these data has been inconsistent, prob-
ably due in part to the heterogeneity of the phenotypes within the autism spectrum.
However, several studies have linked autism with immune-based genes, such as
human leukocyte antigen (HLA)-DRB1*04, interleukin (IL)-4 receptor, and comple-
ment C4B null allele (Lee et al., 2006; Torres et al., 2006; Warren et al., 1992, 1995).
These data are supported by findings showing the higher prevalence of autoimmune
disease in the primary and secondary family members of autistic children when
compared to families with no history of autism (Comi et al., 1999; Money, Bobrow,
and Clarke, 1971; Mouridsen et al., 2007). These data suggest that alterations in the
genes that control the immune system/immune function may act as susceptibility
factors for the development of autism. The role of autoimmunity and immune dys-
function in autism is discussed in detail in Section 14.2.1.
It is most likely that the vast majority of the cases of autism are caused by the
combination of environmental factors and multiple susceptibility genes (Cederlund
and Gillberg, 2004; Glasson et al., 2004). Environmental factors such as congenital
Autism, Gastrointestinal Disturbance, and Immune Dysfunction 279
rubella infection have previously been reported to be associated with autism; 7% of

250 children with congenital rubella later developed autism (Chess, 1971). However,
due to immunization programs, rubella likely accounts for only 0.75% of cases
(Fombonne, 1999). Pre- and perinatal Haemophilus influenzae and cytomegalovirus
infections may also cause autism through significant damage to the immature brain
(Gillberg and Coleman, 2000). Thalidomide (Rodier), an anticonvulsant taken dur-
ing pregnancy, and perinatal hypoxia have also been linked with ASD (Baird et al.,
2003). More recently, associations between increased infection early in life and later
autism diagnosis have been documented (Niehus and Lord, 2006; Rosen, Yoshida,
and Croen, 2007). To date, however, these all remain as mere associations, and no
clear causally linked environmental factors have been established.
14.2 AUTISM AND IMMUNE DYSFUNCTION

As our understanding of the immune system increases, it has become more apparent
that extensive cross talk between the immune and neurological systems exists. Many
of the components of the immune system are capable of directly altering neurologi-
cal pathways and can affect mood and behaviors. In turn, certain neurotransmitters
can modulate the immune response. There have been a number of hypotheses that
discuss the links between the immune system and autism. Indeed, altered immune
function has been described in autism for almost 40 years (Money, Bobrow, and
Clarke, 1971). Alterations of the immune system at the cellular level in autistic indi-
viduals include changes in the number and activation status of B cells, T cells, natu-
ral killer (NK) cells, and monocytes (Denney, Frei, and Gaffney, 1996; Enstrom
et al., 2009; Fiumara et al., 1999; Stubbs and Crawford, 1977; Sweeten, Posey, and
McDougle, 2003; Warren, Foster, and Margaretten, 1987). Autism also appears to be
associated with a shift in the balance of T-helper type 1 (TH1) and TH2 responses, and
associated with autoimmune and atopic diseases (Gupta et al., 1998; Molloy et al.,
2006; Singh, 1996; Singh, Mehrotra, and Agarwal, 1999). As well as possible shifts
in cytokines associated with TH1/TH2 balance, there are also reports of abnormali-
ties in proinflammatory and anti-inflammatory cytokine profiles, such as increased
IL-1, IL-6, and TNFα (Croonenberghs et al., 2002; Jyonouchi, Sun, and Le, 2001;
Vargas et al., 2005).
14.2.1 AUTOIMMUNITY AND AUTISM

In the last several years, there have been a number of reports of the increased his-
tory of autoimmune disease in families of individuals with ASD (Comi et al., 1999;
Mouridsen et al., 2007; Sweeten et al., 2003). One study found that amongst moth-
ers and first-degree relatives of autistic children, there was a 16%–21% incidence
of autoimmunity compared with 2%–4% of control families (Comi et al., 1999).
In this study, type I diabetes, rheumatoid arthritis (RA), hypothyroidism, and sys-
temic lupus erythematosus were the most common autoimmune diseases. Among
the families reporting autoimmune disease, RA was the most common. However,
in a similar study, Molloy and colleagues (2006) reported 70% (108/155) of chil-
dren with regressive autism had a family history (first- or second-degree relatives)
of autoimmune disease versus 55% (81/144) of autistic children with no regression.

When considering only the families reporting autoimmune disease, 36% in the
regressive group and 34% of the non-regressive group reported the family history
of RA, which are similar to the results of the study conducted by Comi et al. (1999).
The study by the Molloy group, however, does not include a control population and
relied on the memory and the accuracy of the caregiver in assessing the autoimmune
status of second-degree relatives (Molloy et al., 2006). The study by Comi et al.
(1999) also relied on recall by family members, which could introduce significant
bias into the study. These factors may mean that the high reports of a familial history
of autoimmune disease need further verification. Sweeten et al. (2003) also identi-
fied an increased frequency of the familial history of autoimmune disease amongst
children with autism and Asperger’s disorder when compared with children who had
an autoimmune disease and with healthy children. In this study, the most frequent
autoimmune disease reported was hypothyroidism. This compares with the findings
of the Molloy group, in which regressive-type autism was associated with thyroid
disease (41% in regressive type versus 27% in non-regressive autism) (Molloy et al.,
2006). Furthermore, in a large study of medical charts of 420 ASD children and
2100 children without the history of developmental delay, an association between the
maternal diagnosis of psoriasis, an autoimmune condition, and the ASD was found
(Croen et al., 2005). Moreover, in mothers diagnosed with allergy or asthma in the
second trimester of pregnancy, there was a two-fold increase in the diagnosis of ASD
in the offspring suggesting that immune disorders or immune dysregulation may be
a common feature during pregnancy in mothers of children who later develop ASD.
It must be noted that although there may be significant bias in family interviews as
compared to chart reviews, in turn, chart reviews rely on doctor reports, which may
underreport some cases of autoimmunity. However, the role of familial autoimmunity
in the etiology of ASD is at present unclear.
Recently, there is growing evidence to suggest that, in some cases, potential
alterations in neurodevelopment could be caused by the placental transfer of maternal
antibodies that interfere with the development of the fetal brain (Braunschweig
et al., 2008; Dalton et al., 2003; Martin et al., 2008; Singer et al., 2008). In a study by
Dalton et al. (2003), blood serum was taken from a 38-year old mother of three chil-
dren. She was the “test mother” of the study, as her first child was a boy with typical
development (9 years), her second child was a girl (8 years) with high-functioning
ASD and no history of regression, and her third child was a boy (6 years) with a
severe language-specific disorder (not autism) after regression at 18 months. For con-
trols, they took serum from four parous women, each of whom had between one and
four typically developing children. When brain tissue from adult rats and neonatal
mice was exposed to serum samples from the five different mothers, immunohis-
tochemical analysis showed that antibodies to neuronal antigens from the test mother
bound more strongly to cerebellar Purkinje cells compared with serum from the
control mothers (Dalton et al., 2003). In addition, the investigators injected serum
samples from the test mother and two of the control mothers into pregnant mice dur-
ing several days of gestation. Subsequent assessment of the mouse offspring showed
behavioral impairment (e.g., reduced exploratory behavior and impaired righting
reflex) and neurochemical changes in the cerebellum in those mice exposed in utero
to serum from the test mother compared with those mice exposed in utero to serum
from the control mothers. Recently, purified IgG isolated from sera of mothers of
children with autism and from sera of control mothers with typically developing
children were injected into pregnant rhesus macaques. The offspring of macaques
injected with IgG from mothers of children with autism displayed significant whole
body stereotypies and behavioral changes that were strikingly reminiscent of autism
and were not present in the offspring of macaques treated with IgG from mothers of
typically developing children (Martin et al., 2008).
In addition to potentially increased familial autoimmunity in relatives of subjects
with ASD, there are an increasing number of reports that show direct indications
of autoimmunity in children with ASD (Cabanlit et al., 2007; Connolly et al., 1999,
2006; Singer et al., 2006; Todd et al., 1988; Weizman et al., 1982; Wills et al., 2007).
The presence of self-reactive antibodies has been extensively reported in autistic
children. In one study, 27% of ASD children had self-reactive IgG autoantibodies
compared with 2% of control children; 36% had self-reactive IgM, compared with
none in the control group (Connolly et al., 1999). Specifically of interest, autoanti-
bodies have been detected toward many components of the central nervous system
(CNS) in some individuals with autism. These include antibodies toward neuron-axon
filament proteins (Singh et al., 1997), myelin basic protein (MBP; Singh et al., 1993),
serotonin receptors (Singh, Singh, and Warren, 1997), brain-derived neurotrophic
factor (Connolly et al., 2006), nerve growth factor (Kozlovskaia et al., 2000), and
brain endothelial cells (Connolly et al., 1999). Although these autoantibodies are not
detectable in all individuals with autism, the number of individuals with autism who
have one or more of these antibodies is significant. In one study of 68 children with
autism, 49% had autoantibodies that were reactive against the caudate nucleus and
18% had antibodies against the cerebral cortex (Singh and Rivas, 2004). In separate
studies, Singh et al. (1993, 1997) found 55%–70% of sera collected from children with
autism were positive for antibodies against MBP and brain-derived neurofilament
protein. However, it is unclear whether these autoantibodies are directly involved in
the induction of autistic behavior or secondary to the innate CNS pathology.
14.2.2 CELL-MEDIATED IMMUNE RESPONSE IN AUTISM

Cell-mediated immunity is a host defense system controlled by several different cel-
lular subsets that act in concert with each other and the humoral immune system, in
an effort to orchestrate an appropriate immune response to pathogenic insult. Current
literature concerning immunological aberrations in ASD has provided valuable
insights into potentially dysfunctional cellular subsets. These include a decrease in
total lymphocyte numbers (Ashwood et al., 2003), a reduced frequency of CD4+ T cells
(Ashwood et al., 2003; Ferrante et al., 2003; Yonk et al., 1990), a decrease in suppres-
sor T cells (Warren et al., 1990), an increase in circulating monocytes (Sweeten et al.,
2004), and an altered frequency of NK cells (Fiumara et al., 1999; Enstrom et al., 2009).
In addition to reports of quantitative differences in immune function between autistic
children and typically developing controls, there have also been reports of qualita-
tive differences. Lymphocytes, in general, have shown a decreased responsiveness
when challenged with mitogenic stimulation (Stubbs and Crawford, 1977). In T-cell
subsets, there have been reports of partial activation, evinced by an increase in cell
surface HLA-DR without the expression of the IL-2 receptor (Plioplys et al., 1994;
Warren et al., 1995). NK cells from autistic children also have reduced lytic activity
compared to controls (Enstrom et al., 2009; Warren, Foster, and Margaretten, 1987).
These studies have provided valuable information on the immune status of autistic
children and implicate several cellular subsets as potential antagonists. However,
many of these studies were conducted under steady state or nonspecific mitogenic
conditions at singular time points in the child’s development. At a steady state,
i.e., non-challenged milieu, immune cells may only slightly differ from neurotypi-
cal controls, but when challenged there may be an altered or inappropriate immune
response and corresponding cytokine profile in autism compared with the controls.
14.2.2.1 Cytokines in Autism

It has been hypothesized that neuro-immune factors such as proinflammatory
cytokine secretion and CNS-directed antibodies directly contribute to the behav-
ioral changes seen in autoimmune diseases and vice versa immune changes seen in
psychiatric disorders. The direct evidence of increased proinflammatory cytokines
profiles in the brain specimens of individuals with autism can be difficult to assay
due to the limited availability of appropriately preserved brain tissue. However,
one study collected human brain tissue and cerebrospinal fluid (CSF) from 11
individuals with autism (ranging in age from 5 to 45 years old) and 6 control indi-
viduals (5 to 46 years old) (Vargas et al., 2005). Microglial and astroglial activa-
tions present in the brain tissue from individuals with autism were consistent with
ongoing inflammatory processes. Furthermore, significant numbers of infiltrating
macrophages and monocytes were present in the inflammatory sites. The brain
specimens from individuals with autism also showed significant proinflammatory
cytokine reactions, including the presence of high levels of transforming growth
factor β1 (TGF-β1), monocyte chemoattractant protein (MCP)-1, MCP-3, thymus and
activation-regulated chemokine, IL-6, IL-10, and eotaxin, among others. Based on
the immunohistochemical staining of the tissue, astrocytes and microglia were the
primary source of cytokine release. The authors also examined CSF samples of both
the control and autism groups and found a 200-fold increase in interferon-γ (IFNγ),
a 30-fold increase of TGFβ2, and a 12-fold increase in MCP-1 present in the subjects
with autism. While some of these features, such as increased IL-6 and IL-10, may
suggest a TH2 response, overall the findings are more consistent with proinflamma-
tory responses. Others researchers have also reported increases in proinflammatory
cytokine profiles in the blood of individuals with autism (Croonenberghs et al., 2002;
Jyonouchi, Sun, and Le, 2001; Molloy et al., 2006; Singh, 1996). The increased pro-
duction of cytokines, especially in the brain and CSF, points to an immune system
involvement in autism that may be relevant to the pathophysiology of autism.
14.3 GASTROINTESTINAL DYSFUNCTION IN AUTISM

Although no prospectively designed epidemiological trials have been conducted,
there is a growing body of clinical evidence that suggests that a subgroup of children
with ASD is prone to persistent gastrointestinal (GI) problems. The prevalence of GI
dysfunction in children with ASD reported by different studies varies greatly, with
existing data putting the overall rate anywhere between 18% and 91% (Afzal et al.,
2003; Horvath and Perman, 2002b; Horvath et al., 1999; Lightdale et al., 2001; Ming
et al., 2008; Molloy and Manning-Courtney, 2003; Niehus and Lord, 2006; Parracho
et al., 2005; Valicenti-McDermott et al., 2006). Although it has been suggested that
GI dysfunction is a more common complaint in children with ASD compared with
typically developing children, few studies have utilized adequate controls, making
it challenging to come to a definitive conclusion as to the true extent of GI dysfunc-
tion in ASD (Erickson et al., 2005; Kuddo and Nelson, 2003; Levy et al., 2007).
Despite this, however, it is clear that a substantial proportion of children with autism
present with abnormal GI function. Symptoms of GI dysfunction include persistent
diarrhea, chronic constipation, alternating bowel habits, vomiting, gaseousness and
bloating, abdominal distention, and pain, with the latter symptom often being severe.
In a retrospective evaluation of non-referred children with autism or ASD (n = 137),
24% had ≥1 GI symptom; diarrhea was the most commonly reported symptom
(12%, n = 17) (Molloy and Manning-Courtney, 2003). In another study, the incidence
of GI symptoms in 385 children with pervasive developmental disorders (PDD) was
compared with the incidence in 48 non-ASD siblings and 102 unrelated controls
(Melmed et al., 2000). Chronic GI symptoms were reported in 46% of children with
PDD: 19% had chronic diarrhea, 19% had chronic constipation, and the remaining 8%
alternated between diarrhea and constipation. In contrast, only 8% of healthy
siblings and 5% of unrelated controls had chronic diarrhea, and 10% of healthy sib-
lings and 5% of controls had chronic constipation. In a recent study of 150 children,
the lifetime prevalence of GI symptoms in ASD was 70% compared with 42% of
children with developmental delay but not ASD, and compared with 28% of typically
developing children (Valicenti-McDermott et al., 2006). The high rates of GI symp-
toms reported in this group may likely be due to a recording of both previous and
current GI symptoms. However, the analysis of more specific symptoms reported
was similar to those seen for other studies. For instance, 22% of children with ASD
and 8% of typically developing children reported chronic constipation, which were
similar to the frequency seen in the study by Melmed et al. (2000).
Children with ASD and GI symptoms are more likely to experience sleep distur-
bance, sudden irritability, unexplained crying, and aggressive behavior than children
with ASD but no GI symptoms (Horvath and Perman, 2002a). In their study, Horvath
and colleagues reported disturbed sleep and night-time wake up in 51% of children
with ASD who had GI symptoms, compared with 14% of children with autism but
no GI symptoms, and compared with only 7% of healthy siblings. The same research
team reported that 15.5% of children with ASD had reflux esophagitis, and that nearly
two-thirds of these children experienced sleep disturbances (Horvath and Perman,
2002b). They also reported that 43% of children who had abnormal esophageal histol-
ogy had episodes of unexplained daytime irritability compared with 13% of children
with ASD who had normal esophageal histology. Observations that GI symptoms may
peak before and are relieved following bowel movements suggest that some autism
behaviors may be exacerbated due to GI dysfunction (Ashwood et al., 2003; Wakefield,
2002). However, the significance of the relationship between more severe autistic
behaviors and the presence of GI symptoms in ASD requires further investigation.
Studies have focused on assessing the underlying pathology of GI dysfunction

in children with ASD. Initial research reported low α-1-antitrypsin concentrations in
children with ASD, suggestive of intestinal protein loss (Walker-Smith and Andrews,
1972). More recent endoscopic, histologic, and immunohistochemical analyses of
the GI tract in children with ASD who have GI complaints has revealed the pres-
ence of a subtle diffuse inflammation of the intestinal tract. For example, in a study
of 36 children with autism and GI disturbance, Horvath et al. (1999) reported reflux
esophagitis in 69% of children, chronic gastritis in 42%, and chronic duodenitis in
67%. However, this study did not include a group of control patients for comparison.
In another study, Wakefield et al. (2000) performed ileocolonoscopies and biopsies
on 60 children with developmental disorders (50 diagnosed with ASD) and GI com-
plaints, and 37 developmentally normal controls who were being investigated for
possible inflammatory bowel disease (IBD). Significantly increased ileal and colonic
lymphoid nodular hyperplasia (LNH) and enterocolitis were reported in ASD chil-
dren compared with controls. There is the general impression that ileocolonic LNH
is a normal finding in children with GI complaints, and that age is a critical factor
in presentation (i.e., that LNH is more frequent in younger children) (reviewed in
Wakefield et al., 2005). Therefore, it might be regarded that LNH does not represent
a significant pathologic finding in autism. In order to address this issue, Wakefield
et al. (2005) assessed the prevalence of ileocolonic LNH in a large group of patients
with ASD (n = 148) and GI symptoms versus developmentally normal controls with
GI symptoms, with or without colonic pathology. The different groups of patients
assessed were well balanced, with equivalent median age and gender split. The study
found that ileocolonic LNH was more prevalent in children with ASD compared
with controls, regardless of whether or not the controls displayed colonic inflamma-
tion. Furthermore, there was no correlation between the presence of LNH in children
with ASD and the age at which the ileocolonoscopy was performed.
In addition to an increased prevalence of LNH, histology, immunohistochemistry,
and flow cytometry studies have consistently shown a subtle pan-enteric infiltration
of immune cells such as lymphocytes, NK cells, and eosinophils into the walls of the
GI tract in children with ASD, compared with typically developing children who had
GI symptoms but no GI pathology. These studies also showed differences in immu-
nopathology in ASD children with GI symptoms compared with typically develop-
ing children diagnosed with other GI diseases such as Crohn’s disease and ulcerative
colitis (Ashwood et al., 2003; Furlano et al., 2001; Torrente et al., 2002, 2004).
Furthermore, increased intestinal lymphocyte-mediated proinflammatory response
(e.g., increased CD3+ TNF-α + cells and CD3+ IFN-γ + cells) and reduced regula-
tory response (e.g., decreased CD3+ IL-10+ cells) were demonstrated in children with
ASD and GI symptoms compared with developmentally normal controls (Ashwood
and Wakefield, 2006; Ashwood et al., 2004). This data suggests that the infiltrat-
ing immune cells may be activated and could contribute to inflammatory processes
within the GI tract. Interestingly, in addition to showing the increased immune cell
infiltration of the gut mucosa in children with autism, Torrente et al. (2004) have
shown a co-localization of immunoglobulin G/complement C1q deposition on the
surface epithelium of the GI tract, which not only indicates chronic inflammation but
also suggests an autoimmune component to the inflammatory response seen.
These studies, therefore, seem to demonstrate the presence of an underlying

subtle chronic inflammatory process in children with autism and GI disturbance,
characterized by LNH and enterocolitis, and the mucosal infiltration of immune
cells along the length of the GI tract. It is also clear from these studies that endoscopy
and histological studies alone may not be sufficient to detect these subtle changes
and that more sophisticated and sensitive immunohistochemistry or flow cytometry
methods are needed.
14.4 DISCUSSION: POSSIBLE LINKS BETWEEN

IMMUNE DYSFUNCTION, GASTROINTESTINAL
DYSFUNCTION AND AUTISM
The GI tract represents one of the primary gateways between the internal and external
environments, allowing nutrients to enter the body. Likewise, it must also provide a
robust barrier against pathogens. A compromise in the integrity of this physical and
immunologic barrier can have major consequences. Increased intestinal permeabil-
ity (sometimes referred to as “leaky gut”) may result from impairment in the integ-
rity of the mucosal barrier. D’Eufemia et al. (1996) reported increased intestinal
permeability in almost half the children with ASD enrolled in their study compared
with no change in healthy controls, despite deliberately excluding from enrollment
any child with autism showing clinical signs of G1 disease. This suggested dam-
age to the gut mucosa in children with autism with no obvious signs of GI disease.
An increase in GI permeability shown in some children with autism has provided
support for the “opioid excess” theory, a theory based on an observation that some
symptoms of ASD (such as social withdrawal, insensitivity to pain, and repetitive,
and stereotyped behaviors) are similar to those found in morphine addicts (Kalat,
1978; Panksepp, 1979). It has been proposed that exogenous opioids from foods may
contribute to behavioral and communicative dysfunctions in ASD, and gliadiomor-
phins and β-casomorphins derived from gluten and casein have been put forward as
potential candidates (White, 2003). Although there are reports, mostly anecdotal, of
improved behavioral effects following the elimination of gluten and casein from the
diet, it has not been rigorously assessed as a therapy (Hyman and Levy, 2005). While
only a limited number of trials have been completed, gluten and casein–elimination
trials have been reviewed (Christison and Ivany, 2006; Millward et al., 2008), and
while the authors noted serious design flaws of the studies, the majority of the stud-
ies reported positive behavioral effects from the diets. The most difficult aspects of
the rigorous scientific validation of these diets include placebo controls and care-
giver bias. To reduce this bias, new studies examining child scores on standardized
tests of cognitive function and behavioral assessment should be utilized rather than
relying extensively on caregiver perceptions of behavioral changes. The most con-
trolled study to date was performed by Knivsberg, Reichelt, and Nodland (2001),
which followed children matched for age and cognitive function who were placed
on regular or elimination diets for 1 year. The major advance of this study was that
the subjects were assessed by standardized testing at the end of the study period,
and the testers were blind to the treatment group of the subject. While they found no
benefit to verbal functioning, they reported significant improvements within the diet
group in total impairment, including attention, nonverbal cognition, motor skills,

and overall behavior. Furthermore, a preliminary study was reported that attempted
a double-blinded clinical trial to assess the efficacy of the gluten and casein–free
diet (Elder et al., 2006). Food was prepared by a dietician to eliminate caregivers
and researchers from identifying the study group each child was in. The children
were either placed on a regular diet or modified diet for 6 weeks and then switched.
Unfortunately, only 13 children completed the study, so results were statistically
insignificant. However, it was noted that there were some children whose verbal
communication skills appeared to improve on the diet and regress off the diet. These
results are promising in that they show the viability of a double-blind trial of this diet;
however, it is unlikely that a short 6 week diet would result in the marked improve-
ment of cognitive and motor functioning. Another factor that must be considered is
whether changing to any structured well-balanced diet would have a positive effect
in these children. Indeed, it has previously been shown that dietary modification
including but not exclusively gluten and casein–free diets resulted in improvement
in GI inflammation (Ashwood et al., 2003). These studies need to be replicated to
see whether a specific dietary change or a more global shift to a well-balanced diet
would be beneficial in addressing GI immunopathology and behavior in children
with ASD and GI symptoms.
The lining of the GI tract contains a host of components important for a nor-
mal immune response (T-lymphocytes, B-lymphocytes, macrophages, neutrophils,
antibodies, etc.), in close proximity to the gut lumen. Increased GI permeability
could trigger an adverse immune response and inflammation in the gut, which in
turn might trigger a change in behavior. Furthermore, an impaired gut barrier may
explain the highly activated immune profiles seen in the GI tract of children with
ASD (Ashwood and Wakefield, 2006; Ashwood et al., 2004). In one study, com-
paring the intracellular cytokine staining of peripheral blood T cells from healthy
controls, children with ASD, and children with Crohn’s disease, increases in the
inflammatory cytokines IFN-γ and TNF-α, pre- and poststimulation, were observed
in children with ASD who had GI symptoms compared with controls (Ashwood and
Wakefield, 2006). Similar findings in children with ASD have been reported else-
where (Jyonouchi, Sun, and Le, 2001; Jyonouchi et al., 2005). Although the T-cell
cytokine profiles seen in ASD children were different from typically developing
noninflamed normal controls, they were not the same as those seen for established
IBDs such as Crohn’s disease. This is important as the data suggest that the cytokine
profiles observed in ASD may be unique. Moreover, these findings suggest that there
may be intrinsic defects of the immune response in ASD children with GI symptoms
that are not seen in developmentally normal children without GI symptoms. It is plau-
sible that increased intestinal permeability in some children with autism, as shown
by D’Eufemia et al. (1996), may result in increased exposure to normally innocu-
ous antigens (e.g., food proteins and typical gut microflora), which may result in
increased immune activity leading to inflammation that could affect behavior. Using
a rodent model of intestinal bowel disease, Welch et al. (2005) reported increased
activity in certain brain areas that are abnormally active in patients with autism fol-
lowing the induction of GI inflammation, and may suggest that the initiation of GI
symptoms could affect behaviors
Although GI disturbance may be a comorbidity of autism, such findings have led

to growing interest in the hypothesis that GI dysfunction may have an integral role
in the etiology of ASD, especially in the regressive phenotype (Wakefield, 2002).
Although symptoms such as irritability, aggressive behavior, and sleep disturbance
are not part of the DSM-IV criteria for autistic disorder, they are common in chil-
dren with ASD and GI symptoms (Horvath and Perman, 2002b), and the paren-
tal reporting of behavioral changes and GI symptoms may occur at approximately
similar times (Lightdale et al., 2001). Additional support for a hypothesis linking
neurodevelopmental abnormalities and GI dysfunction in autism comes from analo-
gous findings of behavioral and neurological problems in other diseases character-
ized by GI dysfunction. For example, an untreated celiac disease is the result of an
overt immune response to dietary gluten and is associated with the inflammation
of the intestinal mucosa. Asperger (1961) reported that children with celiac disease
also often exhibited psychological problems. Anxiety and depression have also been
reported in adult patients with celiac disease (Hallert and Derefeldt, 1982). Cooke
and Smith (1966) reported on the neurological disorders found in adult patients with
celiac disease; these included severe progressive neuropathy, gait ataxia, and limb
ataxia. Postmortem analysis revealed extensive inflammatory changes in the CNS,
with a notable loss of Purkinje cells and gliosis in the cerebellum. A review of reports
from 1964 to 2000 showed that peripheral neuropathy and ataxia were the most com-
mon neurological disorders, with other less common manifestations including myel-
opathy and dementia (Hadjivassiliou, Grunewald, and Davies-Jones, 2002). There is
evidence that disturbances such as ataxia, epilepsy, and depression are also associ-
ated with celiac disease in children and adolescents (Fasano and Catassi, 2005).
It has also been reported that patients with disorders such as irritable bowel syndrome
(IBS) and Crohn’s disease often have psychiatric symptoms (Ringel and Drossman,
2001, 2002). Research suggests that the symptoms of IBS are caused or maintained
by an activated immune system. Increased gut permeability, increased numbers of
T-lymphocytes, and enteroendocrine cells in the mucosa are reported in patients with
IBS following gut infection (Spiller et al., 2000). There are also reports of elevated
intestinal permeability in other diseases characterized by intestinal inflammation
and/or abnormal immune response, such as celiac disease (Bjarnason, Peters, and
Veall, 1983; Cummins et al., 1991).
In addition, a number of disorders involving the CNS have been associated with
immune dysfunction. For example, immune responses to some tumor-derived antigens
can trigger rare neurological syndromes (i.e., paraneoplastic syndromes). The anti-
bodies raised against the tumor are thought to cross-react with neuronal antigens in
the CNS causing neurological dysfunction (Lang, Dale, and Vincent, 2003). Some
patients with lung cancer experience paraneoplastic degeneration of the cerebel-
lum; in a large proportion of these patients, raised levels of antibodies to voltage-
gated calcium channels have been found (Graus et al., 2002). Another example is
Rasmussen’s encephalitis, a progressive inflammatory childhood disease affecting
the cerebral cortex, causing intractable seizures, hemiparesis, and decreased motor
and cognitive function. It has been suggested that this disease might be autoim-
mune in origin, as antibodies to the ionotropic glutamate receptor type 3 have been
demonstrated in some patients (Rogers et al., 1994). Stiff person syndrome (SPS)
is also a disease that displays an autoimmune pathology. This disease is charac-

terized by severe progressive muscle stiffness and spasms triggered by external
stimuli. Depression and anxiety are common comorbidities, and psychiatrists are
often the first to recognize the disease following a referral for psychiatric evalua-
tion (Murinson and Vincent, 2001). Highly specific autoantibodies to the enzyme
glutamic acid decarboxylase are found in most SPS patients, and it is believed that
these inhibit the synthesis of GABA. Plasma exchange and intravenous immuno-
globulin treatment improve symptoms in patients receiving them (Murinson and
Vincent, 2001). There is also some circumstantial evidence that anti-basal ganglia
antibodies may be involved in the pathogenesis of tics (e.g., Tourette’s disorder) and
pediatric obsessive-compulsive disorder. Bacterial infection as a potential cause of
this autoimmunity has been hypothesized, and is implicated in pediatric autoim-
mune disorders associated with streptococcal infections (reviewed by Hoekstra and
Minderaa, 2005). However, the precise involvement of autoimmunity in these motor
and psychiatric disturbances remains open to debate and further research is needed.
Although there is much that remains unknown in this area, such examples suggest
that immune dysfunction can have profound effects on the CNS/brain function and
may impact the patient’s behavior.
The cause of the abnormal GI dysfunction in children with ASD is unknown.
However, interest has been raised in the potential role of zonulin, a protein, which is
likely to be involved in the regulation of gut permeability (Fasano and Shea-Donohue,
2005; Fasano et al., 2000; Wang et al., 2000). The epithelial cells of the intestinal
wall form a semipermeable barrier, and their function is to regulate the passage of
molecules from the lumen into the body. The sites at which the epithelial cells adjoin
each other form “tight junctions,” which act as a gateway allowing molecules to
pass through (Fasano and Shea-Donohue, 2005; Liu, Li, and Neu, 2005). The study
by D’Eufemia et al. (1996) indicated that increased GI permeability in children with
ASD occurred through the paracellular pathway (i.e., increased passage of mole-
cules between the epithelial cells). Tight junctions are formed by a meshwork of
proteins; it is thought that zonulin may induce the disassembly of these proteins,
allowing increased permeability (Fasano, 2001; Fasano and Shea-Donohue, 2005).
Interestingly, zonulin expression is raised in intestinal tissue during the acute phase of
celiac disease (Fasano et al., 2000). In addition, a recent study in diabetic-prone rats
suggests that the zonulin-mediated loss of intestinal barrier function may be involved
in the pathogenesis of type I diabetes (Watts et al., 2005). It is possible, therefore,
that some intrinsic defect in the normal functioning of zonulin may be a contributory
factor in the increased permeability of the GI tract in some individuals with ASD.
However, this remains to be proven.
Another theory as to the role of GI inflammation and neurological in flam-
mation involves increased serotonin levels seen in the blood of approximately
30% of ASD patients (Anderson, 1987; Stahl, 1977). Serotonin, also referred to as
5-hydroxytryptamine (5-HT), is a neurotransmitter, as well as an immunomodulator.
Certain forms of 5-HT are stored and released from enterochromaffin cells of the gut
epithelium, an action important for the nerve cells lining the small and large intes-
tines. Increased levels of 5-HT in the periphery have been associated with IBD, and
use of serotonin reuptake inhibitors result in the alleviation of GI symptoms in many
IBD patients. A correlation between high serum 5-HT and decreased 5-HT recep-
tor sensitivity with the extended HLA haplotype B44-DR4, as well as complement
C4B null allele, has been reported in ASD (Warren, 1996). Furthermore self-reactive
antibodies to 5-HT receptors were identified in the serum of ASD patients (Cook
and Leventhal, 1996; Todd and Ciaranello, 1985). This is consistent with findings
of 80% loss of 5-HT binding in mice upon the addition of sera from ASD patients
(Singh, Singh, and Warren, 1997). Genetic disequilibrium at the 5-HT transporter
gene (SLC6A4) has also been reported in ASD individuals in two separate studies
(Cook et al., 1990; Kim et al., 2002). In healthy individuals, approximately 95%
of peripheral 5-HT is produced in the GI tract with the rest produced in the brain.
Increased levels of 5-HT in the brain have been associated with increased levels of
destructive and aggressive behaviors, while increased levels in the GI tract are
associated with IBD. Furthermore, the usage of 5-HT reuptake inhibitors risperi-
done, clomipramine, and fluvoxamine has been shown to decrease certain aberrant
behaviors, such as anger, compulsive behavior, and ritualized behavior, in ASD indi-
viduals (Gordon et al., 1993; Hellings et al., 1996, 2006; McDougle et al., 1998).
The possible links between GI tract inflammation resulting in, or being caused by,
increased local 5-HT are compelling based on the connection between serotonin, the
immune system, and the nervous system. However, 5-HT in ASD with GI symptoms
has not been extensively studied.
How might increased GI permeability, abnormal immune response, and GI
inflammation trigger behaviors associated with ASD? Perhaps the most plausible
factor may be that of cytokine action. Cytokines and their receptors are distrib-
uted throughout the brain and are known to influence neural development, synaptic
transmission, and behavioral traits, and have been implicated in autism as well as
schizophrenia (Dunn, 2006; Larson, 2002; Licinio, Kling, and Hauser, 1998; Meyer
et al., 2006; Muller and Ackenheil, 1998; Nawa, Takahashi, and Patterson, 2000;
Smith et al., 2007; Tohmi et al., 2004; Vargas et al., 2005; Vereker, O’Donnell, and
Lynch, 2000; Yamada et al., 2000). Importantly, the immunocytochemical analysis
of brain tissue from patients with autism showed the marked activation of micro-
glia and astroglia, most notably in the cerebellum. Cytokine profiling indicated that
MCP-1 and TGF-β1, derived from neuroglia, were the most prevalent cytokines in
brain tissues (Vargas et al., 2005). The authors suggested that the activation of the
CNS innate immune system leading to cytokine release may play a role in the patho-
genesis of the disease. It is possible that altered levels of peripheral cytokines occur
as a result of immune dysregulation in the GI tract. These altered peripheral cytokine
levels may subsequently act directly on neurons within the CNS or may trigger a
CNS-mediated inflammatory response via glial cells. These actions could in turn
affect neurodevelopment and/or elicit behaviors associated with ASD.
Autoimmune responses may also be important in impairing GI function in ASD
and may be another mechanism relevant to ASD pathophysiology. It is possible that
antibodies toward neuronal tissue seen in some patients with ASD may be generated
as a result of secondary inflammation in the CNS. However, it is not known whether
these autoantibodies are pathogenic and are involved in the generation of behaviors
associated with ASD. Vojdani et al. (2002) demonstrated that antibodies against
many neuronal proteins in ASD individuals also cross-react with butyrophilin,
Chlamydia pneumoniae peptide, and Streptococcus M proteins. This is interesting

as some evidence of autoimmune etiology for many neurologic diseases (e.g., mul-
tiple sclerosis and myasthenia gravis) have suggested that “molecular mimicry” may
be a key component. Molecular mimicry is defined as immunologic cross-reactivity
between foreign and self-antigens. Furthermore, in multiple sclerosis, cross-reactivity
has been shown between antibodies against the neuronal peptide myelin oligodendro-
cyte glycoprotein and butyrophilin, a protein found in milk (Guggenmos et al., 2004).
Similarly, the potential mechanisms for how molecular mimicry might result in neu-
ronal damage and/or behavioral effects have been reviewed in more detail elsewhere
(Ercolini and Miller, 2005). One plausible mechanism is that B cells may be found
in gut lymphoid tissues that recognize neuronal self-antigens (not normally present
outside the CNS). These cells form part of a neuroprotective mechanism, designed
to deal with antigens released following neuronal damage. Increased GI permeabil-
ity may lead to the increased exposure of these cells to foreign antigens that share
structural homology with self-antigens. This in turn may result in B cells recognizing
foreign antigens and generating cross-reactive antibodies. Although antibodies do not
normally cross the blood-brain barrier (BBB), there is evidence that inflammatory
cytokines may compromise the integrity of the BBB, allowing the antibodies to cross
into the CNS (Ercolini and Miller, 2005). Once within the CNS, these antibodies
may bind directly to the target tissue and disrupt normal function; alternatively, they
may activate macrophages, which could release mediators that in turn could alter the
function of the neuronal tissue. It should be noted that Vargas et al. (2005) showed
no deposition of IgG, IgA, or IgM immunoglobulins in neuronal or neuroglial cell
populations in specimens from brains of individuals with autism. However, the GI
status of these patients is not known, and one might assume that the patients included
in this study did not have GI inflammation. Therefore, it is not possible to rule out
the action of autoantibodies in patients with autism and GI dysfunction. Another
potential mechanism is the activation of auto-reactive T cells in the inflamed GI tract
of children with ASD. Antigen presenting cells in the gut mucosal tissue may present
sequence-homologous antigen to T cells, which in turn recognize both the foreign
peptide and “self” neuronal peptide. These T cells may upregulate adhesion mol-
ecules, allowing them to cross the BBB. Thus, T cells (including cytotoxic T cells)
could then target neuronal tissue expressing the “self” tissue, resulting in damage to
the CNS tissue. In addition, T cells may release inflammatory cytokines within the
target tissue; in turn, these cytokines may damage neuronal tissue directly or activate
further “self-reactive” immune cells to infiltrate the brain and potentially attack it.
The resulting damage to self-tissue from the actions of T cells and other immune
cells, directly or indirectly, could release further self antigen, thus instigating a self-
perpetuating cycle of autoimmune-mediated tissue damage (Figure 14.1).
In conclusion, although the etiology of ASD is not known in the majority of cases,
there appears to be a strong link between immune dysfunction and ASD in many
individuals; whether the immune dysfunction has a causal relationship to ASD is cur-
rently only speculative. However, in other diseases, neurological abnormalities and
immune dysfunction are closely linked. Furthermore, the association between ASD
and GI dysfunction in many children has been documented. It is not known whether
a genetic susceptibility leading to increased GI dysfunction or leading toward altered
Direct cytokine action in brain

Cross BBB Peripheral cytokines activate CNS
immune response
Neuronal damage mediated by cross-
reactive Ab΄s and/or T cells
Proinflammatory
cytokines (e.g., TNF-α
Molecular mimicry
and IFN-γ)
leading to cross-reactive
Ab΄s and/or T cells Regulatory cytokines
(e.g., IL-10)
Egress of Altered GI permeability

microbial/dietary Innate/adaptive immune
antigens dysfunction
GI inflammation
FIGURE 14.1 (See color insert following page 200.) Possible mechanism by which GI
dysfunction may trigger autistic behaviors in children with autism and GI disturbance.
immune responses, or indeed both, may be important etiologic factors in a sub-

group of children with ASD. The role of immune factors and potential mechanisms
influencing both GI dysfunction and neurodevelopment, including the onset of
autistic behaviors, needs to be studied further. However, research findings to date sug-
gest that therapies, which could improve GI dysfunction and/or immune dysfunction
in patients with ASD, may have a beneficial effect. Further investigation to explore
and to better understand the underlying immune mechanisms in ASD are warranted.
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15 Possible Mechanism
Involving Intestinal
Oxytocin, Oxidative
Stress, and Signaling
Pathways in a Subset
of Autism with
Gut Symptoms
Martha G. Welch1,2* and Benjamin Y. Klein1,2
1Division of Developmental Neuroscience,
Department of Psychiatry and 2Department of

Pathology and Cell Biology, Columbia University
Medical Center, New York, NY 10032, USA
CONTENTS
15.1 Introduction .................................................................................................300
15.2 OT Synthesis ...............................................................................................300
15.3 Expression and Activation of OTR ............................................................. 301
15.4 Oxytocin Levels and Oxytocin Receptor in Autism ...................................302
15.5 Autism and Oxidative Stress ....................................................................... 303
15.6 Possible Contribution of Oxidative Stress to GI Aspects of Autism...........304
15.7 Proposed Cellular Mechanism in Gut/Brain Signaling .............................. 305
15.8 Proposed Role of Low Oxytocin in the Generation
of an Autism Phenotype ..............................................................................307
15.9 Possible Implications for the Treatment of Autism
and Developmental Disorders .....................................................................308
References ..............................................................................................................309
299
15.1 INTRODUCTION
The working hypothesis of our laboratory is that developmental disorders such as
autism arise from the dysregulation of a unified gut/brain system, rather than origi-
nating in the brain alone. Indeed, 70% of autistic children suffer from at least one
chronic gastrointestinal (GI) symptom (Horvath et al., 1999), potentially sorting this
group as a subset of autism.
The question of oxytocin (OT) deficiency as a contributing factor in autism was
raised over 15 years ago (Modahl et al., 1992). Recent findings have established OT’s
importance in modulating stress and predict its importance to gut/brain develop-
ment. OT’s antistress (Uvnas-Moberg and Petersson, 2005) and anti-inflammatory
properties (Iseri et al., 2005; Petersson et al., 2001) are well known. OT is present in
mother’s milk. There is an early exposure of the GI tract to this peptide (Stefanidis
et al., 2008). OT antagonists alter gut motility (Monstein et al., 2004). To understand
how OT deficiency might contribute to the development of autism, one must almost
certainly examine stress physiology in the gut. At the cellular level, this means
oxidative stress.
Oxidative stress is defined as an imbalance between the generation of reactive
oxygen species (ROS) and the decreased antioxidant defense systems. We hypoth-
esize that autism stems from physiological stress, including oxidative stress, which if
unmodulated triggers a cascade of adverse interrelated autonomic, endocrinological,
neurological, and immunological reactions.
This chapter examines how the deficiency of OT combined with oxidative stress
in the gut might dysregulate a unified gut/brain network. It reviews evidence that OT
levels and signaling pathways downstream of the oxytocin receptor (OTR) may be
involved in the excitatory drive of gut/brain signaling and thereby in the pathogen-
esis of a subset of autism. It discusses the role of oxidative stress in autism and the
possible contribution of differential responses to hypoxia in preterm and full-term
infants. Finally, it presents a theoretical framework and cellular mechanism for the
modulation of gut/brain signaling by OT and discusses possible implications for
the treatment of autism and developmental disorders, specifically those with comor-
bid GI symptoms and/or inflammation.
15.2 OT SYNTHESIS
Initially, the site of OT synthesis was considered to be restricted to the hypothalamus,
where it is synthesized as a preprohormone (Brownstein, 1983). The preprohormone
is subsequently cleaved into two bioactive peptides: OT close to the N-terminus and
neurophysin from the C-terminus (Gainer et al., 1977a). The OT and neurophysin
precursors are converted into bioactive peptides during axonal translocation to
the neurohypophysis (Gainer et al., 1977b). Like several other neuropeptides, OT
becomes bioactive and resistant to degradation by having its C-terminus amidated
(Prigge et al., 1997). Additional tissues have been reported as sites of synthesis and
secretion of OT. The mRNA and OT peptide were found in vasculature (Jankowski
et al., 2000) and cardiac myocytes consistent with its involvement in the control of
vessel tonus and the secretion of atrial natriuretic peptide (Gutkowska et al., 2000).
Possible Mechanism Involving Intestinal Oxytocin, Oxidative Stress 301
OT transcription has been detected in the chorio-decidual tissue, increasing

threefold during labor (Chibbar et al., 1993). OT is also synthesized in the uterus dur-
ing pregnancy (Mitchell et al., 1998). This peptide synthesis occurs under estrogen/
progesterone control in two forms, one with an extended C-terminus and very
low receptor binding activity, and the other with an amidated C-terminus, which
is fully active. Such amidation results in improved stability and receptor bind-
ing (Blankenfeldt et al., 1996; Fuhlendorff et al., 1990). During synthesis in the
uterus, OT is not coupled to neurophysin as it is in the hypothalamus (Fuhlendorff
et al., 1990). OT is also synthesized in myoepithelial and mammary epithelial
cells along with OTR (Cassoni et al., 2006). OT and OTR in the mammary gland
are active in milk letdown and in the trafficking of milk proteins to the Golgi
compartment during lactation (Lollivier et al., 2006). OT is thus present in the
milk and could potentially affect intestinal epithelium immediately after birth,
when OTR becomes expressed in the GI tract. OTR mRNA is expressed by the
cells in human amniotic fluid (Stefanidis et al., 2008) and since OT expression
in humans has been detected in the chorion, in the amnion, and weakly in the
placenta (Chibbar et al., 1993), one could expect that during fetal life, the GI epi-
thelial OTR gene is already being primed for expression by exposure to amniotic
OT. The presence of OT in meconium (Leake et al., 1981) further suggests early
GI exposure to the hormone.
Whether OT synthesis occurs in human embryonic gut is yet to be determined.
In adult humans, biopsies taken from intact GI tract tissue showed immunoreactivity
of OT in myenteric and submucosal nerve cells (Ohlsson et al., 2006). These biopsies
showed the presence of OT mRNA by polymerase chain reaction (PCR) and northern
blots (Monstein et al., 2004). This suggests that at least the GI and/or adjacent
tissues express the OT gene. These studies were done on GI tissue from aging
humans (seventh to ninth decades). However, if the newborn GI tract responds to
OT, then OTR is expected to be expressed in the enterocytes.
15.3 EXPRESSION AND ACTIVATION OF OTR

OTR is a seven transmembrane G protein-coupled receptor. The OTR gene promoter
contains a variety of cis elements responsive to estrogen receptor, cyclic adenosine
monophosphate (AMP) binding protein, and activator protein transcription factors
(Bale and Dorsa, 1998). The gene itself responds to interleukins and is under
epigenetic regulation by methylation and histone acetylation (Kusui et al., 2001).
OTR is expressed in tissues targeted by OT, for example, in mammary gland cells
(Cassoni et al., 2006), in cardiovascular tissue (Jankowski et al., 2000), and in the
uterus (Mitchell et al., 1998). Although transcripts of OTR have been identified
in elderly adult GI tissue (Monstein et al., 2004), its protein has not been detected
in intestinal epithelium by immunoreagents (Ohlsson et al., 2006). Thus, it is not
clear in which GI tract compartment the OTR is expressed in elderly humans. It
should also be noted that no data are available for the expression of OTR in human
neonates and young adults.
OTR activation by OT inhibits the growth of cells derived from neural, breast
epithelium, endometrium, and bone tissues (Cassoni et al., 2001a). In contrast,
OT induces growth in trophoblasts (Cassoni et al., 2001b) and small lung cancer
cells (Pequeux et al., 2004). The growth inhibition mediated by OTR was shown to
be transduced via cyclic AMP and protein kinase A, whereas growth stimulation
was transduced via Ca2+ flux and protein kinase C (Cassoni et al., 2001a). This dual
activity of OT could also be due to the differential activation of OTR at varying
concentrations of OT.
OT concentrations in breast milk increase above those in serum several days
postpartum (Leake et al., 1981). Therefore, one would expect the upper GI tract
epithelium of the normal breastfed neonate would be exposed to exogenous OT
and/or its fragments. If the contact between GI epithelium luminal surface and
OT has a physiological significance, then OTR could be expressed somewhere
along the GI tract, at least during the neonatal period if not during the entire
breastfeeding period.
Indeed, our laboratory has recently shown that OTR is expressed in the epithelium
of colon, in the small intestinal villi, in the muscularis mucosa, and in the myenteric
neuronal cells in rat newborns (Welch et al., 2009). We have also shown that OTR
expression is developmentally regulated. In fact, transcripts of both OT and OTR
mRNA peak at postnatal day 7 in rat pups. This pattern of OTR expression in the GI
tract could be induced either by endogenous OT from the gut or other sources and/or
by exogenous OT from breast milk.
We hypothesize from our experiments to date that the pattern of OTR expression
in the mucosal smooth muscles and the autonomous neural plexus implies develop-
mental induction. This expression may also imply the conditioning of gut/brain
sensory networks and later functional maintenance, for example, of peristaltic intes-
tinal movements. However, the biological functional advantage of OTR expression
in the absorptive enterocytes (villus epithelium) is not clear and its elucidation will
require further experiments.
The temporal changes of OTR in the villi suggest that OT/OTR is required for a
brief time window, perhaps to regulate the villus maturation process. In rodents, villi
develop during the first 2 weeks postpartum (Porter et al., 2002). Our data show that
after postnatal day 14, OTR gut expression in rat pups appeared in the crypts and
at the crypt–villus junctions where the immature precursors of the crypts develop
into functioning villus absorptive cells (Welch et al., 2009). Although this distribu-
tion is compatible with the possibility that OT/OTR signaling affects or regulates
epithelial maturation, other crypt cells that might also be regulated include Paneth
cells and secretory cells.
15.4 OXYTOCIN LEVELS AND OXYTOCIN RECEPTOR IN AUTISM

With the rationale that brain functions controlled by OT coincide with those
impaired in autism, OT nasal spray was used to treat autism: preliminary data
reported improvement in autistic adults’ repetitive behavior relative to placebo
(Bartz and Hollander, 2008). The tissue target of OT nasal spray is not known.
In any case, there is no indication that OT has acted via GI OTR. Still in question
is whether OT acts as mediator between gut and brain regarding specific features of
autism. Whatever the target tissue, it is evident that OT plays a role in autism.
There is additional evidence that OT is broadly important in development. OT

levels and OTR distribution have been found to correlate with long-term bonding
between monogamous and polygamous animals (Hammock and Young, 2006).
Single nucleotide polymorphism in the OTR gene ass ociated with autism supports the
importance of OT in the pathology of this syndrome (Wu et al., 2005). Interestingly,
abnormal newborn delivery may be associated with anomalies in OT secretion. For
example, delivery under local anesthesia (Krehbiel et al., 1987) or general anesthesia
during cesarean section (Marchini et al., 1988) is associated with low OT. These
modern in-hospital obstetric and pediatric procedures interfere with infant suckling
responses (Ransjo-Arvidson et al., 2001) and maternal milk let down (Matthiesen
et al., 2001), both of which are related to OT release. These challenges to perinatal
OT release could add to the risk of developmental disorders including autism.
15.5 AUTISM AND OXIDATIVE STRESS

The mechanisms underlying autism are yet to be defined. Autism is diagnosed based
upon various behavioral criteria such as impaired social interaction and communica-
tion, and repetitive behaviors. Current autism research is largely focused on identi-
fying genetic and biochemical abnormalities that will serve as diagnostic markers.
Such markers are needed to facilitate the highly complicated diagnosis of this disorder
and may also lead to understanding its etiology. Several genetic mutations have been
associated with autism spectrum disorders (Sebat et al., 2007), suggesting that these
behaviorally defined diseases share a disruption of a biological process rather than a
single mutation.
Oxidative stress can be identified in a subset of autistic children (Oliveira et al.,
2005). Numerous oxidative stress markers in autism were comprehensively reviewed
by Chauhan and Chauhan (2006) and McGinnis (2004). Lactic acidemia suggested
defects in mitochondrial oxidative phosphorylation and electron transfer complexes
(Filipek et al., 2003; Lombard, 1998). Other investigators found decreases in the ratio
of reduced/oxidized glutathione in the circulation, with abnormal methionine trans-
methylation and transsulfuration pathways (James et al., 2004, 2006). These reveal
a susceptibility to chronic oxidative stress in autistic children. Interestingly, parents
who lack the autistic phenotype have been shown to exhibit markers of susceptibil-
ity to oxidative stress that are similar to their autistic children (James et al., 2008).
These findings suggest that metabolic susceptibility to oxidative stress may be either
a contributor to autism or part of its mechanism. However, they are per se insufficient
to cause clinical manifestations specific to autism.
The presence of ROS and the lack of molecular scavengers that underlies suscep-
tibility to oxidative stress are nonspecific to any single disease. Therefore, one should
search for anomalies specific to autism that associate with oxidative stress. One such
anomaly is the progressive increase of brain volume in autistic infants (Courchesne
et al., 2003; Hardan et al., 2001), presumably resulting from cell packing in the
cortex. The histological basis of this phenomenon has been named “mini columns,”
referring to augmented connections between the neurons in cortical stratifications
(Casanova et al., 2006). In addition, brain blood perfusion is diminished in regions
important for the development of language capability (Ohnishi et al., 2000; Wilcox
et al., 2002). This finding raises the possibility that the hyper-connectivity between
neurons in the cortex associated with cell packing, together with reduced blood
perfusion, may result in hyperactive neurons on one hand and lower oxygenation
on the other. Such partial hypoxia could induce oxidative stress, forming ROS in
mitochondria. This sequence could exemplify a nonspecific marker resulting from
an autism-specific mini-column phenomenon.
A possible anomaly that could connect autism to oxidative stress is a disruption
of the secretion of OT hormone by the newborn hypophysis during labor. Areas
in the newborn brain undergo aglycemia and hypoxia during labor, which can
lead to neuronal death (Carbillon, 2007). Normally, neuronal death is inhibited by
OT-induced switch-off of neuronal firing by gamma aminobutyric acid (GABA) in
the brain (Cossart et al., 2006; Tyzio et al., 2006). However, a shortage of OT or the
dysfunction of its receptor might contribute to oxidative stress. Of interest to our
laboratory is whether the OT/OTR signaling system has a comparable cell-survival
function in peripheral organs, particularly the gut, which might be compromised
during episodes of perinatal hypoxia when blood flow may be preferentially distrib-
uted to the brain (Dyess et al., 1998). Also of great interest is the origin of OT regu-
lating the brain GABA switch. OT was shown to be of maternal origin (Khazipov
et al., 2008; Tyzio et al., 2008), but evidence suggests that endogenous fetal OT may
be induced by perinatal hypoxia (Carbillon, 2007).
It is logical to assume that the excitatory-to-inhibitory switch in GABA receptor
responses to stimulation may not occur by the time of birth in premature infants,
inasmuch as this process is normally completed near the time of full-term delivery
(Tyzio et al., 2008). Given the effects of OT on mechanisms regulating concentra-
tions of intracellular chloride, which in turn moves the GABA receptor bias toward
inhibition (Khazipov et al., 2008), we speculate that the up-regulation of the
premature infant OT system through high nurture may promote the maturation of
this important neurophysiologic process. If so, we would expect to find this evidence
in markers of neural function that would include the normalization of patterns of
electroencephalogram development as well as behavior.
15.6 POSSIBLE CONTRIBUTION OF OXIDATIVE

STRESS TO GI ASPECTS OF AUTISM
Several factors involving oxidative stress, such as low glutathione, prematurity, and
gut inflammation, could individually or collectively predispose an infant to devel-
opmental disorders such as autism. One major finding in autistic patients is low glu-
tathione levels in the circulation (James et al., 2004, 2006), which is a reflection of
its synthetic biochemical pathway. Glutathione is an important scavenger of ROS,
which is generated in excess by complex III of the mitochondrial electron transfer
chain under hypoxic conditions (Klimova and Chandel, 2008). Hypoxia during the
prenatal period could result in oxidative stress to the gut when cerebral and cardiac
blood flow is increased and small-intestinal blood flow is decreased.
During hypoxic stress in a premature animal model, critical brain and heart per-
fusion is maintained by the redistribution of blood flow away from skin, muscle,
and viscera including the gut. While small-intestinal perfusion returns to 97% of
baseline after hypoxia in full-term neonates, perfusion returns to only 50% in preterm
piglets; this differential gut response to hypoxia in full-term versus preterm animals
suggests a mechanism for increased risk of intestinal ischemic disease in preterm
human neonates (Dyess et al., 1998). Perinatal hypoxia also suggests a mechanism
that could contribute to the increased risk of autism with or without diagnosed GI
involvement in a vulnerable preterm population (Limperopoulos et al., 2008).
In addition to premature birth, inflammatory bowel disease can cause hypoxia
to the intestine (Hauser et al., 1988). Since OT has a protective effect against
hypoxia in the brain (Carbillon, 2007), one could reasonably predict its protective
effect against hypoxia in the gut. In such cases, the abnormally low levels of gut
OT could lead to chronic inflammation in gut colitis. Indeed, evidence supporting
this hypothesis may be provided in an organ system that synthesizes OT and OTR.
In a rat model of renal ischemia/reperfusion injury, OT protects against oxidative
stress (Biyikli et al., 2006).
Further evidence supporting the role of OT in colitis is provided by findings in our
laboratory. We have shown that experimental induction of colitis (oxidative stress)
by means of trinitrobenzene sulfonic acid elicited inflammation-related early gene
responses in central nervous system nuclei (Welch et al., 2005). Gut/brain signal-
ing measured by c-fos activation was inhibited by combined OT/secretin treatment
(Welch, unpublished results) in brain areas known to be abnormal in autism (Bauman
and Kemper, 1985, 2003; Buchsbaum et al., 2001; Chugani et al., 1997; Rumsey and
Ernst, 2000; Sparks et al., 2002; Vargas et al., 2005). Since intestinal inflammation
is associated with ROS production in the gut (Ardite et al., 2000), these findings
suggest that abnormal gut/brain interactions might be involved in the mechanisms
underlying autism.
15.7 PROPOSED CELLULAR MECHANISM

IN GUT/BRAIN SIGNALING
ROS has been shown to negatively regulate the Wnt-dependent β-catenin signal-
ing pathway by increasing neuroredoxin (Funato and Miki, 2007). Wnt-dependent
β-catenin signaling pathway is a major player in the control of enterocyte prolifera-
tion and differentiation. After birth, Wnt, which activates Tcf4-induced transcription
(Pinto and Clevers, 2005a), maintains a constant stem cell reservoir at the crypt–
villus junction. Thus, epithelial proliferation induced by Wnt provides a constant
supply of new absorptive enterocytes that migrate to the tip of the villi where they
undergo apoptosis every 3–4 days (Pinto and Clevers, 2005b). Concurrently, Paneth
cells migrate in the opposite direction to the bottom of the crypts.
The Wnt-dependent β-catenin pathway is balanced by the bone morphogenetic
proteins (BMPs). BMPs, secreted by intravillus mesenchymal cells, activate small
mother against decapentaplegic (SMAD) nuclear factors in the epithelium to
attenuate epithelial proliferation (Haramis et al., 2004). Remarkably, during crypt
development, intensities of crypt OTR expression coincide with crypt BMP4 expres-
sion found by others (Haramis et al., 2004). Given the dual effects of OT in activat-
ing or inhibiting cell proliferation (Cassoni et al., 2001a), it is possible that OT is
cooperating with BMP4 in the regulation of Wnt signaling in perinatal structural and
functional development of intestinal villi. Interestingly, BMP4 positively controls the

clustering of enteric neurons during development via the induction of extracellular
matrix protein–specific sialylation (Faure et al., 2007). This BMP4 activity occurs
contemporaneously with OTR protein expression detected in the enteric neurons of
newborn rats (Welch et al., 2009).
In addition to the absorptive enterocytes that line the villus surface, equally
relevant to our hypothesis are the Paneth cells that line the crypts. Biopsies show that
Paneth cells are larger and more numerous in autistic children than in normal con-
trols (Horvath et al., 1999). Paneth cells are part of the innate local immune system.
Like macrophages and granulocytes (Ganz and Lehrer, 1994), they provide defense
against microbes in the small intestine. When exposed to bacteria or bacterial anti-
gens, Paneth cells secrete a number of antimicrobial molecules into the lumen of the
crypt, thereby contributing to the maintenance of the GI barrier. Specifically, Paneth
cells contain granules that store precursors of bactericidal peptides, which upon
trypsin cleavage secrete defensins into the lumen to kill pathogenic microorganisms
(Ghosh et al., 2002). Defensins are even present in Paneth cells of germ-free animals
and therefore may have additional functions besides killing pathogens (Putsep et al.,
2000). According to Putsep et al (2000): “it is possible that on the crypt level, the
release of enteric defensins is induced by bacteria and bacterial components, whereas
on the organism level, neural or hormonal signaling could add additional levels of
regulation of defensin-producing Paneth cells.” We suspect that defensins may have
a central nervous system (CNS)-protective role during gut inflammatory-induced
oxidative stress, based on an in vitro defensin interaction with ganglionic neurons
(Plakhova et al., 2002), as discussed below.
Under conditions of hypoxia, Paneth cells could undergo the inhibition of
degranulation and the accumulation of defensin precursors, due to decreased
matrix metalloproteinase-7 (MMP7). The MMP-7 gene is a transcription target
of β-catenin/Tcf-1 transacting complex factors (Brabletz et al., 1999). This rela-
tionship is reflected by its over-expression in colon cancer (Ougolkov et al., 2002),
where it co-localizes with β-catenin accumulation in the nuclei of dark crypt precan-
cerous cells (Lu et al., 2008). MMP-7 has been shown to induce defensin secretion
in Paneth cells (Satchell et al., 2003), where it cleaves the active defensin from its
precursor (Weeks et al., 2006). It is interesting to note that autistic children with GI
inflammation have 50% more Paneth cells, that are also larger in volume, than do
controls (Horvath et al., 1999). Perhaps not coincidental is the fact that Paneth cells
in cases of necrotizing enterocolitis (NEC) in premature infants are also increased
twofold in number; despite an increase in human defensin 5 (HD5) mRNA, the
intracellular levels of HD5 peptide appeared constant, consistent with the possibility
that defensin mRNA translation is inhibited (Salzman et al., 1998). Additionally, as
we will postulate in autism (vide infra), it is possible that prodefensin is not being
cleaved to defensin. It would be important to examine the data from the cohort of
91 premature infants reported to meet diagnostic criteria for autism spectrum disor-
ders at age 2 (26%) (Limperopoulos et al., 2008) to correlate the relationship, if any,
between NEC and later autism spectrum disorder in that premature population.
It is not known how a change in defensin levels could be connected to the disease
mechanism and associated oxidative stress. However, an intriguing recent observa-
tion may provide a clue. It has been shown in rat dorsal ganglion neuronal culture
that sodium currents are attenuated by defensin interaction with the channel protein
(Plakhova et al., 2002). During chronic gut inflammation and associated oxidative
stress, one can postulate that the augmented firing of ganglionic afferent currents could
strongly stimulate nuclei in the brain. If such stimuli occur immediately after birth under
conditions that inhibit the attenuation of neuronal sodium currents, noxious stimuli may
persist chronically and greatly affect neuronal centers in the brain. The “readout” or
imprinting of such chronic and unattenuated intense stimuli could induce epigenetic
patterns of gene expression that result in an autistic epigenetically induced phenotype.
15.8 PROPOSED ROLE OF LOW OXYTOCIN

IN THE GENERATION OF AN AUTISM PHENOTYPE
Hypothetically, it is possible that in association with hypoxic oxidative stress of
GI inflammation, defensin diffusion to ganglion neural cells is greatly diminished
because low MMP7 expression reduces the processing of defensin precursors to
become active peptides. This could presumably inhibit the release of defensin, which
functions to attenuate the afferent noxious stimuli via ganglionic neuronal channels
(Figure 15.1) in addition to its canonical function as an antimicrobial peptide.
Two pathways could negatively regulate defensins. The first is BMP over-expression
and the second is dysregulated OT levels. BMP4 restricts enteric neuronal num-
bers and negatively regulates the Wnt pathway in GI epithelium during villus/crypt
s
nel
4 MMP-7 Defensin c han
Ion Na+ Brain
5 K+
Pro-defensin Enteric
Nucleus neuron
MMP-7
Paneth
cell
Hypoxia
3
1
β-Catenin
2 ROS
Wnt Gut inflammation

OT
FIGURE 15.1 Possible mechanism in Paneth cell for the modulation of gut inflammation
by OT in normal newborns. Hypoxia from gut inflammation stimulates the production of
ROS within Paneth cell (1). OT inhibits ROS and/or its effect (2), enabling a Wnt-dependent
β-catenin pathway (3). β-catenin in the nucleus transcribes MMP-7, which is then translo-
cated to the cytosol where it catalyzes the cleavage of prodefensin to activate α-defensin (4).
The active α-defensin theoretically permeates to enteric neurons to attenuate currents through
various ion channels (Na+ and K+), thus dampening signaling of hypoxia-induced noxious
stimuli to the brain (5).
s
nel
c han Brain
Ion Na+
K+
4
Pro-defensin Enteric
MMP-7 5 neuron
Nucleus
3
Paneth me
cell te a so
Pro 1
in
redox Hypoxia
β-Catenin e uro
N 2 ROS
Wnt Gut inflammation

Low OT
FIGURE 15.2 Possible mechanism in Paneth cell for the dysregulation of gut/brain signal-
ing under the condition of low OT levels in newborns at risk for autism. Hypoxia from gut
inflammation stimulates the production of ROS within Paneth cell (1). If OT level is inad-
equate to counter ROS production, ROS induces neuroredoxin, which in turn inhibits Wnt
pathway (2). As a result, β-catenin transcription factor becomes tagged by phosphorylation
(small red dot) and degraded by the proteasome (3). Without β-catenin in nucleus, MMP-7
gene is not expressed and defensin precursor peptide granulations (large stars) accumulate
within the cell (4). Without the attenuation provided by defensin, hypoxia-induced excitation
of ion channels in enteric neurons may drive noxious gut/brain signaling pathways (5).
development (Chalazonitis et al., 2008). Over-expression of BMP could inhibit Wnt

and result in intensive afferent stimuli that target brain areas abnormal in autism.
Hypothetically, if unchecked, this pattern of hyperstimulation could provide the
basis for an autistic phenotype with gut symptoms.
Alternatively, in the absence of the adequate OT inhibition of ROS, gut inflammation-
induced oxidative stress could lead to the same outcome as above; although in this
case, it would occur via the inhibition of the Wnt pathway by the ROS up-regulation
of neuroredoxin and then to a similar decrease in defensin expression with failure to
inhibit the excitation of ion channels in enteric neurons that drive afferent stimula-
tion to the brain (Figure 15.2). Moreover, the combined effects of abnormal BMP
expression together with impaired OT/OTR signaling in the presence of ROS from
gut inflammation or hypoxia could set the stage for gut/brain dysregulation in a subset
of autism with gut symptoms.
15.9 POSSIBLE IMPLICATIONS FOR THE TREATMENT

OF AUTISM AND DEVELOPMENTAL DISORDERS
The hypothesized role of oxidative stress and OT in the regulation of gut/brain sig-
naling pathways and in the generation of autism and developmental disorders raises
interesting questions about possible early interventions that could be effective in
the prevention of the diseases. Epigenetic imprinting by nurture might contribute

to the regulation of OT levels through activities that are known to stimulate the
synthesis and release of OT, such as breastfeeding, holding, and touching (Howard
et al., 2006; Uvnas-Moberg, 1996).
This idea is supported by animal models of nurture. Nurturing activities in
humans may be analogous to intense licking and grooming (L/G) in animal models:
L/G of pups by their high nurture mothers provides stress-attenuating afferent sensory
stimuli by demethylation of the steroid hormone receptor gene in an experimental
behavioral animal model of epigenetic imprinting (Champagne and Curley, 2009;
Champagne et al., 2006; Weaver et al., 2007). The offspring of high licking mothers
showed reduced stress profiles, which continued into adulthood.
If gut symptoms in autism are a precursor or marker of the disorder, then it is logi-
cal to propose early interventions aimed at the OT-related mechanism postulated in
this chapter. Such OT-related interventions would most certainly target activities that
are known to regulate OT levels in the newborn infant, for example, natural child-
birth without anesthesia (Krehbiel et al., 1987), mother/infant holding, breastfeeding,
and nurture (Howard et al., 2006; Uvnas-Moberg, 1996). Interestingly, the literature
supports the idea that the absence of these activities of mother/infant nurture is asso-
ciated with a high incidence of autism. For example, preterm infants are by necessity
socially isolated because of the life-sustaining controlled environment of neonatal
intensive care. Though a causal connection between such low nurture and autism
is yet to be proven, a high incidence (26%) of autism spectrum disorders has been
reported in preterm infants, as compared to normal children (0.7%) (Limperopoulos
et al., 2008). Yet another population suffering from very low nurture—Romanian
orphans from the 1980s and 1990s—is similarly associated with a high incidence
(one third) of developmental disorders including autism (Hoksbergen et al., 2005).
Future studies must confirm or refute our hypothesis. However, given the extent of
the autism burden we are facing, we believe there is sufficient evidence at this time
to justify further investigation of the interrelated roles of oxidative stress, OT, and
autism with GI involvement.
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16 Cytokine Polymorphisms
in Autism: Their Role
in Immune Alterations
Fabián Crespo,1,2,* Rafael Fernandez-Botran,3
Christopher Tillquist,2 Lonnie Sears,4
Meghan Mott,1 and Manuel Casanova1
Departments of 1Psychiatry and Behavioral Sciences,
2 Anthropology, 3 Pathology and Laboratory Medicine,
4 Pediatrics, University of Louisville, Louisville, KY 40292, USA
CONTENTS
16.1 Cytokine Biology: A General Introduction............................................... 315
16.2 Cytokine Polymorphisms and Cytokine Expression ................................ 317
16.3 Cytokines and the Brain ........................................................................... 318
16.4 Cytokines and Autism ............................................................................... 319
16.5 Why Study Cytokine Polymorphisms in Autism?
The Underlying Genetic Variability Hypothesis....................................... 320
16.6 Why Study Maternal Cytokine Polymorphisms? ..................................... 320
16.7 Discussion ................................................................................................. 321
16.8 Future Directions ...................................................................................... 322
References .............................................................................................................. 322
16.1 CYTOKINE BIOLOGY: A GENERAL INTRODUCTION

Cytokines are a diverse group of soluble regulatory proteins that function as mediators
in many physiological processes, including inflammation, immunity, and hemopoiesis.
As mediators, they regulate the migration, activation, proliferation, differentiation, and
function of the many types of cells involved in inflammatory and immune responses.
While essential to maintaining protective immunity, cytokines have been implicated
in autoimmune diseases and diseases with a chronic inflammation component (Borish
and Steinke, 2003).
* Corresponding author: Tel.: +1-502-852-2427; fax: +1-502-852-4560; e-mail: fabian.crespo@louis-

ville.edu
315
Cytokines are usually very complex in their activities. Most cytokines are highly
pleiotropic and act on many different cell types exerting different effects. Different
cytokines can have redundant effects, or their activities can be either synergistic or
antagonistic depending on the target cells. Normally, cytokines do not act alone, but
are produced in a cascade fashion, and it is their combined effect that determines the
type of response. The responsiveness of a cell to a particular cytokine is determined
by the expression of specific receptors for that cytokine. The binding of the cytokine to
its receptor then triggers the activation of a variety of signaling mechanisms that lead
to changes at the level of gene expression, resulting in transient- or chronic-altered
cellular proliferation, differentiation, and/or function.
Cytokines play an essential role in the regulation of inflammatory responses and
are involved in the regulation of both innate (natural) and acquired immunities.
The cytokines that function in innate immunity and inflammation are normally
produced by cells of the monocitic and myeloid lineages and several other types of
cells in response to microbial, chemical, physical, and other types of inflammatory
stimuli. The main goal of the cytokines in these responses is the localization and
elimination of the instigating insult, orchestrating the recruitment of both cells and
molecules (accomplished through increased vascular permeability and leukocyte
infiltration) at the local site and a variety of systemic responses that include fever
and acute-phase protein synthesis, and the mobilization of leukocytes from the bone
marrow. Among the main cytokines involved in these responses are tumor necrosis
factor α (TNFα), interleukin (IL)-1, IL-6, IL-12, type I interferons (IFN; IFNα and
IFNβ), and chemokines, a family of cytokines that function to mobilize and attract
different types of leukocytes to inflammatory sites. These cytokines are said to
have a “proinflammatory” activity. If cytokines are secreted in excess as a result
of an overwhelming infection, or in cases where the insult cannot be easily elimi-
nated or the stimulus for cytokine secretion persists, leading to chronic inflammation,
these same cytokines can have pathologic effects leading to the damage of healthy
cells and tissues. Because of its potentially serious consequences, the immune sys-
tem has mechanisms to prevent excessive inflammation, including cytokines with
“anti-inflammatory” activity. These cytokines include transforming growth factor
β (TGFβ) and IL-10, which antagonize many of the effects of the “proinflamma-
tory” cytokines mentioned above. It should be kept in mind, however, that certain
cytokines can have both pro- and anti-inflammatory effects depending on different
factors such as the cell and tissue type and the kinetics of release.
In the case of specific immunity, cytokines play an equally important role. In general,
the type of cytokines produced during an immune response determines the effector
mechanisms that predominate and major expression patterns have been character-
ized (Mosmann and Coffman, 1989). For example, different subsets of helper (CD4+)
T cells that differ in both their cytokine secretion patterns and the effector mechanisms
that they induce have been identified. Cells belonging to the Th1 subset secrete IFNγ,
IL-2, and TNFα/β and are primarily involved in cellular immunity mechanisms and
delayed-type hypersensitivity reactions; cells of the Th2 subset secrete IL-4, IL-5,
IL-10, and IL-13 and are primarily involved in humoral mechanisms and allergic-
type reactions; and cells of the newly described Th17 subset secrete IL-17, IL-22, and
a variety of other proinflammatory cytokines (Harrington et al., 2006). Th17 cells
are thought to be involved not only in immune responses to extracellular bacteria
Cytokine Polymorphisms in Autism: Their Role in Immune Alterations 317
but also in autoimmune diseases. Th1 and Th2 cells affect one another: Th1 cells
trigger macrophage activation using IFNγ, which inhibits the proliferation of Th2
cells, and Th2 cells secrete IL-10, which inhibits the secretion of IFNγ by Th1 cells.
In keeping with the need for balance in the immune system, a different subset of
T cells exists, namely T-regulatory cells (Treg), which act as negative regulators
of the activities of other subsets. These cells act, in part, through the secretion of the
“anti-inflammatory” cytokines TGFβ and IL-10 (Bettelli et al., 2006).
Both in inflammation/innate immunity as well as in specific immunity, the
maintenance of a balance between pro- and anti-inflammatory cytokines or among
the different CD4+ T-cell subsets and their cytokines is essential for homeostasis
and the proper function of the immune system. Disruptions in this balance can
have pathologic implications resulting in excessive inflammation and tissue dam-
age, increased susceptibility to infectious agents, and/or the emergence of autoimmune
conditions (Ollier, 2004). For example, abnormal levels of different cytokines have
been described in many diseases, such as autoimmune hepatitis, rheumatoid arthritis,
asthma, systemic lupus erythematosus, inflammatory bowel disease, and some brain
disorders like schizophrenia and Alzheimer’s disease (Kronfol and Remick, 2000;
Theoharides et al., 2004; Vitkovic et al., 2000). Given that cytokines are key components
in the homeostatic mechanisms regulating the immune system, it is not surprising
that variations in their structure at the genetic or protein level (qualitative) or their
production level (quantitative) have been found to be associated with disease pro-
cesses and/or susceptibility to infections.
16.2 CYTOKINE POLYMORPHISMS AND CYTOKINE EXPRESSION

Cytokines and their receptors are often encoded by highly polymorphic genes; and
these polymorphisms are responsible for the observed interindividual differences
in cytokine production and they likely impact the immune response (Hollegaard
and Bidwell, 2006; Keen, 2002a; Warlé et al., 2003). Cytokine genes and their
receptor genes are highly conserved in their exon sequences, while the majority of
polymorphisms are conserved polymorphisms in the non-translated regions of the
gene, located within the promoter, the introns, or the untranslated-3′ regions (Keen,
2002b). Cytokine polymorphisms continue to be discovered as mutation detection
techniques improved to map the extent of cytokine polymorphisms. Cytokines
that were once thought to be non-polymorphic, such as IL-2, IL-8, and IL-12, are
now being shown to have single nucleotide polymorphisms (SNPs), often within
the 5′ promoter regions (Keen, 2002a). Promoter region polymorphisms impact
the levels of protein expression in several ways. Polymorphisms within the 3′ or 5′
regulatory sequences can affect transcription by altering the structure of the bind-
ing sites for the transcription factors. Intronic polymorphisms can affect mRNA
splicing or the binding of enhancers and silencers (Keen, 2002a). As the amount
of data on cytokine polymorphisms increases and becomes available, there are a
growing number of studies that show an effect of cytokine gene polymorphism on
immune disease susceptibility, severity, and outcome (Keen, 2002b).
It is not surprising that different cytokine genotypes have been associated with a
large number of diseases; however, an important caveat must be noted with respect
to these putative associations. The vast majority of these studies analyze individual
SNPs or a series of SNPs in a particular cytokine gene in a relatively small sample

cohort and control group. A principal concern with this common study design is that
the distribution of genomic variation is a function of population history. The frequency
of the SNPs may vary among populations according to the ethnic or ancestry back-
ground of the disease and control group (Keen, 2002b). Furthermore, results from
the HapMap project and other linkage studies have demonstrated large linkage blocks
on chromosomes, and recent SNP-based analyses have demonstrated whole-genome
phylogeographic effects (Lao et al., 2008). The effect of geographic/genetic correla-
tion is that any given SNP showing an association with a disease may be one of many
associated SNPs—others were not detected because they were not part of the original
study design. The implication of this observation is that while there is a true statistical
association between the SNP under consideration and the clinical endpoint, there is
not necessarily an etiological relationship. This is not a novel observation, and work
using high-resolution linkage disequilibrium clarifies the situation. A complementary
approach to understanding the contribution of immune alterations to human disease
is to consider the immune system as a whole, that is, as a single functional pheno-
type. Investigating the immune response as a single phenotype requires assaying as
many functional polymorphisms as possible within a single cytokine gene (or group of
cytokine genes) along with the use of more appropriate control groups (Keen, 2002a;
Ollier, 2004). This approach will enable the detection of polymorphisms with signifi-
cant functional effects on the expression of the immune system, either in response to
environmental stimuli or as part of the developmental program.
16.3 CYTOKINES AND THE BRAIN

For many years in clinical research, the brain has been considered an immunologi-
cally privileged site, with the assumption that there is a reduced qualitative or dif-
ferent immune response compared to the systemic level. Evidence for this statement
includes the brain’s absence of appropriate lymphatic channels to capture antigens,
the blood-brain barrier protecting the brain from circulating blood, and the failure
to demonstrate the early invasion of macrophages and leukocytes. However, recent
developments in neuroimmunology have challenged this concept. Current data
have shown that the brain can exhibit many of the hallmarks of inflammation in
response to stress or injury, including edema, the activation of resident phagocytes,
the local invasion of the circulating immune cells, and the production of cytokines
(Kronfol and Remick, 2000; Vitkovic et al., 2000). Moreover, several studies have
reported associations between psychological stress, psychiatric illness, and cytok-
ines (Theoharides et al., 2004). Most cytokines can be synthesized and released
within the central nervous system and the majority is produced by microglia and
astroglia; there is also some evidence that neurons also produce cytokines under
certain conditions. Although cytokines are usually secreted in response to specific
stimuli, the low-level expression of specific cytokines appears to be maintained in
blood vessels within the brain (Licinio et al., 1998; Vitkovic et al., 2000).
Considerable progress has been made during the past few years in the under-
standing of the immunological mechanisms that control the immune surveillance
and the inflammation in the brain (Bauer et al., 2001). Cytokines in the brain are
also regulated in cascades through feedback loops, and cytokine receptors have been
detected in the brain (Kronfol and Remick, 2000). Besides providing communica-
tion between neural cells, specific cytokines have a significant role in signaling the
brain to produce neurochemical, neuroendocrine, neuroimmune, and behavioral
changes (Maes et al., 1995). There is suggestive evidence that this signaling is a part
of the comprehensive mechanism to mobilize resources to combat physical and phys-
iological stress in an attempt to maintain relative homeostasis. Because cytokines
are associated with central neurotransmitters and cytokine regulation is affected by
stress, many studies have investigated the possible role for cytokines in psychiatric
disorders. These studies have demonstrated the role of abnormal levels of cytokines in
major depression, Alzheimer’s disease, and schizophrenia (Hanson and Gottesman,
2005; Hopkins, 2007; Maes et al., 1995; McGeer and McGeer, 2001a,b).
16.4 CYTOKINES AND AUTISM

In the last 10 years, workers have reported that increased levels of some proinflam-
matory cytokines (TNF-α, IL1-β, IL-2, and IFNγ) are present in the peripheral blood
mononuclear cells (PBMC) of children with autism spectrum disorders (ASD), fuel-
ing the hypothesis that an abnormal immune response could be another component of
this multifactorial disorder (Chez et al., 2007; Cohly and Panja, 2005; Croonenberghs
et al., 2002; Gupta et al., 1998; Jyonouchi et al., 2001; Korvatska et al., 2002; Molloy
et al., 2006; Stubbs and Crawford, 1977; Vargas et al., 2005). In a cytokine analysis
at the systemic level, a specific increase of IFNγ and IL-12 was detected suggest-
ing a shift toward a Th1 lymphocyte response (Singh, 1996). This hypothesis of a
shift toward a Th1 lymphocyte-dominated response was reinforced with a recent
study where an increase of IFNγ was detected in 29 autistic children (Sweeten et al.,
2004). Moreover, the high levels of systemic IFNγ correlated with elevated circulat-
ing numbers of monocytes in a separate autistic population (Sweeten et al., 2003).
However, in an earlier similar study, no increase in IFNγ was observed (Stubbs
and Crawford, 1977). In another study, focusing on CD4+ and CD8 T cells, a shift
toward a Th2-like response was found in autistic children (Gupta et al., 1998). An
association of ASD and an abnormal immune response has support from data of
the increased prevalence of autoimmune disorders among first-degree relatives of
children with ASD (Kronfol and Remick, 2000), but this suggests a bias toward
a Th17-mediated response. The autoimmune component is likely diverse, varying
among autistics depending on genetic predisposition and environmental contingen-
cies (Lee et al., 2006; Vojdani et al., 2003; Wills et al., 2007). At present, it is not
possible to definitively ascertain characteristic perturbations in the relative balance
between Th1/Th2/Th17 responses. In autistic subjects, it is worth noting that some
of the seemingly contradictory evidence is likely the result of an incomplete under-
standing of the balance between major immune response regimes, as well as meth-
odological issues. With regard to the latter, measures of cytokine expression from
PBMCs estimates expression from all Th1, Th2, and Th17 lineages. It does appear
that autism has an inflammatory component—and contradictory results may reflect
the insufficient classification of autistic endophenotypes of variable etiology, age-dis-
parities between autistics and controls, and drug treatments (Ashwood et al., 2006).
It is not clear, however, whether an abnormal or exacerbated immune response is a

contributing factor for autism or simply a symptom of the disease.
16.5 WHY STUDY CYTOKINE POLYMORPHISMS IN AUTISM?

THE UNDERLYING GENETIC VARIABILITY HYPOTHESIS
Cytokine promoter polymorphisms affect cytokine expression levels; this has been
empirically demonstrated. In the current literature, studies have shown differences
in cytokines levels between different autistic populations. One potential reason for
those differences could be associated with population studies that compare young
ASD patients with adult controls, or populations that have a wide range of ages,
within patients and controls groups (Ashwood et al., 2006). But, contradictory
results obtained until present regarding cytokine expression in different autistic
populations could confirm the presence of different endophenotypes in ASD based
on cytokine genotypes. It is reasonable to infer that the combination of these dif-
ferent genotypes may bias a characteristic immune response, constitutively affect-
ing Th1/Th2/Th17 balance. In the context of an acute inflammatory episode, such
a bias may tip the balance toward a more vigorous—and potentially deleterious—
acute-phase immune response or induce a state of chronic inflammation. At this
time, we do not propose that an abnormal immune response due to specific cytokine
genotypes is necessarily the cause or one of the causes for autism. Moreover, we
do not have enough evidence yet, that an abnormal immune response is a contrib-
uting factor for autism or a simple by-product of the disease. However, growing
evidence is showing that autism may begin with genetic susceptibility or with spe-
cific environmental insults during brain development such as particular infections
or particular antigens in the diet; and a proinflammatory scenario associated with
an abnormal cytokine production would exacerbate the whole process. It may be
useful to hypothesize that there are phenotypes of the immune system predisposed
to stronger or weaker inflammatory immune responses or chronic inflammation
or autoimmunity, but that these phenotypes can manifest from several different
combinations of genotypes of different cytokines genes with variable expression.
To the extent that inflammation and autoimmunity are associated with autism, the
assaying of cytokine genotypes may permit distinguishing endophenotypes within
autistics. The combinations of specific genotypes of multiple cytokines may there-
fore be useful as markers for specific autistic endophenotypes.
16.6 WHY STUDY MATERNAL CYTOKINE POLYMORPHISMS?

Immune activation during pregnancy can impact the neurodevelopment of offspring with
far-reaching behavioral sequelae. It is well known that cytokine levels (IL-1β, IL-6, and
TNFα) are altered in human pregnancies complicated with infection (Depino, 2006),
and maternally generated cytokines can cross the placenta and enter the fetal circulation
(Zaretsky et al., 2004). Thus, it is possible that a maternal abnormal immune response
linked to infection or injury during pregnancy may be one factor for altered neurologi-
cal development in the fetus (Shi et al., 2003). During the last decade, new data have
reinforced this hypothesis, leading several researchers to include maternal infection/
injury as another risk factor for brain disorders like autism (Gilmore and Jarskog, 1997;
Juul-Dam et al., 2001; Maimburg and Vaeth, 2006; Patterson, 2002). The immune
activation of pregnant mice or rats using either lipopolysaccharide (a proxy for bacte-
rial infection) or polyriboinosinic polyribocytidylic acid (a proxy for viral infection)
results in modified cytokine expression in the maternal–fetal tandem pair (Gilmore
et al., 2005; Urakubo et al., 2001). Furthermore, the timing of the insult during ges-
tation is important with regard to the ultimate neurological and behavioral impact
(Smith et al., 2007).
This may have to do with the nature of the link between maternal and fetal immune
systems, and when the fetal immune system is capable of mounting an appropriate
response. It may be inferred that the timing and etiological origin of immune activa-
tion potentiates different neurological and behavioral fetal impacts. The variability
of the effect in both the timing of immune activation and time since activation is
likely linked both to the innate development of the fetal immune system and to an
altered Th1/Th2 balance in the mother. To our mind, variability due to cytokine
expression polymorphisms could either exacerbate or attenuate the degree of both
maternal and fetal immune activations.
16.7 DISCUSSION
Several studies demonstrate that an abnormal cytokine production is present in some
autistic populations, suggesting a more complex scenario where an abnormal immune
response is another component in the patient phenotype. If specific cytokine geno-
types associated with abnormal cytokine expression are linked to autism, this could
be understood as a genetic predisposition in the patient population. However, we must
be careful when trying to understand the meaning of “genetic predisposition” in the
development of this complex disease. Beyond the “cause or effect” paradox applied
to immune alterations in autism, we propose that specific genetic cytokine makeup
could be understood as a risk factor that will exacerbate any immune response that was
triggered by the multiple factors leading to the oxidative stress present in this disease
(Chauhan and Chauhan, 2006). Moreover, if the abnormal cytokine genetic makeup
is also present on the mother, since cytokines can cross the placenta, the convergence
of both phenotypes (mother and child) leading to an abnormal immune response could
significantly increase the risk—during pregnancy—for the development of autism.
Recent findings indicate that during the development of cortical and neuronal
organization, unknown factors influence neuronal and neuroglial cell populations,
disturbing neurodevelopment and producing the characteristic neuroanatomical
changes seen in autism (Vitkovic et al., 2000). This abnormality is best exemplified
in the significantly more narrow cortical minicolumns present in autistic individuals
when compared to controls (Casanova et al., 2006). As an effect of heterochronic
modifications to their developmental program, many autistic subjects have aug-
mented prefrontal cortices. This over-exuberant expansion of the prefrontal cortex
is by the addition of minicolumns. Clearly, there is population variability in cortical
size and minicolumnar density, and there are normal individuals with large brains
and densely packed minicolumns (and indeed autism has a familial component);
so why are some of these individuals autistic and others not? The manifestation of
emergent properties is dynamic, and their expression is a function of sufficient meta-
bolic supplies and active controls (Casanova and Tillquist, 2008).
Here, we argue that the crossing of the threshold leading a disruption of active
controls and impinging upon long-range neural networks, leading to autism, is mater-
nal and/or fetal immune activation. This may have been a single activation, but we
would like to propose the novel idea that maternal and/or fetal immune activation may
permanently alter the fetal Th1/Th2/Th17 balance, predisposing the fetus to a lifetime
of chronic inflammation (or susceptibility to inflammation) or autoimmunity issues.
A major underlying genetic contribution to the balance of Th1/Th2/Th17 populations
must be related to the expression of different cytokines involved in the immune cas-
cades generated during immune activation. Given expression polymorphisms in certain
key cytokine genes, some overall immune phenotypes will be predisposed for stronger
or weaker immune activation, contributing to the etiology or emergence of autism.
16.8 FUTURE DIRECTIONS

1. Pleiotropism and cytokine network considerations. Because of the well-
established pleiotropic and interactive nature of cytokines, we propose a more
comprehensive analysis with increased sample numbers and the consideration
of polymorphic haplotypes.
2. Cytokine expression baseline. Since cytokines can show a broad range of
expression levels affected by different factors as age, gender, ethnicity, and
annual seasons, we propose to determine the baseline of cytokine expression
levels in our populations for those cytokines showing a particular genotype/
haplotype for mothers and/or autistic children.
3. Paternal influence. Given behavioral similarities between autistic males
and their fathers, paternal genetic contribution is likely important for under-
standing autism; so we propose to ascertain whether a characteristic paternal
contribution can be detected in terms of cytokine polymorphisms.
4. Global survey of cytokine polymorphism in human populations. As it has been
detected for other genetic polymorphisms, cytokines SNPs show a heteroge-
neous distribution in human populations; so we propose, and are currently
conducting (Crespo et al., 2008), a global survey for cytokine polymorphisms
in different human populations.
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17 Autism, Teratogenic
Alleles, HLA-DR4,
and Immune Function
William G. Johnson,1,3,* Steven Buyske,4,5
Edward S. Stenroos,1,3 and George H. Lambert2,3
Departments of 1Neurology, 2Pediatrics, 3Center
for Childhood Neurotoxicology and Exposure
Assessment, Robert Wood Johnson Medical
School, University of Medicine and Dentistry
of New Jersey, Piscataway, NJ 08854, USA
Departments of 4Statistics and 5Genetics, Rutgers University,
New Brunswick, NJ 08854, USA
CONTENTS
17.1 Teratogenic Alleles in Neurodevelopmental Disorders............................. 326
17.2 Study Designs that Suggest or Document Action
of a Teratogenic Allele .............................................................................. 329
17.3 Early Examples of Teratogenic Alleles ..................................................... 330
17.3.1 Rh Incompatibility ..................................................................... 330
17.3.2 Maternal Phenylketonuria .......................................................... 331
17.4 A Rationale for Finding Teratogenic Alleles in Autism ........................... 332
17.5 HLA-DR4 and Autism ............................................................................... 332
17.5.1 Major Histocompatibility Complex ............................................ 332
17.5.2 Case–Control Studies of HLA-DR and Autism ......................... 332
17.5.3 Case–Parent Study of Autism .................................................... 334
17.6 Possible Modes of Action by which HLA-DR4
Might Contribute to Autism ...................................................................... 334
17.6.1 Oxidative Stress.......................................................................... 335
17.6.2 Synaptic Pruning and the MHC ................................................. 336
17.6.3 HLA-DR4 and High Relative Birthweight ................................. 337
17.6.4 Maternal Antibodies and Autism ............................................... 337
17.7 Future Studies of HLA-DR4 in Autism ..................................................... 337
References .............................................................................................................. 337
325
17.1 TERATOGENIC ALLELES IN

NEURODEVELOPMENTAL DISORDERS
Neurodevelopmental disorders are complex disorders in which multiple genes
contribute to the clinical phenotype and their effect is modified by environmental
factors. Genes contributing to neurodevelopmental disorders are usually thought of
as acting in the affected individual, i.e., the child or adult with the neurodevelopmen-
tal disorder. In the gene–teratogen model, an additional class of genes is considered,
maternal genes that act in the mother to contribute to the phenotype of their affected
offspring. The alleles of maternal genes that act in this way are termed “teratogenic
alleles” because their effect is in some ways similar to ingested maternal teratogens
that affect fetal development. Teratogenic alleles most likely act in the mother during
pregnancy to modify the development of the embryo or fetus, e.g., brain development
in the affected children, although action on the ovum or in the ovum before conception
is also possible. These maternal genes may interact with fetal genes and with envi-
ronmental factors. These fetal genes are termed “modifying or specificity alleles” in
the gene–teratogen model since they may modify the severity of the phenotype in the
fetus or determine which organ is affected. At present, at least 34 reports of teratogenic
alleles have been published (Johnson et al., 2008, Table 17.1; Johnson et al., 2009).
TABLE 17.1
Reports of Teratogenic Alleles
Teratogenic Allele
Report Disease Analysis By References
Maternal TDT
1. MTR*2756G NTD/spina bifida Mat TDT, log-linear Doolin et al. (2002)
2. MTRR*66G NTD/spina bifida Mat TDT, log-linear Doolin et al. (2002)
3. GSTP1*val105 Autism Mat TDT van Beynum et al.
(2006)
Maternal TDT-equivalent
4. Rh d Erythroblastosis Mat TDT equivalent Westgren et al. (1995)
fetalis
5. PAH mutations Maternal phe- Mat TDT equivalent Rouse and Azen (2004)
nylketonuria
6. Rh d Schizophrenia Mat TDT equivalent, Hollister, Laing, and
log-linear Mednick (1996),
Palmer et al. (2002)
Case–parent log-linear analysis without maternal TDT or equivalent

7. MTHFD1*653Q Spina bifida Log-linear Brody et al. (2002)
8. CYP1A1*6235C Low birthweight Log-linear Chen et al. (2005)
9. NAT1 composite NTD/spina bifida Log-linear Jensen et al. (2006b)
genotypes
10. CCL-2*2518AA NTD/spina bifida Log-linear Jensen et al. (2006a)
Autism, Teratogenic Alleles, HLA-DR4, and Immune Function 327
TABLE 17.1 (continued)

Teratogenic Allele
Regression analysis
11. APOE*E2 Lower LDL-C, apoB, Forward stepwise Descamps et al. (2004)
higher HDL-C, and regression analysis
apoA1 in newborns
12. APOC3*S2 Lower newborn Forward stepwise Descamps et al. (2004)
LDL-C, apoB, regression analysis
HDL-C, and apoA1
13. LPL*S447X Lower newborn Forward stepwise Descamps et al. (2004)
LDL-C, apoB, regression analysis
and TG
14. GSTP1*Val105, Asthma Multiple linear Carroll et al. (2005)
*Val114 regression
Case–control plus case TDT

15. MTHFR*677T, Oral-facial clefting, Case–control, case Martinelli et al. (2001)
CT, TT cleft lip ± cleft palate TDT negative
16. MTHFR 677T Congenital heart Case–control, case van Beynum et al.
disease TDT negative (2006)
17. MTHFR*677TT Down syndrome Case–control, case Rai et al. (2006)
TDT negative
18. HLA-DR*4 Autism Case–control, case Lee et al. (2006)
TDT negative
Case–control
19. C4B*0 Autism Case–control Warren et al. (1991)
20. HLA-DR4 Autism Case–control Warren et al. (1996)
21. GSTP1–1b Recurrent early Case–control Zusterzeel et al. (2000)
pregnancy loss
22. MTHFR*1298C NTD/spina bifida Case–control De Marco et al. (2001)
23. MTRR*66G *GG Down syndrome Case–control O’Leary et al. (2002)
24. MTHFR*677T/ Down syndrome Case–control O’Leary et al. (2002)
MTRR*66GG
25. GSTT1*0 Oral-facial clefting Case–control van Rooij et al. (2001)
26. MTHFR*1298C NTD/spina bifida Case–control Gonzalez-Herrera et al.
(2002)
27. GSTM1*0 Recurrent pregnancy Case–control Sata et al. (2003)
loss
28. DHFR*19bp Spina bifida Case–control Johnson et al. (2004)
deletion
29. CYP1A1*2A Recurrent pregnancy Case–control Suryanarayana,
loss Deenadayal, and
Singh (2004)
(continued)
TABLE 17.1 (continued)

Teratogenic Allele
30. DHFR*19bp Preterm delivery Case–control Johnson et al. (2005)
deletion
31. MTHFR*1298C, CC Down syndrome Case–control Rai et al. (2006)
32. MTHFR*1298C, CC Down syndrome Case–control Scala et al. (2006)
33. RFC1*A80G, GG Down syndrome Case–control Scala et al. (2006)
Source: Adapted and reprinted from Johnson, W.G. et al., Teratogenic alleles in autism and other
neurodevelopmental disorders, in Autism: Current Theories and Evidence, ed. Zimmerman,
A., Humana Press, Totowa, NJ, 2008. With permission.
Notes: Reports of teratogenic alleles are listed: (1) according to the method of analysis used and
(2) according to the date of the report beginning with the earliest. The specific teratogenic allele
is listed on the left, next the specific disease or disorder studied, next the study design, and
finally, the literature reference. The specific teratogenic allele is given in the nomenclature: gene
symbol*allele designation. The names of the genes corresponding to the gene symbol are given
in the text and correspond to the designation in Online Mendelian Inheritance in Man. Gene
symbols are given in uppercase letters and italicized while the corresponding protein symbol is
given in the same uppercase letters but not italicized. Sometimes a genotype is given, as in #31
and 32, where “MTHFR*1298C” refers to the C-allele at position 1298 of the gene “MTHFR”
and CC, the genotype, refers to a double dose of the C-allele, i.e., homozygosity. Human genes are
given in uppercase italics, corresponding proteins in the same uppercase letters that are not itali-
cized. MTR, methionine synthase gene; MTRR, methionine synthase reductase gene; GSTP1,
glutathione S-transferase P1 gene; NTD, neural tube defect; PAH, phenylalanine hydroxylase
gene; Rh, Rhesus factor, a protein that is either present or absent on the surface of human red
blood cells; MTHFD1, methylenetetrahydrofolate dehydrogenase gene; CYP1A1, cytochrome
P450 1A1 gene; NAT1, N-acetyltransferase gene; CCL, monocyte chemoattractant protein gene;
APOE, apolipoprotein E; APOC3, apolipoprotein C3; LPL, lipoprotein lipase; MTHFR, methyl-
enetetrahydrofolate reductase gene; HLA, human leukocyte antigen system; OFC, oral-facial
clefting; CHD, congenital heart defect; C4B, complement component 4B, a blood group antigen;
GSTT1, glutathione S-transferase T1 gene; GSTM1, glutathione S-transferase M1 gene; DHFR,
dihydrofolate reductase gene; and RFC1, reduced folate carrier 1 gene.
Nearly all of these reports of teratogenic alleles involved neurodevelopmental disor-

ders. Their number has more than doubled since the topic was last reviewed in 2003
(Johnson, 2003). It is possible that teratogenic alleles are a specific and even the
defining feature of neurodevelopmental disorders, but too little work has been done
so far to clarify their impact. Since only 34 reports of teratogenic alleles are known
at present, it is possible that these are rare compared with genes acting in affected
individuals themselves and have little overall impact. On the other hand, since the
number of reports of teratogenic alleles has more than doubled in the last 5 years, it
is possible that they are numerous and their ultimate impact may turn out to be quite
large. Clearly, more work needs to be done to assess their importance. In order to
identify more teratogenic alleles, this concept needs to be incorporated into study
designs at the beginning of a project.
17.2 STUDY DESIGNS THAT SUGGEST OR DOCUMENT

ACTION OF A TERATOGENIC ALLELE
To identify a candidate gene for a “modifying or specificity allele,” i.e., the genes
that act in the fetus in the gene–teratogen model, is straightforward because the
ordinary methods of linkage mapping are suitable and well developed, e.g., the lod
score method, sib pair methods, or standard case–parent methods. For this reason,
genes for neurodevelopmental disorders identified by these methods are likely to be
of this type.
However, identifying a candidate gene for a teratogenic allele is not straightfor-
ward since it is the mother of the proband with the neurodevelopmental disorder who
is the one that is affected, i.e., the “genetic patient” for a teratogenic allele. Pedigrees
collected for use with the lod score method often do not contain the right individuals
for study if the pedigree is simply re-coded so that the individuals with the neurode-
velopmental disorder are now “unaffected” and their mothers are “affected.” Sib pair
methods are also problematic for the study of a teratogenic locus, even if parents
are included, because a sib pair contains only one “affected” individual, the mother.
Likewise, in the standard transmission/disequilibrium test (TDT), carried out with
neurodevelopmental probands and their parents, the only affected individuals in the
family are the mothers.
The presence of a teratogenic allele may be first suspected when the examina-
tion of a dataset that contains probands, mothers, and fathers for a neurodevelop-
mental disorder in a case–control study of allelic association shows a significantly
increased frequency for an allele of interest among mothers but not fathers compared
to controls. Increased frequency in probands of an allele-of-interest is also to be
expected because the probands will often inherit an allele from the mother simply
by descent, whether or not the allele acts also in the probands. For a teratogenic
allele, the expected disease allele frequencies in family members are from highest to
lowest mothers > neurodevelopmental probands > fathers = controls. However, allelic
association studies are problematic since the allele may be subject to population
stratification. In that situation, the case families may not be matched to the controls
and the study may be invalid. Since there may be no data on population stratification
for that allele, it may not be realized that the study is invalid.
If maternal allele frequencies are increased and TDT analysis shows the increased
frequency of transmissions to probands from mothers but not fathers, then genomic
imprinting or mitochondrial transmission is likely; the allele should be investigated
to determine if it is subject to imprinting or if the gene is a mitochondrial gene.
However, if no increased frequency of transmissions is found, then the action of a
teratogenic allele is probable.
There are at present three major approaches for documenting the action of a sus-
pected teratogenic allele. One is to use TDT analysis for transmissions from maternal
grandparents to mothers as we and others have suggested (Johnson, 1999; Mitchell,
1997). Since the mother is the genetic patient for a teratogenic locus, the expectation
is that such transmission disequilibrium will occur. This approach has been success-
ful in a study of spina bifida (Doolin et al., 2002) (Table 17.1, #1 and 2) and a study
of autism (Williams et al., 2007b) (Table 17.1, #3). This approach works best for dis-
orders where the individuals with the neurodevelopmental disorder are young, since
living maternal grandparents can more readily be found. This approach is direct and
strongly supports the action of a teratogenic allele, but it does not address the interac-
tion between maternal and fetal genes.
A second approach is to use the case–parent log-linear method (Starr, Hsu, and
Schwartz, 2005; Weinberg, 1999; Weinberg and Wilcox, 1999; Weinberg et al., 1998;
Wilcox, Weinberg, and Lie, 1998). Since this method requires only the trio consisting
of the individual with the neurodevelopmental disorder and the two parents, it may
be the most suitable approach if living maternal grandparents are difficult to find. It
also addresses the question of the interaction between maternal and fetal genes. With
this method, the data is stratified by parental mating type and a modeling term is
included for maternal genotype. For example, a mother with AA genotype and father
with aa genotype represent the same mating type as a mother with aa and father with
AA, but the observation that the former pair occurs more often in parents of affected
offspring constitutes evidence that the AA genotype in mothers is a risk genotype for
offspring. There are reasons to believe that the case–parent log-linear method may
have less power than maternal TDT for the same number of families studied based
upon the one report that used both methods on the same dataset (Doolin et al., 2002)
(Table 17.1, #1 and 2).
A third approach uses “pents,” i.e., families with neurodevelopmental proband, par-
ents, and maternal grandparents (five individuals per family) (Mitchell and Weinberg,
2005). The pent approach has the advantage of estimating both maternal and offspring
genetic effects, and offers increased power, per proband, compared with the log-linear
approaches. Since DNA from maternal grandparents is required, as a practical matter,
it will work best for early onset disorders.
These three analytical approaches are not the only possible ones. Other approaches
have also been presented (Mitchell et al., 2005). Interestingly, some of the genes
identified in affected individuals by conventional approaches, especially allelic asso-
ciation studies, and thought to act in affected individuals may in fact be teratogenic
alleles. This is because these affected individuals frequently receive the teratogenic
alleles from their mothers simply by descent. These affected individuals will thus
themselves have an increased frequency of the teratogenic allele, as discussed ear-
lier, despite the fact that the allele acts in the mothers not in the affected individuals
themselves to contribute the phenotype of the affected individuals.
17.3 EARLY EXAMPLES OF TERATOGENIC ALLELES

Two neurodevelopmental disorders, Rh-incompatibility and maternal phenylketonu-
ria (maternal PKU), are perhaps the earliest disorders shown to result from the action
of teratogenic alleles, although the concept of teratogenic alleles was not recognized
at that time.
17.3.1 RH INCOMPATIBILITY
For Rh incompatibility to occur (Table 17.1, #4), the mother must lack the RhD
antigen on the surface of her erythrocytes and thus be Rh negative (RhD negative);
i.e., she must be a homozygote for the Rh d allele with the d/d genotype. Also the
fetus must carry an Rh D allele that can only be of paternal origin (father is RhD
positive). During the pregnancy, the Rh d/d mother is exposed to the RhD antigen
produced by the fetus. Since the mother lacks this antigen, she makes antibodies
to RhD antigen as the pregnancy progresses. During a subsequent pregnancy with an
RhD positive fetus, maternal antibodies are again produced but in greater amount and
in an accelerated fashion. With further RhD positive fetuses, the mother mounts an
immunological attack upon the fetus who may develop erythroblastosis fetalis and a
developmental disorder. This developmental disorder consists of three clinical syn-
dromes in the fetus and neonate: anemia of the newborn; neonatal jaundice that can
lead to the severe fetal encephalopathy of kernicterus; and the generalized neonatal
edema of hydrops fetalis with massive anasarca, pleural effusions, and ascites. The
teratogenic allele here is Rh d, for which the mother is a homozygote. Each of her
parents carries an Rh d allele and has transmitted an Rh d allele to her. Thus, trans-
mission disequilibrium is present. The mechanisms of fetal damage are unclear, but
cytokine abnormalities have been observed (Westgren et al., 1995).
Rh incompatibility has also been recently linked to another neurodevelopmental
disorder, schizophrenia (Table 17.1, #6). An increased incidence of schizophrenia
has been found in the offspring of pregnancies with Rh incompatibility (Hollister,
Laing, and Mednick, 1996) compared to control pregnancies. This effect was not dem-
onstrated for autism in a similar study (Zandi et al., 2006).
17.3.2 MATERNAL PHENYLKETONURIA

Maternal PKU results from the major intrauterine effect on fetuses of mothers with
PKU. PKU itself is a recessive postnatal disorder. Untreated homozygous PKU moth-
ers and fathers both have elevated blood phenylalanine. However, the heterozygous
offspring of untreated PKU mothers may develop maternal PKU, a disorder different
from PKU, that has an abnormal developmental and neurodevelopmental phenotype
(Abadie et al., 1996; Allen et al., 1994; Koch et al., 1994). Nearly all cases of PKU
and hence maternal PKU are known to result from mutations in the phenylalanine
hydroxylase (PAH) gene. Thus, the mutations in the maternal (PAH) gene, through
the resulting elevation of maternal blood phenylalanine or other metabolite(s) in
the untreated PKU mother, act during pregnancy as teratogenic alleles for the fetus
(Table 17.1, #5).
Infants with PKU (Menkes, 1990) are normal at birth and develop a progressive
metabolic disorder of postnatal onset characterized by vomiting, eczema, mental
retardation, and infantile spasms with hypsarrythmia on the electroencephalogram.
In contrast, infants with maternal PKU (Menkes, 1990) have a congenital nonprogres-
sive disorder of fetal onset characterized by microcephaly, abnormal facies, mental
retardation, congenital heart disease, and prenatal and postnatal growth retardations.
The teratogenic effect in maternal PKU is not dependent upon the fetal genotype,
although the fetus is an obligate heterozygote since the mother is a homozygote for
PKU and the father (usually) has the normal genotype.
Since PKU mothers of maternal PKU offspring are homozygotes for PAH muta-
tions, each maternal grandparent is a heterozygote who has transmitted the mutant
allele to the mother. Thus, if a maternal TDT was carried out in these maternal trios
(maternal PKU, mothers, and their parents), it would show transmission disequi-
librium. However, since the maternal PKU mothers have PKU and are thus known
to be homozygotes for PAH mutations, such a TDT is now unnecessary. Thus, this
documentation of the action of a teratogenic allele in maternal PKU is, in a sense, the
equivalent of a maternal TDT.
17.4 A RATIONALE FOR FINDING TERATOGENIC

ALLELES IN AUTISM
Children with autism have a change in the normal developmental pattern with
impaired social interactions and communication, restricted interests, and repetitive,
stereotyped patterns of behaviour that can be observed before 36 months of age
(American Psychiatric Association, 1994; Rapin, 1997). Clinical genetic studies
suggest the presence of multiple gene loci that interact with each other (Muhle,
Trentacoste, and Rapin, 2004; Szatmari, 2003) and possibly with environmental
and epigenetic factors (Lawler et al., 2004; Szatmari, 2003) that may contribute to
autism. Some of these contributing factors appear to be immune abnormalities.
Neuropathological studies (Rodier et al., 1996; Stromland et al., 1994), cytoarchi-
tectonic studies (Piven and O’Leary, 1999), and minicolumn studies (Casanova et al.,
2002; Casanova, Buxhoeveden, and Gomez, 2003) all support the prenatal origin
of certain brain abnormalities in autism. Consequently, it is possible that maternal
genes, acting during pregnancy, could contribute to the autism phenotype in the fetus.
17.5 HLA-DR4 AND AUTISM

17.5.1 MAJOR HISTOCOMPATIBILITY COMPLEX
The major histocompatibility complex (MHC) is a major collection of immune-
related genes, the largest collection in the genome. The human MHC region contains
over 200 genes that have been grouped as class I, class II, and class III genes. Class I
loci include HLA-A (human leukocyte antigen-A), HLA-B, and HLA-C. Class II loci
include HLA-DR, HLA-DQ, and HLA-DP genes. Class III loci include certain
complement-related genes, e.g., C4A and C4B. These genes are polymorphic, many
of them highly polymorphic. The region has limited recombination in most areas
except for a few recombination hot spots. Since recombination is limited in most
areas of the MHC, linkage disequilibrium is seen over long distances leading to the
presence of MHC-extended haplotypes. Reed Warren’s group studied a number of
MHC genes including the class II gene HLA-DR, particularly the highly polymorphic
β-chain gene, HLA-DRb1 (Daniels et al., 1995; Warren et al., 1992, 1996).
17.5.2 CASE–CONTROL STUDIES OF HLA-DR AND AUTISM

Early studies in autism by Warren et al. (1996) found that certain polymorphic alleles in
the MHC on chromosome 6, including HLA-DR4, were increased in frequency in moth-
ers of individuals with autism. As an explanation of their striking maternal findings,
Warren et al. raised the question of whether a gene acting in the mother during preg-
nancy might contribute to autism in her fetus (Warren et al., 1996) (Table 17.1, #20).
Warren and coauthors studied the frequency of an MHC-extended haplotype that

contains HLA-DR4 in a case–control design. This haplotype, B44-S30-DR4, is com-
posed of HLA-B44, the S allele of the BF gene, the 3 allele of C4A, the 0 or null
allele of C4B, and the DR4 allele. Compared with controls, B44-SC30-DR4 was
significantly increased in both cases and in mothers but not in fathers of individuals
with autism (Warren et al., 1992). Daniels et al. (1995) confirmed this finding in a
subsequent case–control study. Subsequently, Warren et al. (1996) found that certain
alleles of the third hypervariable region of HLA-DRb1 had very strong association
with children with autism especially alleles within HLA-DR4 (Table 17.1, #20).
Torres et al. (2002) carried out a case–control study with individuals with autism
spectrum disorder (ASD) compared with control Caucasian allele frequencies from
the National Marrow Donor Program and found that HLA-DR4 occurred more fre-
quently in children with ASD than control subjects.
Recently, Lee et al. (2006) carried out a case–control study of HLA-DR4 in autism
in families from eastern Tennessee and families from the AGRE repository that
were selected to be from all parts of the United States and in which multiple males
had the diagnosis of autism, and compared them with a control group of healthy
unrelated adults (Table 17.1, #18). Compared with controls, children with autism
in the east Tennessee group and their mothers but not their fathers had a signifi-
cantly higher frequency of HLA-DR4 alleles than did control subjects. The mothers
were 5.54 times (95% confidence interval [CI] 1.74, 18.67) and their children with
autism 4.20 times (95% CI 1.37, 13.27) more likely to have HLA-DR4 than control
individuals (Lee et al., 2006). However, HLA-DR4 frequencies of the children with
autism from the autism genetic resource exchange (AGRE) repository, their mothers,
and their fathers were not significantly different from controls (Lee et al., 2006).
The authors interpreted their findings in the eastern Tennessee group as consistent
with a hypothesis that maternal–fetal immune interaction in utero could affect fetal
brain development; such an immune interaction could conceivably involve both
HLA and related genes in both genetic and epigenetic mechanisms.
Although these studies suggested a maternal effect of HLA-DR4 for autism, all of
them compared mothers with controls in case–control study designs, and none of them
carried out a more direct test such as the maternal TDT that could document the pres-
ence of a maternal allele acting during pregnancy to contribute to the autism pheno-
type (Doolin et al., 2002; Johnson, 1999, 2003; Johnson et al., 2008; Mitchell, 1997).
Case–control studies are powerful and highly useful, but also have certain limita-
tions. One major limitation is that the cases and controls may not be ascertained in
the same way and hence may not be identical. The case–control design presumes
that controls are drawn from the same population as the cases, or from a population
equally at risk. Important as this presumption is, it is often not achieved in practice.
Other study designs, e.g., case–parent designs often used with the TDT, use internal
controls, e.g., by comparing the genotype of the affected child with the untrans-
mitted parental alleles. Hence, population stratification is not a major limitation for
TDT studies. A case–control study is an excellent first step to suggest the action of a
teratogenic allele in a dataset, but such studies need to be confirmed with more direct
method, e.g., one of the study designs discussed earlier: the maternal TDT, the log-
linear method, the method of pentets, or another such design.
The finding of the increased frequency of a polymorphic allele in mothers but not
fathers of affected individuals is an interesting one because it may help identify a factor
that contributes to the disease. There are several possible explanations of this pattern
of increased allele frequency. The known possible reasons for the increased frequency
of an allele observed in mothers of affected individuals include the following:
1. The allele is a teratogenic allele.

2. The allele acts by imprinting and is imprinted in the mother.
3. The allele acts in the affected individual and hence will have increased fre-
quency in the parents—sometimes, by chance the allele will have increased
frequency in mothers but not fathers.
4. The allele is a mitochondrial allele and hence is transmitted only by mothers
to affected individuals and therefore has increased frequency in mothers.
17.5.3 CASE–PARENT STUDY OF AUTISM

A recent study of autism used a case–parent study design to document HLA-DR4 as
a teratogenic allele for autism (Johnson et al., 2009). The study used the maternal
TDT method (Johnson, 1999; Mitchell, 1997) in which transmissions from maternal
grandparents to mothers of individuals with autism were studied for transmission
disequilibrium. HLA-DR4 was transmitted significantly more frequently than was
expected by chance (p = 0.008), documenting the action of HLA-DR4 in mothers of
affected individuals, i.e., action as a teratogenic allele. This appeared to explain the
elevated frequency of HLA-DR4 in mothers of individuals with autism. However, as
a secondary study, other causes of the elevated frequency of HLA-DR4 in mothers
of cases were considered: transmission disequilibrium was not found for transmis-
sions from mothers to individuals with autism excluding action of HLA-DR4 in the
individual with autism. Two tests of genomic imprinting were negative. HLA-DR4
is known not to be a mitochondrial gene. Thus, the other possible explanations for
the elevated allele frequency of HLA-DR4 in mothers of individuals with autism,
discussed in Section 17.5.2, were not supported.
17.6 POSSIBLE MODES OF ACTION BY WHICH

HLA-DR4 MIGHT CONTRIBUTE TO AUTISM
It is unknown how HLA-DR4 could act in mothers to influence the brain develop-
ment of their affected offspring. A number of possible mechanisms are suggested by
the work so far that could lead to further studies. Maternal DR4 could contribute to a
subset of autism cases possibly interacting with other risk alleles for autism and with
environmental factors to perturb pathways affecting brain development in autism.
A possible environmental factor could be the maternal infections during pregnancy
(urinary tract, respiratory, and vaginal) reported previously as more common in
autism mothers compared with controls, although that increase was not statistically
significant (Comi et al., 1999). HLA-DR4 (along with DR3) is a risk allele for type I
diabetes mellitus and appears to modulate the humoral immune response to entero-
virus antigens (Sadeharju et al., 2003).
Interestingly, in mice, maternal immune stimulation during gestation may affect

developmental outcome in offspring. For example, maternal immune stimulation
reportedly ameliorated malformations induced by chemical teratogens (Holladay
et al., 2002), perhaps through the maternal immune regulation of fetal gene expres-
sion, including cell cycle/apoptotic genes (Sharova et al., 2000). Maternal stimula-
tion with IFN-γ (interferon-gamma) decreased the severity of fetal cleft and palate
caused by urethane, while stimulation with Freund’s complete adjuvant reduced
both the incidence and the severity of the lesion (Holladay et al., 2000). Maternal
immune stimulation inducing inflammation increased fetal brain cytokine response,
decreased the number of reelin-immunoreactive cells in certain areas of postnatal
brain of offspring, and altered behavior in adult offspring (Meyer et al., 2006).
Reelin gene polymorphisms have been associated with autism (Serajee, Zhong, and
Huq, 2006; Skaar et al., 2005) and reelin protein levels are decreased in autism in
blood (Fatemi, Stary, and Egan, 2002) and cerebellum (Fatemi et al., 2001).
17.6.1 OXIDATIVE STRESS

It is thought that a state of elevated inflammation is a part of normal pregnancy, involv-
ing both the mother and the placenta; if this inflammation is increased, e.g., during
maternal infection, this could increase the risk of autism (Patterson et al., 2008). Since
the immune system can be a major generator of oxidative stress, e.g., during an
infection, it is possible that HLA-DR4 could act to increase maternal oxidative stress
and thereby alter fetal brain development, thus contributing to autism in the fetus.
Moreover, alterations of proteins that normally diminish oxidative stress by clear-
ing inducers of oxidative stress and products of oxidative stress could potentiate this
effect; such polymorphic mutations have been reportedly associated with autism,
e.g., paraoxonase (Serajee et al., 2004), glutathione S-transferase-P1 (GSTP1)
(Williams et al., 2007a), and GSTM1 (Buyske et al., 2006). A resulting increase in
oxidative stress could perturb mechanisms of brain development in the fetus and in
the neonate.
A number of biochemical changes support this idea (reviewed in Chauhan and
Chauhan, 2006): total glutathione was observed to be significantly decreased and oxi-
dized glutathione significantly increased in autism (James et al., 2004). Also, standard
markers of oxidative stress, such as malondialdehyde, a lipid peroxidation marker in
plasma (Chauhan et al., 2004), and 8-isoprostane in urine (Ming et al., 2005), were
found to be significantly increased in individuals with autism.
Ling et al. (2007) recently demonstrated that a rheumatoid arthritis shared epitope
(SE) acts as a signaling ligand that activates a nitric oxide–mediated pro-oxidative
pathway and blocks a cyclic adenosine monophosphate (cAMP)-mediated antioxidative
pathway, leading to increased vulnerability to oxidative damage. The SE consists of
alleles encoding certain amino acid sequences in positions 70–74 of the HLA-DRb
chain, many of them corresponding to subtypes of HLA-DR4, including the most
common DR4 subtypes. This action of the SE, leading to increased vulnerability to
oxidative damage, could be of relevance to brain development in autism.
A number of studies support the idea that oxidative stress could play a role in
autism (reviewed in Chauhan and Chauhan, 2006). For example, elevated cytokine
levels have been reported in children with ASD (Molloy et al., 2006). Interestingly
increased HLA-DR homozygosity both in pre-eclamptic women and in their part-
ners has been strongly associated with pre-eclampsia (de Luca Brunori et al.,
2000), a disorder in which oxidative stress plays a role. A number of possible
mechanisms exist by which maternal inflammation during pregnancy can affect
neuronal development (Jonakait, 2007). The neuropoietic cytokine family appears
to play an important role in nervous system development as well as in affecting
neuronal plasticity (Bauer, Kerr, and Patterson, 2007). In a rat model using lipopoly-
saccharide (LPS) to initiate maternal inflammation and induce maternal infection
during pregnancy, it has been demonstrated that cytokines may mediate effects of
prenatal infection on the fetus, a finding that has implications for neurodevelop-
mental disorder such as schizophrenia (Ashdown et al., 2006) and autism. Another
approach to inducing maternal infection used maternal poly I:C exposure to cause
maternal inflammation without an infectious agent and found that this regulates
tumor necrosis factor-α, brain-derived neurotrophic factor, and nerve growth factor
expressions in neonatal brain and the maternal–fetal unit of the rat (Gilmore, Jarskog,
and Vadlamudi, 2005). In a rat model, the inflammatory/cytokine response from the
maternal injection of LPS affected both placenta and fetal brain (Bell, Hallenbeck,
and Gallo, 2004). In another report using a rat model, prenatal exposure to mater-
nal inflammation induced by LPS mimicking maternal infection, altered cytokine
expression in placenta, amniotic fluid, and fetal brain (Urakubo et al., 2001).
17.6.2 SYNAPTIC PRUNING AND THE MHC

An interesting alternative to the oxidative stress mechanism is that HLA-DR4 could
play a role in brain development through neural pruning. It has recently been reported
that the immune system plays a role in brain development through synaptic prun-
ing during development; the classical complement cascade appears to mediate the
elimination of synapses in the central nervous system (Stevens et al., 2007). Thus, it
is possible that the immune system may contribute to marking, which synapses are
to be eliminated and to pruning them. Since this mechanism involves the comple-
ment system, it is not clear how HLA-DR4 might contribute. However, portions
of the complement system, class III markers, are in linkage disequilibrium with
HLA-DR4. Moreover, this mechanism has so far been shown to involve only genes
acting in the fetus.
Genes of proteins of the postsynaptic density (PSD) have been shown to be asso-
ciated with autism supporting the idea that synaptic abnormalities contribute to
autism. MHC class I protein co-localizes with PSD-95 postsynaptically in dendrites
and appears to contribute to the regulation of synaptic function during development,
at least in experimental animals (Goddard, Butts, and Shatz, 2007). MHC class I or
Ib antigens are required for the regulation of synaptic pruning on neuronal bodies
undergoing retrograde degeneration after axonal transection (Wekerle, 2005). Again,
it is not clear how HLA-DR4, a class II molecule, might contribute. However, there
is strong linkage disequilibrium between HLA-DR and class I markers. In an experi-
mental animal model, MHC class I molecules were involved in the stripping off of
synapses after an axonal lesion (Thams, Oliveira, and Cullheim, 2008).
17.6.3 HLA-DR4 AND HIGH RELATIVE BIRTHWEIGHT

Autism is associated with growth abnormalities in the brain, e.g., a striking increase in
brain growth after the first year of life. The fetal haplotype HLA-DR4_DQB1*0302,
the haplotype conferring the highest risk for type I diabetes mellitus, is reportedly
associated with intrauterine growth alteration, i.e., increased relative birthweight, in
normal pregnancies (Larsson et al., 2005) as well as in pregnancies of mothers with
type I diabetes (Hummel et al., 2007), an effect that was aggravated by gestational
infections, e.g., gestational fever, gastroenteritis, or both (Larsson et al., 2007).
17.6.4 MATERNAL ANTIBODIES AND AUTISM

Autoantibodies to human fetal brain proteins have been identified in the maternal cir-
culation during pregnancy and may persist for over a year, more frequently in mothers
of individuals with autism than in control mothers (Braunschweig et al., 2008; Morris
et al., 2008). Maternal immunoglobulin G (IgG) is known to cross the placenta and may
also enter the fetal circulation. Certain pathogenic maternal antibodies may cause a
number of neonatal autoimmune diseases (Heuer, Ashwood, and Van de Water, 2008).
It is possible that such transplacental transfer of maternal antibodies could contribute
to the perturbation of fetal brain development (Morris et al., 2008; Zimmerman et al.,
2007). Again, it is not clear how HLA-DR4 might contribute to these processes in
such a way as to contribute to the autism phenotype.
17.7 FUTURE STUDIES OF HLA-DR4 IN AUTISM

A larger study is required to confirm the action of HLA-DR4 in mothers of individu-
als with autism and also to address the question of whether interaction occurs between
maternal and fetal genotypes. Since linkage disequilibrium is present over large areas of
the MHC, it is possible that another gene within the MHC or even an extended haplotype
is responsible for the association observed with mothers of individuals with autism.
However, studies of the action of the maternal immune system are very much worth
pursuing since therapy for immune-related disorders, e.g., autoimmune disorders, is
becoming increasingly varied and effective. If HLA-DR4 acts during pregnancy, this
time is also the earliest opportunity for therapy directed toward the prevention and
cure of autism. In utero therapeutic approaches are actively being developed.
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18 Autism: The Centrality of
Active Pathophysiology
and the Shift from Static
to Chronic Dynamic
Encephalopathy
Martha R. Herbert*
Department of Pediatric Neurology, Massachusetts
General Hospital, Harvard Medical School,
Charlestown, MA 02129, USA
CONTENTS
18.1 Introduction ................................................................................................. 345
18.1.1 Classical Model: Behavioral Syndrome Deriving from
Genetically Determined Static Encephalopathy ............................ 345
18.1.2 Emerging Understanding of Active Persistent Pathophysiology .....346
18.1.3 Does Active, Ongoing Pathophysiology Actively Impact
Functions Central to ASD? ............................................................ 347
18.1.4 Evidence for the Potential for Plasticity and Its Pertinence ........... 349
18.1.5 Rethinking Basic Assumptions ...................................................... 350
18.2 Interrogation of Earlier Assumptions and Prior Findings from Newer
Vantage Point .............................................................................................. 352
18.2.1 Is Autism Purely a Developmental Disorder?
Or Are Its Active and Persistent Pathophysiological
Features Centrally Important? ....................................................... 352
18.2.1.1 Reassessing What We Know .......................................... 353
18.2.1.2 Probable Centrality of Glial Cells in ASD ..................... 358
18.2.1.3 From “Developmental Disorder” to “Chronic
Dynamic Encephalopathy” ............................................ 359
18.2.1.4 Sample Scenario of Pathophysiology-Based
Narrative of Autistic Regression ....................................360
* Corresponding author: Tel.: +1-617-724-5920 ; fax: +1-617-812-6334; e-mail: [email protected]
343
18.2.2 Is Autism Best or Most Usefully Defined at the Behavioral

Level? Multisystem and Multileveled Complexity in Autism........ 362
18.2.2.1 Are Systemic and Somatic Features
Really “Secondary”? ......................................................364
18.2.2.2 Beyond a Behavior-Centered Definition of Autism ....... 365
18.2.2.3 Research Questions for a Whole-Body
Approach to ASD ........................................................... 365
18.2.2.4 Characterizing the Relationship between Brain
and Somatic/Systemic Features...................................... 366
18.2.2.5 Somatic/Systemic Autism Animal Models .................... 367
18.2.3 Is Autism’s Etiology Primarily Genetic? Genes, Environment,
and Epigenetics in Autism ............................................................. 368
18.2.4 Is Autism a Static Encephalopathy? Plasticity in Autism .............. 371
18.2.5 How Does Specificity in Autism Relate to Many of the
Pathophysiological Features That Are Not Unique to Autism?
Non-Specificity of Important Pathophysiological Features in
Autism and Its Implications ........................................................... 376
18.3 Summary and Conclusions ......................................................................... 376
References .............................................................................................................. 377
The purpose of this chapter is to reflect upon the implications of the identification
of active pathophysiological processes in autism spectrum disorders (ASD), and to
reflect back upon prior findings and formulations in the light of these recent dis-
coveries. This chapter articulates challenges posed by these discoveries to deeply
held assumptions about the ASD. These assumptions are embodied in a classi-
cal model framing the ASD as a problem of genes, brain, and behavior, i.e., as a
genetically determined developmental disorder of the brain whose main manifesta-
tion is behavioral alterations based on an indelible static encephalopathy; this model
would not have predicted the growing documentation of pathophysiological distur-
bances. This chapter also describes an emerging pathophysiology-centered model of
autism that can subsume genes, brain, and behavior but also includes much more.
Prior findings and models are reevaluated to support the framing of the ASD as
(1) not only a developmental but also a chronic condition based on active pathophysi-
ology, (2) not only having behavioral but also having somatic and systemic features
that are not secondary but rather intrinsic consequences of underlying mechanisms,
(3) not only genetic but also environmental, (4) not a static encephalopathy but a
dynamic, recalcitrant encephalopathy, and (5) not a set of discrete behavioral fea-
tures neatly mapping to specific genetic mechanisms but a set of emergent properties
dynamically arising from pathophysiological systems whose parameters have been
dramatically and interactively perturbed. It is argued that a research program based
on this approach will incorporate the strengths of the classical model, will encourage
many more routes to investigations with practical and treatment applications, and may
be a much more rapid path to provide much-needed help to affected individuals and
their families.
Autism: The Centrality of Active Pathophysiology 345
18.1 INTRODUCTION
While autism spectrum disorders (ASD) can involve exquisite gifts and unusual
qualities of perception and thought, they can also involve a great deal of suffering,
for the individual on the spectrum as well as family and community. On this account,
a core question in autism work needs to be how to help the most people in the most
effective ways as quickly as possible. The goal of making sense of autism and its
mechanisms needs to be deeply harnessed to this core purpose. Our aim should be to
relieve suffering at multiple levels—from aversive sensory overload, sleep disruption,
recurrent infections, and gastrointestinal (GI) troubles to overcoming obstacles to
communication; to misunderstanding by the non-autistic people of the experiences
of people with autism; to aggression and self-injurious behavior; to the burden of
allocating scarce resources to deliver therapies that may not be optimally designed,
targeted, or implemented; and to acrimonious debate and fiscal drain. Last but hardly
the least, if any part of the impairment of optimal functioning in new cases of autism
is not purely genetically determined, the suffering and severity that is therefore neither
inevitable nor necessary should be prevented or ameliorated.
If we are to help most quickly and with the broadest and greatest effectiveness,
then how do we do so, and how much can we really help? What can we realisti-
cally expect to accomplish? The answers we give to these questions are greatly
conditioned by what we understand autism to be. The main goal of this chapter is
to explain and compare two models of autism that lead to greatly different expec-
tations: (1) a classical model of autism as a genetically determined developmental
brain disorder and static encephalopathy and (2) an emerging model of autism
centered around active systemic environmentally as well as genetically influenced
pathophysiological processes beginning early in life and leading to an chronic
persistent encephalopathy with dynamic features. This comparison will show that
the emerging dynamic pathophysiological model not only includes the strengths
of the classical static model, but also takes account of emerging data that are incom-
mensurable with the older formulations. It will also give support to the argument
that the emerging more inclusive model offers more opportunities for constructive
investigation and intervention.
18.1.1 CLASSICAL MODEL: BEHAVIORAL SYNDROME DERIVING FROM

GENETICALLY DETERMINED STATIC ENCEPHALOPATHY
Autism has until recently been considered to be a developmental disorder originat-
ing in faulty genes that skew early brain development and lead to a devastating and
incurable static encephalopathy. Since this perspective frames autism as directly
deriving from an indelibly fundamental alteration of brain structure and function,
its adherents take the logical next step when they assume that there are fundamental
profound limitations to the potential efficacy of any current therapies. An additional
commonly held assumption of this classical viewpoint is that the core behavioral fea-
tures of autism are specifically determined at the genetic and molecular levels. From
this vantage point, only extremely precise molecular or genetic interventions targeting
some critical aberrant pathways have any chance of unlocking neural functioning,
but these pathways have yet to be discovered and the molecules to target them are yet
to be invented. Therefore, to identify targets and develop effective and safe interven-
tions, an extensive, expensive, and long-term research strategy is necessary in order
even to begin to relieve suffering in any serious way.
The recent framing of autism as heterogeneous, or “autisms” (plural), modifies
this model by suggesting that “autism” is really a collection of different “autisms,”
each with its own mechanism and perhaps even its own gene(s). The research
program derived from this framing would still look for distinctive mechanisms, but
now may implicitly propose multiple parallel searches for mechanisms. If this is
not accompanied by seeking final common pathways that may bridge across these
distinct “autisms” and provide routes of intervention that could be beneficial more
broadly than to any one small subgroup, then the road ahead is even longer.
18.1.2 EMERGING UNDERSTANDING OF ACTIVE PERSISTENT PATHOPHYSIOLOGY

Clearly some kind of atypical brain functions must be going on in ASD in order
for its atypical behaviors to be produced. The very high prevalence of sensory and
sleep problems (Leekam et al. 2007, Tomchek and Dunn 2007) and the high rate of
epilepsy in ASD (Canitano 2007) also support this. Critical questions for which we
have enticing clues but no clearly worked out answers include (1) what are the mech-
anisms underlying the altered brain function and (2) what are the causes? Within the
classical model, a common phrase heard is “genes–brain–behavior.” This suggests
that the genetic alteration of the brain causes autistic behavior, and it also implies
that researching this specific chain of levels and their relationships is sufficient for
understanding ASD.
The trouble with the “genes–brain–behavior” framework is that it promotes over-
simplified thinking about the way genes alter brain and the way brain alters behavior.
Even to use the three words in a string is a problem, because (1) genes themselves do not
directly impact brain but shape other processes that alter brain, (2) these processes
that alter brain are impacted by other things in addition to genes, (3) the combina-
tion of genes and these other processes alter not only brain but also the rest of the
body including systemic molecular and cellular mechanisms that are not organ spe-
cific, (4) there is not unidirectionality but bidirectionality—indeed Web-line network
interconnections—in that the consequences of all of these dynamical alterations can
feed back and alter gene expression, and (5) the outputs of all of this complexity are
not limited to behavior but also include phenomena at many other levels (Herbert
2002, Noble 2008). Therefore, to say “genes–brain–behavior” leaves unspecified
many intermediary levels that need to be explicitly spelled out and investigated.
One formulation more inclusive than “genes–brain–behavior” is “input–
pathophysiology–output” (Herbert 2005a). Input can include genes and also a range
of environmental factors. Pathophysiology can not only include prenatal processes
with early impacts on brain development that modulate fundamental features of brain,
but also processes and impacts at other time points that have other types of effects
on both brain and body. Outputs can include not only behaviors but also medical
illnesses and a host of other functions.
Critical findings in ASD at the level of active, ongoing pathophysiology that

inspire the present volume would not have been predicted by the classical genes–
brain–behavior model. Particularly of note are the phenomena of oxidative stress,
mitochondrial dysfunction, and inflammation that have been identified in a growing
number of studies in a substantial number of individuals with ASD. Evidence of
these processes has been identified in somatic tissue samples with the measure-
ment of alterations in a variety of substances including in membrane phospholipids
(Bu et al. 2006, Chauhan et al. 2004, Vancassel et al. 2001), antioxidant enzymes, and
metabolites in the glutathione synthesis pathway (Chauhan and Chauhan, 2006,
James et al. 2006); and the documentation of both oxidative stress (Evans et al.
2008, Pardo et al. 2008, Vargas et al. 2006) and neuroinflammation (Li et al. 2009,
Vargas et al. 2005) as well as rapid membrane turnover (Minshew et al. 1993) and
altered energy metabolism (Chugani et al. 1999) in brain has also been produced.
Much more is reviewed extensively in this volume.
These phenomena are active, ongoing pathophysiological abnormalities. While
their chronic impacts can be stubbornly persistent, and while they can cause dam-
age that is more stable, their primary mechanisms act on the timescale of hours
and days or even less. They cannot be attributed to genetic errors or early insults in
a simple or straightforward fashion, although those could contribute vulnerability
or get these processes started. It needs to be emphasized that the identification of
active, persistent disturbances of physiological functions, particularly in the brain,
is a landmark in the history of autism science because it adds dimensions to the
parameters we need to include in considering the condition, and also because it
changes the temporality from a playing out of something that happened early on to
a process that is continuingly active.
18.1.3 DOES ACTIVE, ONGOING PATHOPHYSIOLOGY ACTIVELY

IMPACT FUNCTIONS CENTRAL TO ASD?
Even granted the existence of active pathophysiological processes in ASD, a skeptic
from the classical model vantage point might question whether they have any signifi-
cant relevance to ASD. From the classical point of view, it would seem obvious that
these sorts of influences could be little more than small bubbles on the surface of
the genetically determined ocean of profound brain abnormality. To face this chal-
lenge, we need to determine whether the ASD phenotype or any of its components
or contributors could be created or substantially aggravated by neural functioning
alterations that are chronically and actively maintained.
From a pathophysiology-centered point of view, once the chronic persistence and
active character of these processes is recognized, it is not so radical to suggest that
perhaps these phenomena might affect synaptic and neural systems function. In the
literature of neurobiology, there is plenty to suggest that oxidative stress and immune
activity can be neuromodulatory. The immune system, energy metabolism, and oxi-
dative stress are abundantly documented as impacting the central nervous system
and its function (Lowry et al. 2007, Lozovaya and Miller 2003, Mattson 2007, 2008,
Mattson and Liu 2002, Miller, Maletic, and Raison 2009, Wrona 2006). These con-
siderations may be particularly pertinent to the phenomenon of autistic regression,
which generally occurs somewhere around the middle of the second year of life. Even
if “regression” is preceded by a variety of subtle signs of dysfunction, it is occurring
far beyond the most critical periods of brain development and deserves investigation
as a new event and in particular, as a shift in functional/metabolic/neurodynamic state
and not just as an inevitable playing out of early hard-wired brain alterations.
With chronic-active pathophysiology identified systemically and in brain tissue
from individuals with autism, with this active pathophysiology having potential
neuromodulatory effects, and with functional changes such as regression needing
mechanistic explanation, it becomes necessary to consider the possibility that the
biological basis of the autism behavioral phenotype may not be determined “archi-
tecturally” once and for all in utero, but rather may be actively sustained, possibly
even caused or at least substantially aggravated by persistently active pathophysiology
(Anderson, Hooker, and Herbert 2008, Zimmerman 2008).
We can imagine a number of possibilities: (1) inputs (e.g., genes and environmental
factors) create an indelible alteration in prenatal or early postnatal brain development;
1b) these indelible in utero impacts of genes and environmental factors are mediated
by pathophysiological processes such as inflammation or oxidative stress; (2) some
inputs (e.g., genes, teratogens, infections, or immune responses to infections) increase
vulnerability to other inputs that alter early prenatal or early postnatal brain devel-
opment; (3) some inputs increase vulnerability to other inputs (e.g., excitotoxins) or
pathophysiological states (e.g., immune triggers and oxidative stress) that alter neu-
ral function postnatally; and (4) chronic, persistent alteration in neural function (e.g.,
cumulative toxic body burden and/or chronic neuroinflammation having a persistent
excitotoxic impact) can in turn lead to changes in brain tissue (e.g., mitochondrial
damage → cellular dysfunction → cell death), which in turn may feed back to further
affect function.
Once these additional dimensions beyond the genetic determination of altered
brain development join the parameters of concern, how do we assess what the type
of influence and relative weight may be of each class of contributor? How far can this
be pushed? For example, if the excitotoxic modulation of synaptic function is chronic
(i.e., from ongoing exposure or chronic inflammation) and/or persistent (i.e., with
semipermanent effects from even a transient exposure), can we consider whether it
could contribute to a chronic encephalopathy? And could such a chronic encephal-
opathy potentially in some cases not simply modulate the autism but actually be the
autism? Could genetic vulnerability and genetic impacts turn into autism (or more
severe autism) with the onset of these pathophysiological processes? We obviously
do not know the answer, but this chapter reflects on the question.
Insofar as pathophysiological mechanisms can be affected by environmental
input, it is also important to consider potential positive impacts. If there is a for-
mative role for pathophysiology, this suggests that factors like diet, sleep quality,
stress, exercise, autonomic arousal, environmental exposures, and medications all
could be having substantial short-term impacts on symptom severity and the qual-
ity of life. It also suggests that such factors, which include both health-promoting
and health-destroying variants, can have substantial effects over time on the level
of function and the quality of life. On the scale of years, the “ongoing” nature of
pathophysiological activity means that some interventions might be able to provide
major long-term benefits as well.
18.1.4 EVIDENCE FOR THE POTENTIAL FOR PLASTICITY AND ITS PERTINENCE
To make a plausible argument that active, persistent pathophysiology might strongly
modulate or even create core features of autism, there would need to be evidence
of some kind of intraindividual variabilities in the phenotype that occurred in
relationship to pathophysiologically pertinent processes. Such variability (e.g.,
symptom onset, marked worsening, or marked improvement) would suggest that
fluctuations in modulatory processes might have significant impact. As it happens,
such evidence exists.
The idea that physiological modulation could contribute more than marginally
is becoming less far-fetched in the light of published reports of short-term marked
improvements in core features of autism. Investigators recently pursued suggestions
from clinical case reports that behaviors and core capacities in autism may improve
markedly in the setting of fever (Curran et al. 2007)—clinicians were fairly com-
monly hearing from parents that their affected children could relate better, make more
eye contact, and sometimes even talk transiently in the setting of fever—one mother
poignantly described her experience during her child’s fever to the author of the pres-
ent review as “visiting with my son.” A prospective study was thus performed utiliz-
ing the Aberrant Behavior Checklist to rate behavior changes; the study found that
fewer aberrant behaviors were recorded for febrile patients on the subscales of irrita-
bility, hyperactivity, stereotypy, and inappropriate speech compared with control sub-
jects in a fashion that was not associated with the severity of illness. While lethargy
scores were greater during fevers, and all improvements were transient, the behavioral
improvements could not be attributed to the lethargy and the results instead suggested
a genuine improvement in core functions. An earlier paper investigated 11 children
with the history common in ASD of a period of often recurrent infection and antibi-
otic exposure followed by the development of chronic persistent diarrhea and then the
onset of autistic features, or “regression” (Sandler et al. 2000). This common phenom-
enon has spawned research demonstrating abnormal variants of clostridial bacteria
in ASD (Finegold et al. 2002, Parracho et al. 2005, Song, Liu, and Finegold 2004)
and animal models showing nervous system and behavioral impacts of propionic
acid, a metabolic product of clostridia (MacFabe et al., 2007, Shultz et al. 2008a,b)
that are part of a larger ferment of research on the influence of intestinal microecology
(the “microbiome”) on medical and psychiatric health (Alverdy and Chang 2008,
Li et al. 2008, Nicholson, Holmes, and Wilson 2005). This study investigated impact
on the behavior of oral vancomycin, which is a potent antibiotic normally given intrave-
nously and minimally absorbed from the intestine but that devastates intestinal micro-
organisms. They noted significant short-term improvement using multiple pre- and
post-therapy evaluations coded by a blinded clinical psychologist, with the transiency
presumably due to the regrowth of pathogenic intestinal microorganisms after the ces-
sation of antibiotic dosing. In both of these cases, the improvement was notable, rapid
in onset, and short in duration suggesting that the maladaptive physiological setpoint
was insufficiently challenged by fever or transiently altered intestinal microbiota to
shift to a different semi-stable state.
Some challenges prior to conceptions of developmental disorders have also
emerged on the laboratory front. Symptom reversal has recently been reported in
mouse models of developmental disorders—fragile X syndrome (Hayashi et al. 2007),
Rett syndrome (Guy et al. 2007), and tuberous sclerosis (Ehninger et al. 2008), all
considered genetic and incurable—through molecular intervention, including in
older animals. This is striking because it forever undermines the basis for simply
taking for granted that neurodevelopmental disorders are incurable or have only a
narrow critical window after which intervention is pointless. At the other end of the
lifecourse, rapid though transient reduction of Alzheimer’s disease symptoms within
minutes of the administration of perispinal etanercept suggests that chronic-active
and potentially reversible pathophysiology may also contribute to the encephalopathy
in this devastatingly progressive disorder (Tobinick and Gross 2008a,b).
With regard to the autism clinical papers discussed above, it is critical to note
that fever does not create a permanent alteration of immunologic or neurobiological
pathways, and oral vancomycin does not permanently alter intestinal flora, consis-
tent with the changes not being persistent. But it is also critical to note that these
supposedly lifelong core features of autism could be altered even in the short term,
which itself is inconsistent with a “static encephalopathy” model. All of this chal-
lenges us to think outside of the box of irretrievable brain damage in relation to the
encephalopathy of ASD (and other conditions as well). The potential mechanisms
that come to mind are in the domain of active, dynamic pathophysiology (including
but hardly limited to altered gene expression) rather than genetic predetermination,
as the genetic mechanisms causing an in utero disturbance of brain development
would not explain such short-term fluctuations. In the Curran et al. (2007) paper on
improvement with fever, the authors speculated that the phenomenon was driven by
some mechanisms related to immunologic and neurobiological pathways, intracel-
lular signaling, and synaptic plasticity; in the Sandler et al. (2000) paper, the authors
speculated that the oral antibiotic transiently suppressed an enteric microorganism
and its production of a neurotoxin-like substance.
If such marked short-term changes are possible, the idea that the encephalopathy
in ASD is a dynamic (albeit recalcitrant) “state” rather than a wired-in static “trait”
becomes conceivable, and the possibility of identifying the mechanism for and
extending the duration of such changes can be framed as a worthwhile and important
goal for research.
The implication of this is major: it means that we must consider the possibility
that the functional impairments we observe in individuals with autism may be prod-
ucts not so much of innate “deficits” as of the active (and obstinate) pathophysiological
obstruction of capacities for which brain substrate is still at least partly present.
Moreover, given that these processes are known to progressively assault and damage
cellular integrity, and given the evidence suggesting progressive changes in brain
tissue (cellular changes (Bauman and Kemper 2005) and volume loss (Aylward et al.
2002)), the importance of finding ways to medically intervene to slow or stop this
degeneration as early as possible comes into clear focus.
18.1.5 RETHINKING BASIC ASSUMPTIONS

As our understanding of these new dimensions take shape, it starts to seem that
the assumptions underlying the classical model of ASD need to be revisited. With
these features in mind, it becomes possible and necessary to interrogate prior
findings for fresh interpretations. The goal of this chapter is thus to spell out
how emerging fi ndings are revealing limitations in the assumptions of the classi-
cal view, and to outline some core features of a newer more inclusive view. These
emerging findings are elucidating mechanisms suggesting that autism is more than
a developmental disorder, that more than genes are etiologically contributory, and
that the encephalopathy has dynamic features so that it is not strictly static.
We will develop the argument by posing the following questions, and explaining
our rationale for the following responses.
Questions:
1. Is the category of “developmental disorder” adequate for autism?

2. Is autism best or most usefully defined at the behavioral level?
3. Is autism’s etiology primarily genetic?
4. Is autism best described as a static encephalopathy?
5. Is autism a unique and distinct syndrome?
Responses:
1. More than developmental: Autism is more inclusively framed as a chronic

and also dynamical/semi-episodic multisystem condition that begins in
utero or early in life during a period when developmental processes are
greatly sensitive to perturbation and that continues through the lifecourse
with persistent, ongoing, active, and dynamic pathophysiology that may
contribute critically to phenotypic features.
2. More than behavioral: Behavioral criteria alone do not encompass the mul-
tisystem features that are increasingly being appreciated in ASD, which
are so common in affected individuals as to suggest that they may play
central rather than secondary roles and/or reflect shared core underlying
pathophysiological processes.
3. More than genes: Autism is likely to be the result of a complex interaction
of multiple risk factors; neither genes nor environmental agents can a priori
be assumed to be primary in their contributions, and the interaction of con-
tributors persists beyond early development.
4. From static to active dynamic: Within-individual variability in the severity
of core features and the emerging awareness of plasticity and improvement
in autism, alongside of the relative intactness of neural structures in ASD,
suggest that the encephalopathy in autism is recalcitrant but rooted strongly
in dynamic processes, and that framing it as static is inaccurate.
5. From autism as a specific entity with the specific genetic determination of
each of its subcomponents to ASD as an emergent property of a neural and
somatic system altered by physiological challenges during a sensitive period
of early development. From a systems pathophysiology perspective, autism
appears as a complex integration of continuously distributed abnormalities in
multileveled features and has substantial physiological overlap with underly-
ing pathophysiology in many other chronic diseases. It may be that we do not
need to target features specific to autism, but that therapies targeting underly-
ing physiological features that are contributory but not unique to ASD could
lead to altered emergent properties including altered behaviors.
18.2 INTERROGATION OF EARLIER ASSUMPTIONS

AND PRIOR FINDINGS FROM NEWER VANTAGE POINT
18.2.1 IS AUTISM PURELY A DEVELOPMENTAL DISORDER? OR ARE ITS ACTIVE
AND PERSISTENT PATHOPHYSIOLOGICAL FEATURES CENTRALLY IMPORTANT?
The idea that ASD is a developmental disorder seems self-evident. ASD begins in
early childhood, with abnormalities in responsiveness sometimes even evident at
birth. Brain abnormalities have been documented at the neuropathological level con-
sistent with changes occurring in utero. The high heritability and high recurrence
rate also support this framing. The characteristic clustering of behavioral features in
the ASD phenotype suggests some kind of specific causes.
There are other ways of interpreting the above cluster of phenomena. These fea-
tures of early onset, neuropathology changes suggestive of in utero onset, specific
behavioral configuration, and high heritability/high recurrence suggestive of genetic
cause can be at least partly decoupled from the inferences with which they have been
associated. Certainly important events occur at these early stages of development.
The problem arises at the level of drawing implications from these observations about
underlying mechanisms. If one assumes a priori that this is a “developmental disor-
der” in the neurobiological or neurogenetic sense, clinical and research observations
may be given interpretations consistent with the implications of this assumption,
while other potentially valid interpretations consistent with a more chronic model
may be neglected.
The notion of a “developmental disorder” has a number of different connota-
tions. From a developmental psychology point of view, it can connote simply that
because function and capability change with development, a disorder in childhood
will manifest differently at different ages. This is unquestionably true. However,
there are other perspectives carrying more severe connotations. From a medical
and neurobiological vantage point, “developmental disorder” commonly connotes
at least the following four characteristics: (1) that there is a profound, if poten-
tially subtle, alteration in the developmental trajectory of the brain, (2) that the
ensuing developmental brain alterations are primary core targets of the etiological
agent rather than incidental or secondary, (3) that these alterations directly cause
the behavioral phenotype, and (4) that these brain features and the accompanying
encephalopathy are indelibly unchangeable. This “developmental disorder” model
is derived from observations in neurogenetic syndromes and syndromes of brain
malformation where there are clearly observable and classifiable alterations in brain
development based upon a fault in some neurochemical or regulatory processes that
lead to fairly predictable consequences in affected individuals.
While this framing of developmental disorders is most commonly associated with
syndromes having genetic etiologies, the fields of developmental neurotoxicology
and teratology have shown that exogenous substances can target early developmental
processes and lead in a similar fashion to predictable malformations and develop-

mental syndromes, such as fetal alcohol syndrome, fetal valproate syndrome, and
fetal Minamata disease. Similar arguments are also being made about disorders of
later onset such as schizophrenia (Arnold 2001, Opler and Susser 2005).
Given the widespread assumption that autism is not only a developmental disorder
but also a static encephalopathy, it appears that the stronger and more severe model
outlined above has been applied in interpreting the presentation of ASD. But if we
carefully examine the support for inferring the four characteristics connoted by the
medical-neurobiological framing of “developmental disorder” listed above, it turns
out that there are major gaps in our knowledge and more particularly in the evidence
basis for the assumptions we have been making. We have certainly (1) identified a
range of brain alterations that qualify as profound, often as subtle and sometimes
pervasive, including changes in limbic system structures, cerebellum, white and gray
matter volume, corpus callosum, subcortical gray matter structures, and asymme-
try. But we have not (2) shown for all of them that they are primary targets of an
identifiable etiological agent rather than secondary consequences of a pathophysi-
ological process, nor have we (3) conclusively demonstrated that they specifically
cause the autism behavioral phenotype (we have merely shown association and have
not excluded the possibility that some of these changes may be the downstream of
something else that is driving the phenotype), and neither have we (4) proved that
they are unchangeable, or that their unchangeability correlates with the functional
lack of plasticity—for all of these points our “knowledge” is at the level of plausible
narrative, not the empirical elucidation of mechanisms.
18.2.1.1 Reassessing What We Know

In the light of emerging pathophysiological findings, it needs to be considered that the
set of phenomena leading people to consider autism a “developmental disorder”—i.e.,
the brain changes, the early onset, the heritability and recurrence, and the cluster-
ing of behavioral features—individually and together may have potential additional
and/or alternative interpretations. Below are a series of considerations that cannot
be encompassed within the “developmental disease” model as described above.
Individually and together, they put autism in the “chronic active disease” category
and pose challenging questions about what the interfaces may be between chronic
processes that begin very early in life and alterations in development.
1. Weak Spots in “Developmental Disorder” Inferences from Existing Data

a. Brains of autistic individuals are for the most part remarkably normal
looking. An MRI scan of the brain of most individuals with ASD would
be interpreted by a clinical neuroradiologist as normal (and clinical
abnormalities when identified are typically idiosyncratic, possibly inci-
dental, and quite possibly secondary to some other processes), and it
is only by careful quantitative analysis that macroanatomical differ-
ences from brains of neurotypical subjects can be identified—it takes
this level of intensive research-grade measurement because for the most
part, changes are too subtle to be identified by the unaided eye (Caviness
et al. 1999). Indeed, some neuropathological researchers have held that
since the brains lack major dysmorphology, they are unlikely to have
suffered significant insult prior to the late gestational or early postnatal
period (Ciaranello, VandenBerg, and Anders 1982, Coleman et al. 1985,
Raymond, Bauman, and Kemper 1996). The observation has been made
that there is a striking disconnection between the almost indiscernible
white matter tract as well as general structural abnormalities and the
dramatic functional impairments (Conturo et al. 2008).
b. Suggestions that neurodevelopmental disorders can be triggered by
events during the fetal period are supported by a growing body of
literature (Connors et al. 2008, Fatemi et al. 2002, Patterson 2002, Shi
et al. 2003, Smith et al. 2007). There is a huge literature on developmen-
tal neurotoxicity (Slikker and Chang 1998) as well as developmental
immune injury (Dietert and Dietert 2008, Hertz-Picciotto et al. 2008).
However, while these exposures can now be said to increase the poten-
tial for neurodevelopmental disorders, there is no support at this time
for going further—i.e., such exposures have by no means been shown to
be sufficient on their own to cause postnatally emerging developmental
disorders or ASD in particular. Nor have developmental disorders or
ASD in particular have been shown to be necessarily or in all cases
preceded by such events.
c. The model of autism derived from the connectivity literature related
to connectivity impairments underlying impairments in complex pro-
cessing (Just et al. 2004, 2007, Muller 2007) is synchronic—i.e., it can
be marshaled to explain the apparent selective impairment of complex
processing in individuals with fully developed autism at a particular
point in time. It is not a diachronic model—i.e., it does not help at all in
explaining the phenomenon of the development of autism, and particu-
larly the phenomenon of regression into autism. We do not understand
what changes so that a child who was producing behaviors closely con-
sistent with normal developmental milestones either falters, plateaus,
shifts tracks, or in some other ways shifts to slow and/or alter develop-
ment. If one assumes that autism is inborn, then it is possible to construct
a narrative stating that the connectivity problem is innate or prenatal in
origin, but does not show itself until critical processes kick in (or fail to
occur) postnatally at which point the innately altered wiring becomes
a problem. An alternative narrative with a slightly later developmental
timepoint is the idea that there is the “failure of the pruning” of excess
neural processes. We have no direct evidence to prove either narrative,
and in fact imaging evidence as noted in point d below goes against the
idea that there has been a pruning failure.
2. Alternative Interpretations of Prior Findings
d. The brain findings to date contain many suggestions of prenatal events,
but it must be remembered that explaining findings in a fully devel-
oped brain of a child past toddlerhood and particularly of an adult is
an “archaeological” exercise in reading a developmental history from
a snapshot—i.e., it is highly interpretive. At least some of the findings
interpreted as supporting a prenatal onset have alternate possible inter-

pretations. Moreover, given the scarcity of postmortem brain specimens
from people reliably diagnosed with ASD, most neuropathological
observations have been noted in only a small number of cases and
the observations have not always been replicated. The following are
some examples where alternative explanations have been suggested:
• An observed tight packing of a larger number of smaller cells in lim-
bic structures has been interpreted as indicating a mid-gestational
event, but it is also becoming evident that limbic structures are
especially sensitive to immune influences and could be altered in
their cellular structures through other classes of events than the
early developmental events initially considered—with these other
classes of events conceivably occurring at somewhat later times
(Buller and Day 2002, Buller, Hamlin, and Osborne 2005, Nyffeler
et al. 2006).
• Minicolumnar alterations have been interpreted as occurring fairly
early in gestation, but arguments have been advanced for how they
could occur later as well (Gustafsson 2004).
• Purkinje cells appear vulnerable but they may not necessarily be
lost: while they do not pick up Nissl stain, they do appear when
calbinden staining is used. Purkinje cells are highly vulnerable to
excitotoxicity and their failure to be detected by Nissl stain may
reflect chromatolysis or excitotoxic-induced alterations in their
metabolism (Kern 2003).
• Brainstem and inferior olivary findings in ASD earlier interpreted
as indicating the prenatal disturbance of development have upon
restudy been identified not only in ASD but also in control brain
tissue (Thevarkunnel et al. 2004, Whitney et al. 2008), suggesting
that interpretations regarding both developmental trajectory and
specificity need to be rethought.
• Brain enlargement was early on attributed to a “failure of pruning”
(i.e., a failure to eliminate the super-abundance of neurons produced
early in brain development), but magnetic resonance spectroscopy
(MRS) studies have shown reduced (DeVito et al. 2007, Endo et al.
2007, Friedman et al. 2003, 2006, Kleinhans et al. 2007) or unchanged
(Vasconcelos et al. 2008, Zeegers et al. 2007) rather than increased
n-acetylaspartate (NAA) not consistent with neuronal increase.
• Moreover, while NAA is often considered to be a measure of cell
density, it can also be construed as measures of cellular and even
mitochondrial functions, and its reduction may be an indicator not
so much of neuronal loss as of neuronal dysfunction, particularly
given the reversibility of NAA decrements in contralateral tissue
following the surgical resection of epileptic foci (Hugg et al. 1996,
Pan et al. 2008, Serles et al. 2001).
• White matter enlargement has been identified in T1-weighted scans
that offer only macroanatomic measures but no resolution at the
scale of cellular changes; the distribution of this enlargement sug-

gested an increase in short-cortico-cortical fiber density consistent
with local hyperconnectivity and long-distance under-connectivity
(Courchesne and Pierce 2005), although there was no neuropatho-
logical data on the tissue composition of this enlargement. But as
results from diffusion tensor MRI imaging (pertinent to assessing
white matter integrity) and MRS imaging (pertinent to measuring
metabolites) are appearing, this inference is being contradicted by
evidence suggesting that the expanded volume cannot be explained
by increased fiber density and in fact may be due to altered water
properties in the tissue more consistent with alternative pathophysi-
ology such as neuroinflammation (Hendry et al. 2006, Sundaram
et al. 2008, Zimmerman 2008).
The overall point here is not to argue that we have a clear-cut case in every
respect for postnatal or pathophysiology/dynamical-influenced events in
ASD brain development, but simply to say that there remains a fair amount
of ambiguity in the limited data presently available to us.
3. The Restrictive Impact of Poor Communication between Silos of
Hyperspecialization and across Disciplinary Boundaries
e. Functional imaging methods including fMRI (functional magnetic
resonance imaging), EEG (electroencephalography), MEG (magneto-
encephalography), PET (positron emission tomography), and SPECT
(single photon emission computed tomography) have shown alterations
in regional interconnectivity by various methods (e.g., connectivity,
coherence, and covariation) (Herbert 2005b, Muller 2007, Herbert and
Caviness 2006). Some investigators have inferred that this intercon-
nectivity alteration might be linked to structural alterations in white
matter. But demonstrating such a relationship would require coregistering
functional data such as fMRI or MEG or PET with anatomical data
such as diffusion tensor imaging or MRS, to see whether alterations in
white matter integrity occur in a fashion that relates in any consistent
manner with alterations in functional connectivity; while such work is
in progress, few results have been reported to date (Kleinhans et al. 2007,
Just et al. 2007) and so we actually have little evidence-based idea what
tissue changes might be causing alterations in functional connectivity,
EEG/MEG coherence, or interregional covariation. The possibility has
not been tested that an alteration of synaptic function secondary to the
excitotoxic effects of chronic tissue pathophysiology could have systems
impacts on the patterning of neurodynamic activity that could contribute
to altered functional connectivity. In fact, the investigators studying
brain connectivity hardly even mention the emerging pathophysiology
findings—the silo effect where groups of narrowly specialized investi-
gators fail to cross-fertilize outside their own small circles and the
cognitive dissonance effect are both apparently very strong, and the cross-
fertilization between the levels of investigation is quite weak.
f. Several neuropathological investigations of brain tissue in ASD have
found evidence of neuroinflammation and oxidative stress (Evans et al.
2008, Li et al. 2009, Sajdel-Sulkowska et al. 2008, Vargas et al. 2005,

2006). In addition, some neuropathological investigations into the nature
of white matter enlargement are beginning to suggest that there is astro-
glial activation in the enlarged outer, radiate part of the white matter that
is not present in the deeper, non-enlarged white matter, along with micro-
glial activation that is present particularly in the cerebral cortex (Pardo
et al. 2008). These early findings point toward fresh ways of making sense
of both altered synaptic activity and brain hypoperfusion in ASD.
• Regarding synaptic function, an emerging field of literature relates
to the active roles played by glial cells (astroglial, microglial, and
oligodendroglial cells) in signal transmission in the brain (Fields
2006, 2008). These cells are being promoted in our understand-
ing from handmaidens of neurons to active players in a much
more complicated collaborative endeavor; the importance of these
cells has prompted some to say that we should change the name
of the field of study from “neurobiology” to neurogliobiology”
(Peschanski 1991). Astroglial cells participate in a “tripartite syn-
apse” (Halassa, Fellin, and Haydon 2007) as they wrap themselves
around the ends of two synapsing neurons and neurochemically
modulate the synaptic activity between these two cells. In two
different specialized silos of the research world, it is known that
(1) immune-activated astroglial cells behave quite differently neu-
rochemically and (2) astroglial cells are exquisitely sensitive to
toxicant exposures, which can also put them into an activated state.
Apparently however, the impact of activation of glial cells on their
function in the tripartite synapse has not yet been researched—i.e.,
the silos of glial-immune and glial-toxin specialists are still not
communicating and synergizing with the silo of glial-synapse spe-
cialists. So some basic science that we would need to understand
the functional impact of either white matter enlargement or glial
activation simply has not been performed.
• Regarding brain hypoperfusion, it is also known that astroglial cells
become larger when they are activated, and since they encircle small
vessels, this enlargement can reduce capillary diameter by as much as
50% (Aschner et al. 1999); such a reduction is consistent with (though
such consistency does not prove it is the cause of) measures of brain
perfusion in ASD, where the several papers report perfusion reduced
blood perfusion, albeit in different distributions (Herbert 2005b,
Herbert and Caviness 2006). Other possible pathophysiological
contributors to hypoperfusion worth investigating include the modu-
lation of vasoconstriction and platelet activation and aggregation
by oxidative stress (Yao et al. 2006) and the impact of the activa-
tion of microglia encircling cerebral microvasculature (Vargas et al.
2005). Interestingly, the papers reporting hypoperfusion in ASD to
date are almost entirely mute on the subject of the tissue biology or
the pathophysiology of this hypoperfusion, with the interpretations
in the discussion sections of the papers focusing on the psychological
significance of the localization of the hypoperfusion with an unstated

assumption that this phenomenon is stable, static, and persistent.
Here the silo of pathophysiology specialists is not linked with the silo
of psychology specialists.
The point of all of these examples is to give a taste of various ways that the intro-
duction of pathophysiological variables can point to rather different interpretations
of existing brain findings. It also serves to illustrate how much of what we think
we know about the brain in autism is actually a morass of fragments of data being
extrapolated to support inferences based on a priori assumptions. By showing that
when we augment the conceptual input parameters to include chronic pathophysiol-
ogy and not just genetics and brain development, we get as output a substantially
different set of interpretations, I hope I have at least begun to demonstrate how tenu-
ous are the established interpretations. On this basis, I would argue that we have no
solid grounds for excluding or dismissing a research program based on a different
set of assumptions than “developmental disorder of prenatal onset.” On the contrary,
there are many reasons for arguing that it is very important that we pursue a research
program based on these different assumptions, as well as communication and synergy
across specialized silos, and do so aggressively.
18.2.1.2 Probable Centrality of Glial Cells in ASD

The role of glial cell activation in brain dysfunction in autism needs more attention at
the functional as well as at the neuropathological levels (Coyle and Schwarcz 2000,
Giaume et al. 2007). Glial cell activation can be set off by a myriad of triggers, and
many of the downstream consequences are not specific to the initiating agents. While
pathophysiologically oriented investigators have been greatly influenced by the iden-
tification of activated microglia and astroglia in brain tissue from individuals with
autism (Vargas et al. 2005), adherents of the classical “developmental disorder” model
often refuse even to discuss it, some arguing insufficient replication. Since the publica-
tion of the Vargas et al. (2005) paper that identified activated microglia and astroglia in
all 11 of the brains studied, the group has collected at least another 9 brains, one from
someone with Asperger’s syndrome, and all of these subsequent brains also showed
this activation, including the one with Asperger’s syndrome, considered to be a milder
condition (Zimmerman 2008). And now, as mentioned, another group has also identi-
fied central nervous system immune activation in ASD (Li et al. 2009).
Microglia comprise about 10% of the cells in the brain and perform important
functions in both the resting and the activated states. They appear to release trophic
factors during development, some of which have been measured as having different
levels in infants who later develop autism (Nelson et al. 2001). When activated,
microglia synthesize and secrete a range of pro-inflammatory cytokines (Hanisch
2002), several of which are neurotoxic, and in this activated state, they also
promote astroglial overactivation and dysfunction, as well as edema (Orellana et al.
2009). Astroglia are multipurpose cells that not only support neurons but also
perform metabolic and signaling functions (Aschner et al. 1999, Fields 2006, Halassa,
Fellin, and Haydon 2007); astrocytes are highly networked into a syncytium through
gap junctions (Theis et al. 2005) through which depolarization and calcium waves
spread rapidly and interact with neuronal activity; and they are centrally involved
in regulating neurovascular coupling (Koehler, Gebremedhin, and Harder 2006).
It is increasingly appreciated that astrocytes are influenced by inflammation in a
variety of disease states (Kielian 2008); the activation of astroglial and microglial
cells have a wide variety of effects that are arguably consistent with many observed
features of ASD. Microglia activation is associated with vasogenic and cytotoxic
edema associated with hypoperfusion; activated microglia release glutamate that
induces astrocyte edema (Han et al. 2004, Liang et al. 2008). Microglial activation
can occur rapidly in response to insults; when it persists, its neurotoxic impact
progressively increases over time. The astroglial support of neuron chemistry
and the secretion of neuromodulators are altered in the activated state (Aschner,
Sonnewald, and Tan 2002). Astroglia maintain the redox potential including through
the production of glutathione, which they transfer to neurons. In their resting
state, astrocyte networks prevent glutamate excitotoxicity in the brain (Schousboe
and Waagepetersen 2005). In the setting of acute inflammation, these functions are
compromised, leading to increased neuronal vulnerability (Orellana et al. 2009,
Tilleux and Hermans 2007). This might lead to a runaway self-reinforcing vicious
cycle, with microglial activation releasing glutamate and activating astroglia, and the
activation of astroglia reducing their ability to perform their multiple metabolic and
signaling functions. In summary, the activation of these classes of glial cells leads
to a series of pathophysiological phenomena that can be self-perpetuating and also
progressively more excitotoxic and neurotoxic.
Given how insensitive existing in vivo imaging is to neuroinflammation and how
few clinical measures collected to date pertain to these processes, we have no way of
knowing how pervasive these processes are among people with ASD, how they inter-
act with contributory genes, whether the above cascade of cellular and molecular
changes is either sufficient or necessary to produce ASD, or whether genetic vul-
nerability is required. But all of the above raises the possibility that dysfunction in
these cells could be central to ASD pathophysiology and functional impairment,
prominently triggered by noxious environmental influences, and only subordinately
related to genetic influence. These mechanisms suggest that there are substantial
complexities beyond the boundaries implied by the assumption that ASD is simply a
“developmental disorder.”
18.2.1.3 From “Developmental Disorder” to “Chronic

Dynamic Encephalopathy”
I would argue that an alternative to the “developmental disorder” model is that we are
dealing at the core with an alteration of neural function. It would follow from this that
the brain structural changes we observe might very well be to a significant extent a
consequence of the underlying pathophysiology that alters function either in addition to
or rather than being the structural basis for the functional alterations. In terms of this
model, an alteration of cellular function would lead to gradual decrements; at some point,
there would be a “tipping point” with a shift from quantity to quality leading to qualita-
tive alterations in neurodynamics (i.e., interregional connectivity, patterns of oscillation
and synchronization, etc). This shift would manifest itself at the brain systems level as
“underconnectivity” and at the level of behavior as a “regression” process.
What is being offered here is a model of chronic dynamic encephalopathy. It is

different from the classical model in the critical respect of being able to accommo-
date a number of features including the relative gross anatomical normality of most
brains of people with ASD; and in particular, the phenomenon of transient improve-
ments that is increasingly being appreciated. It also can accommodate the highly
common sensory and sleep problems and common epilepsy. It can accommodate
somatic features. It can even accommodate the high intraindividual variability in
many individuals at the level of the intensity of their reactivity. And finally, it can
accommodate autistic regression.
18.2.1.4 Sample Scenario of Pathophysiology-Based Narrative

of Autistic Regression
Many scenarios have been advanced in the literature of how prenatal influences
could set the stage for ASD; many of these are useful, but will not be repeated here.∗
We will instead present an example of a scenario that links existing data into a
chronic pathophysiology-based narrative of autistic regression, since such a discus-
sion is harder to find in the existing literature.
• In utero events (infection, toxicants, radiation, stress, maternal metabolic,

or immune factors), possibly but not necessarily in the setting of genetic
vulnerabilities, have epigenetic effects that increase the responsivity of the
organism to subsequent immune, metabolic, and infectious stressors.
• The infant has a series of exposures or experiences that challenge the system at
the points of vulnerability. These could include infections that the immune
system cannot handle well; antibiotic exposure that disrupts GI micro-
ecology; and the immune and metabolic functions played by this complex
intestinal microbiome, food allergens, toxic exposures, and other stressors.
These exposures alter physiological function, and some of the alterations
have neuromodulatory impact. Repeated exposures may lead to hypersensi-
tivity and maladaptive responses at lower doses.
• Metabolic resiliency is cumulatively challenged: for example, every input
that promotes the development of a pro-inflammatory cytokine profile and/
or the depletion of glutathione and reduction of ability to buffer pro-oxidant
stress and that is not followed by a recovery of a more normal cytokine
profile, the repletion of glutathione, etc. increases the infant’s vulnerability
to subsequent challenging inputs. The weakening of metabolic resiliency
may also be accompanied by subtle signs of impairment of higher cortical
functions as well as by various medical symptoms.
• At some point, the ability of astrocytes to continue to maintain their local
and syncitial support for neuronal function and their appropriate release
of neuromodulatory “gliotransmitters” and glutathione is overcome by
immune activation and toxic and redox challenges. At this point (which
may have gradual or sudden onset), optimal neural systems connectivity
can no longer be maintained. A functional consequence is the sharp curbing
∗ Table 2 in Herbert and Anderson (2008) schematically lays out other possibilities.
of the ability for engagement in activities requiring exquisitely timed and

coordinated mental processing (such as the core behavioral domains of
ASD—sensitivity to social nuance in communication and the ability to be
flexible in the face of transitions). Mitochondrial function, a component of
these physiological networks, is also challenged, which is a major prob-
lem given the enormous energy demands of brain activity; this further
undermines neural systems integration and increases brain irritability and
hypersensitivity. Cerebral microvascular regulation is altered. The system
enters into a self-propelling pathogenic feedback loop that is difficult to
interrupt and leads to a maladaptive “stable state.” The whole process has
many commonalities with mechanisms operative in neurodegenerative
disorders (Standridge 2006).
• Impacts are widespread, including altered neural networks; altered perfusion
patterns; neurotransmitter alterations; and a pattern of potentially progres-
sive inflammation and oxidative stress in the brain causing a chronic state
of excitotoxicity, hyperreactivity, and increased excitation/inhibition ratio,
with consequent electrophysiological disturbances causing the disruption of
sleep and sensory processing, motor tone and coordination decrements, and
the increased onset of seizures with time and exposure to further stressors
(e.g., pubertal hormonal shifts). With all of these system challenges and
breakdowns of optimal neural systems activity and coordination, the child
withdraws from the social universe and seeks a manageable sameness.
• Cellular function in other systems, e.g., GI barrier function, is challenged
by the same mechanisms that are challenging the function of gap junction
and redox buffering in astrocyte syncitial networks, with resultant somatic
symptoms as a consequence. This may be either due to problems with glial
cells or analogs in extra-CNS sites like the GI tract (Ruhl 2005), or to
related physiological vulnerabilities and cascades.
While the details of this scenario could be modified at various places along the
way, and while many linkages have not been tested, the starting points and subse-
quent features for each step of the narrative are taken from existing literature. The
point of presenting this sample narrative is to show that aberrant pathophysiology,
with or perhaps even without genetic vulnerability, could lead to a systems shift in
state that would cause altered brain function that could plausibly produce outputs
including autistic behaviors.
Another very important point is that much of what has been identified in autism
neuropathology and imaging could potentially be caused by rather than the cause
of this cascade. Purkinje cell loss or dysfunction could be due to excitotoxicity
(Blaylock and Strunecka 2009, Kern 2003, Yip, Soghomonian, and Blatt 2008).
White matter enlargement could be due to inflammation (Hendry et al. 2006, Pardo
et al. 2008, Dager et al. 2008). Limbic structure enlargement could also be due to
inflammatory processes particularly given some evidences that these structures have
greater immune sensitivity and vulnerability(Buller and Day 2002, Churchill et al.
2006, Kim et al. 2000). Altered connectivity could be due to an interaction of fac-
tors including reduced perfusion, gap junction closure, mitochondrial dysfunction,
and altered astrocyte metabolic activity as discussed above. Impairment in complex

processing could be a result of the inability of a system whose cellular infrastructure
is energetically and metabolically compromised to optimally coordinate informa-
tion required to pull the components of complexity together in a timely and useful
fashion (Anderson, Hooker, and Herbert 2008).
This chronic dynamic encephalopathy model could in particular accommodate
the way that systemic alterations at the level of pathophysiology (e.g., oxidative
stress, mitochondrial dysfunction, and inflammation) could impact brain function
not only on an ongoing basis but also in a fashion that is malleable—which is poten-
tially consistent with reports of the level of functioning that is dynamic—i.e., that
changes with physiological alterations whether naturalistically or therapeutically
induced (e.g., fever and metabolic treatment). That is, if cells in the CNS can be
supported so that their degree of energetic and metabolic compromise is reduced or
eliminated and damage from chronic persistence of the pathological state is not too
far advanced, the neurodynamic state of the system may be able to qualitatively shift
and allow marked improvements in coordination and integrative function.
I think that this model needs to be built out into a detailed research program that
in particular links cognitive neuroscience questions with pathophysiological consid-
erations, and also includes a systematic re-interrogation of existing data in a fashion
I could only begin to sketch here. Some further suggestions of what this could involve
will appear in later sections below. This chronic dynamic encephalopathy model
can not only incorporate developmental contributors to vulnerability, but it can also
accommodate the interaction of such risk factors with subsequent environmental
triggers, something that the “developmental disorder” approach does less well.
If we sit in the “developmental disorder” model and assume that specific genetically
based developmental mechanisms are in there messing up brain development but we
just have not found them yet, we will intensively orient our research program to seek-
ing these mechanisms and arguing for the causal linkage of candidate mechanisms
when we find them with core components of the behavioral phenotype before we
have elucidated the intermediary mechanisms through all the levels that these can-
didate mechanisms must traverse to impact brain and behavior. The above argu-
ments support a different approach, a pathophysiologically centered neurodynamic
research program that incorporates etiological inputs and behavioral outputs, but that
focuses on core pathophysiological mechanisms and on their potential for dynamic
change. The outcome can be cooperative and collaborative, since this approach
does not lose the strengths of the classical model, but rather reincorporates those
strengths into a more inclusive framework. More strongly, this dynamical model not
only accommodates the observed metastability, variability, and plasticity in ASD but
also allows the investigation of intervention strategies that can be implemented in the
short term with potential substantial reduction severity and suffering.
18.2.2 IS AUTISM BEST OR MOST USEFULLY DEFINED AT THE BEHAVIORAL LEVEL?

MULTISYSTEM AND MULTILEVELED COMPLEXITY IN AUTISM
Autism was initially identified by a psychiatrist (Kanner 1943); and with its promi-
nent behavioral manifestations, it has been studied first as a psychiatric syndrome
and for the last few decades with the accumulation of evidence of brain and ner-
vous system abnormalities as a neuropsychiatric, neurodevelopmental syndrome
(Tuchman and Rapin 2006). At the same time, there has long been a more whole-
body physiological strand in autism research and treatment. Although several early
scattered papers appeared describing measurable changes in somatic and systemic
physiological features, these insights have not been integrated or assimilated into the
dominant model of autism.
There are several reasons for this lack of integration of physiological and behav-
ioral understanding:
1. Many of the physiological studies have been weak: small sample size, meth-
odological problems, and inconsistency of results between studies have
contributed to keeping these findings marginalized.
2. The immaturity of methods of investigation has limited the strength of such
findings and hindered their ability to engender serious interest.
3. The behavioral definition of ASD has made it seem necessary or at least
important to map physiological findings to specific behavioral features of
this syndrome in order to support their significance to the condition, but
attempts to do this have produced weak results, probably because the
systems pathophysiology is unlikely to lead to this kind of specific mecha-
nism-to-behavior mapping.
4. The heterogeneity of ASD is only recently being appreciated, so that most
studies to date have not been designed to tease out distinctive subgroups.
The problem of subgroups is particularly pertinent here: a pathophysiology-
centered approach would emphasize that subgroups may be effectively
distinguished at the physiological level; but at the same time, there is no
guarantee of discerning any one specific measure at the metabolic or
immune level that is present in the majority of a cohort. Thus, physiological
insights, particularly those that could be pertinent to such subgroups, have
not been clearly identified.
In recent years, multisystem and systemic features of autism have been getting
more attention, in part because of research (Herbert 2005a) and also because of the
experiences of patients and the insistence of many such patients and their families
that these are major issues and should not be ignored. Most commonly appreciated
at this point are the GI symptoms (such as chronic constipation, diarrhea, and gas-
troesophageal reflux) (Afzal et al. 2003, Torrente et al. 2002, Valicenti-McDermott
et al. 2006) and the immune abnormalities (such as recurrent infections and chronic
allergies) (Ashwood and Van de Water 2004a,b, Ashwood, Wills, and Van de Water
2006), both of which appear to have high prevalence in individuals with ASD and
sometimes in their family members (Croen et al. 2005). Less widely known but sup-
ported by a growing body of literature are the underlying abnormalities in oxidative
metabolism and sulfur metabolism already discussed above (Chauhan and Chauhan
2006, James et al. 2006). There are also various nervous system manifestations that
are highly prevalent but that fall outside the triad of behaviors that define autism;
these include sensory abnormalities (present in as many as 95% of individuals with
autism) (Tomchek and Dunn 2007), sleep disturbances (Malow 2004, Malow et al.
2006), abnormal autonomic reactivity (Goodwin et al. 2006, Groden et al. 2005,
Ming et al. 2004), epilepsy (Canitano 2007), and various motor and neuromuscu-
lar abnormalities. In parallel with these developments in the ASD literature, there
are analogous developments in other neuropsychiatric fields where the interest is
expanding beyond behaviors to include pathophysiology and systemic biomarkers
(Schwarz and Bahn 2008).
18.2.2.1 Are Systemic and Somatic Features Really “Secondary”?

From the vantage point of framing of autism as a genetically based neurodevelop-
mental syndrome, it is logical to assume that the brain problems come first, that
developmentally rooted alterations in brain structure and function lead to the behav-
iors we observe and use to define autism, and that while we may find other features
in large subsets of autistic individuals, they are secondary and not directly related
to the core brain-based behavioral features. Even so, a growing number of people
holding this classical point of view are acknowledging somatic/systemic features in
ASD; how do they explain the frequent occurrence of these features?
Within the framework of a primarily genetic and developmental neurobiological
model of ASD, there are two main distinct but non-mutually exclusive explanations
for this co-occurrence or “comorbidity” of somatic and neurological problems. One
of them relates to the noxious impacts of physical discomfort: this is the idea that
physical symptoms may create problem behaviors or reduce the level of function;
for example, pain (e.g., from esophageal reflux or constipation) may contribute to
aggression or self-injurious behavior, while sleep dysregulation may reduce atten-
tion and cognitive function (Bauman 2006). The second goes deeper and touches on
cause: this is the important insight that genes may express in multiple systems, so
that genes that impact the brain may also impact the gut or the immune system.
Both of these explanations seem substantially true and important. But they do
not exhaust what needs to be said about the issue of the so-called “comorbidities.”
The pain argument takes for granted that the somatic features are secondary and not
mechanistically related to brain alterations, while the “genes express in multiple sys-
tems” argument assumes that genes are the main effect and ignores environmental influ-
ences and gene–environment interactions (Rutter 2008). Neither explanation promotes
reflection about other mechanisms of brain–body interaction that may be in play,
either developmentally or chronically. Both explanations leave much unexplained.
What if the pathophysiology leading to pain is part of the same disturbance that
is also altering brain function—either developmentally, chronically, or both? And
what if genes are contributory but not the main effect? Both of these are reasonable
questions. If the answer to either is in any way positive, then the above two explana-
tions for the comorbidity of brain and somatic/systemic features must be considered
incomplete.
Because of the notion that autism is a genetically caused brain-based syndrome,
the important clinical insight described above, that physical symptoms may aggra-
vate behavioral problems or reduce levels of function, is often accompanied by an
additional comment or implicit assumption: “but this has nothing to do with the core
autism.” First of all, it needs to be asked, “How do you know it has nothing to do with
the core autism? Where are the documented specific mechanisms proving that your
framing of autism as not only specifically neurobiological but also nothing more than
a genetically caused brain-based syndrome is actually the best framing? Do we have
enough multidisciplinary systems biological phenomic research to prove that there
are really cases of ‘pure autism’ with absolutely no features other than the three core
behaviors? Where are the systematic studies conducting sufficient appropriately sen-
sitive measures in people with apparently nonsystemic, non-somatic presentations
to exclude all implicated dysregulated physiology? Can anyone point to a literature
reporting the systematic investigation and exclusion of the possibility that there may
indeed be a relationship between brain and body features in affected individuals?”
In fact, from the vantage point of a pathophysiology-centered approach to autism,
there are many reasons to expect that there is a vital linkage between body and brain,
and strong reason to disagree with the idea that the somatic and systemic features
are simply secondary to “the autism.” In truth, as mentioned in the introduction,
there are many mechanisms by which brain and body may very well be related in
autism (and in many other settings), and in particular, by which body may signifi-
cantly influence brain, and there are many papers in the non-autism peer-reviewed
literature showing immune–brain and gut–brain relationships via mechanisms that
may very well also be operating in autism. We do not need to remind people that the
notions that such mechanisms are irrelevant or of minimal effect because the brain
is immune-privileged and/or the blood-brain barrier is fully protective are obsolete
(Carson et al. 2006). Particularly pertinent are that both brain and body are known
to be affected by oxidative stress, mitochondrial abnormalities, and inflammation,
mechanisms that growing evidence implicates in autism.
18.2.2.2 Beyond a Behavior-Centered Definition of Autism

Because systematic and phenomic studies of ASD are just beginning, it is premature
to propose a rigorous definition of ASD that includes biological features. But it is time
to treat the behavioral definition with a great deal of circumspection. With a high
prevalence of a range of somatic/systemic features, the behavioral definition of autism
can be appreciated as a starting point that gives some uniformity to subject charac-
terization in research studies. But it should not be assumed that it is directly and spe-
cifically caused by the core underlying biology. This argument was made some years
ago by Morton and Frith (1995), who diagrammed the complex pathways leading
from genes (consistent with dominant genetic determinist biases they did not discuss
environmental influences, but the argument about complex multileveled interacting
cascades of influences would be the same for how any pathogenic factor leads to an
impact on phenotype) to brain tissue changes to brain system changes to behaviors
where the connections were much more likely to be circuitous and interactive than
simple, straight, and direct. In the meantime, systematic work needs to be done to
tackle the question of somatic/systemic–brain–behavior relationships directly.
18.2.2.3 Research Questions for a Whole-Body Approach to ASD

Three of the core challenges facing a pathophysiology-centered approach to autism are
1. To develop study designs that have the capacity to concretely address and
elucidate brain–body–systemic relationships in autism itself, and not merely
by inference from other domains
2. To develop research methods and identify measures optimally sensitive to

the changes at the brain level that may be associated with changes at the
somatic/systemic level in ASD
3. To develop treatment research programs that utilized these sensitive
measures in whole-body, whole person treatment research, and treatment
efficacy tracking (Herbert 2007)
To achieve these goals, we need to work across silos of narrow specialization so

that pathophysiology-centered studies incorporate brain function measures and cog-
nitive neuroscience studies incorporate somatic and systemic measures. We also
need a network of collaborating researchers and infrastructure to pool our data.
All of this requires infrastructure capable of supporting these cross-silo integrative
collaborations.
18.2.2.4 Characterizing the Relationship between Brain

and Somatic/Systemic Features
A number of key questions need to be addressed now that somatic/systemic features
are on the table in ASD.
• Are systemic features really secondary? To study this problem, we will

need not only to look for the presence of systemic and somatic features in
individuals with autism, but also to assess what kinds of relationship these
features may have to the brain. Is there any kind of correlation of somatic
with brain features? Is there any covariation of measures of somatic or sys-
temic symptom severity with the severity of behavior or neurocognitive
impairment?
• Does any kind of treatment of somatic systems measurably alter brain
function?
• In comparing biomedical treatments, is there a difference in measurable
brain impact between treating somatic symptoms as compared with treat-
ing systemic/metabolic root causes? For practitioners holding the classical
“developmental disorder” model, the goal is to relieve symptoms in order
to achieve the reduction of discomfort and improved function by virtue
of the absence of pain, sleeplessness, etc.; there is no goal of achieving
change in the autistic encephalopathy itself. On the other hand, for prac-
titioners with a pathophysiology orientation, targets further upstream
would be sought, with the idea that correcting systemic pathophysiology
would make possible reconfiguring of systems to healthier adaptation in
body and brain together. To test whether it matters how far upstream treat-
ments are targeted, outcomes could be compared between upstream and
symptomatic approaches. For example, for diarrhea, stopping symptoms
by medicating to reduce gut motility would not treat a mechanism that
could drive both body and brain involvement, while treating an inflam-
matory process at an upstream point or removing an inflammatory trigger
(such as by treating and eliminating a chronic infection) might have a
more widespread effect; can this theoretical difference be demonstrated
in practical studies?
• What domains of brain structure or function might be most sensitive to

pathophysiological disturbances and to modulating these disturbances
therapeutically? What neurobiological-dependent variables that can be
measured noninvasively in a living individual (from coherence to sensory
and motor to social and emotional, from auditory to language, and more)
might be most useful to include in brain–body and treatment research in
ASD? It would seem that if we are talking about chronic alterations of
synaptic function, then measures sensitive to activity at the timescale of
synaptic transmission, such as EEG and MEG that have millisecond tem-
poral resolution, would probably be more sensitive than measures looking
at brain activation in anatomical space, such as functional MRI, which has
excellent spatial but poor temporal resolutions.
• What are the implications of tissue pathophysiology for cognitive neurosci-
ence? Here are some questions that have hardly even been posed, let alone
answered:
• Is there any correlation between particular pathophysiological features
and particular behavioral features?
• Are language, communication, and theory of mind impairments a mani-
festation of the psychologically based lack of motivation or of a physiolog-
ically based inability to mobilize cellular activity to drive these functional
systems? That is, are the core “impairments” we see in ASD the conse-
quence of a “deficit” or of a pathophysiology-based heavily reinforced
obstruction of a capacity that is potentially still at least partly present?
• What kinds of vulnerabilities are created by oxidative stress, neuroin-
flammation, and immune dysfunction at the levels of neuronal and glial
functioning, synaptic functioning, and connectivity? Sensory processing
and sleep? Is there any specificity or preferential impact?
• Is autism a cluster of coexisting, comorbid distinct endophenotypic com-
ponents? Or are there underlying mechanistic interconnections between
apparently specific behavioral and somatic/systemic domains? This is a
question that has received significant attention at the level of behavioral
phenotype (Happe and Ronald 2008, Happe, Ronald, and Plomin 2006),
and could also receive fruitful further attention with the inclusion of
somatic and systemic features of autism. The investigation of pathophysi-
ological mechanisms and their response to treatment, when accompanied
by the careful documentation of neurocognitive response, could help
address whether change happens in modules or systemically, or some-
where in between.
Addressing the above research questions will lay the foundation for incorporating
somatic/systemic features into phenotyping and defining autism spectrum disorders,
and help us develop a coordinated research and clinical approach that integrates
somatic and systemic features with brain, behavior, and genetic factors.
18.2.2.5 Somatic/Systemic Autism Animal Models

Alongside human studies, it is possible to utilize a somatic/systemic pathophysi-
ological approach in constructing animal models. A particularly comprehensive
approach to implementing this has been performed by MacFabe, using a propi-

onic acid environmental stimulus. Propionate as mentioned in the introduction is a
short-chain fatty acid produced by clostridial bacteria, abnormal varieties of which
have been documented in stool samples from children with ASD (Finegold et al.
2002, Parracho et al. 2005, Song, Liu, and Finegold 2004). Propionate is also used
as a food preservative. MacFabe injected this substance into the ventricles of mice,
and induced features spanning the levels of autism manifestations, ranging from
stereotypies and social isolation behaviors through electrophysiological spiking to
neuroinflammation and oxidative stress in brain tissue to the upregulation of genes
such as neurexin and neuregulin that have been identified as candidate genes by
genetics researchers (MacFabe et al. 2007, Shultz et al. 2008a,b). This model also
includes reversibility, as the effects of the injection wear off over weeks; on the
other hand, it includes a kindling effect—repeated injections result in prolonged
abnormalities and slower recovery.
The complex multilevel-integrated model MacFabe has constructed could be
repeated for a variety of other environmental stimuli, and would probably again
show the unification of features across the range of complexity characterizing ASD.
It could also be used as a treatment research platform, testing the multisystem effects
and efficacy of treatment interventions.
18.2.3 IS AUTISM’S ETIOLOGY PRIMARILY GENETIC? GENES, ENVIRONMENT,

AND EPIGENETICS IN AUTISM
There is nothing in the formal DSM-IV (Diagnostic and Statistical Manual, 4th
edition) definition of autism relating to etiology except for one thing: to qualify for a
diagnosis of childhood autism, the disturbance cannot be better accounted for by
Rett’s syndrome or childhood disintegrative disorder. Beyond this, there is no exclu-
sion for any specific genetic etiology or for that matter, for any biological etiology
whatsoever. The disorder is defined simply by a constellation of behavioral symp-
toms. This clustering of behavioral symptoms into a diagnosis not unique to ASD but
is standard procedure in the DSM (American Psychiatric Association 1994).
In the early literature, some papers noted that a high proportion of parents of
individuals with autism had occupations that would expose them to potentially toxic
chemicals (Coleman 1979, Felicetti 1981, Rosenberger-Debiesse and Coleman 1986).
But for decades, the emphasis in thinking about etiology has been on genetics. More
recently, there has been increasing openness to considering environmental influences
and gene—environment interactions (Campbell et al. 2006, 2008, D’Amelio et al.
2005, Newschaffer et al. 2007, Persico and Bourgeron 2006, Tsuang et al. 2004). By
now, there are fewer who would maintain that autism is purely genetic, but still many
who would expect that genetic influence is primary and greater, while environmental
influence is lesser and of much smaller effect.
While a variety of genes have been implicated as associated with autism, no gene
identified to date has both high impact and high prevalence. Even developmental or
neurogenetic disorders associated with high rates of ASD, such as fragile X or tuber-
ous sclerosis, do not have anything near a 100% prevalence of ASD amongst affected
individuals (Belmonte and Bourgeron 2006), suggesting that the genetic alterations
underlying these conditions would be better construed as conferring high risk, rather
than being called causal.
The basis for privileging genetics is largely inference from indirect evidence
rather than a solid knowledge of which specific genes are implicated and in what
ways they lead to what we call ASD, since such knowledge does not exist. As with
the discussion of prior points, the issue becomes examining whether the indirect
evidence makes a strong case for a uniquely primary genetic contribution, or whether
it is also consistent with a significant contribution from nongenetic factors.
A full discussion of these issues is beyond the scope of this chapter, and good
coverage of much of this is available elsewhere (Corrales and Herbert in press). The
discussion here focuses on what is most pertinent in the setting of articulating a
pathophysiology-centered approach to autism.
Two key pieces of indirect evidence for a strong or primary role for genetics are
the high monozygotic-twin concordance rates and the high sibling-recurrence rates.
However, a number of factors could contribute to at least somewhat altered interpre-
tations of these numbers:
1. High heritability is often overinterpreted as being exclusive of genetic

influences, whereas in fact, high-genetic contributions can coexist with
high-environmental contributions—i.e., the total percentage can add up to
much more than 100% (Rothman and Greenland 1998, Visscher, Hill, and
Wray 2008).
2. Intriguingly, it has been found that when monozygotic twins are distin-
guished by whether they shared a placenta (were monochorionic) versus
whether they each had their own (were dichorionic), the monozygotic
monochorionic twins averaged 60% concordance for schizophrenia,
whereas for the dichorionic monozygotic twins the concordance was only
10.7% (Davis, Phelps, and Bracha 1995). The investigators suggested that
this implies an infection, probably viral; it could also be due to perfusion
differential imposed between placentas; notably oxidative stress modulates
vascular reactivity (Yao et al. 2006) and there is a particular sensitivity to
this problem later in gestation during periods of rapid growth (McGinnis
2007). To date, a comparison of monochorionic with dichorionic monozy-
gyotic twins has not been performed in ASD research in spite of some
efforts, due to the obstacle of poor delivery-room record-keeping regarding
placenta characteristics in US hospitals (Hallmayr 2008).
3. Some recent as yet unpublished twin studies are showing high-dizygotic
concordance rates, one interpretation of which is that shared uterine envi-
ronment is pertinent. Fetal impacts are being approached in a variety of
ways in the ASD field by a number of investigators (Braunschweig et al.
2008, Connors et al. 2008, James et al. 2008, Smith et al. 2007).
4. It is not clear at all how to quantitate the potential impact of epigenetics that
could be an enormous confound, potentially reflecting environmental influ-
ences over several generations and not just in the current individual.
5. The identification of copy number variant genetic alterations in singleton

children but not in their parents, as well as the increase in autism incidence
with increasing paternal age suggest that environmental influence could
destabilize gene replication or mutate genes (Corrales and Herbert in press).
6. Arguments that increase in ASD prevalence are due to greater awareness
or earlier diagnosis and looser criteria are being challenged by recent
empirical epidemiological studies suggesting a strong role for environment
(Hertz-Picciotto and Delwiche 2009).
In addition, various pathophysiological abnormalities identified in ASD can result

from environmental and not just (or perhaps even more than) genetic contributors.
Some examples are as follows:
• Some studies have identified mitochondrial abnormalities in a significant

fraction of individuals with ASD (Correia et al. 2006, Filipek et al. 2004,
Oliveira et al. 2005). This has raised the question of whether the abnormal-
ity in mitochondria is associated with disease entities (presumably geneti-
cally based and rare) or whether it is a dysfunction that is more common
(Rossignol and Bradstreet 2008). Our appreciation of the complexity of
mitochondrial disease and dysfunction has increased enormously in recent
years. But it has been known for some time that mitochondria are exquisitely
sensitive to dysfunction resulting from the impact of exogenous substances
whether pharmaceutical or xenobiotic—thousands of which target various
phases of mitochondrial metabolism (Wallace and Starkov 2000). Recent
work by Holtzman (2008) identified mitochondrial dysfunction, most com-
monly in Complex I of the electronic transport chain, in 12 out of 12 cell
cultures of individuals with autism as compared with their unaffected
siblings. It is quite unlikely that all of these unrelated individuals carried
the same mitochondrial genes; it is much more conceivable that Complex I
as well as potentially other parts of mitochondrial metabolism are targets
for a diverse array of influences and hence highly vulnerable.
• The activation of microglia and astroglia is well known to be associated
with a large range of environmental influences, including, but not limited
to infections, ultrafine particulate matter, heavy metals, and other pollutants
(Calderon-Garciduenas et al. 2008a,b). The identification of significant
numbers of these activated glial cells in the brains of individuals with
autism suggests an environmental influence.
• Many xenobiotics are known to impact aspects of synaptic development and
activity (Slikker and Chang 1998), although this is not often mentioned in
discussions of “autism as a disorder of the synapse” (Zoghbi 2003). These
impacts could interact with underlying genetic vulnerability and exert their
effects at lower dosage or with more severe effects (Pessah and Lein 2008).
The identification of genes associated with autism being regulated by neuronal
activity (Morrow et al. 2008) raises the question of how the environmental
(and particularly xenobiotic) modulation of this neuronal activity might inter-
act with such genes to lead to the amplification of impact on the phenotype.
In reviewing psychiatric gene–environment interaction literature, Rutter (2008)

notes the apparent contradiction between high heritability and the miniscule
effects of individual genes as assessed through molecular genetic investigation.
He comments
The G×E findings raise the possibility that the mistake has been to assume that all
genetic effects are “main” effects independent of the environment. The truth may be
that there is much more gene–environment interdependence than has been appreciated
up until now (see Caspi and Moffitt 2006, Rutter 2007). Also, it is very striking that
the G×E effects that have been found are of moderate size and by no means are as
small as the main effects of single genes considered independently of the environment
(Rutter 2008).
The chronic features of autism, the fact that environmental triggers are known more
broadly to be associated with much of the pathophysiology identified in ASD (even
if specific linkages have not yet been established in this context) as well as the other
above arguments all point toward the need for a framework that includes environ-
ment as well as genes.
18.2.4 IS AUTISM A STATIC ENCEPHALOPATHY? PLASTICITY IN AUTISM

There is nothing in the formal definition of autism that specifies prognosis. A person
simply has to meet behavioral criteria at a particular point of time. For a diagnosis
of autism disorder, a child has to meet these criteria fully and before the age of 3.
If some criteria are missing or if not all become evident before 3 years of age, there
are variants of spectrum diagnoses available. But there is nothing to say that a
person who meets these criteria at one point and no longer meets them later in life
did not “really” meet them in the first place. Yet it is generally, though not univer-
sally, assumed that autism involves lifelong and irreversible impairment.
It also needs to be remembered that autism is not at all universally accompa-
nied by mental retardation. The IQs of individuals with ASD range from mentally
retarded to highly gifted. The alterations leading to the “autism” are not a function
of intelligence.
The impact of any belief system is to focus attention toward information and
questions consistent with belief, and to filter out the perception of features not per-
tinent or contradictory to the belief system or conceptualization. In the field of ASD
research and treatment, because the improvement or loss of diagnosis has not been
considered conceivable, most outcome studies in autism have not collected data per-
tinent to documenting and discriminating the details of such positive outcomes. In
some questionnaires, a child who started as nonverbal and then becomes verbal will
produce a score that goes down (implying worsening), presumably due to the lack
of anticipation of this outcome in the design of the measure. Reports of improve-
ment have often been met with dismissive and even indignant assertions that the
individual “could not have been really autistic in the first place,” generally without
an acknowledgment that making such a statement goes beyond either the definition
of autism (which does not include prognosis) or the evidence of published outcomes
(which has not been sensitive to the loss of diagnosis).
This dismissive attitude has not made recovery stories go away. A substantial num-
ber of anecdotal reports are circulating that describe transient improvement under
conditions of stress, intense emotion, clear fluid diet in preparation for colonoscopy/
endoscopy, and after anesthesia. In addition, the Internet and YouTube abound in
narrative and video documentations and parent testimonials about recovered autis-
tic children. But for decades, there has also been a small amount of the academic
documentation of improvement, the loss of diagnosis, and recovery. Early reports of
improved outcome include the Case #1 in the 1943 paper by Kanner in which autism
was first described and named. This individual appeared severely affected through
childhood—his parents were in fact told that there was nothing to be done for him
and were advised to let him live with a caring family elsewhere and get on with their
lives. In late adolescence after a severe illness diagnosed as juvenile rheumatoid
arthritis, for which gold salts treatment was administered, he experienced a remis-
sion not only of the arthritis but also of the autistic symptoms, and went on to earn
a bachelor’s degree, live independently, hold down a job, and travel widely (Kanner
1968, 1971, Olmsted 2005). Early documentation of improvement and recovery also
includes papers coauthored by Rutter, Greenfeld, and Lockyer (1967), Rutter and
Lockyer (1967), Gajzago and Prior (1974), DeMyer, Hingtgen, and Jackson (1981),
and Lovaas (1987). Fein and colleagues have produced a further review of outcome
studies and the notion of autism recovery (Helt et al. 2008). Recent reports of the
loss of diagnosis in children rigorously diagnosed with autism according to cur-
rent diagnostic standards suggest that the loss of diagnosis is likely to be accompa-
nied by residual neurodevelopmental impairments such as attention deficit disorder
or language impairment, and that good motor functioning is predictive of optimal
outcome (Fein et al. 2005, Kelley et al. 2006, Sutera et al. 2007). In addition to
recoveries, there are also the transient improvements (e.g., fever or oral antibiotic
associated) and animal model reversals of developmental disorders already reviewed
in the introduction.
The loss of diagnosis and the cases of transient improvement in core features—
as well as the fairly common short-term fluctuation between more lucid days and
days of being more severely “zoned out” that parents can find maddening—raise
intriguing questions about what kinds of underlying neurobiological basic features
and changes could enable such variability and improvement to occur (Herbert and
Anderson 2008). The transient improvements add further intrigue by suggesting
that changes occurring over a very short timescale can lead to significant observable
improvements in the level of functioning, further honing the questions posed by
these phenomena—what kinds of neurobiological mechanisms could be amenable
to such rapid change?
An interesting potentially related phenomenon is an increasing recognition of an
often substantial discrepancy between expressive and receptive language impair-
ments. Many clinicians and parents are observing signs of receptive language abilities
in some individuals with autism far in advance of their expressive language capa-
bilities. Some such individuals test extremely well on IQ tests and can read and use
keyboards to express themselves (sometimes showing great creativity and nuance),
but not produce speech. Some attribute this discrepancy to oro-motor apraxia and
others focus on sensory-processing issues; increasing research attention is being paid
to this phenomenon, which supports the importance of remembering that mental

retardation is not coupled with ASD, and that individuals with ASD can have great
potential. It again suggests that the brain changes causing the autism may not be
about deficit but could be about the difference or alteration or perhaps obstruction of
a potentiality for which brain capacity is present but whose utilization or expression
is heavily obstructed in some fashion.
There are various further clinical findings and ASD-pertinent pathophysiologi-
cal phenomena that suggest either the presence of plasticity or pathophysiological
mechanisms that would be consistent with plasticity potential:
• Certain metabolic disorders that are frequently accompanied by autism are

amenable to treatment where resulting improvement in the metabolic condi-
tions is sometimes accompanied by improvement in autistic features such
as the lessening of severity (Page 2000). Autistic symptoms are reduced in
phenylketonuria by a low phenylanlanine diet (Gillberg and Coleman 2000),
in hyperuricosuric autism by a low purine diet with or without allopurinol
(Coleman 1989, Gillberg and Coleman 2000, Page and Moseley 2002), in
patients with low cerebrospinal fluid (CSF) biopterin by biopterin supple-
mentation (Fernell et al. 1997), in some hypocalcinuric autistic patients
by calcium supplementation (Coleman 1989), in some patients with lactic
acidemia by thiamine and/or ketogenic diet (Coleman 1989), in cerebral
folate deficiency by folinic acid supplementation (Bauman 2006, Moretti
et al. 2005), and in Smith-Lemli-Opitz syndrome by cholesterol treatments
(Natowicz 2004). Some clinicians use vitamin cocktails to treat mitochon-
drial disease and report that when this is done with autistic children, some
show significant improvement in function and reduction in the severity of
autistic-like behaviors (Gold and Cohen 2001).
• Many individuals with ASD and other neurodevelopmental disorders are
noted to have sub-epileptic electrophysiological disturbances on their
EEGs. In addition, some children with autism experience improvement in
core symptoms when treated with antiepileptic medications, even if they
do not have epilepsy. This raises intriguing questions of the extent to which
language impairment, emotional information processing, sensory distur-
bances, and sleep problems may be on a continuous electrophysiological
distribution with seizures and epilepsy. This may well be the level at which
metabolic disturbances and neurophysiological disturbances interact to pro-
duce what is here being labeled “chronic dynamic encephalopathy.” This
issue cannot be addressed by the current standard neurological evaluation
for seizures, which does not include an evaluation for other nervous system
functions governed by brain electrophysiological activity. While the clini-
cal evaluation of EEG studies is generally done by visual inspection only,
the contemporary computational analysis of electrophysiological tracings
has great potential for identifying patterns of disturbance not apparent to
the unaided eye, and their utilization in probing studies of the neurophysi-
ology of sleep and sensory processing in ASD appear to be on a path to
contributing insight beyond seizure diagnosis and expanding the clinical
utility of electrophysiological assessment. This clinical research ferment is

an example of a shift from a disease model (i.e., seizures vs. no seizures)
to a functional pathophysiology model at the level of brain signaling (i.e.,
the examination of the clinical impact of more subtle electrophysiological
disturbances).
• Findings from an MRS study documenting the recovery of reduced NAA
discussed under question #1 in item #4 suggest both that cells are alive
rather than missing and that function can potentially be restored if the irri-
tant is removed. It also raises intriguing research questions, such as what
the impact might be on the cortical connectivity of cellular dysfunction
sufficient to lower NAA, and whether both of these measures might be
expected to improve coordinately in improvement associated with pertinent
pathophysiological change. This is a question that, to be entertained most
appropriately, requires an integration of measures sensitive to pathophysiol-
ogy with sophisticated cognitive neuroscience.
• The administration of lipopolysaccharide, a bacterial cell membrane
component to wild-type mice and elicited an elevation of tumor necrosis
factor-α that subsided in the serum within 9 h and in the liver within a
week but persisted in the brain for 10 months (Qin et al. 2007); a variety
of other pro-inflammatory brain factors also showed increased expression.
This suggests that many triggers of brain inflammation, even if sporadic,
can lead to a chronic inflammatory state. With any kind of reexposure to
triggers, this state can become unremitting and even self-propelling due to its
long persistence. For all intents and purposes, this persistence will create
the impression of a static trait, even though there is an underlying dynamic,
active component.
• The excitotoxic impact of glial activation as well as its impact on gap junc-
tions, which in turn could substantially impact electrophysiology and have
further downstream impacts on electrophysiologically mediated functions
such as sleep, sensory processing, and seizures, suggest that dysfunction in
all of these downstream areas is dynamic rather than static.
• The modulation of glial activation as well as the opening and closing of gap
junctions modulated among other things by glial activation can occur on a
short timescale commensurate with the timescale of the transient improve-
ments reported in Curran et al.’s paper (2007) on improvement with fever
and Sandler et al.’s paper (2000) on improvement with oral antibiotic
discussed in the introduction.
• The dietary depletion of tryptophan, which is a precursor of serotonin (very
frequently documented as abnormal in ASD), has been shown to exacerbate
autistic behaviors (McDougle et al. 1996); tryptophan can also be depleted
by neuroinflammation, which upregulates the tryptophan-dependent
synthesis of kynurenin, thereby depleting the tryptophan available for the
synthesis of brain serotonin (Maes et al. 1997).
• A provocative recent theoretical paper hypothesizes that the strikingly
improved behavior and enhanced communication manifested in some indi-
viduals with ASD during fever suggests the involvement of a pervasive
neural system that can affect relatively rapid changes in the functional
activity of widespread neural networks involved in the core features of ASD
(Mehler and Purpura 2009). The authors specifically suggest that fever
might transiently restore normal function to a dysregulated locus ceruleus–
noradrenergic system (LC/NA). This system is capable of facilitating rapid
and widespread neural network remodeling to behavioral adaptations
to environmental challenges. The authors note that their hypothesis is in
keeping with studies that have failed to find substantive neuropathologi-
cal lesions in the cerebral cortex and other brain sites. Both their specific
hypothesis about LC/NA and the more general notion that a mechanism
performing the rapid regulation of functional remodeling is likely to be
operative are intriguing and will undoubtedly trigger further research on
mechanisms of plasticity and interventions for enhancing it.
Each of these examples suggests in its own way that if some of the pathophysi-
ology or dysfunction can be reduced, there is a potential for clinical improvement
that would not be predicted in the classical “developmental disorder” framing of
ASD. These insights come from a pathophysiology-centered approach to autism
which, not being bound by the model of static encephalopathy, can orient us to
a range of possible mechanisms that could contribute to transient and sustained
improvement. Such an orientation is better suited than a central focus on genetic
alterations of brain development for studying these changeable features that are
more suggestive of dynamic than static encephalopathy. The possibility that we are
not dealing with a developmentally based deficit but with potentially partly or fully
functional domains whose dysfunction is not only or even necessarily structurally
base but also has a significant contribution from obstruction by pathophysiologi-
cal dysfunction can be considered in this fresh framework; this can open a greater
range of approaches to potentially helpful treatments. Moreover even if there is a
developmentally based alteration of brain development, that in itself is not sufficient
reason to exclude the possibilities of (1) brain plasticity and (2) clinically significant
improvement via the amelioration of exacerbating contributors such as metabolic
and energetic dysfunctions.
Finally, investigating dynamic features of autism could contribute to our
understanding of underlying mechanisms. The treatment of pathophysiological
maladaptation (e.g., remediation of inflammation or oxidative stress) could be an
interesting cognitive neuroscience probe. If these treatments have any efficacy
in improving behavioral function, it would be most interesting to document the
nature and distribution of the functional and structural neural systems impacts,
and ask some important questions: Does functional improvement occur one
domain at a time or in an across the board fashion? Might some treatments (e.g.,
the treatment of disrupted gut microbiology) more specifically target repetitive
behaviors, obsessions, and stereotypies while other treatments (e.g., the reduction
of oxidative stress) have more general effects? The answers would tell us a lot
about underlying brain mechanisms; but to get these answers, a real partnership
in research and treatment between pathophysiology and cognitive neuroscience
is necessary.
18.2.5 HOW DOES SPECIFICITY IN AUTISM RELATE TO MANY OF THE

PATHOPHYSIOLOGICAL FEATURES THAT ARE NOT UNIQUE TO
AUTISM? NON-SPECIFICITY OF IMPORTANT PATHOPHYSIOLOGICAL
FEATURES IN AUTISM AND ITS IMPLICATIONS
Neither genetics nor environmental research has to date found any one factor that
seems clearly and dominantly causal in ASD. On the other hand, some of the physi-
ological (including metabolic) changes being identified relate to vulnerability to
a multiplicity of agents and stressors. Methylation and transsulfuration pathways
are vulnerable to a myriad of environmental agents; mitochondria are exquisitely
vulnerable to an enormous number of pharmaceuticals and xenobiotics; and the
immune system shifts being identified are potentially both caused and perpetuated
by a wide range of triggers. In addition, these features are not unique to ASD; very
similar underlying physiological alterations are being identified in an impressive
range of other contemporary chronic illnesses.
Meanwhile, we know that there have been perpetrated an exponentially increasing
number of technological innovations leading to an evolutionarily unprecedented
increase in the range of new-to-nature exposures (Goldman and Koduru 2000,
Grandjean and Landrigan 2006). In effect, a whole panoply of exposures and stres-
sors are converging on a smaller number of physiological pathways, overwhelming
them and altering their systems dynamics. One output of this alteration arguably is
autistic behaviors.
Thus, from a systems point of view, the “outputs” of the pathophysiological
disturbances are “emergent properties” of the system rather than specifically deter-
mined by particular inputs, such as a particular gene or a particular toxin. It is
perhaps fortunate in an ironic kind of way that there appear to be final common
pathways of physiological compromise. This means that interventions might not
need to be so specific, and that the support of the vulnerable physiological functions
could serve to address harm from many inputs, break out of the gridlocked mal-
adaptive state and allow a resetting of the system into a more adaptive homeostasis
(Jones 2005, Rose 2001).
However, our methods for developing and evaluating interventions are based on
a more determinist model, where we look for sensitivity and specificity of both bio-
markers and targets for pharmacological intervention. From this determinist point of
view, it would seem to be an odd notion to use a more generically oriented treatment
(e.g., the treatment of inflammation) to treat a much more specific problem. This
presents an obstacle to the study of systems-oriented treatment research. As research
accumulates to support the systems-oriented active pathophysiological model of
autism described above, it is hoped that methodologies will be developed and find
acceptance that are suitable to the complexity and individuality of ASD pathophysi-
ology, and to its amenability to a range of physiology-supportive interventions.

The complex dynamic pathophysiological features of ASD cannot be encompassed
within a classical view that formulates the condition as a genetically determined
brain-based static encephalopathy. Much information already exists suggesting a
more inclusive model formulating ASD behaviors as one of a range of outputs of

a set of active, persistent dynamic and interactive pathophysiological disturbances
resulting from inputs that include environment as well as genes.
It is important to aggressively pursue approaches to research and treatment based
upon this more inclusive model because there are strong reasons to believe that it will
deliver palpable help sooner and will open the way to a greater range of construc-
tive approaches. Cooperation and collaboration will allow the knowledge and skill
sets of a range of specialists to come together in a synergistic approach to the mul-
tileveled challenges posed by autism (as also by other complex chronic conditions).
In ASD, well-designed demonstrations of environmental influence, of physiological
dimensions, and of physiological interventions regarding whether and how they lead
to demonstrable, measurable brain change are probably the most critical leverage
points to address in helping to redirect the central force of our efforts toward the
helping most effectively.
The identification of pathophysiological disturbances in ASD consistent with
environmental contributors, alongside of an increase in the prevalence of the
condition, raise concerning questions not only about autism but also about much
broader features of our way of life. The need for a transition to a more com-
plex systems pathophysiological approach to autism is paralleled by the need
for a transition to more integrative ecological approaches to healthy sustain-
able production that would reduce the destructive inputs that now appear to be
overwhelming physiological (as well as social, psychological, ecological, and
biogeochemical) systems. That a dynamic pathophysiological is supported by
phenomena consistent with plasticity and malleability is encouraging and should
help speed the advance of our models and the delivery of much needed help to
affected individuals, and also encourage us to look for critical leverage points in
other challenged systems.
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19 AtheReevaluation of
State of Autism
Treatment: The Need
for a Biopsychosocial
Perspective
Eric London*
Department of Psychology, New York State
CONTENTS
19.1 Introduction ............................................................................................... 389
19.2 Biopsychosocial Model ............................................................................. 391
19.3 Scope of the Term “Autism” ..................................................................... 393
19.4 Treatment of Autism ................................................................................. 394
19.5 Recommendations for Autism Treatment ................................................. 398
19.5.1 Primary Care Autism Professional ............................................ 399
19.5.2 Assessment and Treatment Centers ............................................ 401
19.5.3 Funding, Government, and Collaboration ..................................403
19.5.4 Positive Psychology ....................................................................405
19.6 Autism, Anxiety, and Autonomic Regulation ...........................................408
19.7 Treating the Environment ......................................................................... 410
19.8 Conclusion ................................................................................................. 413
References .............................................................................................................. 413
19.1 INTRODUCTION
An anecdote (take 1): My 21-year-old adult son with autism was taken to a klezmer
music concert. The staff of his treatment program reports that it was a wonderful
success. He loved the music, so much so that he got up and jumped up and down
and waved his arms ecstatically. His joy was infectious and made many of the other
389
concert-goers loosen up and enjoy the concert. His treatment team will look for other
concerts, which might also make him that happy.
Anecdote (take 2): My 21-year-old son was taken to a klezmer music concert.
The staff of his treatment program reported that this was not a successful outing.
He became so excited by the music that he could not sit in his seat; he waved his
arms and jumped, which must have disturbed others at the concert. Redirection was
ineffective as he was so excited. The plan will be to take him to more concerts but
perhaps not as stimulating for him so that he can learn appropriate behavior in a
concert setting; however, for the foreseeable future, klezmer music will not be part
of his plan.
These two versions of events are composites of a host of true stories that go on
every day in the world of autism. Aside from being a tactical decision as to whether
or not the treatment team should enforce “appropriate” behavior, the question also
speaks to a whole concept of how society and those with the disease of autism should
interface with each other. To explore this topic will raise a host of questions as to how
the presently established systems, which interface with autism, might be redirected
toward revised roles and relationships with each other.
Autism and its related disorders are among the most severe of the chronic child-
hood onset illnesses. A diagnosis given to a two-year-old will surely be a life-altering
experience. That family will never be the same. Nearly any measure of morbidity
can document the severity of this disease and for that reason, there is a pressing need
for treatment. In the past 20 years, we have seen great progress in the development
of some treatments for autism. These treatments have grown out of existing tech-
nologies such as applied behavior analysis (ABA) and psychopharmacology. These
techniques have been applied to autism, sometimes with a great deal of success and
sometimes not. Looking at the situation broadly however, Howlin et al. (2004) pres-
ent a bleak picture. Only 12% of autistic adults were rated as having a very good
outcome with 46% poor and another 12% were termed very poor outcomes. Some
of the measures used in this report were cognitive, language, social, communication
measures, as well as behavioral problems. Few of those individuals lived alone, had
close friends, or permanent employment. In another report studying a cohort born
in the 1960s through the 1980s, Billstedt et al. (2005) overall outcome was poor in
78% of the cases. It is likely that with more developed and more generally available
treatments, the numbers would be better if measured now; however, the overall con-
clusion must be that a very significant number of autistic individuals have and will
continue to have negative outcomes, which dwarf that of most other diseases.
The great need for treatments has directed the discussion, thinking, and plan-
ning about autism to simple questions as to whether these treatments are effective
and or whether they have side effects that outweigh their benefits. While even these
questions are difficult to answer, there exists little discussion in the literature or at
professional meetings as to what the more overarching goals of autism treatment
ought to be.
The science of the treatment of autism (and the other developmental disabilities)
can reasonably be characterized as being in its infancy. Although the state of the art
is disappointing to those who are in dire need of relief, the more optimistic view is
that we have an opportunity to conceptualize treatment possibilities in a fresh way.
A Reevaluation of the State of Autism Treatment 391
We can learn from some of the mistakes made in other areas of the medical psycho-
logical and social treatments for various diseases and conditions.
The goal of this chapter is to elucidate some of the issues, which have been either
totally ignored or received less than adequate attention by the professional and the
advocacy community. As this is a broad task, many topics will only be alluded to
rather than exhaustively explored. Clearly, there are no simple answers as to the best
way to approach the treatment of autism and with a spirit of humility, we must strive
to understand our predicament. In doing so, we must evaluate our conceptual models
with an attitude of inquiry. Quick fixes and sound bites will only hamper the effort to
provide benefit to those who most need it. I will attempt to challenge some prevailing
concepts around autism treatment and make a list of action items, which might be
used in moving toward a more effective and rational treatment portfolio for autism.
These action items will include the creating of a primary care model for autism treat-
ment, the creation of specialized assessment and treatment centers, the formation of
collaborations to cross disciplines including both the voluntary sector as well as the
government, the recognition and the more explicit treatment of autonomic dysregu-
lation, and the shift in focus of intervening in the environmental factors that have
large effects on the outcomes in autism. The central concept that unifies all of these
modifications is the application of the biopsychosocial model of treatment delivery.
19.2 BIOPSYCHOSOCIAL MODEL

I would like to propose the utility of the biopsychosocial model. This terminology
was introduced and popularized by George Engel (Engel, 1977, 1980, 1992, 1997).
It became an important focus of discussion in the 1970s. His papers were largely
aimed at the medical community although at this point in the twenty-fi rst century,
it appears that his message is relevant for a wider group of readers. As it is likely that
many of the readers will be unfamiliar with Engel’s body of work, I would like to
outline the basic concepts of this model. That will be followed by a description of
some proposed ideas of how this model can be applied to autism.
After World War II and into the 1960s and 1970s, the medical field was moving
dramatically in a direction toward a more and more biologically based practice.
New discoveries and techniques supported the development of more technologically
advanced practitioners. This along with a trend to more urban and suburban living
at the expense of rural living hastened the decline of the G.P., that is, the general
practitioner. Medical students were in greater numbers convinced that to have suc-
cessful and an up-to-date career, it was necessary to specialize and even to sub-
specialize. In addition, the 1960s was a time of social upheaval and activism and
there was a movement away from disjointed and impersonal health care. The new
social consciousnesses lead to the creation of the family practice specialty with its
own training programs. This new specialty had to fight for hospital privileges and
to be recognized as a legitimate specialty. Family practitioners struggled to have
their own departments in medical schools. This movement was largely successful in
capturing the support of the public, primarily due to its philosophy of humanizing
medicine again. There was also a very clearly stated awareness that medicine had
nearly totally focused in the realm of pathology and how to remediate it. Preventive
medicine (except for some of the neater technologic fixes such as vaccinations and
plumbing to produce clean water) was left in the dust.
In reality, another societal force, the introduction of managed health care and cost
containment, helped the family practitioners to prosper at first with the concept of
replacing higher priced specialists with lower cost generalists. Perhaps for the same
reason, they became the next target of cost containment. In 1998, the number of
family practice residencies positions filled by U.S. medical graduates began to fall.
This appears not to be inconsistent with a move back to “expert” technically oriented
care or in other words, back to what has been called the medical model. Preventive
medicine and a more holistic practice continue to be only a minor part of the
physician’s role.
Engle, in describing his theory of biopsychosocial medicine, draws from general
systems theory and outlines a number of interacting “systems.” He describes these
starting with subatomic particles, and progressing to atoms, molecules, organelles,
cells, tissues, organs/organ systems, nervous system, person, two person, family,
community, culture/subculture, society/nation, and biosphere. Looking at the above
list of systems and conceptualizing them one a linear chart, one can subcategorize that
the first few systems from subatomic particles to nervous system are what we usually
term basic sciences. These are the foci of the medical model. The next level of person
up to the family is usually under the prevue of psychology while the systems of com-
munity up to biosphere are generally thought of as social. In our specialized world
of medical care, the physician handles medical model, the psychologist handles the
psychological issues, and the social worker deals with the social issues. If health
problems were to neatly fit into these systems there may be no reason to even do
this level of analysis; however, this is not the case. Each of these systems maintains
a separate existence and separate boundaries only up to a point. There is always a
dynamic interplay between the systems. A molecule, for example, retinoic acid, if
ingested, can lead to a depression, which can lead to family problems, or even rise to
the level of societal problem if the protagonist is in a central enough position. In the
other direction, wars cause emotional trauma, which we now know can alter levels
of the molecules made in the brain. Systems interact with each other not only on
this vertical level, but also a two-person system can interact with other two-person
system. The relationship between an employer and his employee can influence the
relationship between that employee and his wife.
The application of a systems model would change the content and certainly the
emphasis of the scientific literature associated with clinical issues. Scientific method
is inherently reductionist. The more complex the system, the less elegant the science
will be to study it. It is no accident that despite a desire to be scientific, there remains
very little “good science” to prove the benefits of psychotherapy. The evidence that
does exist is largely limited to behavioral therapy and cognitive therapies. Both of
these two therapies lend themselves more easily to manualization and therefore stan-
dardization. Therapies that call for more spontaneity on the part of the therapist are
much more difficult to standardize and study (Frank, 1979). Those therapies with
more concrete outcomes are more easily measured. For example, it is easier to mea-
sure a behavior than an insight. The more factors one includes in their analysis (as is
inevitable in a systems-based approach), the less amenable the therapy becomes to
being researched. There is now a general appreciation that often, randomized con-
trolled clinical trials give results that differ from “real-life” population-based studies.
This is to say that the studies that take into account the larger or more systemic
issues, while being less specific, may offer results closer to the realities of clinical
problems. Effectiveness studies, also known as naturalistic, pragmatic, practical, or
real-life studies attempt to mimic daily clinical practice but often at the cost of
scientific reducibility. Therefore, one could say that clinical studies may suffer if sci-
entific rigor is elevated at the expense of a systemic understanding. It is only recently
that “real-life” studies are appearing in the literature and that funding sources are
acknowledging their necessity.
What is true for research is all the more of an issue in clinical practice. The patient,
who typically identifies a problem (be it on a biological or psychological or social
level), generally choose to go to one practitioner. If that practitioner is limited in his
or her scope, which nearly all practitioners are, this can lead to inferior care and often
to a lack of success in treatment. Despite the wide spread acknowledgment of Engel’s
work (up until 2002, there were 1419 citations of his science article), it appears that
the biopsychosocial model remains on the margins of medicine. Lindau et al. (2003)
believes that the biopsychosocial model has failed to replace the biomedical model
for two major reasons both of which are conceptual. One is because of our being
wedded to a particular model of fighting disease (rather than promoting health) and
the second is our existing institutions are historically rooted in the medical model.
I would like to add that the failure of this model to be more accepted could also be
explained by systems theory itself. Physicians live in multiple systems. Physicians
are also humans who desire to make money to support their families. Managed care
discourages this model as each physician spends only a few minutes with each patient.
If physicians were to delve into the psychological and social spheres with the same zest
that they approach the biological issues, it would take much more time. This would be
financed directly by reducing the physicians’ own income. This is a huge disincentive
to broaden ones view of the patient. Medical schools despite giving lip service to the
biopsychosocial model, I would suspect, continue to prefer the biomedical model, and
therefore, reward students who are more interested in that model. A brief glance at
the National Institute of Health funding patterns should convince anyone that the road
to an academic career (except in rare cases) is not paved through the biopsychosocial
model. Further, with other disciplines staking out their “turf,” it seems safer to fall
back on the skill that makes the practitioner unique, which for the MD is biologi-
cal. Despite the psychiatric establishment being sympathetic to the biopsychosocial
model, a huge political effort was aroused when psychologists tried to get prescribing
privileges thus potentially usurping their biologic role. Despite the ongoing calls of
prestigious institutions, the National Research Council and the Institute for Medicine,
to embrace this model, it appears that this will require much more effort than one can
reasonably expect from our currently empowered institutions.
19.3 SCOPE OF THE TERM “AUTISM”

Over the past few years, very few diseases have gotten the amount of public atten-
tion as the autistic disorders. It is also likely that no other disease sparks the levels of
controversy that autism does at this time. There could be a host of reasons for this;
however, I believe that the heterogeneity of the illness is a major factor. Most clinicians
and investigators are in agreement that autism is the syndromic endpoint of an etiologic
heterogeneous group of diseases (London, 2007). Further, there is a huge heterogene-
ity of the phenotype, which may or may not correlate with the etiologic heterogeneity
(Happe et al., 2006). Autism is present with a huge range of core symptom and severi-
ties. In addition, there is a wide range of associated symptoms with varying severities.
Even in a given individual, one can note an ever-changing set of symptoms, which vary
and sometimes even disappear with age and development. Nosologic considerations
dictate that we continue to use the word “autism” (Miller et al., 2006), yet this leads
to a situation characterized by the elephant and the blind-man paradigm, that is, the
vantage point that one is looking from and even the time frame of when you look will
determine what you see.
With this level of heterogeneity, one might easily understand that the concept of
“treatment” for “autism” is a very broad and often a confusing topic. Many treat-
ments, from many theoretical origins, practiced by many different practitioners; with
a dearth of literature support are the norm at this point. Despite all of these difficul-
ties, it appears to me that autism could be much better treated, and managed with the
application of biopsychosocial principles. Some of the ways we can improve treat-
ment would be by creating specificity about what we are treating, and clearly defining
the treatment goals. Through the inclusion more disciplines as part of a broader
treatment team and through an optimized treatment delivery system, the lives of
autistic individuals and their families can be greatly improved.
19.4 TREATMENT OF AUTISM

At this point in time, the one most proven and accepted treatment for autism is ABA.
Although there seems to be little doubt that, ABA is effective for some people and for
some reasons, the real benefit of ABA and its superiority to other interventions are not
well documented. Foxx (2008) points out that there have been over a thousand peer-
reviewed autism articles describing ABA’s success and that various impartial agencies
such as the New York State health department recommended only ABA. Disturbingly,
there seems to be a surprising lack of critical analysis of ABA by its proponents.
Reviews of ABA (Foxx, 2008; Green, 1996) at times seem to confuse a lack of research
with the proof of the lack of benefit for any other forms of treatment. They conclude that
ABA and only ABA should be instituted as treatment, and even combining ABA with
other treatments will only detract from the outcome. The motivation for this advocacy
of one particular intervention is likely due to the frustration accumulated over many
years of parents choosing untested and often dangerous treatments sometimes to the
exclusion of treatments with more evidence and a much greater likelihood of success.
Ospina et al. (2008) in a recent review arrived at a very different conclusion. They
found that although Lovass therapy showed benefits versus no treatment, they found
no evidence suggesting the superiority of that treatment when comparing it to other
active interventions. The evidence reviewed showed some support for discrete trial
training in terms of motor and functional skills but not for communication skills.
They further found some evidence of benefit of a host of other interventions although
similar to ABA, there was often a lack of quality studies or adequately sized studies.
Their overall conclusion was that based on the lack of hard evidence for ABA as well
as other treatments that interventions be guided by individual needs and the avail-
ability of resources. In a similar vein, Reichow et al. (2008) motivated by the lack of
any treatment (for young autistic individuals) that meets the criterion for evidence-
based practice in autism, suggest better methods of evaluating research so that the
practitioner can have more informed guidance for their clinical work.
It is clear that ABA has much to offer the autistic individual. However, the quan-
titation of how much it offers remains less clear. The argument often invoked by
behaviorists that unproved treatments could crowd out the better—proven behavioral
treatment can be looked upon in a different way. Although the leading treatment at
the moment, the history of medicine also shows that conventional treatments eventu-
ally yield to other types or treatments. We must be vigilant not to exclude other forms
of treatment, which currently have less or no evidence of benefit solely due to a lack
of research to document benefit. These facts call for the necessity of the rigorous
but also creative study of many types of treatments. A blind adherence to orthodoxy
can be almost as dangerous as blind faith in unproven treatments. A case that I had
some personal experience with involved an orthodox ABA school that when con-
fronted with an increase in previously well-treated behaviors, resumed behavioral
procedures to extinguish these behaviors. A few days later the young man died of
a ruptured appendix. The staff was just unaware that not all behaviors should be
treated with ABA and only ABA.
Perhaps an even more confusing situation exists for the use of psychopharma-
ceutical agents in autism. Mandell et al. (2008) in a survey of Medicaid enrolled
children with the diagnosis of autism spectrum disorders found that 56% were on
at least 1 psychotropic medication and 20% were on 3 or more medications. The use
of psychotropics was common even in the <2 age group. These numbers are not out of
line with previous estimates (Aman et al., 2005). Similar to behavioral interventions,
it seems that there can be little serious doubt that psychopharmacology has much to
offer to the autistic population. The specifics, sadly, are again not that clear.
The most established medication for use in autism is risperidone, which has
obtained Food and Drug Administration approval for the indication of irritability
associated with autism (Parikh et al., 2008). Risperidone and virtually all other
medications used in autism target associated rather than the core symptoms of
autism. In contrast, there are no drugs that are commonly used or proven effective in
the treatment of the core social and communication impairments that are hallmarks
of the pervasive developmental disorders (PDDs) (Posey et al., 2008). The lack of a
clear understanding of the pathophysiology of the core symptoms of autism makes
it difficult to identify drug targets for autism.
Therefore, current medications are targeted at symptoms rather than the disease.
It would appear that the attempt to show that a medication can treat as heterogeneous
disease as autism might be a conceptual error. The literature almost invariably frames the
benefit of the medicine in the context of the disease although nearly all medications
in psychiatry cross diagnostic boundaries in their efficacies. Medications that may be
very effective at treating one or more associated symptoms in some individuals may be
rather ineffective at treating the broad group of patients diagnosed with autism.
Another related conceptual problem is the use of the “dual diagnosis” concept,
which has been developed over the years for mental retardation. The concept is
that intellectual disability is not the reason for a behavioral syndrome. Therefore,
there must be a second diagnosis to explain the behavior. This is based on many
assumptions, which are problematic. One is the fact that mental retardation or intel-
lectual disabilities are gross descriptors of a very heterogeneous group. It seems
almost certain that some forms of intellectual disability can explain behavior prob-
lems on a biological level. A second problem is the imposition of Diagnostic and
Statistical Manuel of Disorders (DSM) diagnoses which were formulated and vali-
dated on more “neurotypical” individuals and applying these diagnoses to those who
have various types of brain development abnormalities. Is the repetitive behavior
observed in autism the same (on a neurobiological level) as obsessive symptoms seen
in obsessive compulsive disorder? Are the social difficulties seen in autism the same
as that seen in social anxiety disorder? Although there is likely an overlap between
a developmentally disabled and a more neurotypical persons with these symptoms,
it is also very likely that when the neurobiology is clarified, it will show many funda-
mental differences despite having similar behavioral endpoints. Again, medications
are more likely to be efficiently targeted to specific symptoms, which will cross
diagnostic boundaries; however, in many cases, medications may be more specific if
the underlying pathophysiology is treated rather than the symptom.
Clinicians treating autism, due to a lack of literature support, generally have to
form their own practice styles and call on their clinical experience (which may be of
great value or highly skewed). An example of the problems facing the pharmacologi-
cally oriented clinician, trying to practice evidence-based medicine is highlighted
by a recent publication. Tyrer et al. (2008) compared haloperidol, risperidone, and
placebo in 86 nonpsychotic intellectually disabled subjects having aggressive chal-
lenging behaviors. Remarkably, the placebo group showed the most benefit in reduc-
ing the aggression scale score, with placebo, risperidone, and haloperidol showing
79%, 58%, and 65% improvement, respectively. The author’s conclusion was that
“antipsychotic drugs should no longer be regarded as an acceptable routine treatment
for aggressive challenging behavior in people with intellectual disability.” What was
meant by an acceptable routine treatment is not clear to me. An interpretation of this
conclusion could be that they are contending that this type of patient is being inad-
equately treated on a non-pharmacologic basis although they do not state that explic-
itly. In the same issue of Lancet (Matson and Wilkens, 2008), there is a comment on
that article. These authors point out that in most cases of aggression in people with
intellectual disability, there is an environmental component such as escape from
demands, attracting one’s caregiver, or gaining access to preferred items. They are
taking a more biopsychosocial perspective and suggesting that perhaps a behavioral
intervention or a combination of treatments would be more effective. Scahill et al.
(2008), in response to this study, point out a couple of methodological flaws to the
Tyrer article. These include the use of two active comparators with a small sample
size and the selection criterion used.
Tyrer et al. (2008) themselves also note that their findings were not concordant
with other studies in the literature, which have shown antipsychotics valuable for this
population. It seems that Tyrer et al. (2008) as well as Matson and Wilkins (2008)
were tying to put forth the argument that a psychosocial intervention should be tried
in aggressive persons with intellectual disabilities. The data presented had little to do
with that conclusion. Rather instead of clearly promoting a biopsychosocial approach,
the impression was left that antipsychotics are no better than placebo in this group
that strains credulity. It is striking that a 79% improvement on placebo was not even
commented on by the authors. A 79% improvement on placebo, if taken literally,
might necessitate that all intellectually impaired subjects with aggression problems
be given a trial on placebo despite the ethical issues that might be raised. Another
and perhaps the most important lesson from the above discussion might be acknowl-
edging the enormous complexity in conceptualizing benefit in this population.
The lack of a biopsychosocial model and a lack of attention to the limitations of
reductionist assumptions that permeate the clinical research field continue to make
evidence-based understandings difficult if not impossible for the clinician interested
in autism and developmental disorders. A much more interesting and informative
publication would have been an analysis of why this study was able to document
such a robust placebo effect. The psychopharmacologic literature rarely, if ever, con-
trols or even comments on psychosocial treatments, which the subjects are invari-
ably exposed too (even if only in school). This is true also for the behavioral and
educational literature making little comment on the medications being used by their
subjects. My own experience includes being an MD prescribing medications to autis-
tic children who were in behavioral (ABA) studies, where the medications were not
mentioned in the publications documenting the benefits of ABA. I suppose that I am
not alone in having that experience.
It is estimated that as many as 50%–75% of children with autism may be treated
with complimentary and alternative medicine (CAM) treatments (Levy and Hyman,
2008). Levy et al. (2003) reported that almost one-third of children referred for evalu-
ation were already being treated with dietary therapies by their parents even before
the confirmation of the diagnosis. This should not be surprising in that the National
Center for Complimentary and Alternative Medicine reports that over three-fourths of
American adults use CAM treatments for disease or to maintain health. It is estimated
that 2%–50% of children in the United States are given CAM therapies (Davis and
Darden, 2003), and this is likely to be an underestimate. Children with chronic illness
such as cancer asthma, rheumatoid arthritis, and developmental disorders are even
more likely to be using CAM therapies. Although there are some CAM treatments
with evidence to support its use (such as melatonin for sleep), many other treatments
have been clearly disproved (e.g., secretin), or are disproportionately dangerous
(e.g., chelating agents). The majority of CAM treatments, however, have not been stud-
ied and there is scant evidence to support or deny their use. Levy and Hyman (2008)
comment on the issue of families even reporting CAM use to their clinicians. Although
there may be many reasons for this, I would contend that the lack of a biopsychosocial
perspective on the part of the clinician leads the families to disqualify the practitioners
as consultants on this topic. The assumption is made that the physician is constricted in
their thinking and unable to see past what is an inadequate literature. Often this drives
the families into a counterculture of practitioners who are resistant to examining the
evidence at all and tout their own treatments. These practitioners sometimes make
rather fantastic profits on treatments, which have no medical evidence. A few years
ago when the hormone secretin was reputed to be beneficial for autism, there were
practitioners charging five figure amounts per injection.
I would also hypothesize that a large part of the reason for the sizable anti-medical
and antiscience movements, which have grown around the families of autistic indi-
viduals, is related to the lack of the application of the biopsychosocial model.
An important article “Needs of parents of mentally retarded children” was written
by Murray (1959), a chairperson of the Virginia Association for Mentally Retarded
Children (an organization now know as ARC). In this article, she outlined six basic
problems that the parents faced. One was “coping with inept, inaccurate, or ill-timed
professional advice.” Rather than this being rejecting of need for professionals, she
goes on to say “The greatest single need of parents of mentally retarded (MR) chil-
dren is constructive professional counseling at various stages in the child’s life which
will enable the parents to find the answers to their own individual problems.”
There is a feeling of disconnection that the families feel from their practitioners.
Parents perceive that practitioners tailor treatments to their own theoretical predis-
positions, tout their brand of therapy to the exclusion of others, dismiss other forms
of therapy, and have little understanding or interest in how the families perceive the
problems. Into that vacuum enter other parent advocates, often with little under-
standing of medicine, psychology, or science but with a great deal of understanding
of the problems (often systemic) that they are encountering on a daily basis. The
response has been to take the solutions to these problems into their own hands and
become their own case managers. There is no shortage of “alternative” practitioners
who are willing to lead this overtly angry charge against “conventional” practitio-
ners. A plausible way of framing the issue is that the treatment community, by isolating
themselves from the families issues (bio, psychological, and social) have not only
facilitated the propagation of an industry of dubious practices, but also the spin-off
has been the creation of a dangerous anti-vaccine lobby that is currently threatening
the public health both in America and worldwide (Offit, 2008).
19.5 RECOMMENDATIONS FOR AUTISM TREATMENT

Having analyzed the current state of the thinking around autism treatment, I would
like to now propose some innovations that if implemented might be beneficial. These
will be presented in the context of the biopsychosocial model. Although it is my
intent to be specific, each recommendation should be considered as a direction to go
in and will undoubtedly be subject to refinement and improvement.
One might rightly ask why autism as a disease would need these special consid-
erations. The answer is that our medical model and our educational model are not
good fits for this entity. The medical model is primarily set up around acute illness.
It is struggling today to accommodate the chronic diseases, which are becoming
more prevalent in our world. Alzheimer’s disorder is a good case in point. The ser-
vices needed for Alzheimer’s are heavily loaded toward care giving and nursing.
Medicare does not support nursing homes. It does support acute care hospitals and
very expensive procedures that are for acute care. Therefore, there has been a cost
burden shifted to the families and the states through Medicaid. Medical students are
trained primarily in acute care medicine. Autism is a chronic illness. Even more than
in Alzheimer’s disease, the burden of care giving falls in nontraditional settings, that
is, the families and the school systems. Schools have the legal obligation to provide
an “appropriate education,” a vague term if there ever was one. The educational
system has de facto become the major treatment entity for autism. This role has not
been fondly embraced. Evidence for this is the microscopic funding that the federal
Department of Education spends on autism treatment research, which is at odds
with an astronomical budget that school districts pay for the special education of
autistic children.
Each of the recommendations made involve major changes conceptualizing how
we treat autism. Each one involves multiple systems to be involved in the changes.
Again I would hope that these recommendations are looked upon as conceptual
directions and that further work will need to go into refining the details of how to
facilitate real change and hopefully improvement.
19.5.1 PRIMARY CARE AUTISM PROFESSIONAL

I am calling for the creation of a “primary care autism professional” (PCAP) to serve
the patient and their families. This would be modeled after the role most often associ-
ated with a primary care physician. Although some physicians or other professionals
may be playing this role now, I believe they are rare. It could be debated; however,
I would propose that the primary care professional be a nurse practitioner. Perhaps
sadly, I do not believe that medical practitioners, as the system exists, now would be
able to perform this role adequately. The practitioner will need the time to spend with
the families to work on psychosocial issues as well as the medical issues. The medical
field over the past 30 years has gone away from the biopsychosocial model despite calls
for its implementation as I have outlined earlier. I would doubt that this situation would
change any time in the near future.
The PCAP would be the first line of help for the family. That person would have
to be fluent with the needs of the early diagnosed, middle childhood, adolescence,
and up to geriatric individuals. The goal would be for this person to be in that role
for the long term, with that individual and family, again modeled after the concept
of the family practitioner.
The role would call for a strong knowledge of autism, which will only occur
through the knowledge gained through specialization. The practitioner would need
to be trained in many of the disciplines, which are relevant to helping the patients
and their families and much like a primary care physician would have to know their
limitations and when more specialized care is needed. Postgraduate training pro-
grams for this position would need to be created and should exist in universities.
Some universities have already developed certificate programs beyond the bachelors
or graduate degree that focuses on autism (Swiezy et al., 2008). As these practitioners
would specialize in autism, they will likely over time become very sophisticated in
recognizing the issues encountered and experienced at solving them. The practitio-
ner would need to be competent in medicine (this is the reason I am recommending
a nurse practitioner). This person could do a primary care–type medical visit. Simple
problems could be treated, and more complex ones would be referred on to the super-
vising pediatrician or other physician. Many of the complaints, which will be present
in these cases, will be related to autism symptoms but many will not be. The PCAP
must have the expertise to make that differentiation. Many problems may cross over.
An example might be self-abusive behavior. Although wounds must be tended to on
a medical level, the behavior must also be controlled. The PCAP must also have to
have a working knowledge of behavioral treatments, including ABA, psychopharma-
cology, as well as other emerging treatment modalities. The practitioner would need
to have the ability to institute some treatments and also a good working understand-
ing of places to refer. The practitioner would also need to be conversant with the edu-
cational system and be able to function as a case manager for obtaining the right fit
for an educational placement. As the autistic individual ages toward adulthood, the
PCAP will help council the family as to their options and help to supervise the transi-
tional planning toward the work world. The PCAP would also function in the role that
social workers usually take in geriatrics. That is facilitating the process of obtaining
support systems such as respite. The PCAP, by virtue of being “in the middle” of
these issues, would be able to perform an advocacy role. They would be fully aware
of the services that are not available but badly needed in that community.
People with autism grow up and live roughly normal life spans. It is not a child-
hood disease when the subject becomes an adult. The practitioner should be equally
conversant with the problems encountered in adulthood. The existence of a disci-
pline such as PCAP is more than just a bureaucratic nicety. As the disease of autism
is not likely to be eliminated any time in the near future, a host of treatment-based
decisions must be made. Bergsma and Engel (1988) discuss this issue in reference
to medical decisions, which must be made around the concept of the quality of life
(QOL). Some of these decisions entail when and how much palliative care should
be instituted especially at the end of life. They outline three different models of the
doctor–patient relationship. One is the model of the doctor being authoritative. That
is he or she will basically make the decision due to their knowledge and experience.
A second option is the one where the patients make their own decision, and the phy-
sician’s opinion is only of minor importance. Other sources of information can be used
such as getting second opinions, consulting with friends, relatives, and, nowadays,
the Internet. This leads to “doctor shopping” or going to different doctors until one is
found who takes the position already formulated. Unfortunately, this type of doctor–
patient relationship is not uncommon now in those families afflicted with autism. With
only specialists to consult with (the behaviorist, the occupational therapist, the psy-
chopharmacologist, etc.) families find themselves with professionals who have only
one treatment. Often each one touts his/her own treatment, sometimes to the exclusion
of the other. More perniciously, there are many practitioners who predict dire conse-
quences if their treatment is not done exclusively and around the clock. It is no wonder
that the families at this point regroup and decide that only they can make the choices.
This is often done during times of crisis and under extreme emotional pressure (not
unlike the end of life decisions discussed above). Most families are going through this
issue for the first time and have little of their own experience to fall back on. In mak-
ing these decisions, they can be more influenced by those with the slickest message
promising the best outcome. Parents bond together using the Internet and advise each
other; however, the highly emotionally charged nature of these interactions can readily
be seen. All of this often leads to poor choices or worse dangerous ones. The danger
appears now to spilling over from the autism cases themselves to the community at
large (Offit, 2008).
The third kind of relationship is one characterized by openness and mutual
respect. In this relationship, it is possible to have a dialogue and to arrive at a conclu-
sion together. This type of relationship is unfortunately becoming quite rare in our
society. It calls for trust, which is formed through a personal relationship. In most
cases, this can only be formed through long-term relationships.
Decisions are often multifactorial and often there is no absolute right answer.
Sandler and Hulgus (1992) analyze the clinical application of the biopsychosocial
model and its applicability in clinical situations. They outline three components to
the decision-making process in medicine. The first can be described as having the
scientific knowledge and the experience that the clinician can offer in their role.
The second is the ethical aspect. In this sphere, decisions need to be made based on
values. At times, the values held by the patient or the family might be different than
that of the doctor, that is, “I would rather die than have my legs amputated.” The
biopsychosocially competent practitioner must be able to represent their own values
but at the same time be respectful of the patient’s values. A dialogue about these val-
ues might help the patient (or the doctor) revise their thinking. The third component
is the pragmatic aspect. For example, if a behavioral program is recommended and
is not available or the finances to pay for it are not available, what should be done?
Perhaps a legal consultation is needed or perhaps, an alternative type of therapy
should be explored first. Again the clinician might have a great deal to offer in helping
the family make the very hard decisions of how to proceed.
I would propose the PCAP could serve in these roles for autism. As one who
is knowledgeable about the field as well as the patient and their family, decisions
can be informed by the longitudinal history developed over years. The PCAP must
be trained in systems theory, to understand that these decisions are often not one-
dimensional. If the family needs to hire an attorney to fight for behavioral therapy,
will that mean that the sibling cannot go to summer camp that year? This might
be the real issue, which the family is facing. How often is a family able to express
problem to a clinician? How often does the clinician actually help the family address
the issue? Systems theory might suppose that the stress on the family would lead
to stress on the autistic individual, which could lead to behavioral acting out. This
would lead the psychopharmacologist to suggest a medication. This may have side
effects such as sedation or gastrointestinal problems, which could lead to further
consultations. This cycle could go on and the only way to get a handle on it is for
someone (at this point it is generally the parent) to see the whole chain of events and
work with the system to maximize benefits.
19.5.2 ASSESSMENT AND TREATMENT CENTERS

To compliment a primary care model of autism treatment, it is imperative that there
also be a cadre of well-qualified specialists and it is equally important that these
specialists be accessible to those who are in need of the services. In order to accom-
plish this, I am proposing the formation of assessment and treatment centers. At the
present time, there are many university-based programs, which are fundamentally
functioning as assessment and treatment centers. There is no necessity to reinvent the

wheel; however, there are several considerations that need to be fulfilled to make this
effective. The primary difference between existing programs and these proposed
centers is in the methods of delivering the services. Although many university-
based programs are excellent, they tend to serve only a small portion of the people
in need of services. Further each center at this point might have certain areas of
focus rather than being broad based. In the proposed assessment and treatment
centers, a goal will be to make the specialty services as accessible as possible. This
will be accomplished by a more consultative model rather than being a primary
treatment center.
The concept of assessments should not be confused with diagnosis, although
diagnosis is one function of an assessment center. Rather, assessments need to be
focused on the needs of the patients. Assessments can be done throughout the life
span in order to help guide treatment decisions. Assessments currently are mandated
by school districts for their own considerations, that is, when this information is
needed for educational classification. Assessments could be done when problems
arise or decisions need to be made. Examples might include a young autistic subject
who is not easily managed at school and more routine efforts and remedying the
situation have failed. Another example would be around the beginning of adulthood
to help facilitate the transition to adult programming. These assessments would most
often be multidisciplinary. The team could include behaviorists, medical generalists
or specialists, psychiatrists, speech therapists, educational consultants, physical ther-
apists, etc. The staff of these centers would be specialists in autism and have a great
deal of expertise, which is often not easily found in the community. These assess-
ments would yield treatment recommendations, which could be carried out back in
the community by the PCAP, teachers, job coaches, pediatricians, etc. The centers
should provide ongoing supervision and consultation to the practitioners. This com-
ponent of the plan is more difficult than it might seem at first glance. The ability to
have an active dialogue between these centers and community-based practitioners
might seem simple but is rarely accomplished in the real world. There must be a rec-
ognized budget for the time spent in this dialogue. Creative funding strategies need
to be implemented to accommodate this invaluable communication.
The centers should provide treatments not available in the community. One
example would be the treatment of the very highly behaviorally disturbed autistic
individuals. At the present time, there is an acute lack of service for this individual.
More general psychiatric units are not set up to handle autistic patients. Their treat-
ment modalities used in general psychiatric units such as group therapies are clearly
not pertinent to this group. The staff is taught interventions that are more pertinent
to other conditions such as schizophrenia and mood disorders, which comprise the
overwhelming majority of their patients. There are a very few hospital-based set-
tings that are set up for this group, but they have waiting lists that make delivering
acute care impossible. Often families have to live with ongoing violence for weeks
or months prior to a bed being available. If these individuals are to be reintegrated
in the community, a “step-down” system must be available, with a slowly decreasing
amount of support available and an inclusion of the family and the ultimate commu-
nity service deliverers into the treatment package. This would imply that there also
must be a geographic distribution of these assessment and treatment centers to real-

istically serve the public. Other examples might include medical clinics with medical
specialists who are familiar with autism, and perhaps new types of specialists such
as environmental specialist who can consult about issues such as noise pollution,
which may be having a negative impact on the success of a given individual.
19.5.3 FUNDING, GOVERNMENT, AND COLLABORATION

As a physician, I am not trained nor do I have experience in designing funding issues
around health care. Our society has been struggling with issues around funding for
some time now with no consensus solutions having been reached. With this dis-
claimer in place, it appears that without a simultaneous discussion of funding issues
on the table, any hope of significant changes in the treatment delivery system is futile.
The pragmatic element of what service is available and how to fund that service may
be more important to the outcome of a given case than what medication they are on,
or what type of therapy is instituted. A significant problem is that perhaps more than
any other disease, autism is treated by many players with many different missions,
and their own budgets and funding sources.
The first step in the clinical odyssey of a family with autism is to get a diagnosis.
This is done generally through a pediatrician and is generally paid for with health
care insurance. If diagnosis is done early enough (up to age 2), the next step is an
early intervention program, which also is a medical cost. The next stop is generally
with the educational establishment. For those age 3 and above, there is preschool and
then later the school program, funded largely by the local school district. It is unclear
how much treatment the school districts are responsible for and this is often fought in
courts. It might be worth noting that the educational establishment might recoil at my
even using the word treatment, as they do not see themselves as treatment profession-
als but rather educators. In reality, whether intentional or not, at this point, I would
guess that 80% or the real treatment of autism is going on in the school setting.
Some examples of the interface between education and medicine include such
services as speech therapy. Speech therapy, which may or may not be adequately
available in the school, could be supplemented by speech therapy after school hours
at home. Health insurance often covers only “restorative” therapies. That is, contend-
ing that if the child never had language then it is not restorative. Behaviors are also
an unclear issue. How much and what type of behavioral and cognitive therapies are
needed outside of the interventions done in the school are a source of contention.
Adult services also cross boundaries. The educational systems are usually done with
their responsibility to this group after age 21. States have agencies, which service the
developmentally disabled, and that budget line becomes operative for many of the
needs of that individual. Again there is often a lack of clarity in how far that respon-
sibility must go. Housing is sometimes supported by Medicaid (a health care fund),
and ironically offices of mental retardation at times directly reimburse physicians for
health care. The health care of this group has largely been taken out of the private
sector, as most private physicians are not participants in the Medicaid program as
the reimbursement is very low. Being a very underemployed group (with health care
in our country largely tied into employment), this cohort is in reality served by the
government or agencies directly contracting with the government such as Medicaid

managed care.
Despite my being a physician serving this group as well as being a parent of an
autistic individual, and being interested in the policy issues of the autistic population,
I must confess that I am still very confused by the system. Simply stated, if after a
couple of decades of trying, I still have trouble explaining the system to anyone, it
is just too complex. This complexity, aside from being very frustrating to the com-
munity trying to access services, is ripe for abuses, inefficiency, lacks transparency,
and makes the system nearly impossible to reform.
At the time that this is being written, there is a deep economic recession in the
United States and around the world. For the foreseeable future, it is impractical to
think about increasing services through increased budgets. It is equally unthink-
able that as we progress in our understanding and capabilities of treating disease,
we settle for suboptimal outcomes because we are bogged down in a bureaucratic
quagmire. We must be willing to look at the realities of what we have created and
make the needed changes.
A conceptual solution to this quagmire also involves the understanding and
implementation of systems theory. If the treatment of an autistic individual involves
funding from three or four sources, these agencies need to be in communication
and budgets should be combined or shared to solve problems. A state government
funds their Departments of Education, Office of Developmental Disabilities, and the
Department of Health. If these three agencies are not cooperating, we are wasting
money. An example of this might be to invest hundreds of thousands of dollars into
the education of an autistic individual, train him for a job, and pay for a job coach
but not pay for transportation to the job. The individual then sits at home. Do we ask
the parents to quit their jobs to stay at home with the individual? I can attest that
this is a very real scenario, which I have encountered in my practice many times.
Just as the medical institutions need to reward physicians for being biopsychosocial
physicians, government agencies that can collaborate for the public good must also
be rewarded. The public needs to be aware of the efforts and successes of govern-
ment agencies and they should not be taken for granted. The public often views the
government as a giant pocketbook. As described in Section 19.5.1 in terms of the
doctor–patient relationship, a consultative relationship between the government and
the public could be formed.
In New York State, we are attempting to create such a partnership. The New
York Autism Consortium is being formed. This will comprise all of the elements
invested in the treatment and research of autism. Medical centers, schools, agencies,
and the advocacy community will be organized to tackle problems. Pilot programs
and research can take place with both public and private fundings. This consortium
has a seat at a newly formed interagency autism committee, which is comprised of all
of the state agencies that have an interest in autism. Having a direct line of communica-
tion from government to the private players has enormous potential. A similar concept
involving collaboration has already been started in Indiana (Swiezy et al., 2008). If my
estimate is correct or even partly correct, and 80% of the treatment of autism is going
on in the schools, it would seem unconscionable to fail to acknowledge this and to pro-
mote the most efficient and efficacious programs possible. By bridging the silo effect
of the medical world and the educational world, there would be a synergistic effect of
improving outcome.
Up to this point, this chapter has focused mostly on issues related to service delivery
and the systemic roles of the players. There are specific treatment-oriented items in
the literature, which have received very little attention, and which would have a large
influence on the way autism treatment could be approached.
19.5.4 POSITIVE PSYCHOLOGY

Dykens (2006) in a recent paper proposes the use of positive psychology in the
treatment of mental retardation. This would be a radical departure from the cur-
rent treatment concepts employed in autism. The predominant conceptual guide
to the treatment of autism at this time is behavioral treatment. In no way do I propose
to devalue the need to do treatments to make autistic individuals fit into the world and
enjoy the company of the people in their families and communities. Appropriate
behavior makes it possible for autistic individuals to work, go to the movies and res-
taurants, family gatherings, etc. It could be argued however that all of those accom-
plishments are vehicles in the effort to produce happiness. Behaviorism rarely if ever
asks the question of whether the individual is happy. Happiness is not an easy thing
to measure and code. It is highly subjective. It is even more difficult to measure it
in an individual with language disability. In the field of intellectual disability, there
has been some movement toward a psychology of capitalizing on the strengths and
capabilities of this population although this remains somewhat outside of the main-
stream of the field and the correlations to happiness and to autism are still lacking
(Shogren et al., 2006).
Seligman (2002) states that before World War II, psychology had three distinct
missions. These were curing mental illness; making the lives of all people more
productive; and fulfilling, identifying, and nurturing high talent. Right after the war,
he posits that two major events changed the field of psychology. One was the found-
ing of the Veterans Administration, which had the effect of enticing thousands of
psychologists to make a living treating mental illness. In 1947, the National Institute
of Mental Health was founded. It was grounded in the disease model and grants
were much more available for studying pathology. Seligman acknowledges that
this brought about great benefit, in that huge strides were made in the treatment of
mental illness. The downside of this was that the other roles of psychology were
largely forgotten. He contends that the effect of this extended even to the way we see
ourselves. We see ourselves as fixed entities having biological and environmental
forces assaulting us and if severe enough, making us ill. Our job was to fight them
off, in other words to fix what is wrong. The opposite viewpoint would be to focus
and understand what our strengths are and to nurture and work with them. He goes
on to trace the reintroduction of the awareness of positive psychology to the 1998
American Psychological Association meeting at which prevention of mental illness
was the theme. His contention is that human strengths are the buffers against mental
illness and therefore a powerful tool for prevention. Some of the traits associated
with these strengths include hope, gratitude, engagement, skill, and the ability to
achieve what is termed “flow,” happiness and optimism.
Dykens (2006) reviews the history of treatment in mental retardation with its empha-
sis on “external measures of success and how happiness has been de-emphasized.”
She reviews four major movements. These movements include the QOL movement,
the dual diagnosis movement, the personality motivation movement, and family
research.
There have been some efforts to quantify the “QOL” in developmental disabili-
ties, autism, and Aspergers disorder (Fresher-Samways et al., 2003; Gerber et al.,
2008; Jennes-Coussens et al., 2006) and in their families (Allik et al., 2006; Mugno
et al., 2007). The concept of QOL is in itself an elusive concept (Fresher-Samways et al.,
2003) with 44 different definitions found in 47 studies on the topic. The questions
asked are highly subjective. With a nonverbal population, the studies have had
to rely on proxies to survey (Gerber et al., 2008) with the finding that staff and
families have different opinions and the scores are considerably different. QOL
includes the following dimensions: (1) emotional well-being, (2) interpersonal rela-
tions, (3) material well-being, (4) personal development, (5) physical well-being,
(6) self-determination, (7) social inclusion, and (8) rights. Only 1% of the questions
in QOL surveys related directly to the concepts of happiness, contentment, enjoy-
ment, and zest for life. It is true that QOL items found in the surveys are likely to be
indirectly correlated to happiness; however, caution should be exercised in that we
are not projecting a “neurotypical” slant on what makes an autistic person happy.
Direct measurement of these factors would be very difficult; nevertheless, strategies
need to be developed to accomplish this task. A possible solution will be discussed
below under the topic of autonomic functioning.
The issue of psychiatric problems or the “dual diagnosis” movement is Dykens’
second area of focus. The 40% of MR population has clinically significant psychi-
atric problems (Einfeld and Tongue, 1997) and 52% of autistic individuals are on
psychotropic medications. The most common issues include self-injury, depression,
aggression, anxiety, and stereotypies. It would seem that these behaviors or symp-
toms would have a profound influence on happiness. There remain questions of how
many of these problems are related to biologic or brain-based abnormalities (to be
treated largely with medications and biologic treatments) and how many of them are
related to environmental issues (to be treated with various psychologically based
treatments). A third outlook on the problem and one rarely taken is that the symp-
toms are a response to a lack of happiness and can be treated by the focus and devel-
opment on those factors that will increase happiness such as working toward creating
a sense of pleasure in ones work.
The third movement is described as the personality-motivation movement. This
describes the phenomenon, which aside from the low IQ or brain lesion is causing
problems. These secondary problems may be very significant. Often the MR popu-
lation has a sense of low expectations for success, which leads to a low motivation
to master challenge and ultimately a learned helplessness (which has been tied to
depression). This issue of motivation might be both in the realm of psychology, that
is, learned helplessness, and there is also a biologic overlap, which appears large
in autism. There have been studies on “intrinsic motivation” a concept that due to
a lack of attention, which might be a very profound deficiency in our understand-
ing of autism. The basis of behavioral treatments in autism is to provide artificial
motivators (such as edibles) for children who typically receive intrinsic motivation.
An example is in playing with toys. Autistic children in behavioral programs are
given edibles to induce them to increase the time they might spend playing with a toy
in contrast to the typical child who cannot be kept away from a new toy or in other
words have a powerful intrinsic motivation. While the behaviorist focuses on the
endpoint of the length of time spent in playing, there has been little or no attention
paid to the phenomenon of the intrinsic reward or lack of it, and whether there are
ways to improve the intrinsic rewards.
The last movement described is the family research movement. Systemic fam-
ily therapy has long viewed the system or in this case, the family as the “identified
patient.” With developmental disabilities and perhaps more so autism, there has been
a reluctance to view the family functioning as an object of treatment. The reason for
this is likely due to the early history of autism treatment in which Bettelheim (1967)
and the psychoanalytic movement introduce the concept of the “refrigerator mother”
who was too emotionally cold to raise a healthy child. Autistic children were taken
away from their families and placed in inpatient settings. Much of the early advocacy
in autism was aimed at reversing this notion. In reality, some families do function
better than others and there are correctable systemic issues, which would make the
family happier and most likely the individual also. This could be framed in a positive
psychology format. The clear message should be that the families functioning did not
create the autism but rather the family has the ability to use their discretion to set up
systems to problem solve. Research to examine this is just beginning.
Dykens asks us to ponder the applicability of these formulations to mental retar-
dation. She notes that based on the research, little can be said; as there has not been
“a shred of research” in mental retardation that speaks to the issues that makes peo-
ple thrive and be happy. She then proposes that the same features that help people in
general to thrive and be happy—positive emotions, gratifications, flow, virtues and
strengths—also operate in the context of mental retardation. I agree with Dykins
on this concept. It would seem very improbable if there were not a set of properties
that would be concordant with producing happiness in the developmentally disabled.
It would be hard to argue that producing happiness is not a worthwhile goal. The ques-
tion that appears to be the more controversial might be whether the field of autism
treatment will consciously adopt the happiness of the individual and his systems as
a treatment target to be spoken about, planned for, measured, and researched in order
to improve the technology for that goal. There is no doubt that behaviorists as well as
psychopharmacologists consciously or unconsciously target their treatments toward
creating happiness. Many of the competencies built through behaviorism have been
shown to be a powerful part of the arsenal of the positive psychologist.
Perhaps a more productive way of framing the debate would be about the bal-
ancing of the goals. Referring back to the opening paragraphs of this chapter, let
us review the initial question. Should ecstatic jumping and arm waving be encour-
aged as an integral part of achieving happiness during the music concert, or is the
competency of being able to sit appropriately in the concert hall and be an accepted
part of the crowd more important. Dykens call to research on this topic is more
than an academic exercise. Rather it might speak to the heart of treatment programs
for autism.
A new phenomenon that is going to be present in the next few years will be the
emergence of an unprecedented numbers of autistic adults. This new demographic
group might color the thinking related to these questions. The field has long been
dominated by a rehabilitative, learning model to correct what is “wrong” with the
autistic individual. I suspect that this will continue to be the model adopted and
supported by the parents of young children who are hopeful that the autism will be
“cured.” As the child ages, an alternative viewpoint would be created by the tacit
acknowledgment that these adults are largely going to be who they are now and that
the amount of biologic or internal capacity improvement in older teens or adults is
likely to be minimal. Instead, the most beneficial treatments of these older groups
will be to organize optimal systems around them. The question of how to optimize
these systems is first dependent on our clearly prioritizing our goals. If we do clarify
our priorities, the development of the systems to optimally create happiness will still
be very challenging. I would like to suggest two specific directions that would be
important for this goal, although many more ideas need to be created.
19.6 AUTISM, ANXIETY, AND AUTONOMIC REGULATION

Autism is defined by the triad of symptoms including language disturbance, social
impairment, and the repetitive need for sameness. It is the rare case however, who
has only these three symptoms and no comorbidities. A case can be made to consider
that there is a nearly invariant symptom, which might be described as anxiety or
perhaps more accurately described as autonomic dysregulation.
The interface of autism and anxiety is a complex topic. Anxiety was described
by Kanner in his initial account of autism (Kanner, 1943). The DSM III recognized
the presence of intense and unusual anxieties in a description of childhood onset
PDD (American Psychiatric Association, 1980). Anxiety plays a prominent role in
that diagnostic schema with the criteria including a number of emotional regula-
tion symptoms such as sudden excessive anxiety, catastrophic reactions to everyday
occurrences, inability to be consoled when upset, unexplained panic attacks, unex-
plained rage reactions, and extreme mood liability. DSM IV lists abnormal reactions
such as excessive fearfulness in response to harmless objects as an associated feature
of autism (American Psychiatric Association, 1994). It could also be argued that the
core symptom of “insistence on sameness” is a reflection of anxiety (Sukhodolsky
et al., 2008). Volkmar et al. (1999) made the observation that the interruption of ste-
reotyped behaviors may increase anxiety, tension, and emotional upset in children
with PDD. An attractive hypothesis (Bodfish et al., 2000; Hirstein et al., 2001) is that
repetitive behaviors may be for the purpose of reducing anxiety. Muris et al. (1998)
reported in a series of 44 cases of PDD that as many as 84% of the children had a
diagnosis of at least one anxiety disorder. They found that only 9% had a diagnosable
panic disorder but 57% had regularly experienced isolated panic attacks. Thus, in
addition to its core symptoms, autism may carry a high level of anxiety as an almost
invariant symptom.
Gadow et al. (2004) found in their 6–12 year olds, a wide range of different types
of anxiety disorders and compared them to regular education students. Generalized
anxiety disorder was present in 24% of the sample as opposed to 3% of the controls.
Specific phobias were present in 59% as opposed to 24%, obsessions in 41% as

opposed to 18%, and compulsions in 29% compared to 3%. Sukhodolsky et al.
(2008) found 43% of their sample had met screening criteria of an anxiety disorder
and noted that this was twice as high as the lifetime prevalence of anxiety disorders in
the population.
The causes and significance of these findings of high anxiety are largely unknown
but raise some interesting questions. It has been speculated that the awareness of
social deficits and the legacy of failure may raise the anxiety levels especially in the
high functioning with autism. This hypothesis is attractive and clinicians who work
with this population often hear the angst of the repeated failures; however, there
is no data to support this hypothesis. Chamberlin et al. (2007) contend almost the
opposite, by describing an apparent lack of awareness or caring about social failure.
Another theory advanced by Sukhodolsky et al. (2008) is that children with autism
have weak central coherence and that they might be experiencing everyday situations
as chaotic and therefore frightening. This also has intuitive resonance, but another
study by Burnette et al. (2005) found no association between weak central coherence
and anxiety in a high-functioning group. A third hypothesis concerning the origins
of anxiety in this population is a more biologic one with documented abnormali-
ties of the amygdala (Amaral and Corbett, 2003; Baron-Cohen et al., 2000) being
responsible for the clinically observed anxiety. Juranek et al. (2006) reported signifi-
cant correlations between anxiety/depression scores, the total amygdala volume, and
the right amygdala volume.
The discussion and literature review presented above concerns anxiety and
autism, but it is worthy of consideration that the concept of anxiety may not exactly
describe the phenomena observed in autism, especially in lower functioning indi-
viduals. The National Library of Medicine defines anxiety with the following three
definitions: (1) feelings of distress or apprehension whose source is unknown; (2)
a vague, uneasy feeling of discomfort or dread accompanied by and autonomic
response: a feeling of apprehension caused by the anticipation of danger. It is an
alerting signal that warns of impending danger and enables the individual to take
measures to deal with the threat; and (3) apprehension or fear of impending actual
or imagined danger, vulnerability, or uncertainty. Anxiety is described as a feeling
and is highly subjective. The scales reported on in some of the studies listed above
used items such as “has difficulty rating controlling worries” and “is excessively
shy with peers.” Items like this are hard to interpret in individuals who have little or
no language, little social interest, a severe lack of social awareness, and even a lack
of self-awareness. It would be more scientifically accurate to call the phenomena
observed in autism “anxiety-like.” Another descriptor of these phenomena could be
termed “autonomic dysregulation.”
There is ample evidence that there is autonomic dysregulation in autism
(Althous et al., 2004; Graveling and Brooke, 1978; Hirstein et al., 2001; Hutt et al.,
1975; Jansen et al., 2006; Ming et al., 2005; Toichi and Kamio, 2003; Zahn et al.,
1987). Many of the symptoms found in autism including sleep disturbance, GI
disturbance, and immune system abnormalities all can be plausibly explained by
autonomic dysregulation (Axelrod et al., 2006; Czura and Tracey, 2005; Powley,
2000). Neuroanatomically, abnormal findings in the amygdala, frontal and
temporal cortex, cerebellum, and the brain stem can support a central nervous
system hypothesis of autonomic dysregulation (Courchesne, 1997). Abnormalities
in the oxytocin systems of the brain in autism (Green et al., 2001; Modahl et al.,
1998) and some evidence of symptom improvement with oxytocin (Hollander
et al., 2003) would also fit an autonomic dysregulation hypothesis. Oxytocin has
been shown to reduce the fear-inducing activation of the amygdala, which via a
brain-stem pathway regulates autonomic functioning (Kirsch et al., 2005). These
findings are consistent with the polyvagal theory (Porges, 2006), which predicts
that there will be pathologic findings in a social engagement system in which vagal
functioning is dysregulated.
Studies have shown low cortisol levels in autism or normal levels despite the auto-
nomic hyperactivity and or high adrencorticotropic hormone (ACTH) levels (Curin
et al., 2003; Jansen et al., 2003; Tani et al., 2005) with a slow response to ACTH
(Marinovic-Curin et al., 2008) leading to the speculation that some of the symp-
toms seen in autism are a result of the lack of a normal hypothalamic–pituitary–
adrenal axis and autonomic physiologic functioning that helps the organism cope
with stress.
Despite the substantial amount of evidence that these types of symptoms are very
common in autism, and that they likely have a profound impact on behavior, the
ability to learn and to adjust to various situations and settings, there has been very
little systematic study of the treatment of these problems. Pharmaceuticals have been
studied (Findling, 2005) and no doubt have an important role; however, safety and
tolerability considerations make this option less than optimal. One successful study
of cognitive behavioral therapy with high functioning autism or Aspergers syndrome
has been published (Chalfant et al., 2007). The question of how generalized this is
to the lower functioning group as well as how many of even the higher functioning
group will cooperate with this is unknown. Alternative methods such as relaxation
and yoga have been suggested; however, there is little in the literature to document
its practicality as a treatment.
A concerted effort would need to be made to monitor the extent of this above
discussed phenomena and to prove that treatments are available that could lower the
anxiety, with the likely outcome of improving behavior, improving cognition, and
perhaps even enhancing happiness.
19.7 TREATING THE ENVIRONMENT

Another form of treatment, which is in line with a biopsychosocial perspective, is
that of creating “safe” environments for those with autism or more accurately put,
environments that are perceived as safe. Conceptually, rather than having the autistic
person learn to cope with the environment (which in some cases is never success-
ful), a form of treatment would involve having the environment meet the autistic
individual half way. This is not a new concept and in the 1960s, this concept was
very popular in psychiatry. Often called milieu therapy, this concept appears to have
only taken hold for the addicted population, although the potential of these concepts
should be explored (Karst, 1991). Fifty years ago, the “therapeutic communities”
captured the imagination of the psychiatric community. Partly based on the discovery
of Thorazine for schizophrenia, the concept of deinstitutionalization took root.

This was a very popular concept and the large state-run psychiatric hospitals around
America emptied out and many were closed. The identified alternative to the large
institutions was to be “community psychiatry,” that is, to bring the needed treatments
into the communities of the patients and to have them integrated in their community
rather than being institutionalized. Very much like the fate of the family practitioner,
community psychiatry was never adequately funded and became more of a concept
than a viable alternative. This failure to follow through and support the community-
based treatment programs lead to the homelessness, which became rampant in the
1970s. Despite the apparent lack of success of these movements, it would be grossly
unfair to term the concepts as failures. Rather it appears that the enticement of cut-
ting budget by closing institutions was much more inviting than increasing budget to
support new community-based alternatives.
Aside from the public health issues discussed above, there was a rich theoreti-
cal basis for milieu therapy, therapeutic community, and community psychiatry.
All three concepts were based on the systems approach that the individual is not
an island unto himself but rather his success or failure is largely dependent on the
milieu or system or community that he or she is part of.
Various arguments might be made against the idea of meeting autistic individu-
als half way. Young autistic children often tantrum when the environment is not to
their liking, and behaviorists often argue that the tantrum is a form of avoidance
that if reinforced will lead the autistic individual not to develop coping skills. This
is true up to a point. No doubt, there are individuals who if not confronted with a
challenge, might never develop coping skills to a particular issue. To use a more
physical analogy, one could posit that the use of ramps is not thought of as a way
that physically impaired individuals “avoid” stairs and if forced to deal with stairs,
they would eventually master them. The fact that we cannot perceive the subjective
neurological responses felt by an autistic individual, in no way negates the reality of
their limitations. There is little doubt that people with autism perceive the world dif-
ferently than neurotypical individuals (Grandin, 1991). Anecdotal reports document
sensory distortions, which make the individual uncomfortable (Dunne et al., 2002).
The habilitative model has been the dominant theoretical force in mental retardation
treatment; however, a pertinent question has rarely been asked. When should reha-
bilitation stop and adjustment to the realities of the condition begin? As we deal with
more autistic adults, this will become an important question.
Although it is well known that autistic children can be easily distracted by the
environment, there has been little attention paid to the environments that they are
actually in. Temple Grandin (1991) discusses the fact that noise and confusion at
large gatherings overwhelm her senses and “at times speech reached her brain like
the noise of an onrushing freight train.” Jeffrey Kittay (2008) presents a discus-
sion in more depth about the ambient sounds that autistic and other handicapped
children must cope with and that the field in general has not dealt with. People who
live in rural areas are often astounded by the levels of ambient noise, which city
dwellers must live with. Those who live in New York City accommodate to sirens
and other street noises, which they hear on a constant basis. A question that has
not been answered is the extent to which autistic individuals are sensitive to the
sounds in their environment and a second question is their abilities to accommodate

to those sounds. Some treatment settings have put up sound proofing materials
and have anecdotally reported improvements in overall behaviors of the residents.
The study of this to prove efficacy is lacking. This might place into question the
advisability of the common practice of putting autistic people together as the noises
created by one individual might be less tolerable for another autistic individual than
it is for a neurotypical peer. Again, the extent that this is a problem especially in the
lower functioning nonverbal population is not known. It is possible that attention
to these parameters, despite their “low tech” nature, might have treatment effects,
which could be profound. These arguments, which were made about sound, can be
generalized to many environmental factors including the architectural space, visual
stimulation, food, chemical substances in the environment, temperature, and the
textures of clothing among others.
Of all the environmental influences that might have an impact on the outcome
of an individual, it could be argued that none has as profound an effect as the
caring and love that one receives. This is a very difficult topic to address for even
social scientists and the vagaries of the concept of love and caring make it even more
difficult for the more biological scientist. Yet, there is an emerging science of love
(Esch and Stefano, 2005) and there are attempts to analyze love in terms of its effect
on neurological biologic systems and neurotransmitter systems involved. This topic
is of utmost importance in the understanding of the holistic treatment of autism.
There is a voluminous body of work, which over many years has established the
value of social affiliation and intimacy bonds to the overall functioning of the
organism (Cassidy, 2001). These social functions have wide-ranging effects, includ-
ing emotional and cognitive functioning as well as the production of general physical
well-being resulting in the overall health of the organism. The family is the struc-
tural unit responsible for the formation and sustenance of these bonds for the vast
majority of children both autistic and otherwise. The attention and support that we
give to the caregivers can have a profound effect on the functioning and the ben-
eficial effects, which the family can create. It would seem that supporting the
families in many ways might reduce the need for other mental health interventions
down the road.
In individuals with autism, except for the very rare exception, the path of forma-
tion of intimacy bonds does not follow the typical pattern. Rather than growing and
developing slowly with the formation of autonomy and individuation, the autistic
person generally remains in a dependent and often childlike position. It is only the
very exceptional autistic person who marries and has his or her own family. As the
family of origin ages, affiliative bonding will most likely occur with paid staff of
work programs or residential programs. Kittay (2001) reviews many of the sociologi-
cal and philosophical issues around the attitudes of society toward caregiving and the
caregivers (both paid and family). She proposes that the most effective advocacy for
the one cared for would be to advocate for the caregivers. At this point, caregiving is
usually a minimum or close to minimum wage job (Pollack, 2007), with little in the
way of professional training and little in the way of career growth possible. These
jobs are often a temporary (most direct caregivers stay no more than a few months at
a given job) stopping point for those who are more ambitious and unfortunately may
be a place where some with their own personal problems may find work sometimes
leading to scandalous examples of abuse (Kittay, 2001). Family caregivers likewise
face enormous stress; by 2004, there were an estimated 711,000 Americans with
intellectual disability living with caregivers over the age of 60 (Pollack, 2007).
The goal of public health (Talley and Crews, 2007) is to promote healthy indi-
viduals living in healthy communities, pursuing QOL rather than just the absence of
disease. Caregiving has become an issue that affects the QOL for millions of indi-
viduals. Scientists and practitioners rarely thought of caregiving as a public health
matter. The authors call for the public health systems to understand more about the
caregivers themselves and then to design and implement evidence-based interven-
tions to address the identified needs. Research needs to be done to uncover the pos-
sible risk factors associated with the endless types of caregiving situations. Of all
diseases, progress in this area seems most critical for autism.
19.8 CONCLUSION
To many in society, and even to those in the health professions, autism seems to
have placed itself in our consciousness, as if sneaking in from nowhere. This may
be because the prevalence is actually on the rise or the more likely explanation is
that it is due to redefinition, and the awareness of these symptoms in the population.
In practical terms, there is no debate that there must be a new and improved set of
interventions for this very hard to treat group. Defying easy classification, autism
straddles many systems (i.e., educational and medical) diagnostic entities (psychi-
atric or intellectual disability) and, to some extent, has led the way to an intensified
interest in brain development and early childhood development. It is also true that
those searching for answers do have many bases of knowledge to start from. Many
medications developed for psychiatric indications work fairly well for autism. Much
of the progress in the conceptualization and integration of intellectual disability can
be transferred to autism. Models of care, which have been developed and held out
much hope such as milieu therapy and the biopsychosocial model of medicine, need
to be dusted off and applied more consciously to autism. The new advances in
neuroscience hold out great hope for our understanding, leading to treatments and
prevention for autism and if added to enhance clinical delivery systems, there is great
promise for improvement in the lives of these affected individuals.
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Index
A public health, 413
safe, 410–411
ABA, see Applied behavior analysis tantrum, 411
Active pathophysiology; see also Chronic haloperidol, risperidone and placebo, 396
dynamic encephalopathy illness heterogeneity, 393–394
functions to ASD, 347–348 intellectual disabilities, 397
persistent, 346–347 medical model, 398–399
potential for plasticity, 349–350 model
Age related macular degeneration (AMD), 38 application, 392–393
AGP, see Amino-glycerophospholipids basic concepts, 391
AGRE, see Autism genetic resource exchange biomedical model, 393
Amino-glycerophospholipids (AGP) medicine theory, 392
blood plasma, 179 New York autism consortium,
content, 180 404–405
Animal model, 135–136 PCAP (see Primary care autism professional)
Applied behavior analysis (ABA) positive psychology
benefit, 394 biologic/internal capacity, 408
orthodox, 395 family therapy, 407
PCAP, 400 mental retardation, 405
Asperger syndrome (AS), 62 personality-motivation movement,
Autism colitis 406–407
gastrointestinal (GI) mucosa, 255–256 quality of life (QOL) and dual
proinflammatory cytokines, 256 diagnosis, 406
Autism genetic resource exchange (AGRE), 333 psychopharmaceutical agents, 395
Autistic cerebella 3-nitrotyrosine levels, 49 quagmire, 404
speech therapy, 403–404
B Bipolar disease (BPD)
comorbid conditions, 216
BBB, see Blood–brain barrier SNP markers, 217
BDNF, see Brain-derived neurotrophic factor Blood–brain barrier (BBB)
Biopsychosocial treatment cadmium, 158–159
ABA, 394–395 CVO, 155, 156
anti-medical and antiscience movements, 398 integrity, 233
antipsychotics, 396–397 melatonin, 162
anxiety and autonomic regulation neuroglial cell populations, 228
causes and significance, 409 neurotoxicants, 157–158
cortisol levels, 410 NTS, 164
description, 408 organic mercury, 160
dysregulation, 409–410 paraquat, 159
types, 408–409 TNF, 167
assessment and treatment centers BPD, see Bipolar disease
and diagnosis, 402 Brain-derived neurotrophic factor (BDNF)
psychiatric units, modalities, antioxidant factor, 40
402–403 cord blood, 166
university-based programs, hippocampal, 50
401–402 immunoaffinity chromatography, 51
CAM, 397–398 and neurotrophins
diagnosis, 403 levels, 19
environment NT-3 and NT-4/5, 18–19
children, 411–412 polymorphism, 53
intimacy bonds, 413–414 Rett syndrome, 55
419
420 Index
Brain development CEP, see Carboxyethyl pyrrole

abnormal, 8 Chronic dynamic encephalopathy
BDNF and neurotrophins active persistent pathophysiology
basal forebrain, 19 genes–brain–behavior, 346
NT-3 and NT-4/5, 18–19 somatic tissue samples, 347
brain stem and serotonin assumptions, rethinking
5-HT, 19 classical model, 350–351
serotonergic system abnormalities, responses, 351–352
19–20 behavioral syndrome
neuroimmunity, inflammation and brain development, 345
cytokines and chemokines, heterogeneous, 346
230–231 developmental disorder
immune system, 229–230 autistic regression, 360–362
behavioral manifestations, 362–363
C characteristics, 352
glial cells, centrality, 358–359
Cajal–Retzius cells, 99 knowledge gaps, 353
Calcium signaling abnormality, mitochondrial reassessment, 353–358
component etiology
defects environmental influences, 368
FHM3, 214–215 indirect evidence, 369–370
microarray homozygosity mapping, 215 molecular genetic investigation, 371
TS mutation, 214 pathophysiological abnormalities, 370
DNA mutations, 209 multisystem and multileveled complexity,
dysfunction, neurosecretion defects, 215 362–368
functional mitochondrial defects pathophysiology impact
chromosomal rearrangements, neural functioning, 347
210–211 in utero impacts, 348
lactate/pyruvate ratio, 210 plasticity
SLC25A12 gene, 211 mental retardation, 371
homeostasis and pertinence, 349–350
electron transport, 211 specificity, 376
endoplasmic reticulum, 211–212 Circumventricular organs (CVO); see also
MHS gene, 212 Toxicants
neuronal abnormality, 165
BPD, 217 injury and toxicant accumulation, 157–160
FHM, 216–217 melatonin, 161
presenilin, 217–218 primitive brainstem, 156
seizure and migraine diseases, 216 TNF-positive cells, 167
pharmacogenetic abnormalities, 218 CNS, see Central nervous system
Timothy syndrome, 212–214 CNVs, see Copy number variations
Carboxyethyl pyrrole (CEP) Common disease common variant (CDCV)
formation model, 63
human and rat model, 42–43 Common disease multiple rare variant (CDMRV)
PMI, 41 model, 63
SHR, 41–42 Complimentary and alternative medicine (CAM),
generation, 39 397–398
modifications, 41 Copy number variations (CNVs)
Central nervous system (CNS) de novo/noninherited, 65–66
innate immunity in schizophrenia, 66
chronic inflammation mechanisms, 259 C-reactive protein (CRP), 103
immune cells, 252–253 CVO, see Circumventricular organs
inflammation, 258 Cytokines
neuro-immune network, activities and inflammatory responses, 316
253–254 brain
Treg cells, CNS homeostasis, 253 inflammation, 318
serotonin levels, 79 stress, 319
Index 421
definition, 315 F
polymorphism
and expression, 317–318 Familial hemiplegic migraine (FHM)
genetic variability hypothesis, 320 and autism alleles, 215
maternal, 320–321 mutational lesions, 216
Th1/Th2/Th17 responses, FHM, see Familial hemiplegic migraine
319–320
G
D
GABA, see Gamma-aminobutyric acid
Developmental disorders GALT, see Gut-associated lymphoid tissue
connotations, 352 Gamma-aminobutyric acid (GABA)
knowledge gaps, 353 adult brain, 260
reassessment type 1 diabetes, 261
prior findings, alternative interpretations, Gastrointestinal (GI) dysfunction
354–356 children, 282–283
restrictive impact, 356–358 vs. immune dysfunction
weak spots, 353–354 anxiety and depression, 287
treatment mechanism, 308–309 cognitive function, 285–286
working hypothesis and OT cytokines, 286
deficiency, 300 disorders, 287–288
DHA, see Docohexaenoic acid immunocytochemical analysis, 289
Diagnostic and statistical manuel of disorders mechanism, 291
(DSM), 396 molecular mimicry, 290
DMV, see Dorsal motor nucleus of the vagus opioids, 285
Docohexaenoic acid (DHA) serotonin, 288–289
description, 39 tight junctions, 288
and EPA, 181 infiltration, 284–285
Dorsal motor nucleus of the vagus (DMV) LNH, 284
area postrema (AP), 156 symptoms, 283
NTS, 164, 168 Genetics
PD, 156–157 CNVs
phonation, 163 family types, 66
TNF, 167 frequency of, 65–66
visceromotor fibers, 162 epidemiology, 62
heritability, 63
E integrative, 63–64
noncoding RNA role, 67–68
Eicosapentaenoic acid (EPA) parental age, 66–67
dietary consumption, 181 prenatal maternal stress and, 67
PLA2 concentrations, 182 single-gene mutations
Environmental exposures candidate genes, 64–65
organophosphates (OP), 98 fragile X syndrome overlapping, 64
paraoxonase activity, 99 Glial cells, probable centrality
reelin, 99–100 activation, 358
RELN, 100–101 microglia, 358–359
Enzyme-linked immunosorbent assay (ELISA) in vivo imaging, 359
analysis, 51 Glutathione (GSH)
EPA, see Eicosapentaenoic acid astrocytes, 119
Epilepsy cellular, reduction, 125–126
lesions, 17–18 cysteine residue, 115
pathology, 18 and GSSG ratios, 120
Etiology HCY, 117
autistic regression, 135 human neuronal cells, 124
gastrointestinal (GI) distress, plasma concentration, 38
133–134 synthesis, 115–116
MMR vaccination, 134–135 and transsulfuration, 121
422 Index
GSH, see Glutathione I

Gut-associated lymphoid tissue (GALT)
innate immune responses, 250 Idiopathic autism, 3–4
mucosal immune system, 248 Immune dysfunction
Gut mucosa, innate immunity alterations, 279
effects autoimmunity
environmental factors, 250–251 familial history, 279–280
genetic factors, 250 pregnancy, 280
micronutrients, 251 self-reactive antibodies, 281
epithelium serum samples, 280–281
heparan sulfate proteoglycans, 247 cell-mediated immune response
interferon-β, 247–248 cellular subsets, 281–282
GALT, 248 cytokines, 282
T-cell subsets vs. gastrointestinal dysfunction,
proinflammatory natures, 249 285–291
T-helper (Th), 248 Immune insult
Treg cells, 249 postnatal period
elimination diet, 265
infection-induced exacerbation, 264
H propionic acid, injection, 263
HCY, see Homocysteine in utero
HDL, see High-density lipoprotein maternal infection, 262
Heavy metal-induced autism serotonin levels, 263
cellular consequences Impaired signaling, neurotransmitters
GSH levels, 125–126 Ca2+ signaling, 261
mitochondrial dysfunction, 126 cholinergic neurotransmission, 260
genetic risk factors, 122–123 GABA neurotransmission, 260–261
heavy metal exposure Inflammation
prenatal, fish, 123 acute-phase response, 103
vaccination, 123–124 in CNS, 228–229
mercury, molecular hypothesis, 126 described, 227–228
molecular targets genetic response
intramolecular disulflide bond, 125 MHC role, 236–237
organic mercury bind, 124 neurological disease, 235–236
High-density lipoprotein (HDL) immune imbalance/autoimmune
apoJ and serum amyloid A (SAA), 103 processes, 102
LPS, 102 immunological responses, 228
mediated cholesterol efflux, 95 LPS, 102–103
PON1 and PON3, 92 mechanisms
HLA-DR4 alleles microglia, 166–167
birthweight and maternal antibodies, 337 TNF, 167–168
environmental factor, 334 neuroglia, adulthood, 233–234
HLA-DR, case-control studies Innate immunity
AGRE repository, 333 CNS
frequency, 334 chronic inflammation mechanisms, 259
polymorphic alleles, 332 immune cells, 252–253
maternal immune stimulation, 335 inflammation, 258
MHC, 332 neuro-immune network, 253–254
oxidative stress Treg cells, 253
cytokine, 336 gastrointestinal (GI) system
inflammation, 335 autism colitis, 255–256
synaptic pruning and MHC, 336 dysbiosis and intestinal permeability, 255
Homocysteine (HCY), 116–117 food allergy, 256–258
5-HT, see 5-Hydroxytryptamine gut mucosa
Human accelerated region 1 (HAR1), 68 effector T-Cell subsets, 248–249
5-Hydroxytryptamine (5-HT), environmental factors, 250–251
288–289 epithelium, 247–248
Index 423
GALT, 248 Maternal immune activation (MIA), 103

genetic factors, 250 Membrane and signal transduction abnormalities
micronutrients, 251 AGP, 179
Treg cells, 249 calcium ion channels
neuro-immune interactions functional mutations, 184–185
immune insult, 262–265 glutamate receptors, 185
neurotransmitters, impaired signaling, resting cell, 184
260–261 cytokines, inflammation, 194
redox/methylation hypothesis, 262 lipid rafts and disorders, brain, 195
lipoprotein and cholesterol
L apolipoprotein E, 183–184
in blood, 184
Lethal cardiac arrhythmia syndrome, extracellular signals, 183
see Long QT (LQT) membrane-associated proteins
Lipopolysaccharide (LPS) Bcl2 and p53, 191–192
HDL, 102 cellular signaling, 185–186
in rodents, 103 neurexins, 187
LNH, see Lymphoid nodular hyperplasia neuroligins, 189
Long QT (LQT) phosphorylation, 186
and MHS, 216 Pten and PI3K/Akt pathway,
mutations, 213 188–189
Lovass therapy, 394 reelin, 190
LPS, see Lipopolysaccharide serotogenic receptors, 190–191
LQT, see Long QT SHANK3 gene, 189–190
Lymphoid nodular hyperplasia (LNH) membrane fluidity and fatty acids
autism colitis, 255–256 EPA and DHA, 181
and enterocolitis, 284, 285 insulin receptors, 180
lengths and saturation, 179
M oxidative stress, 192–194
PLA2
Magnetic resonance imaging (MRI) chromosomal linkage studies, 181
brain, volumetry, 6–7 genetic inheritance, 182
cerebellum size evaluation, 16 polyphosphoinositides, 182
functional MRI (fMRI), protein kinases, 183
13–14 Mercury exposure, (MeHg)
hypoplastic brainstem, 161 brain, 138–139
maternal effects data, 84 effects, 138
and postmortem morphometric neural development, 139
studies, 17 trolox, mice
Major histocompatibility complex (MHC) body weight, 140
human, 332 cognitive development,
polymorphic alleles, 332–333 141–144
role of, 236–237 pregnant BALB/c, 139
and synaptic pruning, 336 reflex development, 140–141
Malignant hyperthermia syndrome (MHS) social development, 144–145
channel genes, 216 treatment, 139–140
cytosolic calcium pool, 212 MHC, see Major histocompatibility complex
mutations, 214 MHS, see Malignant hyperthermia syndrome
MAOA, see Monoamine oxidase-A Molecular mimicry, 290
Maternal depression Monoamine oxidase-A (MAOA)
genetic factors, 77 gene as DEP/AUT
MAOA gene and behavior profiles, levels and, 79–80
82–84 serotonin, 78–79
mood and anxiety disorders, 76 uVNTR, 80
PDDBI domain scores, 76–77 maternal effect, gene
risk alleles hypothesized overlap, domain groups, 83–84
77–78 PDDBI, 82–83
424 Index
uVNTR polymorphism repetitive and stereotyped behaviors,

PDDBI domain T-scores, 82 16–17
4-repeat allele, 81 sensorimotor deficits, 15–16
MRI, see Magnetic resonance imaging speech, language and communication, 13
Multisystem and multileveled complexity cohorts and stereology, 5–6
animal models, 367–368 cortical and subcortical
behavior-centered definition, 365 brain structure, neuronal growth, 9–10
brain and somatic/systemic features, 366–367 characteristics, 8–9
integration, lack of, 363 minicolumnar abnormalities, 10
nervous system manifestations, 363–364 early childhood, brain growth
psychiatric syndrome, 362 abnormal, functional consequences, 8
research, 365–366 developmental heterochronicity, 7–8
systemic and somatic features head growth acceleration, 6–7
brain-based syndrome, 364–365 MRI-based volumetry, 6
comorbidities, 364 pattern, 7
immune–brain and gut–brain etiology, 4
relationships, 365 idiopathic/nonsyndromic, 3–4
oxidative stress and metabolic changes,
N 10–13
postmortem studies, 5
Nerve growth factor (NGF) Neuropathology, cortical and subcortical
basal forebrain BDNF level, 51 dysgenesis, lamination defects, migration
neurotrophins, 50 disturbance, 8–9
in women, 53 minicolumnar abnormalities, 10
Neuroglia, neuroinflammation neuronal growth, 9–10
adulthood responses, 233–234 Neurotoxic brainstem impairment
synaptic plasticity and, 234–235 abnormal morphology, 161
Neuroimmunity, inflammation and biochemical abnormalities
brain development melatonin, 161–162
cytokines and chemokines, 230–231 oxytocin, 162
immune system, 229–230 clinical and electrophysiological finding,
chronic reactions, 235 160–161
CNS, immunological response, 228 features, regression
genetic susceptibility, 235–236 diarrhea, 162–163
maternal environment, 231–232 Frank dysarthria, 163–164
MHC role, 236–237 impaired vagal signaling, 163
neuroglia, adulthood, 233–234 NTS, 164
Neuroinflammation proximate CVO, 165
CNS, 228 somatobehavior, 162
innate neuroimmune responses, 228–229 inflammatory mechanisms, 166–168
and oxidative stress oxidative mechanisms, 164–166
cobalamin, 121 regression
genes and SNPs, 122 somatobehavioral, 154–155
plasma levels, 120–121 toxicants, 155–157
Neuroligins toxicant accumulation and CVO injury,
genes, 189 157–160
neurexin and, 187 Neurotrophin 3 (NT-3)
SHANK3 gene, 189–190 autistic and control brains, 51–52
Neuropathological changes and NT-4/5, 51
brain development overexpression, 52
BDNF and neurotrophins, 18–19 and oxidative stress, 53
stem and serotonin, 19–20 Neurotrophins
clinicopathological correlations and brain
behavioral deficits, hypothalamus, 14–15 altered signaling, 52–53
cognitive deficits, 17 dual role of, 53
epilepsy, 17–18 expression, altered, 51–52
face perception, 13–14 role of, 55
Index 425
signaling plasma metabolites, 101–102

altered, mechanism, 52–53 production, lipid peroxides, 192
BDNF and NT-4, 51 redox homeostasis, 101
dual role, 53 role, 136–137
and gender, 54 valproic acid (VPA) and
genetic component, 53–54 behavioral deficits, 137–138
neuronal plasticity, 50 hydroxylation, 137
NT-3 levels, 51–52 treatment, 138
therapy targets, 55 Oxytocin (OT)
NGF, see Nerve growth factor autism phenotype generation
Nonsyndromic autism, see Idiopathic autism gut inflammation, 308
NT-3, see Nerve growth factor Paneth cell mechanism, 307–308
NTS, see Nucleus tractus solitarius OTR and, 302–303
Nucleus tractus solitarius (NTS), 156 synthesis
estrogen/progesterone control, 301
O neurophysin, 300
Oxytocin receptor (OTR)
Opioid excess theory, 285 expression and activation
OT, see Oxytocin epigenetic regulation, 301
OTR, see Oxytocin receptor temporal changes, 302
Oxidative damage, autistic brain oxytocin levels, 302–303
axon, 40–41 protein expression, 306
CEP–adduct formation, 41–43
neurodegeneration, stress, 36 P
peripheral stress, 37–38
stress, 38–40 PAH gene, see Phenylalanine hydroxylase (PAH)
Oxidative stress gene
altered, 137 PAMPs, see Pathogen-derived molecular
autistic brain patterns
antioxidant systems, 38 Paraoxonase 1 (PON1)
CEP, 39–40 activities, 96–97
lipid hydroperoxides and peroxidation, 39 C-108T, L55M and Q192R, 97
species, 38–39 functional SNPs, 96
biomarkers, 136 human serum
ceruloplasmin and transferrin, 193 cardiovascular diseases, 92–93
copper-mediated oxidation, 193–194 coding regions, 95
diagnostic markers, 303 functional perspective, 94
gastrointestinal (GI) aspects phylogenetic analysis, 92
glutathione, 304 physiological role, 94–95
hypoxia, 305 plasma activity, 96
involvement evidence structure, 93
antioxidant defense mechanisms, 49 Parkinson disease (PD)
8-OH-dG, 49–50 DMV, 156–157
role, 48–49 microglial activation, 166
ROS damaging action, 48 paraquat, 159
lipid peroxidation, 102 TNF, 167
mini columns, 303–304 Pathogen-derived molecular patterns (PAMPs)
neurodegeneration, brain, 36 immune insult, 263
neuronal and metabolic changes pattern recognition receptors (PRRs) and,
β-APP and intraneuronal Aβ, 13–14 246–247, 252
glutathione (GSH), 10–11 tolerance and cross-tolerance, 251
lipid peroxidation, 11 PCAP, see Primary care autism professional
lipofuscin, 11–12 PD, see Parkinson disease
peripheral PDDs, see Pervasive developmental
autoantibodies, 38 disorders
digestive pathology, 37 Peripheral blood mononuclear cells
species, 37–38 (PBMCs), 102
426 Index
Pervasive developmental disorders (PDDs) Recycling immunoaffinity chromatography, 51

childhood onset, 408 Redox
core social and communication and heavy metals, mercury
impairments, 395 inhibitory effects, 119
GI symptoms, 283 organomercurials, 118–119
non-PDD cohorts, 77 toxicity molecular mechanism, 119–120
PDDBI human brain, metabolism
classifications, 74 GSH synthesis, 115–116
tools, 74–75 HCY transsulfuration, 116–117
prenatal and postnatal total DAPs, 98 tripeptide glutathione, 115
Pervasive developmental disorders behavior metabolic adaptation, 114–115
inventory (PDDBI), 74–75 and methylation
Phenotypic expression DNA, gene expression, 117–118
MAOA methionine synthase, 116, 117
gene as DEP/AUT, 78–80 RELN gene, 100
and maternal effect, 82–84 Repetitive and stereotyped behaviors
uVNTR polymorphism, 81–82 caudate volumes, 16–17
maternal depression, 76–78 definition, 16
PDD assessment, 74–75 Rett syndrome, 55
Phenylalanine hydroxylase (PAH) gene ROS, see Reactive oxygen species
homozygotes, 332
mutations, 331 S
Phospholipase A2 (PLA2)
lymphoblasts, 182 Serotonin, see 5-Hydroxytryptamine (5-HT)
and oxidative stress, 181 SHR, see Spontaneous hypertensive rat strain
Plasticity Single-nucleotide polymorphisms (SNPs),
aberrant behaviors, 349 122, 236
expressive and receptive language SLOS, see Smith-Lemli-Opitz syndrome
impairments, 372–373 Smith-Lemli-Opitz syndrome (SLOS), 184
improvement documentation, 372 SNPs, see Single-nucleotide polymorphisms
maladaptation, 375 Somatobehavioral regression
mental retardation, 371 behavioral changes, 154
potential, 373–375 gastrointestinal (GI) problems, 155
short-term changes, 350 Speech therapy, 403–404
symptom reversal, 349–350 Spontaneous hypertensive rat strain (SHR), 41
PMI, see Postmortem interval Superoxide dismutase (SOD), 37–38
PON1, see Paraoxonase 1
Postmortem interval (PMI), 41–43 T
Primary care autism professional (PCAP)
biopsychosocial model, 401 TDT, see Transmission/disequilibrium test
family, 399 Teratogenic alleles
models, 400 HLA-DR4
patients decisions, 400–401 case–parent study design, 334
role, 399–400 HLA-DR, case-control, 332–334
Purkinje cells MHC, 332
analysis, brain growth, 47 maternal phenylketonuria
cerebellar abnormality, 15 PAH gene, 331
TDT, 331–332
R neurodevelopmental disorders
impact, 328
Reactive oxygen species (ROS) maternal genes, 326
damaging action, 48 reports of, 326–328
glutathione, 304 Rh incompatibility, 330–331
intestinal inflammation, 305 study designs
oxidative stress, 300, 303 candidate gene identification, 329
and oxidative stress markers, 36 pent approach, 330
Paneth cell, 307, 308 TDT analysis, 329–330
Index 427
Timothy syndrome (TS) Treg cells

calcium ion, 212 characterization, 249
channel inactivation, 214 CNS homeostasis, 253
LQT mutations, 213 Foxp3+, 251
Toxicants Trolox, MeHg exposure
accumulation and CVO injury antioxidant pretreatment,
cadmium, 158–159 139
intramuscular injection, 159–160 attacks, 146
neurotoxicant classes, 157–158 body weighta and hanging
organic mercury, 160 wire, 140
paraquat and MSG, 159 hidden platform water,
regression 141–143
area postrema (AP), 155–156 MeHg Cl/PBS, 139–140
BBB, 155 midair righting and negative geotaxis,
DMV and NTS, 156–157 140–141
plausible mechanisms, 157 reversal learning, 143–144
Transmission/disequilibrium test (TDT) sniffing, 145–146
maternal, 326, 330–334 visible platform, 144
neurodevelopmental probands, 329 TS, see Timothy syndrome
KWWSERRNVPHGLFRVRUJ
1 N
D 6
H1
D
C H2
C
2 B
B
A C H3
A
N
4
(a) (b)
FIGURE 6.2 Overall structure of PON1. (a) View of the six-bladed-propeller from above.
Shown are the N- and C-termini, and the two calcium atoms in the central tunnel of the
propeller (the “catalytic calcium” or Ca1, green (left); the “structural calcium” or Ca2, red
(right)). (b) A side view of the propeller. At the top of the propeller, there are three helices
H1–H3 which determine the PONs’ cell distribution, translocation and secretion (H1), and
protein–lipid and protein–protein interactions (H2 and H3). (Reproduced from Harel, M.
et al., Nat. Struct. Mol. Biol., 11, 412, 2004. With permission.)
High PON1 activity Low PON1 activity

Reelin protease activity
= 7–10 GGC = Exposure to organophosphates (OP)

= ≥ 12 GGC = Threshold for correct neuronal migration
FIGURE 6.3 The gene × environment model for autism. The model involves the Reelin
(RELN) and Paraoxonase 1 (PON1) genes, and prenatal exposure to organophosphates (OP).
RELN variants carrying either “normal” (7−10 repeats) or “long” (≥12 repeats) GGC alleles
genetically determine whether levels of reelin are normal or reduced, respectively. In prin-
ciple, both conditions are compatible with normal neurodevelopment. However, prenatal
exposure to OP can transiently inhibit the proteolytic activity of reelin, which might then fall
below the threshold required for correct neuronal migration, also depending on baseline levels
of RELN gene expression determined genetically and epigenetically. In addition, exposure
to identical doses of OP can affect reelin to a different extent depending on the amount and
affinity spectrum of the OP- inactivating enzyme paraoxonase produced by the PON1 alleles
of each individual. (Reproduced from Persico, A.M. and Bourgeron, T., Trends Neurosci., 29,
349, 2006. With permission.)
Neocortex,
Presymptomatic Symptomatic primary,
phase phase secondary
Neocortex,
high order
association
Mesocortex,
thalamus
Threshold Substantia
nigra,
amygdala
Gain setting
nuclei
Dorsal
motor ×
nucleus
1 2 3 4 5 6 Stages of the
PD-related
(A) path, process
(B)
FIGURE 9.3 The ascending pathology of Parkinson’s disease occurs in six recognizable
stages, beginning in the DMV of the medulla. Progressive shading in table (A) corresponds
to the like-shaded anatomic regions represented in diagram (B). (From Braak, H. et al., Cell.
Tissue Res., 31, 121, 2004. With permission.)
(A) (B)
FIGURE 9.4 Enlargement of Paneth’s cells, as indicated by dark staining of secretory gran-
ules, was a frequent finding in a series of duodenal biopsies from children with regressed
autism (A), as compared to non-autistic controls (B). (Photographs courtesy of Karoly
Horvath MD, PhD, Professor of Pediatrics, Thomas Jefferson University, Philadelphia, PA.)
Growth factor Reelin
Ca2+ Ca2+
Voltage-gated
Growth factor receptor calcium channels
VLDLR ApoER2
Cellular
PI stress
PI 4,5 - P2 PI3K PKB/Akt Dab-1
3, 4, 5 – P3
Ca2+
Pten mTOR
p53
GSK-3β
Bcl2
Tau
Neuronal migration Proliferation Apoptosis
FIGURE 10.2 Participation of growth factor receptors, reelin, voltage-gated calcium chan-
nels, p53, and Bcl2 in neuronal migration, proliferation, and apoptosis. Growth factor binds
to its receptor and activated phosphatidylinositol 3-kinase (PI3K), which converts phosphati-
dylinositol 4,5-bisphosphate (PI 4, 5-P2) to phopsphatidylinositol 3, 4, 5-trisphosphate (PI 3, 4,
5-P3). PI 3, 4, 5-P3 is an activator of protein kinase B (also known as Akt) that triggers prolifera-
tion pathways by activating the mammalian target of rapamycin (mTOR). Akt also activates
phosphatase and tensin homolog on chromosome 10 (Pten), which dephosphorylates PI 3, 4,
5-P3 to PI 4, 5-P2. Pten activates glycogen synthase kinase-3β (GSK-3β), which phosphorylates
tau protein involved in neuronal migration. Reelin binds to very low-density lipoprotein recep-
tor (VLDLR) and apolipoprotein E receptor 2 (ApoER2), which activate Disabled-1 protein
(Dab-1). Dab-1 activates the Akt involved in neuronal migration and proliferation. Cellular
stress, on the other hand, affects the activities of p53 and Bcl2, proteins involved in apoptosis.
Presynaptic vesicle
Neurexin
2+ 2+
Ca Ca
channel channel
Neuroligin
SHANK3/ProSAP
PSD-95 PSD-95
Foderin Cortactin
Actin filament
Postsynaptic vesicle
FIGURE 10.3 Interaction of neurexin and neuroligins. Neurexins participate in synaptic

transmission by affecting calcium channels. They also interact with neuroligins of postsyn-
aptic vesicles. Neuroligin controls the actin assembly by interacting with postsynaptic density
protein 95 (PSD-95), which further interacts with SHANK3. SHANK3/PSD-95 interact with
foderin and cortactin, which bind to actin filament.
Serotonin
Serotonin reuptake
transporter Synaptic gap
Serotonin receptors
FIGURE 10.4 Serotonin and its receptors, and serotonin reuptake transporter. Serotonin
molecules from the presynaptic vesicles are released into the synaptic gap, where they are
internalized by serotonin receptors into the postsynaptic vesicles. Serotonin molecules are
also retaken from synaptic gaps to the presynaptic vesicle by the serotonin reuptake transport-
ers. An imbalance of the uptake and reuptake system in serotonin causes brain dysfunctions.
Direct cytokine action in brain

Cross BBB Peripheral cytokines activate CNS
immune response
Neuronal damage mediated by cross-
reactive Ab΄s and/or T cells
Proinflammatory
cytokines (e.g., TNF-α
Molecular mimicry
and IFN-γ)
leading to cross-reactive
Ab΄s and/or T cells Regulatory cytokines
(e.g., IL-10)
Egress of Altered Gl permeability

microbial/dietary Innate/adaptive immune
antigens dysfunction
Gl inflammation
FIGURE 14.1 Possible mechanism by which GI dysfunction may trigger autistic behaviors
in children with autism and GI disturbance.

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Boca Raton London New York

CRC Press is an imprint of the

© 2010 by Taylor and Francis Group, LLC

No claim to original U.S. Government works

Printed in the United States of America on acid-free paper

International Standard Book Number: 978-1-4200-6881-8 (Hardback)

Library of Congress Cataloging-in-Publication Data

Autism : oxidative stress, inflammation, and immune abnormalities / edited by Abha

Visit the Taylor & Francis Web site at

and the CRC Press Web site at

Chapter 1 Type, Topography, and Sequelae of Neuropathological Changes

Chapter 2 Evidence for Oxidative Damage in the Autistic Brain ....................... 35

Chapter 3 Oxidative Stress and Neurotrophin Signaling in Autism ................... 47

Chapter 4 Genetics of Autism ............................................................................. 61

Chapter 5 Phenotypic Expression of Autism, Maternal Depression,

Chapter 6 Paraoxonase 1 Status, Environmental Exposures,

Chapter 7 The Redox/Methylation Hypothesis of Autism: A Molecular

Chapter 9 Neurotoxic Brainstem Impairment as Proposed

Chapter 10 Abnormalities in Membrane Lipids, Membrane-Associated

Chapter 11 Mitochondrial Component of Calcium Signaling

Chapter 12 Inflammation and Neuroimmunity in the Pathogenesis

Chapter 13 Possible Impact of Innate Immunity on Autism .............................. 245

Chapter 14 Autism, Gastrointestinal Disturbance, and Immune

Chapter 15 Possible Mechanism Involving Intestinal Oxytocin,

Chapter 16 Cytokine Polymorphisms in Autism:

Chapter 18 Autism: The Centrality of Active Pathophysiology and

Chapter 19 A Reevaluation of the State of Autism Treatment:

of β-amyloid precursor protein, enhanced turnover of cell organelle and pigment

Abha Chauhan, PhD

Tel: 718-494-5258; Fax: 718-698-7916

Dr. Chauhan is a member of the editorial board of the International Archives of

Teresa Wierzba Bobrowicz, MD, PhD Ira L. Cohen, PhD

Steven Buyske, PhD Department of Psychiatry

Manuel Casanova, MD Richard C. Deth, PhD

Maria Dronca, PhD Alycia K. Halladay, PhD

Martha R. Herbert, MD, PhD

Rafael Fernandez-Botran, PhD William G. Johnson, MD

Division of Human Genetics Harumi Jyonouchi, MD

Benjamin Y. Klein, MD Woody R. McGinnis, MD

Shuang Yong Ma, MD, PhD Carlos A. Pardo-Villamizar, MD

Sergiu P. Paşca, MD Mark A. Smith, PhD

Jerzy Wegiel, VMD, PhD Carrie L. Yochum, MS

New York State Institute for Basic Research in

* Corresponding author: Tel.: +1-718-494-5231; fax: +1-718-982-4856; e-mail: [email protected]

1.3 Deregulation of Brain Growth in Early Childhood ........................................6

regions, appears to be a key factor determining topography and brain region–

1.2 CLINICAL, ETIOLOGICAL, AND NEUROPATHOLOGICAL

metabolic modifications with abnormal amyloid precursor protein (APP) processing

1.3 DEREGULATION OF BRAIN GROWTH

1.3.1 DEVELOPMENTAL HETEROCHRONICITY

1.3.2 FUNCTIONAL CONSEQUENCES OF ABNORMAL BRAIN DEVELOPMENT

1.4 CORTICAL AND SUBCORTICAL NEUROPATHOLOGY

1.4.2 BRAIN STRUCTURE–SPECIFIC DELAY OF NEURONAL GROWTH

1.4.3 MINICOLUMNAR ABNORMALITIES IN AUTISM

1.5 NEURONAL OXIDATIVE STRESS AND METABOLIC CHANGES

1.5.1 OXIDATIVE STRESS IN AUTISM

and elimination of environmental toxins. Due to the lack of glutathione-producing

1.5.2 LIPOFUSCIN IN AUTISM

1.5.3 b-AMYLOID PRECURSOR PROTEIN AND INTRANEURONAL