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Recent Advances in Parkinson s Disease Basic Research
1st Edition Anders Björklund And M. Angela Cenci
(Eds.) Digital Instant Download
Author(s): Anders Björklund and M. Angela Cenci (Eds.)
ISBN(s): 0444536140
Edition: 1
File Details: PDF, 10.85 MB
Year: 2010
Language: english
 PROGRESS IN BRAIN RESEARCH

VOLUME 183

RECENT ADVANCES IN PARKINSON’S

DISEASE: BASIC RESEARCH

EDITED BY

ANDERS BJÖRKLUND
Wallenberg Neuroscience Centre
Division of Neurobiology
Lund University
Lund, Sweden

M. ANGELA CENCI
Basal Ganglia Pathophysiology Unit
Department of Experimental Medical Science
Lund University
Lund, Sweden

AMSTERDAM – BOSTON – HEIDELBERG – LONDON – NEW YORK – OXFORD

PARIS – SAN DIEGO – SAN FRANCISCO – SINGAPORE – SYDNEY – TOKYO
Elsevier
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 List of Contributors

D.M. Alessi, Departments of Neurology, Pathology and Cell Biology, and the Center for Motor Neuron
Biology and Disease, Columbia University, New York, NY, USA
C. Baunez, Laboratoire de Neurobiologie de la Cognition (LNC), UMR6155 CNRS/Aix-Marseille
Université, Marseille, France
H. Bergman, The Interdisciplinary Center for Neural Computation; Institute for Medical Research Israel-
Canada (IMRIC), Department of Medical Neurobiology (Physiology), The Hebrew University,
Jerusalem, Israel
R.E. Burke, Departments of Neurology, Pathology and Cell Biology, Columbia University, New York,
NY, USA
P. Calabresi, Fondazione Santa Lucia IRCCS, Rome, Italy; Clinica Neurologica, Università degli Studi di
Perugia, Ospedale S. Maria della Misericordia, Perugia, Italy
A.R. Carta, Department of Toxicology, University of Cagliari, Cagliari, Italy
M.A. Cenci, Basal Ganglia Pathophysiology Unit, Department of Experimental Medical Science, Lund
University, Lund, Sweden
S. Chan, Department of Physiology, Feinberg School of Medicine, Northwestern University, Chicago, IL,
USA
M.R. Cookson, Cell Biology and Gene Expression Unit, Laboratory of Neurogenetics, National Institute
on Aging, Bethesda, MD, USA
T.M. Dawson, NeuroRegeneration and Stem Cell Programs, Institute for Cell Engineering, Johns
Hopkins University School of Medicine, Baltimore, MD, USA; Department of Neurology, Johns
Hopkins University School of Medicine, Baltimore, MD, USA; Solomon H. Snyder Department of
Neuroscience, Johns Hopkins University School of Medicine, Baltimore, MD, USA
V.L. Dawson, NeuroRegeneration and Stem Cell Programs, Institute for Cell Engineering; Department of
Neurology; Department of Physiology; Solomon H. Snyder Department of Neuroscience, Johns Hopkins
University School of Medicine, Baltimore, MD, USA
M. Day, Department of Physiology, Feinberg School of Medicine, Northwestern University, Chicago, IL,
USA
R.L.A. deVries, Departments of Neurology, Pathology and Cell Biology, and the Center for Motor
Neuron Biology and Disease, Columbia University, New York, NY, USA
M. di Luca, Department of Pharmacological Sciences, University of Milano, Milano, Italy
S. Elias, Institute for Medical Research Israel-Canada (IMRIC), Department of Medical Neurobiology
(Physiology), The Hebrew University, Jerusalem, Israel
M. Fournier, Laboratory of Molecular Neurobiology and Neuroproteomics, Brain Mind Institute,
The Ecole Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland
M.J. Frank, Department of Cognitive, Linguistic, and Psychological Sciences, Department of Psychiatry
and Human Behavior, and Brown Institute for Brain Science, Brown University, Providence, RI, USA

v
vi

F. Gardoni, Department of Pharmacological Sciences, University of Milano, Milano, Italy

T. Gasser, Hertie Institute for Clinical Brain Research, Department of Neurodegenerative Diseases,
Tübingen, Germany; DZNE, German Center for Neurodegenerative Diseases, Tübingen
T. Gertler, Department of Physiology, Feinberg School of Medicine, Northwestern University, Chicago,
IL, USA
V. Ghiglieri, Fondazione Santa Lucia IRCCS, Rome, Italy
J.A. Goldberg, Department of Physiology, Feinberg School of Medicine, Northwestern University,
Chicago, IL, USA
P. Gubellini, Institut de Biologie du Développement de Marseille-Luminy (IBDML), UMR6216 CNRS/
Aix-Marseille Université, Marseille, France
J.N. Guzman, Department of Physiology, Feinberg School of Medicine, Northwestern University,
Chicago, IL, USA
G. Heimer, Institute for Medical Research Israel-Canada (IMRIC), Department of Medical Neurobiology
(Physiology), The Hebrew University, Jerusalem, Israel
V. Jackson-Lewis, Departments of Neurology, Pathology and Cell Biology, and the Center for Motor
Neuron Biology and Disease, Columbia University, New York, NY, USA
A. Kachroo, MassGeneral Institute for Neurodegenerative Disease, Massachusetts General Hospital,
Boston, MA, USA
C. Konradi, Center for Molecular Neuroscience and Kennedy Center for Research on Human Development,
Departments of Pharmacology and Psychiatry, Vanderbilt University, Nashville, TN, USA
H.A. Lashuel, Laboratory of Molecular Neurobiology and Neuroproteomics, Brain Mind Institute, The
Ecole Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland
I. Martin, NeuroRegeneration and Stem Cell Programs, Institute for Cell Engineering, Johns Hopkins
University School of Medicine, Baltimore, MD, USA; Department of Neurology, Johns Hopkins
University School of Medicine, Baltimore, MD, USA
M. Morelli, Department of Toxicology, University of Cagliari, Cagliari, Italy
A. Oueslati, Laboratory of Molecular Neurobiology and Neuroproteomics, Brain Mind Institute, The
Ecole Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland
J.L. Plotkin, Department of Physiology, Feinberg School of Medicine, Northwestern University, Chicago,
IL, USA
S. Przedborski, Departments of Neurology, Pathology and Cell Biology, and the Center for Motor Neuron
Biology and Disease, Columbia University, New York, NY, USA
M. Rivlin-Etzion, The Interdisciplinary Center for Neural Computation; Institute for Medical Research
Israel-Canada (IMRIC), Department of Medical Neurobiology (Physiology), The Hebrew University,
Jerusalem, Israel
J. Sanchez-Padilla, Department of Physiology, Feinberg School of Medicine, Northwestern University,
Chicago, IL, USA
M.A. Schwarzschild, MassGeneral Institute for Neurodegenerative Disease, Massachusetts General
Hospital, Boston, MA, USA
W. Shen, Department of Physiology, Feinberg School of Medicine, Northwestern University, Chicago, IL,
USA
D.J. Surmeier, Department of Physiology, Feinberg School of Medicine, Northwestern University,
Chicago, IL, USA
X. Tian, Department of Physiology, Feinberg School of Medicine, Northwestern University, Chicago, IL,
USA
 vii

M. Tocilescu, Departments of Neurology, Pathology and Cell Biology, and the Center for Motor Neuron
Biology and Disease, Columbia University, New York, NY, USA
C. Vives-Bauza, Departments of Neurology, Pathology and Cell Biology, and the Center for Motor
Neuron Biology and Disease, Columbia University, New York, NY, USA
T.V. Wiecki, Department of Cognitive Linguistic, and Psychological Sciences, Department of Psychiatry
and Human Behavior, and Brown Institute for Brain Science, Brown University, Providence, RI, USA
 Preface

Research on Parkinson´s disease (PD) is one of the most dynamic fields of modern neuroscience. It is an
excellent example of how clinical and basic research can fruitfully interact and inspire each other in a truly
translational way. During the decades after the discovery of dopamine in the late 1950s the field was
dominated by pharmacological and neurochemical approaches. Since the discovery of the role of alpha-
synuclein and its role in PD pathogenesis in the late 1990s, PD research has entered a new exciting phase
of development, and its scope has broadened to include dynamic molecular and genetic approaches. This
had led to the discovery of further genetic mutations accounting for familial forms of the disease (Parkin,
DJ-1, Pink-1, and leucine-rich repeat kinase 2), and spurred intense molecular biological investigations on
the mechanisms of neurodegeneration in PD. In addition to molecular genetics, other fields of PD
research have undergone a dramatic development during the past 20 years. Significant progress has
been made modelling PD in animals both on a symptomatic level and on a mechanistic perspective. The
current availability of a diversified range of models in different species provides neuroscientists with
articulate tools to study molecular mechanisms, test pathophysiological hypotheses and identify new
treatment principles. The discovery that PD motor symptoms and treatment-induced dyskinesias are
dramatically ameliorated by high-frequency stimulation of some deep basal ganglia nuclei has prompted
efforts on the part of both neurophysiologists and computational neuroscientists to decipher the basic
neural operations of the basal ganglia in health and disease. Finally, technological developments in the
area of brain imaging have provided exciting new opportunities for pathophysiological investigations,
differential diagnosis and treatment monitoring in PD patients.
These two companion volumes of Progress in Brain Research were composed to capture all the richness
and complexity of PD as a topic for basic, translational and clinical investigation. A year ago, when we
approached leading researchers in the different subfields to contribute, the vast majority of the invited
authors enthusiastically accepted the invitation and delivered contributions that turned out to represent
the utmost state-of-the art in each given field. It is with great pleasure and pride that we now present this
collection of review chapters to a broad audience of readers.
The chapters have been grouped into two volumes and five sections. The first volume covers basic and
molecular investigations of the mechanisms of neurodegeneration in PD (Section I: Genetic and
molecular mechanisms of neurodegeneration in PD) and the secondary adaptations that affect the basal
ganglia at both the single cell level and the system level (Section II: Cellular and system-level pathophy-
siology of the basal ganglia in PD). The second volume focuses on translational and clinical aspects of PD
research reviewing animal models of PD from drosophila to non-human primate species (Section I:
Animal models of PD) the very dynamic area of functional neuroimaging (Section II: Exploring PD with
brain imaging) and the most challenging therapeutic developments (Section III: Frontiers in PD
treatment).
Like no other neurological disease, PD is inspiring enormously diversified research themes and
approaches in a way that would have been impossible to foresee some 10 years ago. An increasing number

ix
x

of investigators, also from areas outside neuroscience, have joined the PD research community and are
now contributing to the richness and diversity of this field. Over the last 15 years, several international PD
patient-initiated, non-profit organizations have dramatically improved the funding possibilities for this
area of research which is now advancing at an extremely rapid pace. It is our hope that this formidable
development of knowledge and technologies will deliver novel options for treatment – and eventually a
cure – for all who suffer from this disease.
In closing we would like to express our warmest thanks to all the authors for their outstanding
contributions and to Gayathri Venkatasamy, our Developmental Editor at Elsevier, for her expert and
patient assistance.

Lund, June 11th 2010

Anders Björklund
M. Angela Cenci
 SECTION I

Genetic and molecular mechanisms of

neurodegeneration in PD
A. Bjorklund and M. A. Cenci (Eds.)
Progress in Brain Research, Vol. 183
ISSN: 0079-6123
Copyright
2010 Elsevier B.V. All rights reserved.

CHAPTER 1

Identifying PD-causing genes and genetic

susceptibility factors: current approaches and future
prospects

Thomas Gasser

Hertie Institute for Clinical Brain Research, Department of Neurodegenerative Diseases, T€

ubingen, Germany, and
DZNE, German Center for Neurodegenerative Diseases, T€ ubingen

Abstract: Over the last years, a plethora of genetic findings have completely changed our views on the
aetiology of Parkinson’s disease (PD). Linkage studies and positional cloning strategies have identified
mutations in a growing number of genes which cause monogenic autosomal-dominant or autosomal-
recessive forms of the disorder. While these Mendelian forms of PD are relatively rare, high-throughput
genotyping and sequencing technologies have more recently provided evidence that low-penetrance
variants in at least some of these genes also play a direct role in the aetiology of the common sporadic
disease. In addition, rare variants in other genes, such as the Gaucher’s disease-associated
glucocerebrosidase A, have also been found to be important risk factors at least in subgroups of patients.
Thus, an increasingly complex network of genes contributing in different ways to disease risk and
progression is emerging. These findings provide the ‘genetic entry points’ to identify molecular targets
and readouts necessary to design rational disease-modifying treatments.

Keywords: Parkinson’s disease; Genetics; Genetic risk factors; DNA polymorphisms

Introduction innovations of the 1980s, such as polymerase chain

reaction amplification of DNA fragments and the
The progress of molecular genetic technology over discovery of polymorphic micro-satellite repeat
the last 25 years has revolutionized our understand- elements in the genome (usually consisting of
ing of Parkinson’s disease (PD) and many other repetitive di-, tri- or tetranucleotide sequences
common complex disorders. The technological which proved to be extremely useful as landmarks
(‘DNA markers’) to map the genome) hand in

hand with the development of appropriate statis­
Corresponding author.
Tel.: 07071/2986529; Fax: 07071/294839; tical tools and computer programmes, led to the
E-mail: [email protected] mapping and cloning of a large number of genes

DOI: 10.1016/S0079-6123(10)83001-8 3
4

which cause – when mutated – monogenic dis­ The advent of micro-array technology which
eases, that is inherited disorders following a Men­ provided the opportunity to study hundreds
delian mode of transmission (Gasser, 2009a). of thousands or even millions of genetic variants
Most of these classic neurogenetic disorders such (usually single-nucleotide polymorphisms, ‘SNPs’)
as Huntington’s disease, myotonic dystrophy or in a large cohort of patients and controls has
spinal muscular atrophy, to name just a few, are provided the basis for a new generation of genetic
relatively rare. Nevertheless, with the identifica­ studies, genome-wide association studies (GWAS)
tion of these rare disease genes ‘neurogenetics’ in order to go beyond the analysis of single candi­
has become part of the mainstream of neurology date genes and to systematically analyse, in an
(Harbo et al., 2009). unbiased way, the genetic risk profile of complex
During the 1990s it became apparent that in some diseases. While most GWAS evaluate the role of
rare cases the much more common and typically common genetic variability as risk factors for a
sporadic neurologic disorders such as Parkinson’s disease, this approach has already been success­
disease (PD) (Denson and Wszolek, 1995), fully applied also to quantitative traits such as age
Alzheimer’s disease (AD) (Goate et al., 1991) or of onset in PD (Latourelle et al., 2009) or to
amyotrophic lateral sclerosis (Rosen et al., 1993) laboratory values such as serum levels of uric
could also run in families following a Mendelian acid (Dehghan et al., 2008). Another extension
pattern of inheritance. In fact, it turned out that of this technology is the combination with gen­
many of these monogenic variants of common dis­ ome-wide transcriptome analysis (Elstner et al.,
eases resemble the typical sporadic forms to a large 2009), promising a deeper insight into relevant
degree both clinically (with the exception that age of gene regulation networks.
onset is often younger in patients with inherited The next technological revolution is already
forms of these disorders) and pathologically, sug­ under way. Massive-parallel sequencing will
gesting that the molecular pathways discovered make large-scale whole exome or even whole gen­
through the relevant genes in hereditary forms may ome sequencing feasible. This will be necessary to
also be of importance in sporadic cases. The same identify the suspected multitude of rare genetic
gene identification strategies used in classical neuro­ variants in different genes which are thought to
genetic diseases were successfully used to show that explain another substantial fraction of the genetic
the Mendelian forms of the respective common dis­ risk for common diseases.
orders were also caused by mutations in single Methods are being developed to deal with the
genes. The identification of those disease genes, increasingly complex and vast amount of informa­
such as SNCA, the gene encoding alpha-synuclein tion generated by current and future sequencing
(aSYN) for PD, or APP, the gene coding for technology.
the amyloid precursor protein in AD, was a crucial
step in the elucidation of the chain of molecular
events which lead to neurodegeneration in these Identification of monogenic forms of PD by
disorders. positional cloning strategies
It was then only in recent years that accumulating
evidence suggested that the close resemblance Autosomal-dominant forms of inherited PD
between familial and sporadic forms on the clinical
and pathologic level also has its correspondence on The classic approaches of linkage analysis and
the genetic level: common genetic variants in genes positional cloning have been the uniquely success­
identified in monogenic forms or in genes belonging ful strategies to identify genes causing the autoso­
to the identified pathways have been found to mal-dominantly inherited diseases including the
modify the risk to develop a sporadic disease. major forms of familial PD.
 5

This strategy relies on the availability of large individuals were identified with a relatively early-
and clinically well-characterized families, usually onset form of PD (average age of onset was 46
with at least 8–10 affected family members. By years), a high rate of dementia and an unusually
studying the co-segregation of genetic (DNA)mar­ severe and rapid course (average disease duration
kers (most often highly polymorphic dinucleotide less than 10 years), segregating as an autosomal-
repeat elements, above-mentioned micro-satellite dominant trait (Golbe et al., 1990). Only a year
polymorphisms, were used), the genetic locus of later, the same group identified a putative disease-
the disease-causing gene in a given family can be causing mutation in a known gene of the region,
narrowed down to a region of several million base alpha-synuclein (the gene is abbreviated as
pairs (megabases, Mb) of DNA. The statistical SNCA, the protein as aSYN). It was a single
method to estimate the likelihood that a particular base-pair change (a G to A transition) at position
set of neighbouring DNA markers (a so-called 209 of the coding sequence, leading to the change
haplotype) are co-inherited with a disease gene from alanine to threonine at position 53 of the
as a result of its physical proximity on the chromo­ aSYN protein (A53T) (Polymeropoulos et al.,
some (i.e. that DNA markers and disease gene are 1997). As expected, the mutation co-segregated
‘linked’) is called linkage analysis. The most with the disease in the affected family, but this in
important prerequisite for this type of study, in itself is no proof of pathogenicity. Depending on
addition to the availability of sufficiently large the size of a linked genetic region, the affected
families, is the unequivocal classification of affected members in a family will share a large number of
and unaffected family members. Erroneous classi­ candidate genes and consequently all genetic var­
fication, which in many age-related complex dis­ iants that are located in this region. In fact, the
eases is a real possibility, will lead to false linkage causative role of the SNCA mutation initially was
results (Gasser, 2008). When a disease locus is doubted by some researchers based on the surpris­
identified with sufficient confidence (a so-called ing fact that the highly homologous mouse SNCA
lod score of >3 is equivalent to a genome-wide p- gene contains the threonine thought to be patho­
value of 0.05 and is considered to be significant genic in humans at position 53. It was therefore of
evidence), all the genes in the identified region great importance to find additional independent
have to be sequenced and analysed for potentially sequence variants segregating with PD in other
disease-causing mutations. Of course, not all of the families. Eventually, those variants were found,
identified sequence variants in a linked region are although they are extremely rare: only two further
pathogenic. This means that either the demonstra­ pathogenic point mutations in SNCA have
tion of mutations in several independent families been recognized, leading to the exchange A30P
co-segregating with a disease is necessary (amount­ (Krüger et al., 1998) and E46K (Zarranz et al.,
ing in effect to a replication of the initial finding) or 2004), respectively, each in a single, large, domi­
the careful functional studies in model systems are nant family, reflecting the high penetrance of
required to prove pathogenicity. these mutations. SNCA point mutations are very
rare and have not been found in large cohorts of
patients with sporadic PD (Berg et al., 2005).
PARK1 (alpha-synuclein) Further important insight into the link between
SNCA and PD came from the discovery of gene
It was by classic linkage analysis that Polymero­ multiplication mutations. A family with autoso­
poulos et al. mapped the disease locus in a large mal-dominant parkinsonism, also frequently
Italian family with autosomal-dominant PD to the accompanied by dementia, had been mapped to
long arm of chromosome 4 (Polymeropoulos the short arm of chromosome 4 (4p15) and had
et al., 1996). In this family, more than 40 therefore been thought to be genetically distinct
6

from the families with SNCA mutations (Farrer protein aggregates which had long been recog­
et al., 1999). The affected members resembled nized as the pathologic hallmark in familial as
those with SNCA mutations both clinically and well as sporadic cases of the disease (Fig. 1). The
pathologically to a remarkable degree (see currently favoured hypothesis states that the
below). It was therefore not too surprising when amino acid changes in aSYN lead to an increased
it became apparent that the assignment to chro­ tendency of the protein to form oligomers and
mosome 4p was in fact due to a genotyping error fibrillar aggregates (Goedert et al., 1998; Karpinar
and that the disease also co-segregated with the et al., 2009), eventually resulting in neuronal dys­
SNCA locus in this family. However, direct function and cell death, although the precise rela­
sequencing of the SNCA gene did, however, not tionship between mutations, aggregate formation
reveal any putative pathogenic mutations. Instead, and their deleterious effects on neurons is still
Singleton and co-workers found a triplication of unknown. Many studies favour the hypothesis
the entire locus containing the SNCA gene in the that the mature aggregates (i.e. Lewy bodies and
affected members of this pedigree (Singleton Lewy neuritis detected on the light microscopy
et al., 2003). This finding is of particular relevance level) are not themselves the toxic moiety, but
with respect to the molecular pathogenesis of PD, rather an attempt of the cell to clear much smaller
as it suggests that not only structurally altered toxic oligomers (Cookson and van der Brug,
aSYN can cause PD but that also the wild-type 2007), while the elusive oligomers are the true
aSYN protein is pathogenic, if it is over- toxic moiety (Conway et al., 2000).
expressed. In fact, the triplication is associated Mutation carriers from the first family described
with a roughly twofold over-expression of the with an SNCA mutation (the ‘Contursi’ kindred)
aSYN protein in the brain of affected individuals,
as shown by Western blotting on autopsy material
(Singleton et al., 2003).
Subsequently several additional families with
SNCA triplications, and also with SNCA duplica­
tions, have been found (Ibanez et al., 2009). In
those gene locus multiplication families, a clear
dose dependence of the pathogenic effect was
observed: SNCA duplications, that is a 50%
increase of the gene dosage (three instead of the
usual two gene copies) leads to relatively late-
onset dopa-responsive parkinsonism resembling
typical PD, while triplications are associated with
an earlier disease onset, a high prevalence of
dementia and a rapid disease course. This could
be even shown in a single family in whom different
branches segregated a duplication and a triplica­
tion of a 1.5 Mb genomic fragment containing the
SNCA gene (Fuchs et al., 2007).
The identification of the first SNCA mutations Fig. 1. aSYN immunopositive neuronal inclusions in the
dorsal motor vagal nucleus of a patient with an A30P SNCA
by Polymeropoulos and co-workers soon leads to
mutation. Marked aSYN pathology is obvious with numerous
the discovery that the encoded protein (aSYN) is Lewy bodies (arrowheads) and Lewy neurites (arrows). Figure
the major fibrillar component of the Lewy body kindly provided by Prof. R. Krüger, Hertie Institute for Clinical
and Lewy neurites (Spillantini et al., 1997), the Brain Research, Tübingen.
 7

clinically had relatively early onset of a mostly showed typical L-dopa-responsive parkinsonism
akinetic/rigid form of PD with rapid progression with onset in their fifties (Funayama et al., 2002).
and commonly also suffered from dementia. By positional cloning, missense mutations in the
The clinical spectrum was later confirmed and gene for leucine-rich repeat kinase 2 (LRRK2)
extended in additional families carrying the same (Paisan-Ruiz et al., 2004; Zimprich et al., 2004)
mutation, in whom prominent autonomic distur­ were found to be disease causing. The gene
bances were also noted (Spira et al., 2001). spans a genomic region of 144 kb, with 51 exons
A very similar clinical picture with early dementia encoding 2527 amino acids, and to date, at least six
and autonomic disturbances, thereby more resem­ verified disease-causing mutations have been
bling a variant of PD called ‘dementia with Lewy identified.
bodies’ (DLB) rather than typical PD, was Mutations in the LRRK2 gene are clearly the
described in the family with the E46K mutation most common cause of dominantly inherited PD
(Zarranz et al., 2004). On the other hand, a more discovered so far. In a number of studies across
typical late onset of parkinsonian symptoms with several different populations between 5 and 15%
only late development of relatively mild dementia of dominant families carry mutations in LRRK2
was described in a German family with the A30P (Berg et al., 2005; Di Fonzo et al., 2005). The
mutation (Krüger et al., 1998). Although this single most common mutation, G2019S, is respon­
family is relatively small and therefore this conclu­ sible for familial PD in up to 7% of familial cases
sion rests only on very few cases, there appear to be in different Caucasian populations (Di Fonzo
mutation specific differences in disease et al., 2005; Kachergus et al., 2005; Nichols et al.,
presentation. 2005). This mutation has also been found in about
Pathologically, fibrillar aSYN aggregates (i.e. 1–2% of sporadic patients of European descent
Lewy pathology) were found in all patients with (Gilks et al., 2005), indicating that the mutation
SNCA mutations, both point mutations and multi­ has a reduced penetrance, which was estimated
plications, not only in the substantia nigra but also between 35 and 70% (Goldwurm et al., 2005;
in other brain stem nuclei and widespread in Ozelius et al., 2006) and which must be taken
mesocortical and neocortical neurons, again com­ into account in genetic counselling. Even higher
patible with a diagnosis of DLB (Spira et al., G2019S prevalence rates of up to 40% were found
2001). Interestingly, in mutation-positive cases, in genetically more isolated populations, such as
aSYN pathology was also found in oligodendro­ the Ashkenazi Jewish or the North African Arab
glial cells, a feature thought to be typical for multi­ populations, both in sporadic and in familial cases
ple system atrophy (MSA) (Dickson et al., 1999). (Lesage et al., 2006; Ozelius et al., 2006) due to
This large overlap of pathologic features of PD, genetic founder effects.
DLB and MSA in cases with SNCA mutations Despite its reduced penetrance, the G2019S
strongly supports the close aetiologic link between mutation is usually thought of as a true ‘disease­
these disease entities. causing mutation’, because it is very rare in all
control populations studied so far. Other more
common variants in the LRRK2 gene, on the
PARK8 (LRRK2) other hand, appear to act more like risk factors
of modest effect sizes. The G2385R (Farrer et al.,
Another locus for a dominant form of PD was first 2007) variant, for example, was found not only in
mapped in a large Japanese family to the pericen­ approximately 9% of Chinese patients with PD
tromeric region of chromosome 12 and named but also in about 3% of controls. The same role
PARK8, again by a classic linkage analysis of a risk allele has been suggested for the R1628P
approach. Affected members in this family exchange (Lu et al., 2008; Ross et al., 2008).
8

Clinical signs and symptoms of LRRK2-related PARK2 (Parkin)

disease closely resemble typical sporadic PD.
This is also true for age of onset, which is on It was in autosomal-recessive families with very
average in the late fifties and only slightly below early-onset parkinsonism that the first recessive
that in non-mutation-carrying PD patients (Healy PD gene was mapped to the long arm of chromo­
et al., 2008). However, age at onset as well as some 6 in the vicinity of the gene for superoxide
severity of the disease may be highly variable, dismutase 2 (SOD2) (Matsumine et al., 1997).
even within families. Because of the suspected role of toxic oxygen
Pathological changes in patients with LRRK2 radicals in the pathogenesis of PD, SOD2 was a
mutations are consistent with typical Lewy body plausible candidate gene. However, no sequence
PD in most cases reported so far and also include variants in this gene could be identified. Rather, a
diffuse Lewy body disease, nigral degeneration with­ number of different mutations were found in a
out distinctive histopathology and rarely even pro­ very large neighbouring gene which was then
gressive supranuclear palsy-like tau aggregation. called Parkin (PRKN) (Kitada et al., 1998).
LRRK2 mutations may therefore be an upstream PRKN mutations turned out to be a common
event in the cascade leading to neurodegeneration cause of parkinsonism with early onset, particularly
with different pathologies. Although the natural sub­ in individuals with evidence of recessive inheri­
strate and disease-relevant function of LRRK2 is tance. Nearly 50% of sibling pairs with PD were
unknown, cell culture studies suggest that pathogenic found to have PRKN mutations (Lücking et al.,
mutations seem to be associated with increase, rather 2000), if at least one of them had an age of onset
than a loss, of kinase activity (Gloeckner et al., below 45 years. As recessive diseases often appear
2006), raising the interesting possibility that kinase to be sporadic, particularly in societies with rela­
inhibition may be a potential therapeutic strategy. tively small families, because statistically only 25%
of the offspring of two heterozygous mutation car­
riers will be homozygous for the disease allele,
Autosomal-recessive parkinsonism PRKN mutations are also responsible for the major­
ity of sporadic cases with very early onset (before
The strategies of linkage mapping and positional age 20) and are still common (25–40%) when onset
cloning can also be used to identify loci in genes is between 20 and 35 years. PRKN mutations are
responsible for autosomal-recessive monogenic rare in sporadic cases with onset later than 45 years.
diseases. This mode of inheritance is characterized
typically by the occurrence of the disease in sib­
lings while the parents are obligatory heterozygous Other recessive forms of parkinsonism
mutation carriers and usually remain healthy.
Autosomal-recessive PD has clinically been first Mutations in the PINK1 gene (PARK6) have
recognized and characterized in Japan (Ishikawa been identified as another cause for autosomal-
and Tsuji, 1996). Sibling pairs with PD often have recessive early-onset parkinsonism (Valente
much earlier age of onset compared with patients et al., 2004), again following a linkage mapping
with the sporadic disease, which is why the term approach in several multiplex recessive families
‘autosomal-recessive juvenile parkinsonism’, has (Valente et al., 2001). This gene is particularly
been coined. Since families with a recessive disease interesting within the context of the findings link­
are usually much smaller than multigenerational- ing PD to mitochondrial dysfunction and oxidative
dominant pedigrees, linkage analysis is only stress, as PINK1 encodes a mitochondrially
successful if several families mapping to the same located protein. Mutations in the PINK1 gene
locus are included into a study. are less common than PRKN mutations in most
 9

populations studied and probably account for only While some mutations such as R1441C or Y1699C
1–2% of early-onset cases (Hatano et al., 2004; have so far only been found in families with clear
Rogaeva et al., 2004; Rohe et al., 2004; Valente autosomal-dominant inheritance and thus are con­
et al., 2004). From work with animal models it has sidered to be high-penetrance disease genes, the
become quite clear that PINK1 acts in a common G2019S variant is clearly a disease gene with mark­
pathway with PRKN (Dodson and Guo, 2007). edly reduced penetrance, which is still very rare in
However, the natural substrate of the kinase unaffected individuals, while the variants G2385R
activity of PINK1 is still unknown. or R1628P are found in >1% of the healthy
Mutations in the DJ-1 gene (PARK7) are yet Chinese population and thus must be considered
another rare cause of autosomal-recessive parkin­ relatively ‘common’ genetic variants, which are
sonism (Bonifati et al., 2002; Healy et al., 2004; associated with an approximately threefold
Hedrich et al., 2004). The clinical picture with increased risk to develop PD.
early onset and slow progression is similar to the In addition to the concept that rare mutations
other recessive parkinsonian syndromes. Follow­ cause rare genetic diseases and – on the other
ing the initial discovery of two mutations in an hand – common variants are associated with a mod­
Italian and a Dutch family (Bonifati et al., 2002), erate increase of relative risk for common disorders,
only a few additional bona fide pathogenic muta­ another concept has emerged over the last several
tions [one homozygous (Hering et al., 2004) and years: the rare variant-common disease hypothesis.
one compound heterozygous (Abou-Sleiman It states that multiple rare variants in a potentially
et al., 2003)] have been identified. large number of genes may each be significant (par­
While mutations in the genes named above all tial) causes in a relatively small proportion of
cause a ‘pure’ form of early-onset parkinsonism, patients with a common disease such as PD. This
an increasing number of genes have been found to hypothesis is exemplified in the role of mutations in
cause more complex phenotypes which includes, the gene for glucocerebrosidase (GBA) in PD.
in addition to parkinsonism, often dystonia, spas­ About 10 years ago, astute clinical observation
ticity, and dementia (Klein et al., 2009). suggested that patients with Gaucher’s disease, an
The common denominator for all those cases is autosomal-recessive, usually childhood-onset lysoso­
that they represent Mendelian forms of the dis­ mal storage disease associated with a wide variety of
ease, which are relatively rare, can be tackled by organ manifestations, had a conspicuous tendency to
classic approaches of linkage analysis and posi­ develop PD in later life (Machaczka et al., 1999;
tional cloning, and can be modelled in different Tayebi et al., 2001). Gaucher’s disease is caused by
cellular and animal model systems, which is mutations in the gene for glucocerebrosidase, GBA,
invaluable for a better understanding of the mole­ which is located in chromosome 1q21. GBA is an
cular pathways leading to dopaminergic neurode­ enzyme of the ceramide pathway, and its deficiency
generation (Gasser, 2009b). leads to the excessive storage of its substrate,
glucosylceramide, within lysosomes of many differ­
ent cell types, including neurons and macrophages
Rare genetic variants causing or pre-disposing to PD (Grabowski, 2008). Following the initial reports of a
clinical association of Gaucher’s disease with PD it
While the genes described above are generally was observed that heterozygous, otherwise healthy,
thought of as high-penetrance disease-causing carriers of GBA mutations, that is the relatives of
genes, the genetic epidemiology of LRRK2­ Gaucher patients, also have an increased risk to
associated PD has already made clear that the develop PD. This association, which was initially
boundary between disease genes and risk factors observed in Ashkenazi Jewish families in whom the
is more a matter of semantics than of biology. carrier frequency for GBA mutations is particularly
10

high, was later confirmed in a number of larger develop a disease. Over the years, literally hun­
patient series from different Jewish and non-Jewish dreds of studies have been published, but unfortu­
populations (Aharon-Peretz et al., 2004; De Marco nately, only very few of them have produced
et al., 2008; Mata et al., 2008), in a study comprising robust and reproducible results. The reasons for
autopsy-proven cases (Goker-Alpan et al., 2006) the failure of this approach are manifold:
and, most recently, in a large meta-analysis
(Sidransky et al., 2009). This association is now 1. Most studies were greatly underpowered in
firmly established. For example, a recent study relation to the small increases in the relative risk
showed that the prevalence of GBA mutations in that today are known to be conferred by common
British patients with sporadic PD is 3.7%, while genetic variants (usually the odds ratios are in
the frequency of these variants is less than 1% in the range of 1.2–2, with some exceptions, for
the general population. Mutations in the GBA gene example apolipoprotein E for AD).
therefore are the most common risk factor for 2. The choice of candidate genes was often based
development of PD in this population detected so on rather arbitrary rationales with very weak
far (Neumann et al., 2009). The relative risk experimental or epidemiologic evidence. As the
conferred by heterozygous carrier status for the gene-mapping studies in monogenic diseases
development of PD varies, for different mutations have shown, newly identified genes most often
and in different studies, from about 4 to 20, with the could not have been predicted based on the
large meta-analysis suggesting a relative risk of current knowledge of pathogenesis.
about 4 for the N370S mutation, the most common 3. In most studies only arbitrarily chosen
variant allele in Ashkenazi Jews, and about 6 for individual genetic variants were interrogated;
the most common mutation in non-Jews, L444P thus it was a priori unlikely that the causative
(Sidransky et al., 2009). variant or a variant in high linkage
This estimation of modest but significant rela­ disequilibrium, tagging the risk-conferring
tive risks to develop the disease explains why so variant, would be among those studied.
far no large ‘GBA-families’ have been identified.
As far as it is known today there is no way to Finally, it is not easy to match patient and con­
distinguish PD patients with GBA mutations from trol cohorts with respect to their genetic back­
those without, both clinically and pathologically. ground. Often, due to different recruiting
As a group, GBA-positive patients have a slightly strategies, these cohorts differ in their genetic
earlier age of onset (55 vs. 59 years) and a some­ composition (a problem called undetected popula­
what higher prevalence of dementia (Sidransky tion stratification, which today can be easily
et al., 2009). Neuropathologic examination of 17 resolved in GWAS, see below). Due to different
GBA mutation carriers showed typical PD allele frequencies in different populations, spur­
changes, with widespread and abundant SNCA ious associations can be detected.
pathology, and most also had neocortical Lewy Due to these shortcomings in study design, so far
body pathology (Neumann et al., 2009). not a single association study result concerning PD
truly withstood the test of time and replication, with
the notable exceptions of candidate gene associa­
Common risk variants for PD: candidate gene and tion studies of the SNCA and MAPT genes, two
whole genome association studies genes that initially were identified by mapping
disease genes in monogenic families (see below).
Association studies have been widely used in an Technical advances in sequencing technologies
attempt to identify common genetic variations including a vast increase in speed and accuracy of
that carry a mild to moderately increased risk to genotyping individual sequence changes (SNPs)
 11

together with a rapid increase in knowledge about certain degree of linkage disequilibrium between
the haplotype structure of the human genome those regions, it was not possible to definitively
paved the way for a more systematic study of separate the signals. On the other hand, as a num­
genetic variability and its relationship to common ber of different regulatory mechanisms are likely
diseases such as PD. This is nicely exemplified to determine SNCA expression, it would not be
in the way that variability in SNCA and in the surprising if multiple regions of the gene were
gene for the microtubule-associated protein tau found to be involved.
(MAPT) is now recognized as the major genetic Since then a number of additional association
risk factors in sporadic PD. studies were able to replicate these results in dif­
Since aSYN aggregates are widely accepted to ferent populations (Mizuta et al., 2006; Winkler
be the hallmark neuropathologic change in PD et al., 2007). Most of them found 30 -variants to be
and the role of SCNA point mutations and gene most significantly associated with the disease.
multiplications is clearly established in familial Further evidence for a role of genetic variants in
PD, it was only obvious to search for a possible the SNCA gene came from GWAS (see below).
association of genetic polymorphisms in the The history of the association of MAPT, the
SNCA gene with sporadic PD. SNCA variability, gene encoding the microtubule-associated protein
putatively influencing the level of SNCA gene tau (MAPTau), is less straightforward. The gene
expression, became a particularly plausible candi­ has been studied as a candidate gene for neuro­
date after the discovery that multiplications of the degenerative diseases for many years. Initially,
SNCA locus and thus a gene dosage effect of the MAPTau was identified as the main component
wt aSYN were the cause for dominant PD. of paired helical filaments, the intracellular
Early studies had produced somewhat conflict­ hallmark neuropathology of AD (Goedert et al.,
ing results, because again, in hindsight, those stu­ 1988). Tau aggregates also form the major pathol­
dies were underpowered given the relatively small ogy in several other diseases, the so-called tauo­
odds ratios that are known today. Nevertheless, pathies, which include a subset of patients with a
the majority of association studies, including a dementia syndrome called frontotemporal lobar
large meta-analysis of studies interrogating the degeneration (FTLD), and also atypical parkinso­
‘NACP-REP1’ polymorphism, a complex repeat nian syndromes, such as progressive supranuclear
element located about 10 kb upstream of the palsy (PSP) and corticobasal degeneration (CBD)
SNCA coding region (Maraganore et al., 2006), (Williams, 2006). Point mutations in MAPT,
supported the assumption of a role of SNCA var­ which either affect its amino acid sequence or
iants in sporadic PD. Müller et al. performed the the splicing of its isoforms, were then identified
first systematic study of SNPs in the SNCA gene, to cause a monogenic form of FTLD, called
analyzing more than 50 variants distributed over FTLD-17 or, today, FTLD-Tau (Hutton et al.,
the entire gene in more than 600 patients and a 1998). Given the prominent tau-pathology in
similar number of controls, thereby capturing PSP and CBD, it was not surprising when it was
nearly all of the genetic variability of the gene. reported that genetic variability in MAPT, initi­
They found that the SNCA gene consists of two ally a dinucleotide repeat, was found to be asso­
major haplotype blocks: one comprising the pro­ ciated with these disorders (Conrad et al., 1997).
motor and exon 1–4 and another one spanning A closer study of this genetic region on chromo­
exons 5 and 6 and the 30 -untranslated region of some 17 revealed that this variant was part of an
the gene. The strongest association signal was extended haplotype, spanning about 1.5 Mb. Two
detected with SNPs in 30 -haplotype block, major forms of this haplotype exist, called H1 and
although a second, somewhat weaker signal in H2, with H1, which occurs on about 80% of Cau­
the promotor region was also detected. Due to a casian chromosomes, conferring the higher risk.
12

Despite their large size, H1 and H2 haplotypes do important complex human diseases such as type 2
not recombine, because they are positioned in diabetes, atherosclerosis or AD, to name just a
inverse orientation on the chromosome (Zody few (Harold et al., 2009; Kronenberg, 2008;
et al., 2008). As a consequence, the association Latourelle et al., 2009). Although there are more
cannot be pinned down more accurately by than 3 million single-nucleotide variants compared
recombination mapping. However, Pittman and with the published reference sequence in any given
co-workers identified an H1 sub-haplotype individual genome, most of the genetic variability
(H1c), which showed a stronger association with can be captured by genotyping approximately
PSP than the H1 parent haplotype, and suggested 500 000 carefully chosen SNP markers. This is due
that the risk-conferring variant was located to a to the fact that the human genome consists of
22-kb intronic region of MAPT (Pittman et al., distinct segments, typically 10 000–50 000 base
2005). pairs in length, which are usually inherited en
Although the major pathology of PD is com­ bloc. This so-called haplotype bloc structure of
posed of aSYN and not MAPTau, the MAPT the human genome has been mapped and depos­
gene was studied as a candidate gene for typical ited in publicly available data banks (the interna­
PD, because the genomic region containing tional HapMap Project) and allows to predict, with
MAPT produced positive, although not genome- high probability, the genetic variants at an adjacent
wide significant, lod scores in a linkage study of locus within this bloc.
174 multiplex PD families (Scott et al., 2001). GWAS have been able to reliably identify
Somewhat surprisingly, a rather strong association genetic variants which convey a relative risk
signal was found in a subsequent study (Martin of about 1.2–1.5 if populations on the order of
et al., 2001), a finding later confirmed and refined 5 000–10 000 individuals are analysed. However,
in other studies (Kwok et al., 2004; Skipper et al., based solely on association studies it is usually
2004; Tobin et al., 2008; Zabetian et al., 2007). not possible to determine which of the many
Again, this association was recently confirmed in variants in a haplotype bloc are functionally rele­
genome-wide approaches. vant. Many of the risk-conferring variants are
likely not to alter the amino acid sequence of
one of the genes in the region; rather, biologically
Genome-wide association studies relevant variants will probably influence genetic
regulatory networks, for example gene expression
The advent of array technologies has taken the through alteration of transcription factor binding
analysis of human genetic variability and its influ­ sites, alternative splicing or mRNA stability,
ence on the development of complex diseases to a for example by changing sequences detected by
new level. This technology allows to simulta­ micro-RNAs. The elucidation of the biologic
neously genotype hundreds of thousands of mechanisms underlying an association is therefore
SNPs, the most frequent form of genetic variants, still a challenge for molecular biology.
in a large number of individuals at relatively low GWAS have overcome many of the problems of
costs. In parallel, analytic tools have been devel­ candidate gene association studies that have been
oped to deal with the vast amount of data which described above. For example, the ‘genetic back­
are generated by these techniques. ground’ is easily corrected for by taking into
In recent years GWAS have been extremely account the overall genetic ‘likeness’ of the SNP
fruitful in identifying common (a frequency of profile of the probands in the study. Individuals
more than 5% of a genetic variant in a population who differ with respect to their genetic back­
is considered to be common) risk variants of rela­ ground can be easily excluded from the analysis.
tively low individual effect strength in many A major challenge of GWAS is the fact that
 13

genotyping of 500 000 variants per individual not detect associations that survived stringent
results in a huge number of statistical tests to be Bonferroni correction for multiple testing. How­
performed and thus a considerable problem of ever, SNPs in the SNCA and MAPT gene were
multiple testing. Although not all 500 000 tests among their top hits (Pankratz et al., 2009), with
can be considered to be truly independent, the p-values of 5.5 × 10–5 and 2.0 × 10–5, and odds
mere number of tests leads to a large number of rations of 1.35 and 0.56, respectively. The close
nominally significant associations. Typically sev­ concordance with previous candidate gene studies
eral thousand results with a nominal p-value of of these loci (Martin et al., 2001; Mueller et al.,
10–3 to 10–5 are obtained, the vast majority simply 2005) supported these findings.
by chance. In most of the recent studies, however, Only recently in a still larger two-stage GWAS
this has not been much of a problem. Sufficiently studying a total of more than 5000 PD subjects and
powered studies provided results with p-values of 8000 controls, SNCA and MAPT again showed
genome-wide significance applying stringent Bon­ the strongest association signal and finally this
ferroni corrections (the genome-wide threshold is study was powered sufficiently to identify those
usually at a p-value in the order of 10–7), and regions unequivocally with genome-wide signifi­
results of genome-wide significance have usually cance (p-values < 10–7) (Simon-Sanchez et al.,
proven to be reproducible in subsequent studies. 2009). As in the candidate gene study reported
Therefore, the danger of false-positive results is earlier (Mueller et al., 2005), the most strongly
low if adequate corrections are applied. However, associated SNPs in SNCA were located in the 30 ­
it is unknown how many of the results with nom­ region of the gene, in a haplotype block containing
inally significant p-values below the genome-wide exons 5 and 6 as well as the 30 -untranslated region
threshold are true associations that are presently (30 -UTR) (Fig. 2). The relative risk conferred by
being missed. genetic variability in SNCA was only about 1.3.
In PD, the first GWAS was published in 2005. A However, the risk allele is common (about 40% of
total of 198 345 SNPs were genotyped in 443 sib­ the population), and therefore this variant
ling pairs discordant for PD. In a second stage, the explains about 9% of the disease risk on the popu­
top 1793 PD-associated SNPs (p < 0.01) and 300 lation level. Based on this converging evidence it
genomic control SNPs were typed in 332 matched is now firmly established that genetic variability in
case-unrelated control pairs (Maraganore et al., SNCA influences the risk to develop typical spora­
2005). Given today’s knowledge of the effect dic PD. Very similar results with respect to SNCA
strengths of common risk variants in PD, this have been obtained in a simultaneous study in
study was significantly underpowered and none Japan (Satake et al., 2009).
of the detected associations reached genome- However, it is still unclear how changes in the
wide significance after Bonferroni correction. non-protein coding sequence of SNCA influence
Nevertheless, it demonstrated the feasibility of PD risk. Possible mechanisms are differential
this approach in a complex disorder such as PD binding of enhancers or suppressors of transcrip­
and other studies soon followed. The next GWAS tion (Fuchs et al., 2008), alterations in splicing, or,
again used a relatively small sample, 267 PD particularly with respect to variants in the 30 -UTR,
patients and 270 neurologically normal controls differential binding of micro-RNAs. So far no
(Fung et al., 2006). More than 408 000 unique direct in vivo evidence has been produced that
SNPs were genotyped, without detecting an clearly supports any one of those possibilities.
association signal. Interestingly, the MAPT locus has also been
Increasing the study cohort to almost 900 confirmed with genome-wide significance as a
patients and controls, Pankratz and co-workers, risk factor in sporadic PD in the European, but
using Affimetrix 550K mapping chips, still did not in the Asian, population (Satake et al., 2009;
14

Fig. 2. The genomic and haplotype structure of the SNCA locus (encoding SNCA in light grey) and p-values of SNPs investigated as
part of a genome-wide association study (Simon-Sanchez et al., 2009). Each dot in the upper panel represents a single-nucleotide
variant (SNP) tested for association in a region on chromosome 4, from base pair 90 500 000–91 500 000 (x-axis) with its respective p-
value (y-axis). In the lower panel, the haplotype structure of the region is symbolized. Figure kindly provided by Dr. A. Singleton.

Simon-Sanchez et al., 2009). In Asians, the tau- discussed for a number of protein aggregation
locus is not polymorphic. Whether SNCA and diseases such as the polyglutamine diseases and
MAPT act independently as risk factors as sug­ the prion disorders (Derkatch et al., 2004).
gested by two GWAS (Pankratz et al., 2009; Still larger study populations will probably
Simon-Sanchez et al., 2009) or synergistically, as reveal even more common risk alleles in future
proposed in a candidate gene study by Goris et al. GWAS.
(2007), is still unclear. While evidence has been
provided that the biologic effect of the MAPT
haplotype is mediated by increased transcriptional Future strategies
activity of the risk haplotype (Simon-Sanchez
et al., 2009), this does not seem to be the case Rapid further advances in sequencing technolo­
for SNCA. An interaction on the protein level is gies have already set the stage for next generation
possible, as co-staining of Lewy bodies with anti­ of analysis, which will take the form of whole
bodies to aSYN and MAPTau has been demon­ exome or whole genome sequencing. While these
strated (Duda et al., 2002), and the concept of technologies today are still rather expensive and
‘cross-seeding’ of different proteins has been the bioinformatics to deal with the enormous
 15

amount of data generated are still a formidable the entire exome of two affected members of a
challenge, the pace of technological progress is family (or better two or three families) with a rare
certain to increase in the next years and therefore monogenic disease, who are separated by at least
a significant contribution to the understanding of four to six meiotic events (e.g. first- or second-
complex diseases can be expected. A combination degree cousins) is sequenced, the number of
of technologies, such as whole genome haplotyp­ potentially pathogenic shared novel variants is
ing or sequencing combined with transcriptome relatively limited, particularly if, as in a recessive
analysis or RNA sequencing in tissues and cell disease, loss-of-function mutations are suspected.
types, will generate huge libraries mapping genetic It is conceivable that a significant proportion of
expression networks with so far almost unimagin­ cases even in a late-onset neurodegenerative dis­
able complexity. order such as PD might be due to rare recessive
monogenic causes which could be identified using
this approach.
Whole exome sequencing Another application may be the identification of
relatively rare risk alleles of moderate effect,
Several technologies have been developed to similar to the GBA mutations described above
sequence all expressed exons of the human (Sidransky et al., 2009), which usually escape
nuclear genome. Basically, these technologies detection by whole genome SNP-genotyping
comprise multiple steps, including target enrich­ approaches, because they are, individually, too
ment, actual sequencing, and data analysis. In rare, although in their entirety, they may still
a first step, hybridization techniques, using solid- explain a significant proportion of cases. Although
phase or emulsion-based short sequences repre­ the distinction is of course somewhat arbitrary, by
senting all ~180 000 exons of the human genome, definition, risk alleles (as opposed to ‘disease-caus­
are used to enrich for the desired target ing mutations’) also occur in the general popula­
sequences. Then, the enriched fragments are tion. It will therefore be necessary to genotype a
sequenced in a ‘massive-parallel’ fashion, that is very large number of patients and controls to be
each sequence is usually read multiple times sure of a significant disease association. The quan­
(usually 10- to 30-fold coverage is required). titative contribution of rare variants to a complex
Finally, the sequence data are assembled and disease such as PD is, however, still completely
aligned to produce a consensus sequence. unknown.
So far, whole exome sequencing has revealed a
large number (several thousands) of novel var­
iants in each sequenced individual. It will be a Whole genome sequencing
formidable challenge to devise strategies to eval­
uate the biological relevance of these variations. When the first finished grade human reference
It is expected that whole exome sequencing will genome [NCBI build 36 (International Human
be particularly useful to identify rare variants of Genome Sequencing, 2004)] was published in
moderate to high effect strength. 2004, it was almost inconceivable that only 3
One potential application of whole exome years later the personal genome of one of the
sequencing that has been already successfully pioneers of genomic science J. Craig Venter
applied is the identification of rare autosomal- would be published (Levy et al., 2007). Only a
dominant or autosomal-recessive disease genes year later, the personal genomic sequence of
following an approach that can be considered to James D. Watson was made public (Wheeler
be a ‘shortcut’ to the classic positional cloning et al., 2008). Since then a rapidly growing number
approach described above (Ng et al., 2010). If of individual genomes has been sequenced and
16

published, most recently an example of a medical Conclusion

application, the identification of a mutation in a
patient with a rare form of Charcot–Marie–Tooth The unravelling of the genetic underpinnings of
neuropathy (Lupski et al., 2010). complex diseases, such as PD, has just begun.
In each of the individual genomes sequenced, Known Mendelian forms of PD and known
more of 3 million single-nucleotide variants deviat­ genetic risk factors presently explain at most
ing from the published reference sequence and sev­ about 20% of all cases, on the general population
eral hundreds of thousands of larger structural level. It is often assumed that the common form of
variations usually copy number repeats were sporadic PD results from an ‘interaction of genetic
detected. In a recent study of the entire genome of and environmental factors’. While this is probably
five men from sub-Saharan Africa, 1.3 million novel true if ‘environmental factors’ are defined broadly
DNA differences genome-wide including more and would include, for example, the aging process,
than 13 000 coding changes were identified. Again, which can be seen as the major ‘environmental’
it will be a challenge for years to come to study the risk factor for PD, it is likely that the potential of
functional relevance of all this variability. genetics in explaining the multiple causes of PD is
Whole genome sequencing will certainly far from exhausted.
replace even whole exome sequencing within a
foreseeable future. Today basically the same tech­
nology platforms are used as in whole exome Abbreviations
sequencing, except for the initial target enrich­
ment step. Because of the much larger number APP amyloid precursor protein
of sequences that have to be read, a whole AD Alzheimer’s disease
genome sequence is still estimated to cost more aSYN alpha-Synuclein (protein)
than 100 000 dollars (Metzker, 2009). Therefore GWAS genome-wide association
only a few examples have been published. Never­ studies
theless the ‘1000-dollar genome’ will probably be MAPT microtubule-associated protein
available within the next 3–5 years. tau (gene)
MAPTau microtubule-associated protein
tau (protein)
Low-coverage genome sequencing MSA multiple system atrophy
PD Parkinson’s disease
An international collaborative project called the SNCA alpha-synuclein (gene)
‘1000 genomes project’ aims to establish a catalo­ SNP single-nucleotide
gue of human genetic variants that have a preva­ polymorphism
lence of at least 1% in the populations studied.
This goal is achieved by sequencing a large num­
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expression implicate the MAPT region for Parkinson dis­ of the MAPT 17q21.31 inversion region. Nature Genetics, 40
ease: The GenePD Study. Neurology, 71(1), 28–34. (9), 1076–1083.
A. Bjorklund and M. A. Cenci (Eds.)
Progress in Brain Research, Vol. 183
ISSN: 0079-6123
Copyright
2010 Elsevier B.V. All rights reserved.

CHAPTER 2

The impact of genetic research on our understanding

of Parkinson’s disease

Ian Martin†,‡, Valina L. Dawson†,‡,§,k and Ted M. Dawson†,‡,k,

†
NeuroRegeneration and Stem Cell Programs, Institute for Cell Engineering, Johns Hopkins University School of
Medicine, Baltimore, MD, USA
‡
Department of Neurology, Johns Hopkins University School of Medicine, Baltimore, MD, USA
§
Department of Physiology, Johns Hopkins University School of Medicine, Baltimore, MD, USA
k
Solomon H. Snyder Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, MD, USA

Abstract: Until recently, genetics was thought to play a minor role in the development of Parkinson’s
disease (PD). Over the last decade, a number of genes that definitively cause PD have been identified,
which has led to the generation of disease models based on pathogenic gene variants that recapitulate many
features of the disease. These genetic studies have provided novel insight into potential mechanisms
underlying the aetiology of PD. This chapter will provide a profile of the genes conclusively linked to PD
and will outline the mechanisms of PD pathogenesis implicated by genetic studies. Mitochondrial
dysfunction, oxidative stress and impaired ubiquitin–proteasome system function are disease mechanisms
that are particularly well supported by genetic studies and are therefore the focus of this chapter.

Keywords: alpha-synuclein; LRRK2; PINK1; Parkin; DJ-1; mitochondrial dysfunction; oxidative stress;
ubiquitin-proteasome system

Introduction principally by symptoms of resting tremor, brady­

kinesia, rigidity and postural instability, although
Parkinson’s disease (PD) is the most common neu­ additional dysfunction of non-motor systems is fre­
rodegenerative movement disorder, affecting quently present (Savitt et al., 2006). The primary
approximately 1% of the population at 65 years symptoms, which together constitute parkinson­
of age, increasing to 5% at 85 years (Van Den ism, arise from a profound degeneration and loss
Eeden et al., 2003). Clinically, PD is characterized of dopaminergic neurons in the substantia nigra
pars compacta, which leads to a marked depletion

Corresponding author.
of the neurotransmitter dopamine in the striatum,
Tel.: (410) 614 3359; Fax: (410) 614 9568; a key region of the basal ganglia that regulates
E-mail: [email protected] movement (Dauer and Przedborski, 2003).

DOI: 10.1016/S0079-6123(10)83002-X 21
22

Identifying a role for genetics in PD considered a non-genetic disease caused by envir­

onmental factors and aging (Farrer, 2006). This
The main discoveries that have shaped our under- notion was propagated early-on by the emergence
standing of PD are outlined in Fig. 1. Until the end of parkinsonism in survivors of the encephalitis
of the twentieth century, PD was predominantly epidemic that occurred from 1917 to 1928

Timeline of key discoveries on PD pathogenesis

1817 James Parkinson publishes the first formal description of PD entitled “An Essay on the Shaking
Palsy,” establishing Parkinsonism as a recognized medical condition.

1912 Friedrich Lewy first describes concentric inclusion bodies in nigral cells of patients presenting
with “paralysis agitans” (Obsolete name for Parkinsonism). These inclusions were later named
Lewy bodies and Lewy neurites.

1919 Konstantin Tretiakoff observes a reduction in pigmented cells of the substantia nigra in brains of
PD patients.

1920s The post-encephalitic parkinsonism (PEP) epidemic following a worldwide outbreak of

Influenza in 1918 suggests possible link between PD development and viral exposure.

1950s Arvid Carrlson reports a high concentration of dopamine in the basal ganglia which, in rabbits,
becomes depleted by treatment with reserpine. Reserpine-treated rabbits are incapable of
voluntary movement, similar to patients with severe PD. These symptoms could be alleviated by
administering L-DOPA indicating a role for dopamine deficiency in causing PD and leading to
clinical use of L-DOPA.

1983 Exposure to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) associated with acute-onset

of PD. Chemical similarity of MPTP to the herbicide paraquat leads to studies linking pesticide
exposure to PD.

1997 Identification of α-synuclein mutations as an underlying cause of familial PD. Subsequent studies
suggest that aggregates of misfolded α-synuclein may be toxic to dopamine neurons and impair
α-synuclein’s role in vesicular transport.

1997 Mutations in Parkin linked to AR-JP. Evidence suggests impaired ubiquitin–proteasome function
and mitochondrial dysfunction associated with loss of Parkin E3 ligase function instrumental in
PD pathogenesis.

2003 DJ-1 mutations linked to early-onset PD. DJ-1 is important in protecting against oxidative stress
and cell death illustrating central importance of these factors to PD pathogenesis.

2004 Loss-of-function Pink1 mutations linked to PD and subsequently shown in model systems to
result in mitochondrial dysfunction and cell death.

2004 Pathogenic LRRK2 mutations identified, many later shown to possess increased kinase activity
to generic substrates. Translational inhibitor 4E-BP may be in vivo substrate, suggesting a role
for general protein translation in PD pathogenesis.

Fig. 1. Timeline of key discoveries in PD pathogenesis. Parkinson’s disease was first formally described in 1817 by James Parkinson.
From then until the late twentieth century, advances were made in describing the pathological features of PD and in understanding
possible causes of the disease, such as exposure to pesticides and MPTP. The recent discovery of multiple genetic causes of PD has
generated insight into novel mechanisms of PD aetiology. Studies using genetic models of PD will, no doubt, continue to advance our
understanding of disease pathogenesis.
 23

(Poskanzer and Schwab, 1963) and later fuelled by (LRRK2) contribute to the lifetime risk of sporadic
discoveries from epidemiological studies that par­ PD in the population (Cookson et al., 2005; Satake
kinsonism is associated with exposure to certain et al., 2009). Importantly, recent advances in the
pesticides and to 1-methyl-4-phenyl-1,2,3,6-tetrahy­ genetics of PD have led to cell and animal models
dropyridine (MPTP) via narcotic use (Elbaz and of disease that promote our understanding of mole­
Moisan, 2008; Langston et al., 1983). A major con­ cular pathways underlying the pathogenesis of PD
tribution of heredity to development of the disease (Dawson et al., 2002; Moore and Dawson, 2008).
was thought unlikely due to epidemiological studies Although monogenic and sporadic forms of PD are
which indicated no effect of heritability on lifetime not clinically or pathologically identical, they exhibit
risk of developing PD (Cookson et al., 2005). A role common core features such as parkinsonism and
for genetics in PD was also not supported by cross- loss of nigral dopamine neurons suggesting that
sectional twin studies that suggested low concor­ they share common mechanisms of disease and
dance rates in monozygotic and dizygotic twins that genetics should, therefore, provide clues to
(Marttila et al., 1988; Ward et al., 1983). This was the aetiology of sporadic PD (Cookson et al.,
despite the fact that since as early as the 1880s, 2005). The following section describes the genes
clinicians had been noting familial aggregation of that have been definitively linked to PD, the normal
parkinsonism (Gowers, 1900; Leroux, 1880) and function of their gene products and how pathogenic
that numerous families inherited PD in a Mendelian mutations are thought to impact cell systems. This
fashion (Bell and Clark, 1926) suggesting a genetic outline will form the basis for a subsequent discus­
contribution to disease. For example, studies on sion of how genetics has impacted our understand­
multiple populations in the mid-twentieth century ing of PD pathogenic mechanisms.
indicated that the emergence of many cases of
parkinsonism was consistent with an autosomal
dominant mode of inheritance (Allen, 1937; Dominantly inherited mutations
Mjones, 1949). Finally, over the last decade, linkage
mapping studies have resulted in the identification a-Synuclein
of distinct genetic loci that definitively cause familial
PD (Farrer, 2006) (Table 1). These discoveries have Studies on a large family of Italian descent (the
resulted in a paradigm shift in perceptions towards Contursi kindred), with apparent autosomal
the contribution of genetics to PD that extend dominant PD, led to the discovery of a PD sus­
beyond early-onset familial disease, since variants ceptibility locus on the long arm of chromosome 4
in a-synuclein and leucine-rich repeat kinase 2 (Polymeropolous et al., 1996) that was later

Table 1. Genes underlying familial PD

Inheritance Typical age

Locus Gene pattern of onset Phenotype characteristics

PARK1 and SNCA Dominant 24–65 Parkinsonism (progression related to gene dose) with common
PARK4 dementia and autonomic dysfunction
PARK2 Parkin Recessive 16–72 Slow-progressing Parkinsonism
PARK6 PINK1 Recessive 20–40 Slow-progressing Parkinsonism
PARK7 DJ-1 Recessive 20–40 Slow-progressing parkinsonism sometimes with behavioural
disturbances
PARK8 LRRK2 Dominant 32–79 Classic Parkinson’s disease

Gene variants that segregate with PD are listed. The inheritance pattern, typical age of disease onset and characteristic phenotypes observed are described.
24

identified as the SNCA gene that encodes a-synu­ conformation when associated with membranes
clein (Polymeropolous et al., 1997). The 209G>A (Ferreon et al., 2009).
(Ala53Thr) mutation found in this family The precise function of a-synuclein is unclear,
was followed by the discovery of two further PD- although its association with synaptic vesicles sug­
associated SNCA missense mutations: 88G>C gests a possible role in neurotransmission. Indeed,
(Ala30Pro) (Kruger et al., 1998) and 188G>A studies in yeast and mammalian systems suggest
(Glu46Lys) (Zarranz et al., 2004). Patients with that a-synuclein may regulate synaptic vesicle
SNCA point mutations typically develop promi­ trafficking via binding to lipids (Jenco et al.,
nent dementia and an earlier onset of parkinson­ 1998; Nemani et al., 2010; Outeiro and Lindquist,
ism than in sporadic PD (Farrer, 2006). In 2003). A recent report describes a possible role
addition to point mutations, duplication or tripli­ for a-synuclein in the assembly of soluble NSF
cation of SNCA has been found in kindreds with attachment protein receptors (SNARE) complex
classic PD or sometimes parkinsonism with auto­ between vesicle and pre-synaptic membranes,
nomic dysfunction and dementia (Chartier-Harlin which is crucial for priming and recycling of synap­
et al., 2004; Singleton et al., 2003). The strong tic vesicles (Bonini and Giasson, 2005; Chandra
association between SNCA mutations or multi­ et al., 2005). Deletion of the co-chaperone
plications and PD suggests a central role for cysteine-string protein-a in mice results in neuro­
a-synuclein in PD pathogenesis (Moore et al., degeneration with underlying impairment in
2005). Furthermore, comparison of patients with SNARE complex assembly (Chandra et al.,
duplications and triplications of SNCA reveals 2005). The authors further report that transgenic
that age of onset is younger and disease progres­ expression of a-synuclein attenuates inhibition of
sion faster with gene triplication (which results in SNARE complex formation and prevents neuro­
an approximate doubling of plasma a-synuclein degeneration. Despite this, genetic knock-out
levels) (Farrer et al., 2004; Miller et al., 2004; Ross studies in mice have indicated that the absence
et al., 2008) suggesting that a-synuclein expres­ of a-synuclein has no significant effect on the
sion level and disease severity are related. This pool size of recycling synaptic vesicles, synaptic
correlation between a-synuclein expression levels plasticity or dopamine uptake and release from
and PD susceptibility is further supported by stu­ nerve terminals (Chandra et al., 2004) suggesting
dies in patients with sporadic PD in which allelic that a-synuclein may not be required for regulat­
variability in regions of the SNCA promoter ing synaptic vesicle release or uptake under nor­
(especially the Rep1 region) is associated with mal conditions and may, instead, be protective
risk of developing PD (Farrer et al., 2001; Pals following exposure to certain cell stressors.
et al., 2004; Tan et al., 2000), although this remains Mutations and multiplications in the SNCA
somewhat controversial (DeMarco et al., 2008; gene may cause PD through a gain-of-toxic-function
Spadafora et al., 2003; Tan et al., 2003). mechanism as suggested by their dominant inheri­
a-Synuclein exists mainly as a 140 amino acid tance pattern. When mutated or at elevated concen­
protein whose precise function is unknown trations, a-synuclein has a propensity to develop a
(Moore et al., 2005). a-Synuclein is expressed b-sheet-rich structure that readily polymerizes into
in neurons throughout the mammalian nervous oligomers (Sharon et al., 2003) and higher order
system where it resides predominantly at aggregates such as fibrils (Conway et al., 1998) in
pre-synaptic terminals associated with vesicles cells, animal models and human brain (Lee et al.,
and membranes (Bonini and Giasson, 2005; 2002; Miller et al., 2004; Outeiro et al., 2008; Sharon
Fortin et al., 2004; Kahle et al., 2000). Interest­ et al., 2003). Insoluble a-synuclein fibrils are a
ingly, a-synuclein is natively unfolded in solu­ major component of hallmark PD inclusions called
tion, although it adopts an alpha-helical-rich Lewy bodies and Lewy neurites, present in
 25

perikarya and neurites, respectively. In PD, Lewy models involving viral vectors to deliver a-synuclein
bodies and neurites can be found in both dopami­ to the substantia nigra (Kirik et al., 2003; Yasuda
nergic and non-dopaminergic neurons of the brain­ et al., 2007). Aggregation may be promoted by
stem and in the cortex (Farrer, 2006). Importantly, numerous factors including mitochondrial complex
Lewy bodies are found in a number of neurodegen­ I inhibitors paraquat and rotenone (Manning-Bog
erative diseases involving SNCA mutations includ­ et al., 2002; Sherer et al., 2002, 2003). Evidence
ing PD, parkinsonism with dementia and dementia linking exposure to these compounds with the
with Lewy bodies (Martı́ et al., 2003). This estab­ occurrence of sporadic PD suggests a possible role
lishes a link between these diseases with distinct for a-synuclein aggregates in sporadic disease.
clinical features but shared pathology. Controversy Additionally, oxidative and nitrative damage,
exists as to whether Lewy bodies and neurites are a which accumulate in the brains of many species
cause or consequence of PD, and some evidence including humans during aging, may promote aggre­
suggests that they might actually have a protective gation of a-synuclein to toxic species (Cole et al.,
role by acting to sequester toxic a-synuclein oligo­ 2005; Leong et al., 2009; Ostrerova-Golts et al.,
mers (Olanow et al., 2004; Tanaka et al., 2004). 2000; Qin et al., 2007). Tyrosine nitration of a-synu­
Fuelling this controversy are the findings that cer­ clein is found in the PD brain and has been shown to
tain SNCA mutations (A53T and A30P) promote accelerate a-synuclein aggregation in vitro (Giasson
oligomerization but not fibrillization of a-synuclein, et al., 2000) by a mechanism that may include
and moreover that Lewy bodies are frequently reduced efficiency of a-synuclein degradation by
absent from the brains of PD patients with genetic calpain I and 20S proteasome (Hodara et al.,
mutations (Ahlskog, 2009; Conway et al., 2000; 2004). Lastly, the interaction of a-synuclein with
Gaig et al., 2007). Accordingly, emerging evidence other amyloidogenic proteins such as tau (Giasson
suggests that pre-fibrillar oligomers and protofibrils et al., 2003) or amyloid-b (Masliah et al., 2001) may
are the toxic species responsible for PD pathology synergistically drive fibrillization of these proteins.
(Conway et al., 2000; Danzer et al., 2007; Goldberg For example, amyloid-b peptides were shown to
and Lansbury, 2000; Kayed et al., 2003; Masliah promote intraneuronal a-synuclein aggregation
et al., 2000). a-Synuclein oligomers, akin to in cell cultures and transgenic mice expressing
other amyloidogenic oligomers cause elevated both a-synuclein and amyloid-b neuronally devel­
Ca2þ influx into cells in vitro, possibly by altering oped more a-synuclein-immunoreactive inclusions
membrane stability or permeability by forming than singly a-synuclein transgenic mice. These dou­
membrane pores (Danzer et al., 2007; Demuro ble transgenic mice also exhibited motor deficits
et al., 2005). Elevated intracellular Ca2þ levels and impaired learning and memory before mice
may promote cellular toxicity through increased expressing transgenic a-synuclein only (Masliah
generation of reactive oxygen species and resultant et al., 2001). A clear relevance of this stimulatory
oxidative damage. effect on a-synuclein aggregation applies to patients
Whether the pathogenic a-synuclein species is with clinical and pathological features of both PD
oligomeric, fibrillar or both, it is reasonably clear and Alzheimer’s disease (e.g. those with the Lewy
that aggregates of this protein are toxic in primary body variant of Alzheimer’s disease).
neuronal cultures (Petrucelli et al., 2002; Tanaka While a-synuclein is seen to undergo aggregation
et al., 2001; Xu et al., 2002; Zhou and Freed, and post-translational modifications, and these
2005), invertebrate animal models (Feany and events may lead to its toxicity to neurons, the effects
Bender, 2000; Kuwahara et al., 2006; Lakso of a-synuclein responsible for causing cell death are
et al., 2003; Park and Lee, 2006; Periquet et al., not yet clear. Nevertheless, several theories have
2007) and in rodent (Lo Bianco et al., 2002; been put forth to explain a-synuclein toxicity and
St Martin et al., 2007) and non-human primate are briefly outlined here. As already mentioned,
26

a-synuclein oligomers can form pore-like structures the vesicular monoamine transporter 2 which
and annular rings of a-synuclein were previously may lead to increased cytosolic levels of dopamine
observed in brains of patients with multiple system (lotharius et al., 2002). Pathogenic species of
atrophy, a synucleinopathy. Neural cells expressing a-synuclein are not good substrates for proteaso­
mutant forms of a-synuclein (A53T and A30P) mal degradation and aggregates can directly bind
exhibited non-selective cation pores that increased to 20/26S proteasomal subunits inhibiting proteo­
both basal and depolarization-induced intracellular lytic activity (Snyder et al., 2003). a-Synuclein
Ca2þ levels (Furukawa et al., 2006). Furthermore, may also affect protein degradation through inhi­
cells expressing mutant a-synuclein were more biting lysosomal function (Stefanis et al., 2001)
sensitive to iron-generated reactive oxygen species, and chaperone-mediated autophagy (Cuervo
unless treated with Ca2þ-chelating agents, suggest­ et al., 2004). Recent data suggest that chaperone-
ing that elevated intracellular Ca2þ levels were mediated autophagy is important in the regulation
responsible for the increased vulnerability of these of the neuronal survival factor MEF2D (myocyte
cells to toxic insults. Given that a portion of enhancer factor 2D) and that a-synuclein expres­
a-synuclein has been observed to localize to sion can disrupt this leading to cell death (Yang
mitochondrial membranes of dopamine neurons et al., 2009).
(Li et al., 2007; Nakamura et al., 2008), and the Hence, a-synuclein aggregates appear to exert
central involvement of mitochondria in PD patho­ toxic effects on numerous cell functions. The rela­
genesis, an obvious question is whether such pores tive contributions of these effects to neuronal cell
form in mitochondrial membranes to promote death are not well understood and may vary
mitochondrial dysfunction. Over-expression of depending on cell type and other circumstances
a-synuclein in cells was reported to induce abnor­ such as the type and amount of a-synuclein patho­
mal morphology and dysfunction of mitochondria genic species present. Teasing apart primary
together with increased oxidative stress (Hsu et al., effects of a-synuclein toxicity from secondary
2000). Since there was little change in cell viability, will be important for identifying therapeutic
this suggests that mitochondrial deficits were not targets for preventing cell death (Cookson, 2009).
secondary to cell death, but a direct consequence
of a-synuclein over-expression.
Another potential mechanism of a-synuclein Leucine-rich repeat kinase 2
toxicity supported by the interaction of a-synu­
clein with synaptic vesicles is that increased or Since the first identification of pathogenic LRRK2
mutant a-synuclein expression interferes with mutations in 2004 (Paisan-Ruiz et al., 2004;
synaptic neurotransmission. Wild-type or A30P Zimprich et al., 2004a, 2004b), mutations in this
a-synuclein was shown to impair catecholamine gene are now the most common known cause of
release from chromaffin and PC12 cells associated familial PD worldwide (Webber and West, 2009).
with an accumulation of ‘docked’ vesicles at the Autosomal dominantly inherited LRRK2
pre-synaptic membrane (Larsen et al., 2006). mutations exist in families from diverse ethnic
Furthermore, a-synuclein over-expression to backgrounds and mostly give rise to PD pheno­
levels predicted to result from gene multiplication types that are highly similar to those of typical
impaired neurotransmitter release in mice through late-onset PD. This suggests that understanding
a mechanism involving reduced size of the recy­ the effects of mutant LRRK2 on disease patho­
cling vesicle pool (Nemani et al., 2010). Uptake of genesis has the potential to generate substantial
dopamine into synaptic vesicles may also be insight into sporadic PD mechanisms. Interest­
perturbed by a-synuclein. Mutant a-synuclein ingly, despite consistency of clinical phenotypes,
over-expression was reported to downregulate LRRK2 mutant carriers can exhibit diverse
 27

neuropathology occasionally lacking Lewy bodies, et al., 1999). Phosphorylation of 4E-BP disrupts
even between individuals with the same mutation its interaction with eIF4E and stimuli that affect
(Gaig et al., 2007; Zimprich et al., 2004b). 4E-BP phosphorylation such as oxidative stress
LRRK2 encodes a large, 280 KDa protein with and activation of the mammalian target of rapa­
initial studies indicating potential roles in cytoske­ mycin pathway can impact protein translation
letal dynamics, protein translation control, mito­ indicating that 4E-BP phosphorylation is asso­
gen-activated protein kinase (MAPK) pathways ciated with increased protein translation. Over-
and apoptotic pathways. LRRK2 contains numer­ expression of human LRRK2 in mammalian cells
ous domains, namely ankyrin-like repeats, or a Drosophila orthologue (dLRRK) in flies was
leucine-rich repeats, COR (C-terminal of ROC) shown to result in increased 4E-BP phosphoryla­
WD-40 domain and a catalytic GTPase/kinase tion at two threonine sites (Thr37/Thr46) leading
region. Interestingly, LRRK2 kinase activity to secondary phosphorylation by additional
appears to require a functional GTPase domain kinases at other sites including Ser65/Thr70
(West et al., 2007) and possibly LRRK2 dimer (Imai et al., 2008). The authors also reported
formation (Sen et al., 2009). LRRK2 undergoes that RNAi-mediated silencing of LRRK2 in cells
autophosphorylation and phosphorylates a num­ or loss-of-function dLRRK mutation in flies led to
ber of protein substrates in vitro. Analysis of the a decrease in 4E-BP phosphorylation at these sites
human kinome indicates that the kinase domain of supporting the possibility that 4E-BP is a kinase
LRRK2 and its homologue LRRK1 are most simi­ substrate of LRRK2. Finally, the authors showed
lar in sequence to the receptor-interacting protein that dopamine neuron pathology associated
kinase and death-domain containing interleukin with dLRRK mutations was suppressed via over-
receptor-associated kinase families and to a lesser expression of 4E-BP suggesting that increasing
extent, MAPK kinase (MAPKK) kinases. Several 4E-BP activity might attenuate PD pathology.
of the most clear and common pathogenic muta­ Another recent study in Drosophila has strength­
tions (G2019S, I2020T and R1441C/G) are found ened a potential link between LRRK2, 4E-BP
in the central catalytic region and may result in activity and PD pathology (Tain et al., 2009).
increased kinase activity in vitro (West et al., Increased 4E-BP activity resulting from loss of
2007), although it is important to note that not dLRRK or administration of rapamycin was suffi­
all PD-associated LRRK2 mutations increase cient to suppress pathology in PTEN-induced
kinase activity and only the G2019S mutation has putative kinase 1 (PINK1) and Parkin mutants
consistently been found to increase kinase activity raising the possibility that an involvement of
to date (Greggio and Cookson, 2009). general protein translation in PD pathology
Much effort is currently focused on attempting might be relevant to other PD-associated genes.
to identify kinase substrates and pathogenic Another candidate substrate for LRRK2 kinase
mechanisms linked to altered kinase activity. activity is moesin (Jaleel et al., 2007). Moesin is a
A role for LRRK2 in protein translation control member of the ezrin/radixin/meosin (ERM) pro­
has been put forth by the identification of the tein family whose primary role is to anchor the
translational inhibitor eukaryotic initiation factor cytoskeleton to the plasma membrane. Jaleel et al.
4E-binding protein (4E-BP) as a LRRK2 sub­ found that moesin could be phosphorylated by
strate both in vitro and in a Drosophila model LRRK2 at Thr558 and to a lesser extent at
(Imai et al., 2008; Tain et al., 2009). 4E-BP in its Thr526. One caveat is moesin phosphorylation
non-phosphorylated state interacts with eukaryo­ could only be observed after denaturating moesin
tic initiation factor 4E (eIF4E), preventing activity via heating and even then, phosphate was
of eIF4E within the protein translation machinery minimally incorporated suggesting that moesin
thereby inhibiting protein translation (Khalegpour may be a weak kinase substrate of LRRK2.
28

Despite this, a recent study on developing neurons LRRK2 (e.g. G2019S), it will be important to
supports the possibility that moesin is a LRRK2 assess whether the phosphorylation of any
substrate in vivo (Parisiadou et al., 2009). putative substrate is enhanced in the presence of
Phosphorylated ERM protein accumulated more mutant LRRK2 relative to wild-type LRRK2.
in developing neurons from G2019S LRRK2 Numerous studies show that expression of
transgenic mice and less in LRRK2 knock-out mutant LRRK2 causes cell death. Over-expression
mice than controls (Parisiadou et al., 2009). of mutant LRRK2 in primary neuronal cultures
Furthermore, the extent of ERM phosphorylation leads to rapid cell death possibly by apoptosis,
correlated negatively with neurite outgrowth while comparable expression of wild-type
suggesting that LRRK2 mutations may perturb LRRK2 has only subtle effects on cell viability
normal neuronal development. (Greggio et al., 2006; Ho et al., 2009; Iaccarino
Based on sequence similarity between LRRK2 et al., 2007; MacLeod et al., 2006; Smith et al.,
and MAPKK kinases, which are involved in 2005, 2006; West et al., 2007). A proposed link
the MAPK signalling pathway and important to between increased LRRK2 kinase activity and
cellular stress responses, Gloeckner et al. recently neuronal death in PD requires further investiga­
used in vitro studies to probe MAPK kinases as tion in vivo, although a dominant mode of
potential substrates for LRRK2 kinase activity inheritance (consistent with a toxic-gain-of-func­
(Gloeckner et al., 2009). These studies revealed tion) and preliminary studies in cell culture are
phosphorylation of MKK3, MKK4, MKK6 and supportive of this. Multiple investigators have
MKK7 by LRRK2 and moreover that PD-linked discovered that pathogenic LRRK2 mutants
G2019S or I2020T mutations in LRRK2 exhibit engineered to ablate kinase activity are substan­
increased phosphotransferase activity as well as tially less toxic in cultured cells than kinase-
enhanced autophosphorylation. Whether these active counterparts (Gregio et al., 2006; Smith
changes are due to LRRK2 kinase activity is not et al., 2006; West et al., 2007) indicating that
clear since the authors did not use kinase-dead ver­ kinase activity contributes to cellular toxicity.
sions of LRRK2 as a control. Since phosphorylation One caveat is that not all pathogenic LRRK2
of MAPK kinases within their activation loop is mutations appear to result in elevated kinase
linked to increased downstream phosphorylation activity based on measurements of autopho­
of c-Jun N-terminal kinase (JNK) and c-Jun, it sphorylation or generic kinase substrate phos­
might be expected that increased LRRK2 kinase phorylation. A possibility to consider here is
activity would lead to higher phosphorylated JNK that pathogenic LRRK2 mutations might alter
and/or c-Jun levels. However, this is inconsistent kinase activity towards specific substrates that
with prior studies in cell system (West et al., 2007) are key to LRRK2-mediated toxicity but not a
in which LRRK2 over-expression does not appear universal increase in kinase activity to all sub­
to increase levels of phosphorylated JNK or c-Jun. strates. Perhaps identification of true in vivo
Hence, the simplest explanation here is that there LRRK2 substrates will permit definitive assess­
exists disparity between kinase activity observed in ment of the role of kinase activity in LRRK2­
vitro and that found in intact cells. mediated PD pathogenesis. Despite considerable
For any putative LRRK2 kinase substrate iden­ recent progress, much remains to be understood
tified in vitro, it will be imperative to determine its about the contribution of mutant LRRK2 to PD
relevance as a substrate in vivo where conditions pathogenesis. Given the pervasiveness of these
affecting protein localization, activity and struc­ mutations in PD, unlocking these mysteries will
ture are far more complicated. Moreover, since surely have broad implications in understanding
existing studies largely support an increase in fundamental mechanisms of PD development
kinase activity following certain mutations in and for therapeutic strategies aimed at LRRK2.
 29

Recessive mutations progression often accompanied by early-onset

dystonia. Interestingly, Parkin-linked disease
Compelling evidence implicates loss-of-function may be associated with an absence of Lewy body
mutations in three genes, Parkin, PINK1 and DJ­ pathology, a finding that is inconsistent with these
1, in autosomal recessive PD and some sporadic being causal in disease pathogenesis. However,
cases (Dodson and Guo, 2007). Recent work Lewy body pathology is observed in some cases
has demonstrated key roles for all three gene of Parkin-linked PD.
products in preserving mitochondrial function Parkin encodes a protein of 465 amino acids
and protecting against reactive oxygen species. consisting of an N-terminal ubiquitin-like domain,
This underscores the central role of mitochondrial a central linker region and a C-terminal RING
dysfunction and oxidative stress in PD pathogen­ domain containing two RING-finger domains
esis, reinforcing previous studies linking sporadic (Moore et al., 2005). Parkin demonstrates E3 ubi­
PD cases with mitochondrial poisons such as quitin protein ligase activity, tagging protein lysine
MPTP and paraquat. Recessively inherited residues with ubiquitin. Attachment of polyubi­
mutations in a fourth gene, ATP13A2, are linked quitin chains to proteins via lysine K48 usually
to Kufor-Rakeb syndrome, a pallidopyramidal targets them for degradation via the 26S protea-
syndrome featuring parkinsonism together with some, whereas monoubiquitylation and polyubi­
behavioural and cognitive disorders (Najim quitination through K48 or K63 can influence
al-Din et al., 1994). Large-scale association studies other pathways such as intracellular signalling,
showed that ATP13A2 genetic variants do not DNA repair, endocytosis, transcriptional regula­
segregate with PD within families indicating that tion and protein trafficking (Mukhopadhyay and
these mutations likely do not contribute to disease Riezman, 2007; Sandebring et al., 2009).
risk (Vilarino-Guell et al., 2009). While the majority of the Parkin pool is loca­
lized to the cytoplasm and vesicular structures
(Kubo et al., 2001; Shimura et al., 2000), a portion
Parkin is found associated with the outer mitochondrial
membrane (Darios et al., 2003). Several recent
Mutations in Parkin were originally linked with studies on Drosophila and mice have revealed a
autosomal recessive juvenile-onset parkinsonism key role for Parkin in regulating mitochondrial
in three unrelated Japanese families in 1997 function and protection against oxidative stress
(Kitada et al., 1998). Homozygous loss-of-function (Deng et al., 2008; Greene et al., 2003; Palacino
or compound heterozygous Parkin mutations et al., 2004; Park et al., 2006; Yang et al., 2006),
account for approximately 50% of all familial which is discussed further in the section on mito­
early-onset cases of PD with point mutations chondrial dysfunction and oxidative stress. Most
being the most frequent genetic lesion and with mutations in Parkin appear to impair its E3 ligase
deletions, duplications and exonic rearrangements activity or interactions with E2 enzymes such as
also contributing to Parkin-linked PD (Mata et al., UbcH7 and UbcH8 (Shimura et al., 2000; Zhang
2004). Although the majority of Parkin-associated et al., 2000). Although it is not definitively known
PD is inherited in an autosomal recessive manner, that loss of Parkin’s E3 ligase activity is sufficient
there is some evidence to suggest that Parkin for development of PD, a prominent hypothesis is
haploinsufficiency due to polymorphisms in the that Parkin mutations lead to toxic accumulation
promoter or coding regions may associate with of its substrates due to impaired ubiquitin–protea­
increased susceptibility to late-onset PD (Farrer, some function (UPS) (Dodson and Guo, 2007).
2006). Clinically, patients with Parkin mutations Through extensive in vitro investigations, a num­
are L-dopa responsive and exhibit slower disease ber of putative Parkin substrates have been
30

identified including the aminoacyl-tRNA synthase suggests that PINK1 is important for protection
cofactor p38 (Corti et al., 2003), far upstream ele­ against cell death related to mitochondrial dys­
ment-binding protein-1 (Ko et al., 2006), cyclin E function and oxidative stress (Clark et al., 2006;
(Staropoli et al., 2003), Parkin-associated Deng et al., 2008; Exner et al., 2007; Hoepken
endothelin receptor-like receptor (Imai et al., et al., 2007; Wood-Kaczmar et al., 2008). Although
2001), synphilin-1 (Chung et al., 2001a, Chung several PD-associated mutations reduce PINK1
et al., 2001b), synaptotagmin XI (Huynh et al., kinase activity, it is not clear whether loss of
2003), CDCrel-1 (Zhang et al., 2000) and alpha/ kinase activity is required for PD pathogenesis
beta-tubulin (Ren et al., 2003). From this set, only since disease-associated mutations are found
the p38 subunit of aminoacyl-tRNA synthase and both within and outside of the kinase domain. It
far upstream element-binding protein-1 have been seems, however, that kinase activity is required for
demonstrated to accumulate in the brains of the protective function of PINK1 against pro­
patients with both Parkin mutations and Parkin apoptotic agents since staurosporine-induced cell
null mice (Ko et al., 2005, 2006) highlighting the death was substantially reduced by wild-type
importance of determining the authenticity of all PINK1 over-expression, whereas an equivalent
other putative substrates in vivo. Further studies increase in kinase-inactive PINK1 mutant had no
will also be required to determine the roles of protective effect (Petit et al., 2005). Additionally,
these substrates in PD pathogenesis. recent in vivo data suggest that phosphorylation of
the mitochondrial chaperone TNF-receptor-asso­
ciated protein 1(TRAP1) by PINK1 is important
PINK1 for the protective action of PINK1 against oxida­
tive stress-induced cell death (Pridgeon et al.,
A second locus for autosomal recessive early- 2007). The authors also reported that the ability
onset parkinsonism was discovered initially in a of PINK1 to phosphorylate TRAP1 is impaired by
large Sicilian family mapped to the short arm of PD-associated G309D, L347P and W437X PINK1
chromosome 1p35–p36 (Valente et al., 2001) and mutations suggesting a possible connection
later extended to eight additional families from between these mutations, impaired PINK1 sub­
four European countries (Valente et al., 2002). strate phosphorylation and cell death. PINK1
Subsequent work revealed that within this locus, was recently demonstrated to regulate mitochon­
mutations in PINK1 are linked to PD (Valente drial Ca2þ efflux in mammalian neurons, and loss
et al., 2004). Atypical clinical phenotypes have of PINK1 was associated with mitochondrial Ca2þ
been reported in PINK1-linked PD, including dys­ overload and consequent respiration inhibition via
tonia, psychiatric disturbances and sleep benefit increased reactive oxygen species generation and
(Hatano et al., 2004; Tan and Dawson, 2006; opening of the mitochondrial permeability transi­
Valente et al., 2004). PINK1 is a cytosolic and tion pore (Ghandi et al., 2009).
mitochondrially localized protein kinase which
contains an N-terminal mitochondrial targeting
sequence followed by a predicted transmembrane DJ-1
domain, suggesting that PINK1 may be an integral
transmembrane protein possibly in the mitochon­ Mutations in DJ-1 were originally associated with
drial inner membrane with which it closely associ­ early-onset PD in 2003 (Bonifati et al., 2003) and
ates (Silvestri et al., 2005). However, a recent are known to be very rare, accounting for less than
study indicates that the kinase domain of PINK1 1% of early-onset cases. The DJ-1 protein is a
faces out into the cytosol (Zhou et al., 2008). member of the ThiJ/PfpI family of molecular
Existing evidence from cell and animal models chaperones that are induced by oxidative stress
 31

(Dodson and Guo, 2007). Consistent with this, For example, neuronal a-synuclein accumulation
DJ-1 deficiency in Drosophila increases cell death often in Lewy bodies can be found in a number of
caused by reactive oxygen species (ROS)-generat­ neurodegenerative disease including PD, demen­
ing species linked to PD in humans (Muelener tia with Lewy bodies, multiple system atrophy and
et al., 2005). Additional studies revealed that a pure autonomic failure (Goldstein and Sewell,
conserved cysteine residue in human (Canet- 2009; Kövari et al., 2009; Kramer and Schulz-
Aviles et al., 2004), mice (Andres-Mateos et al., Schaeffer, 2007) suggesting that a-synuclein
2007) and Drosophila (Meulener et al., 2006) of aggregation is a common mechanism in these
DJ-1 is modified under conditions of oxidative diseases. Genetic studies have in some instances
stress, and this modification is necessary for the corroborated pathogenic mechanisms indicated by
protective effects of DJ-1. Furthermore, evidence environmental factors such as a central involve­
in mice indicates that DJ-1 acts as an atypical per­ ment of oxidative stress and mitochondrial dys­
oxiredoxin-like peroxidase to scavenge H2O2 pro­ function in PD aetiology. Additionally, genetic
duced by mitochondria (Andres-Mateos et al., studies have implicated protein mishandling due
2007). Accordingly, DJ-1 knock-out mice exhibit to dysfunction of the UPS in development of PD.
elevated mitochondrial H2O2 and reduced activity Since perturbations in the UPS or mitochondrial
of mitochondrial aconitase activity levels, although function both lead to the same pathological out­
the pathological consequences of this are uncertain come, that is loss of dopamine neurons and devel­
since there was an absence of dopaminergic neuron opment of PD, it is likely that an important
degeneration in these mice (Andres-Mateos et al., relationship exists between these functions that
2007). Hence, DJ-1 may act as a cellular redox converge on dopamine neuron viability. The
sensor, which becomes activated under oxidative model presented in Fig. 2 illustrates molecular
conditions to provide protection against ROS- pathways in PD pathogenesis implicated by
mediated damage. Numerous functions have been genetic studies and how these pathways may be
ascribed to DJ-1, including protease, transcrip­ connected.
tional co-activator and molecular chaperone func­
tions, although which of these, if any, contribute to
its protective role in PD remains to be determined. Mitochondrial dysfunction and oxidative stress

A role for mitochondrial dysfunction in PD patho­

Impact of genetic research on understanding genesis is supported by studies on a number of
mechanisms of PD pathogenesis gene products linked to PD. Knock-out studies in
animals clearly indicate that two PD-linked genes,
Genetic studies over the last decade have resulted Parkin and PINK1, have key roles in preserving
in the identification of pathogenic gene variants mitochondrial function and that loss-of-function
that underlie familial PD and in some cases con­ mutations in these genes can lead to PD. Droso­
tribute to the lifetime risk of developing sporadic phila Parkin null mutants exhibit mitochondrial
PD. By linking these genes to PD and understand­ pathology marked by enlarged size and rarified
ing the biological roles of the products they cristae, as well as enhanced sensitivity to oxidative
encode, a wealth of insight has been generated stress, apoptotic muscle degeneration, significant
into mechanisms of PD pathogenesis. These (albeit slight) degeneration of a subset of dopa­
pathogenic genes also yield understanding of mine neurons and reduced life span (Greene
possible relationships between PD and disorders et al., 2003; Pesah et al., 2004). Parkin null mice,
with overlapping clinical or neuropathological which exhibit nigrostriatal deficits without nigral
features that may share common mechanisms. degeneration, do not have gross changes in striatal
32

Pink1 Parkin

Mitochondrial Ubiquitin­
dysfunction proteasome
system
dysfunction
?

Oxidative α-Synuclein
DJ-1 stress aggregates SNCA
(oligomers/fibrils)

↑[Ca]2+ Impaired vesicular Accumulated parkin

↓ ATP transport substrates

Neuronal
dysfunction & death

?
Aberrant protein
translation

LRRK2

Fig. 2. Pathogenic mechanisms implicated by genetic studies of PD. This model links genetic mutations (autosomal recessive
mutations in blue boxes and autosomal dominant mutations in purple boxes) to neurodegeneration via pathways involving
mitochondrial dysfunction, oxidative stress and impaired UPS. Loss-of-function mutations in PINK1 or Parkin cause PD possibly
through a mechanism involving mitochondrial pathology and dysfunction. Deleterious effects of mitochondrial dysfunction include
reduced ATP generation and oxidative stress due to elevated ROS generation. Loss of Parkin’s E3 ubiquitin ligase activity may also
lead to dopamine neuron toxicity via impaired UPS and accumulation of Parkin’s substrates. Loss of DJ-1 antioxidant function may
promote neuronal oxidative stress, as might reduced respiratory chain function and elevated intracellular calcium influx via pores
created by a-synuclein oligomers. LRRK2 mutations might be linked to PD through altered protein translation further supporting a
role for protein turnover in PD pathogenesis.

mitochondrial morphology but do experience or Parkin mutants can be prevented by over-

mitochondrial dysfunction evidenced by reduced expression of antioxidants (Wang et al., 2006;
activity of multiple respiratory chain complexes Whitworth et al., 2005) supporting the contention
along with decreased antioxidant capacity that that oxidative stress is important to PD pathology.
results in increased oxidative damage (Palacino Indeed, oxidative damage to cell macromolecules
et al., 2004). Intriguingly, complete loss of PINK1 is consistently observed in the substantia nigra of
function in flies led to phenotypes that are highly PD patients (Jenner, 2003), and elevated reactive
similar to Parkin null flies (Clark et al., 2006). oxygen species generation may occur as a result of
The subtle neuronal death observed in PINK1 impaired respiratory chain function.
 33

Several lines of evidence suggest a genetic inter­ neuronal cells expressing mutant a-synuclein
action between PINK1 and Parkin. For example, showed a selective increase in mitochondrial dys­
over-expression of Parkin rescued all phenotypes function and apoptotic cell death when treated
resulting from PINK1 deficiency, although a reci­ with a proteasome inhibitor (Tanaka et al.,
procal rescue effect of PINK1 over-expression in 2001). Inhibition of the mitochondrial respiratory
Parkin mutants was not found (Park et al., 2006). chain complex I, which is caused by pesticides and
Similarly, loss of mitochondrial potential, abnor­ certain other environmental toxins, commonly
mal mitochondrial morphology and reduced cris­ leads to the accumulation of a-synuclein-positive
tae seen in HeLa cells with PINK1 deficits can be inclusions suggesting that a-synuclein aggregation
rescued by increased expression of wild-type but may be a consequence of mitochondrial dysfunc­
not PD-associated mutant Parkin (Exner et al., tion (Betarbet et al., 2000; Manning-Bog et al.,
2007). These lines of evidence have led to the 2002). Interestingly, a-synuclein knock-out mice
hypothesis that Parkin acts downstream of are resistant to the toxic effects of MPTP on neu­
PINK1 to preserve mitochondrial function (Dod­ rons while a-synuclein transgenic mice are more
son and Guo, 2007). Recent studies in Drosophila sensitive indicating that a-synuclein might be
have suggested a possible link between PINK1, necessary for neuronal toxicity associated with
Parkin and mitochondrial function by demonstrat­ impaired complex I activity (Dauer et al., 2002;
ing that both appear to regulate mitochondrial Song et al., 2004). Taken together, this evidence
dynamics by either promoting fission or inhibiting suggests that a-synuclein, likely in aggregate form,
fusion of the organelle (Deng et al., 2008; Poole may have a toxic role both in causing mitochon­
et al., 2008). Genetic manipulations of the fly drial dysfunction and in the deleterious effects
mitofusin homologue (Mfa), Opa1 (optic atrophy resulting from it. Future studies will hopefully
1) or drp1 in favour of mitochondrial fission are elucidate the nature of the relationship between
sufficient to rescue mitochondrial pathology, cell a-synuclein and mitochondrial dysfunction.
death and muscle degeneration in Parkin or
PINK1 mutants (Deng et al., 2008). However,
these data and their relevance to human disease Ubiquitin–proteasome system impairment
should be interpreted cautiously due to discrepan­
cies in mitochondrial morphology abnormalities Compelling evidence from genetic studies links
that exist between fly and mammalian model sys­ UPS dysfunction to development of PD.
tems. Nonetheless, impaired mitochondrial Mutations in Parkin associated with the disease
respiration has been detected in peripheral tissues are widely believed to cause impairment of UPS
taken from human PD patients with PINK1 function with consequent accumulation of poten­
(Hoepken et al., 2007) or Parkin mutations tially cytotoxic proteins that may result in death of
(Muftuoglu et al., 2004) suggesting that the mito­ dopamine neurons (Chung et al., 2001a; Moore
chondrial dysfunction observed in animal models et al., 2005). In support of this, proteasome inhi­
may be relevant to human disease. Hence, consid­ bitors are found to cause a number of phenotypes
erable evidence supports a role for PINK1 and that closely recapitulate those in PD when injected
Parkin in protecting against cell death due to into rats (McNaught et al., 2004). Furthermore,
mitochondrial dysfunction and oxidative stress. genetic knock-out of a 26 proteasomal subunit in
Mutations and multiplications in SNCA may mice impairs ubiquitin-mediated protein degrada­
also promote mitochondrial dysfunction leading tion and leads to intraneuronal Lewy-like inclu­
to neuronal death. Mitochondrial pathology fol­ sion formation and substantial degeneration in
lowing MPTP exposure is exacerbated in a-synu­ the nigrostriatal pathway (Bedford et al., 2008).
clein transgenic mice (Song et al., 2004), and The accumulation of aggregated proteins such as
34

a-synuclein in nigral neuron Lewy bodies in tools to begin to understand the cellular net­
sporadic PD also indicates mishandling of protein work of dysfunction that ultimately results in
turnover perhaps due to impaired UPS function. neuronal demise and manifestation of disease
As previously mentioned, Lewy bodies are often phenotypes. Through the generation of cellular
absent in certain familial forms of PD suggesting and small animal models expressing PD-linked
that they may not be neuropathological in PD but genetic mutations, new insights into the
are, instead, a hallmark of protein aggregation and mechanisms of disease pathogenesis have been
therefore still supportive of a role for protein mis­ achieved. Further study will hopefully lead to
handling in PD pathology. Aggregated a-synu­ new disease-modifying treatments for PD that
clein has been shown to strongly bind to and will provide more than temporary symptomatic
inhibit the 26S proteasome in vitro (Snyder et al., relief.
2003), and over-expression of mutant a-synuclein
in cells was observed to decrease proteasome
function and cause selective toxicity to catechola­ Acknowledgements
minergic neurons (Petrucelli et al., 2002). Interest­
ingly, co-expression of Parkin was reported to This work was supported by grants from
reduce sensitivity of a-synuclein-expressing cells National Institutes of Health, National Institute
to proteasome inhibitors suggesting that Parkin of Neurological Disorders and Stroke P50
protects against mutant a-synuclein-mediated NS38377, NS054207. T.M.D. is the Leonard
toxicity. Similarly, over-expression of the molecu­ and Madlyn Abramson Professor in Neurode­
lar chaperone Hsp70 in flies (Auluck et al., 2002) generative diseases.
or transgenic expression of the yeast chaperone
Hsp104 in rats (Lo Bianco et al., 2008) prevents
degeneration of dopamine neurons caused by Abbreviations
expression of mutant a-synuclein indicating that
chaperones function may protect against PD PD Parkinson’s Disease
pathology related to a-synuclein. Consistent with SNARE soluble NSF attachment
this, Lewy bodies examined from post-mortem protein receptors
human brain were found to contain molecular MEF2D myocyte enhancer factor 2D
chaperones (Auluck et al., 2002), which may indi­ LRRK2 leucine-rich repeat kinase 2
cate that in the PD brain, these fail to effectively eIF4E eukaryotic initiation factor 4E
prevent protein aggregation at physiological 4E-BP eukaryotic initiation factor 4E
levels. Further investigations assessing the ability binding protein
of molecular chaperones to prevent neuronal ERM ezrin/radixin/meosin
toxicity associated with a-synuclein aggregation MAPK mitogen-activated protein
will hopefully lead to new therapeutic strategies kinase
for PD. MAPKK mitogen-activated protein
kinase kinase
JNK c-Jun N-terminal kinase
Conclusion PINK1 PTEN-induced putative
kinase 1
Although the majority of PD is sporadic, the TRAP1 TNF-receptor-associated
discovery of rare familial forms of PD and protein 1
subsequent identification of disease-causing MPTP 1-methyl-4-phenyl-1,2,3,6­
mutations have provided extremely valuable tetrahydropyridine
 35

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Exploring the Variety of Random
Documents with Different Content
He was a splendid reader of human nature, could from his great
experience tell the inner workings of the heart, which the face was
striving to hide, and he saw that Mark Merrill had some bitter cause
of quarrel against Scott Clemmons, deeper by far than the latter
cared to admit or had implied. But the good nature of the young
sailor triumphed, and he said:
“I will accept Mr. Clemmons’ hand in friendship, sir, if he means it in
good faith.”
There was a world of meaning in the words: “If he means it in good
faith.”
The eyes of Mark Merrill looked unflinchingly upon the face of Scott
Clemmons, but he did not meet the gaze, and his face flushed
painfully.
This that keen observer, the commandant, saw, and he read who
had been the transgressor in the past.
“Now, Mr. Merrill, as Mr. Clemmons had just reported when you were
convoyed into port, as Cadet Bascomb expressed it, I will hear what
he was about to say to me and then give my attention to you.”
Mark bowed, while the commandant read a letter from Merchant
Clemmons, whom he had once met, and he took the liberty of
inclosing a liberal check for the use of his son—the same as he
might have done had he been sending him to boarding-school.
“I shall return this check to your father, Clemmons, and explain the
situation of a cadet here, after I have heard whether you pass the
examinations or not, which are before you,” and the commandant
seemed not over-pleased with Merchant Clemmons’ letter.
Then he turned to Mark, and continued:
“Mr. Merrill, I am glad to welcome one to the academy who comes
as you do, and I only hope that you, as well as Mr. Clemmons here,
may not find the physical and mental examination too great a
stumbling-block for you to surmount.
“Commodore Lucien has spoken of you to me, and of what a
devoted son you have been to your mother, and it is just such boys
that make the greatest men.
“The surgeon and examining committee are now ready for you, and
my orderly will conduct you to their quarters.
“I wish you success, young gentlemen,” and the commandant bowed
the two youthful seekers after fame out, placing them under the
guidance of an orderly.
Surgeon Du Bose received the appointees pleasantly, there being
one other youth in his quarters just drawing on his coat after having
learned the sad tidings that his chest expansion was below the
average, and his general physical condition not such as to warrant
his being accepted as a cadet.
The poor fellow cast an envious look at the fine forms of Mark Merrill
and Scott Clemmons, and the latter gave him a pitying look of
almost contempt, as though to wonder how he had dared anticipate
being accepted. Then the usual formula was gone through with,
Scott Clemmons being first examined, and his confident smile
showed that he knew that he, at least, had “passed.”
Then came Mark’s turn, and as he stripped for the ordeal the
surgeon gave a low whistle, a decided expression of admiration of
the lad’s physique.
His name, age, height, weight, chest measure and expansion were
all taken, his muscular developments noted, and the questions asked
regarding having had any broken bones and other injuries of a
harmful character. His bones were as straight as arrows, his eyesight
was put to a crucial test and marked as “phenomenal,” and his
health put down as perfect.
His pendulum of life, the heart, swung with the regularity of
clockwork, and not a flaw was found in his teeth, which were white,
even and firm.
A frown passed over the brow of Scott Clemmons as he noted the
fact that Mark Merrill had stood the test better than he had, proud
as he was of his fine form and handsome face.
“It is seldom, if ever, I meet a youth of your perfection of physique,
Mr. Merrill,” said Surgeon Du Bose, in a complimentary way, and
Scott Clemmons turned his head away to hide his plainly visible
chagrin at the praise bestowed upon the young sailor.
Assured that they had passed the physical ordeal the two youths
went to face the examining committee, who were to decide as to
what they did or did not know.
“Here he will fail,” muttered Scott Clemmons, with malign hope that
such would be the case.
Quickly they were put to the test, and when the hours of alternate
hope and despair were over each knew that the other had passed,
and Scott Clemmons fairly ground his teeth with rage, as he heard
Lieutenant Briggs, one of the examiners, say in reference to Mark
Merrill’s very fine penmanship:
“I saw you run your schooner in, Mr. Merrill, and you handle a pen
as well as you do the tiller. I congratulate you that no barrier is now
between you and your cadetship.”
“Curse him!” muttered Scott Clemmons. “He passed better than I
did; but he shall yet be dismissed in disgrace—I swear it!”
 CHAPTER XX.
THE MIDSHIPMAN.

Having passed both his physical examination and the one to discover
how far he had progressed in “book learning,” Mark Merrill felt happy
at the thought that there was no other barrier between him and his
cadetship.
He had been asked by one of the committee where he had attended
school, for he was well up in all questions asked, wrote an excellent
hand, and answered with a knowledge evidently not acquired for the
occasion.
His reply had been a simple one, and truthful:
“My mother taught me all I know of books, sir, for I never went to
school.”
Reporting to the quartermaster of the post, Mark found there the kit
which Commodore Lucien had gotten for him, and he discovered
that it left no needs to be filled.
His room was a pleasant one, and by a rare stroke of good fortune
he was given a first-rate fellow to be his companion to share it. He
had dreaded that, as Scott Clemmons was also from Maine and
known to be an acquaintance, the two might be roomed together.
In such a case he hoped Clemmons would object, but if he did not
then he certainly should, for he could not bring himself to like the
youth who had shown such an ugly humor toward him in the past.
The moment that he could get away Mark started to go aboard his
little schooner and bid farewell to Captain Crane and his two sons,
and also bring ashore the few things he had brought with him from
home.
As an act of duty he had sought Scott Clemmons and said:
“Mr. Clemmons, my little schooner returns home under Captain
Jasper Crane, whom you must know, and I will be glad to give him a
letter for your people, if you wish.”
Scott Clemmons was in his room, getting his things to rights, and at
the remark of Mark Merrill he laughed rudely.
He was no longer under the piercing eye of the commandant, and
need not act for effect, as he had done when at headquarters.
He had stood the ordeal put upon him, but little less acceptably than
had Mark Merrill.
He was a well-formed fellow, bright in his lessons and all that, but
did not take into consideration that, with all his advantages, he had
not done as well as the “fisher lad” he had sneered at.
“Send a letter by a sailing ship, Merrill? Not I, and you must live
away back in the Dark Ages to think of such a thing in these days of
telegraphs and railroads; but I forget that you know nothing of the
world, living as secluded as you have. No, thank you, I have already
telegraphed my father that I went through with flying colors, and I
congratulate you upon having passed, even if it was by the skin of
your teeth, for, of course, they would not refuse you, Merrill. Wait
until the first year’s examination, which you cannot hope to get
through.”
Mark Merrill’s eyes flashed, but he controlled his temper, and
responded:
“I shall try hard to pass, Mr. Clemmons, for I came here to fight hard
to win my way against all odds that I know are before me. Pardon
me for disturbing you. I did not know but that you might wish to see
Captain Crane and his boys, and send some word by them.”
“No, I do not associate with them at home, you know, and the
telegraph and mails will answer my wants.”
Mark turned away, for he felt that he could not much longer listen to
Scott Clemmons’ insulting words and patronizing manner.
“So he offered his friendship simply to blind the commandant, did
he? I wondered how he could be guilty of such an act of manliness
as he professed; but it was for a purpose, not meant. Well, I know
what to expect from him now, and will govern myself accordingly;
but I have not forgotten a voice I heard one night before I left
home, when a net was set to drown me. I think I shall send Silly
Sam a letter by Captain Crane, for the poor fellow is to be trusted,
and is keen enough in mind when he has an object in view.”
So Mark went on board his schooner to write his letters and give the
joyful news to his mother that she could address his letters to:
“Cadet Midshipman Mark Merrill,
U. S. Naval Academy
Annapolis, M. D.”
 CHAPTER XXI.
SHAKING HANDS WITH THE PAST.

“Well, Master Mark, I congratulate you with all my heart,” said

Captain Jasper Crane, when the youth told him that he had stood
the first test, and crossed the rubicon of his hopes and fears.
The two sons of the skipper also offered their congratulations in
their honest way, and the skipper added:
“Well, it means we must sail back alone, and that we’ll not see you
for many a long day, Master Mark?”
“Not until my graduation leave, Captain Crane, unless business may
call you to this port or Baltimore some time, when you must surely
give me a call.”
“You won’t be too proud to wish to see an old coast skipper, then,
after you get your brass buttons on?” said the skipper slyly.
“If I thought becoming an officer of the navy would change my
nature so as to make me forget old friends, captain, I’d go back with
you now and stick to the life I have been always leading at home.
No, my nature won’t change, I assure you; but I hope the schooner
will earn a fair livelihood for you and mother, for I hope to have her
run on here with old Peggy some day to see me, as I know she will
wish to do.”
“I know she will, and I’ll make the schooner pay every dollar she
can; but there was a sailor here to see you, Master Mark, and
yonder comes a boat, and I guess he’s coming back, for he said he
would, as he wished to see you.”
Mark turned to the gangway as the boat ran alongside, and called
out heartily:
“Jack Judson, my sailor friend of B——, how are you?”
The sailor grasped the extended hand, and said, warmly:
“Well, Master Mark Merrill, and glad to see you again. I recognized
you at the helm of the schooner as she ran in, and I never saw a
craft better handled. Going to stay in port long, young mate?”
“I hope to remain some years, Mr. Judson, for I am launched now as
a cadet midshipman,” was the smiling reply.
Jack drew himself up quickly and saluted, while he said:
“Pardon me, sir, but I did not know that, or I would no have made so
bold; but I am a coxswain on the cruiser yonder, and thought I’d
come over to remind you that I had not forgotten you and your
plucky fight in B——.”
“And I am glad to see you, Coxswain Jack, and I have not forgotten
your great kindness that day in B——, either. But let me tell you that
Scott Clemmons is also a cadet.”
“Then look out for him, for he’s your foe,” blurted out Jack Judson.
“I do not believe he is over friendly,” responded Mark, while Jack
said:
“I must be off, sir, for there’s a difference between us now; but I
wish you success, Master Mark, and if you don’t win, I’ll be mistaken
in my calculations.”
The coxswain saluted, when Mark again put out his hand and said:
“Good-by, coxswain, I guess we’ll often meet now.”
The boat pulled away, the coxswain very thoughtful now, for he
remembered how he had once neglected his advantages and thrown
away the chance of an appointment to the navy.
“I’d have been a lieutenant now, if I had gone in; but I didn’t have
the grit to study, and to-day I am only a coxswain. But that youth
has it in him to work his way upward, and he will; but he must keep
his eye on Scott Clemmons, or he’ll foul him if he can.”
After the coxswain’s departure Mark went into the cabin, wrote his
letters, one to his mother and another to Silly Sam, and he asked
Captain Crane to hand the letter to the youth in person.
“I do not know if he can read or not, Captain Crane, but if he
cannot, you please read it to him, and he’ll understand it. The letter
to my mother I know you will deliver first, as you will run straight for
Cliff Castle harbor?”
“Yes, Master Mark, and if you get time some day drop me a line to
let me know how you are getting along,” said the honest skipper.
“You shall hear from me, captain, and I’ll expect you to see my
mother as often as you can, for you know her home is not a cheerful
one, and she has only old Peggy.”
“Yes, and more pluck than any man I know of, to dwell in that old
Spook Hall.”
Then Mark bade good-by to the captain and his boys, sprang into
the boat he had rowed out, and rested on his oars while the crew
got up anchor and hoisted sail.
He waved his hat as they went down the Severn, Captain Crane
dipping his colors to the farewell of the youth.
For a long while the young sailor watched the retreating vessel, then
rowed ashore, and returned the boat to where he had gotten it.
He sighed as he cast another lingering glance after the little Venture,
returning to the weird old home and scenes he had loved so well,
and murmured to himself:
“There goes the last link to bind me with my life of the past few
years. Now my career is to be so different! The struggle begins—my
hard fight for fame. But I will win. I cannot afford not to do so, for
Scott Clemmons shall never rejoice over my failure.”
“Ah, Merrill, all broken up, I see, at parting with your fisher friends—
strange that you did not stick to the low life that suited you so well.”
It was Scott Clemmons, and Mark felt as though he would like to
have struck him to the earth.
But instead he said, calmly:
“I have shaken hands with the past life, Clemmons, and when I
leave this academy you will be behind me!”
“Never! mark my words, never!” and Scott Clemmons uttered an
oath at Mark’s threat to leave him behind in the race for honors.
 CHAPTER XXII.
DISCIPLINING A “CAPTAIN.”

Mark Merrill entered upon his duties like one who had gone in to
win.
His modest nature recoiled at having been discovered as a hero, for
he had hoped to gain success without there being one thing in his
favor.
He had as a room mate a youth from South Carolina by the name of
Bemis Perry, a quiet, unassuming youth, about Mark’s age, and who
made a pleasant companion.
“You knew Clemmons before you came here?” said Bemis Perry, the
day after the two had become mates.
“Yes, I had met him.”
“They say his father is awfully rich, and the king bee of his part of
the country.”
“Yes, Mr. Clemmons is said to be a very rich and influential man.”
“And Scott is his only heir, I hear.”
“He has a sister, I have heard, who is younger than he is.”
“What has Clemmons got against you?”
“I really do not know,” and Mark did not, for he did not recall having
ever done aught to cause Scott Clemmons to dislike him.
“Well, I’ll tell you that he is not your friend, Merrill.”
“So I am aware, but it is a matter of utter indifference to me.”
Entering upon his duties, Mark was naturally put in the same
“awkward squad” as Scott Clemmons.
The latter had been to a military school for a couple of terms, and
was thus priding himself upon his being well up in drill.
He had, in fact, mentioned that he had been captain of his company
at the military school which he had attended, and in various ways he
had thrown out the hint that his father was enormously rich, and a
man of great influence with the government authorities.
He had also taken occasion to say that Mark Merrill was the son of a
poor widow who, from the charity of the agent in charge of a fine
old house, was allowed to live in one wing of it, while her son had
been a mail-carrier and fisher lad.
Now Herbert Nazro was the cadet midshipman who had the drilling
of the new men, and he had with rare judgment taken in the
characters of those under his command.
He realized that they were all green, some exceedingly modest and
willing to admit their know-nothingness, while others were
determined to “cheek it through.”
Mark reported for duty, and when the cadet officer said: “Well, sir,
what do you know?” he answered, with extreme candor:
“Nothing whatever, sir.”
“Then you can be taught easily,” was the frank reply.
“And you, sir?” he turned to Scott Clemmons.
“I do not understand you,” and Scott Clemmons meant to overawe
the cadet officer.
He made a mistake, and he soon realized it.
“Why were you not paying attention, so that you should know?” was
the stern question.
“You were not addressing me, sir.”
“I am now, and I ask you, what do you know?”
“About drilling?”
“Yes.”
“I am pretty well drilled, though perhaps a trifle rusty from lack of
practice.”
“I’ll get the rust off of you, never fear.”
“I was captain of my company.”
“In the army?”
“No.”
“When you address your superior always use the expression ‘sir.’”
Scott Clemmons flushed at the rebuke, and Cadet Officer Nazro
asked:
“Where were you a captain?”
“At the military school which I attended.”
“What did I tell you about addressing your superior? Be careful not
to err again. Then you have been to a military school?”
“Yes.”
“Yes what?”
“Yes, sir. Am I compelled to speak thus to you?”
“Go ask the commandant.”
“No, sir.”
“If you were a captain, you should have known as much. I see I
shall have a hard time with you, for it is no easy task to teach an old
dog new tricks. Fall in line, sir, and take the position of a soldier.”
Mark Merrill really felt sorry for Clemmons, and the little advice given
the youth he decided to take to heart.
He had seen several military companies parading, and that was all,
but he meant to do his best.
He fell in line, and when shown the “position of a soldier” by the
splendid young drill-master, he determined to keep his mind upon
the duty before him.
In spite of his having been a “captain,” Scott Clemmons was found
more fault with than all the others of the awkward squad.
“You are wrong, sir,” shouted Cadet Nazro. “Just see how you stand.
Your drill master must have been a veteran of 1812. Now these men
can learn, for they know nothing; but you know it all, and like most
know-alls, you give no demonstration of your knowledge. See Merrill
there, how well he stands, and I have not had to correct him a
second time, nor Perry either. Look to it, Captain Clemmons, that I
don’t have to correct you again.”
There were others of the greenhorns who got rebuffs, also, but for
some reason Officer Herbert Nazro seemed to have picked upon
Scott Clemmons for his especial target of ill-natured flings.
“He has only himself to blame for it,” said Bemis Perry to Mark, when
the squad was dismissed, after the hardest work the new men had
ever known.
“Yes, he should have kept quiet about having been captain of his
company,” Mark returned.
“As I did; for I was three years at the military school in Charleston,
but to-day convinced me that the drill there is nothing in comparison
to this naval school. We shall see stars here, Merrill.”
“I have become convinced of that,” was Mark’s laughing response.
 CHAPTER XXIII.
A SECRET FOE.

Of course Scott Clemmons became a mortal enemy of Herbert Nazro

after his first drill in the awkward squad, under the command of that
most efficient young officer.
He dared not come out in open rebellion, as he well knew what that
would mean to him; but he treasured up for Nazro a bitter feeling
and a hope of revenge in the future when the chance should come
in his way.
To be rebuked before Mark Merrill cut him deeper than if it had been
before the entire corps, for he had tried to impress Mark with his
importance.
He had watched Mark’s face for some sign of rejoicing, but even his
ill-nature had failed to detect there any expression of triumph.
Fisher lad though Mark Merrill had been, the spoiled and petted child
of fortune, Scott Clemmons, was intensely jealous of him.
He feared the reserve power of the youth who had gotten an
appointment to the naval school by his own acts, when, with all his
father’s influence, he had found it no easy task to accomplish it.
Then, too, Mark had entered with a kind of hurrah, and more, he
had passed the surgeon and examining committee under flying
colors, while his first drill had been marked by no grave error upon
his part.
There were lads at the academy to toady to the riches and influence
of Scott Clemmons, and so that youth at once found a following
among them.
To his willing “satellites” Scott Clemmons, from a knowledge of his
own nature, judged Mark, believing that the young sailor would
inform his friends of the affair of the toy ship and what followed. He
had told his version of the affair, and soon through the corps went
the story of enmity between the two “men from Maine,” as they
were called.
Had Scott Clemmons been less arrogant, Herbert Nazro would not
have been so severe upon him as he was.
But all new cadets must expect hard times the first year they enter
into Uncle Sam’s service as baby tars.
In his studies Mark went to work with the determination to win, and
a feeling began to creep over the class in which he was that he
meant to be a dangerous man in the race for honors.
Scott Clemmons understood this more keenly than any one else, and
he began to feel his inferiority in spite of his vanity, so he decided
that the only way to beat Mark Merrill was to get him out of the
academy.
He sized up the others of the class, and felt that, with a struggle, he
could lead for honors, but Mark Merrill was dangerous, and intended
to see to it that his threat to leave him behind was carried out.
Demerits against a cadet would upset all standing for good lessons,
perfect drill and attention to duties, and that these ugly little demerit
marks could be readily gotten from the slightest causes Scott
Clemmons soon discovered. He accordingly induced his roommate to
enter into a plot against the unsuspecting young sailor.
When rigged out in his uniform Mark Merrill was certainly a very
handsome and striking-looking lad.
The corps tailor had complimented him by saying he had never
measured a finer formed lad for his clothes, and seldom one his
equal.
Fortunately for the new men, there had recently been several
dismissals from the academy of “hazers,” so that no great indignities
were heaped upon Mark and the others.
Still they came in for their share of petty jokes played upon them, all
of which Mark submitted to as really a part of the discipline of the
institution.
He was universally good-natured, dignified, yet courteous to all, and
on duty and in study hours nothing could move him from what he
deemed right.
He was a favorite with the officers, popular with his comrades, and
yet for all that there seemed to be some mysterious undercurrent
working against him.
Once his cap was missing, and he was absent at roll call, so a
demerit went against him; but he did not report that his cap had
been cleverly taken from his room by some one.
Another time he could not find his shoes for parade, and again a
demerit went down against his name.
A third time his handsome uniform was disfigured by enormous ink
stains, and he knew that he was no more responsible for that than
he had been for his missing hat and shoes.
His books, too, became disfigured in some mysterious way, and one
morning he was reported as having been caught out of his room at
night when he had been fast asleep in bed.
So Mark Merrill, without a word in his own defense, had been put on
the list for a reprimand and punishment.
These constant demerits were counting up sadly against Mark, until
he knew that by the end of his first year they would be so
formidable as to mean dismissal. Yet what could he do to save
himself?
He was innocent of wrong-doing, and though he suspected his
persecutor, he had no proof of it that he was right in his suspicions,
while, if he was, he had too manly a nature to go and report him.
So he determined to suffer in silence, and trust to some good
fortune to make all things even in the end.
 CHAPTER XXIV.
A SECRET FRIEND.

The petty persecutions of Mark Merrill became so persistent, so

annoying, and so frequent that those who knew how matters were
going became confident that, as they all counted against the young
sailor and not against unknown persecutors, he would not be able to
stay his year out at the academy.
It had leaked out that Mark Merrill had been a tough citizen at
home, and was nothing more than a coast fisherman, until brought
into a position above his station by an appointment to the naval
school.
In truth there were a number of rumors about the academy
detrimental to our young hero, and though they reached his ears,
often most unpleasantly from hearing them himself, oftener from
having them told him by his devoted chum, Bemis Perry, he suffered
in silence, making no denials.
At length some who had been his friends grew cold in their greetings
of him, and his popularity began to waver.
“You can’t make a silk purse out of a sow’s ear,” said Scott
Clemmons, one day, in speaking of Mark in a crowd, who had been
referring to his many demerits.
“No, and you can’t ward off the attack of a secret assassin,”
remarked Bemis Perry quietly.
All eyes turned upon the speaker, for he seldom attracted attention
by any outspoken words, and Scott Clemmons, with angry face,
asked:
“Do you mean that for me, sir?”
“I shot at random, Clemmons; and if you got in the way it is your
lookout, not mine.”
“I wish you to explain your ambiguous words,” said Clemmons hotly.
“Permit me to do so,” was the response. “You were pleased to apply
an insulting application to my roommate and friend, Mark Merrill,
and as he has suffered much secret persecution from one who
would stab him in the back, I say that one can no more protect
oneself from a secret assassin than you can make a silk purse out of
a sow’s ear. Now, if the shoe fits you, put it on and wear it.”
“As it does not, there is no cause of quarrel between us,” Scott
Clemmons said, retreating through the exit open to him.
“You are wise,” and with this Bemis Perry walked away, and as he
did so he muttered to himself:
“I will do it.”
An hour after found him in the presence of the commandant, waiting
to be heard by that august personage.
“Well, Mr. Perry, what is it?” said the commandant, somewhat
abruptly.
“I have no complaint to make, commandant, for myself, but I have
an explanation to offer in behalf of another.”
“Well, Mr. Perry, I will hear you.”
The commandant had taken a fancy to the quiet, reserved but
brilliant youth who had become Mark Merrill’s roommate, and he
now saw that he had something more than a favor to ask.
“I wish to make a statement, sir, and hope that you will take what I
have to say as though uttered under oath.”
“So serious as that, is it, Mr. Perry?”
“Yes, sir; but as I said, it is not of myself that I will speak.”
“Who, then?”
“Of my roommate, sir.”
“Ah! Has Merrill gotten out with you, too?”
“On the contrary, I wish to say that Merrill is the noblest fellow I
ever met. I have watched him closely, when he little dreamed I was
paying the slightest attention to his acts, or the actions of others,
and I wish to say, commandant, that the day he missed roll call on
account of not finding his cap, some one had taken it to cause him a
demerit. The ink stains on his uniform were put there by others, and
the night that he was reported as absent without leave from his
room I lay awake, unable to sleep, and he never got out of his cot;
but, whoever it was, gave the name of Merrill instead of his own,
and this I’ll take oath to, sir. In a number of other cases,
commandant, Merrill has been accused and silently submitted, when
I know he was innocent, and thus the demerits roll up against him.
Against these demerits, sir, he stands perfect in lessons, thorough in
drill, and no complaint against the performance of any duty he is put
upon, which, I think, sir, if you will pardon the expression of my
opinion, go to prove that where he has a chance to get perfect
marks he gets them, while others get the demerits against him as
one dangerous to have as a rival for honors.”
“Ah! I see your reasoning, Mr. Perry; but may I ask if Merrill knows
of your coming to me?”
“No, sir, he has not a suspicion of it, for I come on my own
responsibility, knowing the facts.”
“It does you credit, let me say, Perry, and your reasoning is so good
that I shall look into the matter myself.”
“Thank you, sir.”
“But what does Merrill say of the demerits he receives?”
“I have only heard him express himself once, sir, and then he said
that it was not the plain sailing he had hoped to have here, for in
spite of his every effort to win success he seemed to make a dead
failure of it.”
“I see; but do not speak of this visit to Merrill or any one else, and
I’ll see what explanation can be arrived at of his many demerits.”
“Simply, sir, that he has a secret foe,” was the almost blunt assertion
of Bemis Perry.
“Then he is fortunate in having also a secret friend in you, Mr. Perry,”
was the commandant’s smiling response; and Bemis Perry saluted
and retired, satisfied that he had acted as he should have done to
save Mark Merrill from an underhand foe, who meant his dismissal
from the academy.
 CHAPTER XXV.
A CLOUDED RECORD.

Weeks passed away and the strange fact presented itself that the
cadet midshipman, who was devotedly studious, thorough in every
duty devolving upon him, perfect in drill and courteous to all, yet
kept his list of demerit marks steadily increasing against him, a
circumstance that could only end in one way.
Pranks were played, and time and again the guilty one was said to
be Mark Merrill, for he was the one who seemed to be leading two
lives, as it were, secretly a wild one, openly a perfect one.
Half-smoked cigars were found by the officer of inspection in his
room, and when he asserted he never smoked them, as proof
against him was a box of perfectos nearly empty.
Upon another occasion the inspector found a bottle that had
contained whisky in Merrill’s room, and there was enough left in it to
prove that it had contained the real old beverage of the Kentucky
colonels.
In many other ways had seeming proof been brought against Mark
Merrill that he was not all that he professed to be, and many
predicted that he would take his departure from the United States
Naval Academy before very long.
But one afternoon the corps were assembled, and, to the surprise of
all, the demerits against the cadets were read out openly.
Here and there a name was called which held no demerit mark
against it, but when the adjutant came to the name of Mark Merrill
he paused, and a moment of suspense followed.
Then came the reading of the number which was known as the
“Fatal Figures.”
Beyond that number no cadet could go, and Mark Merrill’s face
became deadly pale as he heard the calling out of the fatal figures.
Other names followed, until the whole roll of the corps had been
called, and no one else came within startling distance of the fatal
figures.
“Cadet Mark Merrill to the front!” came the adjutant’s command, for
that officer already had his orders.
Mark advanced promptly until halted.
White-faced but cool, with every eye upon him, he stood awaiting
what was to come as though he were to hear his death warrant
read.
To him it was worse, for he expected ignominious dismissal from the
corps.
“Cadet Merrill, the number of demerits against your name has
reached the limit, the fatal figures which mean dismissal. The
commandant desires to know what you have to say in your
defense?”
“Nothing, sir, for the demerits stand against me, and I submit to the
laws of the academy in silence.”
Every one heard the distinctly uttered reply of the young cadet.
Then the commandant’s voice was heard:
“Adjutant, you are to cancel every demerit that stands against the
name of Cadet Midshipman Mark Merrill.”
In spite of stern discipline a murmur ran down the line, for such a
command could not be understood.
But the explanation was not long delayed, for again the stern voice
of the commandant was heard:
“Cadet Merrill, I have reason to know that when you failed to appear
at roll call, from having lost your cap, that it was taken from your
room to bring about just such trouble for you. I have reason to know
that ink stains were placed upon your uniform to get you into
trouble, and that the night when you were reported absent from
your room without leave, the one who answered the officer of the
guard was not you, but used your name. The bottle found in your
room, also the cigars, were put there by those who meant to get
you into trouble. Against such acts, which are explained away, you
stand perfect in your lessons, in drill and all duties devolving upon
you. Hence I cancel these demerits with the warning to your secret
enemies that, were they known, dismissal should at once follow the
discovery, and if like underhand acts against you, or others, are
perpetrated the guilty ones shall be hunted down and the severest
penalty shall be visited upon them. Return to the ranks, Cadet
Merrill, with your record clear.”
There are no more manly youths in the world, taken as a whole,
than our baby tars of Annapolis and boy soldiers of West Point, and
none more ready to do justice to one of their number wronged, and
so it was that the cadet midshipmen felt assured that the
commandant was doing only justice to Mark Merrill and letting his
persecutors down lightly.
So they gave three rousing cheers for Mark’s “clear record,” and a
groan for his secret foes.
If there were several in the corps who joined in the cheers and
groans it was to hide their own confusion worse confounded.
 CHAPTER XXVI.
THE TELLTALE COIN.

Barney Breslin was not a popular youth in the Naval School.

His nature was somewhat morose; it seemed to go against him to
salute his superiors, and he had never won golden opinions for his
studious habits and strict attention to duty.
He had but one intimate in the corps of cadets, and that one was
Scott Clemmons, his roommate.
Many wondered how it was that Scott Clemmons had gotten in with
Barney Breslin, for, where the one was an aristocrat, the other had
just escaped being born in the Emerald Isle, for his parents had set
foot upon the “land of the brave and the free” only a week when
Barney made his début in life.
The father of the youth had played his cards so well in the
metropolis that he had gotten to be a man of wealth and a politician
of influence, and it had been the dream of the mother’s life to see
her boy an admiral before she died.
An only son, Barney had gone it a trifle rapid for a youngster, and
was sent to the Naval School for training. As he passed his
examinations he had the courage, when a full-fledged cadet, to write
to his father of certain unpaid debts left behind in New York, and
they were promptly settled by the parent, but with an admonition
that not a dollar more should be received from the Breslin bank
account until he had graduated, and if he failed to do this he had
better ship before the mast, and not show up again under the
parental roof tree.
Now, Barney was fond of a game of chance, and when he could find
a congenial spirit to play with, he often indulged in gambling,
generally to his sorrow, for he soon had several I. O. U.’s for various
amounts.
It was supposed that Scott Clemmons helped Barney Breslin in his
studies, for the former was bright and stood splendidly in his classes.
In return it was hinted that Barney did many little favors for
Clemmons, mostly of a menial nature, however.
The inspector always found Clemmons’ wardrobe and half of the
room neat as a pin, while Barney was often “spotted” for disorder.
Cadets generally “size up” a man very correctly, and they decided
that when examination day came and Barney’s displacement was
taken, his tonnage in knowledge would fall short, even though aided
by Scott Clemmons.
In other words, Barney could never “bone” hard enough to step
across the threshold into the third class.
“He’ll bilge, certain,” was the general way of putting Barney’s
prospects by his fellow cadets.
It may, therefore, be inferred that Barney Breslin was as unpopular
as his roommate, Scott Clemmons, was popular, for the latter was
looked upon as a “good fellow all round,” though a trifle too haughty,
perhaps.
From the first Barney had not liked Mark Merrill, and he made no
effort to disguise it.
A tall, heavily formed fellow, he possessed great brute strength, and
was brave from this very reason, feeling his power over weaker
mortals, and inclined to be a bully from nature.
One afternoon the cadets assembled in considerable force in the
gymnasium, and many were giving exhibitions of their prowess as
athletes, and no mean exhibition it was, either, for the training that
they received made iron physiques of the youths.
For some reason an unpleasant feeling rested upon many, which
soon became general when it was known that Scott Clemmons had
lost a valuable coin that morning.
It was a rare coin, what is known as a fifty-dollar gold piece,
octagonal in shape, and always quoted at a large premium on
account of the scarcity of such issues of money.
All who had seen Scott Clemmons with it knew that he called it his
“luck coin,” and that he prized it most highly.
He had changed his clothes that morning, leaving the coin in the
pants he had taken off, and, going for it an hour after, he found it
gone.
Barney Breslin had expressed himself boldly about one whom he
believed had taken the coin, as he had said that he met a cadet
coming out of the room of Scott Clemmons and himself, and unless
the gold piece was returned that night, he would make his
accusation public.
He would not give a hint as to whom he suspected, but said:
“Wait until night, and then I shall accuse the one I deem the thief,”
and he turned away to perform an act which he had won quite a
reputation for, which was to walk around the pedestrian track of the
gymnasium on his hands.
“Can you do that, Merrill?” asked Scott Clemmons, who stood near
him, and there was a sneer in his tone and manner.
“I think so,” was the quiet response, and Mark Merrill threw himself
upon his hands and began to go around the track, when suddenly,
with a loud ring, the missing gold-piece rolled from his pocket amid
almost a roar of amazement from his brother cadets.
 CHAPTER XXVII.
A DOUBLE ACCUSATION.

Barney Breslin had just completed his walk on his hands around the
track of the gymnasium, and the applause with which he had been
greeted had ceased, when Scott Clemmons asked Mark Merrill if he
could accomplish a like feat.
When the gold coin fell from Mark’s pocket and the loud murmur of
amazement was heard, Barney Breslin had sprang forward, and
seizing the piece of gold cried:
“It is your luck coin, Clemmons, as I live!”
“It certainly is, but surely there must be some mistake, for Merrill
could not be guilty of——”
“I tell you now that he is the man I saw leaving our room,” said
Breslin, interrupting Clemmons.
And all this time, unheeding the dropping of the coin from his
pocket, Mark Merrill had continued his hand-walk around the track,
accomplishing the feat with an ease far greater than Barney Breslin
had done.
As he approached the group now, his face flushed from his peculiar
exercise, every eye was upon him, and a death-like silence was upon
all.
“You must speak, Clemmons, for this cannot be allowed to go by,”
said Breslin, breaking the silence.
“Merrill, it seems that you accomplished Breslin’s feat, but you have
also done something that he could not and would not do,” said Scott
Clemmons.
“What is that, may I ask, Mr. Clemmons?”
“You dropped something from your pocket awhile since?”
“Yes, I heard it drop, but as I had no claim to it I paid no attention
to it.”
“You know what it was?”
“Ah! yes; an octagonal coin which Breslin stole from you and placed
in my pocket, hoping to prove me the thief,” was the cool response.
“Ha! you dare accuse me of being a thief?” and, like a mad bull,
Barney Breslin rushed upon Mark Merrill.
Some would have interfered had they had time, and all expected to
see Barney Breslin seize and crush Mark Merrill in his iron grasp.
But instead, they saw the huge bully fly backward with terrific force
and measure his length upon the track of the gymnasium.
He had been dealt a blow by Mark that half-stunned him, and
amazed all, for the young sailor had never before shown what he
could do with his fists, and his latent strength was never once
suspected, unless it was by Scott Clemmons.
With a howl of rage Barney Breslin arose and rushed again upon
Mark, who cried out:
“Back, Breslin, or you will regret it!”
A cry of defiance was Breslin’s only answer, and as the cadet struck
up Mark’s guard, he was enabled to seize him in his long, powerful
arms.
But only for a moment did he retain his hold, for he was raised
bodily from his feet and dashed to the floor with a force that shook
the building, and he lay limp and dazed from the fall.
Though astonished at Mark’s grand exhibition of strength, and glad
as many were to see Barney Breslin punished, the cadets could not
let the charge about the gold coin go by, and several called out:
“Prove that you know nothing about that coin, Merrill, or it will go
hard with you.”