2016 Book GlialCellsInHealthAndDiseaseOf PDF

Advances in Experimental Medicine and Biology 949
Rommy von Bernhardi Editor
Glial Cells in
Health and
Disease of the
CNS
Advances in Experimental Medicine
and Biology
Volume 949
Series editors
Irun R. Cohen, Rehovot, Israel
N.S. Abel Lajtha, Orangeburg, USA
Rodolfo Paoletti, Milan, Italy
John D. Lambris, Philadelphia, USA
More information about this series at http://www.springer.com/series/5584
Rommy von Bernhardi
Editor
Glial Cells in Health

and Disease of the CNS
123
Editor
Rommy von Bernhardi
Faculty of Medicine
Pontificia Universidad Católica de Chile
Santiago
Chile
ISSN 0065-2598 ISSN 2214-8019 (electronic)

Advances in Experimental Medicine and Biology
ISBN 978-3-319-40762-3 ISBN 978-3-319-40764-7 (eBook)
DOI 10.1007/978-3-319-40764-7
Library of Congress Control Number: 2016948593
© Springer International Publishing Switzerland 2016

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Contents
Glial Cells and Integrity of the Nervous System . . . . . . . . . . . . . . . . . . . . 1

Rommy von Bernhardi, Jaime Eugenín-von Bernhardi, Betsi Flores
and Jaime Eugenín León
Part I Glial Cell Function in the Healthy Nervous System

NG2-glia, More Than Progenitor Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
Jaime Eugenín-von Bernhardi and Leda Dimou
Pharmacological Tools to Study the Role of Astrocytes in Neural
Network Functions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47
Fernando Peña-Ortega, Ana Julia Rivera-Angulo
and Jonathan Julio Lorea-Hernández
Microglia Function in the Normal Brain . . . . . . . . . . . . . . . . . . . . . . . . . . 67
Rommy von Bernhardi, Florencia Heredia, Nicole Salgado
and Paola Muñoz
Physiological Functions of Glial Cell Hemichannels . . . . . . . . . . . . . . . . . 93
Juan A. Orellana
Role of Astrocytes in Central Respiratory Chemoreception . . . . . . . . . . . 109
Jaime Eugenín León, María José Olivares and Sebastián Beltrán-Castillo
Purine Signaling and Microglial Wrapping. . . . . . . . . . . . . . . . . . . . . . . . 147
Bernardo Castellano, Mar Bosch-Queralt, Beatriz Almolda,
Nàdia Villacampa and Berta González
Oligodendrocytes: Functioning in a Delicate Balance Between High
Metabolic Requirements and Oxidative Damage . . . . . . . . . . . . . . . . . . . 167
Alejandro D. Roth and Marco T. Núñez
Schwann Cell and Axon: An Interlaced Unit—From Action Potential
to Phenotype Expression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 183
Felipe A. Court and Jaime Alvarez
v
vi Contents
Part II Glial Cell Dysfunction and Pathological Implications

Age-Dependent Changes in the Activation and Regulation
of Microglia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 205
Francisca Cornejo and Rommy von Bernhardi
Astrocyte Dysfunction in Developmental Neurometabolic Diseases . . . . . 227
Silvia Olivera-Bravo, Eugenia Isasi, Anabel Fernández,
Gabriela Casanova, Juan Carlos Rosillo and Luigi Barbeito
Microglia in Cancer: For Good or for Bad? . . . . . . . . . . . . . . . . . . . . . . . 245
Anna Carolina Carvalho da Fonseca, Rackele Amaral, Celina Garcia,
Luiz Henrique Geraldo, Diana Matias and Flavia Regina Souza Lima
Peripheral Inflammation and Demyelinating Diseases . . . . . . . . . . . . . . . 263
Verónica Murta and Carina Ferrari
Regulation of Oligodendrocyte Differentiation and Myelination
by Nuclear Receptors: Role in Neurodegenerative Disorders . . . . . . . . . 287
Adrián Sandoval-Hernández, María José Contreras, Jenny Jaramillo
and Gonzalo Arboleda
The Role of Galectin-3: From Oligodendroglial Differentiation
and Myelination to Demyelination and Remyelination Processes
in a Cuprizone-Induced Demyelination Model . . . . . . . . . . . . . . . . . . . . . 311
H.C. Hoyos, Mariel Marder, R. Ulrich, V. Gudi, M. Stangel,
G.A. Rabinovich, L.A. Pasquini and J.M. Pasquini
Prenatal Systemic Hypoxia-Ischemia and Oligodendroglia Loss
in Cerebellum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 333
Penha Cristina Barradas, Tiago Savignon, Alex C. Manhães,
Frank Tenório, Alan P. da Costa, Marta C. Cunha-Rodrigues
and Juliana Vaillant
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 347
Contributors
Beatriz Almolda Unit of Histology, Department of Cell Biology, Physiology and

Immunology, Institute of Neurosciences, Universitat Autònoma de Barcelona,
Bellaterra, Spain
Jaime Alvarez Center for Integrative Biology, Universidad Mayor, Santiago,
Geroscience Center for Brain Health and Metabolism, Santiago, Chile
Rackele Amaral Instituto de Ciências Biomédicas, Centro de Ciências da Saúde,
Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil
Gonzalo Arboleda Grupo de Neurociencias y Muerte Celular, Facultad de
Medicina e Instituto de Genética, Universidad Nacional de Colombia, Bogotá,
Colombia; Departamento de Patología, Facultad de Medicina, Universidad
Nacional de Colombia, Bogotá, Colombia
Luis Barbeito Neurodegeneration Laboratory, Institut Pasteur, Montevideo,
Uruguay
Penha Cristina Barradas Depto. Farmacologia e Psicobiologia, Instituto de
Biologia—UERJ, Rio de Janeiro, RJ, Brazil
Sebastián Beltrán-Castillo Departamento de Biología, Facultad de Química y
Biología, Universidad de Santiago de Chile (USACH), Santiago, Chile
Mar Bosch-Queralt Unit of Histology, Department of Cell Biology, Physiology
and Immunology, Institute of Neurosciences, Universitat Autònoma de Barcelona,
Bellaterra, Spain
Gabriela Casanova UMET, School of Sciences, UDELAR, Montevideo,
Uruguay
Bernardo Castellano Unit of Histology, Department of Cell Biology, Physiology
Bellaterra, Spain
vii
viii Contributors
María José Contreras Grupo de Neurociencias y Muerte Celular, Facultad de

Medicina e Instituto de Genética, Universidad Nacional de Colombia, Bogotá,
Colombia
Francisca Cornejo Departamento de Neurología, Escuela de Medicina, Pontificia
Universidad Católica de Chile, Santiago, Chile
Felipe A. Court Center for Integrative Biology, Universidad Mayor, Santiago;
Geroscience Center for Brain Health and Metabolism, Santiago; Millenium Nucleus
for Regenerative Biology, Pontificia Universidad Católica de Chile, Santiago, Chile
Marta C. Cunha-Rodrigues Depto. Farmacologia e Psicobiologia, Instituto de
Biologia—UERJ, Rio de Janeiro, RJ, Brazil
Alan P. da Costa Depto. Farmacologia e Psicobiologia, Instituto de Biologia—
UERJ, Rio de Janeiro, RJ, Brazil
Anna Carolina Carvalho da Fonseca Instituto de Ciências Biomédicas, Centro
de Ciências da Saúde, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ,
Brazil
Leda Dimou Physiological Genomics, Biomedical Center, Ludwig-Maximilians-
University, Munich, Germany
Jaime Eugenín-von Bernhardi Graduate School of Systemic Neuroscience,
Physiological Genomics, Biomedical Center, Ludwig-Maximilians-University,
Planegg-Martinsried, Munich, Germany
Jaime Eugenín León Departamento de Biología, Facultad de Química y Biología,
Universidad de Santiago de Chile (USACH), Santiago, Chile
Anabel Fernández Neuroscience Division, Instituto de Investigaciones Biológicas
Clemente Estable, (IIBCE), Montevideo; Comparative Neuroanatomy-Associated
Unit, School of Sciences, UDELAR, Montevideo, Uruguay
Carina Ferrari Instituto de Ciencias Básicas y Medicina Experimental, Instituto
Universitario del Hospital Italiano, Buenos Aires, Argentina
Betsi Flores Departamento de Neurología, Escuela de Medicina, Pontificia
Celina Garcia Instituto de Ciências Biomédicas, Centro de Ciências da Saúde,
Luiz Henrique Geraldo Instituto de Ciências Biomédicas, Centro de Ciências da
Saúde, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil
Berta González Unit of Histology, Department of Cell Biology, Physiology and
Immunology, Institute of Neurosciences, Universitat Autònoma de Barcelona,
Bellaterra, Spain
Contributors ix
V. Gudi Department of Neurology, Hannover Medical School, Hannover,

Germany; Center for System Neurosciences, Hannover, Germany
Florencia Heredia Departamento de Neurología, Escuela de Medicina, Pontificia
H.C. Hoyos Department of Biological Chemistry, School of Pharmacy and
Biochemistry, Institute of Chemistry and Biological Physical Chemistry (IQUIFIB),
Buenos Aires, Argentina; School of Pharmacy and Biochemistry, University of
Buenos Aires and National Research Council (CONICET), Buenos Aires,
Argentina
Eugenia Isasi Cellular and Molecular Neurobiology, Instituto de Investigaciones
Biológicas Clemente Estable (IIBCE), Montevideo, Uruguay
Jenny Jaramillo Grupo de Neurociencias y Muerte Celular, Facultad de Medicina
e Instituto de Genética, Universidad Nacional de Colombia, Bogotá, Colombia
Flavia Regina Souza Lima Instituto de Ciências Biomédicas, Centro de Ciências
da Saúde, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil
Jonathan Julio Lorea-Hernández Departamento de Neurobiología del
Desarrollo y Neurofisiología, Instituto de Neurobiología, Universidad Nacional
Autónoma de México, Querétaro, Mexico
Alex C. Manhães Depto. Ciências Fisiológicas; Instituto de Biologia Roberto
Alcantara Gomes, Universidade do Estado do Rio de Janeiro, Rio de Janeiro, RJ,
Brazil
Mariel Marder Department of Biological Chemistry, School of Pharmacy and
Argentina
Diana Matias Instituto de Ciências Biomédicas, Centro de Ciências da Saúde,
Verónica Murta Laboratorio de Neuropatología Molecular, Instituto de Biología
Celular y Neurociencias, Universidad de Buenos Aires, Buenos Aires, Argentina
Paola Muñoz Departamento de Neurología, Escuela de Medicina, Pontificia
Marco T. Núñez Departamento de Biología, Facultad de Ciencias, Universidad de
Chile, Santiago, Chile
María José Olivares Departamento de Biología, Facultad de Química y Biología,
Universidad de Santiago de Chile (USACH), Santiago, Chile
x Contributors
Silvia Olivera-Bravo Cellular and Molecular Neurobiology, Instituto de

Investigaciones Biológicas Clemente Estable (IIBCE), Montevideo, Uruguay
Juan A. Orellana Departamento de Neurología, Escuela de Medicina, Pontificia
J.M. Pasquini Department of Biological Chemistry, School of Pharmacy and
Argentina; Dpto. de Qca Biol, FFyB-UBA, Bs, Argentina
L.A. Pasquini Department of Biological Chemistry, School of Pharmacy and
Argentina; Dpto. de Qca Biol, FFyB-UBA, Bs, Argentina
Fernando Peña-Ortega Departamento de Neurobiología del Desarrollo y
Neurofisiología, Instituto de Neurobiología, Universidad Nacional Autónoma de
México, Querétaro, Mexico
G.A. Rabinovich Laboratory of Immunopathology, Institute of Biology and
Experimental Medicine (IBYME; CONICET), Buenos Aires, Argentina;
Laboratory of Functional Glycomics, Department of Biological Chemistry, Faculty
of Exact and Natural Sciences, University of Buenos Aires, Buenos Aires,
Argentina
Ana Julia Rivera-Angulo Departamento de Neurobiología del Desarrollo y
Neurofisiología, Instituto de Neurobiología, Universidad Nacional Autónoma de
México, Querétaro, Mexico
Juan Carlos Rosillo Neuroscience Division, Instituto de Investigaciones
Biológicas Clemente Estable (IIBCE), Montevideo, Uruguay
Alejandro D. Roth Departamento de Biología, Facultad de Ciencias, Universidad
de Chile, Santiago, Chile
Nicole Salgado Departamento de Neurología, Escuela de Medicina, Pontificia
Adrián Sandoval-Hernández Grupo de Neurociencias y Muerte Celular,
Facultad de Medicina e Instituto de Genética, Universidad Nacional de Colombia,
Bogotá, Colombia
Tiago Savignon Depto. Farmacologia e Psicobiologia, Instituto de Biologia—
UERJ, Rio de Janeiro, RJ, Brazil; Depto. Farmacologia e Toxicologia, Instituto
Nacional de Controle de Qualidade em Saúde, Rio de Janeiro, RJ, Brazil
Contributors xi
M. Stangel Department of Neurology, Hannover Medical School, Hannover,

Germany; Center for System Neurosciences, Hannover, Germany
Frank Tenório Depto. Farmacologia e Psicobiologia, Instituto de Biologia—
R. Ulrich Department of Pathology, University of Veterinary Medicine Hannover,
Hannover, Germany
Juliana Vaillant Depto. Farmacologia e Psicobiologia, Instituto de Biologia—
Nàdia Villacampa Unit of Histology, Department of Cell Biology, Physiology
Bellaterra, Spain
Rommy von Bernhardi Departamento de Neurología, Escuela de Medicina,
Pontificia Universidad Católica de Chile, Santiago, Chile
Glial Cells and Integrity of the Nervous
System
Rommy von Bernhardi, Jaime Eugenín-von Bernhardi, Betsi Flores

and Jaime Eugenín León
Abstract Today, there is enormous progress in understanding the function of glial

cells, including astroglia, oligodendroglia, Schwann cells, and microglia. Around
150 years ago, glia were viewed as a glue among neurons. During the course of the
twentieth century, microglia were discovered and neuroscientists’ views evolved
toward considering glia only as auxiliary cells of neurons. However, over the last two
to three decades, glial cells’ importance has been reconsidered because of the evidence
on their involvement in defining central nervous system architecture, brain metabo-
lism, the survival of neurons, development and modulation of synaptic transmission,
propagation of nerve impulses, and many other physiological functions. Furthermore,
increasing evidence shows that glia are involved in the mechanisms of a broad
spectrum of pathologies of the nervous system, including some psychiatric diseases,
epilepsy, and neurodegenerative diseases to mention a few. It appears safe to say that
no neurological disease can be understood without considering neuron–glia crosstalk.
Thus, this book aims to show different roles played by glia in the healthy and diseased
nervous system, highlighting some of their properties while considering that the
various glial cell types are essential components not only for cell function and inte-
gration among neurons, but also for the emergence of important brain homeostasis.
R. von Bernhardi (&) B. Flores

Department of Neurology, School of Medicine, Pontificia Universidad
Católica de Chile, Marcoleta 391, Santiago, Chile
e-mail: [email protected]
J. Eugenín-von Bernhardi
Pettenkoferstr.12, 80336 Munich, Germany
Graduate School of Systemic Neuroscience, Ludwig-Maximilians-University, 82152
Planegg-Martinsried, Munich, Germany
J.E. León
Department of Biology, Faculty of Chemistry and Biology, USACH, Santiago, Chile
© Springer International Publishing Switzerland 2016 1

R. von Bernhardi (ed.), Glial Cells in Health and Disease of the CNS,
Advances in Experimental Medicine and Biology 949,
DOI 10.1007/978-3-319-40764-7_1
2 R. von Bernhardi et al.
Keywords Astrocytes
Microglia
Myelin
Development Neuron–glia

crosstalk Neuroimmunity NG-2 cells Oligodendrocyte Schwann cells
Tripartite synapses
Abbreviations
ADNF Activity-dependent neurotrophic factor
ADAM10 A disintegrin and metalloproteinase domain-containing protein 10
AMPA α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid
ATP Adenosine triphosphate
CNS Central nervous system
EAAT Excitatory amino acid transporters
EGF Epidermal growth factor
ER Endoplasmic reticulum
FcR Receptor for the (Fragment, crystallizable) region of antibodies
GABA γ-aminobutyric acid
GDNF Glial cell-derived neurotrophic factor
GFAP Glial fibrillary acidic protein
IGF-I Insulin-like growth factor 1
iNOS Inducible nitric oxide synthase
InsP3 Inositol trisphosphate
MAG Myelin associated glycoprotein
MBP Myelin basic protein
MOG Myelin oligodendrocyte glycoprotein
MS Multiple sclerosis
NCAM Neural cell adhesion molecule
NG-2 Neuron–Glia antigen 2
NMDA N-methyl-D-aspartate
NO Nitric oxide
OPCs Oligodendrocyte precursor cells
PLP Proteolipidprotein/DM20
PNS Peripheral nervous system
PMP22 Peripheral myelin protein-22
P0 Protein zero
ROS Reactive oxygen species
TNFα Tumor necrosis factor α
VGLUT Vesicular glutamate transporters
VNUT Vesicular nucleotide transporters
Glial Cells and Integrity of the Nervous System 3
The Concept of Neuron–Glia Crosstalk
The discovery of neurons as the basic functional unit of the nervous system towards
the end of the nineteenth century was a milestone in neuroscience research in
shaping our view of the functional organization of the nervous system. While
neurons have been studied continuously since then, extensive glial research has
been seemingly in steps, with the greatest understanding developed only over the
last few decades. In the middle of the twentieth century, the first studies on glial
physiology were done by Kuffler and Nicholls (1966). Recordings from the far
smaller microglia came several decades later, and consistent with recent knowledge
of their separate origin from the astrocytes and oligodendrocytes (Ginhoux et al.
2010; Rowitch and Kriegstein 2010), they are physiologically quite distinct. For
nearly a century, the main view was that neurons were the only cells responsible for
nearly all the complex functions of the nervous system, whereas glia were just
relatively passive support cells (Halliday and Stevens 2011). Recent decades have
seen a major shift in understanding of the roles particularly of astrocytes and
microglia.
The development of animal models for many neurological and psychiatric dis-
eases has enabled study of the cellular responses in various pathological stages. It
appears that there are no neuropathological processes that occur without partici-
pation of glial cells, specifically microglia and astrocytes (Halliday and Stevens
2011; Quintanilla et al. 2012; Verkhratsky et al. 2014; Jha et al. 2015). Interest in
glial cells has also been potentiated by the evidence showing glial responsiveness,
leading to gliotransmission for the propagation of information among glial cells
(Zorec et al. 2012) and eventually to neurons (Vesce et al. 1999; Araque 2008;
Hamilton and Attwell 2010). This form of communication is much slower than the
neuronal response; however, glial cells, and astrocytes in particular, appear to be
able to modulate neuronal activity (Jirounek et al. 2002; Araque 2008) and thus
brain function. Evidence gathered over the last couple of decades and data obtained
earlier show that glia have important functions of their own in addition to their
effects on neuronal function.
Based on their broad spectrum of characteristics and functions, glial cells are
probably some of the most versatile cells in our body. Here we will discuss the
participation of the NG2–glia progenitor cells, oligodendrocytes, Schwann cells,
astrocytes, and microglia, both in the central (CNS) and peripheral nervous system
(PNS). All are important players during development and for neural function in the
adult animal. All cell types, including neurons and glia, show both passive and
active support roles for each other (Fields and Stevens-Graham 2002; Ramirez et al.
2005; Vecino et al. 2015). For instance, considerable evidence now indicates that
neurons provide trophic support and regulate glial activation (Ramírez et al. 2008;
Biber et al. 2011).
Astrocytes
Astrocytes, which interact with neurons, blood vessels, and many structures of the
nervous system (Abbott et al. 2006; Cheslow and Alvarez 2016), are the most abun-
dant glial cells in many parts of the CNS (Pakkenberg and Gundersen 1988). The ratio
of glia to neurons varies from one brain region to another (Pakkenberg and Gundersen
1988), and it is still amply discussed (Hilgetag and Barbas 2009). Astrocytes are
involved in synaptic transmission (Newman 2003; Croft et al. 2015; Gittis and Brasier
2015) and the regulation of neuronal processing. They remove extracellular potassium
and neurotransmitters from the synaptic space, and participate in the energy metabo-
lism (Araque et al. 1999; Perez-Alvarez and Araque 2013; Perea et al. 2009;
Reichenbach and Bringmann 2013). They are key partners for synaptogenesis, synapse
function, and synaptic plasticity (Chung et al. 2015; Gittis and Brasier 2015).
Although named for their star-like shape, astrocytes are difficult to characterize.
They can have various shapes and expression of identity markers, including the
characteristic intermediate filament glial fibrillary acidic protein (GFAP).
Astrocytes display a remarkable heterogeneity in their morphology and function
(Oberheim et al. 2012). Conceptually, astrocytes in different brain regions can have
different physiological properties (Bayraktar et al. 2015; Schitine et al. 2015). For
example, only brainstem astrocytes appear to have the ability to sense changes in
PCO2 and contribute as mediators of the respiratory response to hypercapnia in
mammals (Gourine et al. 2010).
Two major morphologies are recognized:

(a) Protoplasmic astrocytes are in gray matter and have many fine processes, most
of them elaborate and complex. Their processes contact blood capillaries,
establishing perivascular endfeet, and contact neurons (Abbott et al. 2006;
Hawkins and Davis 2005). The complex astrocyte–neurons–blood vessel is
known as the neurovascular unit and form the blood–brain barrier (Hawkins
and Davis 2005). Some protoplasmic astrocytes also form subpial endfeet at
the pial surface.
(b) Fibrous astrocytes are in white matter (Hristova et al. 2010). Their processes
are long (up to 300 µm), but much less elaborate than those of protoplasmic
astroglia. The processes of fibrous astrocytes establish several perivascular or
subpial endfeet. Fibrous astrocyte processes also send numerous extensions
(‘perinodal’ processes) that contact oligodendroglia wrapped myelinated
axons at nodes of Ranvier (Butt et al. 1994).
Other regions of the CNS contain distinctive populations of astroglial
cells: velate astrocytes in the cerebellum, where they form a sheath surrounding
granule neurons; interlaminar astrocytes in the cerebral cortex of higher pri-
mates; tanycytes in the periventricular organs, the hypophysis and the raphe
nucleus; pituicytes in the neuro-hypophysis. Perivascular and marginal astro-
cytes are localized very close to the pia mater, where they form numerous endfeet,
establishing the pial and perivascular glia limitans barrier, which assists in isolating
the brain parenchyma from the vascular and subarachnoid compartments.
There are important groups of astroglia, including radial glia (Noctor et al. 2001;
Hansen et al. 2010) in the developing brain, which are bipolar progenitor cells with
one main process forming apical endfeet at the ventricular wall and the other, basal,
at the pial surface (Noctor et al. 2001). Radial glia form a scaffold, which guides
neuronal migration from the ventricular zone (Hartfuss et al. 2001) after they divide
asymmetrically in a self-renewing fashion to generate neurons and then glia—first
astrocytes and then oligodendroglia. In humans, they also give rise to progenitors
that maintain only a basal process that accounts for the majority of neurons and glia
in the cerebral cortex. It is believed that radial glial cells remain in the retina as
Müller glia (Reichenbach and Bringmann 2013), and in the cerebellum
as Bergmann glia. Some astrocytes in neurogenic niches of the adult brain, retain
stem cell properties throughout life and are the source for the adult neuro- and
gliogenesis (Riquelme et al. 2008; Shimada et al. 2012).
Astrocytes have many functions: they generate the brain environment, establish
the microarchitecture of the parenchyma, maintain brain homeostasis, generate,
store and distribute energy substrates, control the development of neurons,
synaptogenesis and synaptic efficacy and maintenance, and participate in brain
defense. Astrocytes define the microarchitecture of the parenchyma in the mam-
malian brain by dividing the grey matter, through the process called “tiling”, into
relatively independent structural units, within the limits of their processes.
Furthermore, the individual astrocytes are integrated into functional syncytia
through gap junctions between their processes (Wallraff et al. 2006).
Astrocytes and the Tripartite Synapsis
The close relation between astrocytes and synapses as well as their functional
expression of relevant receptors prompted the “tripartite synapse” hypothesis
(Volterra and Meldolesi 2005; Santello et al. 2012). In the gray matter, astrocytes
are closely associated with synaptic regions, enwrapping presynaptic terminals and
postsynaptic structures (Vesce et al. 1999). Astrocyte membranes enwrap about
80 % of large synapses, being probably the most functionally active, but only about
half of the small ones. In the cerebellum, nearly all of the synapses formed by
parallel fibers on the Purkinje neuron are covered by the Bergmann glial cells.
According to the “tripartite synapse” hypothesis, synapses are comprised of
three parts, the presynaptic terminal, postsynaptic membrane, and associated
astrocyte. The astrocyte processes possess neurotransmitter receptors. The receptors
expressed by astrocytes match the neurotransmitters released at the synapses they
cover. In the cortex both pyramidal neurons and neighboring astrocytes express
glutamate and purinergic receptors (Koizumi et al. 2005), whereas in the basal
ganglia both neurons and astrocytes are sensitive to dopamine. Neurotransmitters
released by the presynaptic terminal, which themselves have metabotropic receptors
for the transmitters they release, activate receptors in both the postsynaptic neuron
and the associated astrocyte. This results in the generation of a postsynaptic
potential in the postsynaptic neuron and a Ca2+ signal in the astrocyte (Vesce et al.
1999). Ca2+ signals can propagate through the astrocytic syncytium, and could also
trigger the release of neurotransmitters from neighbor astrocytes (Malarkey and
Parpura 2008; Lalo et al. 2014), which in turn will signal onto both pre- and
postsynaptic neuronal membranes.
Astrocytes regulate the extracellular concentration of potassium ions, neuro-
transmitters and metabolites, and water movements (Newman 2003). A key func-
tion of astrocytes is the control of extracellular K+ (Kuffler and Nicholls 1966).
Synaptic activity increases K+ concentration from its resting level (3 mM) to 10–
12 mM under physiological conditions, and to even higher concentrations under
pathological conditions (Wallraff et al. 2006; Hansen and Nedergaard 1988). High
extracellular K+ increases neuronal reactivity.
Simultaneously, astrocytes remove most of the accumulated glutamate released
at the synapses from the extracellular space through excitatory amino acid trans-
porters (EAAT) (Anderson and Swanson 2000). Glutamate is the major excitatory
neurotransmitter in the CNS of vertebrates. When released in excess, glutamate
becomes excitotoxic and triggers neuronal cell death (Foran and Trotti 2009).
Astrocyte uptake of glutamate is crucial for glutamatergic neurons. Glutamate is
enzymatically converted into glutamine by the astrocyte-specific enzyme glutamine
synthetase (Schousboe et al. 2014). Glutamine can be safely transported to presy-
naptic terminals; and after entering the neuron, it is transformed into glutamate by
glutaminase. Astrocytes also possess the enzyme pyruvate carboxylase, and are a
key source for de novo glutamate synthesis. Thus, astrocytes have the machinery to
regulate the availability of glutamate, by both degrading it and generating new
neurotransmitter.
Metabolic Support by Astrocytes
Astrocytes provide some metabolic support for neurons. The glucose–lactate shuttle
hypothesis (see Pellerin et al. 2007) proposes that astrocytes take up glucose,
metabolize it to lactate, and release it as an energy substrate for neurons. In addi-
tion, astrocytes are the only brain cells that synthesize glycogen and thus govern the
energy reservoir.
Moreover, astrocytes are a part of the neurovascular unit (Hawkins and Davis
2005) that coordinates neural activity with local blood flow according to the
metabolic demands. Blood vessels are almost entirely covered by astrocyte endfeet,
with one arm at the blood vessel, and the other at the neuron soma, synapse, or
axon. Increased activity of neurons induces Ca2+ signals in astrocytes, which could
be integrated by the neurovascular unit, leading to the release of vasoactive agents
that regulate local blood flow (Zonta et al. 2003; Takano et al. 2006). Astrocytes
appear to link neuronal activity and blood perfusion, although reports are
contradictory. Some groups report that astrocyte activity leads to local vasocon-
striction, while others report vasodilatation.
Astrocytes in Synaptogenesis and Synaptic Maintenance
Astrocytes regulate formation, maturation, maintenance, and stability of synapses.

They secrete numerous factors, including thrombin, needed for synaptogenesis
(Christopherson et al. 2005; Diniz et al. 2012). Synaptogenesis is also affected by
glial signals regulating the expression of agrin, essential for synapse formation
(Faissner et al. 2010). Later in life, astrocytes participate in the maturation of
synapses. Several soluble molecules released by astrocytes are involved in the
regulation of synapses, including tumor necrosis factor α (TNFα), which regulates
the insertion of glutamate receptors and activity-dependent neurotrophic factor
(ADNF), which increases the density of NMDA receptors in postsynaptic mem-
branes (Slezak and Pfrieger 2003).
Astrocytes appear also to be capable of limiting the number of synapses. They
ensheath the neuronal processes blocking the formation of synapses, as well as
being involved in the elimination of synapses, which is the basis for the final tuning
and plasticity of neuronal communication (Chung et al. 2013, 2015). Elimination of
synapses is achieved by secretion of certain factors and proteolytic enzymes that
degrade the extracellular matrix and reduce the stability of the synaptic contact.
Subsequently, astrocyte processes invade the synaptic cleft and substitute for the
missing neuronal synaptic element. This process is especially robust in neu-
ropathological conditions.
Signaling in Astrocyte Networks and Gliotransmission
Glutamate metabotropic receptors in astrocytes activate intracellular signaling

cascades, providing a cell activation mechanism. Their excitability depends on the
response of the Ca2+ channels on the endoplasmic reticulum (ER): InsP3 receptors
and ryanodine receptors. Stimulation of astrocytes metabotropic receptors induces
formation of InsP3, which in turn triggers Ca2+ release from the ER, increasing
intracellular Ca2+ levels (Hua et al. 2004). These Ca2+ signals are generally asso-
ciated with glial activation. Astrocytic Ca2+ signals can propagate through astrocyte
networks, generating intercellular Ca2+ waves depending on the diffusion of InsP3
through gap junctions, as well as the release of ATP from astrocytes (Beck et al.
2004; Scemes and Giaume 2006). Gap junctions may also be the signaling pathway
in astrocyte networks, which involve various second messengers, metabolic sub-
strates, and other molecules (Scemes and Giaume 2006; Wallraff et al. 2006).
As discussed in Chapters “Pharmacological Tools to Study the Role of

Astrocytes in Neural Network Functions”, “Physiological Functions of Glial Cell
Hemichannels”, and “Role of Astrocytes in Central Respiratory Chemoreception”,
astrocytes as well as other glia release a variety of gliotransmitters (Santello et al.
2012; Petrelli and Bezzi 2016), which include several molecules also utilized by
neurons, such as glutamate, ATP, GABA, and D-serine, but also taurine and
kynurenic acid, which appear to be exclusively released by glia (Rassoulpour et al.
2005). Different mechanisms of release of gliotransmitter have been described
including: (i) diffusion through permeability channels, like volume-activated Cl−
channels, unpaired connexin “hemichannels”, pannexin channels, or P2X7
pore-forming purinoceptors; (ii) through transporters, e.g., by reversal of EAAT or
exchange via the cystine-glutamate antiporter or organic anion transporters; and
(iii) through Ca2+-dependent exocytosis.
Astrocytes have been proposed to undergo exocytosis because they express
proteins involved in exocytosis such as synaptobrevin 2, syntaxin 1, and
synaptosome-associated protein of 23 kDa (Ropert et al. 2015). Astrocytes also
express transporters required for neutotransmitter accumulation in synaptic vesicles,
including the vacuolar type of proton ATPase (V-ATPase), vesicular glutamate
transporters (VGLUTs) 1, 2, and 3, and vesicular nucleotide transporters (VNUT)
(Ni and Parpura 2009; Morel et al. 2014). Exocytotic release of transmitters by
astrocytes is mediated by Ca2+-dependent exocytotic glutamate and can affect
several neuronal responses including increased neuronal Ca2+ influx and generation
in neurons of a slow inward current mediated by NMDA receptors (Montes de Oca
Balderas and Aguilera 2015). Thus, glutamate released from astrocytes can affect
neuronal excitability, modulate synaptic transmission, and synchronize synaptic
events, therefore potentially modifying behavior (Oliveira et al. 2015).
Astrocytes in Neuropathology
By the end of the nineteenth century, the neuropathological potential of glia was
proposed by such prominent neuropathologists, as Carl Frommann, Franz Nissl,
Alois Alzheimer, and Pio del Rio-Hortega. Nevertheless, the participation of glia
remains poorly understood. As described, astrocytes are fundamental for brain
homeostasis, and they also serve an important part of the nervous system defense
system (Posada-Duque et al. 2014). Brain insults trigger a response of astrogliosis
(Pekny and Pekna 2014), which is essential for limiting the area of damage by scar
formation, for post-insult remodeling and for recovery of neural function. Astrocyte
activation and dysfunction are apparent in all types of brain pathologies: acute
lesions (trauma or stroke), developmental neurometabolic disorders (see Chapter
“Astrocyte Dysfunction in Developmental Neurometabolic Diseases”), psychiatric
diseases, and neurodegenerative processes, such as Alzheimer’s disease,
Parkinson’s disease, and multiple sclerosis (MS).
Oligodendrocytes and Schwann Cells
Oligodendrocytes in the CNS and Schwann cells in the PNS produce myelin that
provides insulation for axons (Bercury and Macklin 2015), and are thus responsible
for the high-speed information propagation in axons of vertebrates. Therefore, the
cells are active partners for neurons for the propagation of information. Myelin
sheaths are produced in response to neuronal activity (Grigoryan and Birchmeier
2015). In addition to providing axon insulation, the glia provide trophic support,
affect the structure of axons, and modify their electrical properties by controlling
their diameter, and the clustering of specific ion channels at the node and paranode
region (Poliak and Peles 2003).
In the periphery, Schwann cells also serve key functions for the regeneration of
axons, and at the neuromuscular junction (Love and Thompson 1998; Love et al.
2003; Lee et al. 2016), given the absence of astrocytes in the PNS, undertake
similar functions at the synapses (Poliak and Peles 2003) (see Chapters
“Oligodendrocytes: Functioning in a Delicate Balance Between High Metabolic
Requirements and Oxidative Damage” and “Schwann Cell and Axon: An Interlaced
Unit—From Action Potential to Phenotype Expression” for further reading on
oligodendrocytes metabolism and the functional effect of the interaction axon–
Schwann cell). There are also other specialized cells. Neuronal somata in sensory
sympathetic and parasympathetic ganglia are surrounded by flattened satellite cells,
and neuromuscular junctions are also covered by the terminal glia, a specialized
Schwann cell (Connor and McMahan 1987; Kang et al. 2014).
Myelin insulation is needed for high-speed nerve conduction (up to 200 m/s) in
vertebrates, although in an invertebrate, as the shrimp, could be even higher without
the usual myelin (Xu and Terakawa 1999). Most oligodendrocytes are located in the
white matter to form myelin. However, they are also found in gray matter and possibly
regulate ionic homeostasis similar to astrocytes. Furthermore, axon–glia interaction is
important for neuron regulation also in unmyelinated nerves (Jirounek et al. 2002).
Myelination is observed in all jawed vertebrates and can be traced back to 400
million years ago, and even a few invertebrates show “loose” myelin sheaths. In
fact, the occurrence of myelin in evolution potentiated the neural development of
vertebrates. Myelin plays a key role for allowing neurons to be interconnected in
the complex fashion observed in the vertebrate nervous system (de Hoz and Simons
2015). In general and for biophysical reasons, only axons larger than 1 µ are
myelinated (Rushton 1951; Waxman and Bennett 1972). Recent reports show that
axons signal to the oligodendrocyte to determine the thickness of the myelin sheath
(Bozzali and Wrabetz 2004). A key signaling mechanism provided by the axon is
via the growth factor neuregulin-1, which binds to ErbB receptor tyrosine kinases,
is expressed by oligodendrocytes (Lemke 2006). A similar signaling mechanism
also exists in Schwann cells (Newbern and Birchmeier 2010; Salzer 2015).
Myelin forming oligodendrocytes have up to 40 processes, each forming a
segment of myelin called an internode that is several hundred micrometers long
around separate axons. Internodal segments are separated from each other by
specialized regions known as the node of Ranvier, which spans for around 1 μm. At
the node, the axon is not wrapped by myelin (Eshed-Eisenbach and Peles 2013).
The end of intermodal segment contains more cytoplasm forming the paranodal
loop creating special junctions with the axon that induce specific changes on the
axon in the node region. In addition, astrocyte processes contact the axonal
membrane at the nodal region in the CNS, whereas loose sheaths of Schwann cell
cover the node region in the PNS (Eshed-Eisenbach and Peles 2013).
In contrast to oligodendrocytes that each can form a myelin sheath around
several axons, each myelinating Schwann cell generates one segment of myelin
sheath for only one axon. Due to compaction, myelin dry mass is about 70 % lipids
and 30 % proteins. While the myelin structure formed by oligodendrocytes and
Schwann cells has a similar ultrastructure, it is not composed of an identical set of
proteins (Simons and Trotter 2007; Llorens et al. 2011). There are several highly
specific proteins that are needed for the formation of myelin. The major proteins of
the CNS myelin are myelin-associated glycoprotein (MAG), myelin basic protein
(MBP), myelin oligodendrocyte glycoprotein (MOG), proteolipidprotein (PLP)/
DM20, and peripheral myelin protein-22 (PMP22), whereas CNS and PNS myelin
share the MBP, the PNS lacks MAG and PLP, but expresses the protein zero (P0)
and PMP22 (Simons and Trotter 2007; Llorens et al. 2011).
Myelination and Saltatory Nerve Conduction
Like astrocytes, oligodendrocytes are also interconnected by gap junctions formed

by connexins, although connexin proteins for oligodendrocytes and astrocytes are
distinct. Connexins are key components for the structure and function of myelin. In
fact, mutations in certain connexin proteins lead to hypomyelination and are
involved in several human pathologies (Kleopa et al. 2010).
The axon’s node of Ranvier contains a high density of sodium channels, which
allows for a fast inward Na+ current capable of generating an action potential only
at the node, in what is known as saltatory conduction (Schafer and Rasband 2006).
As discussed in Chapter “Oligodendrocytes: Functioning in a Delicate Balance
Between High Metabolic Requirements And Oxidative Damage”, once the action
potential is triggered at the node, it spreads passively and rapidly to the next node,
where the next action potential is generated. This is not only faster, but consumes
much less energy, since Na+ gradients must be restored only at the node, reducing
the amount of Na+ to be transported out by the Na+/K+-ATPase. At the time of
ensheathment, Na+ channels start to cluster at the site of the future node of Ranvier,
promoted by protein interactions between myelin and the axonal membrane
involving cell adhesion molecules like gliomedin, neurofascin, and neural cell
adhesion molecule (NCAM). K+ channels, involved in repolarization, are concen-
trated very close, in the juxta paranodal region (Scherer 1999; Poliak and Peles
2003). In contrast, in non-myelinated axons, voltage sensitive sodium and potas-
sium channels are distributed along the whole surface of the axon.
Myelination Impairment and Disease
As discussed in Chapters “Peripheral Inflammation and Demyelinating Diseases”,

“Regulation of Oligodendrocyte Differentiation and Myelination by Nuclear
Receptors: Role in Neurodegenerative Disorders”, and “The Role of Galectin-3:
From Oligodendroglial Differentiation and Myelination to Demyelination and
Remyelination Processes in a Cuprizone-Induced”, the most frequent disease affect-
ing oligodendrocytes is MS. It is caused by the loss of myelin in areas of the brain and
spinal cord resulting in the impairment of axonal action potential propagation (Olsen
and Akirav 2015). Remyelination is observed, but often relapses occur leading to
neurodegeneration as the disease progresses. The primary cause for the impairment of
oligodendrocytes is still unknown. Demyelinated regions contain inflammatory cells,
including infiltrating lymphocytes and macrophages, and activated microglia, which
appear to potentiate or even initiate the damage cascade (Tanaka and Yoshida 2014).
Most of the genetically determined pathologies are associated with mutations in
myelin proteins or connexins, the molecular entities forming gap junctions. Similar to
the CNS, mutations in Schwann cell myelin or gap junction proteins lead to neu-
ropathies such as Charcot–Marie–Tooth disease (Kleopa 2011).
NG2–Glia
As further discussed in Chapter “NG2–Glia, More Than Progenitor Cells”, NG2–

glia are progenitor cells in the immature and adult nervous system that give rise to
oligodendrocytes. Therefore, they are also called oligodendrocyte precursor cells
(OPCs) (Lopez Juarez et al. 2015). Although they appear to have the capacity to
differentiate into astrocytes and oligodendrocytes, the main route seems to be
confined to the oligodendrocyte lineage. However, they are found in the brain in
numbers much greater than would be needed for that role, and in brain areas where
oligodrendrogenesis does not often occur.
NG2–glia are a potential source for remyelination in demyelinating diseases
such as MS (Grade et al. 2013). There are distinct markers that allow one to identify
them, such as the transcription factor Olig-2 or the proteoglycan NG-2 (neuron/glia
antigen 2), a membrane-spanning signaling protein that is exclusively found in
NG2–glia in the CNS (Trotter 2005). NG2–glia appear to interact with neurons.
They express glutamate receptors and are capable of sensing neuronal activity. This
appears to be a potential mechanism for neurons to regulate the differentiation of
NG2–glia into oligodendrocytes.
NG2–glia in culture may be induced by certain growth factors to become
multipotent neural stem cells. It is important to note that most glial cells in the CNS
are not neural stem cells, but NG2–glia are a distinct subset. Other investigators
revealed the role of NG-2 in the communication between neurons and NG2–glia
(Sakry et al. 2014). They reported that a NG-2 fragment that is shed into the
extracellular matrix, is produced by activity-dependent cleavage of NG-2 by the

secretase ADAM10 and other secretases. This process generates a large ectodo-
main, containing two neurexin-like domains, which is released to the extracellular
medium, and two smaller pieces, which remain associated with the cell.
The physiological functions of NG-2 in the brain have been determined from
research in mice lacking NG-2. In the absence of NG-2, neuronal AMPA receptors
show altered subunit composition compared with normal mice, which is probably
responsible for the impaired LTP observed in the KO animals (Sakry et al. 2014).
When brain slices from NG-2 KO mice in culture are treated with a recombinant
protein containing the extracellular domain of NG-2 that is shed by cleavage, the
properties of neuronal AMPA receptors in the slices return to normal. Therefore, the
neurexin-like domains appear to be crucial for the modulation of neuronal function
by NG-2 cleavage.
NG-2 KO mice show behavioral deficits that depend on functioning somatosen-
sory cortex (Yang et al. 2013), which shows the relevance of the regulatory crosstalk
between neurons and NG2–glia. Although many questions remain to be answered,
recent evidence indicates that in addition to receiving input from neurons, NG2–glia
might modulate neuronal properties and, therefore, activity. These findings add
weight to the notion that glia are far from being only support cells. At this point, it is
becoming clear that neurons and glia talk constantly to each other.
Microglia
Microglia are the immune cells of the CNS, corresponding to the endogenous brain
defense system, and are responsible for CNS protection against diverse pathogenic
factors (Kettenmann et al. 2011). They derive from progenitors of mesodermal origin
that migrate into the nervous system halfway through development (Ginhoux et al.
2010). After invading the CNS, microglial precursors disseminate throughout the
neural parenchyma and acquire a distinctive phenotype that clearly distinguishes
them from the blood-derived monocytes. They are highly reactive and become
activated in response to any changes in the nervous system (Hanisch and Kettenmann
2007). They migrate to the site of damage, proliferate, and become phagocytes.
Microglia can also interact with the peripheral immune system by antigen presen-
tation and sense the presence of brain tumors (see Chapter “Microglia in Cancer: For
Good or for Bad?” for further reading on microglia–tumor interaction).
Under physiological conditions, microglia are the fastest moving cells in the
nervous system and send multiple thin processes that extend in all directions. Similar
to astrocytes, each microglial cell has its own territory, of around 50 µm diameter,
showing very little overlap between neighboring microglia. The processes of resting
microglial cells are constantly moving through its territory, with a speed of about
1.5 µm/min. In addition, processes also extend and retract small protrusions.
Through these mechanisms, microglia scan through their territory. Considering the
speed of this movement, the brain parenchyma can be completely scanned by
microglia several times each day (Hanisch and Kettenmann 2007). Although
microglia processes motility is not affected by neuronal activity per se, it is sensitive
to activators (ATP) and inhibitors of purinoceptors (Koizumi et al. 2013). In contrast,
focal neuronal damage induces a rapid and concerted movement of microglia toward
the site of lesion (von Bernhardi and Muller 1995; Cornejo and von Bernhardi 2013;
Duan et al. 2009). This injury-induced motility is regulated by activation of
purinoceptors (Honda et al. 2001), and it is sensitive to the inhibition of gap junctions.
Inhibition of gap junctions also affects motility of astrocytes processes, cells that also
extend processes defining specific territories. Thus, both microglia and astrocytes
show sophisticated scanning system that allows them to survey the environment. As
further discussed in Chapter “Microglia Function in the Normal Brain”, a feature that
exemplifies that microglia are capable of cross talk with neurons is the microglial cell
expression of fractalkine receptors. Fractalkine (CX3CL1), a 373 amino acids cy-
tokine protein member of the CX3C chemokine family, is found particularly in
neurons. CX3CL1 is upregulated in the hippocampus by spatial learning and is likely
to regulate glutamate-mediated neurotransmission.
Activation of Microglia
When an insult to the nervous system is detected by microglia, they launch a

specific program that results in the rapid transformation from surveillance typical of
ramified microglia into an ameboid shaped cell that may move toward a nearby site
of injury, a process referred to as ‘microglial activation’, similarly observed in
vertebrates (Raivich et al. 1999; Gerhard et al. 2006; Kettenmann et al. 2011; von
Bernhardi et al. 2015b) and invertebrates, like the leech (Samuels et al. 2013; Dahl
and Muller 2014). Activation involves multiple steps. In terms of morphological
changes, microglia retract their processes, which become fewer and thicker, and
increase the size of their cell bodies. Their motility is increased as they are recruited
to move toward the injury. Microglia reportedly can also proliferate, increasing
their numbers further at the injury site. In terms of function, microglia change the
expression pattern of many enzymes and receptors, and the production of immune
response molecules is induced (Colton 2009). If the damage is strong and persistent
enough, microglia become phagocytes (see Chapter “Microglia Function in the
Normal Brain”). Thus, microglial activation appears to be a complex and highly
coordinated set of changes. Furthermore, in contrast to the traditional view of the
polarized M1-M2 activation of microglia (Aguzzi et al. 2013; Crain et al. 2013), the
process of activation most likely corresponds to a wide spectrum of many specific
substates that will combine specific physiological features (Boche et al. 2003; von
Bernhardi 2007; Kettenmann et al. 2011; Aguzzi et al. 2013; von Bernhardi et al.
2015b). In fact, activated microglia display heterogeneous properties depending on
the pathological conditions and the region of the nervous system that is affected
(Goings et al. 2006), similar to what has been described for astrocytes.
The precise nature of signals triggering microglial activation is not well

understood (Kettenmann et al. 2011, 2013; Dahl and Muller 2014). It probably
depends both on the lack of trophic/regulatory molecules released during normal
CNS activity and the appearance of abnormal molecules (Biber et al. 2007).
Neurotransmitters are good examples of signals regulating microglial activation
(Hanisch and Kettenmann 2007; Pocock and Kettenmann 2007). Microglia express
several neurotransmitter receptors, such as receptors for GABA, glutamate, dopa-
mine, and noradrenaline. In general, activation of the receptors inhibits the
inflammatory activation of microglia. It could be speculated that decreased neuronal
activity could induce an “alerted” state in microglia. The other type of signals are
the appearance of abnormal molecules or abnormal concentrations of physiological
molecules, that indicate tissue deterioration (Inoue 2002; Minghetti et al. 2005;
Biber et al. 2007). Damaged neurons can release high amounts of ATP, cytokines,
neuropeptides, and growth factors. These factors can be sensed by microglia and
trigger activation (Inoue 2002; Duan et al. 2009). Both types of signaling provide
microglia information regarding the status of brain parenchyma (Samuels et al.
2013; Dahl and Muller 2014). Astrocytes (Orellana et al. 2013) and neurons
(Bernhardi and Nicholls 1999; Neumann 2001; Ramírez et al. 2008) provide robust
regulation of microglial activation.
Microglial activation may start with defense-oriented functions to fight off
pathogens or to limit progression of tissue damage after an injury. However, after
activation microglia may continue to show long-lasting changes. Epigenetic
mechanisms organizing long-lasting adjustments may cause previously activated
cells to behave differently after a second challenge (Orellana et al. 2014). Such
microglia might facilitate the development of dysfunction or could result in what is
observed as age-related impairment (Conde and Streit 2006; Block et al. 2007; von
Bernhardi 2007; von Bernhardi et al. 2011). As further discussed in Chapter “Age-
Dependent Changes in the Activation and Regulation of Microglia”, age-related
changes in the regulation of inflammatory activation could facilitate the develop-
ment of neurodegenerative diseases (Minghetti et al. 2005; Hart et al. 2012; von
Bernhardi et al. 2015a, b).
The functional heterogeneity of microglia must be considered. For example, a
myelinated environment is a different setting for microglia than that of gray matter.
White and gray matter microglia show different immunoregulation (Anderson et al.
2007). These differences could affect development and normal function, as well as
the response to inflammation (Hristova et al. 2010; Hart et al. 2012). Regional
heterogeneity of microglia is observed regarding their morphology, proliferative
behavior (Ladeby et al. 2005b; Marshall et al. 2008), transcription, and translation
of various constitutive and inducible molecules, including neurotrophins such as
IGF-I (Elkabes et al. 1996), cytokines, and other inflammatory mediators (TNFα,
IL6, nitric oxide (NO)), and membrane receptors (integrins, CD4, CD11c, CD34,
CD40, CD45, CD86, MHC class II, FcR) (Ford et al. 1995; Ren et al. 1999; Ladeby
et al. 2005a; Wirenfeldt et al. 2005; Sriram et al. 2006; Carson et al. 2007;
Davoust et al. 2008; Kawahara et al. 2009). Exposure to neurotransmitters, prox-

imity to blood vessels, and properties of the blood–brain carrier controlling the
microenvironment (Abbott et al. 2010) might be associated with differences
observed in microglial morphology and function (Davoust et al. 2008).
NO Production and Oxidative Stress
As previously mentioned, activated microglia can produce several inflammatory

mediators, including NO. NO is a lipid soluble radical gas that freely crosses cell
membranes and acts as a signaling molecule, participating in several biological
processes (Govers and Oess 2004; Pacher et al. 2007). NO is synthesized from L-
arginine and molecular oxygen by NO synthases (NOS). There are three isotypes of
NOS: endothelial NOS (eNOS), neuronal NOS (nNOS), and inducible NOS (iNOS).
The first two are constitutively expressed in endothelial cells and neurons, respec-
tively, while the latter is induced in macrophages, microglia, astrocytes, and other
cell types in response to inflammatory mediators including bacterial lipopolysac-
charides (Forstermann and Sessa 2012; Govers and Oess 2004; Pacher et al. 2007).
NO has an extremely short half-life of only a few seconds due to its fast reaction
with other molecules. Many of its oxidation products have been reported to have
cytotoxic effects, and indeed its induction in the presence of bacteria reflects its
bacteriocidal effect. Simultaneous production of NO and superoxide by microglia
can form peroxynitrite, a powerful oxidant able to irreversibly inhibit mitochondrial
respiration, cause DNA fragmentation and lipid oxidation, and induce neuronal
death (Mander and Brown 2005; Moncada and Bolanos 2006; Brown 2010; Brown
and Neher 2010). Importantly, oxidative stress produced by the accumulation of
reactive oxygen species (ROS) increases as organisms age, favoring the onset of
neurodegenerative disorders, as further discussed in Chapter “Age-Dependent
Changes in the Activation and Regulation of Microglia”.
Antigen Presentation
Microglial cells are the principal antigen presenting cells in the CNS. Under basal
conditions the expression of the molecular complex for presenting antigen, the
major histocompatibility complex II (MHCII) and co-stimulatory molecules such as
CD80, CD86 and CD40 are not detected. Upon stimulation the molecules are
highly upregulated (Perry 1998). This upregulation has been observed in several
pathologies (O’Keefe et al. 2002) including MS. By releasing cytokines such as
CCL2, microglial cells are important for recruiting cells into the CNS. As discussed
in Chapter “Microglia in Cancer: For Good or for Bad?”, microglia can interact
with infiltrating T lymphocytes (Ford et al. 1995) and, thus, mediate the immune
response in the brain.
Damage Versus Protection
Although research has mainly focused on the detrimental effects of microglia-

mediated neuroinflammation and oxidative stress, it is now accepted that acute
self-limited activation of microglia is essential for the structural and functional
integrity of the CNS. Microglia participate actively in the remodeling of synapses and
tissue repair (Trapp et al. 2007; Wake et al. 2009). They remove cell debris and
myelin fragments and secrete neurotrophins and cytokines for the survival of injured
neurons (Batchelor et al. 1999; Schwartz 2003). However, they can also produce
cytotoxic factors and are involved in the pathogenesis of several neurodegenerative
diseases (Brown 2010; Kim and de Vellis 2005). Therefore, their activity needs to be
strictly regulated (see Chapters “Microglia Function in the Normal Brain” and
“Purine Signaling and Microglial Wrapping” for further reading).
Recapitulation
For a long time, glial cells were considered to be a cell population subordinated to
neurons. However, this point of view has dramatically changed in recent decades
and growing evidence indicates that each type of glial cell-astrocytes, oligoden-
drocytes, Schwann cells, NG-2 cells and microglia plays key roles in the normal
functioning of the nervous system. It has also been demonstrated that they are
involved in the pathogenesis of neurological diseases. Thus, we can state that every
process carried out in our brain can only be understood if the interaction of neurons
and glial cells are taken into account. This challenges the accepted paradigm that
nervous system function results exclusively from neuronal network activity and
suggests that glial cell activity influences the neuronal activity outcome. Although
there are still several unresolved questions, current research is delivering interesting
and promising results.
Acknowledgments Supported by Grants Fondo Nacional de Desarrollo Científico y Tecnológico

(FONDECYT) 1130874 (JE) and 1131025 (RvB).
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Part I
Glial Cell Function in the Healthy Nervous
System
NG2-glia, More Than Progenitor Cells
Jaime Eugenín-von Bernhardi and Leda Dimou
Abstract NG2-glia are a mysterious and ubiquitous glial population with a highly
branched morphology. Initial studies suggested that their unique function is the
generation and maintenance of oligodendrocytes in the central nervous system
(CNS), important for proper myelination and therefore for axonal support and fast
conduction velocity. Over the last years this simplistic notion has been dramatically
changed: the wide and homogeneous distribution of NG2-glia within all areas of the
developing CNS that is maintained during the whole lifespan, their potential to also
differentiate into other cell types in a spatiotemporal manner, their active capability
of maintaining their population and their dynamic behavior in altered conditions
have raised the question: are NG2-glia simple progenitor cells or do they play
further major roles in the normal function of the CNS? In this chapter, we will
discuss some important features of NG2-glia like their homeostatic distribution in
the CNS and their potential to differentiate into diverse cell types. Additionally, we
will give some further insights into the properties that these cells have, like the
ability to form synapses with neurons and their plastic behavior triggered by
neuronal activity, suggesting that they may play a role specifically in myelin and
more generally in brain plasticity. Finally, we will briefly review their behavior in
disease models suggesting that their function is extended to repair the brain after
insult.
Keywords NG2-glia
Myelination Oligodendrocytes Neuronal activity

NG2-glia neuronal synapse Disease Injury Proliferation Differentiation
Plasticity
J. Eugenín-von Bernhardi (&) L. Dimou (&)

Großhaderner Str. 9, 82152 Planegg-Martinsried, Germany
L. Dimou
Graduate School of Systemic Neuroscience, Ludwig-Maximilians-University,
82152 Planegg-Martinsried, Germany

DOI 10.1007/978-3-319-40764-7_2
28 J. Eugenín-von Bernhardi and L. Dimou
Abbreviations
Ab Amyloid protein b
AD Alzheimer’s disease
AMPA a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid
AMPAR AMPA receptor
Ascl1 Achaete-scute homolog 1
aScTX A-scorpion toxin
BrdU 5-bromo-2′-deoxyuridine
Cavs Voltage-gated calcium channels
CC1 Adenomatous polyposis coli
DNQX 6,7-dinitroquinoxaline-2,3-dione
EAE Experimental autoimmune encephalomyelitis
EdU 5-ethynyl-2′-deoxyuridine
EPSC Excitatory postsynaptic current
GABAAR c-aminobutyric acid receptor
GPR17 G-protein coupled receptor 17
K vs Voltage-gated potassium channels
LPC a-lysophosphatidylcholine
Mash1 Mammalian achaete-scute homolog 1
MCAO Middle cerebral artery occlusion
mEPSC Miniature EPSC
Navs Voltage-gated sodium channels
NBQX 2,3-dihydroxy-6-nitro-7-sulphamoyl-benzo(F)quinoxaline
NG2 Neuron/glia antigen 2
NMDAR N-methyl-D-aspartate receptor
OPCs Oligodendrocyte progenitor cells
PDGF Platelet-derived growth factor
PDGFR PDGF receptor a
PFC Prefrontral cortex
PLP Proteolipid protein
PSD-95 Postsynaptic density protein 95
TeNT Tetanus neurotoxin
TTX Tetrodotoxin
NG2-glia, More Than Progenitor Cells 29
NG2-glia in the Central Nervous System
In the mammalian central nervous system (CNS), oligodendrocytes that build the
myelin, develop from a progenitor cell population during late gestational and early
postnatal life (Miller 1996) (see Section “A Brief Introduction to Oligodendrocyte
Structure and Function” in Chapter “Oligodendrocytes: Functioning in a Delicate
Balance Between High Metabolic Requirements and Oxidative Damage,” for a
description on oliyodendro genesis). These progenitors are known as oligoden-
drocyte progenitor cells (OPCs) during development and as NG2-glia at later
stages, becoming the fourth major group of glial cells in the CNS. The name
NG2-glia derives from the expression of the chondroitin sulfate proteoglycan
neuron/glia antigen 2 (NG2) (Fig. 1a–c) on their cell surface. To distinguish these
over the pericytes of the CNS that also express NG2 (Ozerdem et al. 2001), we use
the term NG2-glia instead of simply NG2-positive cells. NG2-glia can also be
found in the literature as polydendrocytes, because of their branched morphology
revealed by the immunolabeling for NG2 and the platelet-derived growth factor
receptor a (PDGFRa) (Fig. 1a, b and d) and as OPCs due to their association to the
generation and maintenance of the oligodendrocyte population under physiological
and pathological conditions. However, restricting NG2-glia to be simply
Fig. 1 a Confocal microscopy image showing the multiprocess morphology of NG2-glia

co-expressing NG2 and PDGFRa. Scale bar represents 50 µm. b–d Magnification of two
NG2-glia; b co-expressing NG2 and PDGFRa, c expressing NG2 and d expressing PDGFRa.
Scale bar represents 10 µm
oligodendrocyte progenitors does not give them enough credit. In fact, it has not
only been shown that NG2-glia represent the major proliferative cell population in
the healthy adult brain (Psachoulia et al. 2009; Simon et al. 2011; Dimou and Götz
2014) outside the neurogenic niches but also that they can self-renew. In vitro and
in vivo fate mapping experiments suggest that they can also give rise to a sub-
population of astrocytes in the ventrolateral forebrain during development (Raff
et al. 1983; Zhu et al. 2008) and, strongly controversial, to neurons in the adult
brain within restricted areas, like the piriform cortex and the hypothalamus (Guo
et al. 2010; Robins et al. 2013) (Fig. 2). Nevertheless, the neuronal progeny of
NG2-glia has been highly questioned in the field, as these results failed to replicate
in several other mouse models genetically fate mapping NG2-glia (Dimou et al.
2008; Kang et al. 2010) leaving the question open if they are really capable to
differentiate into neurons at least under physiological conditions.
NG2-glia represent around 5–10 % of the total cell population in the developing
and adult brain and they are evenly distributed within the cerebral and cerebellar
gray and white matter (Dawson et al. 2003) (Fig. 1a). Furthermore, NG2-glia can
proliferate and differentiate into mature oligodendrocytes throughout life, although
both their proliferation and differentiation rates depend on the area of the nervous
system and decrease with age (Psachoulia et al. 2009; Kang et al. 2010; Zhu et al.
2011). Previous studies have supported the idea that NG2-glia can divide inde-
pendently of extracellular signals to maintain their population and that they are
Fig. 2 Scheme representing the different cell fates of NG2-glia. Canonically, NG2-glia have the
capability to proliferate and differentiate into oligodendrocytes in the immature and mature brain.
However, NG2-glia can also differentiate into astrocytes in the ventrolateral forebrain during
development. Additionally, some studies have suggested that, in the mature brain, NG2-glia could
also differentiate into neurons, nevertheless, these claim is still under strong criticism
capable to proliferate and migrate short distances within the intact brain par-
enchyma, processes that are highly regulated by self-repulsion among NG2-glia
(Hughes et al. 2013). Moreover, when a NG2-glia differentiates into an oligoden-
drocyte or after focal laser ablation of individual NG2-glia, neighboring NG2-glia
proliferate and/or migrate in order to fill the “NG2-glia-free gap” in the mammalian
and zebrafish CNS (Kirby et al. 2006; Hughes et al. 2013; Birey and Aguirre 2015).
Furthermore, NG2-glia can divide asymmetrically giving origin to one NG2-glia
and one oligodendrocyte (Hill et al. 2014), a common mechanism that progenitors
have to differentiate without altering their total number. This feature, that is
homeostatically controlled, opens several questions, e.g., why is a constant popu-
lation of NG2-glia needed in the adult brain, even in areas where only few oligo-
dendrocytes exist? The assumption that NG2-glia solely act as progenitors for
oligodendrocytes has therefore been strongly questioned over the last years and the
hypothesis for a further active and functional role of NG2-glia in the healthy and
diseased brain has been supported.
Unexpectedly, other important features have been assigned to NG2-glia that
until some years ago were thought to be exclusive for neurons. In vitro and in vivo
studies could show that NG2-glia express voltage-gated sodium channels (Navs)
(Karadottir et al. 2008; De Biase et al. 2010) sensitive to tetrodotoxin (TTX),
voltage-gated potassium channels (Kvs), as well as low and high voltage-gated
calcium channels (Cavs) on their processes that get downregulated during their
maturation into oligodendrocytes (for review see Verkhratsky and Steinhauser
2000), revealing these channels to be required solely during their progenitor stage.
This wide expression of voltage-gated channels in NG2-glia suggests that these
cells may have dynamic electrical properties, and theoretically they have all the
components needed to generate and propagate action potentials. As a matter of fact,
electrophysiological characterization of white matter NG2-glia in the rat has
brought a thorough discussion regarding their possible ability to generate action
potentials. The study of Karadottir et al. (2008) could show the existence of two
subpopulations of NG2-glia within the white matter of the rat brain, proposing a
classification of spiking and non-spiking populations and suggesting the existence
of NG2-glia capable of generating action potentials (Karadottir et al. 2008).
However, spiking NG2-glia have not been found in other species besides the rat
white matter (Bergles et al. 2000; Karadottir et al. 2008; De Biase et al. 2010;
Clarke et al. 2012), suggesting that this feature could be specific for NG2-glia in
certain species or during certain developmental stages or both (Clarke et al. 2012).
Therefore, generation and propagation of action potentials by NG2-glia appear to
have no plausible role for the moment. Nevertheless, NG2-glia with different
electrical properties rise an interesting point in the field, the NG2-glia
heterogeneity.
NG2-glia Heterogeneity
Despite the even distribution of NG2-glia in the brain and their similar morphology,
it is accepted in the last years that NG2-glia represent a highly heterogeneous
population with diverse intrinsic properties and probably also distinct roles and
functions. The heterogeneous nature of NG2-glia could be shown regarding dif-
ferent aspects of these cells, e.g., their morphology as well as their differentiation
and proliferation properties. For instance, NG2-glia in the adult cerebral white
matter can differentiate into oligodendrocytes faster and more efficiently than those
located in the gray matter (Dimou et al. 2008). Subsequent homo- and heterotopic
transplantation experiments revealed that these diverse differentiation properties
between white and gray matter NG2-glia are the result of mainly intrinsic hetero-
geneity between these cells (Vigano et al. 2013). Interestingly, also their mor-
phology and process distribution show differences between NG2-glia in these two
areas (Vigano et al. 2013) (Fig. 3). NG2-glia in the white and gray matter have also
been described to be different in regard to their cell cycle length. Although all
NG2-glia can divide, gray matter cells have a longer cell cycle length than their
white matter counterparts, further supporting the idea of the heterogeneity of
NG2-glia (Psachoulia et al. 2009). Moreover, their response to extracellular signals
is also variable. For example, NG2-glia in the white matter show a stronger pro-
liferative response to platelet-derived growth factor (PDGF) than in the gray matter,
despite that both populations express comparable levels of the receptor PDGFRa
(Hill et al. 2013). Interestingly, in the last years NG2-glia heterogeneity has been
suggested not only between different regions but also within the very same brain
area. Indeed, some studies described the distinct expression of proteins only in
subsets of NG2-glia located in the same region. For example, only 50 % of
Fig. 3 NG2-glia are heterogeneous in regard to their morphology. Magnification of confocal

images showing NG2-glia from a the gray matter and b white matter of the brain, highlighting
clear morphological differences between both populations. Scale bar represents 10 µm
NG2-glia in the cortical gray matter express the transcription factor achaete-scute
homolog 1 or mammalian achaete-scute homolog 1 (Ascl1 or Mash1), an important
factor for neuronal fate determination (Parras et al. 2007). However, by now no
distinct roles could be assigned to these two populations. In the same line, also the
G-protein coupled receptor 17 (GPR17), that is involved in the differentiation of
NG2-glia (Boda et al. 2011; Chen et al. 2009), is only expressed in a subset of
NG2-glia in different regions and at different ages. Interestingly, it could be shown
that GPR17-positive cells represent a population of NG2-glia that differentiates
very slowly in the intact brain. However, after a cerebral damage, they rapidly react
and undergo maturation, suggesting a role as a “reserve pool” of adult progenitors
that are maintained for repair processes (Vigano et al. 2016). Whether all these
described differences between NG2-glia in distinct or even in the same area are the
result solely of an intrinsic program or micro-environmental influences restricting or
providing the cells special properties, is not clear; and the existence of evidences in
favor and against both ideas keeps the question still unanswered (for review see
Vigano and Dimou 2016).
Synapses Between NG2-glia and Neurons
In addition to the controversial possibility of generating action potentials, NG2-glia

have shown other features, which are also shared with neurons. The formation of
synapses between NG2-glia and neurons is a captivating observation. These
synapses were first observed in the mouse, where stimulation of neurons located in
the CA3 region of the hippocampus triggered an evoked excitatory postsynaptic
current (EPSC) of a sodium current nature in NG2-glia located in the CA1 region
(Bergles et al. 2000). Furthermore, the spontaneous fusion of transmitter-filled
vesicles that occurs at individual excitatory synapses between NG2-glia and neu-
rons resulting in miniature EPSCs (mEPSCs) has also been shown, a feature not
exclusively present in the hippocampus, but also in several other brain areas
(Bergles et al. 2000; De Biase et al. 2010). In the same line, the brief application of
the neurotoxin pardaxin, which enhances the frequency of vesicular release from
nerve terminals, caused the appearance of high frequency bursts of mEPSCs in
NG2-glia (Bergles et al. 2000), revealing a correlation between vesicular release
and the electric properties of NG2-glia. In both studies, these currents were abol-
ished by the application of the noncompetitive a-amino-3-hydroxy-5-methyl-4-
isoxazolepropionic acid (AMPA)/Kainate receptor antagonist 2,3-dihydroxy-6-ni-
tro-7-sulphamoyl-benzo(F)quino-xaline (NBQX) (Bergles et al. 2000; De Biase
et al. 2010), demonstrating the glutamatergic nature of these signals. Finally, the
existence of synapses between NG2-glia and neurons has also been confirmed by
transmission electron microscopy that revealed presynaptic release sites in the axon
membrane close to NG2-glia membrane (Bergles et al. 2000; Ziskin et al. 2007).
Notably, the complexity of these NG2-glia-neuron synapses is not limited to the
expression of the AMPA receptor (AMPAR), but extends to a wide variety of
typical postsynaptic receptors such as N-methyl-D-aspartate receptor (NMDAR)

(for review see Dzamba et al. 2013), acetylcholine receptor (De Angelis et al.
2012), c-aminobutyric acid receptor (GABAAR) (Von Blankenfeld et al. 1991;
Williamson et al. 1998) and others. Moreover, not only receptors can be found in
NG2-glia, but as transcriptome data also suggest, they may also express proteins
important for the establishment of a postsynapse like the postsynaptic density
protein 95 (PSD-95) (Sakry et al. 2011), one of the major components of the
postsynaptic density and important for synapse formation, maturation, and
remodeling (El-Husseini et al. 2000; Marrs et al. 2001). Nowadays, many questions
still remain unanswered regarding these synaptic glia-neuron structures. First, it is
neither clear if these synapses are only sending information from neurons to
NG2-glia in an unidirectional fashion or if they could also be part of a bidirectional
communication mechanism, nor if these synapses are present in all or in just a
subpopulation of NG2-glia. For example, it could also be that NG2-glia express
different channels and form synapses with neurons only at a specific timepoint of
their life when they need neuronal signals to perform specific functions like, e.g.,
differentiation or neuronal support. Moreover, neither the molecular composition of
these synapses nor the main functions that these glia-neuron structures may have,
have been elucidated (for review see Dimou and Gallo 2016).
Myelin Plasticity
Myelination of the nervous system enables fast electric conduction along the
myelinated axon and reduces the metabolic cost of neuronal activity; features
making myelination important for the proper function of the nervous system in
gnathostomates vertebrates and some invertebrates (Davis et al. 1999). Myelin
formation changes the electrical properties of axons by reducing the capacitance
and increasing the peripheral resistance of the surrounded axon. Myelin sheaths do
not cover the complete axon, but they are absent in short segments, structures
known as nodes of Ranvier, that express high levels of Navs, important for the
generation of action potentials in this area. This particular arrangement of myelin
along axons provides neurons with the structural basis for the saltatory action
potential propagation (Nave and Werner 2014).
In vertebrates, there are two cell types that have the unique ability to synthesize
large amounts of membrane that wrap and compact around axons to form myelin:
Schwann cells in the peripheral nervous system (PNS) and the oligodendrocytes in
the CNS. Although both cell types have the capability to myelinate axons, they
differ among other features enormously in regard to their developmental origin,
their protein composition, the number of myelinated axonal segments each cell can
establish, and the myelin periodicity. Therefore, this apparent structural and func-
tional similarity of PNS and CNS myelin is a superb example of convergent cellular
evolution of the nervous system (see Chapters “Oligodendrocytes: Functioning in a
Delicate Balance Between High Metabolic Requirements and Oxidative Damage”
and “Schwann Cell and Axon: An Interlaced Unit—From Action Potential to

Phenotype Expression” for further reading on oligodendrocytes and Schwann cells).
In the past, the two-dimensional nature of electron microscopy images had
promoted the notion that myelin is a static structure in our nervous system.
Nevertheless, lately it has be shown that myelin in the rodent brain is constantly
remodeling during lifespan, giving us a new perspective of highly plastic processes.
Different signals trigger changes in the compaction of myelin sheaths, in the
internode length (the areas of the myelinated axons flanked by the nodes of
Ranvier), in the number of sheaths surrounding one axon, and in the number of
axons that one oligodendrocyte can myelinate (Nave and Werner 2014).
Neuronal activity has shown to be an important factor in the remodeling of the
myelin along the axon. The inhibition of action potentials with TTX, which blocks
Navs currents, leads for example to a decrease in myelination in vitro and in the
mouse optic nerve in vivo (Demerens et al. 1996). Conversely, the stimulation of
neuronal activity with a-scorpion toxin (aScTX), which delays the inactivation of
Navs, or direct electric stimulation of neurons with an electrode, triggers the
increase of myelination in mixed oligodendrocyte-neuronal cultures (Demerens
et al. 1996; Gary et al. 2012). Furthermore, social deprivated adult mice showed
impaired myelination in the prefrontal cortex (PFC), an area which has been
associated with complex emotional and cognitive behavior (Liu et al. 2012).
Notably, neuronal activity dependent changes could also be observed in human
white matter, a structure that primarily consists of myelinated axons, cells of the
oligodendrocytic lineage, other glial cells, and no neuronal cell bodies. Diffusion
tensor imaging (DTI) studies have shown that various complex visual-motor tasks,
such as juggling or extensive piano playing, trigger changes in the white matter
architecture, by significantly increasing its size (Bengtsson et al. 2005; Scholz et al.
2009). Amazingly, it appears that neuronal activity is not just limited in increasing
the myelination of vertebrates’ axons. Recent studies in the zebrafish larvae spinal
cord have indeed shown that neuronal activity additionally provides a signal bias
for which axons must be myelinated. By in vivo time lapse imaging of the zebrafish
spinal cord, it was shown that while the initial axonal myelin wrapping is axon
activity independent, the stabilization and extension of the prospective myelin
sheaths depends on the activity-dependent secretion of axonal factors, like neuro-
transmitters and neurotrophic factors (Hines et al. 2015). Furthermore, blocking the
synaptic vesicle release with tetanus neurotoxin (TeNT) decreases the number of
myelinated axons and the total number of myelin sheaths per oligodendrocyte in the
zebrafish spinal cord (Mensch et al. 2015). Interestingly, similar results could be
obtained in rodents, where an activity independent myelination could be observed
first, followed by an activity dependent myelination triggered by neuregulin
(Lundgaard et al. 2013). Together, these studies show that neuronal activity has a
direct effect on the oligodendrocytes population, defining which and how many
axons must be myelinated.
Thus, one obvious potential role for NG2-glia-neuron synapses could be to
regulate the proliferation and differentiation of NG2-glia. The outcome of these
changes probably aims at improving or even modulating myelination of certain
Fig. 4 Scheme of the effect of neuronal activity on NG2-glia behavior. In response to a neuronal
activity, b neurons could release signals secreted from the axon or the soma; or secreted into the
synaptic cleft formed between neurons and NG2-glia. These signals could manipulate NG2-glia
c proliferation or d differentiation into oligodendrocytes. It is also possible that e after proliferation
the newborn NG2-glia differentiate. If new oligodendrocytes are generated, they could potentially
f myelinate surrounding axons and g change the electrical properties of neurons and therefore
modify the properties of the neuronal network
circuits, therefore enhancing the computational properties of networks required to

improve performance in certain specific tasks (Fig. 4).
NG2-glia Behavior and Dynamics Modulated

by Neuronal Activity
Although many studies have been performed regarding the influence of neuronal
activity on the cells of the oligodendrocyte lineage, it is still unclear whether this
happens in a direct or indirect manner. Early work showed that intraocular injection
of TTX diminishes the proliferation of NG2-glia close to axons of the retinal

ganglion cells by preventing the neuronal activity-dependent release of mitogens
(Barres and Raff 1993). NG2-glia can express a wide variety of purinergic receptors
and it has additionally been shown that neuronal activity induces adenosine release
by neurons and decreases NG2-glia proliferation (Stevens et al. 2002). It is there-
fore possible that other extra-synaptic molecules, whose release is activity depen-
dent, like growth factors, also play a significant role in the modulation of the
proliferation and differentiation of NG2-glia.
Apparently NG2-glia that are proliferating and differentiating could be also
modulated by signals provided directly by the neuro-glia synapses. It has been
shown that neurotransmitter release may have an important role in the development
of the oligodendrocyte lineage. Experiments in organotypic cultures of cerebellum
slices obtained from P6 mice have shown that the exposition to glutamate receptor
agonists like kainate and AMPA decrease the proportion of NG2-glia positive to the
proliferation marker 5-bromo-2′-deoxyuridine (BrdU), a thymidine analogue
incorporated into the cell during the S-phase of the cell cycle. In contrast, admin-
istration of the kainate and AMPA receptor antagonist 6,7-dinitroquinoxaline-2,3-
dione (DNQX) lead to an increase in cell proliferation (Yuan et al. 1998).
Interestingly, this evidence indicates that neuronal activity promotes changes in
the behavior of NG2-glia in both, a direct and indirect way. Unfortunately, the
investigation of this idea in in vivo experimental models can be very challenging. It
could be shown that high-frequency electrical stimulation applied in the medullary
pyramids of rats increases the proliferation and differentiation of NG2-glia in the
contralateral dorsal corticospinal tract (Li et al. 2010). However, the experimental
approach in this last study required the implantation of electrodes to promote
neuronal activity in vivo that inevitably resulted in injury and subsequently in
inflammation. This brings serious complications in the interpretation of these results
as NG2-glia show morphological and functional changes in response to damage,
by, e.g., increasing their proliferation rate, becoming hypertroph, polarizing toward
the injury, and migrating into the injury (Dimou et al. 2008; Simon et al. 2011;
Hughes et al. 2013; for review see Dimou and Götz 2014). Therefore, most
experiments in this field have been performed by promoting neuronal activity
through indirect methods.
Physical stimuli, provided by keeping mice under enriched environment con-
ditions, e.g., by adding running wheels or other toys into cages for two weeks,
trigger an increase in the differentiation of NG2-glia into oligodendrocytes, in the
motor and somatosensory cortex and in the amygdala (Simon et al. 2011; Ehninger
et al. 2011). Another strategy implemented in this field has been stimuli depriva-
tion, which also leads to changes in the behavior of NG2-glia. NG2-glia from the
barrel cortex of layer IV are functionally innervated by thalamocortical fibers
coming from the ventral basal thalamus, determining the distribution of these cells
in the walls of the barrel cortex and not in its core (Mangin et al. 2012). After
cauterizing mouse whiskers at birth, NG2-glia show an aberrant homogenous
distribution in the whole barrel cortex. Additionally, an increased fraction of
NG2-glia in the core of the barrel cortex is positive for Ki67, an active proliferation
marker. These results suggest the hypothesis that the thalamocortical fibers are
inhibiting the proliferation of NG2-glia in the core of the barrel cortex (Mangin
et al. 2012), resulting in the particular distribution of NG2-glia in the layer IV.
Furthermore, in another experimental approach, clipping the whiskers of adult mice
unilaterally induced a decrease in the total number of oligodendrocytes and an
increased expression of activated caspase-3, a marker for apoptosis, in oligoden-
drocytes derived from proliferating NG2-glia in the somatosensory cortex ipsilateral
to the clipped side (Hill et al. 2014). These results suggest that stimuli deprivation
not only compromises the differentiation of NG2-glia into oligodendrocytes but
also their survival (Hill et al. 2014).
New strategies to test how neuronal activity affects the dynamics of NG2-glia
in vivo are emerging, promising to provide data while avoiding the collateral effects
caused by CNS damage. The development of optogenetics, a technique in which
light-sensitive ion channels can be selectively expressed in a specific subpopulation
of neurons, which then can be activated through their exposure to a specific light
wavelength, could result in great advances in this area. A recent study using a
transgenic mouse line expressing the channel rhodopsin in neurons with an active
Thy1 promoter, allowed the stimulation of neurons located in the cortical layer V
and the analysis of the effects of this artificial stimulation in the dynamics of the
oligodendrocyte lineage (Gibson et al. 2014). Indeed, stimulation of neurons lead to
an increase in the number of cells positive for the thymidine analogue and prolif-
eration marker, 5-ethynyl-2′-deoxyuridine (EdU), in the neighborhood of the
stimulated neurons. A fraction of these EdU-positive cells were positive for Olig2, a
transcription factor used as a marker for the oligodendrocyte lineage (Gibson et al.
2014), for PDGFRa, a marker for NG2-glia (Dimou and Gotz 2014), and for the
adenomatous polyposis coli (CC1), a marker for mature oligodendrocytes (Gibson
et al. 2014). These results highlight the relation between the experimental increase
of firing rate and the increase of the proliferation and differentiation dynamics of the
oligodendrocyte lineage. Moreover, the study went even further and showed that
increased stimulation of neurons lead to an increase in myelination, specifically of
the axons from the stimulated neurons, leading to the improvement of
behavioral-motor function of these animals (Gibson et al. 2014).
The above described data, and the additional growing evidence consistently
show that neuronal activity, triggered by different physiological environmental or
artificial stimuli, efficiently promotes proliferation and differentiation of NG2-glia
in in vitro as well as in in vivo models. It has also been shown by correlation that
the modulation of this behavior can result in an improvement in the myelination of
axons belonging to neurons with increased activity and, therefore, improvement of
the fine tuning of neuronal networks which subsequently ameliorates tasks per-
formance depending on this specific network. However, although signals provided
by neurons to induce proliferation and differentiation and the consequences related
to the change in the NG2-glia behavior have been identified, the cellular and
molecular mechanisms being triggered by neuronal activity in the NG2-glia still
remain unclear.
NG2-glia reaction towards Central Nervous System

Pathology
As we briefly mentioned before, NG2-glia change their dynamics and behavior

when the CNS is damaged. Moreover, changes in NG2-glia in different models of
injury and demyelination have suggested that NG2-glia may play an active role in
repairing the brain under pathological conditions. Hypomyelinating mutant mouse
lines like the jimpy, in which oligodendrocytes die as the result of a point mutation
in the most abundant CNS myelin protein, the proteolipid protein (PLP), or the
shiverer, in which oligodendrocytes are unable to form compact myelin sheaths due
to the deletion of the myelin basic protein (MBP), have shown an increase in the
proliferation and differentiation of NG2-glia in the spinal cord white matter in
comparison to the control wild type groups (Wu et al. 2000; Bu et al. 2004). This
shows an association between myelination defects and change of physiological
NG2-glia behavior. Unfortunately, due to the mutation in important myelin pro-
teins, both models are incapable to demonstrate if there is remyelination after
NG2-glia reaction toward aberrant myelination. However, transplantation of heal-
thy human NG2-glia into the shiverer mouse model resulted in improved
remyelination of the brain, hinting into the capability of NG2-glia to repair
demyelinated areas (Windrem et al. 2008) and therefore a probable strategy for the
therapy of demyelinating diseases in the CNS (Franklin and Ffrench-Constant
2008) (see Chapter “Peripheral Inflammation and Demyelinating Diseases” for
further reading on demyelinating diseases).
Other strategies have also been performed in this field to specifically injure brain
white matter tracts and study the behavior and the remyelination properties of
NG2-glia. Cultured cortical slices exposed to a-lysophosphatidylcholine (LPC),
show damage in the corpus callosum, and subsequent demyelination of the affected
area. After this acute lesion, an increase in proliferation of NG2-glia (Garay et al.
2011) and in their differentiation into myelinating oligodendrocytes (Gensert and
Goldman 1997) could be observed. Two-photon in vivo imaging of postnatal
cortical slices further confirmed these results and showed that this acute injury
model changes the differentiation behavior of NG2-glia by increasing the asym-
metric division of these cells, giving rise to one NG2-glia and one oligodendrocyte
(Hill et al. 2014). Additionally, in the experimental autoimmune encephalomyelitis
(EAE) mouse, a classic model for multiple sclerosis (MS; a disease that promotes
demyelination and axonal loss in the CNS), or in antibody-induced demyelination,
the number of NG2-glia also increased in those areas where extensive demyeli-
nation occurs, while the population of NG2-glia did not change in the tissue sur-
rounding the lesions (Keirstead et al. 1998; Di Bello et al. 1999).
The role of NG2-glia during demyelination and the subsequent remyelination
appears to be clear. The decrease in myelin triggers the proliferation and differ-
entiation of NG2-glia in order to reestablish the loss of myelin in the CNS, making
NG2-glia an excellent target for regenerative therapy. However, it is still unclear
what the trigger for the reaction of NG2-glia after demyelination could be; it is e.g.
possible that NG2-glia possess the special capability to sense the lack of oligo-
dendrocytes/myelin or that NG2-glia react to the aberrant neuronal activity pro-
moted by the demyelination. It is known that the CNS of MS patients fails to
remyelinate with disease progression. Interestingly, postmortem NG2 and PDGFRa
immunolabeling of brains of these patients have revealed that NG2-glia are more
frequently present in areas of the white matter undergoing active inflammatory
demyelination, where commonly remyelination occurs, than in chronic lesions
(Wilson et al. 2006), suggesting that NG2-glia are subjected to certain limitations
for effective remyelination in long-term pathology, probably provided by an
antagonistic environment or by an exhaustion of the NG2-glia limiting them to
further proliferate and differentiate.
In contrast to de-/remyelination, the role of these cells in other brain insults like
acute injury and neurodegeneration is not clear yet. After a stab wound injury in the
cerebral cortex an increase in the proliferation and density of NG2-glia surrounding
the lesion could be observed (Dimou et al. 2008; Simon et al. 2011). Moreover, in
the APPPS1 mice, a mouse model for Alzheimer’s disease (AD), where also
demyelination occurs, NG2-glia react with increased proliferation and differentia-
tion (Behrendt et al. 2013) and become hypertrophe in the gray matter cerebral
cortex (Sirko et al. 2013). Li et al. (2013) have also reported that NG2-glia cluster
around the amyloid plaques and as already suggested for other glial cells that they
are capable of engulfing the amyloid protein b (Abeta) and degrade it by an
autophagy-lysosomal pathway playing a role in clearing it from the affected brain
(Li et al. 2013). Although the specific function of NG2-glia remains unknown, it
could be possible that they may serve as orchestra directors recruiting and modu-
lating the function of other glial and immune cells in the CNS in order to reestablish
the homeostasis of the brain.
Another important question addressed in the field is whether the whole popu-
lation or only a subset of NG2-glia are actively participating in repairing the brain
damage. As discussed above, an interesting subset of NG2-glia that expresses
GPR17 seems to resemble NG2-glia specialized for repair. GPR17 is a deorpha-
nized receptor for both uracil nucleotides and cystein leukotrienes, cysLTs (e.g.,
UDP-glucose and LTD4) (Lecca et al. 2008). Both ligands for GPR17 are secreted
after brain injury and their extracellular concentration is increased suggesting a role
of this receptor in “sensing” brain damage (Lecca et al. 2008). It has been shown
that GPR17-positive NG2-glia in the adult brain increase their density after acute
cortical stab wound injury in the gray matter surrounding the lesion and in the white
matter underneath the lesion (Boda et al. 2011). Moreover, fate mapping studies of
GPR17-positive cells have shown that although they have a limited differentiation
capacity under physiological conditions, after stab wound injury or middle cerebral
artery occlusion (MCAO), a model of ischemic stroke, these cells strongly differ-
entiate into oligodendrocytes probably in order to repair the injured tissue.
Interestingly, in the above described APP/PS1 mouse line, GPR17-positive
NG2-glia were responsive specifically in the gray matter, but not in the white
matter (Boda et al. 2011). This result highlights once again the potential hetero-
geneity of these cells and their functional differences in the brain.
Concluding Remarks
We have shown in this chapter that NG2-glia are a population of cells that are
widely represented within the CNS during the late stage of development and in the
whole adult lifespan. Their potential to possibly originate different cell types, their
long cell cycle length and maintenance of their own population size resembles more
the characteristics of neuronal stem cells rather than simple oligodendrocyte pro-
genitors. Moreover, special features like the expression of channels and receptors,
their response to neurotransmitters and growth factors and the synapse formation
with neurons make them a unique group of glial cells. Additionally, the capability
of NG2-glia to modulate their behavior and dynamics in response to neuronal
activity and disease, suggest an important role not only for myelin maintenance and
remodeling under physiological but also for repair under pathological conditions.
There are still many mysteries around the specific NG2-glia functions in the healthy
and diseased brain. The understanding of these cells may lead to significant
improvement not only of our global knowledge of the complexity of the brain, but
also of the treatment of traumatic and neurodegenerative diseases and the
improvement of the quality of life during normal aging, which also has been related
to an impairment of myelin (Sturrock 1976; Peters 2002; Lasiene et al. 2009; Lu
et al. 2011; Lu et al. 2013).
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Pharmacological Tools to Study the Role
of Astrocytes in Neural Network Functions
Fernando Peña-Ortega, Ana Julia Rivera-Angulo

and Jonathan Julio Lorea-Hernández
Abstract Despite that astrocytes and microglia do not communicate by electrical

impulses, they can efficiently communicate among them, with each other and with
neurons, to participate in complex neural functions requiring broad
cell-communication and long-lasting regulation of brain function. Glial cells express
many receptors in common with neurons; secrete gliotransmitters as well as neu-
rotrophic and neuroinflammatory factors, which allow them to modulate synaptic
transmission and neural excitability. All these properties allow glial cells to influence
the activity of neuronal networks. Thus, the incorporation of glial cell function into
the understanding of nervous system dynamics will provide a more accurate view of
brain function. Our current knowledge of glial cell biology is providing us with
experimental tools to explore their participation in neural network modulation. In
this chapter, we review some of the classical, as well as some recent, pharmaco-
logical tools developed for the study of astrocyte’s influence in neural function.
We also provide some examples of the use of these pharmacological agents to
understand the role of astrocytes in neural network function and dysfunction.
Keywords Astrocyte Microglia Aconitase Fluoroacetate Fluorocitrate

Glutamine synthetase
Abbreviations and Acronyms

ADP Adenosine-diphosphate
ATP Adenosine-triphosphate
BAPTA-AM 1,2-Bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetrakis
(acetoxymethyl ester)
BBB Blood–Brain Barrier
cAMP Cyclic adenosine monophosphate
F. Peña-Ortega (&) A.J. Rivera-Angulo J.J. Lorea-Hernández

Departamento de Neurobiología del Desarrollo y Neurofisiología,
Instituto de Neurobiología, Universidad Nacional Autónoma de México,
Boulevard Juriquilla 3001, Querétaro 76230, Mexico

DOI 10.1007/978-3-319-40764-7_3
48 F. Peña-Ortega et al.
CGa Cystine–glutamate antiporter

CNS Central Nervous System
Cox-2 Cyclooxygenese-2
Cx30 Connexin 30
Cx43 Connexin 43
FA Fluoroacetate
FC Fluorocitrate
GABA Gamma-aminobutyric acid
GFAP Glial Fibrillary Acidic Protein
GLAST Glutamate Aspartate Transporter
GLT-1 Glial Glutamate Transporter 1
GS Glutamine Synthetase
iNOS Inducible Nitric Oxide Synthase
L5 Lumbar segment 5
L-AAA L-alpha-aminoadipic acid
LTP Long-Term Potentiation
MCT1 Monocarboxylate Transporter 1
MCT4 Monocarboxylate Transporter 4
MPTP 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine
MSO L-methionine-S-sulfoximine
ONO-2506 (2R)-2-Propyloctanoic acid
PAR1 Protease-activated receptor 1
pH -log [H+]
S100B S100 Ca2+-binding protein B
TCAC Tricaboxylic acid cycle
TeNT Tetanus Neurotoxin
TgAPP(sw) Transgenic mice carrying the amyloid precursor protein with the
Swedish mutation
TFLLR L-threonyl-L-phenylalanyl-L-leucyl-L-leucyl-L-argininamide
Introduction
Neurons are specialized to produce various firing patterns and synaptic responses
that allow them to interact with each other and to produce complex neural network
dynamics (Ramirez et al. 2004; Peña-Ortega 2012). However, glial cells (mainly
astrocytes and microglia), which do not communicate using electrical impulses, are
well suited to participate in complex neural functions requiring broad spatial
integration and long-term temporal regulation (Peña-Ortega 2012; Fields et al.
2014). Glial cells have biological properties that influence all types of neuronal
networks, including the respiratory rhythm generator that produces stereotyped
network outputs (Hülsmann et al. 2000; Lorea-Hernández et al. 2016) and cortical
Pharmacological Tools to Study the Role of Astrocytes … 49
networks involved in complex neuronal processing (Perea et al. 2014). Astrocytes,

for instance, can modulate synaptic transmission and may couple multiple neurons
and synapses into functional assemblies (Sasaki et al. 2014), whereas microglia can
remove synapses in an activity-dependent manner, thereby altering neural network
activity in real time (Tremblay and Majewska 2011). Thus, the incorporation of
glial cell function into the understanding of nervous system function may provide a
more accurate view of brain function.
The ratio of glial cells to neurons has increased during evolution (Reichenbach
1989; Nedergaard et al. 2003). For instance, nematodes have 302 neurons and 50
glial cells (a ratio of 6–1) (Sulston et al. 1983; Nedergaard et al. 2003). In rodents
there are only 2–3 neurons per glia (Bass et al. 1971; Ren et al. 1992; Nedergaard
et al. 2003; Bandeira et al. 2009). In contrast, in primates there might be equal
numbers or even slightly more glial cells than neurons (Dombrowski et al. 2001;
Nedergaard et al. 2003; Sherwood et al. 2006; Azevedo et al. 2009). Glia have
many of the same receptors as neurons, secrete neurotransmitters and neurotrophic
and neuroinflammatory factors, control clearance of neurotransmitters from
synaptic clefts, and are intimately involved in synaptic plasticity (Van Wagoner
et al. 1999). The prevailing view of the synapse as a structure involving pre- and
postsynaptic connections between two or more neurons has limited our thinking
about synaptic function and plasticity, and their role in cognition. The emerging
concepts of the ‘‘tri-partite’’ synapse (Araque et al. 1999) (see Chapter “Glial Cells
and Integrity of the Nervous System”) and even the ‘‘quad-partite’’ synapse
(Tremblay and Majewska 2011) may better describe recent evidence indicating that
multiple cell types work together at synapses and contribute to network function
(Araque et al. 1999; Tremblay and Majewska 2011; Sasaki et al. 2014). Our
understanding of glial cell biology is providing us with the experimental tools to
explore glial participation in neural network function. In this chapter, we review
some of the classical, as well as some recent, pharmacological tools developed for
this purpose (Hülsmann et al. 2000; Henneberger et al. 2010; Bélanger et al. 2011;
Sasaki et al. 2014; Carlsen and Perrier 2014), without ignoring the fact that the use
of animals with specific genetic modifications is now also contributing to this effort
(Theis et al. 2003; Suzuki et al. 2011; Pannasch et al. 2011, 2014; Perea et al.
2014).
Astrocytes and the Pharmacological Tools to Study Them
Virchow described the so-called “neuroglia,” which originally were thought to

provide physical support for neurons (Somjen 1988). Later, it was established
that astrocytes constitute an integral part of the blood–brain barrier (BBB),
helping to limit the influx of potentially toxic factors into the CNS (Janzer and
Raff 1987). Astrocytes also absorb excess potassium ions and excitotoxic neu-
rotransmitters such as glutamate (Ridet et al. 1997; Nedergaard et al. 2003;
Ransom et al. 2003) and help control the homeostasis of surrounding synapses,
with a fundamental role in energy metabolite supply (Hassel et al. 1995;
Westergaard et al. 1995; Allaman et al. 2011). Moreover, astrocytes enveloping
synapses also control the volume of extracellular space, and hence, the extra-
cellular levels and diffusion of neuroactive substances (Nagelhus and Ottersen
2013) (see Chapter “Glial Cells and Integrity of the Nervous System” for further
reading on astrocyte functions).
Today, we know that one astrocyte can contact many thousands of synapses
via its processes; it has been estimated that a single astrocyte can influence up to
140,000 synapses in the adult hippocampus of rodents (Bushong et al. 2002) and
from 270,000 to 2 million synapses in the human cortex (Oberheim et al. 2009).
Moreover, we know that astrocytes express receptors for many different
neurotransmitters, and in response to the activation of few of them, astrocytes use
Ca2+ signaling to propagate their excitation (Duffy and MacVicar 1995; Bezzi
et al. 1998; Shelton and Mccarthy 2000; Araque et al. 2002; Agulhon et al.
2008). This provides a mechanism by which astrocytes can monitor and respond
to ongoing synaptic transmission by releasing gliotransmitters, such as glutamate,
ATP, D-serine, growth factors, and cytokines (Santello et al. 2012; Ji et al. 2013)
(Fig. 1).
Astrocytes are interconnected with each other through connexin 30 (Cx30) and
connexin 43 (Cx43) (Pannasch et al. 2011, 2014), which help them to communicate
via Ca2+ waves (Barres et al. 1990; McCarthy and Salm 1991, Perea and Araque
2005) that subsequently control the coordinated release of the gliotransmitters
(Moraga-Amaro et al. 2014) (see Chapters “Physiological Functions of Glial Cell
Hemichannels” and “Role of Astrocytes in Central Respiratory Chemoreception”
for further information) (Fig. 1). Interestingly, inhibiting gliotransmission by sleep
deprivation attenuates neural network activity and impairs cognition (Yoo et al.
2007), as does genetic removal of Cx30 and Cx43 (Theis et al. 2003; Suzuki et al.
2011; Pannasch et al. 2011, 2014). Stimulation of astrocytes has also been achieved
mechanically (Liu et al. 2011) or through the intra-astroglial release of caged Ca2+
(Agulhon et al. 2010; Belanger et al. 2011; Sasaki et al. 2014). In contrast, Ca2+
buffering can be used to inhibit astrocyte activity (Henneberger et al. 2010;
Belanger et al. 2011; Sasaki et al. 2014; Carlsen and Perrier 2014), but the phys-
iological relevance of such treatments, and even of their influence on gliotransmitter
release, is still debated (Hamilton and Attwell 2010).
There have been several attempts to assess behavioral effects of genetically
manipulating the major astroglial proteins (Roder et al. 1996; Shibuki et al. 1996;
Nishiyama et al. 2002; Suzuki et al. 2011). The effects of over-expression of S100
Ca2+-binding protein B (S100B) (Fig. 1), previously known as SB100β, on
exploratory behaviors were studied in transgenic mice. A significant difference in
the spatial and temporal exploratory pattern was observed between control and
S100B mutants (Roder et al. 1996). Mutant mice with a deletion of S100B
developed normally, with the cytoarchitecture of the brain preserved, but they
exhibited enhanced long-term potentiation (LTP) in the hippocampal CA1 region,
better performance in the Morris water maze, and greater contextual fear
Fig. 1 Cellular and molecular targets of the drugs used for astrocyte modulation. Astrocytes
(represented by the blue cell) can be activated by the application of L-threonyl-L-phenylalanyl-
L-leucyl-L-leucyl-L-argininamide (TFLLR), which is an agonist of the protease-activated receptor
1 (PAR1). Note that both microglia (represented by the green cell) and neurons (represented by the
purple cell) can also express PAR1. Astrocytes can be killed by the toxin L-alpha-aminoadipic
acid (L-AAA); it is selectively incorporated into astrocytes by the cystine–glutamate antiporter
(CGa), which is not present either in microglia or in neurons. Astroglial function can also be
evaluated by using L-methionine-S-sulfoximine (MSO) to inhibit glutamine synthetase (GS),
which is mainly expressed in astrocytes. Astroglial metabolism can also be inhibited by
fluoroacetate (FA) and fluorocitrate (FC), which affect the tricaboxylic acid cycle (TCAC) by
inhibiting the enzyme aconitase. FA and FC are selectively taken into astrocytes by the acetate
transporter, which is absent in both microglia and neurons. Arundic acid, also known as ONO-256,
inhibits the production and release of the S100 Ca2+-binding protein B (S100B), which is mainly
produced by astrocytes but can also be produced by some populations of neurons
conditioning (Nishiyama et al. 2002). Affecting astroglial function through the

disruption of the astroglial lactate transporters, the monocarboxylate transporter 4
(MCT4) or 1 (MCT1) produced amnesia and LTP impairment, which was rescued
by L-lactate but not by equicaloric glucose (Suzuki et al. 2011). Consistent with
this, inhibition of astroglial glycogenolysis with 1,4-dideoxy-1,4-imino-d-arabinitol
impaired memory, and the impairment was rescued by lactate (Newman et al.
2011). Also, the selective photostimulation of astrocytes in genetically modified
mice with channelrhodopsin-2 enhanced both excitatory and inhibitory synaptic
transmission in the primary visual cortex, which either increased or decreased
baseline visual responses together with complementary changes in orientation

selectivity (Perea et al. 2014). The selective expression of tetanus neurotoxin
(TeNT) in astrocytes significantly reduced the duration of carbachol-induced
gamma oscillations in hippocampal slices without affecting synaptic transmission
(Lee et al. 2014). Moving from the great advantages of using genetically modified
mice to understand astrocyte physiology and its role in brain function, this chapter
shall now focus on pharmacological approaches to study the role of glial cells, in
this case astrocytes, in neural network function.
One of the pharmacological strategies to study astrocytes involvement in neural
network function is the use of the agonist for the protease-activated receptor 1
(PAR1), L-threonyl-L-phenylalanyl-L-leucyl-L-leucyl-L-argininamide (TFLLR;
Lalo et al. 2014a, b) (Fig. 1). TFLLR is considered a useful astroglial tool since
PAR1 localizes preferentially in glial fibrillary acidic protein (GFAP) positive
astrocytes (Wang et al. 2002a, b; Boven et al. 2003; Sorensen et al. 2003; Junge
et al. 2004; Lee et al. 2007; Shigetomi et al. 2008; Hermann et al. 2009; Han et al.
2011; Shavit et al. 2011) (Fig. 1). PAR1 activation triggers astrocyte proliferation
(Grabham and Cunningham 1995; Wang et al. 2002a, b) and stellation (Scarisbrick
et al. 2012). Interestingly, PAR1 activation considerably increases astroglial Ca2+
concentration (Wang et al. 2002a, b; Lee et al. 2007; Shigetomi et al. 2008;
Vandell et al. 2008; Hermann et al. 2009; Han et al. 2011; Oh et al. 2012; Wang
et al. 2013) (Fig. 1), which can later increase the [Ca2+] in neurons by releasing
glutamate (Lee et al. 2007; Hermann et al. 2009; Oh et al. 2012; Han et al. 2013)
(Fig. 1). In fact, astroglial release of glutamate induced by TFLLR occurs via the
Ca2+-activated anion channel Bestrophin 1 (Oh et al. 2012; Woo et al. 2012; Han
et al. 2013), and the neuronal Ca2+ influx takes place through the NMDA receptor
(Lee et al. 2007; Woo et al. 2012; Han et al. 2013). It is also known that PAR1
receptor-mediated astrocyte excitation causes gliotransmitter release by exocytosis
(Bowser and Khakh 2007). Activating PAR1 receptors with TFLLR reduces
synaptic transmission by releasing astroglial ATP, which is extracellularly
transformed into adenosine that activates presynaptic A1 receptors (Carlsen and
Perrier 2014).
Despite some evidence that PAR1 is expressed neither in neurons (Hermann
et al. 2009) nor in microglia (Ishida et al. 2006), there are other reports that PAR1 is
expressed in subpopulations of neurons (Yang et al. 1997; Gingrich et al. 2000;
Striggow et al. 2001; Ishida et al. 2006; Vellani et al. 2010; Han et al. 2011) (Fig. 1)
and that it can be detected in microglia after chronic insults (Henrich-Noack et al.
2006; Laskowski et al. 2007; Pompili et al. 2011) (Fig. 1). In fact, there is evidence
that PAR1 participates in microglia activation (Suo et al. 2002) and that PAR1 is
functional in microglial cultures (Möller et al. 2000; Suo et al. 2002; Balcaitis et al.
2003; Fabrizi et al. 2009) (Fig. 1). In spite of the evidence that TFLLR can increase
the intracellular Ca2+ concentration in dentate neurons as well as in non-neuronal
cells (Han et al. 2011), it was demonstrated that this increase can be prevented by
adding the gliotoxin fluoroacetate or by blocking astroglial Ca2+ waves with
BAPTA-AM (Shigetomi et al. 2008), both of which are known to specifically affect
astroglial function (Fonnum et al. 1997; Liu et al. 2004).
Arundic acid ((2R)-2-propyloctanoic acid) is becoming a promising pharmaco-

logical tool to study astrocyte function (Asano et al. 2005). Also known as
ONO-2506, arundic acid was identified as an astroglial modulator through
screening tests searching for an agent that could inhibit astroglial S100B (Asano
et al. 2005) (Fig. 1). S100B is an acidic Ca2+-binding protein found in the cyto-
plasm and nucleus of astrocytes (Fig. 1). Released S100B produces dual effects
(Rothermundt et al. 2003; Yasuda et al. 2004). At nanomolar concentrations, it
stimulates the outgrowth of neural processes and enhances the survival of neurons
after tissue damage or ischemia (Rothermundt et al. 2003; Yasuda et al. 2004), and
at micromolar levels, it stimulates expression of proinflammatory cytokines and
induces apoptosis (Petrova et al. 2000; Lam et al. 2001; Rothermundt et al. 2003).
The S100B protein is commonly found in the CNS in astrocytes (Ludwin et al.
1976; Friend et al. 1992; Reeves et al. 1994; Muramatsu et al. 2003;
Romero-Alemán Mdel et al. 2003; Kortvely et al. 2003; Vives et al. 2003; Shapiro
et al. 2008) (Fig. 1), but never in microglia (Lillo et al. 2002; Muramatsu et al.
2003; Shapiro et al. 2008; Trias et al. 2013). Despite the evidence that neurons lack
S100B (Reeves et al. 1994; Romero-Alemán Mdel et al. 2003; Kortvely et al.
2003), there are some reports that S100B can be found in certain neurons (Friend
et al. 1992; Vives et al. 2003) (Fig. 1) and in oligodendrocytes (Gonçalves et al.
2008). As mentioned, arundic acid can inhibit the production and release of S100B
protein from astrocytes (Hu and Van Eldik 1996; Asano et al. 2005; Mori et al.
2005; Wajima et al. 2013; Wang et al. 2013; Hanada et al. 2014) (Fig. 1). Arundic
acid inhibits spontaneous epileptic discharges in Cacna1atm2Nobs/tm2Nobs mice,
without affecting maximal electroshock seizures or pentylenetetrazole-induced
seizures, by increasing basal glial release of kynurenic acid but not of L-glutamate,
D-serine or GABA (Yamamura et al. 2013). Furthermore, arundic acid inhibits
excitation-induced release of L-glutamate, D-serine, GABA, and kynurenic in a
fluorocitrate-sensitive (thus, astrocyte-dependent) manner (Yamamura et al. 2013).
The previous findings suggest that arundic acid can be a good anticonvulsant since
it enhances glial inhibitory transmitter release without affecting excitatory trans-
mitter release at rest and it also inhibits glial transmitter release induced by
hyperactivation (Yamamura et al. 2013). In addition, arundic acid inhibits the
expression of cyclooxygenese-2 (Cox-2), nerve growth factor-beta, and inducible
nitric oxide synthase (iNOS) induced by lipopolysaccharide in glial cultures
(Shimoda et al. 1998; Tateishi et al. 2002; Shinagawa et al. 1999). Arundic acid
does not act on neuronal death in pure neuron cultures, but in cultured astrocytes it
suppresses injury-induced changes, such as the increase in S100B content, the
secretion of nerve growth factor, the reduction of glutamate transporter (GLT-1 and
GLAST) expression, and the disappearance of GABAA receptors, in a
dose-dependent manner and without affecting GFAP expression (Katsumata et al.
1999; Shinagawa et al. 1998, 1999). On the other hand, arundic acid administration
significantly inhibits both S100B and GFAP overproduction in the brain of spon-
taneous hypertensive rats (Higashino et al. 2009).
By suppressing astroglial activation and the production of S100B, the admin-
istration of arundic acid reduces the expression of GFAP in activated astrocytes and
reduces infarct volume in injured brain (Tateishi et al. 2002; Mori et al. 2005;
Asano et al. 2005; Higashino et al. 2009). Administration of arundic acid improves
motor function and reduces S100B production while inhibiting the expansion of
secondary injury in rats with subdural hematomas (Hanada et al. 2014). Arundic
acid decreases the expression of S100B protein produced by activated astrocytes
around ischemic lesions (Asano et al. 2005; Mori et al. 2005; Wajima et al. 2013),
and it prevents the depletion of dopamine in the striatum and the loss of
dopaminergic neurons in the substantia nigra in a mouse model of Parkinson
Disease caused by the administration of the toxin 1-methyl-4-phenyl-1,2,3,6-
tetrahydropyridine (MPTP) (Kato et al. 2003). Furthermore, arundic acid can
ameliorate neurological deficits caused by the MPTP treatment (Kato et al. 2004).
Arundic acid can reduce the beta-amyloid deposits and the increase of soluble
amyloid-beta peptide, S100B, and beta-amyloid plaque-associated reactive gliosis
(astrocytosis and microgliosis) in the transgenic mouse TgAPP(sw), which exhibits
some features of Alzheimer’s Disease (Mori et al. 2005).
Aside from astrocyte “modulators,” a broad variety of “gliotoxins” have been
used to probe the role astrocytes in brain function by either killing them (Olney
et al. 1971) or by just inhibiting their tricarboxylic acid cycle (Clarke et al. 1970;
Hassel et al. 1995) or their glutamine synthesis (Tanigami et al. 2005a, b).
L-alpha-aminoadipic acid (L-AAA) is a six-carbon homologue of L-glutamate that
has gliotoxic effects both in vivo and in vitro (Olney et al. 1971; Huck et al. 1984;
Xu et al. 2004; Leffler et al. 2006; Banasr and Duman 2008) (Fig. 1). The initial
observation that L-AAA can kill astrocytes was made by Olney et al. (1971), and
later it was reported that astrocytes, but not neurons, can take up L-AAA through
the cystine-glutamate antiporter (Huck et al. 1984; Pow 2001) (Fig. 1). Once in the
astroglial cytoplasm, L-AAA induces cell death by an unknown mechanism (Brown
and Kretzschmar 1998) (Fig. 1). As expected, astrocyte death is reflected as a
reduction in cerebral glutamate and glutamine (Lee et al. 2013) and as a loss of
astroglial integrity at the ultrastructural level (Takada and Hattori 1986; Rodríguez
et al. 2004). Immunohistochemical staining also shows that L-AAA decreases the
number of astrocytes but not of neurons (Sun et al. 2013). Astroglial death after
intracerebral injection of L-AAA induces anhedonia, anxiety, and helplessness
(Banasr and Duman 2008; Domin et al. 2014). L-AAA also affects attention,
working memory, and reversal learning (Lima et al. 2014). At the cellular level,
L-AAA enhances tonic NMDA responses and neuronal activity in vitro (Fleming
et al. 2011) and can alter the somatosensory-evoked potentials and the multiunit
sensory-evoked spike rates in the barrel cortex (Sun et al. 2013).
Fluoroacetate (FA) and its toxic metabolite fluorocitrate (FC), both aconitase
inhibitors (Clarke et al. 1970) (Fig. 1), are preferentially taken up by glial cells via
their acetate transporters (Clarke et al. 1970) (Fig. 1), and they specifically inhibit
the glial tricarboxylic acid cycle (Clarke et al. 1970; Paulsen et al. 1987; Clarke
1991; Martín et al. 2007) (Fig. 1). Both substances have been extensively used to
study the function of glial cells in the nervous system (Clarke et al. 1970; Paulsen
et al. 1987; Clarke 1991; Hülsmann et al. 2000; Martín et al. 2007; Huxtable et al.
2010; Lorea-Hernández et al. 2016). Although FA does not affect neuronal
ultrastructure, it causes reversible ultrastructural alterations in astrocytes (Paulsen

et al. 1987). These structural changes are accompanied by a temporary decrease in
glutamine, glutamate, and aspartate levels and an increase in alanine concentration
in the brain (Paulsen et al. 1987). Accordingly, with its astrocyte-specific effect, FC
does not affect the activity of other cellular enzyme markers, such as choline
acetyltransferase, GABA-2-oxoglutarate amino-transferase, glutamic acid decar-
boxylase, glutamine synthetase, acetylcholinesterase, and cholinesterase (Paulsen
et al. 1987). Even at high concentrations (up to 20 mM), FA only starts to affect
pyramidal cells after 1 h of incubation (Canals et al. 2008). This specificity for glial
cells arises from the uptake of FA and FC by the acetate transporter, which is
selectively expressed by glial cells and not by neurons (Clarke et al. 1970). FA
causes a marked increase in extracellular adenosine concentration, exceeding 1 μM,
which is sufficient to almost completely inhibit synaptic transmission by activating
A1 receptors (Canals et al. 2008; Wall and Dale 2013). It has been reported that this
increase in adenosine occurs in the absence of neurons, that it can be observed in
pure glial cultures, and that it results from the blockade of the tricarboxylic acid
cycle within glial cells (Canals et al. 2008) (Fig. 1). Interestingly, injection of FA in
chicks affects long-term memory (Gibbs and Bowser 2009; Gibbs et al. 2011) and
abolishes the increase in memory induced by a PAR1 agonist (Gibbs et al. 2011).
Inhibition of astroglial metabolism with FA also causes spike-and-wave discharges
and absence seizures in various species, including mice, cats, dogs, and rabbits
(Ward 1947; Chenoweth and St. John 1947; Goldberg et al. 1966; Hornfeldt and
Larson 1990). In addition, FA can inhibit the respiratory rhythm generation in
brainstem slices (Hülsmann et al. 2000; Huxtable et al. 2010). This effect can be
reversed by glutamine (Hülsmann et al. 2000; Huxtable et al. 2010).
FC is a metabolite of FA that also inhibits aconitase (Clarke et al. 1970) (Fig. 1)
and suppresses the tricarboxylic acid cycle in glia (Clarke et al. 1970) (Fig. 1),
causing citrate accumulation, reducing glutamine production, and depriving astro-
cytes of energy (Hassel et al. 1995; Fonnum et al. 1997). FC also interferes with the
glutamate–glutamine cycle (Paulsen et al. 1987; Swanson and Graham 1994; Largo
et al. 1996). As demonstrated for FA, FC affects just astrocyte metabolism and not
that of neurons (Keyser and Pellmar 1994; Willoughby et al. 2003) (Fig. 1).
Accordingly, FC reduces extracellular kynurenic acid (Yamamura et al. 2013) and
glutamate levels (Tanahashi et al. 2012) without affecting levels of GABA
(Yamamura et al. 2013). Moreover, FC reduces the K+-evoked release of kynurenic
acid (Yamamura et al. 2013) and D-serine (Tanahashi et al. 2012) but not GABA
release (Yamamura et al. 2013). Experiments conducted in astrocyte-brain
endothelial cell co-cultures and brain tissue cultures show that the primary effect
of FC is on astrocytes (Gesuete et al. 2011; Sá Santos et al. 2011) (Fig. 1). Thus, FC
prevents the gliosis induced by oxaliplatin but not the alterations in neuronal
morphology or the microgliosis produced by this compound (Di Cesare et al. 2014).
Interestingly, FC treatment decreases the levels of several memory-related proteins,
such as AMPA receptor GluR1/2, postsynaptic density protein 93/95, Arc, and
phosphorylated cAMP response element binding proteins (Shang et al. 2015), while
it increases synaptophysin and synapsin I levels in the hippocampus (Shang et al.
2015). FC treatment also increases the levels of phosphorylated Tau at multiple,

Alzheimer-related phosphorylation sites (Shang et al. 2015). These effects correlate
with the activation of glycogen synthase kinase-3beta and the inactivation of pro-
tein phosphatase-2A (Shang et al. 2015). Similar effects are also observed in the
primary hippocampal neurons cultured with the conditioned media from FC-treated
primary astrocytes (Shang et al. 2015). Accordingly, intracerebroventricular
injection of FC impairs memory (Wang et al. 2009b). FC abolishes the uptake of
sulforhodamine 101 and reduces GABAergic transmission (Christian and
Huguenard 2013) and some other types of synaptic transmission (Bonansco et al.
2011; Christian and Huguenard 2013). FC also alters tissue pH and respiratory
output (Erlichman et al. 1998; Holleran et al. 2001). Recent evidence suggests that,
in addition to inhibiting astrocyte function, FC may also reduce satellite glial cell
activation and function (Liu et al. 2012). Infusion of FC into the dorsal root gan-
glion (L5) inhibits satellite glial cell activation and reduces spinal nerve
injury-induced pain behavior, suggesting the participation of these cells in the
genesis of neuropathic pain (Liu et al. 2012).
L-methionine-S-sulfoximine (MSO) is a glutamate analogue that inhibits glu-
tamine synthetase (GS) (Tanigami et al. 2005a, b), which is present only in
astrocytes (Norenberg and Martinez-Hernandez 1979; Tanigami et al. 2005a, b).
Thus, the effects of MSO can be counteracted by exogenous application of glu-
tamine (Bacci et al. 2002; Blin et al. 2002; Gibbs and Hertz 2005; Tanigami et al.
2005a, b; Liang et al. 2006; Okada-Ogawa et al. 2009). GS is the only enzyme in
mammals known to effectively synthesize glutamine (Petroff et al. 2002), which is
critical for producing the most abundant excitatory and inhibitory neurotransmit-
ters: glutamate and GABA (Petroff et al. 2002). GS is also important for the
clearance of glutamate released during excitatory synaptic transmission and for the
metabolism of brain ammonia (Albrecht and Jones 1999; Martinez-Hernandez et al.
1977). A large proportion of extracellular glutamate is taken up by high-affinity
excitatory amino acid transporters present on the plasma membrane of astrocytes
(Danbolt 2001). Once in the astrocyte, glutamate binds to GS along with ammonia
and ATP for enzymatic conversion to glutamine and ADP (Otis and Jahr 1998). The
stoichiometry of glutamate transport across the astroglial plasma membrane sug-
gests that rapid metabolism of intracellular glutamate via GS is a prerequisite for
efficient glutamate clearance from the extracellular space (Otis and Jahr 1998).
A loss of GS in astrocytes is therefore likely to perturb the homeostasis of glu-
tamine, glutamate, GABA, and ammonia in the brain.
Blockade of GS not only prevents the synthesis of glutamine from glutamate, but
also results in the loading of astrocytes with glycogen (D’Amelio et al. 1987;
Gutierrez and Norenberg 1977; Hevor et al. 1985). After systemic administration of
MSO, brain astrocytes show cytoplasmic swelling, a doubling in the number of
mitochondria, and glycogen deposition, whereas neurons and oligodendroglia show
no ultrastructural abnormalities (Gutierrez and Norenberg 1977). MSO does not
affect neuronal levels of choline acetyltransferase or glutamate decarboxylase
(Somers and Beckstead 1990). Interestingly, hyperammonemia increases the
number of swollen astrocytes in the cortex, and MSO reduces this increase to
control values (Tanigami et al. 2005a, b). Furthermore, the number of

GFAP-immunopositive cells in the cortex is greater in hyperammonemic rats, and
the increase in superficial cortical layers is attenuated by MSO (Tanigami et al.
2005a, b).
In contrast to the finding that MSO reduces epileptiform discharge in hip-
pocampal slices (Bacci et al. 2002), there are reports that MSO reduces GABAergic
transmission (Liang et al. 2006) and produces convulsions, hypothermia, and ataxia
(Stransky 1969; Ginefri-Gayet and Gayet 1988). Furthermore, chronic infusion of
MSO in the hippocampus produces an epileptic phenotype (Wang et al. 2009a;
Dhaher et al. 2014). MSO also induces depressive behavior in animals, which is
reversed by glutamine (Lee et al. 2014), and it induces inhibition of memory
consolidation in a glutamine-sensitive manner (Gibbs and Hertz 2005). Similar to
the other gliotoxins, MSO also inhibits respiratory rhythm generation in vivo
(Young et al. 2005) and in vitro (Hülsmann et al. 2000; Huxtable et al. 2010),
which is reversed by glutamine (Hülsmann et al. 2000; Huxtable et al. 2010).
In summary, we have presented several pharmacological tools that either increase
(activate) or inhibit astroglial function and found that despite some possible non-
specific effects, these drugs have been very useful for the study of astrocytes’
contribution to neuronal network activity. In our opinion, the controlled use of these
pharmacological tools in combination with the transgenic animals that allow
astroglial function to be modified in a more specific manner will be particularly
powerful for the understanding of astroglial modulation of brain function. This
approach will support the idea that astrocytes are important regulators of neural
network physiology and pathology. Furthermore, the use of these pharmacological
tools to modulate astroglial function is likely to reveal cellular and molecular targets
to treat diseases caused by astrocytes or that are sustained by them.
Acknowledgments We thank Dr. Dorothy Pless for editorial comments. Ana Rivera-Angulo and
Jonathan-Julio Lorea-Hernández are graduate students at UNAM and received fellowships from
CONACyT. This study was supported by CONACyT Grants 235789, 24688, 117 and 181323; and
by DGAPA-UNAM Grant IN200715.
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Microglia Function in the Normal Brain
Rommy von Bernhardi, Florencia Heredia, Nicole Salgado

and Paola Muñoz
Abstract The activation of microglia has been recognized for over a century by
their morphological changes. Long slender microglia acquire a short sturdy ramified
shape when activated. During the past 20 years, microglia have been accepted as an
essential cellular component for understanding the pathogenic mechanism of many
brain diseases, including neurodegenerative diseases. More recently, functional
studies and imaging in mouse models indicate that microglia are active in the
healthy central nervous system. It has become evident that microglia release several
signal molecules that play key roles in the crosstalk among brain cells, i.e., astro-
cytes and oligodendrocytes with neurons, as well as with regulatory immune cells.
Recent studies also reveal the heterogeneous nature of microglia diverse functions
depending on development, previous exposure to stimulation events, brain region
of residence, or pathological state. Subjects to approach by future research are still
the unresolved questions regarding the conditions and mechanisms that render
microglia protective, capable of preventing or reducing damage, or deleterious,
capable of inducing or facilitating the progression of neuropathological diseases.
This novel knowledge will certainly change our view on microglia as therapeutic
target, shifting our goal from their general silencing to the generation of treatments
able to change their activation pattern.
Keywords Central nervous system Cytokines Development Glia

Neuroinflammation
Abbreviations
5-HT Serotonin
Aβ β-amyloid
ALS Amyotrophic lateral sclerosis
R. von Bernhardi (&) F. Heredia N. Salgado P. Muñoz

Escuela de Medicina. Departamento de Neurología,
Pontificia Universidad Católica de Chile, Marcoleta 391, Santiago, Chile

DOI 10.1007/978-3-319-40764-7_4
BDNF Brain derived neurotrophic factor

CNTF Ciliary neurotrophic factor
DAMPs Damage- or Danger-associated molecular patterns
EP2 Prostanoid receptor subtype 2
GABA Gamma aminobutyric acid
GDNF Glia derived neurotrophic factor
GM-CSF Granulocyte/macrophage colony stimulating factor
HIV-1 Human immunodeficiency virus
IFNγ Interferon gamma
IGF1 Insulin-like growth factor 1
IL1 Interleukin 1
InsP3 Inositol trisphosphate
LPS Lipopolysaccharides
LTP Long time Potentiation
M-CSF Macrophage colony-stimulating factor
MHC Class I molecules of histocompatibility major complex
NGF Nerve growth factor
NMDA N-methyl-d-aspartate
NO Nitric Oxide
NT Neurotrophin
PAMPs Pathogen-associated molecular patterns
PGE2 Prostaglandin E2
PRRs Pattern recognition receptors
RANTES Regulated on activation, normal T cell expressed and secreted—
chemokine CCL5
RNS Reactive nitrogen species
ROS Reactive oxygen species
Rs Receptors
SDF-1α Stromal cell-derived factor
SIRPα Signal regulatory protein α
SRs Scavenger receptors
TGFβ Transforming growth factor-β
TLRs Toll-like receptors
TNFα Tumor necrosis factor α
TSPs Thrombospondins
Microglia Function in the Normal Brain 69
Introduction
Microglia are the resident immune cells of the central nervous system (CNS),
accounting for approximately 10 % of the total cell number in the healthy mam-
malian brain (Prinz and Priller 2014). They derive from myeloid progenitors, being
related to peripheral monocyte-macrophages (Ginhoux et al. 2010). Microglial cell
progenitors originating in the yolk sac migrate and colonize the CNS during
embryonic development, before the blood–brain barrier is established, differentiat-
ing and becoming confined into the CNS. Throughout the life, microglia appear to be
capable of local self-renewal. Adult healthy animals show very little exchange
between blood and brain parenchyma (Mildner et al. 2007). Thus, maintenance of
their population normally does not depend on recruitment of circulating progenitors.
However, monocytes invading the brain have been observed under pathological
conditions, such as blood–brain barrier damage by trauma or severe inflammation
and ischemic vascular damage (Ajami et al. 2007; Casano and Peri 2015; Kierdorf
et al. 2013), where they transform into microglia with a ramified phenotype (Mildner
et al. 2007). The environment provided by the brain parenchyma appears to be key
for microglia phenotype. Astrocytes-conditioned medium induces morphological
and functional changes of microglia and blood monocytes in culture (Sievers et al.
1994; Ramirez et al. 2005; Tichauer et al. 2007; von Bernhardi and Ramírez 2001;
Orellana et al. 2013), an effect that is at least partly mimicked by adenosine
triphosphate (ATP) or adenosine. Other mediators capable of modifying microglia
activation are cytokines released from astrocytes, including transforming growth
factor β (TGFβ), macrophage colony-stimulating factor (M-CSF), and
granulocyte/macrophage colony stimulating factor (GM-CSF) (Schilling et al. 2001;
Alarcón et al. 2005; Flores and von Bernhardi 2012; Herrera-Molina et al. 2012;
Tichauer et al. 2014; Tichauer and von Bernhardi 2012).
Microglia, as member of the monocyte-macrophage family, function as
mononuclear phagocytes, recognizing and scavenging dead cells, pathogens and
several endogenous, and exogenous compounds. As mentioned in Chapter “Glial
Cells and Integrity of the Nervous System,” under physiological conditions and in
the absence of inflammatory stimuli, microglia are found in a “surveillance state,”
morphologically defined by having a small soma with long fine-ramified processes.
Surveillance microglia are highly dynamic, retracting and extending their processes
in response to environmental cues, interacting with blood vessels, neurons,
ependymal cells, and other glial cells (Nimmerjahn et al. 2005; Ramirez et al. 2005;
Chen and Trapp 2015; Heneka et al. 2015). They express constitutive markers like
Iba-1, and several other markers, such as MHC-I, MHC-II, FcR, CD68, depending
on the environmental cues they sense (Fig. 1), being involved in antigen presen-
tation, cytotoxic activation, phagocytosis, antibody-associated phagocytosis, etc.
An astrocyte labeled with antibody against glial fibrillary acidic protein (GFAP) is
also included to compare their morphologies. Microglial cell surveillance is highly
relevant for CNS development and function throughout life.
Fig. 1 Labeling of glia by

activation markers.
Immunohistochemical
detection of glia activation
markers in hipocampal
cryosections obtained from
unstimulated adult mice
counterstaining with Harris
Haematoxylin. Iba-1 and
GFAP antibodies identified
microglia and activated
astrocytes, respectively.
MHC-I, MHC-II, FcR, and
CD68 identify microglia
populations that are
functionally different,
showing differences in their
morphology as well as in the
labeling pattern. Scale
bar = 10 µm
Microglia Motility and Migration
Microglia exhibit two types of motility: the active movement of their processes,
sensing the environment, and their translocation in the brain parenchyma. Migration
is frequently observed during development, when invading cells migrate into the
CNS, and when recruited after an insult and migrate to the site of injury/stimulation.
As discussed in Chapters “Glial Cells and Integrity of the Nervous System and
Purine Signaling and Microglial Wrapping,” many molecules appear to signal for
microglia migration, including ATP, cannabinoids, chemokines, lysophosphatidic
acid, bradykinin, ion channels, and transporters (Davalos et al. 2005; Walter et al.
2003; Schwab 2001; Rappert et al. 2002; Schilling et al. 2004; Ifuku et al. 2007).
Although under nonstimulated conditions they do not migrate, real-time imaging
reveals that microglial processes are constantly moving (Davalos et al. 2005;
Nimmerjahn et al. 2005). Processes move rapidly toward an injury. Time-lapse
microscopy of brain slices from adult mice shows extensive migration of microglia
24 h after an injury (Carbonell et al. 2005).
Recruitment of microglia to a lesion involves several factors including

chemokines released from both neurons and glial cells, among others. Most of the
chemokines are released as soluble factors that form chemotactic gradients for cell
migration, although CX3CL1 (fractalkine) occurs also as a surface-bound molecule.
Microglial activation following neuropathological challenges affects the expression
of chemokine receptors (Kremlev et al. 2004), acting as a source and a target of
chemokines in an auto/paracrine fashion. Activation of CXCR3 receptor by che-
mokine CCL21 is linked to microglial migration (Rappert et al. 2002), and the
CCL2/CCR2 system appears to be crucial for the recruitment of peripheral
monocytes to the CNS, where they become microglia (Davoust et al. 2008; Mildner
et al. 2007; Prinz and Priller 2010). Expression of CCR, receptor for CCL2 ligands,
identifies functional subsets of microglia. The CX3CR1, the receptor for fractalk-
ine, is also a key molecule for the CNS-relevant macrophage subclassification
(Prinz and Priller 2010).
It is especially interesting that chemokines including CCL2, CCL21, or CX3CL1
also appear to serve as signals from endangered neurons to microglia (Biber et al.
2008). It has been suggested that CX3CL1 expressed by neurons could provide a
constitutive calming influence on CX3CR1-expressing microglia, thus representing a
neuron-to-microglia signaling system similar to those described for CD200/CD200R
or CD47/SIRP-1α. Interruption of this regulatory mechanism could facilitate
enhanced responses to activating signals. In fact, deficiency in fractalkine signaling
results in enhanced severity of CNS damage in several disease models (Cardona et al.
2006; Prinz and Priller 2010). Similarly, activation of CCR5 by the chemokine CCL5
“regulated on activation, normal T cell expressed and secreted” (RANTES),
supresses lipopolysaccharide (LPS)-induced expression of inflammatory cytokines,
such as interleukin (IL)1β, IL6 and tumor necrosis factor (TNF)α, and inducible nitric
oxide synthase (iNOS) in microglia. In contrast, motor neuron death after nerve
injury is accelerated in CCR5 knock-out animals, suggesting that CCR5-mediated
suppression of microglia toxicity protects neurons (Gamo et al. 2008).
Microglia-Mediated Phagocytosis
Microglia are the professional phagocytes of the CNS. Phagocytosis is a key

function during development as well as in the normal and pathological adult brain
(Neumann et al. 2009). During development, microglia remove apoptotic cells,
mediated by an “eat me” signal produced by apoptotic cells to microglia
(Marin-Teva et al. 2004). They are also involved in synapse removal (Stevens et al.
2007) and in pruning synapses in the developing and postnatal brain (see
Chapter “Purine Signaling and Microglial Wrapping” for a complete description on
microglial wrapping).
Phagocytosis depends on different mechanisms (Table 1). Pathogens are rec-
ognized by Toll-like receptors (TLRs), and apoptotic neurons are recognized by
various receptor systems, including asialoglycoprotein-like-, vitronectin-, and
phosphatidylserine receptors (Witting et al. 2000). Multiple factors regulate

phagocytosis, including ATP, through the metabotropic P2Y6 receptor (Inoue et al.
2009). The P2Y6 receptor is upregulated when neurons are damaged and could be a
trigger for phagocytosis (Koizumi et al. 2007). In contrast, activation of P2X7
receptors suppresses phagocytosis, whereas inhibition of P2X7 expression by
shRNA or oxATP/BBG restores phagocytosis (Fang et al. 2009). The ciliary
neurotrophic factor (CNTF), glia derived neurotrophic factor (GDNF), and M-CSF
potentiates phagocytic by microglia (Chang et al. 2006; Lee et al. 2009;
Mitrasinovic and Murphy 2003). Substrate-bound complement component C1q
enhance both FcR and CR1-mediated phagocytosis (Webster et al. 2000), whereas
the prostanoid receptor subtype 2 (EP2), downregulates phagocytosis (Liang et al.
2005; Shie et al. 2005).
Table 1 Receptors and regulatory molecules associated with microglial cell functions
Function Microglial Regulatory References
receptors molecules
Phagocytosis Apoptotic cells Asialoglycoprotein- Witting et al. (2000)
like-,vutronectin- &
phophatidyiserine Rs
Metabotropic P1 ATP Inoue et al. (2009);
adenosine Rs, Koizumi et al. (2007);
metabotropic P2Y Fang et al. (2009);
& ionotropic P2X Kirischuk et al. (1995);
purinoRS Lalo et al. (2008)
GDNF Rs GDNF, NO Chang et al. (2006)
CNTFRα CNTF Lee et al. (2009)
Pathogens TLRs Inflammatory Olson and Miller
cytokines and (2004)
chemokines
Development Neurogenesis TLRs IL-1β, IL-6, IFNγ Shigemoto-Mogami
(genesis, et al. (2014); Aarum
differentiation & et al. (2003); Walton
migration) et al. (2006); Nakanishi
et al. (2007); Cepko
et al. (1996)
Programmed cell TNFα Rs1 NGF, superoxide Frade and Barde
death (phagocytosis) (TNFR1) ions, TNFα (1998); Marin-Teva
et al. (2004); Sedel
et al. (2004)
Synaptogenesis IL-10 receptors TSPs, Chamak et al. (1995);
anti-inflammatory Moller et al. (1996);
cytokine IL-10 Lim et al. (2013)
Synaptic maturation KARAP\DAP12 Roumier et al. (2004)
Synapse removal Fractalkine CX3CL1 Paolicelli et al. (2011)
(synaptic pruning) receptor
(CX3CR1)
Complement Rs3 MHC1, Corriveau et al. (1998);
(CR3) complement Goddard et al. (2007);
components (C3, Schafer et al. (2012);
C1q) Stevens et al. (2007)
(continued)
Table 1 (continued)
receptors molecules
Adult life Modulation of Rs fir Cytokines & RNs Li et al. (2012);
neuronal activity beurotransmitters, (TGFβ-1, NO) Herrera-Molina and
neuropeptides & von Bernhardi (2005);
neuromodulators Tichauer et al. (2007)
Neuronal Fractalkine Rs ATP & gap Davalos et al. (2005);
surveillance (CX3CR1), junction proteins Liang et al. (2009);
purinergic Rs Haynes et al. (2006)
P2Y12
Synaptic plasticity Fractalkine CX3CL1 Paolicelli et al. (2011);
(involved in learning receptor Rogers et al. (2011)
& behavior) (CX3CR1)
NT, inflammatory Schmid et al. (2009);
cytokines (IL-1β, Goshen et al. (2007);
TNFα) Beattie and Malenka
(2002); Loscher et al.
(2003); Avital et al.
(2003); Labrousse et al.
(2009)
Neurogenesis in Neurotransmitter NT & regulatory Butovsky et al. (2006);
adult brain Rs cytokines (IGF1, Parkhurst et al. (2013);
BDNF, IL4) Ribeiro Xavier et al.
(2015)
Inflammatory Ribeiro Xavier et al.
cytokines (IL1-β, (2015); Ben-Hur et al.
IL-6 TNFα) (2003); Monje et al.
(inhibition) (2003); Koo and
Duman (2008)
TLRs (TLR2, Rolls et al. (2007)
TLR4)
Synaptic stripping MHC class F NGF, NT-4/5, Nakajima et al. (2007);
receptors TGFβ1, GDNF, Trapp et al. (2007);
FGF, IL-3 Oliveira et al. (2004);
Huh et al. (2000)
TNFα, IL-6, NO Nakajima et al. (2005)
Pathophysiological Neurodegeneration SRs Chemokines Rappert et al. (2004);
conditions (phagocytosis, (CCL2, CCL21, Koenigsknecht and
production factors CX3CL1, Landret (2004);
with inflammatory CXCL10, Alarcón et al. (2005);
and CXCL12) Murgas et al. (2012);
immunoregulatory Bezzi et al. (2001);
effect) Stewart et al. (2010);
van Weering et al.
(2011)
TLRs (TLR2, Inflammatory Murgas et al. (2012);
TLR4,TLR9) cytokines (IL1-, Bezzi et al. (2001);
IL-6, TNFα, IFNγ) Mount et al. (2007);
Chakrabarty et al.
(2010)
(continued)
Table 1 (continued)
receptors molecules
Hypoxia, cerebral Chemokine Rs ROS Block et al. (2006)
ischemia, (CXCR3, CCR3,
autoimunity CCR5, CXCR4)
Cytokine Rs RNS (NO) Murgas et al. (2012)
M-CSFR Mitrasinovic and
Murphy (2002)
TLRs Stewart et al. (2010);
Lotz et al. (2005);
Tahara et al. (2006)
As the resident immune cells of the CNS, microglia are the first line of defense
against exogenous threats. The pattern recognition receptors (PRRs), abundantly
expressed in microglia, detect infectious agents and assist in the control of the
adaptive immunity and the cooperative activities of effector cells (Beurel et al.
2010; Hanisch et al. 2008; Padovan et al. 2007). In addition to pathogen detection
by pathogen-associated molecular patterns (PAMPs), several PRRs, including
TLRs, also bind endogenous molecules that are generated or modified upon tissue
injury. These molecules are classified as damage- or danger-associated molecular
patterns (DAMPs) (Bianchi 2007; Kono and Rock 2008; Matzinger 2007). The
TLRs 1–9 and co-receptors, like CD14 are widely expressed in cells of the innate as
well as adaptive immune system, but also in nonimmune cells (Schaffler et al.
2007). In the brain, TLRs are mainly expressed in glia, although some has been
detected in neurons (Aravalli et al. 2007b; Carpentier et al. 2008; Hanisch et al.
2008; Okun et al. 2009; Konat et al. 2006).
The stimulation of TLRs triggers various programs of microglial activation and
activates secretion of cytokines and chemokines (Aravalli et al. 2007b; Okun et al.
2009). Several reports indicate the importance of TLRs in various CNS diseases
including infection, trauma, stroke, neurodegeneration, and autoimmunity (Babcock
et al. 2006; Caso et al. 2007; Lehnardt et al. 2002; Nau and Bruck 2002; Nguyen
et al. 2004).
Participation of Microglia in Development
Microglia are intimately involved in the development of the nervous system

(Table 1). They have roles both in neurogenesis and neuronal death. Microglia
appears to have both detrimental and supportive effects on neurogenesis (Ekdahl et al.
2009), which could depend in the activation state of microglia (Schwartz et al. 2006).
Differentiation of neural precursors in culture requires the presence of soluble factors

secreted by microglia (Nakanishi et al. 2007; Walton et al. 2006). Those factors are
also involved in directing migration of newly generated neural cells (Aarum et al.
2003).
The role of microglia for neuronal loss by programmed cell death during
development has been described in several brain regions, including the retina,
where the pro-apoptotic action of microglia is mediated through nerve growth factor
(NGF) (Frade and Barde 1998). Similarly, microglia induce apoptotic death of
Purkinje neurons by releasing superoxide ions (Marin-Teva et al. 2004), and
motoneurons apoptosis via secretion of TNFα in the embryo (Sedel et al. 2004).
In early postnatal development, elimination of excess synapses—known as
synaptic pruning—appears also to be a microglia-mediated mechanism. Mice
lacking fractalkine receptor (CX3CR1), have reduced numbers of brain microglia,
and show impairment of synaptic pruning, resulting in an abnormally high number
of synaptic spines (Paolicelli et al. 2011).
On the other hand, microglia are also involved in the formation of new
synapses, especially in the early postnatal brain. Microglia stimulate synaptoge-
nesis by secreting the extracellular matrix proteins thrombospondins (TSPs)
(Moller et al. 1996), which are also produced by astrocytes (Christopherson et al.
2005). TSP1 interacts with the integrin-associated protein CD47, which is regu-
lated by signal regulatory protein (SIRP)α, a transmembrane protein expressed by
neurons and macrophages (Matozaki et al. 2009). The SIRPα-CD47 complex is
involved in the regulation of migration and phagocytosis, immune homeostasis,
and neuronal networks, playing homeostatic roles in the immune system, and
participating in synaptic patterning (Umemori and Sanes 2008). Microglia also
serve roles on the functional maturation of synapses (Paolicelli and Gross 2011).
Behavioral abnormalities, including impairment of social interaction and
autistic-like behavior (Tang et al. 2014; Zhan et al. 2014) have been reported on
several models of microglial cell dysfunction.
Participation of Microglia in Adult Life
As the name implies, surveillance microglia actively survey the parenchyma, to

rapidly activate upon appearance of a threat to the CNS. Microglial activation in
response to various stimuli correlates with conspicuous morphological changes.
Microglia reduce the complexity and shortens their branched processes (Fig. 2).
Several stages can be identified, including process withdrawal, and formation of
new processes allowing mobility in the tissue (Lynch 2009; Stence et al. 2001;
Streit et al. 2005).
Microglia is a nonhomogeneous population, their activation being a highly
regulated process. Thus, activated microglia can acquire distinct functional states
(Hanisch and Kettenmann 2007; Perry et al. 2007; Perry and Holmes 2014;
Schwartz et al. 2006). Activation is not an all-or-none process, but varies depending
Fig. 2 Inflammatory activation-dependent morphological changes of microglia.

Immunohistochemical labeling of the constitutive identity marker Iba-1 and the phagocytic
activation-specific marker CD68, and counterstaining with Harris Haematoxylin, of hipocampal
cryosections obtained from inflammatory unstimulated and stimulated young mice. Low (4×)
magnification microphotographs of hippocampal section labeled with CD68 show slender-shaped
microglia evenly distributed. In contrast, the distribution of microglia in the hippocampus becomes
more cluster-like. At high magnification, activated microglia shows shorter sturdier processes.
CD68-labeled microglia show an amoeba-like shape with a big cell body and very short processes,
whereas Iba-1 shows many cells with long, although studier processes than those observed in
unstimulated animales. Scale bar = 100 µm in the right panel and 10 µm in the higher
magnification microphotographs at the left
on the stimulation context (Hanisch and Kettenmann 2007; von Bernhardi et al.
2015b; Areschoug and Gordon 2009). Multiple signals converge to maintain or
change their functional state and to regulate their specific functional repertoire
(Table 1). Activation is triggered when microglia detect the appearance, abnormal
concentration, or altered format of molecules that serve as signals (Block and Hong
2005; Block et al. 2007; Hanisch and Kettenmann 2007). The involvement of two
signaling systems has been proposed, an “on” receptor-mediated signaling corre-
sponding to a novel molecule that is recognized by microglia triggering activation;
and an “off” receptor-mediated signaling that persistently signals to maintain
microglia in a certain default activation state (Biber et al. 2007; Hanisch and
Kettenmann 2007; Kettenmann et al. 2011).
“On” signals include structures associated with bacterial cell walls, viral
envelopes, or their DNAs and RNAs, typically identified as signs of infection.
Pathogen structures are sensed through PRRs, such as TLRs (Hanisch et al. 2008)
and Scavenger Receptors (SRs) (Ozeki et al. 2006; Godoy et al. 2012; Murgas et al.
2014). Molecules released after tissue damage are also signals, and they induce
especially robust microglial responses (Nimmerjahn et al. 2005; Lu et al. 2010;
Napoli and Neumann 2009). Intracellular proteins or serum factors can activate
microglia when they are induced upon stress, appear in new compartments or suffer
biochemical modifications (Hanisch et al. 2008; Lehnardt et al. 2008; Rubartelli and
Lotze 2007), as well as some neurotransmitters indicating impaired neuronal
activity (Boucsein et al. 2003; Haynes et al. 2006). This, both pathogen- and
damage-associated molecular patterns (PAMPs/DAMPs, respectively), activate
microglia.
The “off” receptor-mediated signaling is due to the loss of constitutive control
signaling in the normal CNS, as observed with ligand-receptor systems
CD200-CD200R, CX3CL1- CX3CR1, and CD172a-CD47 (Barclay et al. 2002;
Brooke et al. 2004; Cardona et al. 2006; Hoek et al. 2000). Thus, the “on signals”
are identified as a sign of threat to the CNS homeostasis. Whereas in the “off
signal,” the loss of regulation is the signal.
The CNS show regional variations in glial and neuronal cell populations as
well as in their environment. For example, the different vulnerability of CA1
versus CA3 neurons depends on the regional microglia response upon stimulation
(Hanisch and Kettenmann 2007), with hippocampal neuronal cell death and glial
activation depending on the chemokine/receptor system CXCL10/CXCR3
(van Weering et al. 2011).
As discussed in Chapter “Glial Cells and Integrity of the Nervous System,”
acute self-limited activation of microglia should be deemed as protective, given
microglia primarily support and protect the structural and functional integrity of the
CNS. Although research has mostly focused on the detrimental consequences of
microglia-mediated neuroinflammation, and their potentiation of neuronal damage,
it is now accepted that microglia activation is important for protection and repair of
the diseased and injured brain. However, the final outcome will depend on the
environmental context and timeframe of action (Hellwig et al. 2013; Kierdorf and
Prinz 2013; von Bernhardi et al. 2015b). When encountering a mild injury or
impairment, microglia could act immediately to repair and offer trophic support,
and even reduce activating synaptic input by remodeling synapses (Trapp et al.
2007; Wake et al. 2009). However, the everyday activity of microglia is very
difficult to assess (Hanisch and Kettenmann 2007). Thus, in general there is much
more evidence on the failure and harmful contributions of microglia than on their
physiological roles.
Microglia serve several functional roles, modulating neuronal activity and via-
bility in culture and in the adult brain through direct contact with neurons (Li et al.
2012; Kohman et al. 2013) and through their release of soluble mediators, including
cytokines and reactive species (Herrera-Molina and von Bernhardi 2005; Ramírez
et al. 2008; Ramirez et al. 2005; Tichauer et al. 2007; von Bernhardi and Eugenin
2004; Glass et al. 2010; Di Filippo et al. 2010; von Bernhardi and Eugenin 2012).
Microglia play an active role in the functional integrity of the CNS and its
normal physiological performance even affecting learning and behavior (Ziv et al.
2006; Ziv and Schwartz 2008), through their effect, together with T cells, at various
levels. Both synaptic contacts and neuron trophism could depend on factors pro-
duced by activated microglia. Microglia express several neurotrophins (Elkabes
et al. 1996; Kim and de Vellis 2005; Ferrini and De Koninck 2013), releasing many
factors with powerful neurotrophic actions (Morgan et al. 2004). A number of

cytokines appear to have roles in the maturating CNS. In the adult CNS, IL1 drives
astrocytes proliferation in response to injury (Giulian et al. 1988).
Microglia also contribute to the plasticity of the CNS through support of neu-
rogenesis in adult individuals (Butovsky et al. 2006; Ziv et al. 2006; McPherson
et al. 2011; Ekdahl et al. 2003), which appears to depend on certain subpopulations
of microglia (Ribeiro Xavier et al. 2015). Microglia have neurotransmitter receptors
and are responsive to serotonin (5-HT) (Pocock and Kettenmann 2007) and cyto-
kine levels, and can influence precursor cells, showing a positive regulation of
neurogenesis by 5-HT and a negative regulation by stress and elevated glucocor-
ticoids (Kempermann 2002; Kempermann and Kronenberg 2003). Thus, under
certain conditions, microglia can adopt a pro-neurogenic phenotype, which involves
the expression of neurotrophins and regulatory cytokines, such as insulin-like
growth factor 1 (IGF1), BDNF, and IL4 (Parkhurst et al. 2013; Chen and Trapp
2015; Ribeiro Xavier et al. 2015). However, in inflammatory activation states,
microglia consistently appears to inhibit neurogenesis (Monje et al. 2003;
Nakanishi et al. 2007).
Similar to the synaptic pruning observed during development, microglia keep a
structural role in circuit refinement throughout life. The role of microglia in
removing synapses, is known as “synaptic stripping.” It is also observed in response
to focal inflammation (Trapp et al. 2007). The “stripping” predominantly removes
excitatory glutamatergic synapses, thus limiting neuronal excitability and glutamate
excitotoxicity (Linda et al. 2000). Microglia scan synapses, establishing contacts
with them that last a few minutes. In ischemia, contacts become longer, lasting for
around an hour (Wake et al. 2009). These long lasting interactions often result in
the disappearance of that synaptic contact. Any abnormalities in synaptic perfor-
mance could activate microglia. However, the signal for microglia to remove a
synapsis is poorly understood. The specificity of this action is associated with major
histocompatibility complex (MHC) class F receptors, which are present in both
neurons and microglia (Cullheim and Thams 2007) (see Chapter “Purine Signaling
and Microglial Wrapping” for further reading on synaptic stripping).
Participation of Microglia in Pathophysiological Conditions
Both the absence of protective functions served by microglia, or their abnormal or

excessive activation (von Bernhardi 2007; von Bernhardi et al. 2015b), could led to
functional impairment and eventually to development of a disease of the CNS. The
relevance of microglia activation and subsequent proliferation in aging, in which
condition they adopt an “activated-like” morphology (Fig. 3; see Chapter “Age-
dependent Changes in the Activation and Regulation of Microglia” for further
reading on aging) (Conde and Streit 2006; Gavilan et al. 2007; von Bernhardi 2007;
von Bernhardi et al. 2015b) as well as in many pathological contexts have been
discussed over the past years (von Bernhardi et al. 2010; Heneka et al. 2014; Perry
Fig. 3 Activation of hippocampal microglia with aging. Hippocampal cross section obtained from
9-, 13-, and 16-month old mice were labeled for Iba-1 and counterstaining with Harris
Haematoxylin, a monocyte-macrophage identity marker that labels constitutively microglia, to
compare the morphological features as the animal ages. At 9-months-old animals, microglia have
long and ramified processes, which persisted at 13 months of age. In contrast, microglia from
16-month-old mice begins to shorten their processes, which become sturdier and the cell body
increases in size. Scale bar = 10 µm
and Holmes 2014; Heneka et al. 2015; Hu et al. 2015; von Bernhardi et al. 2015a;
Yirmiya et al. 2015).
Any disturbance on brain homeostasis, as observed in infection, trauma, ische-
mia, altered neuronal activity, and both acute and chronic neurological injuries and
diseases, induces profound changes in microglial cell shape, gene expression and
functional behavior in which is defined as microglial cell activation (Hanisch and
Kettenmann 2007; Block et al. 2007; Colton and Wilcock 2010; Colton 2009;
Davoust et al. 2008; Graeber and Streit 2010; Streit et al. 2005; van Rossum and
Hanisch 2004). Activated microglia show enlarged cell bodies and short and sturdy
processes (Fig. 2). They can become motile and be actively recruited to the injury
site following chemotactic gradients, and can also increase their proliferation. This
phenotype is also correlated with functional changes occurring in complex and
broad spectrum responses (Table 1). The range of microglial cell activities covers
induction and release of multiple factors with inflammatory and immunoregulatory
effects, phagocytotic activities to clear debris, damaged cells, or pathogens, pro-
duction of neurotrophins and interaction with damaged neurons. Inflammatory
response goes from responses centered around the production and release of
inflammatory cytokines, such as TNFα, IL1β, and IL6 to release of factors with an
anti-inflammatory effect (Casano and Peri 2015; Hu et al. 2015; Chen and Trapp
2015). Although some authors consider inflammatory microglia as detrimental, and
anti-inflammatory regulatory microglia as neuroprotective, this rigid classification
fails to recognize the complexity of microglial cell function and regulation (Fenn
et al. 2014). Furthermore, regulatory microglia do not show always neuroprotective
effects (Cherry et al. 2014).
The role of microglia-mediated phagocytosis in neurodegeneration has been
established by several experimental approaches. Microglia are needed for removal
of the dendritic trees of interneurons in the dentate gyrus after entorhinal cortex
lesions (Rappert et al. 2004). In response to the lesion, microglia accumulate at the
molecular layer in the dentate gyrus, mediated by signaling through the chemokine
receptor CXCR3. Deletion of CXCR3 results in the failure of microglia recruitment,
and the dendritic trees of interneurons are preserved.
Microglia also phagocytose molecules and debris such as myelin or amyloid
deposits. Several studies report that Aβ is taken up by microglia in culture through
mechanisms depending on Scavenger Receptors (Koenigsknecht and Landreth
2004; Alarcón et al. 2005; Cornejo and von Bernhardi 2013; Murgas et al. 2012),
among others.
As discussed in Chapter “Age-dependent Changes in the Activation and
Regulation of Microglia,” there is increasing evidence for altered chemokine sig-
naling in diverse CNS diseases such as Alzheimer’s disease (AD) or multiple
sclerosis (Gebicke-Haerter et al. 2001; Trebst et al. 2008) which may involve
microglia activation (Stewart et al. 2010). Microglial cells from AD brains may
have elevated levels of CCR3 and CCR5 receptors (Gebicke-Haerter et al. 2001).
CXCL10 and its receptor CXCR3 have been linked to various CNS pathologies
(van Weering et al. 2011). Studying the mechanisms by which this system mediates
N-methyl-d-aspartate (NMDA)-induced neuronal toxicity in the hippocampus, the
authors demonstrated that astrocytes and microglia cooperate to deliver the effect
and that the deficiency in either the ligand or the receptor diminished or enhanced
cell death depending on the tissue subregion and that microglia was the responsible
cellular element by which this difference in neuronal vulnerability is organized.
A mechanism involving microglia, astrocytes, and chemokines has been pro-
posed for glutamate toxicity (Bezzi et al. 2001). Binding of CXCL12 (stromal
cell-derived factor, SDF-1α) to its receptor CXCR4 in astrocytes, results in Inositol
trisphosphate (InsP3) production, [Ca2+]i increase, and release of TNFα. The
binding of TNFα to its receptor triggers signaling, through autocrine a paracrine
mechanism that causes prostaglandin E2 (PGE2) production. The PGE2, in turn,
induces the release of glutamate, which can participate in glia-glia or glia-neuron
communication, but can also initiate neurotoxicity. In the latter situation, SDF-1α
would also act on microglia, thus driving enhanced TNFα release from both glial
populations and ultimately causing massive glutamate release.
AD is associated with a significant elevation of TLR expression in the brain
(Letiembre et al. 2009; Walter et al. 2007). Treatment with Aβ potentiated TLR2
and TLR4-mediated responses, while inhibiting TLR9 in mouse microglia cultures,
(Lotz et al. 2005). At the same time, all three receptors (TLR2, TLR4, and TLR9)
stimulated the uptake of Aβ by microglia (Tahara et al. 2006). The levels of TLRs
in CNS are generally upregulated in many neurodegenerative diseases, including
multiple sclerosis, Parkinson’s disease, and amyotrophic lateral sclerosis
(ALS) (Okun et al. 2009).
Microglia activation, in turn, upregulates the synthesis of TLRs (Kielian et al.
2005; McKimmie and Fazakerley 2005). Similarly, the levels of TLRs in microglia
are increased following hypoxia (Ock et al. 2007) and cerebral ischemia (Ziegler
et al. 2007) and by inflammatory processes; for example, TNFα stimulates
expression of TLR2 in cultured mouse microglia (Syed et al. 2007).
TLRs also regulate microglial death following pathological activation. TLR4

triggers microglial apoptosis via autocrine production of interferon gamma (IFNγ),
whereas TLR2 is coupled to caspase-8-dependent apoptotic pathways (Lehnardt
et al. 2007). Similarly, TLR2 participate in microglial apoptosis following human
immunodeficiency virus (HIV-1) infection (Aravalli et al. 2007a, 2008).
A link of neurodegenerative processes in AD to microglial TLR4 is suggested
because Aβ fibers bind to CD14, the co-receptor for LPS signaling via TLR4
(Fassbender et al. 2004). CD14 and TLR-dependent mechanisms appear to promote
Aβ clearance and participate in inflammatory responses of microglia (Fassbender
et al. 2004; Landreth and Reed-Geaghan 2009; Reed-Geaghan et al. 2009; Tahara
et al. 2006). Pronounced CD14 immunoreactivity is observed in microglia close to
AD lesion sites in AD brains (Liu et al. 2005). Importantly, a microglial
CD36-TLR4-TLR6 complex appears to promote inflammation in response to Aβ
(Stewart et al. 2010).
However, TLR signaling can also be neuroprotective, by both driving clearance
of infectious agents, and by organizing CNS-intrinsic as well as immune
system-mediated support of neural cell survival, tissue preservation, and CNS
functioning (Glezer et al. 2007; Hanisch et al. 2008). Thus, a critical issue is to
understand the mechanisms by which TLRs could engage in detrimental or in
beneficial programs.
Concluding Remarks
Microglia affects the development, structure, and function of neuronal networks.

They constantly monitor the status of synaptic contacts and receive information
from neuronal activity. Multiple activation states of microglia may allow for the
existence of microglia with different functions, which dynamically interact with
neurons and potentiate their plastic capabilities. Furthermore, they appear to be also
able to remodel neuronal connectivity and thus participate in physiological
processes.
Commitment to distinct reactive phenotypes depending on their activation
profile would then have a variable effect on neurons. It will be important to identify
the nature of such instructing signals as they govern functional orientations of
microglia. Little is also known about the heterogeneity of microglia, i.e., the dif-
ferences in functional capacities of individual microglial populations within dif-
ferent CNS regions. Finally, in pathological situations with blood-derived
monocytes/macrophages infiltrating the CNS, features and functions of resident
microglia and the newly invading cells may complement each other, with both
detrimental and beneficial consequences (Shechter et al. 2009; Simard et al. 2006).
Understanding this various issues will be especially interesting to develop
migroglia-based strategies for the management of several impairments of the ner-
vous system.
Acknowledgments This work was supported by grant FONDECYT 1131025 to RvB.
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Physiological Functions of Glial Cell
Hemichannels
Juan A. Orellana
Abstract The brain performs exceptionally complex and dynamic tasks that
depend on the coordinated interaction of neurons, glial cells, endothelial cells,
pericytes, smooth muscle cells, ependymal cells, and circulating blood cells.
Among these cells, glial cells have emerged as crucial protagonists in the regulation
of synaptic transmission and neural function. Indeed, these cells express a wide
range of receptors that enable them to sense changes in neuronal activity and the
microenvironment by responding locally via the release of bioactive molecules
known as gliotransmitters. In the central nervous system (CNS), a novel mechanism
that allows gliotransmission via the opening of hemichannels has been proposed.
These channels are composed of six protein subunits consisting of connexins or
pannexins, which are two highly conserved protein families that are encoded by 21
and 3 genes, respectively, in humans. Typically, glial cell hemichannels exhibit low
levels of activity, but this activity is sufficient to ensure the release of a broad
spectrum of gliotransmitters, including ATP, D-serine, glutamate, adenosine, and
glutathione. Here, we briefly review the current findings regarding the effects of the
hemichannel-dependent release of gliotransmitters on the physiology of the CNS.

Keywords Connexins Pannexins (ATP) adenosine triphosphate Astroglial

signaling Gliotransmitters
Abbreviations and acronyms

Arc Arcuate
BLA Basolateral amygdala
[Ca2+]i Intracellular free Ca2+ concentration
CSF Cerebrospinal fluid
Cx26 Connexin26
J.A. Orellana (&)

Departamento de Neurología, Escuela de Medicina, Pontificia Universidad
Católica de Chile, Marcoleta #391, Santiago, Chile

DOI 10.1007/978-3-319-40764-7_5
94 J.A. Orellana
Cx36 Connexin36
Cx43 Connexin43
GJCs Gap junction channels
kDa Kilodalton
KO Knockout
NAD+ Nicotinamide adenine dinucleotide
MAP2 Microtubule-associated protein 2
MBH Mediobasal hypothalamus
MI Metabolic inhibition
Panx1 Pannexin1
Panx2 Pannexin2
PGE2 Prostaglandin E2
RTN Retrotrapezoid nucleus
siRNA Small interfering ribonucleic acid
VMH Ventromedial hypothalamic nuclei
VMS Ventral medullary surface
TDCS Transcranial direct current stimulation
Introduction
To achieve a coordinated response to an external stimulus, many phyla have

developed complex and integrative neural structures that progressively enabled the
sophisticated analysis of information. Indeed, as the central nervous system
(CNS) evolved from a basic netlike structure to compacted ganglia and centralized
brains, a new cell type with nonneuronal features emerged in several organisms, that
is the glia (Barres 2008). Possibly driven by positive Darwinian selection, glial cells
have persisted in high densities and have acquired greater diversity in high mammals.
Indeed, they are the most abundant brain cell population—10 times more frequent
than neurons—and constitute approximately 50 % of the total cell mass of the CNS
(Verkhratsky and Toescu 2006). There are two major classifications of the glial
family, macroglia (oligodendrocytes, astrocytes, NG2 cells, and ependymoglial cells)
and microglia, which belong to neuroectodermal and mesenchymal origin,
respectively.
Although often overlooked for much of the twentieth century when discussing
brain function, in the last two decades, glial cells have emerged as crucial pro-
tagonists in the processing of highly complex information. This is particularly true
for astrocytes, which constitute a far-reaching syncytial network that anatomically
and functionally connects neuronal synapses with brain blood vessels (Volterra and
Meldolesi 2005). Through its processes, each astrocyte may contact a numbers of
Physiological Functions of Glial Cell Hemichannels 95
neuronal chemical synapses that range from thousands to millions depending on the
mammalian species (Oberheim et al. 2006). Thus, in a delicate physical and
functional interaction, astroglial processes, together with pre- and postsynaptic
neuronal structures, comprise the “tripartite synapse” (see Chap. 1). Embedded in
this structure, astrocytes sense neuronal activity and respond locally through the
release of bioactive molecules termed “gliotransmitters” (e.g., glutamate, adenosine
triphosphate [ATP] and D-serine) (Perea et al. 2009). In addition to surrounding the
synaptic cleft, astrocytes project specialized terminal processes, which are known as
“endfeet”, toward capillaries, intracerebral asterioles, and venules and thus cover
99 % of the abluminal vascular surface (Simard et al. 2003). This resulting com-
munication with neurons and vascular cells provides astrocytes with an incompa-
rable architectural advantage that facilitates the local and long-distance release of
gliotransmitters and vasoactive factors and thereby modulates different neuronal
circuits and networks.
Microglia are not far behind astrocytes regarding the regulation of neuronal
chemical synapses. In addition to their well-known roles on brain immunity and
inflammatory responses, microglia are now recognized as essential players in the
integration and consolidation of neuronal circuits. Various studies have shown that
microglia constantly extend toward and retract from synapses, participating in
broad range of previously undiscovered functions, including neuronal surveillance,
synapse elimination, regulation of cell death, and others (Schafer et al. 2013;
Tremblay et al. 2010; Wake et al. 2013). Indeed, some authors have proposed a
shift of the current concept of tripartite synapse to a “quad-partite synapse” (Schafer
et al. 2013). Interestingly, neurotransmitter release by neurons can modify various
aspects of glial cell function, including cellular migration, phagocytosis, intercel-
lular Ca2+ wave generation, metabolic coupling, blood flow control, gliotransmitter
release, and others (Fields and Burnstock 2006; Fields and Stevens-Graham 2002;
Inoue et al. 2007). This reciprocal modulation closes a permanent feedback loop of
interactions between neurons and glial cells denominated “neuron-glia crosstalk”.
As previously discussed in Chap. 1, gliotransmission is part of the basis of
neuron–glia crosstalk. Multiple mechanisms of the mediation of gliotransmitter
release have been described, including Ca2+-dependent exocytosis (Bezzi et al.
2004; Imura et al. 2013; Zhang et al. 2004), carrier membrane transport (Rossi et al.
2000) and the opening of a wide range of channels, such as P2X7 channels (Duan
et al. 2003; Hamilton et al. 2008; Suadicani et al. 2006), volume-regulated anion
channels (Kimelberg et al. 1990; Lee et al. 2010; Rudkouskaya et al. 2008; Takano
et al. 2005) and connexin hemichannels (Fig. 1); (Iglesias et al. 2009; Stout et al.
2002; Ye et al. 2003). Each hemichannel is composed of six protein subunits
termed connexins. The latter belong to a highly conserved protein family encoded
by 21 genes in humans and 20 in mice, with orthologs in other vertebrate species
(Fig. 2); (Abascal and Zardoya 2013). Connexins are abundantly expressed in cells
of the CNS (Orellana et al. 2009), and they are named after their predicted
molecular mass expressed in kilodaltons (kDa), for example, connexin43 (Cx43)
has a molecular mass of *43 kDa.
96 J.A. Orellana
Fig. 1 Mechanisms of gliotransmitter release. Glial cells release gliotransmitters (e.g., glutamate,
D-serine and ATP) through Ca2+- and SNARE-dependent exocytosis (1) in addition to the release
that occurs through alternative non-exocytotic pathways (2–8). Depolarization, reductions in
extracellular divalent cation concentrations, increases in intracellular Ca2+ and posttranslational
modifications might result in the opening of connexin and pannexin hemichannels (HCs) and thus
allow the release of gliotransmitters (2). Long-lasting activation of P2X7 by ATP might lead to the
appearance of large currents and the rapid exchange of large molecules, including the release of
gliotransmitters (3). One theory states that P2X7 receptor conductance dilates over the time and
thereby facilitates the passage of large molecules; however, another theory states that ATP
activates a second non-selective permeabilization pathway (Baroja-Mazo et al. 2012). Recently, it
was shown that Panx1 hemichannels might mediate this permeability for large molecules in
astrocytes (4) (Iglesias et al. 2009). Additionally, gliotransmitter release may occur through
volume-regulated anion channels (VRAC) (5) and different carriers and/or co-transporters acting
normally or in reverse (6) (e.g., excitatory amino acid transporters, the cystine-glutamate antiporter
and the D-serine/chloride co-transporter)
For many years, the main function attributed to connexin hemichannels was
providing the building blocks of gap junction channels (GJCs), which allow direct
but selective cytoplasmic continuity and molecular exchange between contacting
cells (Saez et al. 2003); (Fig. 2). Nonetheless, in the last decade, the presence of
functional connexin hemichannels in “nonjunctional” membranes has been
demonstrated by several experimental approaches (Saez and Leybaert 2014). These
channels act like aqueous pores that are permeable to ions and small molecules and
thus provide a diffusional route of exchange between the intra- and extracellular
milieu (Saez et al. 2010; Wang et al. 2013). Therefore, these channels allow the
cellular release of relevant quantities of autocrine and paracrine signaling molecules
(e.g., ATP, glutamate, nicotinamide adenine dinucleotide [NAD+] and pros-
taglandin E2 [PGE2]) to the extracellular milieu (Saez et al. 2010).
Recently, another gene family encoding a set of three membrane proteins termed
pannexins was identified (Bruzzone et al. 2003); (Fig. 2). Currently, the majority of
Fig. 2 Basic structure and function of connexin- and pannexin-based channels. a Pannexins and
connexins share a similar membrane topology that includes four a-helical transmembrane domains
(M1-M4) connected by two extracellular loops (E1 and E2) and one cytoplasmic loop (CL) in
which both the amino (NH2)- and carboxy (COOH)-termini are intracellular. The relative positions
of the extracellular loop cysteines and glycosylated asparagines are also shown. b Vertebrate
hemichannels are formed from six connexins or pannexins consisting of six subunits each.
Recently, a band pattern that is more consistent with an octamer than with a hexamer was observed
for Panx2 in cross-linking studies and native gels of purified homomeric full-length and C-terminal
truncation mutants (Ambrosi et al. 2010). Each hemichannel is formed by connexins or pannexins
that oligomerize laterally, resulting in a central pore in the activated state. Under resting
conditions, the hemichannels preferentially remain closed, but they may be activated by diverse
physiological and pathological conditions to provide a diffuse transmembrane route between the
intra- and extracellular milieu. The middle center of the schematic shows connexin gap junction
channels (GJCs) in close contact between two cells. These channels permit the intercellular
exchange of metabolites, second messengers and ions and allow for the transmission of
intercellular Ca2+ waves, the spread of electrotonic potentials, local blood flow regulation and
spatial buffering of ions and metabolites. Although most evidence indicates that pannexins do not
form GJCs, one recent study suggested that Panx1 and Panx3 can form GJCs with
pharmacological and permeability properties that are distinct from those of channels comprised
of Cx43 (Sahu et al. 2014)
98 J.A. Orellana
the evidence indicates that pannexins support the formation of single membrane
channels, similar to connexin hemichannels (Sosinsky et al. 2011); however, a
recent study has questioned this idea and proposed that pannexin channels also
function as GJCs (Sahu et al. 2014). Although connexins and pannexins do not
share significant amino acid sequences, they have similar secondary and tertiary
structures, with four a-helical transmembrane domains that are connected by one
cytoplasmic and two extracellular loops in which both the N- and C-termini are in
the intracellular compartment. Unlike the other pannexins and connexins that
oligomerize into hexameric hemichannels, pannexin2 (Panx2) seems to form
octamers (Ambrosi et al. 2010).
It is broadly accepted that impairments of the permeability properties of
hemichannels might be critical to the initiation and maintenance of the homeostatic
imbalances observed in diverse brain diseases (Davidson et al. 2013; De Vuyst et al.
2011; Fasciani et al. 2013; Orellana et al. 2009, 2012b; Salameh et al. 2013, 2009).
How might hemichannels contribute to cell damage? At one end, uncontrolled
opening of hemichannels may facilitate the excessive release of molecules that are
toxic to neighboring cells, such as glutamate and ATP (Orellana et al. 2011;
Takeuchi et al. 2006); (Fig. 3). On the other hand, disruptions of the regulatory
properties of hemichannels might lead to an uncontrolled influx of potentially toxic
molecules (e.g., Ca2+) (Fig. 3). Indeed, given that hemichannels are permeable to
Ca2+ (De Bock et al. 2012; Fiori et al. 2012; Sanchez et al. 2009; Schalper et al.
2010) and that their opening is controlled by intracellular free Ca2+ concentration
([Ca2+]i) (De Vuyst et al. 2006, 2009; Ponsaerts et al. 2010), it is possible that
hemichannel activity in concert with N-methyl-D-aspartate [NMDA] or P2 receptor
activation might result in Ca2+ overload and the subsequent generation of free
radicals, lipid peroxidation, and plasma membrane damage (Fig. 3). Another
important mechanism of cell damage that is triggered by hemichannel opening
could rely on the osmotic and ionic imbalances induced by the uncontrolled influx
of Na2+ and Cl−, which would result in further cell swelling and plasma membrane
breakdown (Islam et al. 2012; Paul et al. 1991); (Fig. 3).
Currently, the majority of the evidence supports the notion that hemichannels
play important roles in physiological brain functions, including ischemic tolerance
(Lin et al. 2008; Schock et al. 2008), establishment of adhesive interactions (Cotrina
et al. 2008), fear memory consolidation (Stehberg et al. 2012), synaptic transmis-
sion (Chever et al. 2014; Klaassen et al. 2011; Prochnow et al. 2012), spontaneous
electrical activity (Moore et al. 2014), glucose sensing (Orellana et al. 2012a),
chemoreception (Wenker et al. 2012), blood–brain barrier permeability (De Bock
et al. 2011; Kaneko et al. 2015), neuronal migration (Liu et al. 2010, 2012), and
metabolic autocrine regulation (Kawamura et al. 2010). Given that previous articles
have extensively reviewed the role of hemichannels under pathological conditions
(Bosch and Kielian 2014; Davidson et al. 2013; Shestopalov and Slepak 2014), we
focus here on presenting a brief collection of the most recent findings regarding the
physiological implications of hemichannels in the CNS.
Fig. 3 Hemichannel opening is associated with cell death. Under normal conditions, hemichan-
nels (yellow channels) exhibit low levels of activity that are sufficient to ensure the release of
several paracrine molecules. However, upon exposure to pathological stimuli, the hemichannels
undergo dysregulation and an exacerbated opening that results in cellular damage via the following
different mechanisms: a Ca2+ entry via hemichannels may activate phospholipase A2 and
subsequently induce the generation of arachidonic acid and the activation of the cyclooxygenase/
lipoxygenase pathways, which further leads to increased levels of free radicals, lipid peroxidation
and plasma membrane damage; b Na2+ and Cl− entry via hemichannels triggers cellular swelling
due to an increased influx of H2O via aquaporins (red channels); and c the release of large amounts
of transmitters via hemichannels (e.g., glutamate and ATP) might reduce the viability of healthy
neighboring cells
100 J.A. Orellana
Hemichannel Function in the Physiology of the CNS
Brain Ischemic Tolerance
Although molecular mechanisms of ischemic tolerance have been widely investi-

gated (Gidday 2006), most of these studies have focused on neurons. Nevertheless,
recent findings suggest that astroglial signaling via hemichannels may be relevant to
this process. Typically, brief episodes of sublethal ischemia known as precondi-
tioning induce resistance to subsequent more severe insults (Gidday 2006). This
phenomenon is often called ischemic tolerance and represent a physiological pro-
cess that provides robust neuroprotection and remains as a key medical challenge to
the understanding of neuronal repair after brain injury. Pioneering studies by Lin
and colleagues showed that preconditioning reduces the degradation of Cx43 in
astrocytes, leading to a prominent increase in surface levels of Cx43 hemichannels
(Lin et al. 2008). Underscoring the involvement of hemichannels in preconditioning
responses, Cx43 null mice have been found to be insensitive to hypoxic precon-
ditioning, whereas wild-type littermate mice exhibit prominent reductions in infarct
volume after the induction of preconditioning via the occlusion of the middle
cerebral artery (Lin et al. 2008). The implicated mechanism involves the release of
ATP through astroglial Cx43 hemichannels and the subsequent hydrolyzation of
this ATP to adenosine, which results in a potent neuroprotective effect.
In the same year of the above study, it was proposed that connexin36 (Cx36)
hemichannels could participate in the ischemic tolerance phenomenon (Schock
et al. 2008). This study showed that prior depolarization with KCl reduces the
neuronal death triggered by metabolic inhibition (MI) in an in vitro model of
ischemia that is characterized by the suppression of anaerobic glycolysis and
mitochondrial oxidative phosphorylation (Schock et al. 2008). Prior depolarization
with KCl triggers a Cx36 hemichannel opening in cerebellar granule neurons that
underpins the release of ATP, and the action of ATP on P2Y receptors provides a
potent neuroprotective effect against MI (Schock et al. 2008). Interestingly, phar-
macological blockade or downregulation of Cx36 reduces the KCl-induced
ischemic tolerance and ATP release and thus reveals a crucial role of Cx36
hemichannels in this response. Although Panx1 hemichannels have been linked to
ischemic tolerance in the heart and to the release of cardioprotectants (Vessey et al.
2010, 2011), their involvement in brain preconditioning responses remains
unknown.
Brain Chemoreception
When the nervous system detects changes in the peripheral pH (or CO2), it triggers
a coordinated response that modulates breathing control to maintain arterial CO2
levels. This phenomenon is typically known as central chemoreception and
involves the activation of acid-sensitive neurons via several as-yet unidentified

acid-sensitive ion channels in different hindbrain sites, including the retrotrapezoid
nucleus (RTN), medullary raphe and locus coeruleus (Funk 2010) (see Chap. 6).
A few years ago, a crucial role of glial cell hemichannels in the central control of
breathing was uncovered. Findings from Huckstepp and colleagues demonstrated
that Cx26 hemichannels participate in the CO2-dependent release of ATP in the
ventral medullary surface (VMS) (Huckstepp et al. 2010b). Using ex vivo dye
uptake measurements, these authors observed that a glial fibrillary acidic protein
(GFAP) positive population but not a microtubule-associated protein 2 (MAP2)
positive population exhibited a Pco2-dependent increase in hemichannel activity in
the VMS and that this increase was correlated with high levels of connexin26
(Cx26) immunoreactivity. Indeed, the CO2-dependent dye uptake occurred in the
subpial astrocytes and the astrocytes that ensheath the penetrating blood vessels; all
of these cells exhibited prominent Cx26 expression. Interestingly, hemichannel
blockers diminished the CO2-dependent increase in the breathing response, as
measured with phrenic nerve discharge recordings (Huckstepp et al. 2010b). These
data suggest that a CO2-sensitive mechanism that is linked to Cx26 hemichannel-
dependent release of ATP may play an important role in central respiratory
chemosensitivity. In agreement with this idea, recent in vitro experiments in HeLa
cells showed that Cx26 transfection is sufficient to confer the ability to release ATP
and exhibit hemichannel current events upon CO2 stimulation to these cells
(Huckstepp et al. 2010a). Further studies found that a carbamate bridge between
Lys125 and Arg104 might act as a CO2 sensor in Cx26 (Meigh et al. 2013).
Notwithstanding this evidence, further pharmacological (e.g., mimetic peptides)
and molecular [e.g., tissue-specific inducible knockouts (KO)] studies are required
to completely elucidate the contributions of glial Cx26 hemichannels to central
chemoreception and breathing control.
Brain Glucose Sensing
The hypothalamus controls energy homeostasis and feeding/satiety behaviors based

on its ability to sense peripheral glucose levels. A plethora of recent studies have
focused on the unexpected role of glial cells in these processes (Tonon et al. 2013).
The majority of these studies have focused on astrocytes (McDougal et al. 2013),
and the involvement of other glial cells in hypothalamic glucose sensing has
received little attention. Tanycytes are among the glial cells that have recently been
proposed to mediate the above-mentioned phenomena (Langlet 2014). These cells
are ependymoglial cells that are localized in the III–V ventricle of the basal
hypothalamus, and their cellular processes contact the cerebrospinal fluid (CSF) and
neurons from regions involved in energy balance, such as arcuate (Arc) and ven-
tromedial hypothalamic nuclei (VMH) (Rodriguez et al. 2005). Pioneering studies
from Frayling and colleagues (Frayling et al. 2011) showed that non-metabolizable
analogues of glucose increase [Ca2+]i in hypothalamic tanycytes, specifically those
102 J.A. Orellana
located in the most dorsal regions of the hypothalamus. Moreover, this phe-
nomenon is dependent on P2Y1R activation, indicating the involvement of ATP or
its metabolites (Frayling et al. 2011). Supporting this idea, it was recently reported
that glucose stimulation evokes the release of ATP in tanycytes, which triggers
changes in [Ca2+]i that depend on a glucose sensing mechanism similar to that
observed in b-pancreatic cells (Orellana et al. 2012a). Ethidium uptake and
whole-cell patch clamp recordings have been used to demonstrate that acute glu-
cose stimulation triggers the opening of Cx43 hemichannels, which results in the
further release of ATP and autocrine activation of P2Y1Rs (Orellana et al. 2012a).
In agreement with the notion that Cx43 participates in glucose sensing and feeding
behaviors in the brain, recent in vivo experiments have shown that its expression in
the mediobasal hypothalamus (MBH) is decreased by fasting and is augmented by
glucose injection-induced hyperglycemia (Allard et al. 2014). Interestingly, small
interfering ribonucleic acid (siRNA) injection-induced downregulation of Cx43 in
the MBH results in an impaired insulin response in rats that were subjected to an
intracarotid glucose load (Allard et al. 2014). Further in vivo experiments are
required to determine how Cx43 hemichannels regulate the insulin response to
peripheral glucose and the mechanisms involved in tanycyte-hypothalamic neuron
communication.
Cognition and Behavior
The majority of cognitive and behavioral studies have centrally positioned neurons
as the primary cells that are responsible for higher brain processing. Nonetheless,
evidence strongly suggests that glial cells might crucially participate in cognitive
tasks and behavior and even contribute to different psychiatric disorders (Banasr
et al. 2010; Perea et al. 2009). Recently, Stehberg et al. (2012) determined that the
release of gliotransmitters via Cx43 hemichannels is critical for fear memory
consolidation in the basolateral amygdala (BLA), which is a brain region that is
essential for anxiety and emotional memory processing. These authors microin-
jected the BLA with TAT-L2, a peptide that specifically inhibits Cx43 hemichan-
nels without affecting gap junctional communication among neighboring astrocytes
(Ponsaerts et al. 2010). After 24 h of an auditory fear conditioning protocol, the rats
treated with TAT-L2 peptide exhibited impaired fear memory consolidation and
complete amnesia toward the auditory fear conditioning training (Stehberg et al.
2012). These responses were transitory and did not influence short-term memory,
locomotion, or shock reactivity. Importantly, the TAT-L2-induced inhibition of fear
memory consolidation was blunted by co-injection of TAT-L2 with an exogenous
cocktail of the following gliotransmitters: glutamate, glutamine, lactate, D-serine,
glycine, and ATP (Stehberg et al. 2012).
A few studies have linked pannexins with a physiological role in cognition and
behavior. For example, Prochnow and colleagues recently showed that pannexin1
(Panx1)−/− mice exhibit cognitive alterations that include exacerbated anxiety
behaviors, deficient object recognition, and impaired spatial learning memory

(Prochnow et al. 2012). Followup work in Panx1−/− mice employing cookie-finding
tests, revealed that their reduced ability to detect odorant cues is possibly linked to
impairments in learning capabilities, because the processing of olfactory informa-
tion appeared to be unaffected in these animals (Kurtenbach et al. 2014). Despite
these observations, Panx1−/−/Panx2−/− mice do not exhibit changes in anxiety or
exploratory behaviors compared with their wild-type littermates (Bargiotas et al.
2012). Heterologous expression of Panx2 in Xenopus oocytes does not produce
functional hemichannels (Bruzzone et al. 2001), but neurons seem to express these
channels even in the absence of Panx1 (Bargiotas et al. 2011). The latter finding has
motivated speculation that, as a consequence of posttranslational modifications or
dimerization with other partners, Panx2 can form hemichannels in mammalian cells
in the absence of Panx1 (Bargiotas et al. 2011). Whether compensatory regulations
between Panx1 and Panx2 account for the differences in the behaviors of Panx1−/−
and Panx1−/−/Panx2−/− mice is a subject for future investigations, and the use of
cell tissue-specific KO of Panx1 and/or Panx2 (e.g., neurons versus astrocytes) will
be required for such studies.
Pannexins have been implicated in alterations in cognition and behavior during
brain recovery after injury. Indeed, Panx1 hemichannel opening worsens seizures
and behavioral manifestations of status epilepticus in vivo (Santiago et al. 2011).
Santiago and colleagues determined that Panx1 hemichannels underpin membrane
permeabilization and ATP release during kainic acid-induced seizures. Relevantly,
pharmacological (mefloquine) and genetic approaches (Panx1−/− mice) have been
used to elucidate the contribution of Panx1 hemichannels to the amelioration of
seizure outcomes (Santiago et al. 2011). These data agreed with the protective
effects of pharmacological Panx1 hemichannel blockade in a pilocarpine-induced
seizure model (Kim and Kang 2011). The beneficial effect observed in Panx1−/−
mice may result from reduced ATP release, which might limit the activation of
excitatory P2X receptors and thus the progression of status epilepticus. Others
researchers have proposed that reduced Panx1 function might prevent the total loss
of cellular ATP and thus impair recovery from prolonged seizures in animals
(Santiago et al. 2011). Accordingly, the inhibition of Panx1 hemichannels with
probenecid ameliorates the spatial learning deficits of aged rats (Mawhinney et al.
2011), whereas transcranial direct current stimulation (TDCS), which is a thera-
peutic approach for several neurological disorders (Williams et al. 2009), decreases
Panx1 expression and augments spine density following brain ischemia (Jiang et al.
2011). Moreover, recent observations have shown that Panx1−/−/Panx2−/− mice
exhibit improved functional outcomes and smaller infarcts than wild-type mice
when subjected to ischemic stroke (Bargiotas et al. 2011). Followup work revealed
that Panx1−/−/Panx2−/− mice exhibit reductions in impairments in parameters such
as exploration, anxiety, sensorimotor function, and behavioral symmetry compared
with their wild-type littermates (Bargiotas et al. 2012). Because some evidence
indicates that Panx1 likely forms GJCs (Sahu et al. 2014), future efforts to uncover
the roles of pannexins in behavior need to consider that the molecular ablation of
Panx1 might affect the functions of both channels.
104 J.A. Orellana
Concluding Remarks
The initial characterization of non-junctional hemichannel currents involved large

depolarizations and cell lysis; thus, hemichannel opening was considered to be
incompatible with normal cell life (Paul et al. 1991). Despite these early beliefs,
current evidence supports the notion that hemichannels seem to be active under
physiological conditions. Apparently, these channels are less open in normal than in
pathological states, but they are sufficiently open to ensure paracrine communica-
tion in different tissues, including the CNS. Do the permeability properties of
hemichannels remain unaltered during inflammation? How do changes in the per-
meabilities of hemichannels to Ca2+ and different gliotransmitters influence brain
diseases? Which posttranslational modifications are responsible of these changes?
These are some of the puzzling questions that future studies should attempt to
address. Characterization of the primary elements that specifically regulate
hemichannel function in physiological and pathophysiological conditions will
enable the identification of future therapies for neurological disorders.
Acknowledgments This work was partially supported by the Fondo Nacional de Desarrollo
Científico y Tecnológico (FONDECYT) Grant 11121133 (to JAO), 1160710 (to JAO), the
Comisión Nacional de Investigación Científica y Tecnológica (CONICYT) and Programa de
Investigación Asociativa (PIA) Grant Anillo de Ciencia y Tecnología ACT1411 (to JAO). We
apologize to the authors and groups whose work we did not cite due to space limitations.
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Role of Astrocytes in Central Respiratory
Chemoreception
Jaime Eugenín León, María José Olivares

and Sebastián Beltrán-Castillo
Abstract Astrocytes perform various homeostatic functions in the nervous system

beyond that of a supportive or metabolic role for neurons. A growing body of
evidence indicates that astrocytes are crucial for central respiratory chemoreception.
This review presents a classical overview of respiratory central chemoreception and
the new evidence for astrocytes as brainstem sensors in the respiratory response to
hypercapnia. We review properties of astrocytes for chemosensory function and for
modulation of the respiratory network. We propose that astrocytes not only mediate
between CO2/H+ levels and motor responses, but they also allow for two emergent
functions: (1) Amplifying the responses of intrinsic chemosensitive neurons
through feedforward signaling via gliotransmitters and; (2) Recruiting
non-intrinsically chemosensitive cells thanks to volume spreading of signals (cal-
cium waves and gliotransmitters) to regions distant from the CO2/H+ sensitive
domains. Thus, astrocytes may both increase the intensity of the neuron responses
at the chemosensitive sites and recruit of a greater number of respiratory neurons to
participate in the response to hypercapnia.

Keywords Respiratory rhythm Central chemoreception Raphe nuclei Locus

coeruleus nuclei Retrotrapezoid nuclei Brainstem Glia Gliotransmitters
Astrocytes
Abbreviations
5-HT 5-hydroxytryptamine (Serotonin)
ACh Acetylcholine
aCSF Artificial cerebrospinal fluid
AMPAR α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor
ANP Atrial natriuretic peptide
J.E. León (&) M.J. Olivares S. Beltrán-Castillo

Departamento de Biología, Universidad de Santiago de Chile (USACH),
PO 9170022, Santiago, Chile
e-mail: [email protected]; [email protected]

DOI 10.1007/978-3-319-40764-7_6
110 J.E. León et al.

CA Carbonic anhydrase enzyme
CCHS Central congenital hypoventilation syndrome
CNO Clozapine-N-oxide
CO2 Carbon dioxide
CNQX 6-cyano-7-nitroquinoxaline-2,3-dione—competitive AMPA/kainate
receptor antagonist
cNTS Caudal nucleus tractus solitarius
cVLM Caudal ventrolateral medulla
cVRG Caudal ventral respiratory group
Cx Connexins
DRC Dorsal respiratory columns
EPSP Excitatory postsynaptic potentials
GABA γ-aminobutyric acid
KF Pontine Kölliker-Fuse nucleus
KO Knock out
LC Locus coeruleus
LDT Laterodorsal tegmental nucleus
LPBR Lateral parabrachial nucleus
LTP Long-term potentiation
mRVLM Medial portion of the rostral ventrolateral medulla
MS Methionine sulfoximine
NK1R Neurokinin 1 receptor
NMDAR N-methyl-D-aspartate receptor
NO Nitric oxide
NTS Nucleus tractus solitarius
PaCO2 Partial arterial pressure of carbon dioxide
PCO2 Partial pressure of carbon dioxide
PaO2 Partial arterial pressure of oxygen
PF-LHA Perifornical-lateral hypothalamic area
PPADS Pyridoxal-phosphate-6-azophenyl-2=,4=-disulfonate
PPT Pedunculopontine tegmental nucleus
preBötC PreBötzinger Complex
ORX Orexin
ORX-KO Prepro-orexin knockout mice
RN Medullary raphe nucleus
RPG Respiratory pattern generator
RTN/pFRG Retrotrapezoid/parafacial respiratory group
RVL Nucleus reticularis rostroventrolateralis
Role of Astrocytes in Central Respiratory Chemoreception 111
RVLM Rostral ventrolateral medulla

rVRG Rostral ventral respiratory group
SERT Serotonin transporter
SIDS Sudden infant death syndrome
SP Substance P
SSP-SAP Saporin–substance P conjugate
TH Tyrosine hydroxylase
TIRF Total internal reflection fluorescence
TRH Thyrotropin releasing hormone
TRP Channels Transient receptor potential channels
TS-eEPSCs Tractus solitaries-evoked excitatory postsynaptic currents
VLM Ventrolateral medullary surface
VMS Ventral medullary surface
VRC Ventral respiratory columns
VRG Ventral respiratory group
The Respiratory Network
The neural network responsible for generating the respiratory rhythm, the respira-
tory pattern generator (RPG), is composed of neurons preferentially discharging
during inspiration or expiration and distributed along the ventral (VRC) and the
dorsal (DRC) respiratory columns (Fig. 1) (Feldman et al. 2003; von Euler 1986).
The RPG projects into respiratory motoneurons located at different cranial nerve
nuclei (V, VII, IX, X, XII), which innervate muscles controlling airway flow and
resistance. In addition, the RPG sends projections and synapses on various spinal
cord motoneurons, particularly the phrenic motoneurons (C3–C5), which innervate
the diaphragm muscle, and intercostal motoneurons (T1–T10), which innervate
intercostal muscles. The RPG imposes on these motoneurons a synchronic and
rhythmic activity responsible for generating a sequence of inspiratory,
post-inspiratory, and expiratory phases observable in recordings from phrenic,
abductor laryngeal, and internal intercostal nerves, respectively (Richter and Spyer
2001). The coordinated activation of these motoneurons results in a sequence of air
pressure gradients commanding the inspiratory and expiratory phases of ventilation.
At the RPG, within the VRC, at least two oscillators can be recognized: at the
rostral area of the VRC, the pre-inspiratory retrotrapezoid/parafacial respiratory
group (RTN/pFRG), arising from Phox2b expressing progenitors (Guyenet and
Mulkey 2010; Onimaru and Homma 2006; Onimaru et al. 2006, 2009; Stornetta
et al. 2006; Wang et al. 2013; Takakura et al. 2014; Dubreuil et al. 2009b; Abbott
et al. 2011), and at the caudal portions of the VRC, the inspiratory preBötzinger
Complex (preBötC), which is derived from Dbx1 progenitors and considered
Fig. 1 Schema of the respiratory neural network and central respiratory chemoreceptors. The
dorsal respiratory column (DRC) is represented by the nucleus tractus solitarius (NTS), while the
ventral respiratory column (VRC), by the retrotrapezoid/parafacial respiratory group (pFRG), the
Bötzinger nucleus, the pre-Bötzinger complex (preBötC), and the rostral and caudal ventral
respiratory group (rVRG and cVRG). Input and output to the central respiratory network are
indicated with white and black arrows, respectively; note that the output was represented by
respiratory motoneurons localized in cranial and phrenic nuclei. The phrenic nerve (Ph n.) controls
the diaphragm muscle, main responsible for generating air pressure gradients during breathing.
The main peripheral input is provided by vagal mechanoafferents and peripheral arterial
chemoreceptors. Central chemosensitive sites (NTS, preBötc, LC, RTN, raphe) containing cells
that sense changes in pH or PCO2 in the interstitial or the cerebrospinal fluid of the brainstem are
indicated in red. Note that the raphe and LC provide inputs into the respiratory network, while
NTS, preBötc, and RTN belong to the respiratory network. KF, pontine Kölliker-Fuse nucleus;
LC, locus coeruleus; LPBR, lateral parabrachial nucleus; VII, facial nucleus; XII, hypoglossal
nucleus
essential for generating the inspiratory activity (Fig. 1) (Smith et al. 1991; Feldman
et al. 2003; Gray et al. 2010).
The RPG receives input from several central nervous system (CNS) structures,
including cortex, cerebellum, hypothalamus, and brainstem nuclei, and from the
peripheral nervous system (PNS), including vagal mechanosensory afferents and
peripheral arterial chemoreceptors (Feldman 1986; von Euler 1986). Peripheral
arterial chemoreceptors (carotid and aortic bodies) sense changes in PaO2, PaCO2,
pH, osmolarity, temperature, and flow of blood circulating through great arteries
(Eyzaguirre et al. 1983). In contrast, central chemoreceptors (Fig. 1 indicated in
red) are activated by changes in pH or PCO2 in the interstitial or the cerebrospinal

fluid of the brainstem (Nattie 1999). In fact, CO2- and H+-sensitive neurons are a
major source of the tonic input that drives the mammalian respiratory pattern
generator (Nattie 1999).
Central Chemoreception
Central chemoreception can be defined as: “the detection of CO2/pH at sites within
the central nervous system and the resultant effects on ventilation” (Nattie and Li
2010), or in more systemic terms as “the feedback process whereby changes in the
brain CO2 (or pH) bring about adaptive (homeostatic) changes in breathing to
maintain arterial CO2 (or pH) near steady-state levels” (Funk 2010). Central
chemoreception is crucial for matching breathing to physiological demands relative
to H+ or CO2 elimination. In addition, it appears essential for generating and
maintaining the respiratory rhythm (Eugenin 1995), allowing brainstem respiratory
neurons to be coordinated and excitable in an optimal manner (Nattie and Li 2012).
In fact, CO2- and H+-sensitivities are a major source of the tonic drive that sustains
the activity of the RPG (Nattie 1999; Nattie and Li 2012). For example, in en bloc
preparations from newborn opossum and mice, alkaline superfusion of the brain-
stem arrests the respiratory rhythm (Eugenin and Nicholls 1997; Infante et al. 2003;
Eugenin et al. 2006).
Localization of Central Chemoreceptors
Various strategies have been used to localize chemosensitive sites in the brain.
Detection of c-Fos protein as a marker of neuronal activity revealed that those nuclei
in which the number of c-Fos positive neurons increase after exposure to hyper-
capnia also contain neurons with electrophysiological responses to hypercapnic
acidosis (Belegu et al. 1999; Mulkey et al. 2004; Ritucci et al. 2005; Wang and
Richerson 1999; Wickstrom et al. 2002; Teppema et al. 1997). In these nuclei we can
find neurons that fire in association with or in correlation with the respiratory
response to hypercapnia. On the other hand, destruction (Akilesh et al. 1997;
Biancardi et al. 2008; da Silva et al. 2011; Dias et al. 2007), genetic ablation (Hodges
et al. 2011; Dubreuil et al. 2009b; Ramanantsoa et al. 2011), inactivation, or synaptic
inhibition (Nattie and Li 2000; Curran et al. 2001) of specific nuclei reduces the
ventilatory response to hypercapnia. More direct evidence of the existence of
chemosensitive sites was obtained by focal acidification. Either local application of
acetazolamide (Coates et al. 1993), an inhibitor of the enzyme carbonic anhydrase,
or reverse microdialysis of artificial cerebrospinal fluid equilibrated with high CO2
within specific CNS areas, increased ventilation (Li et al. 1999; Nattie and Li 2001,
2002a; Li and Nattie 2002; Dias et al. 2008; da Silva et al. 2010; Krause et al. 2009;
Kuwaki et al. 2010; Coates et al. 1993). Notably, as illustrated in Fig. 1 (nuclei in
red), central respiratory chemosensitivity was localized to multiple sites, including
such nuclei belonging to the RPG as the pFRG/RTN, preBötC, and nucleus tractus
solitarius (NTS), and such nuclei or identified regions outside the RPG but pro-
jecting into as the medullary raphe (RN), locus coeruleus (LC, A6), ventrolateral
medullary surface, hypothalamus, and fastigial nucleus (Ballantyne and Scheid
2001; Coates et al. 1993; Mitchell et al. 1963; Nattie 2001; Oyamada et al. 1998;
Wang and Richerson 1999; Li et al. 2006, 2013; Guyenet et al. 2005; Nattie and Li
2006, 2009, 2010; Xu et al. 2001; Xu and Frazier 1995; Martino et al. 2007; Krause
et al. 2009). The contribution of specific groups of cells within chemosensitive
nuclei were evident from the effects of lesions of neurokinin-1 receptor expressing
cells in the RTN, or serotonergic cells in the RN, or catecholaminergic cells in the
LC. In all these specific lesions, the CO2 response decreased by 15–30 % during
both sleep and wakefulness (Nattie and Li 2008).
Roles of the RTN and Raphe RN Neurons in Central

Chemoreception
RTN Neurons
RTN neurons are glutamatergic, chemosensitive, express the transcription factor

Phox2b, provide excitatory projections to other sites in the central respiratory
network, and when stimulated activate breathing (Mulkey et al. 2004; Onimaru
et al. 2008; Stornetta et al. 2006; Wang et al. 2013; Guyenet and Mulkey 2010;
Goridis et al. 2010; Dubreuil et al. 2009b).
Inhibition of RTN neurons by muscimol dialysis or their chemical (kainic acid
injection) or electrical destruction reduces basal ventilation and the ventilatory
responses to hypercapnia in anesthetized rats (Nattie and Li 1994). More selective
lesions restricted to RTN neurons expressing the neurokinin 1 receptor (NK1R),
obtained with a saporin–substance P conjugate (SSP-SAP), impairs ventilatory
response to hypercapnia in rats (Nattie and Li 2002b). In anesthetized rats, elimi-
nation of at least 70 % of Phox2b+ tyrosine hydroxylase negative (TH−) RTN
neurons is required for a significant increase of the apnea threshold, but does not
affect the sensitivity of the subsequent responses to hypercapnia (Takakura et al.
2008). Allatostatin inhibition of RTN Phox2b-expressing neurons transformed with
a lentiviral construct to express the G-protein-coupled Drosophila allatostatin
receptor did not affect the basal respiratory activity in unanesthetized, conscious rats
(Marina et al. 2010). Nevertheless, allatostatin reduced the amplitude of the phrenic
nerve discharge and the CO2-evoked ventilatory responses in anesthetized rats, in
in situ preparations, and in conscious rats with denervated or intact peripheral
chemoreceptors (by 28 and 60 %, respectively) (Marina et al. 2010; Ramanantsoa
et al. 2011).
In contrast, photostimulation of RTN neurons expressing channel rhodopsin-2

under the control of the Phox2-responsive promoter PRSx8, increases ventilation in
both anesthetized and conscious animals (Abbott et al. 2009, 2011; Kanbar et al.
2010; Burke et al. 2015).
The human disease called central congenital hypoventilation syndrome (CCHS)
shows a spectrum of defects comparable with the ontogenic defects of the auto-
nomic nervous system in Phox2b mutant mice (Brunet and Pattyn 2002; Pattyn
et al. 1999). CCHS is a life threatening human disease characterized by
hypoventilation periods or apnea during sleep and a variable reduction of ventila-
tory response to hypercapnia, from moderate to severe. CCHS was attributable to a
mutation consisting of a polyalanine expansion in the Phox2b transcription factor
(Amiel et al. 2003, 2009). Moreover, genetic generation of a knock-in mouse
having the most frequent of the CCHS-mutations, the Phox2b27Ala allele, resulted
in the selective ablation of glutamatergic neurons in the RTN and a CCHS-like
phenotype. These mice showed gasping behavior, cyanosis, disruption of the res-
piratory chemo reflex at birth and, in contrast to human CCHS patients, they died
during the first hours of postnatal life from respiratory failure (Dubreuil et al. 2008,
2009a, b; Goridis et al. 2010; Ramanantsoa et al. 2011).
Raphe Nucleus Neurons
In brainstem slices, CO2/H+ responsive neurons can be found in the midline Raphe
nucleus (RN) (Richerson 1995; Wang et al. 1998). As mentioned above, ventilation
increases with focal acidification of the midline RN by microinjection of acetazo-
lamide in anesthetized rats or by reverse microdialysis of acidified cerebrospinal
fluid (CSF) in conscious rats or goats (Nattie and Li 2001; Hodges et al. 2004a, b).
Inhibition of RN neurons by microdialysis of muscimol (Taylor et al. 2006), by
administration of 5-hydroxytryptamine (5-HT)1A autoreceptor agonist
(8-OH-DPAT), which inhibits serotonergic neurons, or by microinjections of
lidocaine or ibotenic acid significantly decreased the response to hypercapnia in
piglets (Messier et al. 2002, 2004; Dreshaj et al. 1998). In the unanesthetized
juvenile rat brainstem preparation perfused in situ, 5-HT2 receptor antagonism with
ketanserin or 5-HT1A autoreceptor activation with 8-OH-DPAT blunted the respi-
ratory response (Corcoran et al. 2013). In rats, injections of a monoclonal antibody
against the serotonin transporter (SERT) conjugated to saporin into the RN
specifically killed serotonergic neurons, and as result decreased the average CO2
response (Nattie et al. 2004). In addition, hypercapnic ventilatory response
decreased by 50 % in adult knock out (KO) mice (Lmx1bf/f/p and Pet-1 knockout
mice) with near complete absence of central 5-HT neurons (Hodges et al. 2008,
2011) or with absence of the 5HT transporter (Li and Nattie 2008). Egr2-null mice
have, among others defects, altered serotonergic progeny, low respiratory rate, and
severe apneas, dying perinatally due to respiratory insufficiency.
Selective hyperpolarization of Egr2 expressing neurons or 5HT neurons was

achieved by clozapine-N-oxide (CNO) activation of the synthetic Gi/o protein–
coupled receptor Di expressed selectively on 5-HT neurons using conditional
intersectional genetics. Hyperpolarization of Egr2 neurons reduced the ventilatory
response by 63 % (Ray et al. 2013). Hyperpolarization restricted to serotonergic
neurons reduced the ventilatory chemoreflex in vivo by almost 50 % and reduced
the CO2-induced firing rate increase of 5HT neurons in culture (Ray et al. 2011).
When Di expression was targeted to a specific subtype of 5HT neuron, the
Egr2-Pet1 serotonergic subgroup was found to contribute most to the ventilatory
response to hypercapnia and acidosis. Egr2-Pet1 neurons project to other
chemosensory areas and show intrinsic chemosensitivity firing in response to a
hypercapnic stimulus (Brust et al. 2014).
Relative Contribution of Chemosensitive Sites

to the Overall Response
Determination of the relative contribution of each chemosensory site to the full

expression of chemosensitivity has been elusive. Pronounced effects after unilateral
chemical or electrolytic lesion of the RTN, NTS, or RN led to the notion that each
nucleus provides an essential, indispensable, and singular contribution to the full
expression of central chemosensitivity (Berger and Cooney 1982; Nattie and Li
1994). However, these deleterious effects caused by lesion of chemosensitive
nucleus were strongly influenced by anesthesia (Nattie and Li 2012; Nattie 2011).
In fact, lesion-related impairment of the responses to systemic hypercapnia largely
disappeared with recovery of consciousness (Berger and Cooney 1982). Thus,
under anesthesia, destruction of the rat RTN reduced the integrated baseline activity
of the phrenic nerve and the respiratory response to hypercapnia (Nattie and Li
1994). In contrast, in conscious, unanesthetized rats, similar unilateral lesions of
RTN produced minor effects on baseline ventilation and the respiratory response to
hypercapnia (Akilesh et al. 1997). In agreement with these results, the magnitude of
the ventilatory effects evoked by acidification of chemosensitive areas using reverse
microdialysis in conscious, unanesthetized animals was lower than that observed in
anesthetized animals (Nattie and Li 2012; Nattie 2011). The reduction in ventilatory
effects observed in conscious animals may be explained in part by an enhanced
clearance of focal stimulus as a result of an increased cerebral blood flow in
unanesthetized mammals.
The relative contributions of chemosensory nuclei in the conscious animal has
been studied using either focal inhibition of chemosensitive sites or focal acidifi-
cation by reverse microdialysis of artificial cerebrospinal fluid (aCSF) equilibrated
with high CO2 (Nattie and Li 2009). Assuming that the contributions of
chemosensitive sites are independent, the overall respiratory response does not
appear to be the result of simple additive interactions of individual contributions
(Nattie and Li 2010). Clear synergisms could be inferred, as for example, observing
the ventilatory depression when RTN and caudal RN were simultaneously inhibited
(Li et al. 2006). More direct evidence of this synergism was obtained with
simultaneous focal acidification of the RTN and caudal RN (Dias et al. 2008).
However, unrealistically complex experiments with multiple probes stimulating
each chemosensory area individually or several simultaneously during wakefulness
and sleep seem to be necessary to fully address this question.
Interestingly, the full expression of central chemoreception also depends on the
peripheral chemoreceptor input. In fact, in unanesthetized awake dogs the venti-
latory responsiveness to four progressively increasing levels of central hypercapnia
depended on the degree of carotid body inhibition or stimulation with respect to
basal eupneic conditions (normoxic, normocapnic carotid body perfusion). The
increase in carotid body activity via carotid body perfusion with a hypoxic, nor-
mocapnic perfusate increased the ventilatory response to hypercarbia by 223 %
respect basal conditions. By contrast, silencing of carotid bodies activity with
hyperoxic, hypocapnic perfusate reduced the ventilatory response to hypercarbia by
81 %. This interdependence between peripheral and central chemoreception sug-
gests that the whole system of central and peripheral chemosensory structures are
functionally interrelated and integrated.
Central Chemoreception Dependency on Functional

State of the Respiratory Network
Special attention should be focused to the fact that contribution of each

chemosensitive site to the overall response to hypercapnia depends on the func-
tional state of the respiratory network. Such dependency not only may give account
of the differences between conscious and anesthetized animals that are mentioned
above, but also of differences in ventilation and ventilatory responses between
wakefulness and sleep (Newton et al. 2014). Studies in rats with focal acidosis by
reverse microdialysis along the sleep–wake cycle have shown that acidification of
the RTN or the perifornical-lateral hypothalamic area (PF-LHA), where orexin
neurons are found, or the caudal ventrolateral medulla (cVLM) increased ventila-
tion predominantly in wakefulness (Li and Nattie 2002; Li et al. 2013; da Silva
et al. 2010). By contrast, acidification of rostral RN increased ventilation pre-
dominantly in sleep (Nattie and Li 2001) while focal acidification of the NTS
increased ventilation in both wakefulness and sleep (Nattie and Li 2002a).
Orexin neurons are good candidates to be the link between arousal state and
chemoreceptive properties at the brainstem (Nattie and Li 2010, 2012). Orexin
neurons are critical for generating wakefulness (Ohno and Sakurai 2008; Sakurai
2014; Alexandre et al. 2013) and controlling breathing (Nakamura et al. 2007; Li
et al. 2013; Li and Nattie 2010; Dias et al. 2010; Terada et al. 2008; Dutschmann
et al. 2007; Deng et al. 2007; Young et al. 2005b; Toyama et al. 2009). They are
sensitive to H+/CO2 (Williams et al. 2007; Li et al. 2013; Sunanaga et al. 2009) and
their firing rate is maximal during wakefulness (Lee et al. 2005) and minimal during
sleep.
As mentioned above, focal acidification of the hypothalamic area containing
orexin neurons increased ventilation up to 15 % only in wakefulness but not in
sleep (Li et al. 2013). In prepro-orexin knockout mice (ORX-KO) basal ventilation
is not affected along the sleep–wake cycle. Neither their ventilatory responses to
hypercarbia during sleep period nor their ventilatory responses to hypoxia during
wake–sleep cycle when compared with those in wild type mice. However,
ORX-KO mice have a ventilatory response to hypercapnia reduced to the half of
that in wild type mice during quiet wakefulness. The ventilatory response to
hypercapnia was partially restored in ORX-KO mice administered intracere-
broventricular with orexin-A or orexin–B, the two orexin subtypes derived from
prepro-orexin (Deng et al. 2007).
Such results are compatible with those obtained by dialyzing the rat RTN with
SB-334867, orexin receptor-1 antagonist that reduced the hyperventilation caused
by hypercapnia by 30 % during wakefulness and 9 % during sleep. A much smaller
effect (16 % reduction of hypercapnia-induced hyperventilation) was observed
when microdialysis of SB-334867 was performed into rostral RN during wake-
fulness in dark period and null effect in the ventilatory chemo reflex when
administered during sleep (Dias et al. 2010). In addition, almorexant, antagonist of
both orexin receptor-1 and orexin receptor-2, administered orally reduced the
ventilatory response to hypercapnia by 26 % only in wakefulness during the dark,
active period of the diurnal cycle (Li and Nattie 2010). Then, we can conclude that
projections of orexin-containing neurons to the RTN and rostral RN contribute, via
orexin receptor-1, to the hypercapnic chemoreflex control during wakefulness and
to a lesser extent during sleep (Dias et al. 2009). However, a possible role for orexin
neurons as a “wakefulness” driver of chemosensitive properties is still uncertain.
Astrocytes
Astrocytes are not mere intermingled cells of the CNS that outnumber neurons. As
already described in Chapter “Glial Cells and Integrity of the Nervous System”,
they serve multiple functions: structure of the nervous tissue, trophism, metabolic
support as for example the lactate shuttle, energy storage in the form of glycogen,
ionic and water homeostasis, homeostasis of the synaptic environment buffering the
concentration of extracellular potassium and the excess of extracellular neuro-
transmitters and release of gliotransmitters and neurotransmitters (most of them
influencing synaptic strength, Table 1), formation and remodeling of synapses,
defense against oxidative stress, scar formation, and tissue repair. Even more, as-
trocytes are involved in complex processes like neural network plasticity, inflam-
mation, and neurodegeneration (Belanger et al. 2011; Grass et al. 2004;
Rodriguez-Arellano et al. 2015).
Table 1 Substances released by astrocytes

Neuroactive substance Reference
Neurotransmitters—neuromodulators
Amino acids L-glutamate Parpura et al. (1994) and Kimelberg et al. (1990)
L-aspartate Kimelberg et al. (1990)
Taurine Kimelberg et al. (1990)
D-serine Schell et al. (1995)
γ-aminobutyric acid Bowery et al. (1976)
(GABA)
Non-amino Dopamine Chen et al. (2005)
acids ATP Guthrie et al. (1999)
Adenosine Albrecht et al. (1991)
Nitric oxide (NO) Murphy et al. (1990)
Met-enkephalin Shinoda et al. (1989)
Somatostatin Mercure et al. (1996)
Atrial natriuretic peptide Krzan et al. (2003) and Guček et al. (2012)
(ANP)
Metabolic precursors
Lactate Pellerin and Magistretti (1994)
Glutamine Yudkoff et al. (1994)
α-ketoglutarate Westergaard et al. (1994)
Malate Westergaard et al. (1994)
Succinate Westergaard et al. (1994)
Growth factors
BDNF Caravagna et al. (2013)
NGF-β Furukawa et al. (1986)
IGF-I Kadle et al. (1988)
IGF-II Kadle et al. (1988)
Inflammatory factors
Prostaglandins D2, E2, Gebicke-Haerter et al. (1988) and Hartung and
I2 Toyka (1987)
Thromboxane Hartung et al. (1988)
IL-1β Corsini et al. (1996)
IL-6 Wu et al. (2005)
IL-10 Wu et al. (2005)
TGF-β Constam et al. (1992)
TNF-α Selmaj et al. (1990)
There is a remarkable heterogeneity among astrocytes, being their phenotype

largely a function of both local anatomy and regional functional demands
(Oberheim et al. 2012). They are in intimate contact with most of the structures of
the nervous system being largely responsible of its compartmentalization.
Astrocytes send end-feet processes that enwrap blood vessels and interact with
endothelial cells determining the formation of the blood brain barrier. Astrocytic
end-feet processes express, among others, glucose transporters and aquaporin 4.
They are involved in the cerebral neurovascular coupling regulating the
microvascular flow for matching this to synaptic activity (Iadecola and Nedergaard
2007).
On the other hand, astrocytes send processes that ensheath most synapses. These
perisynaptic processes express receptors for cytokines and growth factors. In
addition, they express different kind of neurotransmitter receptors, transporters, and
ion channels as expected of an active participant in the homeostasis of the synapse.
Thus, at the synaptic compartment, astrocytes can sense the synaptic activity by
means of neurotransmitter receptors activation (Araque et al. 2014), regulate the
levels of neurotransmitters at the synaptic cleft influencing their recapture and
release (Hamilton and Attwell 2010), modulate the synaptic transmission through
gliotransmitters release, and modulate the neuron excitability by extracellular
potassium buffering (Perea et al. 2014).
In hippocampus and cortex from rodent and humans, astrocytes are organized in
discrete spatial domains (Oberheim et al. 2012). Each astrocyte extends its pro-
cesses on a defined territory without important overlap between adjacent astrocytes.
On other terms, all cellular structures in a territory (blood vessels, perikarya and
synapses) interact with processes from a single astrocyte only (Oberheim et al.
2009). It is estimated that a single spatial domain for a protoplasmic astrocyte in
rodent contains 20,000–120,000 synapses, while that in humans contains the
extraordinary amount of 270 thousand to 2 million synapses (Oberheim et al. 2009).
A particular feature of astrocytes is that each one of them is coupled to others,
through gap junction channels forming an extensive functional syncytium. In
hippocampus, each astrocyte forms gap junctions with 11 others astrocytes, in
average (Xu et al. 2010). This syncytium offers a route of low electrical resistance
for propagation of electronic signaling and ionic currents and for cell-to-cell
propagation of second messengers. This syncytium represents a huge sink for
buffering the changes in potassium composition of the extracellular space. In
addition, this syncytium allows the spreading of calcium waves, which, in humans
reach the speed of 37–43 μm/s (Cornell-Bell et al. 1990; Oberheim et al. 2009),
into neighboring astrocytes. Thus, astrocytes can be sequentially activated and
recruited for performing a common task. Since each astrocytic domain represents
an elementary glio-neuronal unit for monitoring the changes in activity of con-
tiguous synapses, the existence of a functional syncytium implies the capability of
influencing other astrocytic domains and the spreading of a potential astrocytic
response to domains placed far away from an immediate neighborhood.
This organization of highly organized and interconnected anatomical domains will
allow the recruitment of distant domains, which in turn will influence a larger
number of synapses within a neural network. As a consequence, a more intense,
and may be, a more synchronized response will arise.
Calcium management of one astrocyte can affect many thousands of excitatory
synapses nearby as shown by clamping intracellular Ca2+ experiments. In these,
clamping of calcium in individual hippocampal astrocytes is made through a
whole-cell pipette while an extracellular field excitatory postsynaptic potential
(EPSP) recording is done with an extracellular electrode placed either in the
immediate vicinity of the clamped astrocyte or in a more distanced CA1 pyramidal
cells group. Astrocytic Ca2+ clamping blocked long term potentiation
(LTP) induced by tetanic stimulation of Schaeffer collaterals, at nearby, but not far
away positions (Henneberger et al. 2010).
Astrocytes are ideally located to sense synapse activity with the perisynaptic
processes and metabolic supply from blood vessels with the end feet processes. In
fact, they mediate the response consisting in the modification of the local blood
flow as function of synaptic or neuronal activity. It has been shown that astrocytes
respond to increased neuronal activity by consuming more glucose and producing
more lactate, this latter transferred into neighbor neurons as fuel during hyperac-
tivity. As previously mentioned in Chapter “Glial Cells and Integrity of the
Nervous System”, this is known as the “astrocyte-neuron lactate shuttle” hypothesis
(Pellerin et al. 2007).
Astrocytes in the PreBötzinger Complex (preBötC)
The preBötC is the main generator of the inspiratory activity and a chemosensitive
nucleus (Solomon 2003; Solomon et al. 2000). Fluctuations of the extracellular
potassium concentrations are induced by the occurrence of rhythmic bursts of
action potentials (Richter et al. 1978), which in turn are associated to fluctuation in
the neurotransmitter release. Since astrocytes express K+ channels (Kir4.1;
KCNJ10), fluctuations in potassium concentrations generates fluctuations in the
resting membrane potential, which can induce fluctuations in intracellular calcium
concentration in astrocytes. Using whole-cell recordings from astrocytes and
two-photon calcium imaging from rhythmic slices, none coupling between respi-
ratory neuronal activity and astrocytic calcium signals was observed. The absence
of correlation between respiratory neuronal activity and astrocytic calcium fluctu-
ation (Schnell et al. 2011) indicates that astrocytic release of gliotransmitters is not
commanding the respiratory like activity in neurons. Likely, one role of astrocytes
in the preBötC is the control of extracellular levels of neurotransmitters and ions,
both largely influencing the excitability of respiratory neurons.
Astrocytes in Central Chemoreception
Over the last two decades, multiple pieces of evidence revealed that astrocytes can
contribute to central chemoreception. Such contribution may be accomplished by
astrocytes directly playing a role as H+/CO2 sensors or as part of the mechanisms
underlying the cholinergic and glutamatergic hypothesis. Reduction in chemosen-
sitivity of astrocytes may be involved in the pathogenesis of Rett syndrome and
may explain the deficit in ventilatory responses to hypercapnia in these patients
(Turovsky et al. 2015). Also, it has been proposed that astrocytes can play a
modulatory role of the network in charge of the respiratory pattern generation by
controlling the extracellular ion and transmitter concentrations (Neusch et al. 2006;
Szoke et al. 2006; Ballanyi et al. 2010; Erlichman and Leiter 2010). Likely,
astrocytes in different chemosensitive regions also differ in their contributions to
central chemoreception and the mechanisms underlying such contribution.
Astrocyte Chemosensitivity
As illustrated in Fig. 2, several molecular mechanisms by which astrocytes detect

H+/CO2 have been proposed
Fig. 2 Astrocytes may sense acidosis or hypercapnia through different molecular sensors.
Inwardly rectifying potassium (Kir) heteromeric channels Kir4.1–Kirk5.1 are inhibited by CO2
resulting in depolarization of astrocytes (Wenker et al. 2010); carbonic anhydrase (CA) enzyme,
the Na+-HCO3− cotransporter, Na+/H+ exchanger and the Na+-dependent or Na+-independent Cl−/
HCO3− antiporters contribute to pH regulation (Brookes 1997; Baird et al. 1999; Makara et al.
2001; Schmitt et al. 2000; Deitmer and Rose 1996); connexins with a carbamylation motif (Cx26,
Cx30, and Cx32), a site for binding CO2 to induce the opening of connexin hemichannels (Meigh
et al. 2013) endows cells with CO2-sensitivity and the capacity for releasing ATP as a function of
PCO2 at constant extracellular pH (Huckstepp et al. 2010a); TRP channels endows to astrocytes
with the ability for responding to hypercapnic but not isocapnic acidosis (Hirata and Oku 2010). It
is possible that TRP activation could be given by extracellular acidification (Cui et al. 2011)
(1) Inwardly rectifying potassium (Kir) heteromeric channels Kir4.1–Kirk5.1.

These channels contribute to the extracellular potassium regulation and are
expressed in brainstem nuclei, including, among others, the LC, the ventro-
lateral medullary (VLM) area, the RTN, and the NTS (Wu et al. 2004). Kir4.1,
and Kir5.1 channel subunits are observed in astrocytic processes contacting
the pia mater, blood vessels, and synapses associated to PDZ domains con-
taining syntrophins (Hibino et al. 2004). Depolarization of astrocytes by CO2
would involve inhibition of heteromeric Kir4.1–Kir5.1 channels and contri-
bution of Na+-HCO3− cotransporter (Wenker et al. 2010).
(2) Carbonic anhydrase enzyme and, in addition to the Na+-HCO3− cotransporter,
several other transporters that contribute to pH regulation like the Na+/H+
exchanger and the Na+-dependent or Na+-independent Cl−/HCO3− antiporters
(Brookes 1997; Baird et al. 1999; Makara et al. 2001; Schmitt et al. 2000;
Deitmer and Rose 1996).
(3) Connexins presenting a carbamylation motif (Cx26, Cx30, and Cx32), a site
for binding CO2 to induce the opening of connexin hemichannels (Meigh et al.
2013) (see Chapter “Physiological Functions of Glial Cell Hemichannels” for
further information on hemichannels). In particular, connexin 26 is abundantly
expressed at the ventral medullary surface and its CO2 sensitivity is within
physiological range having a steep change in conductance centered around
40 mmHg PCO2 (Huckstepp et al. 2010a, b). It is known that heterologous
expression of Cx26 endows HeLa cells with CO2-sensitivity and the capacity
for releasing adenosine triphosphate (ATP) as a function of PCO2 at constant
extracellular pH (Huckstepp et al. 2010a). Accordingly, connexin hemichan-
nel blockers reduce both the ATP release and the ventilatory response induced
by hypercapnia in vivo and the ATP release induced by hypercarbia in vitro
(Huckstepp et al. 2010b).
(4) Transient receptor potential (TRP) channels endows to astrocytes with the
ability for responding to hypercapnic acidosis. This was assayed in enriched
glia cells cultures using intracellular calcium- and pH-imaging in addition to
perforated patch-clamp methods (Hirata and Oku 2010).
Astrocyte Involvement in Respiratory Rhythm Modulation
Specific glial metabolic inhibitors have been used to evaluate astrocyte contribution
to the ventilatory process. Fluorocitrate or fluoroacetate at low doses, are incor-
porated selectively by astrocytes and block the tricarboxylic acid (Krebs) cycle by
inhibiting the enzyme aconitase. Administration of fluorocitrate into the RTN in
either anesthetized mechanically ventilated or conscious adult rats increased the
respiratory output (Erlichman et al. 1998; Holleran et al. 2001). This response can
be explained on basis of the fluorocitrate-induced ATP and tissue pH decrease.
Inhibition of Krebs cycle reduces ATP levels, which in turn, reduces Na+-
K+ATPase activity. Pump inactivation increases the extracellular potassium con-
centration and, subsequently, depolarizes, among others, chemosensitive neurons.
Since chemosensitive neurons also respond to the acidification of the medium, and
at the end, as overall result, the respiratory output is increased (Erlichman and
Leiter 2010).
In contrast to in vivo experiments, fluoroacetate as well as methionine sulfox-
imine (MS), an inhibitor of glutamine synthetase, an enzyme present only in as-
trocytes that catalyzes the synthesis of glutamine from glutamate (see
Chapter “Pharmacological Tools to Study the Role of Astrocytes in Neural
Network Functions”), reduced the amplitude and frequency of the integrated
inspiratory burst recorded from rhythmically active brainstem slices. At a first
glance, these results suggest that astrocyte metabolic support or astrocyte functions
depend on Krebs cycle and are necessary for the maintenance of the respiratory
rhythm (Hulsmann 2000). In brainstem slices, evoked depolarization of the
hypoglossal neurons by electrical stimulation of the ventral respiratory column
(measured by optical imaging using voltage-sensitive dye) was reduced and delayed
after fluoroacetate administration which is compatible with metabolic inhibition of
fast synaptic transmission (Hulsmann et al. 2003). Accordingly, after fluoroacetate
or MS treatment of brainstem slices, addition of glutamine restored the respiratory
rhythm indicating that likely, the respiratory effects of both inhibitors were related,
essentially, to impairment of the glutamate neurotransmission. In fact, fluoroacetate
also impairs the astrocytic uptake of glutamate and the formation of glutamine
(Swanson and Graham 1994)
In vivo administration of MS reduces basal ventilation and the ventilatory
response to hypercapnia in conscious neonatal rats (Young et al. 2005a). By con-
trast, fluorocitrate administered into the RTN in vivo did not affect the respiratory
response to acidosis or hypercapnia (Erlichman et al. 1998). Likely, the effects of
fluorocitrate-induced reduction in ATP tissue pH oppose and predominate to the
impairment in glutamate neurotransmission.
The hypothesis that astrocytes contribute to H+/CO2 sensitivity concatenates
several steps: first, a subset of glial cells is depolarized in response to acidification
(Fukuda et al. 1978; Fukuda and Honda 1975; Ritucci et al. 2005). Second, and
derived from glial cell depolarization, intracellular Ca2+ increases, which is required
also for the inter-cellular propagation of calcium waves in glia (Guthrie et al. 1999);
third, as a consequence of the intracellular Ca2+ increase, ATP is released from
astrocytes, likely through connexin hemichannels (Huckstepp et al. 2010b). In fact,
electrochemical sensors placed at the ventral medullary surface can detect high
levels of ATP (3.8 ± 0.9 µM) during hypercapnia in anesthetized rats
(Spyer et al. 2004; Gourine et al. 2005). Activation of glial purinoceptors by ATP
can initiate self-propagating calcium waves that are proposed to influence local
network excitability (Fiacco and McCarthy 2006). Finally, ATP, or other neu-
roactive molecules, will activate central chemoreceptor neurons such as those found
in RTN/pFG (Spyer et al. 2004; Gourine et al. 2005).
ATP can act by binding to 7 subtypes of ionotropic P2X receptors (P2X1–7Rs)

and eight subtypes of metabotropic P2YRs (P2Y1,2,4,6,11–14) (North 2002;
Abbracchio et al. 2009).
According to this sequence of events, purinoceptor antagonists should impair the
respiratory effects evoked by CO2 stimulation. In fact, reduction and even abolition
of ATP induced respiratory responses have been observed in vivo and in vitro
(Thomas and Spyer 2000; Gourine et al. 2005; Zwicker et al. 2011; Gourine and
Kasparov 2011; Gourine et al. 2010). Hypercapnia induces the release of ATP from
the ventral surface of the medulla (Gourine et al. 2005). Further, application of ATP
into the most rostral ATP-releasing site, corresponding likely to the retrotrapezoid
nucleus, stimulated respiratory output, whereas application of ATP receptor
antagonists like PPADS (pyridoxal-phosphate-6-azophenyl-2′,4′-disulfonate) to this
area reduced CO2 respiratory responses (Gourine et al. 2005). Hypercapnia-induced
ATP-mediated excitation of the respiratory rhythm in rats is likely to involve a
potent P2Y1R- activation of the preBötC (Lorier et al. 2007; Gourine et al. 2010).
Such P2Y receptor-dependency has also found in vitro RTN neurons (Mulkey et al.
2006). Since pH sensitivity of RTN neurons in bicarbonate-free HEPES medium is
not affected after purinergic receptor blockade with PPADS, ATP would play a role
of modulator of the activity of pH-sensitive neurons, amplifying their responses to
hypercapnia (Mulkey et al. 2006, 2004).
New insights of astrocyte contribution in modulating the function of respiratory
neuronal circuits arise from application of molecular and electrophysiological
methodology in conjunction with genetically engineered optical stimulation and
Ca2+ imaging tools. In an elegant work, Gourine et al. (2010) tested the hypothesis
that rat astrocytes residing in RTN/pFRG behave as pH sensors, and trigger the
respiratory response through the release of ATP. Astrocytes were genetically
encoded with a Ca2+ indicator associated to the promoter for glial fibrillary acidic
protein (GFAP). They could confirm that these astrocytes, but not those from
cerebral cortex, responded to physiological decreases in pH with elevations in
intracellular Ca2+ and ATP release. Accordingly, studies of vesicular fusion using
total internal reflection fluorescence (TIRF) microscopy show that 35 % of astro-
cytes from rat brainstem in dissociated cultures respond to acidification with exo-
cytosis of ATP-containing vesicles. Vesicles were visualized with fluorescent dyes
quinacrine, an acridine derivative with very high affinity for ATP, and MANT-ATP,
an ATP analogue esterified by the fluorescent methylisatoic acid. Vesicular exo-
cytosis requires intracellular Ca2+ signaling and was independent of autocrine ATP
actions (Kasymov et al. 2013). By contrast, ATP was necessary to propagate
astrocytic Ca2+ excitation, since elimination of ATP by the ATP-hydrolyzing
enzyme, apyrase, reduced importantly the CO2-evoked astrocytic calcium responses
(Gourine et al. 2010). In addition, ATP activating P2Y1Rs excited chemoreceptor
neurons leading to the increase in the respiratory rhythm frequency (Gourine et al.
2010). Optogenetic stimulation of astrocytes expressing channelrhodopsin-2 asso-
ciated under the command of GFAP promoter, resulted in a robust increase in
breathing, associated to the increase in intracellular Ca2+ in astrocytes. This opto-
genetic stimulation mimicked the hypercapnia and acidosis induced activation of
chemoreceptor neurons via an ATP-dependent mechanism (Gourine et al. 2010). In

agreement with these results, disruption of purinergic signaling decreases CO2
sensitivity of RTN neurons by 25 % (Wenker et al. 2010) as well as gap junction
blockers, which decrease CO2-evoked ATP release in the RTN, reduced the
whole-animal ventilatory response to CO2 also by 25 % (Huckstepp et al. 2010b). In
addition, fluorocitrate-induced depolarization of astrocytes evoked a reversible
increase in firing rate of RTN neurons. This increase in neuronal firing rate was
abolished by the presence of P2 receptor antagonists (PPADS or suramin)
(Wenker et al. 2012) suggesting that a purinergic signaling was a mediator.
Purinergic blockade also blunted the hypercapnic ventilatory response in vivo and
the firing rate response of RTN neurons to hypercapnic stimulus of slices (10–15 %
CO2) (Wenker et al. 2012).
Regional Differences in Contribution of Astrocytes

to Central Chemoreception
As mentioned above, neurons responding to CO2 with increased firing rate can be
found, among other sites, at the RTN (Nattie et al. 1993a), RN (Iceman et al. 2013),
and the caudal portion of the NTS (Dean et al. 1990; Nichols et al. 2009).
Furthermore, focal acidification either by injection of acetazolamide within these
three regions in anesthetized cats (Coates et al. 1993) or by microdialysis within
these nuclei in unanesthetized awake or sleeping rats (Li et al. 1999; Nattie and Li
2001, 2002a) increases ventilation. Since RTN and RN neurons in culture have
intrinsic CO2-pH-sensitivities (Wang et al. 1998; Wang and Richerson 1999; Wang
et al. 2013), glia would play a coadjuvant, synergic role in these chemoreceptive
nuclei. There is not any study detailing the cytoarchitecture and properties of as-
trocytes at the different areas of the brainstem. Hitherto, the degree of cell-to-cell
interconnections, the extension of astrocyte domains, and the differential expression
of receptors, gliotransmitters, are mostly unknown. Since in other regions of the
CNS, the population of astrocytes is heterogeneous in shapes and functions
(Oberheim et al. 2012), it would not be strange that astrocytes belonging to different
chemosensory nuclei at the brainstem differ in their structure and properties.
Therefore, it is possible that the mechanisms through which astrocytes interact with
the respiratory network at different nuclei could also be different.
As expressed before, data obtained at the RTN suggest the existence of a cascade
of events triggered by hypercapnia or acidosis: depolarization of astrocytes, cyto-
plasmic calcium increase, ATP release, and ATP activation of respiratory neurons.
It is worth to remember that this constitute the glial pathway for RTN neurons
activation since RTN neurons are chemosensitive themselves. At that respect, the
glial pathway appears as intensifier of the RTN neurons response to hypercapnia.
At the NTS and the RN, there is some controversial evidence pointing to the role
of ATP as mediator of the response to hypercapnia. P2 receptors are expressed in
the NTS and with less intensity, at the RN (Yao et al. 2000).
The administration of ATP or its analogues into the NTS, in awake rats produced
cardiorespiratory responses (Antunes et al. 2005; De Paula et al. 2004). On the
other hand, the injection of P2 receptor antagonists into NTS reduces the
sympatho-excitatory response to peripheral chemoreflex activation (Braga et al.
2007; Boscan et al. 2002). Microinjection of ATP into the raphe magnus reduces
the respiratory activity while that into the raphe pallidus increase it in anesthetized
and artificially ventilated rats (Cao and Song 2007). The injection of the P2X
broad-spectrum antagonist, PPADS, into the rostral medullary raphe blunted the
ventilatory response to hypercapnia in conscious rats (da Silva et al. 2012), while
this unaffected ventilation when injection was placed into the caudal RN of con-
scious rats (da Silva et al. 2012) or when it was done into raphe magnus or pallidus
in anesthetized rats (Cao and Song 2007).
To test whether an astrocytic ATP-dependent mechanism was involved in central
chemoreception at the RN and NTS, ATP antagonists were applied into these nuclei
while chemoreflexes were evaluated in vivo as in vitro (Sobrinho et al. 2014). ATP
injections into the caudal NTS (cNTS) increased cardiorespiratory activity in
anesthetized rats (Sobrinho et al. 2014) confirming results obtained with the rat
working heart-brainstem preparation (Antunes et al. 2005). By contrast, the injection
of broad range purinergic receptor antagonists like PPADS or suramin into the cNTS
did not affect basal ventilation or the ventilatory responses to changes in CO2/H+ as it
does at the RTN (Sobrinho et al. 2014). In the case of RN the results were more
negative, because both the injections of ATP or PPADS in anesthetized rats did not
affect neither the basal ventilation nor the responsiveness to H+/CO2 (Sobrinho et al.
2014). Cell-attached NTS neurons recorded from brainstem slices increased their
firing rate in response to ATP, while P2 receptors antagonists (PPDAS or suramin)
did not modified NTS neurons response to hypercarbia. Likewise, the firing rate of
RN neurons were not modified by ATP and their responses to changes in PCO2/pH
were unaffected by ATP-receptor blockade (Sobrinho et al. 2014).
Sobrinho et al. (2014) results are unexpected from previous reports indicating
the existence of P2 receptors, and the respiratory-related effects of ATP agonist and
antagonist injected into the NTS or RN. In fact, it is known that ATP in NTS plays
a role in modulating the glutamatergic excitatory transmission as evidenced by the
reduction in the amplitude of tractus solitaries-evoked excitatory postsynaptic
currents (TS-eEPSCs) by purinergic antagonist (iso-PPADS). The glial cells are the
source of ATP released by tractus solitarius electrical stimulation is suggested by
the reduction in this TS-eEPSCs induced by the glia toxin, fluoroacetate
(Accorsi-Mendonca et al. 2013). Likely, the inconsistency in results may be partly
due to methodological differences, for example the use of anesthesia or the use of
broad-spectrum antagonists which are weakly effective for blocking specific subset
of P2 receptors. However, it remains possible that astrocytes contribute to the

CO2/H+ responsiveness of cNTS and RN neurons, perhaps by an ATP-independent
mechanism.
Other Gliotransmitters
It is possible that other gliotransmitter, different to ATP, could serve as mediator in

NTS or RN. A good candidate is D-serine. D-serine is a D-amino acid synthesized
from L-serine by a pyridoxal 5′-phosphate-dependent serine racemase
(SR) enzyme, which is present in neurons and astrocytes (Rosenberg et al. 2010;
Wolosker 2011). D-serine binds with high affinity to the co-agonist (glycine) site of
the N-methyl-D-aspartate (NMDA) glutamate receptor. D-serine effects have not
been evaluated in the respiratory network, despite of NMDAR activation increases
the respiratory frequency in vivo (Connelly et al. 1992) and in vitro (Greer et al.
1991). Preliminary data from our laboratory indicates that in en bloc preparations
from neonatal mice, D-serine applied into the superfusion bath increases the
respiratory rhythm of neonatal mice (Fig. 3).
Fig. 3 Increase of respiratory frequency induced by D-serine. a integrated inspiratory burst

recorded from C4 ventral root in en bloc preparation obtained from CF1 mouse neonate at the third
postnatal day before (basal), during, and after (recovery) the superfusion with aCSF containing
D-serine 10 μM. b Instantaneous respiratory frequency measured cycle-to-cycle before, during
(indicated by horizontal bar), and after the superfusion with aCSF containing D-serine 10 μM in
the preparation from (a)
Astrocytes and Cholinergic-Glutamatergic Hypothesis

of Central Chemoreception
Historically, two neurotransmitters have been involved in central chemoreception,

acetylcholine and glutamate, what is known as “the cholinergic and glutamate
hypothesis of central chemoreception”.
Cholinergic (ACh) hypothesis: Cholinergic neurons form part of input and
output of the respiratory network. They are found at the NTS (Ruggiero et al. 1990;
Armstrong et al. 1988; Gotts et al. 2015), the hypoglossal nuclei, facial nuclei,
ambiguous nuclei (Kang et al. 2007), within the RN (Tatehata et al. 1987; Ruggiero
et al. 1990), the nucleus reticularis rostroventrolateralis (RVL), and the ventral
medullary surface (VMS); although cholinergic neurons are also detected in other
localizations of the brainstem, like those in the medial portion of the rostral ven-
trolateral medulla (mRVLM), these would not be involved in cardiorespiratory
events (Stornetta et al. 2013). The most important cholinergic inputs to the brain-
stem are originated from the pedunculopontine tegmental (PPT) and laterodorsal
tegmental (LDT) nuclei. These inputs, as well as those provided by the serotonergic
RN and the noradrenergic LC may be a clue for understanding pathogenia of
respiratory dysfunctions associated to sleep–wake cycle, like sudden infant death
syndrome (SIDS).
Muscarine or nicotine applied on the ventral surface of rostral and caudal
medulla increase ventilation in anesthetized cats (Dev and Loeschcke 1979a, b). An
endogenous cholinergic drive of the respiratory rhythm is revealed with acetyl-
cholinesterase inhibitors (physostigmine, eserine) within rostral and caudal medulla
(Dev and Loeschcke 1979a). In part, the respiratory cholinergic drive is exerted on
the preBötC where activation of M3 and α4β2 nicotinic receptors increases the
frequency of the respiratory rhythm in neonatal rats and mice slices (Shao and
Feldman 2005; Shao et al. 2008; Shao and Feldman 2009). A tonic cholinergic
respiratory drive in the mouse en bloc preparation is revealed by application of
atropine, a muscarinic receptor antagonist, which reduces the amplitude and fre-
quency of the respiratory rhythm (Coddou et al. 2009).
That a cholinergic relay may be involved in central chemoreception at the sur-
face of ventral medulla is derived from the fact that acetylcholine-sensitive areas
and H+- or CO2-sensitive areas overlapped. In addition, application of cholinergic
agonists on these sensitive areas elicits similar patterns of respiratory responses than
those evoked by acidic stimulation (Loeschcke 1982; Eugenin and Nicholls 1997).
Furthermore, central chemoreception and muscarinic cholinergic neurotransmission
are strongly linked (Loeschcke 1982) as indicated by the brainstem distribution of
muscarinic receptors (Nattie and Li 1990; Nattie et al. 1994; Mallios et al. 1995).
Application of atropine to the rostral and caudal medulla decreases ventilation and,
at the same time, reduces importantly the ventilatory response to CO2 (Dev and
Loeschcke 1979a; Nattie et al. 1989). Muscarinic blockade also reduces and,
sometimes, abolishes the respiratory responses induced by H+ or CO2 in in vitro
preparations from neonatal rats (Monteau et al. 1990), newborn opossum (Eugenin
and Nicholls 1997), and neonatal mouse (Coddou et al. 2009). Microinjection of
muscarinic M3 antagonist on the rostral ventrolateral medulla (RVLM) has a great
efficacy for inhibiting respiratory CO2-evoked response (Nattie and Li 1990).
Interestingly, the arcuate nucleus, which is the human homologue of the RVLM,
shows decreased muscarinic binding in SIDS infants (Kinney et al. 1995). Such
probable reduction of the muscarinic binding in SIDS is compatible with the
reduction of the muscarinic contribution to the chemosensory responses in en bloc
and slices preparations from P0-P3 nicotine-exposed neonates by the
prenatal-perinatal nicotine exposure (Coddou et al. 2009; Eugenin et al. 2008).
Unexpected results were obtained when muscarinic receptor knockout
(KO) mice were challenged with hypercapnia (3 and 5 % CO2). M1 single KO
mice showed normal, while M3 single KO mice showed reduced VT response slope
to hypercapnia (Boudinot et al. 2004). Surprisingly, M1/3R or M2/4R double-KO
mice showed unaltered chemosensory ventilatory responses (Boudinot et al. 2008).
These results are puzzling and will require future research with conditional KO
mice to evaluate muscarinic contribution to chemo reflexes in adults in absence of
possible compensatory mechanisms exerted during development.
Glutamate (Glu) hypothesis: Excitatory glutamate neurotransmission predomi-
nates within the mammalian RPG, and the ventral surface of medulla is not an
exception. Injection of glutamate into the RVLM increases ventilation in anes-
thetized cats (Li and Nattie 1995; Nattie and Li 1995). By contrast, microinjection
of kynurenic acid, a nonselective glutamate receptor antagonist, or AP5, an NMDA
receptor antagonist, or CNQX, a non-NMDA receptor antagonist, into the RVLM
region decreased both the amplitude of the integrated phrenic nerve activity and the
CO2 sensitivity in a dose-dependent manner in anaesthetized cats (Nattie et al.
1993b). In contrast to in vivo experiments (Connelly et al. 1992), blockade of
NMDARs in brainstem slices had a negligible effect on respiratory rhythm
(Morgado-Valle and Feldman 2007; Greer et al. 1991), while the blockade of
AMPARs completely abolished the rhythm. Similarly, NMDA receptor R1 subunit
(NMDAR1) mutant mice were completely unresponsive to NMDA applications and
showed a respiratory rhythm almost identical to that of controls. These results
indicate that NMDA receptors are not relevant for generating the rhythm and for the
development of circuits in charge of it (Funk et al. 1997). As for muscarinic
receptors, the effects of glutamate antagonists have not been demonstrated to be
specific for chemoreception.
Till now, acetylcholine (ACh) or glutamate (Glu) actions on chemosensitive
areas are attributed to direct effects on neurons and a probable contribution of
astrocytes in such responses has not been evaluated. Numerous studies demonstrate
that astrocytes in different CNS regions express functional neurotransmitter
receptors, which allow them to be sensitive to neurotransmitters like ACh and Glu
(Perea and Araque 2010; Halassa and Haydon 2010; Ben Achour and Pascual 2010;
Paixao and Klein 2010; Attwell et al. 2010; Sidoryk-Wegrzynowicz et al. 2011;
Stipursky et al. 2011; Haydon and Carmignoto 2006; Erlichman et al. 2010). It is
worth noting that astrocytes in the ventral respiratory group (VRG) express
receptors for 5-HT, substance P (SP), and thyrotropin releasing hormone (TRH).
So, projections from chemosensitive RN neurons may modify the activation of

astrocytes within the respiratory network (Hartel et al. 2009).
Astrocytes in the respiratory network respond to prevailing neuromodulators
with an increase of intracellular calcium concentration (Huxtable et al. 2010;
Gourine et al. 2010; Hartel et al. 2009). Besides, astrocytes are also capable of
synthesizing and releasing neuro- and glio-transmitters such as ACh, Glu,
ATP/adenosine, and D-serine (Haydon and Carmignoto 2006; Hamilton and
Attwell 2010; Carmignoto et al. 1998; Araque et al. 2002; Hosli and Hosli 1994b;
Hosli et al. 1988). So, theoretically, astrocytes may be involved in mediating or
amplifying the ventilatory response to cholinergic and glutamatergic inputs by
releasing gliotransmitters able of modifying the activity of the respiratory network.
In addition, astrocytes can remove neurotransmitters from the synaptic cleft so they
may participate in the control of the synaptic neurotransmitter concentration
(Carmignoto et al. 1998; Araque et al. 2002; Hosli and Hosli 1994b; Hosli et al.
1988; Haydon and Carmignoto 2006). For example at the NTS, acidification can
depolarize astrocytes by inhibition of both K+ channel current and voltage-sensitive
glutamate transporters (Huda et al. 2013). Therefore, as consequence of acidifica-
tion at the NTS, the inhibition of this glutamate transporter, increases the levels of
glutamate at the synaptic cleft affecting the excitatory synaptic transmission
(Huda et al. 2013).
In the human infant, about 95 % of the arcuate nucleus neurons (corresponding
to the chemosensitive RVLM in cats and rats) are glutamatergic. A large number of
astrocytes in the ventral medullary surface express the vesicular glutamate trans-
porter 2 and low levels of 5-HT1A and kainate (GluR5) receptors. So, it is rea-
sonable to propose that astrocytes, which can also express muscarinic and nicotinic
receptors (Gahring et al. 2004; Hosli et al. 1994; Hosli and Hosli 1994a, b), may
store and release glutamate, possibly in response to stimulation by 5-HT, by ACh,
or by glutamate itself (Paterson et al. 2006) affecting, in addition to the inhibition of
glutamate uptake, the levels of glutamate at the synaptic cleft.
In addition, astrocytes play an essential role in glutamatergic synapses.
Glutamate in the synaptic space is uptaken by astrocytes, converted by them into
glutamine, and then transferred as glutamine to the presynaptic terminals for
renewal of the glutamate presynaptic pool (Haydon and Carmignoto 2006). In the
RPG, most of the excitatory synapses are glutamatergic; interestingly, 5 mM
fluoroacetate or 0.1 mM methionine sulfoximine, both glial metabolic toxins,
reduce the increase in respiratory frequency induced by ATP in brainstem slices,
but they do not affect substance P evoked increase, suggesting that astrocytes
contribute to the purinergic drive of the inspiratory rhythm generating network
(Huxtable et al. 2010).
Fig. 4 Schema of astrocyte contribution to the chemosensory response. Astrocytes enwrapping

blood vessels or exposed to the CNS environment are continuously monitoring pH and PCO2. As
consequence of their activation (astrocyte depolarization) by acidosis or hypercapnia, there is an
increase in intracellular calcium concentration, which may trigger the release, among others, of
ACh, Glu, ATP, or D-serine and calcium waves that travel from astrocyte-to-astrocyte influencing
the behavior of astrocyte according other astrocyte domains. Thus, the action of gliotransmitters at
the local chemosensitive site may enhance the response of chemosensitive cells in the immediate
environment. In addition, by volume diffusion of gliotransmitters and by activation of faraway
astrocytes influenced by calcium waves more neurons of the respiratory network may be recruited
Concluding Remarks
Astrocytes have diverse roles in modulation of the respiratory rhythm. These

involve controlling neural network excitability through potassium buffering, regu-
lation of synaptic transmitter concentrations via their synthesis, reuptake and
release; in particular, at glutamatergic synapses, astrocyte is the source of glu-
tamine, essential for replenish synaptic vesicles of glutamatergic neurons. Respect
to respiratory central chemoreception, astrocytes have the ability of monitoring

PCO2 and pH and release gliotransmitters like ATP in the RTN, in response to
changes in CO2 and H+ . In addition, they contribute to the regulation of the
extracellular pH either by generating acidic substances derived from metabolic
coupling (lactate shuttle) leading to amplification of hypercapnic stimulus or
through proton buffering (transporters and channels).
On basis to the discussed properties of astrocytes (calcium waves, coupling of
astrocytic domains through gap junctions, regulation of neurotransmitters and
release of gliotransmitters) we propose that astrocytes may play two emergent roles
in central respiratory chemoreception. A first role, as amplifiers of the responses of
intrinsic chemosensitive neurons through feedforward signaling via gliotransmitters
and a second role as recruiter of non-intrinsic chemosensitive cells thanks to vol-
ume spreading of signals (calcium waves and gliotransmitters) to regions far away
the CO2/H+ sensitive domains (Fig. 4).
Acknowledgments Support from Grants Fondo Nacional de Desarrollo Científico y Tecnológico

(FONDECYT) #1130874 (JE), Comisión Nacional de Ciencia y Tecnología (CONICYT)
#21140669 (MJ Olivares), Comisión Nacional de Ciencia y Tecnología (CONICYT) #21120594
(S Beltrán-Castillo). DICYT-USACH (JE).
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Purine Signaling and Microglial Wrapping
Bernardo Castellano, Mar Bosch-Queralt, Beatriz Almolda,

Nàdia Villacampa and Berta González
Abstract Microglial cells are highly dynamic cells with processes continuously
moving to survey the surrounding territory. Microglia possess a broad variety of
surface receptors and subtle changes in their microenvironment cause microglial
cell processes to extend, retract, and interact with neuronal synaptic contacts. When
the nervous system is disturbed, microglia activate, proliferate, and migrate to sites
of injury in response to alert signals. Released nucleotides like ATP and UTP are
among the wide range of molecules promoting microglial activation and guiding
their migration and phagocytic function. The increased concentration of nucleotides
in the extracellular space could be involved in the microglial wrapping found
around injured neurons in various pathological conditions, especially after
peripheral axotomy. Microglial wrappings isolate injured neurons from synaptic
inputs and facilitate the molecular dialog between endangered or injured neurons
and activated microglia. Astrocytes may also participate in neuronal ensheathment.
Degradation of ATP by microglial ecto-nucleotidases and the expression of various
purine receptors might be decisive in regulating the function of enwrapping glial
cells and in determining the fate of damaged neurons, which may die or may
regenerate their axons and survive.

Keywords ATP Adenosine CD39 ‘Eat-me’ signals Neuronal degeneration

Nerve injury Microglial migration Phagocytosis Purine receptors Axotomy
Abbreviations
PAMPs Pathogen associated molecular patterns
DAMPs Damage associated molecular patterns
TLRs Toll-like receptors
B. Castellano (&) M. Bosch-Queralt B. Almolda N. Villacampa B. González

Unit of Histology, Torre M5, Department of Cell Biology, Physiology and Immunology,
Institute of Neurosciences, Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain

DOI 10.1007/978-3-319-40764-7_7
148 B. Castellano et al.
SAMPs Self-associated molecular patterns

TREM2 Triggering Receptor Expressed on Myeloid cells 2
PPT Perforant path transection
ECM Extracellular matrix
“Resting” Microglia in the Healthy CNS and Their

Interaction with the Microenvironment
The term “quiescent” or “resting” microglia, usually used to designate nonactivated

microglia in the normal adult central nervous system (CNS), might lead one to think
that these cells are in a dormant state with no apparent movement and function.
However, nothing could be further from the truth. The combined use of in vivo
time-lapse transcranial two-photon microscopy and transgenic mice with green
fluorescent protein in resident CNS microglia has made it possible to see microglia
interacting with other cortical elements (Davalos et al. 2005; Nimmerjahn et al.
2005). Microglial cells are the most dynamic cells in the healthy CNS, as their
morphological changes far exceed those of both neurons (Holtmaat et al. 2008;
Knott and Holtmaat 2008) and astrocytes (Hirrlinger et al. 2004). Thus, in the
healthy brain, microglial cells are continuously remodeling their shape by extending
and retracting their processes, surveying the local microenvironment to scan the
surface of the surrounding cells and the interstitial fluid (Davalos et al. 2005;
Nimmerjahn et al. 2005) (see Chapters “Glial cells and Integrity of the Nervous
System” and “Microglia Function in the Normal Brain”). Under normal conditions,
each microglial cell seems to be responsible for checking its own territory, and its
highly dynamic processes do not overlap or enter in the territory of neighboring
microglial cells. While the microglial soma and main branches remain stable in the
nervous parenchyma, with few signs of movement and without any clear rela-
tionship to other cells or blood vessels, its motile processes are continuously
making direct contacts with nearby neuronal cell bodies, macroglia, and blood
vessels (Nimmerjahn et al. 2005; Wake et al. 2009; Tremblay et al. 2010).
Although it might appear at first glance that motility of microglial processes is
random (Nimmerjahn et al. 2005), a wide range of studies indicates that microglial
cells express a broad variety of surface receptors that allows them to sense subtle
changes in the microenvironment (Kierdorf and Prinz 2013). In particular, in the
healthy adult brain, movement of microglial processes seems to be closely related to
local concentration of some neurotransmitters, neuropeptides, and neuromodulators
(Pocock and Kettenmann 2007). Although not conclusive, the current data suggest
that microglial motility is increased by global excitatory neurotransmission and
decreased by global inhibitory neurotransmission (Nimmerjahn et al. 2005;
Fontainhas et al. 2011; Eyo and Wu 2013).
Purine Signaling and Microglial Wrapping 149
Fig. 1 In the healthy normal brain, ramified microglia is a very dynamic cell and their processes
are continuously extending and retracting, monitoring the surface of neurons and having a special
predilection for excitatory synapses. Interaction between inhibitory receptors in microglia with
both specific ligands in the neuronal surface and neuronal released molecules keeps microglia in a
nonactivated state. These signals that are expressed constitutively are know as “Off-signals”
Furthermore, electron microscopic studies demonstrate that, under normal con-

ditions, microglial cell processes directly contact presynaptic and postsynaptic
elements and have a special predilection for excitatory synapses (Fig. 1) although
the existence of microglial cell interactions with inhibitory synapses under normal
physiological conditions remains yet unknown (Perry and O’Connor 2010; Siskova
and Tremblay 2013). Microglial cell processes contact synapses about once per
hour, remain in a close proximity to presynaptic boutons for 5 min, and then retract
(Wake et al. 2009). The interactions between microglia and synapses depend on
neuronal activity and, therefore, the frequency of contact declines with decreased
synaptic transmission (Wake et al. 2009).
Signaling Mechanisms Involved in Activation of Microglia
As previously discussed in Chapters “Glial cells and Integrity of the Nervous

System” and “Microglia Function in the Normal Brain”, microglia are activated by
various changes in their microenvironment caused by acute insults and chronic
disease states (Kettenmann et al. 2011; Chen et al. 2014; Gonzalez et al. 2014).
Transformation of the finely branched resting microglia into enlarged cells with
short and stout processes is a hallmark of microglial cell activation (Kettenmann
et al. 2011). In addition to morphological changes, microglial activation involves a
stereotypical pattern of changes, including proliferation and migration to sites of
injury, increased or de novo expression of cytokines and growth factors and, in
some circumstances, the full transformation into phagocytes capable of clearing
damaged cells and debris (Kettenmann et al. 2011). There is a wide range of
molecules promoting microglial activation that can be classified as two main types:
PAMPs (Pathogen associated molecular patterns) and DAMPs (damage associated
molecular patterns). PAMPs warn of the presence of exogenous material, such as
components of bacterial cell walls or repeats of bacterial or viral nucleic acids,
whereas DAMPs warn of internal damage to the cells of the own organism and
include molecules released by injured cells or modified as a consequence of tissue
damage, such as oxidized lipoproteins or fragments of extracellular matrix mole-
cules (Bianchi 2007; Matzinger 2007). Microglial cells possess a wide range of
surface molecules, such as toll-like receptors (TLRs) (Lehnardt 2010), scavenger
receptors (Husemann et al. 2002) and numerous cytokine and chemokine receptors,
whose interaction with DAMPs and PAMPs results in a rapid activation of resting
microglia to become motile effector cells (Kierdorf and Prinz 2013).
However, it would be a mistake to think that activation of microglia is a simple
event; on the contrary, it is complex and includes still unidentified signaling
mechanisms. In the healthy CNS, microglia exhibit a deactivated phenotype due to
the interaction of inhibitory receptors (“Off receptors”) in their plasma membrane,
with the corresponding ligands (“Self-associated molecular patterns” or SAMPs)
located on neurons and glial cells that keep microglia in a resting or nonactivated
stage (Biber et al. 2007; Eyo and Wu 2013; Kierdorf and Prinz 2013) (Fig. 1).
Some of the proposed inhibitory receptors in microglia are CX3CR1 and CD200R,
which interact with their respective ligands, CX3CL1 (fractalkine) and CD200 on
the surface of healthy neurons (Chertoff et al. 2013; Eyo and Wu 2013). Another
proposed microglial inhibitory system is CD45/CD22. Recognition of CD22 on the
surface of neurons by CD45 on microglia dampens microglial activation (Mott et al.
2004). Moreover, in addition to displaying membrane bound “Off-signals,” neurons
also release soluble Off-signals into the extracellular space, such as Transforming
growth factor (TGF) b, neurotransmitters and neurotrophins including NGF, BDNF
and NT-3 (Biber et al. 2007). If any of these Off-signals are lost, due to changes in
the microenvironment, or are downregulated, as may occur in pathological condi-
tions, microglial activation is triggered.
In contrast to Off-signals, which are expressed constitutively in the healthy adult
brain, “On-signals” are produced on demand to initiate either a pro- or
anti-inflammatory microglial activation program (Kettenmann et al. 2013) (Fig. 2).
Some of the On-signals are the so-called “help-me/find-me” molecules (Marin-Teva
et al. 2011; Panatier and Robitaille 2012; Xing et al. 2014). When neurons are
overactive, impaired or endangered, they release these “alert” signals (Noda et al.
2013) which include nucleotides such as ATP and UTP (Sperlagh and Illes 2007);
chemokines such as CCL21 and CXCL10 (Rappert et al. 2004; de Jong et al. 2005);
Fig. 2 “On signals” are produced when neurons are damaged and include “Help-me”/“Find-me”,
“Do-not-eat-me”, and “Eat-me” signals. Endangered neurons may release a wide range of alert
signals (Help-me/Find-me) including nucleotides that promote microglial activation, process
retraction and migration towards neuronal somata. Microglial wrapping in one hand facilitates
contact-dependent neuron-microglia interactions but also isolates damaged neurons leaking
nucleotides. If Do-not-eat-me signaling predominates, phagocytosis is inhibited and neurons are
able to survive. If, on the contrary, Eat-me signaling prevails, an increase in the phagocytic ability
of microglia takes place and damaged neurons are removed. Note the importance of the
ecto-enzymes CD39 and CD73 regulating the levels of nucleotides and nucleosides in the
extracellular space around injured neurons
cytokines like Interleukin (IL) 1 (Cartier et al. 2005); neuropeptides such as

bradykinin (Ifuku et al. 2007), endothelin (Fleisher-Berkovich et al. 2010), galanin
(Ifuku et al. 2011) and neurotensin (Martin et al. 2005); neurotransmitters such as
glutamate, adrenaline, and dopamine (Farber et al. 2005; Liu et al. 2009); and
cannabinoids (Walter et al. 2003) and morphine (Takayama and Ueda 2005). In
response to help-me/find-me signals, microglia approach to the source of these
molecules and develop either a close surveillance or a phagocytic function,
depending on the presence of additional signals in the damaged neuron. If the
receptor SIRP-alpha (CD172a) in the membrane of microglia interacts with the
ligand CD47 on neurons, a “Do-not-eat-me” signal is presented to microglia (Biber
et al. 2007; Ravichandran 2010). However, if the microglial receptor TREM2
(Triggering Receptor Expressed on Myeloid cells 2) recognizes its
still-unknown-ligand on the surface of the damaged neuron, this interaction is
interpreted as an “Eat-me” signal and therefore the microglial cell is able to initiate
an intracellular signaling cascade, through the adaptor protein DAP12, leading to
phagocytosis (Linnartz and Neumann 2013). TREM2 expression has been sug-
gested to regulate not only phagocytic but also the migratory capacity of microglia
(Melchior et al. 2010).
Migration of Microglia Is Guided by Purinergic Signaling
Release of danger signals that act as chemoattractants at the site of damage, initiates
microglial activation and stimulates migration. Time-lapse two-photon imaging
demonstrates that, for example, after a small laser ablation in the cerebral cortex, all
microglial cells located in the surroundings respond within minutes by enlarging
and extending their processes towards the damaged site, converging and forming a
spherical shaped containment around it, but without migration of the somata
(Davalos et al. 2005; Nimmerjahn et al. 2005). Quick extension of microglial
processes to the site of injury without significant displacement of the cell body was
previously described using histological sections (Jensen et al. 1994). In this work
we showed that, a few hours after a perforant path transection (PPT), microglial
cells located in the inner zone of the dentate molecular layer polarize and extend
their processes towards and into the denervated PP zone, and it is not until 2–3 days
after PPT when microglial cell bodies move to the denervated PP zone, where they
accumulate and proliferate (Jensen et al. 1994). Therefore, migration of microglial
cells is probably a complex process that involves two stages: a first phase of
reconnaissance and damage assessment by microglial cells processes and, if dam-
age persists and is important enough, a second phase where the entire cell body
migrates. An intense cross talk, involving the signaling mechanisms referred in the
previous section, between extended microglial processes and damaged neurons and
glial cells, will determine this microglial cell migration.
Purine nucleotides are among the most potent molecules involved in the
migration of microglia. In fact, Davalos et al. (2005) demonstrated that ATP or
ADP microinjection in the brain parenchyma was able to mimic the rapid
chemotactic response of microglial processes observed following laser ablation.
Moreover, lowered ATP extracellular concentration results in reduced microglial
cell process movements, whereas increased ATP gradients stimulate their motility
(Haynes et al. 2006).
In the healthy brain, release of ATP to the extracellular space is a common
phenomenon, as this nucleotide and its derivatives act both as primary transmitter
and as co-transmitter released with other neurotransmitters and peptides in many
synapses. The mechanism by which intracellular ATP is released by neurons is a
matter of intense debate (Cisneros-Mejorado et al. 2015), because in addition to
being released by exocytosis, ATP leakage can also take place through large pores
and transporters. Moreover, not only neurons but also glial cells, in particular
astrocytes, can release ATP (Butt 2011; Cisneros-Mejorado et al. 2015).
Under pathological conditions when neurons are overexcited, injured or stressed
in acute or chronic neurological disorders, a massive release of ATP takes place into
the extracellular space (Braun et al. 1998; Melani et al. 2005). As elevated con-
centrations of extracellular ATP can cause cell death (Matute et al. 2007; Arbeloa
et al. 2012), ATP released from endangered or dying cells may aggravate the extent
of the ongoing damage. In addition, increased extracellular levels of ATP may over
activate the P2X7R in neurons and trigger signaling cascades leading to neurode-
generation (Le Feuvre et al. 2003).
The concentrations of ATP, ADP, AMP and adenosine in the extracellular space
are regulated by the activity of ecto-nucleotidases that are located in the plasma
membrane of microglia and whose expression is dependent on the development and
activation stage of these cells (Dalmau et al. 1998). One of these ecto-nucleotidases
is CD39, also called Ecto-nucleoside triphosphate diphosphohydrolase 1
(E-NTPDase1), whose expression in the CNS is restricted to microglial cells and
vascular endothelium (Braun et al. 2000). CD39 plays a main role hydrolysing
extracellular nucleoside 5′-triphosphates to nucleoside 5′-diphosphates (NTPase
enzymatic activity), as well as nucleoside 5′-diphosphates to nucleoside 5′-mono-
phosphates (NDPase enzymatic activity). Nucleoside 5′-monophosphates are fur-
ther hydrolysed to adenosine by CD73, an ecto-5′-nucleotidase also found, among
other cells, in the membrane of microglia (Dalmau et al. 1998; Bulavina et al.
2013). Therefore, microglial cells could be considered as the cells responsible for
the regulation of purinergic signaling in the CNS as they can control the rate, extent
and timing of nucleotide degradation.
On the other hand, we should consider that microglial cells have several types of
purine receptors on their surface (Ohsawa and Kohsaka 2011) whose interactions
with changing concentrations of extracellular nucleotides and nucleosides (ATP/
adenosine balance) may regulate microglial behavior, including process extension
and retraction, microglial migration and even phagocytosis.
Purine receptors are divided into P1 (adenosine receptors) and P2 (ATP recep-
tors). Microglia express the four subtypes of P1 receptors (A1, A3, A2A and A2B)
and only some of the different subtypes of P2 receptors cloned, which are divided
into ionotropic (seven subtypes: P2X1-7) and metabotropic (eight subtypes: P2Y1,
−2, −4, −6, −11, −12, −13, and −14) (Kettenmann et al. 2011). Simultaneous
costimulation of P1 and P2 receptors seems to be required for microglial migration
(Farber et al. 2008). In particular, microglial process extension is dependent upon
ATP/ADP sensed through microglial P2Y12 receptors (Ohsawa and Kohsaka

2011), which are constitutively expressed on microglia in normal conditions (Sasaki
et al. 2003) and upregulated when activated (Tozaki-Saitoh et al. 2008). P2Y12
receptors activate integrin-ß1, which accumulates in the tips of microglial pro-
cesses, facilitating the adhesion of extended microglial processes with the extra-
cellular matrix (ECM), which is a requisite for subsequent directional microglial
migration (Haynes et al. 2006; Kurpius et al. 2007). Further activation of microglia,
probably due to continuously elevated levels of ATP and ADP, or both (Kurpius
et al. 2007), leads to upregulation of A2A and P2X4 receptors, whereas P2Y12
receptors are downregulated (Haynes et al. 2006; Orr et al. 2009). Signaling
through P2X4 receptors enhances migration of microglia. As microglial activation
involves increased expression of the ecto-enzymes CD39 and CD73 (causing
ATP/ADP degradation), the abnormally increased levels of ATP generated by the
pathological situation are gradually reduced, while the adenosine concentration
increases and activates A2A receptors. Notably, adenosine causes retraction of
microglial processes (Ohsawa and Kohsaka 2011). Therefore, gradually increased
levels of adenosine may be the basis of microglial transformation from ramified
cells into amoeboid migratory morphologies, usually found in various pathologies.
Microglial Wrapping and Synaptic Stripping
As discussed in Chapters “Glial cells and Integrity of the Nervous System”

and “Microglia Function in the Normal Brain”, it has been widely reported that
activated microglia migrate and accumulate near injured neurons in various
pathological conditions. In addition, in certain circumstances, the somata, proximal
dendrites and axons of injured neurons become ensheathed by microglia. Microglial
wrapping of neuronal cell bodies is one of the most prominent features after
peripheral nerve axotomy (Fig. 3). Indeed, the phenomenon of microglial wrapping
has been widely described in various CNS areas in several situations involving
peripheral nerve axotomy, including the facial nucleus (Moran and Graeber 2004),
the hypoglossal nucleus (Sumner and Sutherland 1973; Yamada et al. 2011), the
dorsal motor nucleus of the vagus nerve (Masui et al. 2002), and in the spinal cord
after sciatic nerve axotomy (Gehrmann et al. 1991). Also, this phenomenon has
been reported in experimental models where peripheral nerves are not affected such
as hippocampal organotypic cultures after an ischemic insult (Neumann et al.
2006), in the cerebral cortex during either acute focal inflammation (Trapp et al.
2007) or following intraperitoneal LPS injection (Chen et al. 2012), and in the
spinal cord after experimental autoimmune encephalomyelitis (EAE) induction
(Almolda et al. 2009). Microglial wrapping occurs in parallel with a significant
reduction of axosomatic synapses. It was Blinzinger and Kreutzberg (1968) who
first described, following facial nerve axotomy, the displacement of presynaptic
terminals from the injured motor neuron surface by the interposing of microglial
pseudopods and named this phenomenon as “synaptic stripping”. Although some
Fig. 3 Microglial wrapping in the facial nucleus of the mouse after facial nerve axotomy. In the
normal, non-lesioned facial nucleus, microglia stained with different markers including NDPase
histochemistry and immunohistochemistry for Iba1, CD39 and CD11b (a, c, e, g), show a
ramified morphology (arrows) without any particular association with neuronal motor neuron
somata (asterisks). After axotomy (b, d, f, h), microglia enwrap motor neuron somata (asterisks).
In (a)–(d), sections are counterstained with toluidine blue. In (g) and (h) nuclei are stained in blue
with DAPI. Scale bar = 20 lm
authors claim that reactive microglia spread on the surface of motor neurons to
physically disconnect synapses (Moran and Graeber 2004; Yamada et al. 2008), it
is still not totally clear whether synaptic stripping is either the cause or the con-
sequence of microglial wrapping. As microglial wrapping and synaptic stripping are
associated with motor neuron regeneration, it has usually been considered to be a
neuroprotective process (Kreutzberg 1996). As we will discuss below, recent
studies support this neuroprotective view (Chen et al. 2014), whereas others suggest
that microglial wrapping may reduce neuronal survival (Yamada et al. 2011).
Accumulating evidence indicate that synaptic stripping of either inhibitory or
excitatory synapses is beneficial to damaged neurons. Since microglia wrap neu-
ronal cell bodies and the majority of synapses terminating on projection neuron
somata in the cerebral cortex are GABAergic inhibitory synapses, it has been
proposed that inhibitory axosomatic synapses are preferentially stripped after focal
inflammation or peripheral immune challenge (Trapp et al. 2007; Chen et al. 2012).
Evidence of microglia-mediated stripping of inhibitory GABAergic presynaptic
terminals from cortical neurons in adult mice has been recently confirmed by 3-D
electron microscopy (Chen et al. 2014). Reduced axosomatic GABAergic inner-
vation protects neurons against noxious insult (Hardingham et al. 2002) by
increasing synchronization of neuronal firing (Woo and Lu 2006), which is critical
for synaptic NMDAR-mediated neuronal survival through CREB activation and by
increasing neuronal expression of anti-apoptotic and neuroprotective molecules
(Hardingham and Bading 2003).
However, it is nowadays clear that microglial wrapping is not always specifically
directed to disconnect inhibitory synapses because in other locations, such as the
facial nucleus in the rat after nerve axotomy (Raslan et al. 2014) and the spinal cord
after either intramedullary axotomy in the cat (Linda et al. 2000) or sciatic nerve
transection in the rat (Arbat-Plana et al. 2015), the outcome of microglia-mediated
synaptic stripping is the preferential disconnection of excitatory glutamatergic
synapses. Removal of the glutamatergic input to the axotomized motor neurons is
considered relevant for neuronal survival, as glutamate may exert deleterious
excitotoxic effects on nerve cells (Mehta et al. 2013). In support of this possibility,
blocking of the NMDA-type glutamate receptor has been reported to increase motor
neuron survival after neonatal axotomy in the rat (Mentis et al. 1993). Even
assuming that any changes in the synaptic input, either inhibitory or excitatory, to
the lesioned neurons may reduce their stress and be beneficial for survival and
repair, the question of whether microglia actively participate in this process or if
instead nerve terminals simply retract from the surface of neurons remains unsolved
(Linda et al. 2000).
It is generally accepted that synaptic stripping does not inevitably mean that the
disconnected terminals have to be immediately engulfed by microglia, as they
remain in the vicinity of ensheathed neurons and only after axotomized motor
neurons regenerate their axons, synapses are restored (Navarro et al. 2007).
However, some work indicates that, several weeks after nerve transection, restored
synaptic inputs are not normal (Raslan et al. 2014). The usual prevalence
of inhibitory over excitatory terminals seems to be shifted for surviving lesioned
motor neurons in various locations (Borke et al. 1995; Linda et al. 2000; Raslan
et al. 2014). Although microglia has been suggested to play a main role in regu-
lating these synaptic rearrangements (Raslan et al. 2014), astrocytes might also be
involved (Tyzack et al. 2014).
In the healthy brain, neurons, including their synapses, are generally ensheathed
by fine processes of astrocytes that participate in the regulation of synapse for-
mation, stability, and elimination. Coverage of synapses by astrocytic processes
may change under various physiological conditions (Theodosis et al. 2008; Chung
et al. 2013; Perez-Alvarez et al. 2014). Specifically, in the facial nerve of the mouse
two weeks after axotomy, thin lamellar astrocyte processes begin to replace
microglial wrapping around damaged motor neurons, and by 3 weeks they com-
pletely cover the neuron soma (Moran and Graeber 2004). Some authors have
suggested that this delayed astrocyte behavior might contribute to synaptic
remodeling by engulfing some disconnected presynaptic terminals (Chung et al.
2013) and promoting the rearrangement of synaptic inputs on axotomized motor
neurons (Tyzack et al. 2014).
Microglial Wrapping: Detrimental or Beneficial?
Glial wrapping, whether microglial, astroglial or both, may not only cause deaf-
ferentation, but might also facilitates contact-dependent neuron–glia interactions
that prevent neuron death and promote regeneration. After facial nerve axotomy in
the mouse, for example, about 65 % of axotomized neurons regenerate axons and
survive, whereas 35 % of neurons degenerate. Research in our laboratory per-
forming facial nerve axotomy on transgenic mice with astrocyte-targeted expression
of either IL6 or IL10 in order to investigate how the local expression of those
cytokines may affect microglial activation, showed that in addition to changes in the
microglial reactivity pattern, there is an altered survival/death ratio of motor neu-
rons (Almolda et al. 2014; Villacampa et al. 2015). Interestingly, higher motor
neuron survival in IL10 transgenic mice was not associated with significant changes
in microglial wrapping (Villacampa et al. 2015) although increased motor neuronal
death in IL6 transgenic mice coincides with reduced microglial wrapping (Almolda
et al. 2014). Moreover, ongoing studies performed on IRF8 KO mice indicate that
incomplete microglial wrapping of individual axotomized motor neurons correlates
with increased motor neuron death (Xie et al. 2014). In agreement with this, some
evidence suggests that defects in microglia-neuron attachment after facial nerve
axotomy, as occurs in microglial cathepsin deficient mice (Hao et al. 2007) and
TGFb1 deficient animals (Makwana et al. 2007), might lead to more neuron death.
These observations support the hypothesis that the intimate association between
glial cells and neurons has a neurotrophic rather than neurotoxic function. The close
physical proximity of microglia to injured neurons may facilitate the continuous
supply of growth factors and other required molecules, thus supporting survival and
regeneration (Trapp et al. 2007).
There is however an opposing view holding the possibility that prolonged

contact of microglial cells with enwrapped neurons is detrimental (Yamada et al.
2011). Some studies have demonstrated that the survival ratio of injured motor
neurons is markedly influenced by the species and the age of animals used (Moran
and Graeber 2004; Kiryu-Seo et al. 2005). Interestingly, in this context, facial nerve
axotomy in neonatal rats and mice kills damaged motor neurons within a week of
lesion. Nevertheless, axotomized motor neurons in adult rats are able to survive,
whereas in adult mice there is a slow and progressive motor neuron death after
lesion (Kiryu-Seo et al. 2005). Some authors have suggested that these differences
among adult rats and mice are due to differences in the ratio of microglial/astroglial
wrapping (Yamada et al. 2011). If the astrocytic wrapping predominates, as found
in the rat, some protective effects are exerted on axotomized motoneurons, whereas
if the wrapping is mainly microglial, as observed in mouse, a slow apoptotic cell
death of motor neurons might take place (Yamada et al. 2011).
Microglial wrapping may be the result of a continuous release or leakage of
purine nucleotides that act as find-me signals (Fig. 2). Neuron ensheathment by
activated microglia expressing ecto-nucleotidases in their plasma membrane
effectively isolates damaged neurons leaking purine nucleotides and contributes to
their rapid degradation to adenosine around neurons. Increasing concentrations of
extracellular adenosine may develop a potentially neuroprotective function on
neurons through P1 adenosine receptors (Stone 2002). In addition, adenosine can
impair the phagocytic function of peripheral macrophages by binding to the
P1 adenosine receptors expressed on their membrane (Hasko et al. 2007). Also,
microglial phagocytosis seems to be regulated by purinergic signaling (Bulavina
et al. 2013). It has been shown that activation of P1 receptors by a non-hydrolysable
analog of adenosine decreases microglial phagocytosis (Bulavina et al. 2013). In the
opposite way, activation of P2Y12 receptor by ADP, activation of P2Y6 by UDP
and activation of P2Y2/P2Y4 receptors by UTP markedly increase microglial
phagocytosis both in vitro and in vivo (Koizumi et al. 2007; Fang et al. 2009).
Therefore, the increasing concentration of these nucleotides around injured neurons
may be an eat-me signal for wrapping microglia. In agreement with this, CD39-
deficient animals presented higher microglial phagocytic activity (Bulavina et al.
2013), suggesting that an increased concentration of extracellular ATP/ADP and
UTP/UDP, due to the lack of CD39 enzymatic activity, leads to a chronic stimu-
lation of the microglial phagocytic activity. However, other studies indicate that
activation of P2X7 receptors by exposure to ATP induced inhibition of microglial
phagocytic activity even if microglia are cotreated with UDP (Fang et al. 2009).
Taken together, these observations suggest that a fine control of the levels of
nucleosides and nucleotides in the extracellular space around injured neurons
together with a fine regulation of purine receptors may be decisive to control
phagocytosis and hence in determining the fate of damaged neurons wrapped by
microglia.
Concluding Remarks
The meaning of microglial wrapping around injured neurons is not completely

understood. Microglial wrapping partially isolates endangered neurons from the
adjacent neuropil, leading to an important deafferentation from synaptic inputs.
Besides, the wide area of contact between microglia and neuronal surfaces enables
an intense exchange of molecular signals between them. Injured neurons circum-
scribed by microglia may survive or die and their fate will depend on a plethora of
signals. In this scenario, nucleosides and their phosphorylated nucleotides may play
a key role, as they can be involved in regulation of apoptosis, in the synthesis and
release of different trophic factors by astrocytes (Rathbone et al. 1999), in pro-
motion of axonal growth (Heine et al. 2006), and in modulation of microglial
phagocytosis (Inoue 2008). Although programmed neuronal cell death can result
from axonal injury, cell regeneration and axonal outgrowth programs are also
activated (Raivich and Makwana 2007; Kiryu-Seo and Kiyama 2011). The putative
involvement of microglia and astroglia in the activation of these regenerative
programs are still poorly understood and will be a challenge for researchers in the
coming years.
Acknowledgments This work was supported by the Spanish Ministry of Science and Innovation
BFU2011-27400 and BFU2014-55459-P.
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Oligodendrocytes: Functioning
in a Delicate Balance Between High
Metabolic Requirements and Oxidative
Damage
Alejandro D. Roth and Marco T. Núñez
Abstract The study of the metabolic interactions between myelinating glia and the
axons they ensheath has blossomed into an area of research much akin to the
elucidation of the role of astrocytes in tripartite synapses (Tsacopoulos and
Magistretti in J Neurosci 16:877–885, 1996). Still, unlike astrocytes, rich in
cytochrome-P450 and other anti-oxidative defense mechanisms (Minn et al. in
Brain Res Brain Res Rev 16:65–82, 1991; Wilson in Can J Physiol Pharmacol.
75:1149–1163, 1997), oligodendrocytes can be easily damaged and are particularly
sensitive to both hypoxia and oxidative stress, especially during their terminal
differentiation phase and while generating myelin sheaths. In the present review, we
will focus in the metabolic complexity of oligodendrocytes, particularly during the
processes of differentiation and myelin deposition, and with a specific emphasis in
the context of oxidative stress and the intricacies of the iron metabolism of the most
iron-loaded cells of the central nervous system (CNS).
Keywords Myelination
Ensheathment Reactive oxygen species (ROS)
Oligodendrocyte precursor cells (OPCs)
A Brief Introduction to Oligodendrocyte Structure

and Function
Oligodendrocytes were initially described in 1921 by Pio del Río Hortega, who
demonstrated their neuroectodermal origin and association to axons (see a historical
review by Pasik and Pasik 2004). Historically unlike other glial cells, oligoden-
drocytes have attracted a lot of attention and are probably one of the best-studied
cell lineages in the CNS, reflecting the importance myelin plays in multiple diseases
and in the inhibition of axonal regeneration. At the same time, and in contradiction
to what would be predicted from their complex morphology and intimate
A.D. Roth (&) M.T. Núñez

Department of Biology, Faculty of Science, University of Chile, Santiago, Chile

DOI 10.1007/978-3-319-40764-7_8
168 A.D. Roth and M.T. Núñez
association to axons, oligodendrocytes have proven to be remarkably adaptable to

in vitro culture, as purified precursor cells can achieve terminal differentiation in the
absence of neurons or other glial cells, extending huge membrane sheets and
expressing characteristic myelin markers like myelin basic protein (MBP), prote-
olipid protein (PLP), myelin-associated glycoprotein (MAG), and myelin oligo-
dendrocyte glycoprotein (MOG) (Pfeiffer et al. 1993; Espinosa-Jeffrey et al. 2009).
Oligodendrocytes originate from oligodendrocyte precursor cells (OPCs) which
appear late in embryonic development at different areas of the ventral neural cord
and sub-ventricular zone (SVZ) (Baumann and Pham-Dinh 2001; Miller and Mi
2007). Here, we must note that in rodent telencephalon oligodendrocytes originate
in three waves from increasingly dorsally localized niches and, as each OPC wave
arises, it displaces the previous cells, resulting in a postnatal predominance of
dorsally derived oligodendrocytes (Kessaris et al. 2006; Rowitch and Kriegstein
2010; Tomassy and Fossati 2014). As these highly motile cells (Noble et al. 1988)
migrate away in response to chemoattractants and chemorepulsants (e.g., Netrin-1)
(Jarjour et al. 2003), they undergo multiple rounds of division before differentiating
into oligodendrocyte progenitors (pre-OLGs) which present lower motility, a higher
number of cell processes, and which scan the surrounding area for axons. At this
stage, pre-OLGs undergo terminal differentiation into immature oligodendrocytes,
developing a large number of highly branched cell processes which, in vitro and in
the absence of axons, flatten on the substrate, express characteristic protein markers,
and extend large membrane sheets that recapitulate the biochemical composition of
myelin, and thus are referred to as “mature oligodendrocytes.” It is important to
note that in vitro differentiation and maturation renders these cells highly dependent
on extrinsic survival factors, reflecting their requirement on trophic factors that are
provided by axons (Barres et al. 1992, 1993). In vivo, as oligodendrocytes are the
last neural cell lineage to arise, myelin deposition starts shortly before birth (in
rodents) and proceeds to expand in a rostral to caudal fashion in the ventral spinal
cord, while expanding through the dorsal spinal cord both rostrally and caudally
starting from the cervical enlargement throughout the first weeks of life (Foran and
Peterson 1992). In humans, myelination is initiated close to the 17th week of
gestation and continues throughout life (Tosic et al. 2002; recently reviewed in El
Waly et al. 2014). It should be noted that the onset of embryological myelination
marks the damage window of hypoxic-ischemic injury to the periventricular cere-
bral white matter (periventricular leukomalacia, between 23 and 32 weeks), which
results in the most common brain damage to premature infants: cerebral palsy (Back
et al. 2001; Volpe 2001) (see Chapter “Prenatal Systemic Hypoxia-Ischemia and
Oligodendroglia Loss in Cerebellum” for further reading on hypoxic-ischemic
injury and cerebral palsy).
Terminal oligodendrocytes maturation can be separated into three major steps:
(i) Cell spreading and axon recognition; (ii) Axon–glial association and Initial
wrapping; (iii) Membrane extension and myelin compaction. Once an OLG process
has aligned and spiraled around the axon, it spreads laterally, covering the axon and
the innermost spirals, an extensively studied event that has given rise to multiple
models as to how it is achieved (see review by Bauer et al. 2009). Then, as multiple
Oligodendrocytes: Functioning in a Delicate Balance … 169
plasma membrane lamella have wrapped around and axon, a process of cytoplasm
extrusion brings the inner faces of the plasma membrane close together, displacing
the cytoplasm to the loops at the sides of the internode (see reviews by Pedraza
et al. 2001; Ffrench-Constant et al. 2004; Simons and Trotter 2007). Through this
process of compaction, the myelin sheath is divided into two structurally and
biochemically different domains: compact myelin, which surrounds the internode
and provides radial electrical resistance, and the non-compacted channels,
cytoplasm-rich areas that connect compact myelin domains to the cell body and
provide metabolic support for the compacted myelin domains and the underlying
axon (see Court et al. 2008 and an extensive review by Nave 2010). It should be
stressed that compact myelin lamella corresponds to a particularly lipid-enriched
plasma membrane, where cholesterol (26 %), galactolipids (31 %), and plasmalo-
gens (20 %) overshadow the overall protein content (30 %) (see Chrast et al. 2011).
Here, in comparison to the rest of the CNS, unsaturated long-chain fatty acids are
unusually concentrated. These lipidic characteristics lend myelin a distinct density
when compared to other CNS membranes, simplifying its purification and its
proteomic and lipidomic characterization (Taylor et al. 2004; Roth et al. 2006; Ishii
et al. 2009; Jahn et al. 2009; Dhaunchak et al. 2010; Gopalakrishnan et al. 2013). At
the same time, this highly stable lipid composition, the organization of the
hydrophobic motifs, and the long half-life of myelin components have led to the
interesting suggestion that myelin organization arises through an intrinsic
self-organization and self-assembly into what has been compared to a lipid crystal
(see Aggarwal et al. 2011 for a comprehensive discussion on this topic).
As described in Chapter “Glial Cells and Integrity of the Nervous System,”
myelination itself divides axons into two discrete domains: the myelin free, elec-
trically active nodes (nodes of Ranvier) and the myelin ensheathed internodes,
which separate one node from the next. At a molecular level, nodes present a
characteristic concentration of voltage-gated sodium channels that are central to
saltatory conduction (estimated at 1000–1500 channels/µm2) (Rosenbluth 1976),
while internodes are divided into three specific domains: paranodes, juxtaparan-
odes, and internodes, whose description escapes the scope of this work (interested
readers should seek out Rasband et al. 1998; Poliak et al. 1999; Pedraza et al. 2001,
2009; Traka et al. 2002; Poliak and Peles 2003).
Oligodendrocyte Metabolism and Axonal Support
Studies on myelin tend to concentrate in the acceleration of neural communication,

for the concept of response time is easily conveyed and the expression “the quick
and the dead” is echoed in nature by the myriad of adaptations that hasten response
to stimuli by reducing the time required for an action potentials to transverse the
length of an axon. Nevertheless, adaptations to increase speed may be metabolically
expensive and require considerable resources, as is evident for the giant axons in
squids, where the excitable membrane is depolarized and repolarized throughout the
whole length of the axon, and the ion gradient must be maintained by greater action
of the Na+/K+ pump. Axon myelination induces saltatory conduction of action
potentials, thereby increasing conduction speed up to a hundredfold while main-
taining small cross-sectional axonal diameters and reducing the areas of the exci-
table membrane that are depolarized during each action potential (reviewed by
Hartline and Colman 2007). In this sense, the importance of axonal ensheathment is
highlighted by the convergent evolution of myelin-like structures in annelids and
crustacea, which do not share myelinated common ancestors with vertebrates (Xu
and Terakawa 1999; Wilson and Hartline 2011). Furthermore, although most of
these myelin-like structures are the result of axonal ensheathments by glial cell,
recent observations in calanoid copepods show the construction of myelin-like
structures from the axon itself, without the participation of an accompanying glial
cell (Wilson and Hartline 2011). Multiple authors have put forth the idea that
myelination is advantageous, being metabolically “cheap” for axons, as it mini-
mizes the energy required to reset the axonal membrane to its chemo-electrically
polarized state, to the point that some have suggested that this energy saving was
the original function of myelin sheaths, and that action potential acceleration
emerged later as a response to increases in depredation (Stiefel et al. 2013). Still,
these ideas must be tempered by the impressive metabolic cost this places upon the
myelinating glial cells, which not only must deposit and maintain huge plasma
membrane extensions, but must also provide protection and metabolic support to
axons (see review by Hirrlinger and Nave 2014), a condition highlighted by the
plethora of demyelinating diseases (adrenoleukodystrophy, Charcot–Marie–Tooth
disease, multiple sclerosis (MS), and amyotrophic lateral sclerosis), all of which
have bad prognosis and present incapacitating effects, particularly when axonal
damage ensues (Sauer et al. 2013) (see Chapter “Peripheral Inflammation and
Demyelinating Diseases” for further reading on demyelinating diseases). In this
context, considering the effects on mice lacking myelin-specific genes has led some
authors to suggest that some neurodegenerative diseases could result from problems
intrinsic to the myelinating cells (Popko 2003).
Hypoxia and Oxidative Stress
Myelination requires that each oligodendrocyte first constructs and then maintains
an extensive plasma membrane, for which a vast supply of both precursor mole-
cules and oxidative substrates is required. Thus, the availability of these resources is
ensured through oligodendrocyte secretion of pro-angiogenic factors [Hypoxia
inducible factors, HIFs (Yuen et al. 2014)], events that underscore the involvement
of oligodendrocytes in the construction of the vascular tree of the CNS. In this
context, it is notable that hypoxia-mediated damage to white matter in periven-
tricular leukomalacia occurs precisely during the early phases of CNS myelination
(Back et al. 2001; Volpe 2001). While the underlying causes of periventricular
leukomalacia are a matter of ongoing controversy (Dammann and Leviton 2004;
Edwards and Tan 2006; Khwaja and Volpe 2008; Volpe et al. 2011; Hu et al.
2013), it has been shown that the blood flow to white matter areas is slower when
compared with gray matter areas in preterm babies (Greisen and Borch 2001), a
condition that probably reflects an incomplete maturation of the vasculature (Volpe
et al. 2011). White matter damage has been associated to excitotoxic-mediated
death (Matute et al. 2007) and reactive oxygen species (ROS), to which OPCs are
particularly sensitive as they are poor in antioxidant defenses and their maturation is
inhibited by these reactive species (French et al. 2009; Volpe et al. 2011) (see
Chapter “Prenatal Systemic Hypoxia-Ischemia and Oligodendroglia Loss in
Cerebellum”). At the same time, areas damaged in periventricular leukomalacia
present increased levels of OLG-associated ROS generating 12/15-lipoxygenase
(Haynes and van Leyen 2013), and lipid peroxidation markers of oxidative damage
(Back et al. 2005).
As the CNS matures, these events become less pronounced, for oligodendrocytes
are better protected from oxidative stress by the presence of higher levels of
Glutathione (Back et al. 1998) and by their switch from an oxidative to a glycolytic
metabolism (Funfschilling et al. 2012); events that probably reflect the long half-life
of myelin proteins (Toyama et al. 2013) and the metabolic support of axons by
myelin sheaths (Brown et al. 2001; Morland et al. 2007; Funfschilling et al. 2012;
Hirrlinger and Nave 2014). Nonetheless, as reviewed recently (van Meeteren et al.
2005), oligodendrocytes are vulnerable to oxidative stress (Smith et al. 1999),
particularly in the context of autoimmune-mediated inflammatory injury, as occurs
in multiple sclerosis (Guan et al. 2014), where oxidative stress markers are observed
in the cerebral spinal fluid of MS patients (Sbardella et al. 2013; Mir et al. 2014).
Likewise, current models of X-linked adrenoleukodystrophy have associated to the
loss of peroxisomal ATP-binding cassette transporter D (ABCD1), which allows
the incorporation of very long-chain fatty acids (VLCFA) into this organelle, to an
increase in ROS production and generalized oxidative stress (Baes and Aubourg
2009; Baarine et al. 2012).
Iron Homeostasis in Oligodendrocytes—Importance

and Perils
Oligodendrocytes and iron have a love affair. Indeed, in areas acknowledged as

iron-rich, namely substantia nigra, striatum, and cerebellar nuclei, the cells that
stain most prominently for iron are oligodendrocytes (Hill et al. 1985; Dwork et al.
1988; Benkovic and Connor 1993; Connor and Menzies 1996; Todorich et al.
2009). Likewise, white matter, rich in oligodendrocytes, stains more strongly for
iron than gray matter (LeVine and Macklin 1990). Oligodendrocytes have a par-
ticular need for iron during myelination because of iron’s participation in the
synthesis of cholesterol, an indispensable component of myelin membranes (Beard
and Connor 2003; Saher et al. 2005; Badaracco et al. 2010), and its participation as
a cofactor in a large number of enzymes involved in myelination and re-myelination

(reviewed in Stephenson et al. 2014).
One of the major symptoms of iron deficiency in young children is a decline in
cognitive capacity characterized by poorer cognition, decreased school achieve-
ment, and other behavior problems (Grantham-McGregor and Ani 2001; Felt et al.
2006). In a number of studies, the clinical symptoms of iron deficiency have been
associated to hypomyelination (Oski et al. 1983; Kretchmer et al. 1996; Lozoff et al.
1996). Despite the evident importance of an adequate iron status for the process of
myelin synthesis, particularly in regards to the enzymes involved (see review by
Todorich et al. 2009), knowledge on the specific elements responsible for iron
homeostasis in oligodendrocytes is incipient.
With regard to iron uptake, while the two typical routes have been reported,
namely transferrin endocytosis and direct membrane uptake by iron transporters, a
third and less common route has been recently been proposed: the endocytosis of
the H-rich isoform of ferritin (HFn) (Fig. 1).
Besides providing iron for cell needs, transferrin (Tf) enhances oligodendrocyte
progenitor differentiation, as the presence of Tf in the culture medium increases
proliferation of oligodendrocyte precursors, an effect mediated at least in part by Tf
receptor 1 (TfR1) (Guardia Clausi et al. 2010). Similarly, mice overexpressing Tf in
the brain undergo faster oligodendrocyte lineage maturation than animals
expressing regular levels of Tf (Sow et al. 2006). In in vitro experiments, Tf
enhanced the proliferation rate of OPCs as apoTf added either to the culture
medium or by overexpression of Tf in an immature oligodendrocyte cell line
(Silvestroff et al. 2013). Likewise, Tf-induced neural stem and progenitor cells
toward the oligodendroglial lineage and promoted oligodendrocyte maturation from
OPCs (Silvestroff et al. 2012). Taken together, the evidence supports the notion
that, besides its important role as an iron donor, Tf is a trophic factor that accel-
erates oligodendrocyte commitment and differentiation.
Both TfR1 and H-ferritin have been shown to be up-regulated during in vitro
oligodendrocyte maturation, indicating that both proteins might contribute to the
supply of iron in the process of maturation and myelination of OPCs (Li et al.
2013). Nevertheless, it appears that upon maturation, the contribution of Tf endo-
cytosis as a mechanism of iron uptake losses relevance. Cultured OPCs express low
levels of transferrin receptors (Espinosa de los Monteros and Foucaud 1987), and
upon differentiation into mature oligodendrocytes, transferrin receptor protein
expression becomes undetectable by Western blot (Todorich et al. 2009). Likewise,
histochemical assessment of TfR1 distribution in the brain shows just a slight
staining of TfR1 in mature oligodendrocytes (Giometto et al. 1990; Connor and
Menzies 1995). Given the iron requirements by mature oligodendrocytes, it is likely
that these cells possess an iron acquisition mechanism complementary to
TfR1-mediated uptake, for, as noted, mature oligodendrocytes are the brain cells
that most strikingly stain for iron.
A second form of iron incorporation into cells occurs through the direct transport
across the plasma membrane by the inwards iron transporter protein DMT1. Still, in
normal and Belgrade rats (which express a mutated form of DMT1 with a low
Oligodendrocyte
precursors
Microglia
apoTf
Fe+3
DMT1
holoTf FPN1
Ferritin Hep or CP
Fe+2
DMT1 Lipid metabolism
Ferritin
Myelinating
Oligodendrocyte
Fe+2
Transferrin
Ferritin
Fig. 1 Overview of oligodendrocyte iron metabolism. Differentiating oligodendrocyte precursors

accumulate iron through three different pathways: Transferrin (Tf) or ferritin endocytosis and
direct membrane uptake by iron transporters (DMT1). While both endocytic-dependent pathways
converge into lysosomes, where vesicular iron transporter (also DMT1) allows iron to exit to the
cytoplasm, each holotransferrin (holoTf) molecule transports two iron atoms, while one ferritin
molecule holds up to 4000. Intracellular iron (Fe+2) is mobilized to mitochondria, where it
participates as an enzyme cofactor in oxidative phosphorylation, or becomes bound to enzymes
participating in lipid metabolism. Intracellular iron excesses are stored as intracellular ferritin or
secreted by ferroportin (FPN1), a process which requires further oxidation into Fe+3 by the
membrane-associated ferroxidases Hephaestin (Hep) or Ceruloplasmin (CP). This secreted Fe+3 is
bound by apotransferrin (apoTf) resulting in endocytable holoTf
transport activity), there is a slight staining of DMT1 in oligodendrocytes at the

subcortical white matter, and an overall sparse presence of DMT1 positive oligo-
dendrocytes (Burdo et al. 2001). If this low DMT1 immunostaining pattern is a
characteristic of mature oligodendrocytes, then direct iron uptake mediated by
DMT1 can be predicted to be of a relative low magnitude, consistent with the need
for alternative mechanisms of iron uptake. Unlike DMT1, the immunostaining for
the iron export transporter Ferroportin 1 (FPN1) is relatively strong in subcortical
oligodendrocytes (Burdo et al. 2001). The strong presence of FPN1 suggests an
active iron efflux from oligodendrocytes, which will allow for decreased iron
accumulation. For effective iron efflux, FPN1 must partner with a ferroxidase,
needed to oxidize outcoming Fe2+ to Fe3+ prior to the binding of Fe3+ to transferrin.
Oligodendrocytes express both a membrane-bound form of the ferroxidase ceru-
loplasmin and hephaestin, a ferroxidase initially described in intestinal epithelia
(Schulz et al. 2014), thus iron export, as a form to alleviate iron overload, is most
probably at work in oligodendrocytes.
The third, and least conventional, form of iron incorporation into oligodendro-
cytes is through the endocytosis of the iron storage molecule ferritin in its H-rich
isoform (HFn), which in its hollow center can contain up to 4500 atoms of iron
(Arosio and Levi 2010). The endocytosis of HFn could turn out to be a highly
efficient manner of iron uptake, for the uptake of one ferritin molecule could yield
thousands of cytoplasmic iron atoms.
The evidence for the involvement of HFn endocytosis as a mechanism of iron
uptake by oligodendrocytes is compelling. OPCs bind and internalize 125I-labeled
HFn through a process consistent with clathrin-mediated endocytosis (Hulet et al.
2000). The iron originally present in exogenous HFn is incorporated into the
intracellular iron pool, since incubation of oligodendrocyte progenitor cells with
HFn for 16 h results in a decreased activity of the iron-deficit sensors: Iron regu-
latory proteins 1 and 2 in an amount similar to that observed after incubation for
16 h with ferric ammonium citrate (Hulet et al. 2000). Importantly, HFn endocy-
tosis does not seem to be regulated by cell iron status, since pretreatment of the
oligodendrocyte precursors for 16 h with either unlabeled HFn (Fe loading) or the
iron chelator Desferal (iron depletion) did not affect the amount of 125I-HFn uptake
(Hulet et al. 2000).
The search for the putative receptor for HFn in oligodendrocytes resulted in the
identification of T cell immunoglobulin and mucin domain containing protein-2
(Tim-2) as a likely candidate. In contrast to an earlier study (Hulet et al. 2000), the
study of Todorich et al. (2008) reported that in CG4 cells Tim-2 expression was
responsive to iron, decreasing with iron loading and increasing with iron chelation.
Which is the source of the HFn that supports oligodendrocyte’s needs? The
answer seems to be the microglial cells, which in the CNS serve as iron stores (Erb
et al. 1996; LeVine 1997; Mehlhase et al. 2006; Oshiro et al. 2008). HFn is secreted
by iron-loaded microglia, while conditioned media from microglia increases the
survival of oligodendrocytes. Transfecting microglia with siRNA for HFn blocks
the trophic response of conditioned media on oligodendrocytes (Zhang et al. 2006).
Interestingly, the activation of iron-loaded microglia with lipopolysaccharide results
in the increased release of the proinflammatory cytokines tumor necrosis factor-a

and interleukin-1 (see Chapters “Microglia Function in the Normal Brain” and
“Purine Signaling and Microglial Wrapping”), and the decreased release of HFn
(Zhang et al. 2006). These results establish a putative link between inflammation
and a deficient iron supply by microglia to oligodendrocytes.
The triad of low DMT1—high FPN1—high ferritin predicts a tight management
of intracellular redox-active iron. Nevertheless, the high content of iron in oligo-
dendrocytes raises the question of how tightly is iron homeostasis regulated in these
cells. Do oligodendrocytes have a tight iron homeostasis system that maintains just
enough intracellular iron levels as to satisfy their metabolic needs? Alternatively, do
oligodendrocytes behave like iron-starving cells with a poor homeostatic mecha-
nism to control for iron excess? These questions wait for further experimentation.
Iron Homeostasis in Oligodendrocytes—Perils
Iron is a redox-active metal that in the intracellular reductive environment and the
presence of oxygen generates the highly damaging hydroxyl free radical (Nunez
et al. 2012). The high iron content of oligodendrocytes may be a time bomb, as
shown in neurodegenerative diseases like MS. In patients with MS, iron released by
oligodendrocytes accumulates in macrophages and microglia around the rim of
lesions (Hametner et al. 2013). In turn, iron-loaded macrophages and activated
microglia can release redox-active iron into the extracellular space, damaging axon
integrity at the border of the lesions (Stephenson et al. 2014).
In MS, iron homeostasis mechanisms in oligodendrocytes seem to be operative.
A substantial decrease of iron in the normal-appearing white matter, which corre-
sponded with disease duration, was found in chronic MS patients. This decrease of
iron in oligodendrocytes and myelin was probably mediated by the upregulation of
iron-exporting ferroxidases (Hametner et al. 2013). Nonetheless, iron released from
dying oligodendrocytes accumulated in astrocytes and axons, leading to waves of
iron liberation, which may propagate neurodegeneration together with inflamma-
tory bursts (Hametner et al. 2013). Thus, the data indicates that MS oligodendro-
cytes probably have an active iron export system, and when killed, their iron
content is released at the site of the lesion.
Concluding Remarks
Oligodendrocytes and neurons operate close to the brink, for not only are these cells
extremely large and ramified, requiring de novo synthesis of huge amounts of lipids
and proteins, but they also extend and maintain extremely long cell processes
(axons and dendrites) or enormous plasma membrane sheets. In this sense, con-
sidering the rate at which myelin proteins and membranes are produced (Barbarese
and Pfeiffer 1981; Pfeiffer et al. 1993; Kramer et al. 2001), it is not surprising that
oligodendrocytes are particularly susceptible to chaperone inhibition (Alcazar and
Cid 2009) or endoplasmic reticulum stress derived from missense mutations of the
predominant myelin proteins (Numasawa-Kuroiwa et al. 2014).
As if this were not enough, oligodendrocytes participate in the maintenance and
support of the axons to which they attach. While these conditions must burden the
metabolism of these cells, they have low levels of antioxidant defenses, which
instead in the CNS are mostly associated with astroglial cells. This paints a picture
of intrinsic instability, which is probably offset by switching from oxidative to
glycolytic metabolism. Still, the fact remains that these cells present the highest iron
content in the CNS. Whether the high iron content is a response to the requirements
of cholesterol synthesis and other metabolic processes or to intrinsic peculiarities of
the iron homeostasis in these cells is unknown. Nevertheless, the high iron content
entails a great peril in cases of oligodendrocytes death, since upon its release to the
surroundings it will be taken up by macrophages and microglia, which, in turn, will
transform this iron accumulation into oxidative and inflammatory bursts, which can
easily spiral out of control and, unlike other types of tissue damage, the conse-
quences for neural tissues are for the most part irreversible.
Acknowledgments This work was made possible in part by grant PIA-CONICYT ACT1114 and
a support grant 2014 from the Faculty of Science of the University of Chile.
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Schwann Cell and Axon: An Interlaced
Unit—From Action Potential
to Phenotype Expression
Felipe A. Court and Jaime Alvarez
Abstract Here we propose a model of a peripheral axon with a great deal of

autonomy from its cell body—the autonomous axon—but with a substantial
dependence on its ensheathing Schwann cell (SC), the axon-SC unit. We review
evidence in several fields and show that (i) axons can extend sprouts and grow
without the concurrence of the cell body, but regulated by SCs; (ii) axons syn-
thesize their proteins assisted by SCs that supply them with ribosomes and, prob-
ably, with mRNAs by way of exosomes; (iii) the molecular organization of the
axoplasm, i.e., its phenotype, is regulated by the SC, as illustrated by the axonal
microtubular content, which is down-regulated by the SC; and (iv) the axon has a
program for self-destruction that is boosted by the SC. The main novelty of this
model axon-SC unit is that it breaks with the notion that all proteins of the nerve
cell are specified by its own nucleus. The notion of a collaborative specification of
the axoplasm by more than one nucleus, which we present here, opens a new
dimension in the understanding of the nervous system in health and disease and is
also a frame of reference to understand other tissues or cell associations.
Keywords Axoplasm Exosomes mRNA Microtubular density Wallerian

degeneration
F.A. Court (&)

Center for Integrative Biology, Universidad Mayor, Santiago, Chile
F.A. Court
FONDAP Center for Geroscience, Brain Health and Metabolism, Santiago, Chile
F.A. Court J. Alvarez
Millenium Nucleus for Regenerative Biology, Pontificia Universidad
Católica de Chile, Santiago 8331150, Chile

DOI 10.1007/978-3-319-40764-7_9
184 F.A. Court and J. Alvarez
Early Model
A decade ago, we proposed a model for the reciprocal interaction between Schwann
cells (SC) and axon at the cell biology level, which configured an inextricable
functional unit (Alvarez 2001; Court and Alvarez 2005). In this article, we will
present the contribution of our group to the understanding of this unit. The main
features of this model are, on the side of the axon, (i) the axon contains a sprouting
or growth program fully present in it; (ii) it contains a destruction program; (iii) the
axoplasm synthesizes its intrinsic proteins; and (iv) the axon regulates the SC, in
particular, the axon induces its differentiation. On the side of the SC, its role for the
axon changes radically from the differentiated to the dedifferentiated condition.
While the differentiated SC (v) regulates the phenotype of the axon, and (vi) re-
presses the growth program, the proliferating or dedifferentiated SC (vii) promotes
growth (Fig. 1). The supporting evidence will be summarized below.
Sprouting Program of Axons and Its Repression

by Schwann Cells
It is well known that adult axons have a rather conserved anatomy along the space
axis (trajectory) and along the time axis. Perturbations of axons, e.g., severance of
fibers, result in a destruction of the distal domain and, at the proximal domain, the
Fig. 1 Model of the axon–Schwann cell unit. Parallel bars represent the membrane of the axon;
inside, the bomb is the destruction program, the trident/fork is the sprouting program, and the
beaded string is machinery for protein synthesis. The upper structure is the Schwann cell. Thick
arrows pointing up and down indicate intercellular regulation between axon and Schwann cell.
The thin transcellular arrow spanning from Schwann cell to axon indicates transfer of
macromolecular component bypassing the extracellular space. Present status of our model adds
the transcellular transfer to the previous one (Alvarez et al. 2000; Court and Alvarez 2005)
Schwann Cell and Axon: An Interlaced Unit … 185
severed end of axons develops a regenerative response. We will examine first the
regenerative response. Cajal (1928) with the optic microscope and later Zelena et al.
(1968), with the electron microscope, showed that severed axons a few hours after
injury developed membranous extensions at their cut ends, which suggested that
axons had the ability to start a remodeling/regenerative response without direct
involvement of the cell body. Later, with the pervading notion that all proteins of
the axon originated in the neuronal cell body, it was assumed the cell body
developed a regenerative response triggered by the arrest of ongoing retrograde
signals, or by new signals coming from the lesion. The observed delay between the
lesion—usually a crush—and the beginning of the axonal elongation—1 or 2 days
post crush—was considered the time required for the processes linking the lesion to
the regrowth response developed in the cell body (Bisby and Keen 1985;
McQuarrie and Jacob 1991). We contested this view when we found that a nerve
injected with inhibitors of proteases, such as aprotinin, leupeptin, APP with the
Kunitz insert, and others, resulted in a local sprouting by otherwise uninterrupted
axons. At the same time, SCs proliferated (Fig. 2) (Alvarez et al. 1992, 1995;
Moreno et al. 1996). We reasoned that the axon started to sprout at the treated zone
Fig. 2 Sprouting response of intact axons and dedifferentiation of Schwann cells. Sural nerves of
rats treated with intraneural injections of aprotinin, a serine antiprotease. Left panel, the Schwann
cell presents a normal axon encased in a normal myelin, but is proliferating as shown by the
chromosomes floating in the cytoplasm (dark masses). Right panel, the Schwann cell is
surrounded by its basal lamina, the myelin looks normal and encases an axon while a second
axonal profile loaded with mitochondria (dark bodies) is located between the myelin sheath and
the basal lamina. This axonal profile is a sprout of the encased axon. Proliferation of Schwann cells
and sprouting of axons precede the breakdown of myelin
of its own accord, i.e., the cell body could not cause the initial extension of these
sprouts. From these observations, we surmised that (i) the axon contained a
sprouting program and (ii) repressed by the differentiated but not by the prolifer-
ating SC.
In the light of the hypothesis that the differentiated SC suppresses the axonal
sprouting program, we re-examined the delay of regeneration. Instead of retrograde
signals from the lesion to the cell body, we assumed that the delay of regeneration
was due to the time taken by the SC of the distal stump to proliferate, a response
secondary to the degeneration of the severed axon. In other words, we conjectured
that the delay was caused by the time taken for a change in the territory to be
invaded by regrowing axons instead of signals going from lesion to cell body. In
this scenario, we predicted that the delay of regeneration should be reduced by
treating the distal stump appropriately. When we treated the sciatic nerve locally
with aprotinin to induce SC proliferation and crushed the nerve in this region, the
delay was obliterated. Moreover, when the distal stump was frozen to kill all cells,
the delay was obliterated as well (Fig. 3) (Court and Alvarez 2000; Tapia et al.
1995). These observations supported the hypothesis that local mechanisms, prob-
ably involving the SC of the distal stump, were repressing the axonal growth. As a
consequence, the role of the cell body in the initial stages of axonal regrowth
needed re-evaluation.
To further explore the repression of the growth program of axons by SCs, we
took advantage of the inability of axons of Wlds mice to regenerate. In this strain,
severed fibers do not degenerate for several weeks, and surviving axons do not
Fig. 3 Delay of regeneration is locally controlled. a Rat sural nerves. Ordinates, elongation of
axons in mm, post crush; pinch test. Abscissas, time post crush, in days. Diagram: One side was
frozen over a span of 4–5 mm (thick black line) (open circle) and the other was manipulated
mechanically (filled circle), before the crush (vertical line). The treatment obliterates the delay but
does not change the rate of growth (modified from Tapia et al. 1995). b CD1 albino mice, and
Wlds mice. Regeneration after 3 days of pretreatment with A, actinomycin D; V, vehicle. N, naive,
for comparison. Diagram shows the infiltrated region (thick line) and the crush (vertical line).
Vertical arrows pointing to abscissas indicate the intercepts of the regression lines. Notice that
CD1 nerves reduce the delay of regeneration after pretreatment with A or V, and that Wlds nerves,
which do not spontaneously regenerate, do so after actinomycin D pretreatment, with a short delay
(modified from Court and Alvarez 2000)
regenerate into the distal stump either. We surmised that SCs of the distal stump—
the territory to be invaded—were impairing the ingrowth of regenerating axons. In
Wlds, SCs were destroyed in a restricted zone of the sciatic nerve with actinomycin
D, and the crush performed in that region. This treatment normalized the elongation
of regenerating axons, which otherwise do not regrow (Fig. 3) (Benavides and
Alvarez 1998; Court and Alvarez 2000). Finally, to rule out the involvement of the
cell body in the sprouting response of axons, we used the distal domain of severed
nerves of Wlds mice. The sciatic nerve was cut and the central stump eliminated.
Several mm of the distal stump were crushed to kill all cells. Therefore, the blind
end of surviving Wlds axons disconnected from their cell bodies was next to an
acellular nerve domain. This domain was invaded by sprouts regrowing from axons
isolated from their cell bodies (Fig. 4) (Iñiguez and Alvarez 1999). From these
observations we conclude that axons start sprouting as soon as the ongoing
repression is removed, a process that neither requires interruption of axons nor
involvement of the cell body, and that the differentiated SC represses the axonal
growth. Since the freezing of the SC unleashed the sprouting program of axons with
no delay, we surmise that the repression is the result of an active process requiring a
functional SC; in particular, the repression is not due to components of the extra-
cellular matrix or myelin products. More generally, this view emphasizes the
Fig. 4 Cell bodies are unnecessary for axons to grow. a Diagram illustrates the Wlds preparation
in vivo. A segment of sciatic nerve was removed, the distal segment received an extensive crush,
4–5 mm (dotted line), and the preparation was examined a few days later. Letters in the diagram
correspond to those of micrographs. b The surviving distal nerve looks normal, which is a
characteristic of this strain. c The crushed domain presents axonal profiles (arrowheads) amidst
cell debris and cytoplasmic remnants (arrows). d the surviving nerve was cut 3 days post lesion
(arrow in diagram of panel a) and exposed to horseradish peroxidase. The reaction product is seen
in the axonal profile within membranous structures (arrows). e The fixed nerve was cut (arrow in
diagram of panel a) and exposed to DiI. Dissociated fibers; the blind ends of surviving axons
exhibit sprouts emerging from them (thin threads). Inset, whole nerve at the junction between
surviving fibers and crushed region (from Iñiguez and Alvarez 1999)
presence of ongoing local regulatory mechanisms, what we have called the

autonomous axon, diminishing the importance of the cell body as the “mastermind”
of the neuron. In other words, the axon has a repertoire of behaviors regulated on a
local basis that are not under the direct command of the cell body.
In invertebrates, an even more pushed set of observations has been reported for
axons separated from their cell bodies. They survive unusually long periods, initiate
regrowth, and may fuse with the blind central segments; axons deprived of their cell
body can regenerate to make synaptic contact with a postsynaptic apparatus, intact
axons divested of their ensheathing glial cells extend sprouts, to mention a few
(Bittner and Mann 1976; Hoy et al. 1967; Krasne and Lee 1977; Mason and Muller
1982; Masuda-Nakagawa et al. 1993). These observations suggest that these
properties of nerve fibers appeared way back in evolution, and that vertebrates have
conserved a reduced set compared with invertebrates.
Let us focus now on the SC. In mature nerve fibers, the SC is in a post-mitotic
condition. When the axon dies, its widow SCs proliferate, implying that the axon
was preventing this proliferation. However, the SC enters the cell cycle when
extracellular proteases are inhibited, despite the fact that the axon remains alive and
functional. Therefore, the cascade that represses the cell cycle of SCs involves at
one stage the activity of an extracellular protease. The group of Shubayev
(Chattopadhyay and Shubayev 2009; Liu et al. 2010) has identified the matrix
metalloproteinase 9 (MMP-9) as a candidate to carry on this control of the SC.
Moreover, proliferation of SCs has been shown to be associated with the neuregulin
cascade (Hayworth et al. 2006), as also with an increase of the mRNA coding for
the zinc finger protein Zipro1 (Ellerton et al. 2008), which they suggest to be
involved in the activation of SCs.
Regulation of the Phenotype of the Axon by the Schwann

Cell: Microtubular Content
As mentioned in Chapter “Glial cells and Integrity of the Nervous System,” it is

well known that the glial cell regulates some features of the axonal phenotype. For
example, the caliber of the axon increases upon myelination in cultured peripheral
neurons (Windebank et al. 1985). In the central nervous system, optic nerve axons
acquire myelin past the lamina cribrosa, and at the same time increase in caliber
(Hernandez et al. 1989). In peripheral myelinated fibers, Yokota (1984) reported the
occurrence of intercalated unmyelinated segments where the axon was thinner.
These observations point to a local and ongoing control of the anatomy of the axon
by the glial cell. In this paragraph we will focus on axonal microtubules. In ver-
tebrates, the microtubular content of axons is an extremely constant feature. The
density (microtuble/μm2 of cross-sectional area of axon) exhibits an inverse cor-
relation with the caliber, but for a given caliber, the density is the same, whether the
axon is motor, sensory, or autonomic, myelinated or unmyelinated, central or
peripheral, in adulthood, during regeneration or development, whether the animal

species is minute as a lizard or large as a cow, in wasted or well-nourished animals,
warm- or cold-blooded, whether the terminal field of the axon is extensive or very
restricted, and even if the caliber varies along its course, the microtubular density of
the axon accords with the local caliber (Alvarez et al. 1982; Faundez et al. 1990;
Friede and Samorajski 1970; Pannese et al. 1984b; Vergara et al. 1991; Fadic and
Alvarez 1986; Alvarez and Zarour 1983; Espejo and Alvarez 1986; Saitua and
Alvarez 1989; Pannese et al. 1988; Smith 1973). This tight correlation, though,
changes abruptly in the ventral and dorsal roots, where the microtubular density is
one half that observed in a peripheral or central axon of the same caliber (Fig. 5a)
(Lopez and Alvarez 1990; Pannese et al. 1984a; Fadic et al. 1985; Saitua and
Alvarez 1989).
What makes this structural feature change in this manner? Motor and sensory
axons are continuous from spinal cord, through roots, to the peripheral nerve. Their
cell bodies alone cannot specify an axonal microtubular density for the spinal cord
domain, reduce it by half in the root, and resume the original microtubular density
in the peripheral nerve. A local cue seems necessary. The nodosal ganglion of the
vagal nerve lies in the neck, so the central branches of these neurons are in the
periphery before becoming root fibers inside the skull. The central branches of the
nodosal sensory neurons share the phenotype of sensory radicular fibers and have a
low microtubular density compared to peripheral branches of the same caliber.
When nodosal central branches regenerate along their anatomical course, the
microtubular density remains unchanged, but when they regenerate along the
hypoglossal peripheral nerve, the original low microtubular density increases to
match the higher density typical of peripheral axons (Fig. 5b) (Serra and Alvarez
1989). In brief, the molecular architecture of the axon does not accord with its being
a central branch but with the local environment.
The best candidate to provide this local regulation was the SC. To explore this
conjecture, the SC was crippled with actinomycin D, which blocks transcription and
as a consequence the cell runs down. The drug was applied to a short 4-mm span of
nerve for a few days with a sleeve. Under the electron microscope, SCs debris was
seen throughout while axons were conserved albeit with abundant microtubules. In
unmyelinated fibers, the inverse correlation between microtubular density and axon
caliber changed to a constant density across the range of cross-sectional areas. The
density corresponded to the highest value observed in the normal nerve, that of the
smallest axons. In brief, in the normal condition, the larger the encased axon is, the
greater the Schwann cell down regulates its microtubular density (Fig. 5c). A few
mm away from either side of the sleeve, axons and SCs appeared as usual (Bustos
et al. 1991). We highlight that the molecular make-up of the axon varies from one
point to the next, depending on local cues. In this scenario, the microtubular density
of axons appears to arise from an internal axonal program whose default set point is
very high but is down-regulated by an external agent—we surmise it is the glial cell
—and this repression is more effective the larger is the axon. In this view, the SC of
Fig. 5 Control of the axonal microtubular content by the Schwann cell. a Rat. Diagram shows the
preparation and corresponding symbols. Microtubular density (µt/µm2) of peripheral and radicular
unmyelinated axons as a function of the cross-sectional area of axons. Notice that for each caliber,
the density is about one half in the root compared to that of the peripheral branch. b Cat.
Diagram shows the nodose ganglion (NG), supranodosal (sn) and vagal nerves. When sn
regenerated along its anatomical course, microtubular densities were low, i.e., similar to intact sn
or radicular fibers. When sn regenerated along the hypoglossal nerve, microtubular densities
increased to the level of peripheral fibers. c Sural nerve of rat. A 4-mm cuff loaded with
actinomycin D was placed around the nerve. A week later, at the cuff, the nerve exhibited no
healthy Schwann or resident cells, while axons were filled with microtubules (Act D), whereas
4-mm distal to the treated segment, the nerve appeared normal (4 mm distal). The graph shows
that the crippling of resident cells abrogated the inverse correlation between caliber of axon and
microtubular density, which was replaced by constant values for all calibers, corresponding to the
highest density of control axons
the root must repress this axonal microtubular program even more than its
peripheral sister does.
What is the biological significance of the constant microtubular density of axons
and its drastic reduction at the roots? We do not have an answer. The microtubular
density of axons follows an architectural feature of axons, their caliber. On the other
hand, other features of the axons, e.g., length, transport demand, axoplamic mass,
and size of the terminal, field do not correlate with the microtubular density. The
mechanism by which the SC regulates this axonal feature remains conjectural (see
below).
Destruction Program of the Axon and Its Regulation

by the Schwann Cell
A number of agents trigger axonal destruction, including those associated with

genes, toxins, and mechanical damage to neurons. By far, a crush lesion to a nerve
trunk is the most used perturbation to trigger nerve fiber destruction, also referred to
as Wallerian degeneration. In Wallerian degeneration, axonal mechanisms lead to
the activation of axoplasmic proteases. Generation of reactive oxygen species
(ROS), the activation of the mitochondrial permeability transition pore (mPTP), and
calcium rise in the axoplasm are instrumental for the activation of caspases resulting
in the destruction of the axoplasm (Court and Coleman 2012). Cultured neurons
free of other cell types can lose neurites in a manner resembling thus Wallerian
degeneration (Barrientos et al. 2011; Villegas et al. 2014). This similarity has led to
the notion that axonal destruction following disconnection with the cell body is
essentially an internal program of axons. In Wallerian degeneration, the SC par-
ticipates in the clearing of axonal remnants—a late stage of degeneration—and in
its own remodeling.
In Wallerian degeneration, the myelin sheath breaks into ovoids at the
Schmidt-Lanterman incisures, 2–3 days post lesion. We are currently studying
the role of SCs in the early stages of Wallerian degeneration, and are focused on the
process of ovoid formation by SCs as a possible promoter of axonal degeneration.
Preliminary results suggest that the pharmacological impairment of ovoid formation
delays the destruction of the axoplasm (Catenaccio and Court, unpublished), sug-
gesting that early stages of axonal degeneration are executed by the SC in a non-cell
autonomous mechanism, followed by a later axonal destruction stage involving
mitochondrial dysfunction and calpain activation.
Synthesis of Protein in Axons and Its Dependence

from Schwann Cells
When we proposed our model of the axon-SC unit over a decade ago (Alvarez
2001; Court and Alvarez 2005), the model axon synthesized its own proteins. This
view broke with the current notion at the time that the cell body supplied the bulk of
axoplasmic proteins by a slow transport mechanism. Although evidence for axo-
plasmic protein synthesis has a long history (Edström 1966; Koenig 1984; Alvarez
et al. 2000), only recently has become an established notion (Lin and Holt 2008),
making the slow transport as supplier of most axoplasmic proteins a seriously
flawed model (Court and Alvarez 2011). Mature axons have a small complement of
ribosomes (Kun et al. 2007; Court et al. 2008), so small that they went unnoticed by
electron microscopists (Palay and Palade 1955) despite the fact that micrographs
did record polyribosomes (see Fig. 5-2 in Peters et al. 1991; Court and Alvarez
2011). This low ribosomal content accords with the low rate of amino acid
incorporation into axoplasmic proteins, 1–4 % that of the cell body per unit of
volume, as reported for the Mauthner neuron of the goldfish. However, owing to the
large volume of the axoplasm, its contribution to the proteins of the neuron as a
whole could be greater than that of the cell body (Alvarez and Benech 1983).
What came as a surprise is that a population of ribosomes in axons originated in
the SC. In severed sciatic nerve of Wlds mice, the distal surviving axons exhibit an
enormous amount of ribosomes as assessed with the electron microscope. These
ribosomes could not come from the cell body because of the anatomical disconti-
nuity of axons. In Wlds mice, SC infected with a lentivirus coding for a tagged
ribosomal protein expressed the tagged protein in their cytoplasm, which
co-localized with other ribosomal markers. This co-localization was also observed
in the axoplasm after a crush (Fig. 6) (Court et al. 2008) and during axonal
regeneration (Court et al. 2011). These observations support the idea that SCs
supply ribosomes to axons. In the axoplasm, ribosomes were seen singly and as
polyribosomes, the morphological correlate of several ribosomes translating an
mRNA. Ribosomes were also seen densely packed inside multimembrane vesicles
and in broken vesicles. These morphological structures suggest that ribosomes are
transferred by way of vesicles (see below).
Our finding that ribosomes are transferred from one cell to its neighbor breaks
with the notion that all components of a cell originate in the same cell, which is
seldom stated explicitly but always present in the back of the mind. It is unlikely
that this transfer mechanism appeared during evolution only in the axon-SC unit.
Therefore, it is important to bear in mind such a transcellular tranfer when standard
mechanisms appear to be insufficient to account for a set of results.
Further Developments of Axonal Protein Synthesis and Its

Support by Schwann Cells
Transfer of proteins between cells is an old notion. In the nervous system protein
transfer from glia-to-axon has been previously proposed in the squid giant axon.
Recently, intercellular transfer of proteins and RNA by means of extracellular
vesicles has been described as a novel mechanism for cell-to-cell communication. In
the 80s, experimental evidence indicated that during reticulocyte maturation, vesi-
cles were released to the extracellular space (Harding et al. 1983). These vesicles,
named exosomes, were produced in the endosomal compartment, moved into
Fig. 6 Transfer of ribosomes from Schwann cell to axon. Wlds sciatic nerve axons severed for a
week. In this strain, severed axons survive for several weeks. Upper panel, electron micrograph;
myelin and axoplasm are conserved; the dark particles in it are ribosomes (inset). Lower panel,
immunostained teased fiber; red, ribosomal marker; green, P0, a myelin glycoprotein; blue,
neurofilament. Notice the red puncta in an ocean of neurofilaments. Since axons were interrupted,
this large amount of ribosomes presumably appeared in the axoplasm after its disconnection from
the cell body; hence, these ribosomes did not originate in the neuronal cell body (modified from
Court and Alvarez 2011)
multivesicular endosomes and secreted after fusion of these multivesicular bodies

with the plasma membrane (Théry et al. 2002). A second class of vesicles, produced
by evagination of the plasma membrane, were named microvesicles (Cocucci et al.
2009; Théry et al. 2009). Nowadays, exosome secretion has been described in vitro
and in vivo. Physiological body fluids (plasma, urine, CSF, milk, and saliva) contain
exosomes secreted by different cell types, and their molecular cargoes are rather
specific. This field is currently under intense investigation to detect early biomarkers
for pathological conditions (Russo et al. 2012; Skog et al. 2008).
Transfer of ribosomes from SC-to-axon implies the transfer of mRNAs, as the

subunits require an mRNA to assemble into a ribosome. We have gone a step
farther and studied the role of exosomes as a transfer vehicle in the SC–axon
system. Vesicular-mediated molecular cargoes and exosome secretion have been
described in glial cells from the CNS and PNS (Lopez-Verrilli and Court 2012,
2013). As described above, we have shown that ribosomes are transferred by way
of vesicles from SCs to axons in vivo after axonal damage as well as during axonal
regeneration (Court et al. 2008, 2011). Since mRNAs can be stored in a dormant
state in the distal axon until needed (Yoo et al. 2010), SCs could supply mRNA
transcript to axons for storage and translation in response to acute stimuli
(e.g., nerve damage) or the stimulus itself could trigger the transfer. In our quest to
identify the vesicle type involved in the transfer, we found that exosomes con-
taining proteins and mRNAs were secreted by SCs and taken up by axons both
in vitro and in vivo (Lopez-Verrilli et al. 2013). It is known that exosomes deliver
mRNAs that can be translated in the recipient cells and also can deliver micro
RNAs (miRNA) (Baj-Krzyworzeka et al. 2006; Valadi et al. 2007; Skog et al.
2008). In fact, elongation factors needed for mRNA translation have been found in
exosomes from oligodendrocytes and microglial cells (Valenzuela et al. 2012;
Kramer-Albers et al. 2007; Rigaud et al. 2008), as well as from Schwann cells
(Picou et al., unpublished).
Summing up, through exosomes SCs might provide an efficient, specific, and
highly localized support to axons for their maintenance and responses to challenges,
including regenerative responses. Secreted vesicles interact specifically with the
target cell (Cocucci et al. 2009; Lopez-Verrilli et al. 2013), supplying a variety of
macromolecules; this allows SCs to regulate axonal functions without immediate
involvement of the neuronal cell body. Our preliminary studies reveal SC-derived
exosomes contain mRNA for neuronal-specific proteins. Distal to a nerve lesion,
mRNA for neurofilament, a transcript for a neuron-specific intermediate filament, is
detected in SCs including SCs of intact sciatic nerves, although the protein itself is
absent from SCs (Roberson et al. 1992; Fabrizi et al. 1997). These findings were
initially not considered relevant, since axons were believed unable to synthesize
proteins, but that long-held dogma has been shown to be untenable (Alvarez et al.
2000; Twiss and Fainzilber 2009; Jung et al. 2012). Therefore, Schwann cells
appear to supply ribosomes to axons and a set of mRNAs to be translated in the
axoplasm.
SC Response After Nerve Injury and Vesicular Transfer

During Axonal Regeneration
Axonal regeneration is a subject of intense investigation, as it is one of the

underlying processes of functional recovery after nervous system damage (Rigaud
et al. 2008; Wang and Sun 2010). In the mammalian nervous system, the
regenerative capabilities of neurons show regional variability. In the PNS, axons

efficiently regenerate after nerve damage, a process that is supported by SCs (Chen
et al. 2007). In contrast, in the CNS the regenerative capability is poor due to both
neuronal intrinsic limitations and glial responses that restrict regeneration (Filbin
2003; Blesch and Tuszynski 2009).
In the PNS, axonal degeneration due to trauma, toxic agents, or genetic muta-
tions triggers the dedifferentiation of Schwann cells, which support axonal regen-
eration in contrast to differentiated SCs that repress axonal growth (vide supra). In
fact, dedifferentiated SCs proliferate, upregulate regeneration-associated genes, and
secrete trophic factors that promote axonal growth (Jessen and Mirsky 2008). In
addition, SCs play an active role in removing axonal and myelin debris and secrete
cytokines and chemokines that recruit immune cells to the injured region, which
further eliminate myelin debris (Glenn and Talbot 2013). This leads to an efficient
removal of myelin-associated proteins that are inhibitory for regeneration. At a later
stage, dedifferentiated SCs align under the basal lamina forming endoneurial tubes,
which are efficient substrates for the regrowing axon. Together, these tissue changes
generate a highly supportive environment for axonal regeneration in the PNS (Chen
et al. 2007).
In contrast, the response of glial cells to injury in the CNS (oligodendrocytes and
astrocytes) generates a mechanical and molecular barrier which greatly limits
regeneration (Yiu and He 2006). SCs have been used as a tool for coaxing CNS
axons to regenerate. In fact, optic nerve axons regrow through a sciatic nerve
implant as a conduit to synapse onto their CNS targets (Aguayo et al. 1990).
Subsequent work has largely focused on the extracellular matrix and growth factors
secreted by Schwann cells. Recently, we showed that SC exosomes, which contain
mRNA and miRNA, are selectively internalized by axons, increase neurite growth
substantially (Fig. 7), and greatly enhance axonal elongation in vitro and in vivo
(Lopez-Verrilli et al. 2013). The main novelty of the mechanism we found is that a
complex array of macromolecules—the cargo of exosomes—simultaneously affects
a broad spectrum of processes of the targeted axon.
The use of exosomes in patients is emerging, which adds a clinical relevance to
this field. For example, SC-derived exosomes can be used by exploiting their
endogenous pro-regenerative activity or by loading them with specific transcript or
proteins using modified glial cells (Zhang et al. 2010; Schmitte et al. 2010);
neuronal-targeted exosomes obtained in vitro have been electroporated with specific
siRNAs, and after intravenous injection, they specifically knock-down their target
gene in brain neurons (Alvarez-Erviti et al. 2011). Thus vesicle-mediated delivery
of biological agents to specific targets appears to be a potentially valuable tool in
clinical medicine.
Intercellular regulatory mechanisms are generally thought to rely on such agents
as neurotransmitters, hormones, trophic factors, cytokines, and adhesion molecules,
which trigger signaling cascades in the target cell, even modulating its genetic
programs. But now in the nerve, composed of glial cells and their associated axons,
we have unveiled a regulatory mechanism that goes one step further. A cell, by
delivering RNAs, specifies the protein architecture of the recipient cell. More
Fig. 7 SC-derived exosomes stimulate axonal growth. Sensory neurons from dorsal root ganglia
(DRGs) explanted in vitro, after 1 day, SC-derived exosomes or vehicle solution (PBS) were
supplemented in a daily basis. Micrograph shown axonal growth of DRGs during 5 days. DRGs
are stained for acetylated tubulin (Ac-Tub, green), phalloidin rhodamine (Phall, red) and nuclei
with DAPI (blue). Impressively, at 5 days, exosome treatment increased the regeneration rate
compared to vehicle-treated explants (mean ± SEM, 41 ± 4 vs. 26 ± 3 mm2/day compared to
vehicle, *p < 0.05, linear regression) suggesting that SC exosomes stimulate axonal regeneration
in vitro. Scale bar, 50 µm (modified from Lopez-Verrilli et al. 2013)
generally, the phenotype of one cell is completed by the genome of a second cell.
This mode of interaction provides a new dimension to the understanding of inter-
cellular regulation; we foresee that several phenomena of the nervous system still
poorly understood will be recast under this new light. For example, synapses are
exquisite associations involving various cell types. In the motor endplate, the
mutual regulation enlists at least three main leads, presynaptic axon, muscle fiber,
and terminal Schwann cell, each one with an elaborated part (Hayworth et al. 2006;
Kang et al. 2014; Li and Thompson 2011; Smith 1973), adding to the complexity of
the system. It is likely that the mechanisms operating in the Schwann cell–axon
system presented here are extensive to synapses.
Perspectives and Concluding Remarks
The axon–glia unit proposed over a decade ago is now better understood. Axons
have internal programs under local control, and the organization of their
cytoskeleton and their ability to sprout, to grow, and to destroy themselves, as well
as other programs, are controlled by their associated glial cells. Moreover, Schwann
cells supply axons with ribosomes and mRNA to synthesize their proteins.
Therefore, the molecular architecture of axons relies also on the genome of their
ensheathing glial cells. Since proteins underlie nearly every conceivable cellular
function, both the axon as a structure as well as its repertoire of functions are the
collaborative efforts of the neuronal nucleus and a string of associated glial cells.
The discovery of this mechanism opens a wide avenue of research. The old idea,
still prevalent, that the cell body supplies all axoplasmic proteins, has to be revised;
this notion was put forth to explain a model in which axons are unable to synthesize
proteins, which does not apply to real axons.
SCs also control ongoing functions of axons. They regulate the axonal
cytoskeleton, repress or promote the ability of axons to grow, and promote the
destruction of doomed axons—processes which are important in nerve repair. The
axon–glia unit requires that our understanding of the axon includes its inextricable
association with its supporting cell. This also implies that overt malfunction of a
nerve fiber, which we may call its “clinical scream,” will be always uttered by the
axon even if the primary alteration is seated in the glial cell, as the latter is clinically
mute. This model is particularly relevant to the understanding of axonopathies, as
the primary disturbance may be seated in the inconspicuous glial cell while the
boisterous axon is altered indirectly. We foresee that our model will be rewarding in
the study of neurological disorders in general.
Finally, a comment on vesicular-mediated intercellular transfer is discussed. We
surmise that the transfer of RNAs via exosomes (or other extracellular vesicles) is a
general mechanism for fine tuning between partner cells in order to optimize their
coupling and operation. By extension, the transfer of RNAs plays a pivotal role in
the organization of tissues.
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Part II
Glial Cell Dysfunction and Pathological
Implications
Age-Dependent Changes in the Activation
and Regulation of Microglia
Francisca Cornejo and Rommy von Bernhardi
Abstract As we age, a large number of physiological and molecular changes affect

the normal functioning of cells, tissues, and the organism as a whole. One of the
main changes is the establishment of a state of systemic inflammatory activation,
which has been termed “inflamm-aging”; a mild chronic inflammation of the aging
organism that reduces the ability to generate an efficient response against stressor
stimuli. As any other system, the nervous system undergoes these aging-related
changes; the neuroinflammatory state depends mainly on the dysregulated activa-
tion of microglia, the innate immune cells of the central nervous system (CNS) and
the principal producers of reactive oxygen species. As the brain ages, microglia
acquire a phenotype that is increasingly inflammatory and cytotoxic, generating a
hostile environment for neurons. There is mounting evidence that this process
facilitates development of neurodegenerative diseases, for which the greatest risk
factor is age. In this chapter, we will review key aging-associated changes occurring
in the central nervous system, focusing primarily on the changes that occur in aging
microglia, the inflammatory and oxidative stressful environment they establish, and
their impaired regulation. In addition, we will discuss the effects of aged microglia
on neuronal function and their participation in the development of neurodegener-
ative pathologies such as Parkinson’s and Alzheimer’s diseases.
Keywords Aging
Cytokines
Microglia Neuroinflammation

Neurodegenerative diseases Oxidative stress
F. Cornejo R. von Bernhardi (&)

Departamento de Neurología, Escuela de Medicina, Pontificia Universidad
Católica de Chile, Marcoleta 391, Santiago, Chile
F. Cornejo

DOI 10.1007/978-3-319-40764-7_10
206 F. Cornejo and R. von Bernhardi
Introduction
Aging is a functional decline that affects all living organisms in a time-dependent

fashion. It is characterized by several cellular and molecular hallmarks, such as
genomic instability, epigenetic modifications, loss of protein homeostasis, mito-
chondrial dysfunction, and cellular senescence among others (López-Otín et al.
2013).
Aging concerns society and the world’s economy, particularly because the
human lifespan has drastically extended without reduced disease load (Vaupel
2010). As it is common for many aging cell systems, the brain goes through a
certain loss-of-function process during aging. This has led to scientific research
focused on several neurodegenerative diseases with aging as their main risk factor,
such as Alzheimer’s disease (AD) and Parkinson’s diseases (PD) (Friedrich 2014).
It has been widely reported that brain aging results in several cognitive changes:
information processing and long-term memory show a mild reduction, the speed of
processing becomes slower, and both the working memory and inhibitory functions
played by the brain appear to be to some extent impaired (Denise et al. 1996;
Hultsch 1998; Park et al. 2002). However, there are also some reports suggesting
that some older adults keep their cognitive capabilities and they may have even
better memory function than young individuals (Gefen et al. 2015). The Baltimore
longitudinal study of aging of more than 2000 individuals followed for a decade,
showed a broad interindividual variability in cognitive decline with age (Shock
et al. 1984). Cognitive changes are due to age-related physiological and molecular
changes in the brain that depend mostly on inflammatory process and oxidative
stress induced as the CNS ages (Berr et al. 2000; reviewed in von Bernhardi et al.
2015b). Many of those changes have microglia as the main responsible effectors,
leading to several alterations that favor neurodegenerative diseases (Dröge and
Schipper 2007; von Bernhardi et al. 2010, 2015a; Tsurumi and Li 2012; Tichauer
et al. 2014).
Main Age-Related Changes in the CNS
Several phenotypic changes are reported to occur as our brains age. The Baltimore
longitudinal study of aging, in which non-demented subjects of 65–85 years old
were evaluated with regular Magnetic Resonance Imaging analysis (Driscoll et al.
2009), revealed regional changes in brain volume. There are structural changes,
including shrinking of nuclei and cortical areas such as the caudate nucleus,
cerebellum, hippocampus, and prefrontal cortex (Salat et al. 2004; Raz et al. 2005).
Age-related differences in white matter integrity accompany a conspicuous reduc-
tion in frontal areas of the brain, establishing an anterior-posterior gradient (Head
et al. 2004), and gray matter of prefrontal, parietal, and temporal cortices shrinks
(Courchesne et al. 2000; Good et al. 2001; Ge et al. 2002). There are also
Age-Dependent Changes in the Activation and Regulation … 207
characteristic metabolic changes associated with aging: glucose metabolism decli-

nes in the prefrontal, anterior cingulate, ventral and dorso-lateral cortex, the medial
prefrontal and the pre-central areas (Zuendorf et al. 2003; Kalpouzos et al. 2009;
Hsieh et al. 2012). Age-dependent anatomical changes of the brain affect cognitive
skills (Faith and Naftali 2003; Rosen et al. 2003; Rodrigue and Raz 2004), and as
glucose metabolism declines, so do synaptic density and structural integrity
(Kochunov et al. 2009).
Increasing evidence supports the “free-radical theory of aging” (Harman 1956),
which proposes that an organism’s lifespan depends on its regulation of antioxi-
dants. In terms of brain function, age-related memory impairment is correlated with
a decreased level of antioxidant moieties in plasma (Perrig et al. 1997; Perkins et al.
1999; Berr et al. 2000). Also, glutathione, the most abundant endogenous antiox-
idant, is reduced in several regions of aged rodent brains, including the hip-
pocampus (Calabrese et al. 2004; Balu et al. 2005; Zhu et al. 2006). In addition,
some studies have shown that the increased oxidative stress is due to reduced
antioxidant enzyme activity in aged brains, such as glutathione peroxidase
(Rodrigues Siqueira et al. 2005), catalase (Tian et al. 1998) and Mn- and Cu,
Zn-superoxide dismutase (Gupta et al. 1991; Navarro et al. 2004). Dysregulation of
the homeostatic oxidative response would induce oxidative protein damage in the
aged brain (Fig. 1), impairing its function, which would support the theory that
age-induced oxidative stress (von Bernhardi and Eugenin 2012) is associated with
the impairment of cognitive skills (Forster et al. 1996).
Oxidative stress, associated with the accumulation of reactive oxygen species
(ROS), increases as organisms age. The brain is particularly susceptible to
ROS-induced damage, given that it demands very high amounts of oxygen and has
low levels of antioxidants molecules compared with other tissues (Perluigi et al.
2014). The major source of cellular ROS in non-macrophage cells is mitochondria.
ROS are generated as a consequence of oxidative phosphorylation in the mito-
chondrial inner membrane (Balaban et al. 2005). It has been shown that mito-
chondrial integrity is reduced with age due to inefficient electron transport, reducing
generation of energy rich molecules (ATP) and favoring the formation of reactive
oxidants (Shigenaga et al. 1994). The mitochondrial DNA (mtDNA) is the first
target; mtDNA is particularly vulnerable to oxidative damage because of the
proximity of mtDNA to the main ROS source and the absence of histones to protect
it, causing mtDNA to have a higher mutation rate than nuclear DNA (Richter et al.
1988). Mutations in mtDNA can affect the expression of cytochromes, altering the
electron transport chain, which in turn would increase ROS production, favoring a
vicious circle leading to oxidative stress (Fig. 1) (Dröge and Schipper 2007),
destabilizing the mitochondrial membrane and promoting cell death.
At a molecular level, microarray studies have shown age-related changes in
genome-wide gene expression in the brain. Changes are most conspicuous after age
70. The principal genes that are downregulated are those associated with cellular
processes related to memory and learning, calcium signaling, vesicle-mediated
protein transport, and mitochondrial function. At the same time, genes related to
Fig. 1 Cellular changes induced in the aged brain. Schematic representation of the main
alterations induced by age in brain cells, and their consequences for ROS production, cell viability,
and gene expression. Aged brain cells have reduced antioxidant mechanisms and increased
intracellular ROS levels that affect mtDNA structure, inducing mutations and leading to a
reduction in mitochondrial integrity, which in turn induce more ROS production. ROS
accumulation and reduced activity of DNA-repair systems in aged cells damage DNA. In
addition, aged cells have altered microRNA expression, several histone modifications, and reduced
DNA methylation enzyme activity, shifting the methylation state of some promoter genes. These
age-induced cell alterations produce changes in gene expression and increase the oxidative stress
on the brain
antioxidant functions, DNA repair and the inflammatory response are upregulated
(Lu et al. 2004; Fraser et al. 2005; Erraji-Benchekroun et al. 2005).
Some of the changes in gene expression could depend on DNA damage or
mutations in DNA-repair genes. In fact, ROS have deleterious effects on DNA
structure and protein function (Fig. 1), among others (Fraga et al. 1990). The most
common oxidative DNA lesions observed in the aged brain are single base
modifications of DNA, which accumulate in the promoter region of most genes that
are downregulated during aging (Lu et al. 2004). In addition, there is also evidence
showing that DNA-repair systems become impaired with aging (Fig. 1), which
directly affects age-induced functional decline. There are several mutations in
DNA-repair genes that induce accelerated aging phenotypes in mice and humans,
characterized by organism deterioration and neurodegeneration, commonly known
as progeroid syndromes (Lombard et al. 2005).
Increasing evidence show that age-related changes in gene expression could also
occur due to epigenetic effects, such as histone modifications and DNA methylation
(Gravina and Vijg 2010). It has long been known that aged tissues, including in
brain, heart, and spleen, show a global methylation reduction associated with
changes in the activity of DNA methylation enzymes (Vanyushin et al. 1973).
These changes are the basis of a novel epigenetic clock that can be used to measure
tissue age from its DNA methylation (Horvath 2013). Furthermore, several
age-specific modifications have been observed on histones, such as methylation
(Thakur and Kanungo 1981; Wang et al. 2010) and acetylation (Pruitt et al. 2006),
together with changes in the expression of some micro-RNAs also observed in aged
tissues (Fig. 1) (Cencioni et al. 2013). In addition, changes in DNA methylation
and posttranslational histone modifications cause alterations in normal chromatin
structure, inducing global heterochromatin loss with aging, which has been estab-
lished as the main cause of the deleterious processes observed in aged tissues
(Tsurumi and Li 2012).
Inflammation in the Aging Brain
As observed in the aging organism as a whole, the aging brain is also characterized
by the presence of a chronic mild inflammation, which reduces neuronal dendritic
and axonal branching, synapse density, dendritic spines, and presynaptic markers
(Yankner et al. 2008).
The term “inflamm-aging” was coined in 2000 (Franceschi et al. 2000) to
describe the reduced capability of the organism to deal with stressor stimuli and the
progression to a more inflammatory state as individuals age. It is postulated that the
phenomenon is induced by the continuous stress and antigenic load to which the
organism is subjected throughout life, and that the persistent exposure to this
inflammatory condition could predispose the organism to develop several
age-related diseases, such as atherosclerosis, AD, PD, osteoporosis, and diabetes
(Franceschi et al. 2000). Age-related immune alterations, known as immunosenes-
cence (Larbi et al. 2008), might also be induced by chronic mild inflammation. It has
been shown that changes in gene expression related to inflammation and immune
response (Lee et al. 2000; de Magalhães et al. 2009), increased plasma levels of
inflammatory cytokines (Singh and Newman 2011), and the activation of inflam-
matory intracellular pathways such as NFĸB occur with aging (Helenius et al. 1996).
NFĸB is the main modulator of immune-related gene expression, and its relation
to aging has been a recent focus of research. Genetic blockade of NFĸB causes
reversion of the aged phenotype and shifts the gene expression pattern of aged mice
to that of young mice (Adler et al. 2007). Accordingly, blocking expression of a
NFĸB subunit acting as a repressor of inflammatory gene transcription, induces a
progressive low-grade inflammation, and as a consequence, mice undergo prema-
ture aging (Jurk et al. 2014). Furthermore, there is evidence that appears to link
aging directly with inflammation. Rap1, a protein that is part of the mammalian
telomeric complex, acts as a potent modulator of NFĸB activity by establishing a
macromolecular complex with IĸB kinases (IKKs), driving NFĸB transcriptional
activity (Teo et al. 2010). The evidence suggests that as we age, telomere short-
ening could increase free Rap1 in the cytoplasm, which in turn could increase IKKs
activity, increasing the transcriptional activity of NFĸB. This sequence might
explain the link between aging and “inflamm-aging” related changes (Arka Subhra
and Vinay 2010) with telomere shortening having a direct effect on the NFĸB
inflammatory pathway (Teo et al. 2010). Moreover, microglia, as one of the few
CNS cells with significant mitotic potential, are susceptible to telomere shortening
and have low telomerase activity (Flanary and Streit 2004).
On the other hand, increased levels of TGFβ have been reported in the brains of
aged individuals (Bye et al. 2001; Werry et al. 2010). Non-neuronal cells appear to
be responsible for the increased production of TGFβ, since TGFβ transcripts are
reduced in aged neurons (de Sampaio e Spohr et al. 2002). In the brain, TGFβ
favors cell survival, is neuroprotective (Dhandapani and Brann 2003), and reduces
activation of microglia and their production of oxidative agents (Herrera-Molina
and von Bernhardi 2005; Abutbul et al. 2012).
There are age-related changes in TGFβ signaling at several levels. As the
response to inflammatory stimulation appears to become more oxidative and
potentially more cytotoxic in aged animals (Tichauer et al. 2014), there is an
increased secretion of TGFβ, but a reduced activation of its canonical signaling
pathway, Smad. Both age and chronic inflammation reduce the activation of the
Smad3 pathway in mice hippocampus (Tichauer et al. 2014). The activation of the
Smad pathway in young animals could depend on the induction of TGFβ1 by
inflammatory stimulation (Wynne et al. 2010) and the induction of Smad3 by the
activation of MAPK1 (Ross et al. 2007). In adult mice, increased basal levels of
TGFβ1 (Colangelo et al. 2002; Lukiw 2004) maintain elevated Smad3, which
becomes unresponsive to new inflammatory stimulation. Increased levels of TGFβ1
with an inhibited Smad signaling can result in an impaired regulation of inflam-
matory activation by TGFβ1 (Schmierer and Hill 2007). Other changes depend on
the interaction of TGFβ with other inflammatory mediators or their transcription
factors, such as IFNγ and NFκB, or on regulatory components, such as MKP-1 (von
Bernhardi et al. 2015b). Furthermore, considering that the non-Smad TGFβ1
pathways MAPKs and PI3K, are not abolished in aged mice, inhibition of Smad
could abolish the regulatory anti-inflammatory effect of TGFβ1 on inflammation,
facilitating the cytotoxic activation of glia (von Bernhardi et al. 2015a).
Age-Induced Changes in Microglial Cells
The main mediators of inflammatory responses in the CNS are microglia, the
resident immune cell of the brain, which are responsible for the detection of dis-
turbances and orchestrate the innate immune response after any event. As
previously discussed in Chapters “Glial Cells and Integrity of the Nervous System”,
“Microglia Function in the Normal Brain” and “Purine Signaling and Microglial
Wrapping”, the microglial cell immune response is activated after neuronal damage
by the release of ATP, growth factors, and cytokines, by changes in the extracel-
lular ionic balance, and by the absence of molecules normally released by healthy
neurons, such as neurotransmitters (Hanisch and Kettenmann 2007; Pocock and
Kettenmann 2007; Block et al. 2007). Initially, it was proposed that the microglial
cells fluctuate between two extreme phenotypes: the classically activated pheno-
type, M1, which appears to be more inflammatory, and the alternatively activated
phenotype, M2, a “regulatory” state (Cherry et al. 2014). However, recent studies
have shown that microglia can be activated in several ways, with a number of
adaptive responses to different stimuli (Hanisch and Kettenmann 2007). When
microglia detect pathogen-associated molecular patterns (PAMPs) or
danger-associated molecular patterns (DAMPs) through their pattern-recognition
receptors (PRRs), they shift their activity state to a more “reactive” phenotype,
which is pro-inflammatory (Lucin and Wyss-Coray 2009). Normally, Toll-like
receptors (TLRs) play an important role in PAMPs and DAMPs recognition, ini-
tiating the immune response mediated by microglia (Janeway and Medzhitov
2002). The intracellular signaling of TLRs activates NFĸB and transcription of
numerous pro-inflammatory genes encoding cytokines, chemokines, complement
proteins, enzymes, adhesion molecules, and immune receptors such as the major
histocompatibility complex II (MHCII) and complement receptor 3 (CD11b)
(Nguyen et al. 2002; Hanisch and Kettenmann 2007; Rozovsky et al. 1998).
In the absence of an inflammatory stimulus, microglia are actively searching for
pathogenic signals (Nimmerjahn et al. 2005), whereby they have been considered to
be in a “surveilling” instead of “resting” state (Hanisch and Kettenmann 2007),
having reduced nitric oxide production and increased anti-inflammatory cytokine
release (Mantovani et al. 2004).
As our brain ages, microglia become more activated and irregularly distributed
in several cortical and subcortical areas (Schuitemaker et al. 2012; Tremblay et al.
2012; von Bernhardi et al. 2015b), with diverse cell morphologies and decreased
process length and complexity (Fig. 2a, b), resulting in a smaller volume of par-
enchyma being probed by a single cell, and appearing more granular due to
increased phagocytic inclusions (Tremblay et al. 2012; Vaughan and Peters 1974).
In addition, microglial cell process movement speed, and the dynamics of the cell’s
response to tissue injury decline with age (Hefendehl et al. 2014). Age-induced
changes in microglia phenotype suggest that, rather than being in an over-activated
state, aged microglia have a reduced ability to develop a normal immune response,
becoming dysregulated (Sheng et al. 1998; von Bernhardi 2007; von Bernhardi
et al. 2015a).
It has been reported that aged microglia show a higher proliferation rate in
response to nerve injury in murine models, suggesting a less-regulated proliferative
response under pathological conditions than that of young microglia (Conde and
Streit 2006; Tremblay et al. 2012). However, there is some agreement about the
effect of aging alone (non-pathological aging) on microglial cell replication: there is
Fig. 2 Microglia morphological, physiological and molecular changes in the aged brain.
Microglial cells of young (a) and old (b) mice were labeled in hippocampal cryosections for
Iba1, a microglia identity marker, to compare their morphological features. a Microglia have large
and ramified processes in 2 month old mice, whereas b microglia from 18 month old mice have a
big cell body with shorter processes. c Schematic representation of functions lost and gained by
aged microglia. Some of the main functions lost involve impaired processes integrity, reduced
expression of the fractalkine and TLR9 inflammatory mediators, and impaired proteolytic and
phagocytic activity. On the other hand, several inflammatory markers are up regulated as microglia
age, such as cytokines, TLRs and several other membrane receptors involving the gain of
inflammatory features
no significant increase in the number of microglia as the brain ages (VanGuilder

et al. 2011), owing to an age-induced reduction in microglia replication. Decreased
replication implies a reduced cell turnover that could result in having fewer healthy
microglia and more aged and dysfunctional cells in aged brains (Mosher and
Wyss-Coray 2014).
As summarized in Fig. 2c, aged mice have a transcriptional profile characteristic
of activated microglia (Godbout et al. 2005) with an increased expression of mRNA
for the inflammatory cytokines Tumor Necrosis Factor α (TNFα), Interleukin 1β
(IL1β), Interleukin 6 (IL6), and Interferon γ (IFNγ), a decrease in the
anti-inflammatory cytokines Interleukin 10 (IL10) and Transforming Growth Factor
β (TGFβ) (Sierra et al. 2007; Vaughan and Peters 1974; Frank et al. 2006), and
increased inflammatory receptors MHCII (Henry et al. 2009) and CD86 (Frank
et al. 2006), suggesting a shift to a more inflammatory phenotype.
Recently, an RNA-seq study has shown that several activation markers can
either be upregulated or downregulated in aged microglia. Whereas, 31 % of genes
involved in the “sensing” activity of microglia are downregulated, 13 % are sig-
nificantly upregulated with age; where 81 % of deregulated genes encode for
proteins involved in sensing endogenous ligands and 62 % of the upregulated genes
are related to sensing infectious microbial ligands (Hickman et al. 2013). Because
sensing is one of the main functions of microglia, these changes in the microglial
“sensome” induced by age will alter its homeostatic functions, perhaps inducing a
pathological microglial phenotype, less protective and more cytotoxic (Yan et al.
2014; von Bernhardi et al. 2010, 2011).
Some epigenetic changes have also been observed in aged microglia. The his-
tone H3K27me3 demethylase Jumonji domain 3 (Jmjd3), which is essential for
microglial activation, is reduced in the midbrain of aged mice, resulting in an
elevated level of histone methylation, exacerbating the reactive microglia inflam-
matory response (Tang et al. 2014). Also, it has been recently shown that aged
microglia CpG sites on the IL1β promoter are hypomethylated, inducing high IL1β
expression (Cho et al. 2015). Both epigenetic alterations in aged microglia seem to
contribute to the development of an activated microglial phenotype and the asso-
ciated neuroinflammation observed in the aged brain.
Aged microglia also have membrane markers (Fig. 2c) that in young cells are
either poorly expressed, such as TLR1, TLR2, TLR4, TLR5, TLR7, and CD14
(Letiembre et al. 2007), or even absent, such as MHCII, CD11b, CD14 (Perry et al.
1993). Some of the markers are increased in certain regions of the brain; CD11b,
CD68, CD11c, F4/80, and FcγRI increases in aged microglia in both the white
matter and caudal areas and CD11c in the white matter (Hart et al. 2012). In
contrast, the fractalkine receptor (CX3CR1), critical for microglia migration and
activation, and TLR9 have a reduced expression in aged microglia (Fig. 2c)
(Wynne et al. 2010; Letiembre et al. 2007).
Class A scavenger receptor (SR-A) has recently been proposed as another target
for microglial activation (Cornejo and von Bernhardi 2013). SR-A is a PRR and has
an important role in the phagocytosis of Aβ and other anionic molecules. Thus,
SR-A inhibition could increase Aβ burden in the brain of AD patients, potentially
promoting neurotoxic effects and disease progression (Frenkel et al. 2013).
Nevertheless, SR-A also modulates glial cell activation (Murgas et al. 2014). It has
been reported that treatment with SR-A antagonists appears to improve the phe-
notypic features of AD (Handattu et al. 2009) by reducing activation of microglia
(Handattu et al. 2013). These pieces of evidence have led to the idea that the
modulation of SR-A activity could be a potent mediator of glial activation, such that
SR-A inhibition could reduce key neuroinflammatory effects secondary to the
microglial cell dysfunction observed in aging.
Cytokine release is also altered in aged microglia (von Bernhardi et al. 2015b),
with increased release of IL1α (Sheng et al. 1998), IL6, and TNFα (Njie et al. 2012).
Aged microglia challenged with an inflammatory stimulus release more IL1β than do
young cells (Combrinck et al. 2002; Cunningham et al. 2005; Sierra et al. 2007;
Henry et al. 2009).
Effects of Aged Microglia on Neuronal Activity
Several investigators report that the neuroimmune response impairs memory and
cognition, since inflammation due to illness or injury impairs cognitive and memory
tasks (Hudetz et al. 2009; Selnes et al. 2003; Shapira-Lichter et al. 2008). In
agreement with in vivo studies, in vitro work shows that conditioned medium
derived from LPS-stimulated microglia reduces the number of synapses in neuronal
cultures (Moraes et al. 2014), with the inflammatory cytokine IL1β being the main
cytokine responsible for the regulation of synaptic activation and long-term
potentiation (LTP) (Bellinger et al. 1993; Cunningham et al. 1996; Moraes et al.
2014).
In aged rats, several inflammatory cytokines are upregulated in the hippocampus,
accompanied by progressive deficits in LTP (Griffin et al. 2006). In fact, direct
administration of IL1β to the CNS impairs memory consolidation (Rachal Pugh
et al. 2001), and inhibition of IL1 receptor activity in the hippocampus promotes
short-term and long-term memory retention (Depino et al. 2004). It has been pro-
posed that the effect of IL1β could involve downregulation of BDNF (Barrientos
et al. 2003, 2004) or reduction of glutamate release in the hippocampus (Gonzalez
et al. 2013). However, it has also been shown that IL1β acutely impairs neuroge-
nesis (Goshen et al. 2008; Wu et al. 2012; Zunszain et al. 2012). Thus,
IL1β-induced neuroinflammation would both modulate synapses and impair neu-
rogenesis in the hippocampus.
IFNγ is also upregulated in the aged hippocampus and induces microglial
activation when injected in rat hippocampus, diminishing LTP (Maher et al. 2006).
This demonstrates that inflammatory cytokines released by microglia subjected to
aging-induced changes have a role both in neuronal homeostasis and in the mod-
ulation of synapse integrity and neuronal function. This identifies activated
microglia as important participants in the genesis and progression of neurological
diseases, as previously mentioned in Chapter “Microglia Function in the Normal
Brain”.
Aged Microglia in the Development of Neurodegenerative

Diseases
Microglia have been linked to the pathology and disease progression of several
neurodegenerative disorders, including prion diseases (Perry et al. 2002), multiple
sclerosis (Takeuchi et al. 2006), amyotrophic lateral sclerosis (McGeer and McGeer
2002), Huntington’s disease (Sapp et al. 2001), Pick’s disease (Schofield et al.
2003), and HIV-associated dementia (Sopper et al. 1996). Similarly, age-induced
microglial dysfunction appears to have an important role in the onset of age-related
neurodegenerative diseases, such as PD and AD (von Bernhardi 2007).
PD is a disease characterized by the loss of the ascending nigrostriatal

dopaminergic projections, resulting in motor dysfunctions such as rigidity, tremor,
slowness, difficulty to initiate movements, and loss of balance. One main cause of
dopaminergic neuron loss is activated microglia. Activated microglia are observed
associated with degenerating dopaminergic neurons in the substantia nigra
(McGeer et al. 1988) and in the hippocampus, trans-entorhinal cortex, cingulate
cortex, and temporal cortex of PD patients (Imamura et al. 2003). Microglial cells
are activated by α-synuclein aggregation, involving NADPH oxidase, leading to
persistent and progressive nigral neurodegeneration in PD (Zhang et al. 2005). This
selective loss of dopaminergic neurons in the nigrostriatal pathway might also
depend on the high density of microglia that exists in this region (Kim et al. 2000).
It has been reported that IFNγ-mediated death of dopaminergic neurons is observed
only in the presence of microglia (Mount et al. 2007), with TNFα also having a role
in PD progression (McCoy et al. 2006; Sriram et al. 2002).
As already discussed, one of the main changes associated with CNS aging is
aberrant activation of microglia, generating cells that are incapable of developing a
normal immune response (von Bernhardi 2007). In AD, one of those abnormal
responses of aged microglia is their loss of the ability to deal with amyloid beta
peptide (Aβ) aggregates (Fig. 2c), so that aged microglia internalize less Aβ
(Floden and Combs 2011; Njie et al. 2012) and have less capacity to process it
(Hickman et al. 2008; Mawuenyega et al. 2010; Nixon et al. 2001). Studies in AD
mouse models show that aged microglia have fewer scavenger receptors and
Aβ-degrading enzymes, in addition to increased inflammatory cytokines, as already
mentioned. IL1β and TNFα appear to be the main agents responsible for the
reduced expression of scavenger receptors (Hickman et al. 2008), leading to the
idea that the increased levels of inflammatory cytokines observed in aging down-
regulate the expression of genes involved in Aβ clearance and promote Aβ accu-
mulation. This Aβ accumulation not only induces neurotoxicity, but also affects
neuronal function, inhibiting LTP in aged brains (Lynch et al. 2007). Additionally,
Aβ accumulation induces activation of microglia and the expression of the glyco-
protein of human histocompatibility complex HLA-DR in the vicinity of neuritic
plaques (McGeer et al. 1987; Rogers et al. 1988; Xiang et al. 2006), increases iNOS
(Dheen et al. 2005), produces NO and superoxide anion (Ii et al. 1996; Qin et al.
2002), increases expression of TNFα (Meda et al. 1995), leads to microglial cell
dystrophy, and exacerbates telomere shortening induced by aging in microglial cells
(Flanary et al. 2007). These observations imply that as microglia get older, their
Aβ-phagocytic activity declines. This facilitates the accumulation of Aβ aggregates
in the aged brain, which in turn leads microglia to a more reactive phenotype,
establishing a vicious circle leading to neurodegenerative changes. In fact, injection
of Aβ is neurotoxic only in aged individuals, but not when injected in the brains of
young monkeys, suggesting that Aβ-induced neurodegeneration is a pathological
response of the aged brain (Geula et al. 1998).
It has been reported that Aβ reactivity and phagocytic activity of microglia are
regulated by astrocytes, which attenuate their cytotoxic activation (DeWitt et al.
1998; von Bernhardi and Ramirez 2001). However, this regulation is not observed
when primed microglia are exposed to Aβ (von Bernhardi and Eugenín 2004).
Compared with nonactivated microglia, primed microglia show increased cyto-
toxicity, Aβ precursor protein (APP) synthesis and Aβ aggregation, as well as
impairment of the uptake and degradation of Aβ (Rogers et al. 2002; Ramírez et al.
2008; von Bernhardi 2007).
TGFβ1 secreted by hippocampal neurons and astrocytes has been identified as an
important modulator cytokine, attenuating the release of pro-inflammatory mediators
(Chen et al. 2002; Mittaud et al. 2002; Herrera-Molina and von Bernhardi 2005) and
promoting microglia-mediated Aβ phagocytosis and degradation (Wyss-Coray et al.
2001). We have recently shown that these regulatory effects are mediated by a
Smad3-dependent mechanism (Tichauer and von Bernhardi 2012), the TGFβ1
signaling pathway that is inhibited in aged brain, as discussed above. Interestingly,
the Smad signaling pathway is also impaired in the brains of AD patients and mouse
models for AD, even though TGFβ1 levels are elevated in the cerebrospinal fluid of
these patients (Blobe et al. 2000). Thus, impairment of Smad signaling appears to
result in Aβ accumulation, Aβ-induced neurodegeneration, and neurofibrillary tan-
gle formation (Tesseur et al. 2006; Ueberham et al. 2006), suggesting it could play a
role in the genesis of AD, and eventually be considered as a therapeutic target.
Concluding Remarks
As we age, several morphological and metabolic changes occur in our brain. Many
of those changes are a consequence of alterations in the normal functioning of brain
cells and how they deal with stressor stimuli, leading to genetic and epigenetic
changes. Here we have discussed two theories that aim to explain age-induced
molecular changes: the “free-radical theory of aging,” in which the mediator for
aging is ROS, and the “inflamm-aging theory” that proposes that cumulative
inflammatory signals direct age-related impairment. The latter is particularly
striking because it directly associates aging and telomere shortening with inflam-
matory activation through Rap1 and IKK interaction.
Microglia are the cells most prone to ROS damage and telomere shortening in
our brains, and both theories finger microglia as the main cell type affected by
aging. Aged microglia show a dysregulated inflammatory response associated with
several changes in cytokines and PRR expression compared with young cells.
These changes not only affect immune homeostasis of the brain but also neural
function and cognitive skills.
Among the molecular signaling pathways affected by aging in microglia, the
TGFβ pathway appears to be one of the most relevant, because it modulates
microglial cell reactivity and activation state (Herrera-Molina and von Bernhardi
2005; Boche et al. 2006; von Bernhardi et al. 2010). Study of the TGFβ pathway as
a possible target for age-related diseases should reveal new approaches to promote
the well-being of elderly people, especially those affected by neurodegenerative
pathologies such as AD.
Acknowledgments This work was supported by grant FONDECYT 1131025 and fellowship
CONICYT 21120013.
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277
Astrocyte Dysfunction in Developmental
Neurometabolic Diseases
Silvia Olivera-Bravo, Eugenia Isasi, Anabel Fernández,

Gabriela Casanova, Juan Carlos Rosillo and Luigi Barbeito
Abstract Astrocytes play crucial roles in maintaining brain homeostasis and in

orchestrating neural development, all through tightly coordinated steps that coop-
erate to maintain the balance needed for normal development. Here, we review the
alterations in astrocyte functions that contribute to a variety of developmental
neurometabolic disorders and provide additional data on the predominant role of
astrocyte dysfunction in the neurometabolic neurodegenerative disease glutaric
acidemia type I. Finally, we describe some of the therapeutical approaches directed
to neurometabolic diseases and discuss if astrocytes can be possible therapeutic
targets for treating these disorders.

Keywords Astrocytes Astrocyte dysfunction Neurodevelopmental diseases

Neurodegeneration Myelin damage
S. Olivera-Bravo (&) E. Isasi

Cellular and Molecular Neurobiology, Instituto de Investigaciones Biológicas
Clemente Estable (IIBCE), Montevideo, Uruguay
E. Isasi
A. Fernández
Neuroscience Division, IIBCE and Comparative Neuroanatomy-Associated
Unit to the School of Sciences, UDELAR, Montevideo, Uruguay
G. Casanova
UMET, School of Sciences, UDELAR, Montevideo, Uruguay
J.C. Rosillo
Neuroscience Division IIBCE, UDELAR, Montevideo, Uruguay
L. Barbeito
Neurodegeneration Laboratory, Institut Pasteur Montevideo, Montevideo, Uruguay

DOI 10.1007/978-3-319-40764-7_11
228 S. Olivera-Bravo et al.
Abbreviations
ALS Amyotrophic Lateral Sclerosis
AQP4 Aquaporin 4
BBB Blood–Brain Barrier
GA Glutaric Acid
GA-I Glutaric Acidemia Type I
GCDH Glutaryl CoA Dehydrogenase
GDNF Glial-Derived Neurotrophic Factor
Icv Intracerebroventricular
IEM Inborn Errors of Metabolism
MAPK Mitogen-Activated Protein Kinases
MLC Megalencephalic Leukoencephalopathy with Subcortical Cysts
NPC Niemann-Pick type C Disease
NVU Neurovascular Unit
PC Pyruvate Carboxylase
VWM Vanishing White Matter
Introduction to Astrocyte Functions
As previously discussed in Chapters “Glial Cells and Integrity of the Nervous

System,” “Pharmacological Tools to Study the Role of Astrocytes in Neural Network
Functions,” and “Role of Astrocytes in Central Respiratory Chemoreception,” as-
trocytes are indispensable for neuronal survival and activity. They play many key
regulatory functions in brain homeostasis, including glutamate uptake that is vital for
maintaining a physiological balance and protecting against neurotoxicity (Rothstein
et al. 1996). Astrocytes are the only cells in the CNS that store and process glycogen.
They thus contribute to influx of glucose and energy intermediates into neurons, and
are also a significant source of neurotrophins and antioxidant defenses (Maragakis
and Rothstein 2006). Moreover, astrocytes are the nearly unique neural cells that
express glutamine synthase, the only brain source of endogenous glutamine, which is
the major amino moiety donor for glucose precursors and nitrogenated compounds
and the preferred metabolite for inter-organ transport and temporary storage of
nitrogen (Haberle et al. 2005, 2011). Astrocytes also participate in blood–brain
barrier formation and maintenance (Abbott et al. 2006) and have a unique role in the
clearance of brain solutes along veins (Iliff et al. 2013). Mediated by a gap
junction-coupled network that allows the direct cytoplasmic passage of ions and
small molecules through significant distances, astrocytes communicate with each
other and modulate the activity of adjacent cells (Maragakis and Rothstein 2006;
Verkhratsky et al. 2012, 2015). In addition, astrocytes have receptors to a wide range
Astrocyte Dysfunction in Developmental Neurometabolic Diseases 229
of neurotransmitters, peptides, hormones, and cytokines that regulate their own

functions and also influence neurons, oligodendrocytes, and microglia (Maragakis
and Rothstein 2006; Verkhratsky et al. 2012, 2015). Although astrocytes are not
electrically excitable, they are capable of detecting and modulating neuronal activity
by releasing neuroactive substances (glutamate, ATP, D-serine) that can feed back
onto presynaptic terminals or stimulate postsynaptic neurons (Araque 2008;
Verkhratsky et al. 2012; Pekny and Pekna 2014). Moreover, as each astrocyte domain
covers up to 2 106 synapses, and human astrocytes enhanced learning and memory
skills in transplanted rodents (Han et al. 2013), important roles in integrating and
processing complex cognitive have been proposed for astrocytes (Araque 2008; Han
et al. 2013).
Finally, in damaging conditions, astrocytes can suffer early alterations that elicit
downstream mechanisms that have the potential to irreversibly damage neurons and
oligodendrocytes. Among the most important astrocyte alterations are included the
decreased expression of glutamate transporters and subsequent delayed clearance of
synaptic glutamate, impaired glutamate-glutamine cycle, depletion of glutamine
and neurotransmitter precursors, reductions in glutathione levels as well as induced
expression of nitric oxide synthase and pro-inflammatory cytokines (Maragakis and
Rothstein 2006; De Keyser et al. 2008; Verkhratsky et al. 2012, 2015; Pekny and
Pekna 2014).
Astrocyte Dysfunction and Development: A Focus

on Inborn Errors of Metabolism
Astrocytes play crucial roles during development by actively contributing to the

finely coordinated steps required to establish functional neural networks (Clarke
and Barres 2013; Chung et al. 2013; Sloan and Barres 2014). Moreover, normal
synaptic development and pruning requires this tightly regulated timing and com-
munication between astrocytes and neurons (Helmuth 2001; Eroglu et al. 2009;
Dodla et al. 2010). In turn, astrogenesis depends on the signaling of newly formed
neurons (Barnabe-Heider et al. 2005). In this context of mutual influence it has been
proposed that alterations of astrocyte differentiation or function may irreversibly
unbalance the coordinated developmental events, thereby producing neurodevel-
opmental disorders. Moreover, as most glial cells arise from clonal divisions of
early differentiated astrocytes (Ge et al. 2012), if damage occurs early during
astrogenesis or in the first astrocytes, it might be sustained and amplified by the
clonal descendants, thus perpetuating astrocyte dysfunction (Sloan and Barres
2014) which can further account for neurodevelopmental diseases. In this regard,
we will review as astrocyte dysfunction affects neurons and brain development in
some inborn errors of metabolism (IEM) with a further focus on glutaric acidemia I,
an IEM belonging to the group of neurometabolic diseases because of the very
predominant CNS damage over the rest of the body.
Astrocyte Dysfunction-Associated IEMs: Impact

on Neuron Survival
The term inborn errors of metabolism (IEMs) encompasses an enormous group of

rare disorders (more than 750) that in common affect about 1:2000 newborns. IEMs
are produced by genetic mutations that alter biochemical pathways producing an
excess of a damaging substance or deficiency of a normal biochemical compound,
both causing toxic effects in the brain and/or the rest of the body (Scriver et al.
1995). Brain and neurons are significantly affected either by a direct toxicity or
indirectly by a primary glial cell dysfunction. In the IEMs in which biochemical
pathways are primarily altered in astrocytes instead of neurons, astrocyte damage
elicit downstream mechanisms such as glutamate receptor-mediated excitotoxicity,
lactic acidosis, energetic deprivation, oxidative/nitrosative stress, or neuroinflam-
mation that acting alone or together have the potential to kill neurons (Maragakis
and Rothstein 2006; De Keyser et al. 2008; Verkhratsky et al. 2012, 2015; Pekny
and Pekna 2014). Furthermore, the imbalance of critical and exclusive astrocytic
enzymatic routes such as the glutamine synthase may trigger different neu-
rometabolic diseases associated to either excess or lack of the enzymatic product. In
this regard, hepatic encephalopathy (HE) is an IEM characterized by mild to very
severe neuropsychiatric manifestations that are attributed to glutamine overload
produced by the activity of astrocytic glutamine synthase in response to the brain
increased uptake of ammonia produced by liver failure (Butterworth 2010).
Excessive brain glutamine causes significant astrocyte swelling that in turn triggers
a complex signaling cascade which relies on NMDA receptor activation, elevation
of intracellular calcium, and glutamate exocytosis, all together resulting in a
self-amplifying signaling loop that causes excitotoxicity and increased oxidative
stress that trigger neuron death for one side and sustain astrocyte activation for the
other (De Keyser et al. 2008; Butterworth 2010).
Conversely to what is observed in HE, the loss of function of glutamine synthase
produces the congenital glutamine synthase deficiency, a very rare IEM that usually
causes neonatal death associated with severe brain malformations, including
abnormal gyration and white matter lesions. Glutamine starvation impairs both the
synthesis of nitrogenated compounds and the nitrogen storage and transport among
organs. It is believed that the combination of all of these distorted events produces a
systemic collapse which is the main responsible for unviable brain malformations
(Haberle et al. 2005, 2011).
Lack of pyruvate carboxylase activity (PC), another metabolic pathway confined
to astrocytes, causes the rare autosomal recessive IEM known as PC deficiency,
whose neonatal form is characterized by multi-organ metabolic imbalance, lactic
acidemia, and significant neurological dysfunction. PC catalyzes the conversion of
pyruvate into oxaloacetate which is crucial for the replenishment of citric acid cycle
intermediates, gluconeogenesis, synthesis of glycogen, maintenance of the antiox-
idant glutathione system, and anaplerotic support to neurons (Robinson et al. 1984;
Garcia-Cazorla et al. 2006). As the main pathological findings correlate with
impaired astrocytic anaplerosis, it has been proposed that loss of PC leads to

generalized hyperammonemia and death in the first few months of life. Likely, this
happens because of the impaired astrocyte ammonia buffering and support to
microvascular morphogenesis and myelination (Robinson et al. 1984;
García-Cazorla et al. 2006). Moreover, as PC is involved in the synthesis of myelin
lipids (De Keyser et al. 2008), an altered astrocyte–oligodendrocyte communication
may underlie the paucity of myelin and white matter lesions observed in patients.
Niemann-Pick type C disease (NPC) is an autosomal recessive lipid-storage IEM
characterized by progressive neurodegeneration, hepatosplenomegaly, and general
organ dysfunction. It may be caused by mutations in the NPC-1 gene which in brain
is predominantly present in the astrocytic processes closely associated to the nerve
terminals (Patel et al. 1999). In NPC animal models, astrocytes become activated,
and show decreased gap junctional communication and increased hemichannel
activity that might predispose the surrounding neurons to death by either neu-
roinflammation and/or reduced astrocyte mediated-spatial buffering (Saez et al.
2013). In accordance with a crucial astrocyte role in the disease, rescuing NPC1
expression in astrocytes delays neuronal loss and prolongs life span in NPC1−/−
mice; and simultaneous NPC1 recovery in neurons and astrocytes exhibited better
protection than the sole recovery in neurons (Borbon et al. 2012).
Aceruloplasminemia is another IEM linked to primary astrocyte dysfunction
with neuron death as a secondary phenomenon. Its neuropathological hallmarks
include excessive iron deposition, astrocyte perivascular endfeet deformation, and
progressive neuronal loss (Oide et al. 2006; Miyajima 2015). All defects are
attributed to the lack of astrocytic ceruloplasmin ferroxidase activity which medi-
ates the ferrous ion oxidation needed for transferrin-dependent iron efflux. Impaired
ferroxidase activity leads to prominent redox-active iron accumulation in astrocytes
that causes significant endfeet deformation, lipid peroxidation, and hydroxyl radical
formation as well as impaired transferrin/iron shuttle to neurons. Thus, neurons will
die either by increased oxidative stress, decreased astrocyte protection, and/or iron
starvation (Oide et al. 2006; De Keyser et al. 2008; Miyajima 2015).
Astrocyte Dysfunctions and Leukodystrophies: Effects

on Myelin Formation
The term leukodystrophy comprises all of the diseases genetically determined as

white matter disorders characterized by abnormal myelin formation (Hagemann
et al. 2009; van der Knaap et al. 2006). Astrocyte disturbed functions may also be
the leading cause of several leukodystrophies. Alexander disease, a paradigmatic
leukodystrophy, clearly manifests as a primary astrocyte dysfunction that com-
promises myelin development and integrity. It is considered a primary
astrogliopathy with myelin alterations as secondary damage (Messing et al. 2012).
The only pathological causes known in Alexander disease are the several mutations
in the glial acidic fibrillary protein (GFAP) gene that lead to a toxic gain of function.
Astrocytes but no other cells show the defining features of the disease that include
hypertrophic cell bodies with GFAP-positive processes and characteristic intracy-
toplasmic multiprotein aggregates named Rosenthal fibers. As myelin failure and
oligodendrocyte loss correlate with increased presence of astrocytic Rosenthal
fibers, it is proposed that an altered pattern of the astrocyte signals that promote
myelination is the main pathological underlying mechanism (De Keyser et al. 2008;
Hagemann et al. 2009; Liem and Messing 2009; Messing et al. 2012) (see Chapter
Requirements and Oxidative Damage” for further reading on oligodendrocytes and
myelination).
Astrocytes also play a central role in the pathogenesis of megalencephalic
leukoencephalopathy with subcortical cysts (MLC), which commonly affects
infants, lead to progressive spasticity and ataxia, and exhibit myelin splitting and
intra-myelin vacuole formation as characteristic features (De Keyser et al. 2008;
Ridder et al. 2011). The disease is directly linked to mutations in the MLC1 gene
that is mainly expressed in astrocyte perivascular endfeet (Boor et al. 2005); and
defective astrocyte MLC1 is enough to elicit myelin damage (Duarri et al. 2011).
These findings lead to the proposition that disturbances in astrocytic MLC1 may
irreversibly affect myelin by impeding the transport of essential molecules for
oligodendrocytes or myelin formation (Duarri et al. 2011). Another paradigmatic
leukodystrophy associated with astrocyte dysfunction is the vanishing white matter
(VWM) disease that is caused by alterations in the genes that encode for the
subunits of the eukaryotic initiation factor eIF2B that is expressed in astrocytes and
oligodendrocytes (van der Knaap et al. 2006; Bugiani et al. 2011). VWM is pre-
dominantly manifested in early childhood, and usually presents with a rapid clinical
decline triggered by stress-related events that may lead to death (van der Knaap
et al. 2006). Brain patient samples from patients show astrocytes with an increased
proliferation, immature phenotype, abnormal composition of intermediate filament
network, and metabolic stress (Bugiani et al. 2011). Loss of white matter may be at
least partially explained by excessive astrocyte proliferation at the expense of
oligodendrocytes that may occur due to aberrant specification of glial progenitors,
as has been shown in astrocytosis linked to myelin alterations found in children
born prematurely or surviving cerebral ischemia (Bain et al. 2010).
A Novel Mechanism: Astrocyte Dysfunction Sustaining

the Pathogenesis of the Neurometabolic IEM GA-I
In spite of abundant evidence directly linking abnormal astrocyte pathways to

neurodevelopmental disorders, the participation of astrocyte dysfunction in com-
plex multifactorial IEMs that selectively affect the brain is much less known.
Among these particular IEMs, usually named neurometabolic diseases because the
almost exclusive CNS damage over the rest of the body (Scriver et al. 1995),
organic acidemias and acidurias comprise a predominant group that includes all
abnormal amino acid catabolism pathways. Although most acidemias are charac-
terized by an acute life-threatening stage, some of them present a significant and
progressive neurodegenerative component that cannot be directly linked to the
accumulation or lack of the metabolites transiently produced by the altered path-
ways. Moreover, as has been shown for glutaric acidemia type I (GA-I), that is,
among the most prevalent organic acidemias/acidurias, the accumulated metabolites
may not be directly toxic to neurons (Freudenberg et al. 2004; Olivera-Bravo et al.
2011; Jafari et al. 2013), so the underlying mechanisms remain mostly unknown.
GA-I is characterized by extensive acute striatal and progressive cortical neu-
rodegeneration together with white matter and vascular abnormalities, causing
permanent motor and cognitive sequels. The disease is caused by a loss of function
of glutaryl-CoA dehydrogenase (GCDH), a mitochondrial enzyme involved in the
catabolism of lysine, hydroxylysine, and tryptophan. It results in accumulated
millimolar concentrations of glutaric acid (GA) and related metabolites in fluids and
tissues (Goodman et al. 1977; Strauss et al. 2003; Funk et al. 2005). In spite of
having a strong and progressive neurodegenerative profile, even high concentra-
tions of GA-I metabolites did not directly account for neuron death (Freudenberg
et al. 2004); and the GCDH−/− mice characterized by permanent high levels of
GA-I metabolites do not suffer any neurological deficit (Koeller et al. 2002, 2004).
Therefore, based on previous data that showed an altered glutamate uptake in
astrocytes exposed to GA (Magni et al. 2009), we propose that instead of neurons,
astrocytes are the earliest cell targets of GA-I accumulated metabolites. We have
found that astrocytes, but not neurons or oligodendrocytes, respond to patho-
physiological concentrations of GA by increasing their proliferation that is medi-
ated by activation of the MEK/ERK pathway, augmenting oxidative stress, and
significantly decreasing both their mitochondrial potential and glutathione pro-
duction (Olivera-Bravo et al. 2008, 2011). Astrocyte increased proliferation was
also found when newborn rat pups were injected intracerebroventricularly (icv)
with 5 mM GA. Remarkably, astrocytes pretreated with GA were toxic to
co-cultured neurons even many days after removing the acid, and GA-injected
animals presented delayed striatal neuronal death that became significant only many
days after the peak of astrocytosis (Olivera-Bravo et al. 2011). Astrocyte response
to GA also preceded delayed oligodendrocyte differentiation, which resulted in a
significant and progressive demyelination. Interestingly, striatal axon bundles from
GA-injected animals exhibited not only significantly low levels of myelin, but also
significant decreases in expression of myelin-associated glycoprotein and
myelin-binding protein, suggesting significant white matter damage (Olivera-Bravo
et al. 2014). Transmission electron microscopy of the striatum of GA-injected
animals showed oligodendrocytes with significant endoplasmic reticulum stress,
altered myelination, and thinner axons (Olivera-Bravo et al. 2014), all resembling
the progressive myelin alterations reported in affected patients (Bähr et al. 2002;
Funk et al. 2005). At the ages analyzed, GA-induced myelination failure was
restricted to the basal ganglia, the most vulnerable brain area in GA-I patients
Fig. 1 Striatal damage elicited upon astrocyte response to GA. a Sustained astrocytosis and c
axonal bundle shrinkage. Newborn rat pups received an icv injection of 1 lg/g GA or vehicle and
were sacrificed 30 days postinjection (DPI). At that time, GA-injected rats showed increased
number of S100b astrocytes, which present enlarged bodies and stronger processes (white arrows)
together with reduced phosphorylated neurofilaments (pNF) axonal bundle areas. Inset shows the
region analyzed at all conditions and ages. b Delayed neuronal death. Pictures show an increased
labeling of the marker of degenerating neurons, Fluoro-Jade C (FJC), in GA-injected rats. The
inset shows absence of significant signal in 21 DPI controls when neuron death became significant
in GA-injected animals. c Altered neurovascular unit (NVU) in GA-injected animals.
Platelet-derived growth factor b-receptor (PDGFRb) labeled positive pericyte bodies (*) and
processes contacting blood vessels (white arrows) in 30 DPI vehicle-injected animals. In
age-matched GA-injected rats, PDGFRb immunoreactivity was confined to blood vessels and
there appeared abundant Evans Blue positive cells, indicating abnormal permeation through the
blood–brain barrier. d Quantitation of alterations observed in GA-injected animals. Charts show a
significant and early astrocytosis that precedes increasing neuron damage and decreasing
expression of NVU components such as pericytes and astrocyte endfeet that were recognized with
PDGFRb and aquaporin 4 (AQP4), respectively. All data are related to corresponding age-matched
controls. Calibration: 40 lm in (a), 100 lm in (b), 20 lm in (c). Asterisks indicate p < 0.05
(Goodman et al. 1977; Strauss et al. 2003, 2007; Funk et al. 2005). On the other
hand, GA did not induce acute damage to oligodendrocyte precursors in neonatal
pups suggesting the delayed oligodendrocyte cytopathology found several days
after a single exposure to GA was produced by an indirect neurotoxic mechanism
instead of a direct action. Recently, we have found that the perinatal icv adminis-
tration of GA caused significant long-lasting alterations in the permeability of the
maturing brain–blood barrier (BBB) to substrates of low molecular weight, such as
Evans Blue and a significant reduction in the expression of markers of critical
components of the neurovascular unit (NVU), including astrocyte podocytes, per-
icytes, and basal lamina. These findings were statistically significant in the striatum,
and a similar trend was described in parietal cortex (Isasi et al. 2014) (Fig. 1),
confirming the striatal vulnerability to GA-I metabolites. In summary, our data
indicate that a transient increase in GA levels in the CNS triggers an early but
long-lasting astrocyte dysfunction that leads to delayed myelination, altered BBB
permeability, and NVU failure together with significant loss of striatal neurons. The
single exposure to GA not only caused an acute astrocyte response but also a
sustained altered phenotype that seems to elicit the sequential events that account
for most of the GA-I neurological features (Fig. 2). Thus, GA-induced neurotoxi-
city in our hands appears to require a complex cellular interplay that, after an initial
trigger elicited by toxic levels of GA, can perpetuate itself in ensuing weeks,
leading to progressive damage that resembles the alterations seen in patients
independently on the occurrence of encephalopathic crises (Bähr et al. 2002; Funk
et al. 2005). On the other hand, whereas the genetic model of GA-I expected to
reproduce the human disease fails to develop neuronal loss (Koeller et al. 2002,
2004), even after overload with high amounts of lysine to boost endogenous GA
production (Seminotti et al. 2012), our icv GA injection model moderately repro-
duces the encephalopathic crisis, striatal neuronal loss, myelination, and BBB
defects reported in patients.
Our evidence also supports a crucial role of astrocytes in GA-I. Astrocytes are
preferentially vulnerable because they can actively take up GA (Magni et al. 2009),
which leads to mitochondrial dysfunction, increased proliferation, and oxidative
stress, all causing a poorly differentiated phenotype with high S100b and low
GFAP expression (Olivera-Bravo et al. 2008, 2011). Such inability of astrocytes to
fully differentiate may critically compromise their supportive functions, as proposed
in Fig. 2. Accordingly, astrocytes from GCDH−/− mice, which suffer permanently
elevated levels of GA-I metabolites, cannot offer normal anaplerotic support to
neurons (Lamp et al. 2011), and hyperammonemia caused by selective vulnerability
of astrocytes to GA-I metabolites is a leading neurotoxic mechanism in a

three-dimensional embryo cultures that also contain neurons and oligodendrocytes
(Jafari et al. 2013).
On the other hand, as multiple trophic factors produced by astrocytes are
required for the survival of developing oligodendrocytes (Barres et al. 1993; Nash
et al. 2011), even small changes in astrocytic function may be detrimental to
oligodendrocyte survival and lead to defective myelination, as has been shown in
Alexander disease (Liem and Messing 2009; Messing et al. 2012). Regarding BBB
and NVU failures, it has been demonstrated that astrocytes are key players in BBB
maturation and stabilization (Abbott et al. 2006). The progressive decrease of
AQP4 close to blood vessels in GA-injected animals is suggestive of altered
astrocyte differentiation and polarization at the NVU and probably of a deficiency
of AQP4 in the neurovascular coupling. Moreover, defective laminin and pericytes,
both influencing AQP4 polarization, indicate altered communication among NVU
components long after GA injection (Isasi et al. 2014). In summary, the “toxic”
process has evolved independently of increased GA levels but is tightly dependent
on astrocyte dysfunction. Although these events should be studied in GA-I patients,
astrocytes must be taken into account not only as a main player in GA-I patho-
genesis but also when exploring therapies for this up to now incurable disease.
Current Therapeutic Approaches For IEMs
Early diagnosis and systemic treatments for IEMs and neurometabolic disorders in
particular have in many cases improved quality of life and increased life expec-
tancy. Perinatal or gestational diagnosis pursues disease detection before the
occurrence of precipitating crises or worsening of symptoms to allow the preser-
vation of the systemic equilibrium and neurological performance. Disease man-
agement at this stage is directed to obtain some amounts of lacking products or
avoid the accumulation of toxic compounds that becomes the organism prone to
suffer encephalopathic crises (Ruiz Pons et al. 2007). Unfortunately, perinatal
screening is available for very few IEMs and most patients are often diagnosed
when some symptoms become evident or after the occurrence of crises that usually
cause an acute life-threatening stage where the CNS is exposed to huge amounts of
toxic compounds. At this time, emergency management looks for the restoration of
general homeostasis including the rescue of glucose, oxygen, and pH levels. For
example, glucose supplementation provided adequate preferred energetic sub-
strates, and reduced both the demand for alternate substrates and the amino acid
turnover in the liver all favoring the return of systemic parameters close to normal
values (Zinnanti et al. 2007). The acute life-threatening stage produced during
encephalopathic crises is followed by a sub-acute progressive neurodegenerative
disorder that currently lacks effective medical treatment. In this period, neurological
symptoms become more pronounced and worsening is attributed to the progressive
destruction of motor, mental, and/or perceptual abilities, including loss of function
Fig. 2 Temporal course of astrocyte response to GA and a gliocentric hypothesis for GA-I. a The
scheme shows the early and long-lasting astrocyte response to GA, which was followed by delayed
oligodendrocyte differentiation, which in turn preceded significant neuronal death. Significant
decreases in the expression of the NVU components PDGFRb, AQP4, and laminin close to blood
vessels occurred later. b Proposed pathological events that occur after perinatal exposure to GA
and are mediated by the consequent astrocyte dysfunction, rather than direct effects on
oligodendrocytes, neurons, and NVU components. We hypothesized that astrocyte dysfunction
may trigger (i) delayed oligodendrocyte (OL) differentiation and altered myelination, probably by
a yet unknown imbalance of the astrocyte signals that influence myelination; (ii) astrocyte
oxidative stress and mitochondrial dysfunction may account for the progressive neuron death
observed; (iii) altered AQP4 polarization or imbalanced astrocyte–endothelial and/or astrocyte–
pericyte communication may explain defective NVU composition and increased permeability of
blood–brain barrier observed in GA-injected animals. NVU colors: red (endothelial cells), cyan
(basal lamina), yellow (pericytes), gray (astrocyte endfeet), blue (neuronal processes).
Abbreviations: BMP bone morphogenetic protein; CNTF ciliary neurotrophic factor; ERK
extracellular signal-regulated kinases; FGF2 fibroblast growth factor 2; LIF leukemia inhibitor
factor; MEK kinase of ERK; Ox/ER stress, oxidative and endoplasmic reticulum stress
and seizures that commonly leads to early death. At this stage, all of the actions are
directed to impede the amplification of damage and the recurrence of crises and
catabolic states that will produce major sequels. Dietary treatment is the most
important tool to avoid recurrence and worsening. It includes all of the manipu-
lations needed to avoid the accumulation of toxic metabolites either by potentiating
or inhibiting remaining enzyme action, using alternate substrates, inhibiting its
precursors, or administering cofactors that favor enzyme activation such as cobal-
amin, thiamine, biotin, and riboflavin (Ruiz Pons et al. 2007). In some diseases such
as hepatic encephalopathy, the dietary management aimed at reducing nitrogenous

load is accompanied with non-absorbed agents (rifamixin) or enhancement of
ammonia removal, all attempting to reduce the levels of blood ammonia and other
gut-derived toxins (Butterworth 2010). In GA-I if dietary management starts in the
newborn period—before the occurrence of encephalopathic crises—most of the
patients remain asymptomatic (Kölker et al. 2012). GA-I usual diets are lysine-free,
tryptophan-reduced, and fortified in arginine that competes with lysine to reduce
enzymatic substrate availability (Zinnanti et al. 2007; Kölker et al. 2012).
A combination of diet with carnitine and emergency treatment has been demon-
strated to be effective in preventing neurological disease when the GA-I patient is
thought to be at risk (Kölker et al. 2012).
In addition to diet management, as oxidative stress may contribute to the
pathophysiology of many IEMs, antioxidant treatment is a common complementary
therapeutic approach. It is directed to reduce the brain vulnerability against the
attack of oxygen and nitrogen reactive species generated by abnormal metabolites
or subsequent mitochondrial dysfunction. In this regard, antioxidants such as
ascorbic acid, N-acetylcysteine, folic acid, lipoic acid, a-tocopherol, and L-carnitine
prevented learning/memory deficits and convulsions in animal models of organic
acidemias (Ribas et al. 2014) or hepatic encephalopathy (Butterworth 2010). In
aceruloplasminemia, the direct attack of iron-mediated lipid peroxidation and
oxidative stress with a systemic iron chelation alone or together with oral zinc
sulfate administration ameliorated the neurological symptoms (Miyajima 2015).
Symptomatic therapies are usually employed in neurometabolic diseases. For
example, antiepileptic drugs sometimes are used to keep patients free of seizures for
prolonged periods. Tricyclic antidepressants or CNS stimulants have been shown to
ameliorate cataplexy, whereas dystonia and tremor are usually treated with anti-
cholinergic drugs or gamma-aminobutyric acid derivatives when dystonia is
advanced (Ruiz Pons et al. 2007).
In summary, in spite of some advances in treatment and development of special
diets, IEMs remain as yet incurable diseases with scarce disease-specific therapies
and only general approaches that try to preserve overall systems avoiding recur-
rence of encephalopathic crises.
Could Astrocytes Be Therapeutic Targets in IEMs

and Developmental Neurometabolic Diseases in Particular?
The fact that astrocytes accomplish both adaptive and pathological functions makes
targeting them difficult. The astrogliosis observed in most neurodevelopmental or
neurological diseases is a good example of this problem, whereas glutamate uptake
is beneficial because of its antioxidant effects and associated release of neuropro-
tective agents; reactive astrocytes release inflammatory cytokines, produce reactive
oxygen species, and impede axonal growth (Maragakis and Rothstein 2006;
Verkhratsky et al. 2012, 2015; Pekny and Pekna 2014). Another complication in
targeting astrocytes is the fact that many of the molecules that astrocyte secrete may
be detrimental during some phases of damage, yet advantageous during others. For
example, vascular endothelial growth factor and matrix metalloproteinase inhibitors
acutely increase edema and stroke volume, but many days later both are critical for
angiogenesis and recovery (Zhao and Rempe 2010). In spite of these problems,
some neuroprotective attempts have been focused on astrocytes. It has been shown
that astrocyte-derived neuroprotective factors, such as glial-derived neurotrophic
factor (GDNF), reduce neuronal death in animal models of Parkinson Disease,
ALS, and stroke (Rappold and Tieu 2010; Vargas and Johnson 2010; Zhao and
Rempe 2010). However, side effects and conflicting results in human trials have
diminished the enthusiasm for GDNF as a potential treatment (Rappold and Tieu
2010). Similarly, erythropoietin, a neuroprotective molecule released by astrocytes
that reduces stroke volume in animal models showed promising results in phase 2,
but in phase 3 of clinical trials did not show efficacy (Ehrenreich et al. 2009). The
potentiation of astrocyte glutamate uptake by increasing the expression of glutamate
transporters has also been explored with promising effects in ALS animal models
(Vargas and Johnson 2010), but results in humans are still lacking. Arundic acid
reduces the production of S-100b, a calcium-binding protein proposed as indicator
of CNS damage, in activated cultured astrocytes (Vargas and Johnson 2010) but
was not effective for the treatment of stroke (Zhao and Rempe 2010).
Fewer approaches focused on astrocytes have been made in neurodevelopmental
disorders, probably because we are far from having a complete knowledge of
astrocyte roles in these diseases. In hepatic encephalopathy, targeting of astrocytic
glutamine synthase is proposed as a potential treatment to avoid brain ammonia
accumulation (Brusilow et al. 2010), but more results are needed. Compounds that
had the ability to reduce GFAP expression alone or together with antioxidant effects
or enhanced glutamate astrocytic uptake were employed to ameliorate the GFAP
alterations found as the main pathological features of Alexander disease. Whether
these findings obtained in cellular models can be easily translated into in vivo
treatments, and eventually to humans, remains to be seen (Cho et al. 2010; Messing
et al. 2012). Our experience in GA-I—that we propose as another primary
astrogliopathy—indicates that abrogating the initial astrocyte response to GA with
neuroprotective iron porphyrins (Wu et al. 2003) or MAPK inhibitors offers sig-
nificant neuroprotection in animal models (Olivera-Bravo et al. 2008). Although we
do not know the astrocyte pathways that are targeted by these compounds, the early
astrocyte response to GA could offer a potential therapeutic window for a selective
focus on astrocytes before the occurrence of significant myelin or neuronal damage.
Furthermore, as most IEMs are diagnosed after the whole CNS is damaged, it
makes unlikely that a pharmacological strategy focused on a single cell type can be
successful.
Concluding Remarks
The diversity and number of IEMs that have already been associated with astrocyte
dysfunction are remarkable. However, more significant research is needed to
understand what types of astrocyte dysfunction or how astrocytes themselves
contribute to the pathogenesis of each of these conditions. During development,
adequate neurogenesis and gliogenesis represent only the initial steps for nervous
system formation. Furthermore, a close neuron–astrocyte correlation is needed to
allow proper development and CNS function. Therefore, no therapeutic interven-
tion or pharmacological manipulation of astrocytes can be assumed solely to affect
astrocytes. Instead, each therapeutic approach should target astrocytes in concert
with neurons, other glial cells, and the vasculature, because all together will sculpt
the response to each strategy and finally determine its efficacy. Therefore, it is
necessary to consider the complex cell interactions in the search for efficacious
treatments for neurodevelopmental diseases, which represents an enormous
challenge.
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Microglia in Cancer: For Good
or for Bad?
Anna Carolina Carvalho da Fonseca, Rackele Amaral, Celina Garcia,

Luiz Henrique Geraldo, Diana Matias and Flavia Regina Souza Lima
Abstract Glioblastoma is a malignant tumor of astrocytic origin that is highly

invasive, proliferative and angiogenic. Despite current advances in multimodal
therapies, such as surgery, radio- and chemotherapy, the outcome for patients with
glioblastoma is nearly always fatal. The glioblastoma microenvironment has a
tremendous influence over the tumor growth and spread. Microglia and macro-
phages are abundant cells in the tumor mass. Increasing evidence indicates that
glioblastoma recruits these cell populations and signals in a way that microglia and
macrophages are subverted to promote tumor progression. In this chapter, we
discuss some aspects of the interaction between microglia and glioblastoma, con-
sequences of this interaction for tumor progression and the possibility of microglial
cells being used as therapeutic vectors, which opens up new alternatives for the
development of GBM therapies targeting microglia.
Keywords Microglia Glioblastoma Central nervous system Therapy
A.C.C. da Fonseca R. Amaral C. Garcia L.H. Geraldo D. Matias F.R.S. Lima (&)
Instituto de Ciências Biomédicas, Centro de Ciências da Saúde,
Universidade Federal do Rio de Janeiro, Avenida Carlos Chagas Filho,
373, Rio de Janeiro, RJ 21949-590, Brazil
A.C.C. da Fonseca
R. Amaral
C. Garcia
L.H. Geraldo
D. Matias

DOI 10.1007/978-3-319-40764-7_12
246 A.C.C. da Fonseca et al.

IL Interleukin
TNF Tumor necrosis factor
GBM Glioblastoma
MMP Matrix metalloproteinase
VEGF Vascular endothelial growth factor
DLL4 Delta-like ligand 4
NO Nitric oxide
MCP-1 (CCL2) Macrophage chemoattractant protein 1
CCR2 CCL2 receptor
TNFR1 TNF receptor 1
IkBa Nuclear factor of kappa light polypeptide gene enhancer in
B-cells inhibitor, alpha
NF-kB Nuclear factor of kappa light polypeptide gene enhancer in
B-cells
uPA Urokinase-type plasminogen activator
STI1 Stress-inducible protein 1
Hsp Heat shock protein
MG CM Microglial conditioned medium
MYD88/TLR8 Myeloid Differentiation Primary Response 88/Toll-like
receptor 8
TLR Toll-like receptor
MT1-MMP Membrane-type-1 MMP
GDNF Glial cell-line-derived neurotrophic factor
GFRa GDNF receptor
EGFR Epidermal growth factor receptor
EGF Epidermal growth factor
poly [I:C] Polyinosinic-polycytidylic acid
TRAIL TNF-related apoptosis inducing ligand
mTOR Mammalian target of rapamycin
MIF Macrophage migration inhibitory factor
STAT Signal transducer and activator of transcription
RAGE Receptor for Advanced Glycation End products
S100B S100 calcium binding protein B
CpG-ODN Oligodeoxynucleotides containing CpG motifs
TROY/TNFRSF19 Tumor necrosis factor receptor of mouse embryo
GSC Glioma stem cell
MRI Magnetic resonance imaging
GCV Gancyclovir
Microglia in Cancer: For Good or for Bad? 247
Tumor Microenvironment and the Microglia
The central nervous system (CNS) is composed of several cell types, including
neurons, astrocytes, oligodendrocytes, ependymal cells and microglia. Each cell
type has distinct and essential roles for the optimal functioning of the CNS. As
discussed in Chapters “Glial Cells and Integrity of the Nervous System,”
“Microglia Function in the Normal Brain” and “Purine Signaling and Microglial
Wrapping,” microglia are the resident immune cells in the CNS, but they are
increasingly recognized to play diverse roles. Their embryonic origin is mesoder-
mal, unlike other CNS cells, which have ectodermal origin. It appears that microglia
progenitors come from the yolk sac early in development (Ginhoux et al. 2010).
Phagocytosis of microorganisms, antigen presentation to lymphocytes, phagocy-
tosis of cell debris, transient or aberrant axons and apoptotic cells during devel-
opment, and secretion of neurotrophic factors are some of microglia functions in
CNS (Vilhardt 2005; Mallat et al. 2005; Lima et al. 2010). Following lesions,
microglia become active and assume an amoeboid phenotype and a high metabolic
rate, synthesizing and secreting several cytokines, such as interleukin (IL)6, IL1b
and tumor necrosis factor a (TNFa) (Vilhardt 2005; Yang et al. 2010).
Among all CNS pathologies, one of the deadliest is glioblastoma (GBM). This
malignant tumor of astrocytic origin is highly invasive, proliferative and angio-
genic. Its invasive nature explains the high recurrence even after surgical resection
(Lima et al. 2012). Survival is commonly about 14 months despite all efforts (Stupp
et al. 2005). The GBM microenvironment has a tremendous influence over the
tumor growth and spread. In a still not completely defined way, GBM subverts cells
to act in its favor. Astrocytes were shown to have an increase in MMP-2 (matrix
metalloproteinase-2) expression and to convert pro-MMP-2 to active form only in
the presence of glioma cells (Le et al. 2003; Gagliano et al. 2009), suggesting a
pro-tumor role of astrocytes. Besides, GBM cells produce VEGF (vascular
endothelial growth factor) and DLL4 (delta-like ligand 4), which stimulate the
angiogenesis that sustains tumor survival and growth (Bao et al. 2006; Li et al.
2007).
Microglia and macrophages are abundant cells in the tumor mass. GBM recruits
these cell populations (Fig. 1) and signals in a way that microglia and macrophages
are subverted to promote tumor progression. Moreover, GBM establishes an
immunosuppressed niche, favoring even more its survival, growth, and spread (da
Fonseca and Badie 2013). A current and important topic regarding these
glioma-associated microglia and macrophages is the existence of two phenotypi-
cally distinct cell populations classified as M1 and M2. M1 macrophages are
classically activated, developing an anti-tumor response through the activation of
the immune system and production of reactive oxygen species, nitric oxide
(NO) and proinflammatory cytokines, such as TNF. M2 macrophages are alterna-
tively activated, performing immunosuppressive roles, such as release of IL10 and
tumor promotion, as well as inducing metastatic processes by promoting angio-
genesis and extracellular matrix degradation (Sica et al. 2008; Yang et al. 2010;
Fig. 1 Xenotransplanted tumor produced from human GBM cells injected into the caudate
putamen of mouse brain. In this study, we used the human tumor cell line GBM95, established in
our lab (Faria et al. 2006). After 15 days, the brains were perfused with fixative 4 %
paraformaldehyde, cut into slices and immunostained with rabbit anti-mouse Iba1 (green) antibody
(Confocal Microscope/Leica TCS-SP5), a marker of microglia/macrophages cells. Nuclei were
stained with DAPI (blue). a Tumor mass. b Contralateral hemisphere, without tumor cells. Note
the presence of activated microglia/macrophages in the tumor mass (a) and ramified resident
microglia cells in the contralateral hemisphere (b). Bar 40 lm. This study was approved by the
Ethics Committee of the Health Sciences Center at the Federal University of Rio de Janeiro
(Protocol no. DAHEICB 015) and by the Brazilian Ministry of Health Ethics Committee (CONEP
no. 2340). The “Principles of laboratory animal care” (NIH publication no. 85‐23, revised 1996)
guidelines as well as The Code of Ethics of EU Directive 2010/63/EU were strictly followed for all
experiments
Albesiano et al. 2010; Herrera et al. 2013). Indeed, the escape of tumor cells from
the immune system has been related to a change of M1 to M2 phenotype during
tumor progression (Schmieder et al. 2012). Similar to macrophages, glioma-
associated microglia also present the M2 phenotype (Komohara et al. 2008;
Fonseca et al. 2012; Gabrusiewicz et al. 2011).
Microglia-Glioblastoma Interaction
Malignant gliomas, particularly GBMs, the most aggressive astrocytoma, contain

high levels of microglia infiltrates; about 30 % of tumor mass is composed of
glioma-associated microglia and macrophages which has led to the hypothesis that
microglia may have a role in GBM immunology (Badie and Schartner 2000; da
Fonseca and Badie 2013; Yang et al. 2010). Indeed, evidence strongly suggests that
microglia contribute to the immunosuppressive environment of GBMs and may
promote tumor growth (Schartner et al. 2005; Yi et al. 2011). In this context, the
accumulation of microglia in GBM tissue is due to local production of growth
factors and chemoattractants, such as CCL2, by GBM cells (Prat et al. 2000).
CCL2, also recognized as MCP-1 (macrophage chemoattractant protein 1), is one of
the most highly expressed chemokines in many CNS injuries and exerts its bio-
logical function by binding to its high affinity receptor CCR2, which is expressed
by microglia, astrocytes and brain microvascular endothelial cells (Yao and Tsirka
2014). The interaction between CCL2 and CCR2 triggers IL6 release by microglia,
which is associated with GBM aggressiveness (Li et al. 2010; Rolhion et al. 2001).
IL6 is implicated in many aspects of tumorigenesis, and it is identified as a growth
factor for glioma stem cells (Wang et al. 2009). In addition, IL6 has been found to
increase microglia production of MMP-2, facilitating tumor migration (Li et al.
2010). Markovic et al. (2005), using cultured brain slices where microglia were
previously depleted with clodronate-filled liposomes, showed that injected glioma
cells had decreased infiltrative capacity compared with glioma cells injected into
control slices, possibly because of the decrease in MMP-2 levels, produced by
microglia. Moreover, Platten et al. (2003), using a rat model of intracerebral glioma
cell line implant, demonstrated that the glioma cell line that recruited more
microglia resulted in a larger tumor mass, and they attributed this effect to
CCL2/CCR2 pathways. Another important factor during GBM progression is
TNFa, a proinflammatory cytokine widely secreted by microglia (Rivest 2009),
which stimulates the secretion of several molecules including CCL2, IL6, IL1b, and
NO (Allan and Rothwell 2001; D’Mello et al. 2009; Nadeau and Rivest 2000).
On TNF receptor 1 (TNFR1) activation, IjBa, a protein that blocks NFjB signaling
in resting cells, gets phosphorylated and degraded, leading to p65/p50 nuclear
translocation and transcriptional activation of NFjB target genes, including TNFa
itself (Baker et al. 2008; Tchoghandjian et al. 2013). The constitutively activated
NFjB has been associated with invasive behavior and malignancy of GBM
(Raychaudhuri et al. 2007; Tsunoda et al. 2005). NFjB activation triggers tran-
scriptional activation of pro-migratory genes, like CXC chemokines, urokinase-type
plasminogen activator (uPA) and matrix metalloproteinases, contributing to inva-
siveness of GBM (Tchoghandjian et al. 2013; Wu and Zhou 2010).
In addition to the factors released in the tumor microenvironment, proteins
constitutively expressed by microglia may be implicated in GBM maintenance.
Stress-inducible protein 1 (STI1) is a 66 kDa protein described as a co-chaperone
that binds to both Hsp70 and Hsp90 and regulates their activities (Chen and Smith
1998; Song and Masison 2005). Our group has shown that STI1 released by
microglia promotes tumor proliferation, modulates MMP-9 activity and stimulates
the migration of human GBM cell lines in vitro (Fonseca et al. 2012). We
demonstrated that microglial conditioned medium (MG CM) stimulated prolifera-
tion and migration of the GBM cell lines, and this effect was reversed when the
anti-STI1 antibody was added to the MG CM or STI1 was removed by
immunodepletion from the MG CM. Furthermore, the addition of STI1 antibody to

MG CM significantly decreased MMP-9 activity (Fonseca et al. 2012). These data
suggest that STI1 is an important factor for glioma progression. Using a glioma
model of intracranial and subcutaneous implant of GL261, a murine glioma cell
line, Fonseca et al. (2012) have also shown that STI1 expression increased with
tumor progression, and was also upregulated in glioma-associated microglia and
macrophages and infiltrating lymphocytes. On the other hand, STI1 expression did
not significantly change in circulating leukocytes, and even decreased in leukocytes
that infiltrated tumors propagated in the subcutaneous tissue. Therefore, these
results demonstrated that STI1 expression is modulated by the brain tumor
microenvironment, and for the first time correlated STI1 expression and glioma
progression (Carvalho da Fonseca et al. 2014). Altogether, we conclude that
microglia-GBM interaction determine the degree of GBM invasion, opening the
way for the development of new therapeutic approaches.
Microglial cells are substantial producers of MMPs and inducers of GBM
invasiveness (Alves et al. 2011; Hu et al. 2014). Hu et al. (2014) showed that
MMP-9 levels were expressed by Iba1+ cells in human tumor samples, indicating
that glioma-associated microglia were responsible for MMP-9 local production.
Also, they demonstrated that GBM cells released soluble factors that induced the
MMP-9 expression in glioma-associated microglia via Myeloid Differentiation
Primary Response 88/Toll-like receptor 8 (MYD88/TLR8) signaling pathway.
Interestingly, in this study, when microglial cells were treated with their inhibitor
minocycline, levels of MMP-9 and TLR2 (Toll-like receptor 2) in
glioma-associated microglia decreased, and consequently tumor invasion declined
(Hu et al. 2014). A previous study had shown that GBM cells induced the
expression of membrane-type-1 MMP (MT1-MMP) in glioma-associated micro-
glia, promoting tumor invasion via the TLR2 signaling pathway. In this work,
GL261 murine glioma cells were injected into TLR2 knockout mice, resulting in
smaller tumors and reduced MT1-MMP levels in glioma-associated microglia
(Vinnakota et al. 2013). Thus, it seems important to explore the role of MMPs and
TLRs in microglial cells, since they can stimulate tumor progression.
Another factor that contributes to microglial recruitment during tumor progres-
sion is the glial cell-line-derived neurotrophic factor (GDNF). GDNF is a neu-
rotrophic factor involved in dopaminergic neuronal survival, but it can also
contribute to tumor progression. High levels of GDNF have been observed in GBM
cells (Ng et al. 2009; Wiesenhofer et al. 2000); however, little is known about the
correlation of GDNF and the attraction of microglia during tumor progression. Ku
et al. (2013) have recently demonstrated that GDNF is expressed in GBM cells and
plays an important role in microglia recruitment during tumor progression They
showed that microglia expressed both GDNF receptors, GFRa-1 and GFRa-2, and
then they used transwell assays to understand how GDNF could modulate the
migration of microglia. For this, they used GBM cell conditioned medium depleted
of GDNF, by using a specific shRNA, and observed that there was reduced
microglia migration. In addition, they injected GBM cells not expressing GDNF
into mouse brain and, after 2 weeks, observed lower levels of Iba1+ microglia
infiltration and a reduced tumor size. So, GDNF is indeed an important factor
expressed by GBM cells for microglia attraction (Ku et al. 2013).
Epidermal growth factor receptor (EGFR) also plays a significant role during
GBM invasion and aggressiveness (Ohgaki and Kleihues 2007; Sangar et al. 2014).
It is possible that microglia cells stimulate GBM invasion through the EGFR sig-
naling (Nolte et al. 1997). In particular, Coniglio et al. (2012) demonstrated in vitro
that microglia secreted EGF, which, in turn, activated the EGFR on GBM cells and
consequently induced tumor migration.
Change in the Microglial Profile May Be Useful
Studies by Penfield (1925) led to the suggestion that microglial cells fight tumors
(for review see Charles et al. 2011; Li and Graeber 2012). However, other inves-
tigators continue to believe that microglial cells behave as expected for macro-
phages and are able to promote antigen presentation, release of cytokines and
phagocytosis even in the presence of GBM.
Kren et al. (2010) observed the expression of HLA-G and HLA-E,
immune-modulatory nonclassical molecules with anti-tumor activity, by
glioma-associated microglia and macrophages in most cases of human GBM of 26
samples analyzed. The role of these molecules in GBM is not well described. Even
so, their observations go against the hypothesis that microglial cells and macro-
phages may be attracted to the tumor site and promote tumor invasion through
inhibition of the cytolysis by NK-cell and T-cell, once they consider that the
detected expression of HLA-G and HLA-E in glioma-associated microglia and
macrophages indicates a role in immune functions (Kren et al. 2010). In this sense,
an in vitro study showed that the conditioned culture medium of microglia pro-
moted apoptotic cell death of glioma cells; when microglial cells were previously
treated with LPS or IFNc (Interferon c), this effect was more pronounced.
Proteomic analysis was used to identify the secreted proteins, and several cathepsin
proteases were found to be expressed, especially cathepsin B, as was NO, sug-
gesting a microglial role in tumor cytotoxicity (Hwang et al. 2009). Despite these
observations, much clinical evidence and many in vitro studies indicate that
microglia and macrophages that infiltrate the brain tumor have pro-tumor functions,
promoting cell growth and migration (Li and Graeber 2012; Alves et al. 2011; da
Fonseca and Badie 2013). On the other hand, after treatment with
polyinosinic-polycytidylic acid (poly [I:C]), an agonist for Toll-like receptor 3,
glioma-associated microglia obtained from patients with GBM started to secrete
toxic soluble factors when cocultured with different GBM cell lines. This was also
true when they used the supernatant of glioma-associated microglia previously
stimulated with poly (I:C). Interestingly, these factors had toxic effects only on
tumor cells, since astrocytes and neurons cultures were not affected (Kees et al.
2012). Thus, Kees et al. (2012) demonstrated that it is possible to change the
behavior of microglial cells from a tumor-supporting role to a tumor-suppressing
function after poly (I:C) exposure. In other words, switching the M2 profile
described for glioma-associated microglia, to an M1 profile led to gaining
anti-tumor activities. In the same way, Chiu et al. (2011) proposed that in vitro
microglial anti-tumor functions could be reestablished with treatment with IL12.
Indeed, after IL12 stimulation, microglia increased the levels of TRAIL
(TNF-related apoptosis inducing ligand) releasing and phagocytic activity.
In addition, Lisi et al. (2014) demonstrated that the inhibition of mTOR
(mammalian target of rapamycin), which is activated in gliomas by many dereg-
ulated pathways, polarizes glioma-activated microglia to an M1 profile conferring
cytotoxic functions upon microglial cells and preventing the M2 state that is
involved in tumor establishment. In fact, iNOS was increased followed by a
decrease in IL10 gene expression after treatment with rapamycin and its analog
RAD001 in microglia (Lisi et al. 2014).
A recent in vivo study showed that MIF (macrophage migration inhibitory
factor) is highly expressed on glioma cells, whereas its receptor, CD74, is expressed
only in glioma-associate microglia. In this study, GBM cells and glioma-associate
microglia were isolated from primary human tumors. A higher level of
CD74-positive glioma-associate microglia was associated with increased patient
survival, representing a positive prognostic parameter associated with the
anti-tumor M1 profile (Zeiner et al. 2014).
In face of all these studies, we can conclude that microglia are a potential
therapeutic target for the treatment of GBM. Certainly, the more we know about the
interaction between microglia and GBM cells, the more we will know about the
tumor biology. Data showing microglial cells promote tumors are substantial,
especially from studies mentioned above that have attempted to manipulate the
activation state of microglia, rescuing their M1 profile instead of M2 profile to fight
against tumors.
Possible Therapies Using Microglia as a Therapeutic Target

in the Fight Against Cancer
Currently, the state of the art therapy for GBM consists of surgical resection of the
tumor, followed by chemotherapy with Temozolomide and radiotherapy. Despite
the aggressive therapy, median survival remains only 14.6 months after diagnosis
(Stupp et al. 2005). Many preclinical and clinical studies are trying to improve
patient survival, but without success. Some strategies focus on the interaction of the
tumor cells with the microenvironment; we will discuss some of the strategies that
are being currently developed focusing on the microglia-glioma interaction.
The STAT 3 (signal transducer and activator of transcription 3) pathway is
constitutively expressed in high-grade gliomas (Yu et al. 2007; Weissenberger et al.
2004) and has already been implicated in GBM pathogenesis, progression and
immune evasion (Takeda et al. 1999; O’Farrell et al. 1998; Lang et al. 2002). STAT
3 is also upregulated in microglial cells under glioma influence (Zhang et al.

2009a), at least in part by interaction between glioma S100B and IL-6 with
microglial RAGE (Receptor for Advanced Glycation End products) and IL6R
(Bromberg and Wang 2009; Zhang et al. 2009b). The activation of this pathway
inhibits macrophage activation and reduces expression of co-stimulatory molecules
necessary for antigen presentation by naive T-cells; it increases the secretion of
immunomodulatory cytokines IL6 and IL10, while reducing lymphocyte-
stimulating cytokines (IL2, IL4, IL12 and IL15) (Cheng et al. 2003; Walker
et al. 2003; Hussain et al. 2007). Consistent with this, STAT 3 inhibition in glioma
cells using siRNAs reverses the cytokine expression profile, leading to mi-
croglia/macrophage activation and tumor growth inhibition in a mouse model
(Zhang et al. 2009a). Pharmacological agents such as small STAT 3 inhibitors that
penetrate the CNS have an anti-proliferative and proapoptotic effect on glioma cell
lines (Takeda et al. 1999; O’Farrell et al. 1998; Lang et al. 2002; Iwamaru et al.
2007). Apart from that, these agents are capable of reversing the immune tolerant
microenvironment by activating microglial cells, through production of
lymphocyte-stimulating cytokines (IL2, IL4, IL12, IL15 and CXCL10) and
upregulation of co-stimulatory molecules (CD80 and CD86), and also by stimu-
lating T-cell proliferation and inducing a Th1-response (Iwamaru et al. 2007; Cheng
et al. 2003; Hussain et al. 2007). There are on-going phase I and II clinical trials
with STAT 3 and its more important activator IL6 for several malignancies for
which this pathway is important, such as head and neck cancer, multiple myeloma
and prostate cancer (Sansone and Bromberg 2012). These trials might be translated
into a new therapeutic option for malignant gliomas, where this pathway was also
recently shown to be important.
The role of the immune system in the treatment of the CNS tumors gained
prominence with clinical trials using oligodeoxynucleotides containing CpG motifs
(CpG-ODN) for GBM patients. CpG-ODNs are strongly immunostimulating
agents, activating both innate and specific immunity. Biological effects of
CpG-ODN are mediated by Toll-like receptor 9 (TLR9) (Klinman 2004; Krieg
2004), mainly expressed by B-lymphocytes and plasmacytoid dendritic cells in
humans, and also by microglial and glioma cells (Ribes et al. 2010; El Andaloussi
et al. 2006). In pre-clinical models, local treatment with CpG-ODN injections,
either alone or combined with radiation therapy, reduced tumor size, with no
toxicity to brain parenchyma (Carpentier et al. 2000; Auf et al. 2001; Meng et al.
2005). It was shown that tumor rejection was due not only to direct toxicity in
tumor cells, but also to modulation of microglia/macrophages and induction of a
Th1 response (El Andaloussi et al. 2006; Carpentier et al. 2000; Auf et al. 2001).
This new therapy was so promising in preclinical models that it was rapidly
translated into phase I and II clinical trials. After a promising phase I study, with the
few side effects limited to transient worsening of neurological condition and fever
(Carpentier et al. 2006), phase II trials presented at the 2009 American Society of
Clincal Oncology (ASCO) annual meeting showed only modest activity in the
6-month progression-free survival (PFS) of the cohort, with only a few cases
showing radiological response (Carpentier et al. 2010; Ursu et al. 2009). This trial
did not define the clinical or molecular characteristics of patients who might have
benefitted from this trial, and this therapy was not continued.
Apart from these innovative strategies, old medications with newly discovered
functions are also being investigated. Some glioma drugs reduce tumor growth in
preclinical models by modulating microglial activity. The first, minocycline, a
semi-synthetic broad spectrum tetracycline antibiotic described as capable of
counteracting microglial activation into a proinflammatory phenotype by
p38-MAPK inhibition (Suk 2004), reduces tumor growth in vitro and in vivo by
inhibiting microglial MT1-MMP expression (Markovic et al. 2011). Another is
propentofylline, an atypical synthetic methylxanthine with CNS glial modulating
and anti-inflammatory actions (Si et al. 1996, 1998), described as capable of
decreasing tumor growth in preclinical GBM models by a direct effect on microglial
cells (and not in tumor-infiltrating macrophages) through tumor necrosis factor
receptor of mouse embryo (TROY/TNFRSF19) inhibition (Jacobs et al. 2012a, b).
And, more recently, Sarkar et al. (2014) demonstrated that microglial cells derived
from non-glioma human subjects can markedly reduce the sphere-forming capacity
of glioma stem cells (GSCs) by inducing cell-cycle arrest, reducing proliferation
and inducing differentiation, most likely through IL-8 and MCP-1. Apart from that,
Amphotericin B (a polyene antifungal drug) stimulates glioma-associated microglia
through TLR signaling, reducing GSC survival and sphere-forming capacity in a
manner resembling the action of microglia from healthy subjects. Daily treatment of
mice harboring intracranial GSCs with non-toxic doses of Amphotericin B also
substantially prolongs mouse survival (Sarkar et al. 2014).
Aside from pharmacological approaches, strategies have also been developed
using microglia as vehicles for gene therapy in conjunction with MRI tracking.
Ribot et al. (2007) labeled microglial cells with MRI contrast agents to ascertain
that the injected cells were migrating to the tumor mass. The labeled cells were also
transfected with a thymidine kinase suicide gene, which causes cell death after
administration of its substrate, gancyclovir (GCV). This system is suitable because
it induces a bystander effect: first, monophosphorylated GCV passes through
intercellular gap junctions and thereby triggers the death of cells that have not been
transduced; second, apoptotic bodies released by dead cells are taken up by adjacent
viable cells which then die, amplifying this phenomenon (Caruso et al. 1993; Qiao
et al. 2000; Burrows et al. 2002). Thus, a small quantity of enzyme and a low level
of transduction are sufficient to cause tumor regression (Spencer 2000) under
pharmacological control, since intracellular signaling occurs only if GCV is
administered. The investigators demonstrated that the injected microglial cells are
attracted to the tumor mass and that suicide gene activation with GCV reduces
tumor growth and prolongs survival in a preclinical model of human GBM in nude
mice (Ribot et al. 2007; Caruso et al. 1993; Qiao et al. 2000; Ribot et al. 2011).
Recent knowledge of microglia’s effects on the microenvironment of malignant
gliomas has led to the discovery of several pathways that are promising therapeutic
targets and a new prognostic molecular marker. This time, instead of a mutation or
protein expression in tumor cells, the prognostic marker is a polymorphism in a
microglial chemokine receptor gene associated with cell migration: the
CX3CR1-I249 allele. This allele variant is associated with prolonged mean survival
of GBM patients (23.5 v 14.1 months; P < 0.0001) and with reduced tumor infil-
tration by microglia (Rodero et al. 2008). For the first time, a microglial marker has
been characterized as an independent, favorable prognostic factor and might be
useful in predicting survival in GBM patients.
Since the phase III trials of Temozolomide in 2005, many options have been
studied for the treatment of malignant gliomas without much success, including
anti-angiogenic therapy with Bevacizumab (Avastin®) (Chinot et al. 2014) and
such other chemotherapy regimens as Procarbazine, Lomustine (CCNU), and
Vincristine (PCV) (Brada et al. 2010). Also, many strategies that seemed promising
in preclinical trials, such as the CpG-ODNs, are rather disappointing in clinical
trials. Thus, understanding of the biology of the CNS tumors and of the microen-
vironment’s influence on tumor progression is becoming increasingly important for
developing new therapeutic strategies for this deadly disease.
Concluding Remarks
GBMs are the most aggressive tumors of astrocytic lineage. Despite significant
progress in cancer research, which has led to the development of more effective
therapies for some types of solid tumors, there is no effective treatment for GBM. In
this chapter, we discussed some relevant properties of microglia in contact with
GBM. Better understanding of the interactions between the tumor and its
microenvironment, particularly microglial cells, is important for combating GBM.
In this sense, the development of new therapies targeting the microglia or the
factors produced by them that are specifically related to tumor progression may be
an effective alternative.
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Peripheral Inflammation
and Demyelinating Diseases
Verónica Murta and Carina Ferrari
Abstract In recent decades, several neurodegenerative diseases have been shown to

be exacerbated by systemic inflammatory processes. There is a wide range of lit-
erature that demonstrates a clear but complex relationship between the central ner-
vous system (CNS) and the immunological system, both under naïve or pathological
conditions. In diseased brains, peripheral inflammation can transform “primed”
microglia into an “active” state, which can trigger stronger pathological responses.
Demyelinating diseases are a group of neurodegenerative diseases characterized by
inflammatory lesions associated with demyelination, which in turn induces axonal
damage, neurodegeneration, and progressive loss of function. Among them, the most
important are multiple sclerosis (MS) and neuromyelitis optica (NMO). In this
review, we will analyze the effect of specific peripheral inflammatory stimuli in the
progression of demyelinating diseases and discuss their animal models. In most
cases, peripheral immune stimuli are exacerbating.
Keywords Demyelinating diseases

Systemic inflammation Microglia
Multiple sclerosis
Neuromyelitis optica
Experimental autoimmune
encephalomyelitis

AQP4 Aquaporin-4
BBB Blood–brain barrier
CCL2 Chemokine CC motif ligand 2
CCR2 Chemokine CC motif receptor 2
CD Cluster of differentiation
V. Murta
Laboratorio de Neuropatología Molecular, Instituto de Biología
Celular y Neurociencias, Universidad de Buenos Aires, Buenos Aires, Argentina
C. Ferrari (&)
Instituto de Ciencias Básicas y Medicina Experimental,
Instituto Universitario del Hospital Italiano, Buenos Aires, Argentina
e-mail: [email protected]; [email protected]

DOI 10.1007/978-3-319-40764-7_13
264 V. Murta and C. Ferrari

CXCR2 CXC motif chemokine receptor type 2
GC Glucocorticoids
HPA Hypothalamic-Pituitary-Adrenal
HPG Hypothalamic-Pituitary-Gonadal
IFN Interferons
IgG Immunoglobulin G
IL Interleukin
MHC Major histocompatibility complex
NMO Neuromyelitis optica
PMN Polymorphonuclear
PPMS Primary progressive MS
RRMS Relapsing remitting multiple sclerosis
SGK1 Serum glucocorticoid kinase 1
SPMS Secondary progressive multiple sclerosis
TGF-β Transforming growth factor beta
Th T helper
TLR Toll-like receptors
TNF-α Tumor necrosis factor α
WBC White blood cells
Peripheral Inflammation and Neurodegenerative Diseases
Inflammation can be viewed as one of the primary responses of the immune system
to infections or body injury. Systemic inflammation is associated with several
chronic diseases, including obesity, type 2 diabetes, atherosclerosis, liver disease,
and cancer (reviewed in Wilson et al. 2010; Fung et al. 2012). Additionally, it may
also be associated with an acute stimulus, such as infection, surgery, and acute organ
injury (Ottani et al. 2009). Systemic inflammatory stimuli that circulate in the blood
may induce the synthesis of cytokines in the central nervous system
(CNS) (Besedovsky and del Rey 1996; Pitossi et al. 1997; Combrinck et al. 2002;
Dantzer et al. 1998, 2008; Londono and Cadavid 2010). In a diseased brain, this
production of proinflammatory molecules exacerbates ongoing brain damage in
several neurodegenerative diseases, such as Alzheimer’s disease, multiple sclerosis
(MS), Parkinson’s disease, prion disease, and stroke (Perry et al. 2002; Cunningham
et al. 2005a, b; McColl et al. 2007; Palin et al. 2008; Ferrari and Tarelli 2011; Murta
and Ferrari 2013). In this review, we will discuss the influence of specific systemic
Peripheral Inflammation and Demyelinating Diseases 265
proinflammatory stimuli on different demyelinating diseases and animal models, and

the role of several cells and molecules in this phenomenon.
Microglia as a Mediator of Systemic Inflammation

and Neurodegenerative Diseases
Microglia are the resident immune cells of the CNS; their main role is monitoring
the local environment and triggering an immune response after specific stimuli in
the nervous tissue. As discussed in Chapters “Glial cells and Integrity of the
Nervous System”, “Microglia function in the normal brain”, and “Purine Signaling
and Microglial Wrapping“, microglia activation is characterized by morphological
and physiological changes such as secretion of proinflammatory and
anti-inflammatory cytokines. Therefore, microglia can exert either cytotoxic or
repairing actions, and these are referred as the M1-like and M2-like responses
(Samad et al. 2001).
Resting microglia have a ramified morphology and represent a more quiescent
basal state of this cell type. Systemic infections or mild central neurodegenerative
processes can activate and prime the resting microglia. Priming of microglia
precedes a further neurotoxic activation, which a secondary inflammatory stimulus
can transform into an “active” state (Samad et al. 2001; Cunningham et al. 2005b;
McColl et al. 2007). Microglia activation to an M1 phenotype increases neuro-
toxicity and, therefore, contributes to neurodegeneration through the release of free
radicals such as superoxide radicals and nitric oxide (through the action of inducible
nitric oxide synthase, iNOS) (Minghetti et al. 1999; Czlonkowska et al. 2002;
Arimoto and Bing 2003), and immunomodulatory cytokines such as interleukin
(IL) 1β, tumor necrosis factor α (TNFα), IL6, IL8, IL12, IL15, and IL10 (Kim and
de Vellis 2005; Dilger and Johnson 2008; Henry et al. 2009). Therefore, ongoing
inflammatory degenerative processes can be accelerated by systemic inflammation
through the stimulation of “primed” microglial cells toward a more aggressive state,
which in turn exacerbates damage in the nervous tissue (Fig. 1).
Communication Between the Periphery and the CNS
The brain used to be considered an “immune-privileged” organ isolated from the

peripheral immune system. Nowadays, it is well known that a bidirectional pathway
between the brain and the peripheral immune system exists.
Circulating cytokines and other inflammatory molecules can affect the brain
through several routes, mainly through the neural or humoral pathways. The neural
pathway is mainly related to the transmission of peripheral inflammatory signals
Fig. 1 Schematic diagram showing the relationship between peripheral inflammation and
demyelinating diseases. Demyelinating diseases are characterized by microglia activation; in
which microglia change their morphology from resting (ramified) towards an activated
round-shaped stage. The intermediate stage, “primed microglia,” represents the microglial stage,
which precedes a further neurotoxic microglial activation as a consequence of a secondary
proinflammatory stimulus. The peripheral stimuli come from the periphery either through neural or
humoral pathways and influence microglia activation. Activated microglia releases proinflamma-
tory cytokines which can, in turn, act on myelin sheath integrity, thereby inducing demyelination,
axonal loss and neurodegeneration
through the vagal afferent nerve (Perry et al. 2003; D’Mello et al. 2009; Gautron and
Laye 2009; Teeling and Perry 2009; Campbell et al. 2010). The humoral pathway
involves the direct action of peripheral proinflammatory cytokines (e.g., IL1β,
TNFα, and IL6) and type I interferons (IFNα and IFNβ) that can initiate the syn-
thesis of cytokines within the CNS, through blood–brain barrier (BBB) dependent
or independent pathways (Perry et al. 2003; Teeling and Perry 2009).
Demyelinating Diseases
As mentioned in Chapter “Glial cells and Integrity of the Nervous System”,

demyelinating diseases are a group of neurodegenerative diseases characterized by
inflammatory lesions associated with demyelination, which in turn induces axonal
damage, neurodegeneration, and progressive loss of function. Among them, the
most important are MS, neuromyelitis optica (NMO), acute demyelinating
encephalomyelitis, multifocal leukoencephalopathy, Guillain Barré syndrome, and
acute disseminating encephalomyelitis. This review will mostly focus on MS and
NMO, which are the most frequent in humans, and the most studied.
Multiple Sclerosis
MS is a chronic inflammatory disease characterized by multifocal and repeated

inflammatory events associated with demyelination–remyelination and axonal
damage, which leads to poor conduction of the nervous impulse and eventual loss
of sensory and motor function.
MS follows a varied clinical course, but most patients exhibit a course of
repeating exacerbation and remission from the onset (relapsing/remitting MS or
RRMS) eventually leading to secondary progressive multiple sclerosis (SPMS),
which worsens the patients’ quality of life (Playfair and Chain 1979; Neumann
et al. 1998). A minority of patients exhibit primary progressive MS (PPMS), which
is characterized by a constant decline from the onset with no recovery in neuro-
logical function (Playfair and Chain 1979; Loddick and Rothwell 2002).
Despite the fact that BBB breakdown is a major MS hallmark (McQuaid et al.
2009; Larochelle et al. 2011), some components of the inflammatory response
contribute to the pathology even with an intact BBB (Buljevac et al. 2002;
Lindquist et al. 2011). Although it has been proposed that in RRMS BBB break-
down allows the invasion of inflammatory cells, in the progressive forms inflam-
mation remains enclosed behind an intact BBB (Playfair and Chain 1979).
Relapsing and Remitting MS
RRMS is the prevalent clinical type of MS and is characterized by recurrent episodes

of new or worsened symptoms. Exacerbations or relapses are followed by periods of
partial or complete remission, with apparent clinical stability between relapses.
Relapsing episodes are unpredictable; however, peripheral inflammation may exac-
erbate these events (see below). Infections and other proinflammatory events have
been postulated as possible triggers of the pathology and/or of relapsing episodes, and
some authors have hypothesized that the autoimmune response could be a conse-
quence of a primary central proinflammatory event (Barnett and Prineas 2004).
Progressive Multiple Sclerosis
The progressive forms of MS lead to a continuous and irreversible evolution of the

disease, inducing decline of the quality of life either from the onset (PPMS) or after
a course of relapsing and remitting episodes (RRMS), named SPMS. SPMS is
diagnosed as a worsening after relapsing-remitting phases, with or without acute
exacerbations during the progressive stage (Wagner 1996). PPMS is a distinct,
non-inflammatory, or less inflammatory pathologic form of MS. The progressive
forms of MS are characterized by gray matter atrophy, which could be involved in
physical and cognitive disability (Rivest et al. 2000; Pocock and Kettenmann 2007;
Qian et al. 2012). Cortical lesions have peculiar inflammatory and demyelinating
hallmarks, characterized by lack of BBB disruption, differential inflammatory
process, and reactive microglia, suggesting different immunopathogenic mecha-
nisms (Vitkovic et al. 2000). However, anti-inflammatory or immunomodulatory
therapies have no effect on neurodegeneration and cognitive impairment in the
progressive forms of MS (O’Connor et al. 2005; London et al. 2013). This could be
related to the fact that in progressive MS, the inflammation creates an environment
that favors retention of inflammatory cells within the lesions (Konsman et al. 1999;
Godbout et al. 2005).
Neuromyelitis Optica
NMO, or Devic’s disease, is a demyelinating disease characterized by inflammatory

demyelinating lesions mainly in the spinal cord and optic nerve, potentially leading
to paralysis and blindness. It used to be considered a subtype of MS, but the
pathology and clinical features make them different diseases (Mosher et al. 2001).
NMO is characterized by seropositivity for immunoglobulin G (IgG) antibodies
against the astrocytic water channel aquaporin-4 (AQP4), and secondary inflam-
mation with granulocyte and macrophage infiltration, BBB disruption, and oligo-
dendrocyte injury. Therefore, an adaptive immune response to AQP-4 underlies the
chronic demyelinating in NMO.
The etiology of the disease is still unclear, but infections and BBB permeabi-
lizing factors could be involved in triggering the overproduction of AQP4-IgG, and
its access to the CNS (Schafer et al. 1999; Galiano et al. 2001). Uzawa et al. (2010)
demonstrated a significant difference in the levels of some cytokines/chemokines
(e.g. IL-6 for NMO) in the cerebrospinal fluid (CSF) of patients with NMO or MS,
supporting the view that different immunological and pathophysiological mecha-
nisms exist between them.
Current NMO therapies are directed toward reducing the inflammatory response
and the NMO–IgG load, such as B cell depletion and plasmapheresis. However,
most MS treatments, such as IFNβ, fingolimod, and natalizumab, exacerbate NMO.
Therefore, it is necessary to better comprehend the diseases’ underlying mecha-
nisms and differentiate NMO from MS.
MS and Peripheral Inflammation
MS is a neurodegenerative disease mainly characterized by inflammatory processes.

Activation of systemic immunity affects primed microglia in the CNS, reactivating
lesions and increasing parenchymal inflammation. Although relapsing episodes in
RRMS are unpredictable, most relapses are concomitant with peripheral inflam-
mation (Buljevac et al. 2002). RRMS patients show increased serum levels of IL1β,
IL2, IL4, IL12p70, IFNγ, and TNFα during the relapse phase (Nathan 2006;
Edwards et al. 2011; Trenova et al. 2011), as well as higher numbers of IL1β, IL6,
and TNFα secreting cells (Ysrraelit et al. 2008), and increased levels of T helper
(Th)17 and Treg cells in the periphery (Edwards et al. 2011). Moreover, a change in
CSF cytokine profile is observed during relapses; ranging from high levels of IL1β,
TNFα, and transforming growth factor beta (TGFβ) to lower levels of IL-10
(Hauser et al. 1990; Edwards et al. 2011). However, the treatment of MS patients
with TNFα inhibitors results in the exacerbation of central lesions (reviewed in
Perry et al. 2003).
Differences in cytokine expression patterns are described when comparing
progressive MS and RRMS (during relapses). SPMS patients present elevated
levels of chemokine CC motif receptor 2 (CCR2) in T cells, increased serum/CSF
levels of chemokine CC motif ligand 2 (CCL2) (Brinkmann et al. 2004), and
decreased plasma/CSF values of TNFα and IL4 (Schmitz and Chew 2008).
Peripheral blood mononuclear cells of both remitting RRMS and SPMS patients
express low levels of IL10 mRNA, which return to basal levels during relapses in
the RRMS form (Berkenbosch et al. 1987). Additionally, the progressive forms are
characterized by a permanent peripheral type 1 immune activation, which could
contribute to CNS damage during the progressive phase of the disease (Playfair and
Chain 1979; Hampton et al. 1998). Thus, the peripheral blood of SPMS patients
seems to reflect the inflammatory response accumulated in the CNS (Playfair and
Chain 1979). On the other hand, RRMS is characterized by waves of T helper (Th)1
and Th17 cells, which are recruited into the brain causing the attacks
(Neumann et al. 1998).
Inflammatory Stimuli Associated with MS
Although there is a clear association between systemic inflammation and the onset
or progression of different neurodegenerative pathologies, the particular nature of
these inflammatory phenomena is also relevant. Numerous studies, cited below,
have investigated the role of specific systemic proinflammatory stimuli including
acute or chronic stimuli, physiological imbalances, or external infections and
injuries. A summary of the roles of distinct proinflammatory stimuli in MS will be
addressed in the following section.
Obesity
During the last few years, a strong connection between metabolism, immunity, and
inflammation was described. Obesity is considered an inflammatory disease,
associated with metabolic and cardiovascular complications. Adipocyte tissue acts
as an endocrine organ releasing adipocytokines, and is associated with increased
levels of tissue and circulating inflammatory biomolecules (Oh et al. 1998).
Excessive adipose tissue increases the number and activity of macrophages, mast
cells, neutrophils, and lymphocytes (Ott et al. 1994; Kossmann et al. 1995).
Moreover, leptin (an adipocyte-derived cytokine) has a role in regulating both
innate and adaptive immunity (Bradl and Lassmann 2009), promoting the pro-
duction of cytokines such as TNFβ, IL6, IL12, IL15, and granulocyte
colony-stimulating factor in macrophages, and increasing their phagocytic activity,
as well as inducing the chemotaxis of neutrophils (Bradl and Lassmann 2009; Lee
et al. 2011; Golde et al. 2013; Procaccini et al. 2014). High levels of leptin have
also been reported in both active inflammatory lesions and serum of MS patients
(Batocchi et al. 2003).
Clinical data have demonstrated that obesity worsens the onset and progression
of most autoimmune diseases, such as rheumatoid arthritis, systemic lupus ery-
thematosus, inflammatory bowel disease, MS, type-1 diabetes, and psoriasis.
Additionally, it impairs a positive response to the treatments usually given for these
diseases (Cardona et al. 2008). Data show that 18-year old obese people are twice
as likely to develop MS as their normal weight age mates (Banisadr et al. 2005).
However, even if it seems quite clear that obesity and diet may influence the
progression of MS, few studies have linked a caloric restriction diet to reduced MS
progression (Procaccini et al. 2014).
Aging
Aging processes induce a generalized proinflammatory state in the organism. This

change is induced by increased immune responses in the periphery, disruption of
the periphery-CNS immune communication, and an increment in “primed” mi-
croglia, which increases CNS reactivity (reviewed in Veenstra and Ransohoff
2012). Microglia in aged brains exhibit upregulated major histocompatibility
complex (MHC) class II, complement receptors, toll-like receptors (TLR) 4, and
cluster of differentiation (CD) 14 expression (see Chapter “Age-Dependent
Changes in the Activation and Regulation of Microglia” for further reading).
Therefore, peripheral innate immune stimulation induces microglial cells in aged
brains to have an exaggerated inflammatory response compared with younger
cohorts (Sly et al. 2001; Dilger and Johnson 2008).
PPMS and SPMS manifest around 10 years later than RRMS, therefore the
timeline at which patients develop neurological deficit in PPMS and SPMS is
remarkably similar, and both include aging as a major risk factor for MS pro-
gression (reviewed in Kutzelnigg et al. 2005).
Infections
Infectious pathogens have been described as important factors involved in the

development of MS. Moreover, clinical studies revealed an association between
infections and relapses, which worsen neurological damage even after the infection
is gone (Buljevac et al. 2002; Panitch 1994). Pathogens associated with the exac-
erbation include bacteria (such as Mycoplasma pneumonia, Chlamydia pneumo-
niae, and Staphylococcus aureus-produced enterotoxins), virus (Epstein-Barr virus
and human herpes virus, and human endogenous retrovirus), and the protozoan
(Acanthamoeba castellanii). Viral infections that trigger MS episodes can be
reduced with IFN-γ treatment (Panitch 1994; Andersen et al. 1993).
Studies of MS patients and of animal experimental models have demonstrated
the influence of these infectious agents on the development and/or exacerbation of
MS (Krieger et al. 1992). However, not all infections cause progression of MS,
since it has been reported that infections with some parasites, such as helminthes,
can protect against the exacerbation phase of the disease (Correale and Farez 2011a,
b; Krieger et al. 1992). This protection is associated with the induction of CD4+,
CD25+ T cells secreting IL10, and TGFβ (Correale and Farez 2011a).
Immune Regulation by the Neuroendocrine System
The neuroendocrine system exerts its action on the immune system through finely
tune regulation. Glucocorticoids (GCs) induce the production of pro- and
anti-inflammatory cytokines, specifically causing a shift from Th1 to Th2 immune
response. GCs inhibit the production of Th1 related cytokines (IL1 and IL6, IL2,
IL12, IFNγ) and increase the secretion of anti-inflammatory Th2 cytokines (IL4 and
IL10) (Haak et al. 2009). However, GCs can increase both peripheral and central
inflammatory responses to a systemic challenge if they are administered before the
peripheral stimuli (Sorrells and Sapolsky 2010; Frank et al. 2010). Therefore, GCs
can prime the immune response and, as a consequence, increase proinflammatory
cytokine production and exacerbation of MS symptoms.
The Hypothalamic–Pituitary–Adrenal (HPA) Axis
Clinical and experimental studies have demonstrated that abnormalities in the HPA
axis, which influences the immune response, may exacerbate MS symptoms
(Hofstetter et al. 2005; Seo et al. 2013). Thus high cortisol levels are often
correlated with acute relapses (Hofstetter et al. 2005; Seo et al. 2013), whereas
prolactin increases the peripheral production of IFNγ and IL12 by T cells
(Du and Dreyfus 2002).
The Hypothalamic–Pituitary–Gonadal (HPG) Axis
MS affects predominantly women in comparison with men, therefore, considering

that gender affects the course of autoimmune diseases, the influence of sex hor-
mones is critical (Dunn et al. 2015a, b). In particular, 17β-estradiol induces an
increase of Th2 cytokines (IL10 and IL4) and a decrease of Th1 cytokines (TNFα
and IFNγ) (van Riemsdijk et al. 2001; Janik et al. 1997). Estrogens, in addition to
their anti-inflammatory effects, appear to be neuroprotective in CNS diseases, such
as MS and Alzheimer’s, disease (Nicot 2009; Gao and Tsirka 2011). Additionally,
both clinical symptoms and relapse rates of MS are decreased during pregnancy,
whereas the postpartum period increases the risk for exacerbation of the disease
(Ling et al. 1997).
The increased secretion of estrogen, progesterone, and cortisol during pregnancy
is associated with increased production of Th2 cytokines and decreased production
of Th1 cytokines (Takii et al. 1992, 1994). Additionally, progesterone also inhibits
NFκB and increases IL4 production, demonstrating its anti-inflammatory effect
(Piccinni et al. 1995; Nishiyori et al. 1997). Male hormones, such as testosterone,
also inhibit both innate and adaptive immune systems by enhancing the production
of IL5 and IL10, and decreasing IFNβ secretion, thus promoting a Th2 response
(Murphy and Sturm 1923).
Environmental Factors and Peripheral Inflammation
Environmental factors have influence on most autoimmune diseases.

Epidemiological risk factors for MS, including low vitamin D and elevated salt
intake, are associated with peripheral inflammation. Recent studies have shown that
components of the daily diet and gut microbiota can strongly affect the levels of
effector T cells in the gut (Ransohoff et al. 2007).
On the other hand, high sodium chloride concentrations induced expression of
serum glucocorticoid kinase 1 (SGK1) in T cells, which in turn stimulate the
induction of Th17 cells from CD4+ T cells, promoting autoimmune diseases
(Glabinski et al. 1997). However, direct correlation between salt intake and inci-
dence of autoimmune disease is yet to be demonstrated (Tsai et al. 2002).
Vitamin D plays an important role in the regulation of the immune responses
(Semple et al. 2010), modulating many inflammatory mechanisms including:
(a) the regulation of inflammatory mediators, such as cytokines (IL1β, TNFα,
IL6, TGF1β) and cyclooxygenases, (b) the interference with transcription factors,
such as NFκB, and (c) the activation of signaling cascades, such as MAP kinases
(Xia and Hyman 2002; Bakshi et al. 2011; Semple et al. 2010; Perry and Teeling
2013). MS exacerbation correlates with low levels of Vitamin D, whereas vitamin
D supplementation has a protective effect (Aubert et al. 1995; Romeo et al. 2001;
Varvel et al. 2012).
NMO and Peripheral Inflammation
There is not much evidence for peripheral inflammation affecting NMO, in contrast
with MS. However, recent work shows that the peripheral immune system affects
the progression of NMO. The CSF of NMO patients shows white blood cells
(WBC) ≥50 cells/mm3 or ≥5 neutrophils/mm3 compared to control patients,
whose counts are <5 WBC/mm3 (Campbell et al. 2008). Additionally, removing
inflammatory mediators from the blood of MNO patients alleviates the symptoms
(Okada et al. 2006).
NMO can occur concomitantly with systemic autoimmune disorders such as
Sjogren’s syndrome and systemic lupus erythematous, which likely reflects an
underlying predisposition for these patients to develop autoimmune disorders.
Moreover, the presence of other systemic disease can increase the mortality rate in
relapsing NMO patients (Krady et al. 2008).
Finally, the seropositivity for NMO–IgG represents a key factor for predicting
future relapses; indeed, it is a prognostic marker for NMO. Additionally, humoral
immune mechanisms, including the activation of B cells and the complement
pathway, have been said to play a role in NMO pathogenesis (Quan et al. 2013;
Kim et al. 2011).
Experimental Models of Demyelinating Diseases
Experimental models of demyelination help in understanding the pathophysiology

of such demyelinating diseases as MS (Denic et al. 2011) and NMO (Linington
et al. 1992). Animal models can be divided into two groups: those which attempt to
replicate the disease as accurately as possible and others that provide a reductionist
approach to the diseases by studying demyelination and remyelination processes
(e.g., ethidium bromide, lysolecithin, and cuprizone) (reviewed in Blakemore and
Franklin 2008). For MS, the most common models have been virus-induced
encephalomyelitis and various forms of Experimental Autoimmune
Encephalomyelitis (EAE) (reviewed in Dai et al. 2003).
A clear distinction between NMO and MS only became possible in the past
decade, and nowadays the most frequently used NMO models are NMO/EAE,
NMO-IgG/complement intracerebral injection, and cytokine-injection NMO
(Linington et al. 1992). Some of the main features present in these experimental
models are summarized in Table 1. Consistent with the human diseases, animal
Table 1 Summary of the main features of MS and NMO experimental models

Experimental model Disease features Reference
Lysolecithin and MS and NMO Blakemore
Ethidium Bromide Demyelination–Remyelination (2008)
Demyelination NO autoimmune component
Microglial and Astroglial activation
Cuprizone induced MS and NMO Wilkins et al.
Demyelination Demyelination–Remyelination (2001)
No autoimmune component
Cytokine mediated inflammatory response
Growth factors involved in remyelination
Virus- induced MS Grigoriadis and
Encephalomyelitis BBB breakdown Hadjigeorgiou
Demyelination (2006)
Axon pathology
Cytokine upregulation
Central and systemic inflammatory response
Autoimmune component
Involvement of different immune cells (T cells,
B cells)
EAE (active, passive, MS Dai et al.
or transgenic models) Relapsing-remitting and progressive forms (2003)
BBB breakdown
Demyelination (sometimes remyelination)
Axon pathology
Cytokine upregulation
Central and systemic inflammatory response
Autoimmune component
Involvement of different immune cells
(depending on the model): T cells, B cells,
granulocytes
NMO/EAE NMO Linington et al.
Autoimmune component (AQP4 IgG) (1992)
BBB breakdown Bradl and
Loss of AQP4 expression Lassmann
Presence of astrocyte destructive lesions (2014)
Neutrophil and T cell infiltration to the CNS
Activation of microglia/macrophages
Demyelination
Oligodendrocyte death
NMO-IgG/complement NMO Linington et al.
Autoimmune component (AQP4 IgG) (1992)
BBB breakdown
Loss of AQP4 expression
Presence of astrocyte destructive lesions
Neutrophil and macrophage infiltration to the
CNS
Demyelination
Neuronal death
(continued)
Table 1 (continued)
Experimental model Disease features Reference
Cytokine-injection NMO Linington et al.
NMO Autoimmune component (AQP4 IgG) (1992)
BBB breakdown
Loss of AQP4 expression
Presence of astrocyte destructive lesions
Neutrophil infiltration to the CNS
Demyelination
Activation of microglia/macrophages
experimental models show the influence of peripheral inflammation on the

progression of the disorders.
The importance of humoral components of the immune system is evident in
EAE. For example, a specific cytokine profile appears during the different phases of
acute the EAE model: decreased IL21 expression on the peak phase and high IL22
expression during the induction phase that decreases during recovery (Almolda
et al. 2011). Additionally, systemic TNFα causes clinical signs to recrudesce and
induces relapses in EAE (Crisi et al. 1995).
MS Animal Models and Systemic Inflammation
Obesity
Immunomodulatory effects of leptin, the adipocyte-derived hormone, are involved

in the induction and progression of EAE (Matarese et al. 2001, 2008). In this
context, the use of leptin antagonists improved the course of EAE (De Rosa et al.
2006). Moreover, the leptin-deficient (ob/ob) mice do not develop EAE; however,
exogenous leptin treatment renders ob/ob mice susceptible to EAE development
(Matarese et al. 2001). On the other hand, caloric restriction, (associated with low
levels of leptin in plasma) can significantly increase the overall survival in several
experimental animal models of autoimmune diseases (Oka et al. 2007).
Infections
Peripheral infection with enterotoxin A or B exacerbates clinical signs and induces

relapses in EAE (Brocke et al. 1993; Crisi et al. 1995; Schiffenbauer et al. 1993).
A single dose of peripheral LPS can induce increased inflammatory, demyelinating
and axonal damage in EAE lesions (Serres et al. 2009; Moreno et al. 2011) as well
as CD4+ cells activation (Nogai et al. 2005). Additionally, respiratory tract

pathogens (Streptococcus pneumonia and Chlamydia pneumonia) aggravate EAE
symptoms (Du et al. 2002; Herrmann et al. 2006; Tauber et al. 2007).
On the other hand, some data have been published demonstrating beneficial
effects of peripheral LPS. In those studies, pretreatment with LPS prior to EAE
induction lead to a delay in the onset of the disease by suppressing antigen pre-
sentation and altering the expression of inflammatory mediators (Buenafe and
Bourdette 2007).
The presence of blood-derived peripheral polymorphonuclear neutrophils
(PMN) expressing CXC chemokine receptor type 2 (CXCR2) is requisite for
oligodendrocyte death, demyelination, and BBB breakdown in both EAE and
cuprizone models (Liu et al. 2010; Carlson et al. 2008). Peripheral PMN are con-
sidered the first key effector leukocytes in the pathogenesis of EAE; they produce
cytokines and chemokines that in turn induce lymphocyte and monocyte activation
(Carlson et al. 2008).
Moreover, the importance of PMN neutrophils for the development of a
demyelinating lesion in the CNS of rats has been seen in a model of chronic
neuroinflammation and demyelination in response to a sustained expression of IL1β
in the CNS (Ferrari et al. 2004). Furthermore, a relapsing-like lesion was achieved
in the same model by inducing a peripheral sustained expression of IL1β (Murta
et al. 2015). Here, the involvement of CXCR2 + PMN neutrophils from the
periphery was also proven central for the development of the relapse.
Immune Regulation by the Neuroendocrine System
Estrogen inhibits clinical and histological symptoms of EAE, and pretreatment with
low doses of 17beta-estradiol (E2) diminishes the symptoms of EAE by inhibiting
cell migration into the CNS and promoting axon and myelin survival (Wolswijk
1998; reviewed in Murta and Ferrari 2013). Moreover, in EAE animals proges-
terone decreases proinflammatory cytokine secretion (IL12, IL17), increases IL10
production, and increases the CD19 + and CD8 + populations (Chang et al. 2002).
Environmental Factors
Diet also represents an important factor in experimental animal models. In EAE

mice, a high salt diet increases the number of Th17 cells, worsening the disease
(Tsai et al. 2002). Conversely, vitamin D (or its metabolite 1.25-dihydroxyvitamin
D3) reverses the EAE symptoms by inhibiting chemokine and inducible nitric oxide
synthase (iNOS) synthesis, and CD11b + monocyte trafficking into the CNS
(Moynagh 2005). Moreover, this vitamin also suppresses EAE female selectivity
(Byravan et al. 1994).
Another environmental factor associated with EAE progression is UV irradia-
tion: several authors have shown that UV irradiation suppresses EAE by inducing
immunosuppression through an alteration of dendritic and regulatory T cells,
independently of vitamin D production (Hauser et al. 1984; Waxman 1998;
Lappe-Siefke et al. 2003; Ng et al. 2013).
Moreover, the influence of the microbiome on different pathological conditions
has been investigated. In some models of EAE, gut microflora-free animals are
resistant to the induction of RR-EAE and have decreased Th17 and B cell responses
(Tsunoda and Fujinami 2002; Tsunoda et al. 2003; Huitinga et al. 2000).
Concluding Remarks
Systemic inflammatory insults are risk factors in both the etiology and progression
of demyelinating diseases. The interaction between damaged brain and systemic
inflammation may be responsible for the progression of neurodegenerative diseases.
However, certain systemic stimuli may be beneficial for both disease progression
and repair. Primed microglial cells in the diseased CNS are viewed as one of the
key components in the exacerbation of central damage due to systemic inflam-
matory stimuli in most CNS diseases. Additionally, the peripheral immune system
contributes significantly to the pathophysiology of the demyelinating diseases
discussed in the present review and their animal models, and the environment
appears also to be important. Better understanding of the mechanisms of CNS and
immune system communication should improve therapeutics for immune mediated
diseases.
Acknowledgments Carina C. Ferrari and Verónica Murta are members of the Research Career of
the National Council of Scientific and Technological Research (CONICET), Argentina. CF is
supported by CONICET (PIP 2012-2014, 11220110100560) and National Agency of Science and
Technology of Argentina (ANPCyT) (PICT 2012-2014).
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Regulation of Oligodendrocyte
Differentiation and Myelination
by Nuclear Receptors: Role
in Neurodegenerative Disorders
Adrián Sandoval-Hernández, María José Contreras,

Jenny Jaramillo and Gonzalo Arboleda
Abstract During development and through adulthood, differentiation of diverse

cell types is controlled by specific genetic and molecular programs for which
transcription factors are master regulators of gene expression. Here, we present an
overview of the role of nuclear receptors and their selective pharmacological
modulators in oligodendrocytes linage, their role in myelination and remyelination
and their potential use as a therapeutic strategy for demyelinating diseases. We
discuss several aspects of nuclear receptors including: (1) the biochemistry of
nuclear receptors superfamily; (2) their role on stem cells physiology, focusing in
differentiation and cell removal; (3) the role of nuclear receptor in the oligoden-
drocytes cell linage, from oligodendrocyte progenitors cells to mature myelinating
cells; and (4) the therapeutics opportunities of nuclear receptors for specific
demyelinating diseases.
Keywords Nuclear receptors Oligodendrocytes Demyelinating diseases

Alzheimer’s disease
Abbreviations
Ab Amyloid-b
ALDRP Adrenoleukodystrophy-related protein
AMN Adrenomyeloneuropathy
aNSCs Adult neural stem cells
AR Androgen receptor
CAR Constitutive androstane receptor
CBP cAMP response element binding protein-binding protein
A. Sandoval-Hernández M.J. Contreras J. Jaramillo G. Arboleda

Grupo de Neurociencias y Muerte Celular, Facultad de Medicina
e Instituto de Genética, Universidad Nacional de Colombia, Bogotá, Colombia
G. Arboleda (&)
Departamento de Patología, Facultad de Medicina, Universidad Nacional
de Colombia, Bogotá, Colombia

DOI 10.1007/978-3-319-40764-7_14
288 A. Sandoval-Hernández et al.
CNPase 2′3′-cyclic nucleotide 3′-phosphodiesterase

CNTF Ciliary neurotrophic factor
COUP-TF Chicken ovalbumin upstream promoter-transcription factor
DBD DNA binding domain
DG Dendate gyrus
ER Estrogen receptor
ERR Estrogen-related receptor
FXR Farnesoid X receptor
GFAP Glial fibrillary acid protein
GR Glucocorticoid receptor
HNF4A Hepatocyte nuclear factor 4A
IGF-1 Insulin-like growth factor-1
LBD Ligand binding domain
LRH-1 Human liver receptor homologue-1
LXR Liver X receptor
MCP-1 Monocyte chemoattractant protein 1
MR Mineralocorticoid receptor
MPZ Myelin protein zero
N-CoR Nuclear receptor corepressor
NR2E1 Orphan nuclear receptor subfamily 2 group E member 1 NPCs neural
progenitor cells
NRs Nuclear receptors
NSCs Neural stem cells
Nurr1 Nuclear receptor related 1 protein
OL Oligodendrocytes
OPCs Oligodendrocytes progenitor cells
PDGF Platelet-derived growth factor
PGC-1 Peroxisome proliferator-activated receptor-gamma coactivators 1
PGJ2 Prostaglandin J2
PLP Myelin proteolipid protein
PPAR Peroxisome proliferator-activated receptor
PMP22 Peripheral myelin protein 22
PPREs Peroxisome proliferator response elements
PR Progesterone receptor
PXR Pregnane X receptor
KD Krabbe disease
RA Retinoic acid
Regulation of Oligodendrocyte Differentiation and Myelination … 289
RAR Retinoic acid receptor

RE Response element
ROR Retinoid-related orphan receptor
RXR Retinoid X receptor
SF-1 Steroidogenic factor-1
SMRT Silencing mediator of retinoid and thyroid hormone receptorsT3
triiodothyronine
TH Thyroid hormone
TLX A homolog of Drosophila tailless gene
TR Thyroid hormone receptor
TNFa Tumor necrosis factor alpha
X-ALD X-linked adrenoleukodystrophy
X-CALD X-linked cerebral adrenoleukodystrophy
VEGF Vascular endothelial growth factor
VDR Vitamin D receptor
Introduction
Transcriptional regulation determines the destiny of each stem cell during differen-
tiation. Nuclear receptors (NRs) are transcription factors that are activated by inter-
acting with small molecules such as steroid hormones, fatty acids and oxysterols. This
superfamily of proteins regulates gene transcription through dynamic interactions
with multiple protein complexes called coregulators. These coregulators are proteins
with diverse enzymatic activities that facilitate or repress the gene activity by means of
various mechanisms. In this way, NRs regulate the biological processes in different
cell types in the central nervous system (CNS) including oligodendrocytes (OLs). In
the CNS, mature OLs extend processes and ultimately form sheet-like myelin around
the axons of neurons, the principal component in the white matter. Myelination
involves many highly regulated steps controlling such physiological events as pro-
liferation, migration and differentiation of oligodendrocyte progenitor cells (OPCs)
(see Chapters “NG2-Glia, More Than Progenitor Cells” and “Oligodendrocytes:
Functioning in a Delicate Balance Between High Metabolic Requirements and
Oxidative Damage”). NRs regulate the transcription of specific genes and promoting
the survival and differentiation of neuronal stem cells (NSC), the migration of OPCs
and the maturation of OPCs to myelinating OLs. In this chapter, we review the current
knowledge of the role of NRs in oligodendrogenesis, it also focuses on NRs as
therapeutic targets for pharmacological modulation, using synthetic molecules that
are candidates for treating demyelinating diseases and such other neurodegenerative
disorders as Alzheimer’s disease.
Nuclear Receptors Superfamily
NRs are part of a gene superfamily of transcription factors regulated by structurally

related and evolutionarily conserved ligands that act through a common mechanism
(Chen 2008). These proteins are related to the regulation of many basic cellular and
molecular processes, such as proliferation, differentiation and cellular homeostasis
(Germain et al. 2006; Chambon 2005; Sonoda et al. 2008), and they cover a wide
variety of biological functions involved in development, reproduction, physiology
and pathology, therefore they are useful in therapeutics (Murphy et al. 2005). NRs
modulate transcription through several mechanisms involving gene activation,
repression and trans-repression. Such mechanisms can be ligand-dependent or
independent, or either genomic or non-genomic (Germain et al. 2006).
Additionally, such modulation depends not only on the NR per se, but on cofactors
(corepressors and coactivators) that are recruited after the interaction with specific
ligands and are generally tissue-specific (Chen 2008). All of this makes their sig-
naling complex.
In humans, 48 NRs have been described, and 49 in mice. In the adult mouse
brain, most NRs are expressed differentially in particular regions, having 40 and 36
NR express in the cortex and hippocampus respectively (Gofflot et al. 2007). In
general, these soluble proteins are located in the nucleus or cytoplasm whose
known natural agonists are steroid hormones (estrogens, progestins, androgens,
glucocorticoids and mineralocortocoids), thyroid hormones, lipophilic vitamins
(vitamin D and cis-retinoic acid) and cholesterol metabolites (bile acids, oxys-
terols), among others (Burris et al. 2013).
There are various ways of grouping members of the NR superfamily.
Mangelsdorf et al. (1995) proposed four classes according to their ligand, their
DNA binding site and their dimerization properties. Class I, or steroid receptors:
some are in the cytoplasm and include estrogen receptor (ER), glucocorticoid
receptor (GR), androgen receptor (AR), progesterone receptor (PR) and mineralo-
corticoid receptor (MR), which after binding with their respective ligand form
homodimers that translocate to the nucleus to bind to a DNA response element
(RE) composed of palindromic repeats. Class II, or retinoid X receptor (RXR): in
contrast to the Class I receptors, these remain in the nucleus forming heterodimeric
complexes with corepressor molecules, and bind to the RE formed by direct repeats.
In this group are the thyroid hormone receptors (TRs), the vitamin D receptor
(VDR), the retinoic acid receptors (RARs) and the peroxisome proliferator-
activated receptors (PPARs). Class III: similar mechanism to Class I, but they bind
to the RE formed by direct repeats as homodimers. Among them, we can find RXR
ultraspiracle, chicken ovalbumin upstream promoter COUP and HNF-4A. Class IV,
monomeric orphan receptors: bind to a unique sequence in the RE as monomers.
Another way to classify the NRs is according to their ligands in three categories
[(Mangelsdorf 2010; Sonoda et al. 2008) and references therein]. (1) Endocrine
receptors are those activated by hormones such as estrogen receptor, androgen
receptor, progesterone receptor, glucocorticoid receptor, mineralocorticoid receptor

and thyroid hormone receptor and vitamins as retinoic acid receptor and vitamin D
receptor. The steroid hormone receptors function as homodimers, whereas TR,
VDR, and RAR form heterodimers with RXR. (2) Adopted orphan receptors are
NRs whose ligands were identified after being discovered. They include diet lipids
and xenobiotics, and they function as heterodimers with RXR. They regulate the
metabolism of lipids and glucose, thus they are potential therapeutic targets for
metabolic disorders, including the farnesoid X receptor (FXR), liver X receptor
(LXR), and peroxisome proliferator-activated receptor (PPAR). This group includes
receptors that, despite having an identified ligand, have an unknown physiological
role. They include the estrogen related receptors (ERRs), the retinoid-related orphan
receptor (ROR), the constitutive androstane receptor (CAR), the steroidogenic
factor-1 (SF-1), the human liver receptor homologue-1 (LRH-1), and the hepatocyte
nuclear factor 4A (HNF4A). (3) Orphan receptors are those that do not yet have a
known ligand and whose regulation depends mainly on co-regulators, on the
receptor expression or on covalent modifications more than on the ligand itself.
NRs have additional mechanisms of action. Some NRs such as PPARs, LXRs,
and FXR stimulate transcription minimally above the basal level in the absence of
the ligand and in some cases may inhibit transcription in the absence of ligand by
binding to a negative RE. For example, LXR knockout mice show elevated tran-
scriptional activity (Landrier et al. 2003; Konopleva et al. 2011). Within the tran-
scriptional regulatory mechanisms described, trans-repression is involved in the
immune response (Pascual and Glass 2006). Trans-repression refers to all tran-
scription repression mechanisms dependent on ligand-binding to a NR but do not
involve direct binding to an RE. The most studied process of this type is the direct
interaction of NRs with NFkB and AP-1, which results in their sequestration and
decreased transcriptional activity, inhibiting a cytokine-mediated inflammatory
response, involving tumor necrosis factor alpha (TNFa), interleukins and metal-
loproteinases (Xiao and Ghosh 2005; Karin 2006). In addition, promoters for dif-
ferent inflammatory genes contain REs for NFkB and AP-1, which allow them to
act synergistically (Wagner and Eferl 2005; Shimizu et al. 2006; Rannou et al.
2006).
It has been observed that ligand-free PPARc can be attached to the silencing
mediator of retinoid and thyroid hormone receptors (SMRT) co-repressor, which is
released upon ligand binding so it can interact with STAT3, inhibiting its tran-
scriptional activity. STAT3 can be activated by IL6, IL10, IL11, IL21, and IL23,
and it positively regulates the expression of IL17, IL23, BCL-X, BCL-2 and VEGF
(vascular endothelial growth factor), which control cell proliferation, anti-apoptosis,
angiogenesis and metastasis (Yu et al. 2009). These are important in carcinogenesis
caused by hepatitis C or Helicobacter pylori infection. In a similar way, PPARb
acts with BCL-6, a transcriptional repressor factor involved in the development of B
cell lymphoma. BCL-6 is bound to ligand-free PPARb increasing the transcription
of different proteins including the monocyte chemoattractant protein 1 (MCP-1)
(Toney et al. 2000).
Nuclear Receptors in Neural Stem Cells

and Oligodendrogenesis
In the CNS, myelin is the main component of the white matter. As discussed in
Chapters “Glial cells and Integrity of the Nervous System” and “Oligodendrocytes:
Functioning in a Delicate Balance Between High Metabolic Requirements and
Oxidative Damage”, it is generated by highly specialized cells known as OLs,
whose function is to wrap the axons of neurons and contribute to the efficiency and
speed of conduction of action potentials (Sherman and Brophy 2005). In verte-
brates, myelination occurs mainly during the postnatal period; it requires multiple
steps and highly coordinated and regulated signals to control the proliferation,
migration and differentiation of OPCs into mature or myelinating OLs. This process
is called oligodendrogenesis (Emery 2010; Miller 2002).
OPCs are generated during embryonic development from multipotent stem cells
of the ventral cortex, specifically the medial ganglionic eminence, the anterior
entopeduncular area and the subventricular zone (SVZ) (Kessaris et al. 2006; Rakic
and Zecevic 2003; Zuccaro and Arlotta 2013). During the postnatal period, most
OPCs after a defined number of cellular divisions differentiate into postmitotic OLs.
Approximately 5 % of OPCs maintain their potential for proliferation and migration
in the adult brain, both in the white and the gray matter for the ongoing maintenance
of myelin. Thus OPCs serve as a primary source of remyelination when facing
demyelinating lesions, because they can migrate to the area of lesion, proliferate
and differentiate into myelinating OLs (Franklin and Ffrench-Constant 2008;
Kondo and Raff 2000; Nishiyama et al. 2009; Zawadzka et al. 2010). Furthermore,
it has recently been proposed that OPCs could be categorized as adult stem cells
(Crawford et al. 2014) because of their ability to self-renew, exhibit mitotic qui-
escence, and produce differentiated mature progeny (Young et al. 2013) while
having multipotentiality (Kondo and Raff 2000; Zawadzka et al. 2010). Moreover,
in the adult brain new OLs can emerge from adult neural stem cells (aNSCs) located
in the SVZ of the lateral ventricle (Kriegstein and varez-Buylla 2009).
Recently, several cellular mechanisms that lead to oligodendrogenesis, myeli-
nation and remyelination have been described (Emery 2010; Tyler et al. 2009;
Chong and Chan 2010; Fancy et al. 2009; Gibson et al. 2014). These include
extrinsic factors such as growth factors (insulin-like growth factor-1, IGF-1;
platelet-derived growth factor, PDGF; ciliary neurotrophic factor, CNTF), axonal
surface ligands (LINGO-1, PSA-NCAM, Jagged1), secreted molecules and neu-
ronal activity. And they include intrinsic factors such as several transcription factors
(MRF, Olig1/2, Ascl1, Nkx2.2, Oct4, Sox5/6, Sox10, YY1, Tcf4, Id2/4, APC,
b-catenin, among others), chromatin remodeling (HDAC) and microRNAs (dif-
ferentiation: miR-219 and miR-338; OPCs: miR-17-92 via PTEN and AKT).
Therefore, the search for therapies for demyelinating diseases is aimed at inducing
NSCs and OPCs to generate myelinating OLs.
During development and in adults, NRs are part of the molecular network of
transcription factors that control neurons, astrocytes and OL differentiation. It has
Fig. 1 Scheme describing the role of nuclear receptors in differentiation and maturation of
oligodendrocytes. Green arrows RARs, TRs and PPARc are mainly involved in the maturation of
OPC to immature oligodendrocytes; PPARc, LXR, and COUP-TFI are particularly important in
the full maturation of oligodendrocytes. Pink arrow TLX is involved in the suppression of
oligodendrocyte differentiation
been shown that during embryogenesis DAX-1, ERRb, SF-1 and LRH-1 regulate
the expression of genes such as Oct3 and Oct4, which confer pluripotency char-
acteristics of embryonic stem cells (Jeong and Mangelsdorf 2009; Tokuzawa et al.
2003). An example is the NR Errb, which confers pluripotency characteristics
independently of Oct4, Sox2, and Nanog (Ivanova et al. 2006; Luo et al. 1997), and
its deficiency generates abnormalities in the blastocyst during embryogenesis
(Ivanova et al. 2006). Different NR ligands have started to be explored as potential
therapeutic targets in demyelinating diseases with the purpose of inducing oligo-
dendrogenesis (Fig. 1). In this chapter some findings that involve the participation
of different NRs will be described.
Role of RARs, RXRs and TRs
OPC differentiation can be controlled by retinoic acid (RA) and thyroid hormone
(TH) (Ahlgren et al. 1997). RA is a natural agonist for members of the RAR and
RXR subfamily (Mark et al. 2006). Besides, the TH and the RA promote higher
affinity interactions between members of the PPAR, RAR and RXR family (Qi et al.
1995), where RARs are essential for embryo viability (Mark et al. 2006). Goncalves
et al. (2005) showed that RARa and RARc promote the differentiation of neural
progenitor cells (NPCs) to OLs and astrocytes, as RARa reduces the expression
levels of SF-1, LRH-1, DAX1 and Oct4 (Gu et al. 2005; Niakan et al. 2006; Yang
et al. 2007), while RARb agonists induce neuronal differentiation (Goncalves et al.
2009). By contrast, during the early stages of embryogenesis RA was observed to
inhibit OL differentiation in the spinal cord, in order to allow dispersion of OPCs in

that area (Noll and Miller 1994). TH acts through binding to TR receptors (TRa and
TRb). In oligodendroglial-enriched rat cultures, triiodothyronine (T3), a type of TH,
was proven to induce oligodendroglial maturation, particularly regarding the
intracellular localization of mature OL markers, such as 2′3′-cyclic nucleotide 3′-
phosphodiesterase (CNPase) and myelin basic protein (MBP) (Younes-Rapozo
et al. 2006). This was also demonstrated in in vivo models. Hypothyroidism-
induced rats have an increase in proliferation of OPCs and NSCs in the olfactory
bulb and on the SVZ, and a reduction of PDGFa mRNA and MBP levels in the
optic nerve, which could suggest a delay in differentiation. On the contrary,
hyperthyroid rats exhibit an increase in the NG2 marker in the olfactory bulb and
increased maturation of OLs (Fernandez et al. 2004). It is also suggested that TRa1
may act in a bimodal way in OL differentiation, for during early postnatal stage it
promotes secretion of neurotrophic factors, which act on Purkinje neurons and
astrocytes to induce differentiation of OPCs. However, in later stages, TRa1 stops
OPC proliferation by cell cycle arrest (Picou et al. 2012).
Regarding RXRc, it is proposed that its activation stimulates differentiation of
OPCs to OLs and enhances remyelination in multiple sclerosis (MS) (Diab et al.
2004; Gallo and Chew 2011; Huang et al. 2011). Knockdown of RXRc by RNA
interference or RXR-specific antagonists severely inhibits OL differentiation in
culture. In mice that lack RXRc, adult OPCs efficiently repopulate lesions after
demyelination, but show delayed differentiation into mature oligodendrocytes.
Besides, in experimental autoimmune encephalomyelitis (EAE), agonists of RXR
such as 9-cis-retinoic acid and 15-deoxy-12,14-prostaglandin J2 reduce the severity
of the disease. On the other hand, Huang et al. (2011) report no differential
expression of PPAR, but that RXRc may form a heterodimer with RXRa, RXRb,
LXRa, chicken ovalbumin upstream promoter-transcription factor 1 (COUP-TFI)
or Nurr1 to allow differentiation of OPCs after demyelination.
Role of LXRs
There are two members of the LXR subfamily of NRs: LXRa and LXRb; they
share *80 % homology (Wojcicka et al. 2007). Their natural agonists are oxidized
derivatives of cholesterol called oxysterols. LXRa is expressed in liver, spleen,
kidney, intestine and adipose tissue, whereas LXRb is expressed ubiquitously at
low levels (Auboeuf et al. 1997). Few synthetic agonists of LXRs are known. The
most studied are T0901317 and GW3965, which bind to LXRa and LXRb with an
EC50 of approximately 20 nM. GW3965 preferentially binds to LXR while
T091317 also acts as an agonist for FXR and pregnane X receptor (PXR) (Houck
et al. 2004; Schultz et al. 2000; Shenoy et al. 2004).
In the CNS, LXRs have been described as inhibitors of astrogliogenesis and
inducers of neurogenesis. Mice that do not express LXRs have lower midbrain
development due to a decrease in the number of dopaminergic neurons.
Furthermore, in in vitro experiments it was observed that NSCs easily differentiate

to dopaminergic neurons when exposed to oxysterols, (Sacchetti et al. 2009).
Similarly, LXRs are important regulators of myelination, as they interact with
Wnt/b-Catenin signaling, which regulates transcription of myelin protein zero
(MPZ) and peripheral myelin protein 22 (PMP22) (Makoukji et al. 2011;
Shackleford et al. 2013). Thus, LXRa/b−/− knockout mice express lower levels of
MPZ and PMP22 and have fewer layers in their myelin sheaths (Makoukji et al.
2011).
Xu et al. (2014) showed that LXRb−/− knockout mice have hypomyelination of
the corpus callosum and the optic nerve. They also observed a deficit in the pro-
duction and maturation of OLs, perhaps because LXRb is involved in cholesterol
homeostasis, an essential component of myelin, and cholesterol availability may
limit the rate of maturation of OLs and myelination (Saher et al. 2005). Similarly, in
primary cultures of OLs treated with LXR agonist T0901317, the ABCA1,
ABCG1, ApoE, and LDLR genes, which are associated with cholesterol home-
ostasis, are expressed, and optimal myelination and remyelination are promoted
(Nelissen et al. 2012). It is suggested that LXRb is also essential in differentiation
of radial glial cells to OPCs in the dorsal cortex, which is a newly discovered route
for the production of OPCs, myelination and remyelination. Likewise, LXRb plays
an important role in brain lamination during corticogenesis (Fan et al. 2008; Xu
et al. 2014).
Role of PPAR
Another subfamily of NR involved in regulation of the oligodendrogenesis is that of

PPARs. There are three known members: PPARa, PPARb and PPARc. PPARa is
mainly expressed in tissues with high metabolic rates of fatty acids such as liver,
muscle and heart. PPAR-c is expressed in microglia, astrocytes, OLs and neurons.
PPARs regulate gene expression by recognizing and binding to the so-called
“peroxisome proliferator response elements” (PPREs) present in the promoter
region of target genes. PPARs play an important role in regulating the expression of
lipid metabolism genes: PPARa regulates genes involved in cholesterol metabo-
lism, while PPARc regulates the metabolism of fatty acids (Alaynick 2008; Huang
and Schulman 2009; Makishima 2003). PPARc agonist ligands that belong to the
thiazolidinedione, or TZD (rosiglitazone and pioglitazone, among others), family
are used to treat type-2 diabetes and dyslipidemias by increasing insulin sensitivity
and improving glucose metabolism; PPARa agonists are another type of drug, such
as fibrates (fenofibrate and gemfibrozil), which act as hypo-lipidemics and
anti-atherosclerotic agents and aid in lipid metabolism (Barbier et al. 2002; Etgen
and Mantlo 2003; Fajas et al. 2001; Jay and Ren 2007).
PPARs are also involved in cell differentiation in a wide variety of tissues
(Barbier et al. 2002; Fajas et al. 2001), including the regulation of OL differentiation
and maturation (Bernardo et al. 2009; Heneka and Landreth 2007; Roth et al. 2003;
Saluja et al. 2001). PPARb/d, a PPAR subtype that predominates in the CNS and is
strongly expressed in immature OLs (Heneka and Landreth 2007), has been shown
in vitro when activated to increase mRNA of MBP and myelin proteolipid protein
(PLP) and increase survival and differentiation of OPCs, but it does not have a role in
OPC proliferation (Saluja et al. 2001). PPARc has been associated with induction of
lipids synthesis, which is important in the differentiation of OPCs and myelination.
PPARc is also associated with an increase of alkyl-dihydroxyacetone phosphate
synthase, a peroxisomal enzyme involved in the synthesis of myelin-rich lipid
plasmalogens in B12 neural cells and isolated spinal cord OLs (Roth et al. 2003).
Additionally, treatment with PPARc agonists in rat primary cultures of OPCs pro-
motes the differentiation of these cells and increases their antioxidant activity by
increasing the levels of catalase and superoxide dismutase copper-zinc (Bernardo
et al. 2009). Similarly, it was shown that pioglitazone accelerates OL differentiation
and increases complex IV mitochondrial respiratory chain activity as well as the
response to increased calcium and such environmental cues as ADP (De et al. 2011).
PPARc also regulates growth and differentiation of several cell types during post-
natal and prenatal development. For example, Kanakasabai et al. (2012) found
in vitro that mouse NSCs treated with PPARc agonists stop proliferating and sig-
nificantly increase immunoreactivity for O4 and NG2 OPC markers. Paintlia et al.
(2011) in an in vitro MS model, demonstrated that agonists of PPARc/d protect
OPCs against the cytotoxicity conferred by TNFa and IL17.
Recently, it has been confirmed that PGC-1 (peroxisome proliferator-activated
receptor-gamma coactivators 1, which includes PGC-1a, PGC1-b, and PGC-1-
related coactivator) interact with the NR PPARc, PPARa, ERR, LXR and HNF-4a.
Such activators are involved in mitochondrial biogenesis and in lipid and energy
metabolism (Shao et al. 2010; Sugden et al. 2010; Wareski et al. 2009). PGC-1a is
expressed in OLs, and its genetic deletion leads to dysfunction in the metabolism of
sphingolipids and changes in the lipid composition of their cell membranes,
including decreased ceramides, galactosylceramides, lactosylceramides, phos-
phatidylglycerol, phosphatidylethanolamine and phosphatidylserine, and accumu-
lation of phosphatidylcholine. Although, these changes are not directly related to
alterations in the structure of myelin, they do delay the maturation of OLs. Indeed,
PGC-1a is able to coordinate the differentiation of OPCs, as it regulates the
expression of Nkx2.2, the transcription factor related to MBP and PLP (Camacho
et al. 2013). It is important to add that deletion of PGC-1a in mice causes neu-
rodegeneration in the striatum, cortex and hippocampus, reduction of myelin-
associated proteins and alteration in cholesterol homeostasis (Kiebish et al. 2012;
Xiang et al. 2011).
Role of the COUP-TFs
The chicken ovalbumin upstream promoter-transcription factor (COUP-TF) NR

subfamily is involved in the maturation of OLs, as it directly regulates
myelin-regulatory proteins. This family has two members: COUP-TFI and

COUP-TFII, which share 80 % homology. It was shown in vivo that in early stages
of development there is an increase in expression of these NRs in NSCs, leading to
gliogenesis, while their absence keeps the glial fibrillary acid protein (GFAP)
promoter epigenetically silent. Furthermore, in vitro knockdown of COUP-TF
reduced neuronal phenotypes in advanced stages of neurospheres and generated
mainly glial cells (Naka et al. 2008). It was also shown that animals that do not
express COUP-TFI die perinatally; the mutant embryos develop alterations in
cranial ganglia and nerves, with abnormal axonal projections and arborization.
Therefore, COUP-TFI is required for normal fetal development (Qiu et al. 1997).
Regarding the OL lineage and myelination, COUP-TFI regulates differentiation
by regulating other transcription factors, including SCIP/Oct-6/Tst-I, which is an
important regulator of myelination (Yamaguchi et al. 2004). Also in the mutant
COUP-TFI mice, it has been shown that there is normal proliferation and migration
of OPCs. Otherwise, mature OL’s markers like MBP are reduced considerably in
the white matter and stratium, compared to control mice.
Role of TLX
The NR homolog of the Drosophila tailless gene (TLX) is an inhibitor of OL

differentiation and is critical for neurogenesis. TLX is exclusively expressed in the
CNS (Monaghan et al. 1995). It is expressed predominantly in the neurogenic
niches in the subgranular zone of the hippocampal dendate gyrus (DG) and in the
SVZ of the lateral ventricles. Its maximum expression is associated with increased
neurogenesis and its absence causes a thinning of the neocortex and the forebrain,
as well as a reduction in the number of NSCs and progenitors (Li et al. 2008). TLX
also inhibits astrogenesis and maturation during development (Land and Monaghan
2003; Li et al. 2008; Roy et al. 2004; Shi et al. 2004; Sun et al. 2007; Zhang et al.
2008).
Nuclear Receptors as Potential Therapeutics

for Neurodegeneration
Nuclear Receptors and Demyelinating Diseases
As discussed in Chapters “Glial cells and Integrity of the Nervous System” and
“Peripheral Inflammation and Demyelinating Diseases”, demyelinating diseases
include a heterogeneous group of CNS and peripheral nervous system
(PNS) diseases, both from the clinical and from the pathophysiologic standpoint
(Kassmann and Nave 2008; Dubois-Dalcq et al. 2008).
MS is a genetically linked autoimmune disease of the CNS that specifically

affects the white matter (Hafler et al. 2005). It is characterized by a progressive
deterioration of motor skills (Stathopoulou et al. 2010). The autoimmune hypoth-
esis indicates that it is an immune reaction against myelinated axons reducing
myelin and causing axonal degeneration (Dhib-Jalbut 2007).
X-linked adrenoleukodystrophy (X-ALD) is a neurodegenerative disease that
affects the white matter of the brain, spinal cord, peripheral nerves, adrenal cortex and
testicles (Ferrer et al. 2010). When it starts in childhood, it is known as X-linked
cerebral adrenoleukodystrophy (X-CALD); when it starts later in life, it affects axon
tracts and is known as adrenomyeloneuropathy (AMN) (Powers et al. 1992; Powers
and Rubio 1995; Powers et al. 2000, 2001; van Geel et al. 2001). In general, X-ALD is
the most common peroxisomal disorder, and it is characterized biochemically by the
accumulation of very long chain fatty acids (VLCFAs) in these tissues (Singh 1997;
Wanders 1999; Moser et al. 1984; Singh et al. 1984; Kemp and Wanders 2010). It is
caused by mutations/deletions of the ABCD1 gene, which is responsible for trans-
porting VLCFAs to the peroxisome for degradation (Ferrer et al. 2010).
Krabbe disease (KD) is a fatal neurological disease with autosomal recessive
inheritance. The pathological features of the disease include axon loss, myelin loss,
astrogliosis and the presence of PAS-positive globoid cells (Suzuki 2003). The bio-
chemical defect responsible for the pathogenesis of KD is deficiency of galactosyl-
cerebrosidase, a lysosomal enzyme, which leads to the progressive accumulation of
psychosine in globoid cells (Suzuki 1983, 2003; Suzuki and Taniike 1995).
Psychosine is highly toxic and produces pro-inflammatory agents while inhibiting
plasmalogen enzymes (Haq et al. 2006; Khan et al. 2005). In addition, when it
accumulates in lipid rafts it alters the architecture of the membrane and disrupts
signaling pathways involved in cell survival and myelination (White et al. 2009).
PPAR has been evaluated as a potential therapeutic target in some models of
neurodegenerative demyelinating diseases for its capacity to regulate lipid meta-
bolism, oligodendrocyte proliferation and differentiation (Robinson and Grieve
2009), and for its anti-inflammatory role by inhibiting the NFjB signaling pathway
(Ko et al. 2008; Remels et al. 2009).
In animal models of MS including EAE, treatment with agonists for PPARa
(Gocke et al. 2009) or PPARc (Drew et al. 2008) is effective at inhibiting the
release of pro-inflammatory cytokines from microglia cells and astrocytes.
Similarly, treatment with fibrates increases expression of the ABCD2 gene, which
encodes for adrenoleukodystrophy-related protein (ALDRP) (Rampler et al. 2003);
PPARb agonists seem to have a protective effect against inflammatory processes
that are dependent on gamma interferon (IFNy) and lipopolysaccharide
(LPS) (Defaux et al. 2009). In glioma cells, treatment with PPARc agonists reduces
cell proliferation, migration and invasion. Furthermore, activation of PPARb pro-
motes the differentiation of rat pre-OLs (Saluja et al. 2001).
In the EAE model, PPARs ameliorates pathological manifestations through
mechanisms associated with anti-inflammatory activity, reducing infiltration,
migration and proliferation of Ag-specific T cells for the MBP (Lovett-Racke et al.
2004; Polak et al. 2005; Niino et al. 2001; Natarajan and Bright 2002;
Fig. 2 Effects of rosiglitazone and WY14643 on differentiated MO3.13 cells exposed to

psycosine. IGF-1 and the PPARc agonists, Rosiglitazone and WY14634, protect against
psycosine-induced MO3.13 cell death
Diab et al. 2002; Feinstein et al. 2002; Raikwar et al. 2006; Peiris et al. 2007). In
models of X-ALD oligodendrocytes, the effects of PPAR agonists have not yet been
evaluated but will be important to study because of PPAR’s capacity to regulate
lipid homeostasis and the expression of peroxisomal proteins.
In KD, some effects of PPAR agonists are known. GalC knockdown mice,
known as Twitcher, have lower levels of transcriptional activity and PPARa
expression, which could explain the decrease in peroxisomal proteins (Haq et al.
2006). In our laboratory, PPARc agonists partially protect from psychosine-induced
MO3.13-cell death (Fig. 2).
Nuclear Receptors and Alzheimer’s Disease
Alzheimer’s disease (AD) is a complex and chronic neurodegenerative disorder

characterized by the presence of beta amyloid deposits, altered lipid metabolism,
loss of cholinergic neurons and neuroinflammation leading to memory loss and
cognitive decline (Perrin et al. 2009; Mattson 2004) (see Chapter “Age-Dependent
Changes in the Activation and Regulation of Microglia”). Because NRs can control
diverse physiological processes, including those associated with inflammation and
lipid homeostasis, they are a plausible therapeutic target for this disease (Zolezzi
et al. 2014; Burris et al. 2013; Bensinger and Tontonoz 2008).
Such PPARc agonists as rosiglitazone that do not readily cross the blood–brain
barrier improve cognition in Alzheimer’s transgenic mouse models, reducing Tau
phosphorylation and the amount of soluble amyloid-b (Ab) without increasing the
levels of NEP, IDE or ApoE. However, rosiglitazone slightly increases ABCA1,
which is associated with a decrease in Ab plaques (Escribano et al. 2010).
Rosiglitazone is reported to reverse morphological changes in microglia, retaining
their phagocytic activity needed for removing the Ab plaques (Zhao et al. 2009).
The low ability of rosiglitazone to cross the BBB suggests that it acts by increasing
peripheral insulin sensitivity, which is indeed altered and a risk factor for AD (Haan
2006; Martins et al. 2006). Pioglitazone, a PPARc agonist that readily crosses the
BBB, reduced Ab levels through a decrease in the amyloidogenic processing of
APP by reducing the transcription of BACE1 (Sastre et al. 2003, 2006). It also
reduced proinflammatory markers (Yan et al. 2003). Clinical studies in which
PPAR agonists were used showed small cognitive improvements that depend on the
ApoE genotype (Risner et al. 2006). However, these drugs have been removed from
the market as they increase the risk of heart attack and bladder cancer (Chen 2008).
LXR is widely expressed in the brain. It binds oxysterols and regulates
cholesterol homeostasis (Calkin and Tontonoz 2012; Schulman 2010) and
inflammation (Steffensen et al. 2013). LXR agonists induce cholesterol efflux from
neurons and glial cells by a positive regulation of the cholesterol transporter
(ABCA1) and by induction of apolipoprotein E (ApoE) expression, which has been
associated with a reduction in Ab load (Burris et al. 2013). In transgenic animal
models of AD, LXR agonist TO91317 generates cognitive improvement and
histopathological changes, depending on the dose, duration of treatment and the age
of the animals. These beneficial effects were associated with an increase of ABCA1
and ApoE levels and with a decrease in Ab40 and Ab42 amyloid load, and in some
cases there was a reduction in the amyloidogenic processing favoring a-secretase
cleavage (Koldamova et al. 2005; Vanmierlo et al. 2011; Riddell et al. 2007).
Treatment of 12 month old Tg2576 mice with the LXR agonist GW3965
(33 mg/kg a day for 4 months) increases levels of ApoE and ABCA1 proteins,
associated with an in vitro increase in Ab degradation and a significant decrease
(approximately 65 %) in amyloid plaques (Jiang et al. 2008). Importantly, cognitive
impairment improves in 20-week-old Tg2576 mice treated with GW3965 50 mg/kg
a day for 6 days. From the same investigators, GW3965 modulates the immune
response, and double gene deletion of LXRa and LXRb in the humanized mouse
model of AD (TgAPPswe/PS1_E9) produces a more severe amyloid pathology
without interfering with the phagocytic activity of microglia. In addition, in vitro
and in vivo studies show increased expression of APOE, ABCA1, ABCG1, ApoC2,
and SREBP1c as well as reduced proinflammatory mediators such as TNFa and
Mip1b (Zelcer et al. 2007). Additionally, the cognitive and biochemical recovery
following GW3965 treatment in a mouse model of AD (APP/PS1) and its failure in
knockout of ABCA1 (ABCA1−/−), indicates that formation of lipoproteins is
required for the beneficial effects of this LXR agonist, and suggesting that the
anti-inflammatory component in this model may have a secondary role (Donkin
et al. 2010).
With regard to myelin in AD, few studies have considered alterations in myelin
as an important mechanism in the pathophysiology of this disease. The first
Fig. 3 LXR agonist increases myelination in the entorhinal cortex of WT and 3xTg-AD mice.
Micrograph showing myelination by using MBP staining after 12 weeks treatment with the LXR
agonist GW3965 in 3xTg-AD mice. Left panel, MBP immunoreactivity increased significantly in
the GW3965-treated animals as compared to untreated WT or 3xTg-AD mice. Data expressed as
mean ± S.E.M. Statistical analysis performed by one-way ANOVA followed by Bonferroni post
hoc test. *P < 0.01 compared with WT; #P < 0.05 compared with untreated 3Xtg-AD
detectable change in the triple transgenic mice model (3xTg) of AD is the loss of
myelin markers and demyelination, starting as early as 2 months of age (Desai et al.
2010). In addition, in vitro studies show that treatment with Ab induces OLs death
by activating the neutral sphingomyelinase-ceramide pathway (Lee et al. 2004).
These observations of alterations in myelin suggest the hypothesis that demyeli-
nation may underlie AD, which deserved to be further analyzed.
Recently, we have demonstrated that treatment of 3xTg-AD and wild type mice
with the specific agonist of LXR (GW3965) increases OPC markers (O1 and O4) in
DG of the hippocampus and also an increases myelin basic protein immunoreac-
tivity in the entorhinal cortex (Fig. 3, unpublished results). Our results indicate that
the pharmacological modulation of LXR may stimulate the remyelination process
in AD.
Conclusions and Perspectives
Nuclear receptors are emerging as a plausible therapeutic strategy in diverse dis-

orders associated with myelin alterations, including dysmyelinating and demyeli-
nating disorders. However, the cellular and molecular mechanisms for beneficial
therapies involving these molecules need to be explored further.
Acknowledgments This work was supported by grants from COLCIENCIAS (110145221189),

DIB-Universidad Nacional de Colombia (20101007590, 20201009689) and the Facultad de
Medicina, Universidad Nacional de Colombia.
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The Role of Galectin-3: From
Oligodendroglial Differentiation
and Myelination to Demyelination
and Remyelination Processes
in a Cuprizone-Induced
Demyelination Model
H.C. Hoyos, Mariel Marder, R. Ulrich, V. Gudi, M. Stangel,

G.A. Rabinovich, L.A. Pasquini and J.M. Pasquini
Abstract The aim of this work was to combine our previously published results
with our new data to show how galectin-3 (Gal-3) controls myelin integrity and
function, promotes oligodendroglial cell differentiation, and regulates microglial
responses to limit cuprizone- (CPZ)-induced demyelination and foster remyelina-
tion. In this study, 8-week-old Gal-3-deficient (Lgals3−/−) and wild type (WT) mice
were fed a diet containing 0.2 % CPZ w/w for 6 weeks, after which CPZ was
withdrawn in order to allow remyelination. Our results show that remyelination was
less efficient in Lgals3−/− than in WT mice. Electron microscopic images from
remyelinated sections in Lgals3−/− mice revealed collapsed axons with a defective
myelin wrap, while remyelinated WT mice had normal axons without relevant
myelin wrap disruption. MMP-3 expression increased during remyelination in WT
but not in Lgals3−/− mice. The number of CD45+, TNFa+ and TREM-2b+ cells
decreased only in WT mice only, with no alterations in Lgals3−/− mice during
demyelination and remyelination. Therefore, Gal-3 influences remyelination by
L.A. Pasquini and J.M. Pasquini contributed equally to this work and should be considered as
co-senior.
H.C. Hoyos M. Marder L.A. Pasquini J.M. Pasquini

Department of Biological Chemistry, School of Pharmacy and Biochemistry, Institute of
Chemistry and Biological Physical Chemistry (IQUIFIB), Buenos Aires, Argentina
H.C. Hoyos M. Marder L.A. Pasquini J.M. Pasquini
School of Pharmacy and Biochemistry, University of Buenos Aires and National Research
Council (CONICET), Buenos Aires, Argentina
R. Ulrich
Department of Pathology, University of Veterinary Medicine Hannover, Hannover, Germany
V. Gudi M. Stangel
Department of Neurology, Hannover Medical School, Hannover, Germany

DOI 10.1007/978-3-319-40764-7_15
312 H.C. Hoyos et al.
mechanisms involving the tuning of microglial cells, modulation of MMP activity,

and changes in myelin architecture.
Keywords Galectin-3 Myelination Demyelination Remyelination

Cuprizone Microglia Oligodendrocytes MMPs
Abbreviations
MMPs Matrix metalloproteinases
Gal-3 Galectin-3
Lgals3−/− Gal-3-deficient
WT Wild type
CPZ Cuprizone
CRD Carbohydrate-recognition domain
OLG Oligodendrocyte
EAE Experimental Autoimmune Encephalomyelitis
CC Corpus callosum
OPC Oligodendrocyte precursor cells
PBS Phosphate buffered saline
PFA Paraformaldehyde
SVZ Subventricular zone
EM Electron Microscopy
IOD Integrated optical density
V. Gudi M. Stangel
Center for System Neurosciences, Hannover, Germany
G.A. Rabinovich
Laboratory of Immunopathology, Institute of Biology and Experimental Medicine (IBYME;
CONICET), C1428 Buenos Aires, Argentina
G.A. Rabinovich
Laboratory of Functional Glycomics, Department of Biological Chemistry, Faculty of Exact
and Natural Sciences, University of Buenos Aires, C1428 Buenos Aires, Argentina
L.A. Pasquini J.M. Pasquini (&)
Dpto. de Qca Biol, FFyB-UBA, Junín 956, C1113 Bs as, Argentina
The Role of Galectin-3: From Oligodendroglial Differentiation and Myelination … 313
Introduction
Galectins belong to a family of b-galactoside-binding lectins. Although they lack

specific receptors, they can form multivalent complexes by binding to cell surface
glycoconjugates containing suitable oligosaccharides and generate intracellular
signals in order to regulate cell survival and differentiation (Rabinovich et al. 2007;
Yang et al. 2008). In the immune system, galectin-1 and -3 (Gal-1and Gal-3,
respectively) often have antagonistic roles in the modulation of adaptive immunity,
with Gal-3 being predominantly proinflammatory and Gal-1 anti-inflammatory
(Rabinovich and Toscano 2009). Gal-3, a chimeric protein with many actions,
including modulation of innate and adaptive immunity, has unusual tandem repeats
of proline and glycine-rich in short stretches fused to a carbohydrate-recognition
domain (CRD) (Rabinovich and Croci 2012).
Although the function of Gal-3 in central nervous system (CNS) immunity has
not been elucidated, most in vitro and in vivo studies hint at a proinflammatory role
in the promotion of immune cell activation, migration and inhibition of apoptosis
(Rabinovich et al. 2007), although negative regulation of lipopolysaccharide-
induced inflammation has also been proposed (Li et al. 2008). Our group first
studied the relevance of galectin–glycan lattices in oligodendrocyte (OLG) physi-
ology and identified an essential role for galectin–glycan interactions in regulating
OLG differentiation, leading to control of myelin integrity and function. We found
that astrocytes and microglia have high expression of both Gal-1 and -3. In contrast,
while immature OLGs but not differentiated OLGs highly expressed Gal-1, dif-
ferentiated OLGs expressed Gal-3. Matrix metalloproteinases (MMPs) activity
increased, thereby processing Gal-3 during OLG differentiation and regulating its
biological activity. Recombinant Gal-3 treatment accelerated OLG differentiation in
a dose- and carbohydrate-dependent manner, in accord with the “glycosylation
signature” of immature versus differentiated OLGs. Furthermore, conditioned
media from Gal-3-expressing, but not Gal-3-deficient (Lgals3−/−) microglia,
induced OLG differentiation. Supporting these findings, morphometric analyses
revealed a significant reduction in the number of myelinated axons and myelin turns
(lamellae), as well as in the g-ratio of Lgals3−/− mice. Moreover, myelin sheaths
were more loosely wrapped around axons in Lgals3−/− mice. Lgals3−/− mice had
lower anxiety levels, like those during early cuprizone (CPZ)-induced demyelina-
tion. In addition, neurospheres isolated from WT but not from Lgals3−/−mice
favored commitment to oligodendroglial fate. Together, these results indicate that
glial-derived Gal-3, but not Gal-1, promotes oligodendroglial differentiation and
thus contributes to myelin integrity and function, which has critical implications in
the recovery of inflammatory demyelinating disorders (Pasquini et al. 2011).
Within the CNS, Gal-3 is upregulated by inflammatory stimuli and is harmful in
prion-infected brain tissue (Mok et al. 2006, 2007; Riemer et al. 2004). On the other
hand, Gal-3 mediates the activation and proliferation of microglia in a focal cerebral
ischemia model in mice (Lalancette-Hébert et al. 2012). In experimental autoim-
mune encephalomyelitis (EAE), an animal model of CNS demyelination in which
mice are immunized with myelin OLG glycoprotein, Jiang et al. (2009) reported a
decreased severity in Lgals3−/− animals. In contrast, administration of CPZ in the
diet of young adult mice causes extensive demyelination in several areas of the
CNS, particularly in the corpus callosum (CC), independently of pathogenic T cells
(Matsushima and Morell 2001). CPZ-induced demyelination increases the number
of resident microglia and the presence of few peripheral macrophages
(Masuda-Nakagawa et al. 1993; von Bernhardi and Muller 1995; McMahon et al.
2002). These microglia have been reported to phagocytize myelin, associated with
the upregulation of phagocytic receptors among which TREM-2b is the most
prominent (Voß et al. 2011). Remarkably, considering that myelin can inhibit the
differentiation of oligodendrocyte precursor cells (OPC), this phagocytosis has been
proposed to play a key role in the onset of remyelination (Kotter et al. 2006).
We have recently published a manuscript comparing CPZ-induced demyelina-
tion in 8-week-old Lgals3−/− and WT mice. Our findings showed that Lgals3−/− and
WT mice are similarly susceptible to CPZ until treatment week 5, as evaluated by
myelin basic protein (MBP) immunelabeling and electronic microscopy. However,
OPCs generated in CPZ-treated Lgals3−/− mice showed diminished arborization,
which suggests a decrease in differentiation capability. Surprisingly, while WT
mice experienced spontaneous remyelination by week 5, even though the CPZ diet
was maintained to week 6, Lgals3−/− mice lacked this capacity and remained
demyelinated to week 6, with pronounced astroglial activation. Behavioral studies
of WT and Lgals3−/− mice found lower innate anxiety after 2 weeks of CPZ
treatment, but only Lgals3−/− mice had decreased locomotor activity and impaired
spatial working memory. Gal-3 expression increased during CPZ-induced
demyelination in microglia but not in astrocytes. Interestingly, microglial activa-
tion, ED1 expression, and phagocytic receptor TREM-2b levels increased only in
CPZ-treated WT mice. In contrast, CPZ-treated Lgals3−/− mice showed an
increased number of microglia with caspase-3 activation. Taken together, our
results indicate that Gal-3 is expressed in microglial cells to modulate their phe-
notype during CPZ-induced demyelination (Hoyos et al. 2014).
Multiple sclerosis (MS) is a CNS disease leading to the demyelination of white
and gray matter (Stadelmann 2011) (see Chapter “Peripheral Inflammation and
Demyelinating Diseases”). Remyelination is the regenerative response to de-
myelination (Franklin and Kotter 2008); it is characterized by thinner axons and
myelin sheaths. Although the complex biological interactions underlying remyeli-
nation are still not clear, the process is known to involve the proliferation of OPCs,
their subsequent migration toward demyelinated axons and their final differentiation
(Franklin and Ffrench-Constant 2008), all steps regulated by intrinsic and extrinsic
factors.
MMP are a family of zinc-dependent endopeptidases which, together with
endogenous tissue inhibitors of MMP, play essential roles in tissue remodeling.
MMPs can degrade myelin proteins in vitro (Chandler et al. 1995, 1996; Shiryaev
et al. 2009; Hansmann et al. 2012) and are suggested to be involved in the initiation
of demyelination in vivo (Hansmann et al. 2012; Ulrich et al. 2006; Skuljec et al.
2011). Furthermore, both MMP and their inhibitors have been implicated in
postnatal myelination, myelin maintenance, and remyelination (Skuljec et al. 2011;

Ulrich et al. 2005).
In this work, to further analyze the role of Gal-3 in the control of the glial cell
response to a demyelination insult, we have assessed the involvement of Gal-3 in
the control of the remyelination process after CPZ-induced demyelination. To this
end, we have analyzed the expression of different markers of the oligodendroglial
lineage and performed morphometric analyses of electron micrographs. We have
also evaluated the astroglial response, as well as changes in the microglial phe-
notype and the expression of MMP-3 in the CC during remyelination.
Experimental Procedures
Animals and Experimental Model
Lgals3−/− mice (C57BL/6 background) were generously provided by Dr. Fu-Tong

Liu (University of California, Davis, USA) and generated as previously described
(Hsu et al. 2000). Animals were housed in groups of 4 in a controlled environment
(20–23 °C) with free access to food and water and maintained in a
12 h/12 h day/night cycle, with light on at 6 am. All animal protocols were
approved by the Institutional Review Board of the University of Buenos Aires and
animal experimentation was in accordance with the National Institutes of Health
Guide for the Care and Use of Laboratory Animals.
Experimental demyelination was induced by feeding 8-week-old male mice with
0.2 % (w/w) CPZ (bis-cyclohexanone oxalhydrozone; Sigma Aldrich, Saint Louis,
USA) mixed into standard ground rodent chow (Matsushima and Morell 2001).
These mice were fed CPZ for 6 weeks and then allowed to remyelinate for 2
additional weeks. Studies, including weight measurements, were done fot 5 weeks
(the point of maximum demyelination) and during the remyelination period. The
demyelination–remyelination protocol is summarized in Fig. 1a.
Slice Preparation and Brain Section Selection
Animals were anesthetized with a xylazine–ketamine mixture and intracardially

perfused with 30 ml phosphate buffered saline (PBS), pH 7.4, followed by 4 %
paraformaldehyde (PFA, Sigma Aldrich, Saint Louis, USA) in PBS, pH 7.4. Brains
were carefully dissected out, post-fixed in the same solution overnight and later
thoroughly washed in PBS and cryoprotected in 30 % sucrose in PBS. All brain
slices (25-lm width) were kept at −20 °C in a PBS-glycerol solution (1:1) until
used for immunofluorescence studies.
Previous to immunostaining, brain slices were selected using the Allen Mouse
Brain Atlas as a reference. The corpus callosum (CC) and subventricular zone
(SVZ) were studied in brain sections at the level of coronal slices 44–52 of the
Atlas. After selection, brain slices were kept in PBS for immunohistochemical
b Fig. 1 a Schematic illustration of the experimental design. Two evaluation points were
determined (5th week of demyelination and 8th week—2 weeks after CPZ withdrawal from the
diet). b Representative sections showing MBP expression in stained CC sections at the 5th week of
treatment and 2 weeks after CPZ withdrawal (8th week). c Immunoreactive signal measured by
IOD in a grid of ten 1-mm2 rectangles, as detailed in the scheme (box) using Image Pro Plus 5.5
software. Blanks for IOD were calculated in the zones indicated by white boxes in the
representative image. Images (magnification 10) were obtained from 5 mice per treatment per
time point. d Demyelination score at the 5th week measured by MBP IOD (WT is 100 %).
e Remyelination rate during treatment of WT CPZ relative to WT mice and Lgals3−/− CPZ relative
to Lgals3−/− mice. f Western blot analysis of MBP expression during remyelination. Samples were
taken from CC at the 8th week. b-tubulin was used as a loading control. Values represent the
mean ± SEM of five independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 using
one-way analysis of variance (ANOVA) followed by Bonferroni post hoc tests
studies. For electron microscopy (EM) and flow cytometry (isolation of microglia),
the CC was dissected out from brains kept on ice using a sterile razor blade.
Dissection coordinates were +1.32 mm to +0.74 mm from Bregma, according to
The Mouse Brain Library. The isolated CC was immediately subjected to proce-
dures required for each particular assay.
Immunohistochemistry
Cryosections were rinsed twice with PBS (pH 7.4) and then treated with an
antigen-blocking reagent composed of 5 % FCS and 0.1 % Triton X-100 in PBS.
Primary and secondary antibody dilutions were prepared in 1 % FCS and 0.1 %
Triton X-100 in PBS. Primary antibody incubations were done overnight at 4 °C.
The primary antibodies used were rabbit anti-MBP (1/600; generously provided by
Dr. A. Campagnoni, UCLA, Los Angeles, USA); rabbit anti-CAII (1/400; gener-
ously provided by Dr. W. Cammer, A. Einstein College of Medicine, New York,
U.S.A.); mouse anti-CC1 (1/100, Abcam, Massachusetts, USA); goat anti-PDGFRa
(1/100, Neuromics, Edina, USA); chicken anti-Glial Fibrillary Acidic Protein
(GFAP) (1/100, Neuromics, Edina, USA); mouse anti-MMP-3 (1/100,
Calbiochem), generously provided by Dr. Alicia Jawerbaum (CEFYBO, UBA,
Buenos Aires, Argentina); and goat anti-Iba-1 (1/100, Abcam, Massachusetts,
USA). Incubation with Hoechst 33342 (Sigma Aldrich, Saint Louis, USA) and
various fluorescent secondary antibodies (Alexa 488, Alexa 649, Cy2 and Cy3,
Jackson Immunoresearch Lab, Argentina) was done for 2 h at 37 °C with agitation.
Slides were mounted and covered with Mowiol. Microphotographs were taken with
an Olympus BX50 epifluorescence microscope connected to a CoolSnap digital
camera.
Image Pro Plus software (version 5.5) was used for image analysis. For MBP
and MMP-3, integrated optical density (IOD) was measured in CC. Figure 1c
illustrates the measurement method. Ten squares (surface equal 1 mm2) were dis-
played per image and IOD was calculated and averaged in each square for each
image. For the remaining markers, the number of positive cells was counted in each
case using Image J software and validated through manual count by an experi-
menter who was blind to the experimental design.
For CAII, CC1, PDGFRa, MMP-3, and Iba-1 images, observations were carried
out using an Olympus Fluorview FV1000 MPE multiphoton microscope coupled to
a Zeiss LSM 510 laser scanner. Merged versions and composite images were
obtained with FV10-ASW1.7 viewer software (Olympus). Filament plots of
PDGFRa and Iba-1 cells were obtained from z-stack scanning of cells at a slice
distance of 0.75 µm using the IMARIS 6.3.1 program (Bitplane Sci Software) as
previously described (Hoyos et al. 2014).
Western Blot Analysis
MBP isoforms and GFAP were evaluated in CC cell extracts. Samples were
resuspended and lysed in RIPA 1 (NaCl 300 mM, TRIS 20 mM, pH.7.4, SDS
0.2 %) extraction buffer with a complete EDTA-free protease inhibitor cocktail
(Roche). Equal amounts of protein were separated on SDS-PAGE and transferred
onto PVDF membranes for Western blot analyses. Membranes were incubated with
anti-MBP (1/1000) and anti-GFAP (1/1000) antibodies, followed by incubation
with HRP-conjugated antibodies. Quantification was done by densitometry with the
Gel Pro Analyzer 4.0 system.
Electron Microscopy
Four to six animals per group were decapitated. CC were dissected out as described
above, fixed and immediately prepared for EM. Ultrathin cuts were examined using
a Zeiss Leo 906 E electron microscope equipped with a Zeiss Megaview III digital
camera. Parameters assessed included: (a) percentage of correctly myelinated axons
per field; (b) g-ratio (the ratio between the axon’s diameter and the axon’s diameter
wrapped with myelin); and (c) number of myelin turns around an axon. Images
were analyzed by experimenters who were blind to the experimental design. Eight
images were obtained for each experimental condition.
Isolation of Microglia
Isolation of microglia was carried out as described by Hoyos et al. (2014).

May Grünwald Giemsa Staining
Coronal brain slices of remyelinated Lgals3−/− and WT mice were washed in

distilled water to remove PBS/glycerol, preserved and mounted on glass. When
slices were completely attached to glasses, they were washed twice again and
incubated for 90 s with May Grünwald reagent (Biopack, Buenos Aires,
Argentina). Afterwards, slices were washed four times for 2 min each, then incu-
bated 20 min in Giemsa dye (Biopack, Buenos Aires, Argentina) and rewashed four
more times for 2 min each. Both reagents were generous gifts of Dr. Miriam
Lardone (Hospital de Clínicas “José de San Martín”, Buenos Aires, Argentina).
Behavioral Tests
Assessment of behavioral performance following remyelination was carried out as

described by Hoyos et al. (2014).
Statistical Analysis
Graph-Pad Prism software was used for data analysis. Results were presented as the
mean ± standard error of the mean (SEM). Comparisons were performed using
unpaired two-tailed Student’s t-test or two-way analysis of variance (ANOVA),
followed by Bonferroni’s post hoc tests when appropriate. Values of p < 0.05 (*),
p < 0.01 (**), p < 0.001 (***) were considered significant.
Data from behavioral assays were analyzed by two-way ANOVA, considering
CPZ treatment and animal type as two main factors, and post hoc comparisons were
made using Bonferroni’s post-test. When a significant interaction was observed,
subsequent one-way ANOVA and Newman-Keuls Multiple Comparison post hoc
test were applied. A value of p < 0.05 was considered statistically significant.
Results
Remyelination Starts Earlier in WT CPZ Than in Lgals3−/−

CPZ Mice but Reaches the Same Endpoint
To study the impact of Gal-3 deficiency on the remyelination process, WT and

Lgals3−/− mice were submitted to demyelination through a diet containing 0.2 %
(p/p) CPZ for 6 weeks and then studied after 2 weeks on a normal diet (Fig. 1a).
Animals were sacrificed at the critical point of demyelination (5th week) and after 2
weeks’ remyelination (8th week).
MBP immunelabeling was carried out in WT and Lgals3−/− mice at the critical
point of demyelination and at the end of remyelination (Fig. 1b). MBP IOD
measurements during remyelination relative to WT showed that WT CPZ mice
experienced spontaneous remyelination from the 5th week of treatment (when CPZ
was still in the diet). In contrast, Lgals3−/− CPZ mice exhibited remyelination only
after CPZ removal (Fig. 1c). Similar results were obtained in the analysis of each
CPZ-treated group relative to its own control, i.e., WT CPZ relative to WT C mice,
and Lgals3−/− CPZ relative to Lgals3−/− C mice, which indicates that data are not
altered by basal hypomyelination (Fig. 1e) and that both WT CPZ and Lgals3−/−
CPZ mice reach the remyelination levels of their respective C groups at the end of
the process (Fig. 1e). Western blot analyses of MBP expression provided support
for immunohistochemical data and highlighted that not all MBP isoforms were
equally altered, the 21.5 and 17.4 kDa isoforms being most affected (Fig. 1f).
For a deeper study of the remyelination process, changes in the numbers of CAII+
and CC1+ cells were analyzed as markers of different stages in oligodendroglial
differentiation. In brain coronal slices from CC (Fig. 2a, b), the number of CAII+
cells decreased in both CPZ-treated groups during demyelination, with Lgals3−/−
mice starting at a lower level than WT mice. During remyelination, the number of
CAII+ cells recovered only by 50 % both in WT and Lgals3−/− mice (Fig. 2b). The
same tendency was observed for CC1+ cells during demyelination, although
recovery during remyelination appeared to be more efficient in Lgals3−/− mice
(Fig. 2c, d), with the number of CC1+ cells reaching a higher WT CPZ/WT C ratio
than did Lgals3−/− CPZ/Lgals3−/− C in week 8. It is worth pointing out that although
there were no significant differences between WT CPZ and Lgals3−/− CPZ in the
numbers of CAII+ and CC1+ cells at the peak of demyelination (week 5), these
numbers were greater in WT CPZ than in Lgals3−/− CPZ mice 2 weeks after CPZ
removal from the diet (week 8).
In turn, the number of OPCs labeled for PDGFRa was higher in CC in Lgals3−/−
CPZ compared to WT CPZ during demyelination, as well as in Lgals3−/− C
compared to WT C, which suggests a higher oligodendroglial proliferative response
in Lgals3−/− mice, possibly due to their basal hypomyelination and their higher
degree of demyelination. When both groups were allowed to remyelinate after CPZ
removal, the number of PDGFRa+ cells decreased in both groups, but was more
pronounced in WT mice, which reached levels nonsignificantly different from
Lgals3−/− mice (Fig. 2e).
Interestingly, these cells were stellate, with a higher number of multipolar pro-
cesses in the CC of WT mice both during demyelination and remyelination, which
indicates a decreased ability of Lgals3−/− cells to differentiate. Quantitative support
was obtained through the numbers and lengths of branches and terminal processes.
OPCs from WT mice showed more branching, more ramification and, conse-
quently, more ramification terminals than Lgals3−/− mice at weeks 5 and 8
(Fig. 2f).
Fig. 2 a Identification of CAII+ cells in CC during demyelination and remyelination.

b Quantification shows a decrease in the number of CAII+ cells during demyelination in both
experimental groups, the decrease being greater in Lgals3−/− CPZ mice. Remyelination was not
complete, as values were nonsignificantly corrected in comparison to demyelination. c,
d Identification and quantification of CC1+ cells following the same procedures as in (a, b),
respectively. Values represent the mean ± SEM of five independent experiments. *p < 0.05,
**p < 0.01, ***p < 0.001 and ****p < 0.0001 using two-way ANOVA followed by Bonferroni
post hoc tests. e OPCs identified as PDGFra+ cells in the CC. Quantification revealed an increase
in the number of PDGFra+ cells in CC of Lgals3−/− CPZ mice versus WT CPZ during
demyelination, with an important decrease in both conditions after remyelination. f Filament plot
was performed for each image from CC sections, comparing PDGFra+ cell arborization between
demyelination and remyelination. Quantitative assessment of the filament plot exhibited greater
arborization in WT CPZ than Lgals3−/− CPZ mice, both during demyelination and remyelination.
Values represent the mean ± SEM of five independent experiments. *p < 0.05 and **p < 0.01
using two-way ANOVA followed by Bonferroni post hoc tests
Ultrastructural changes evaluated by EM in CC sections (Fig. 3a) showed that

myelin was more loosely wrapped around axons in Lgals3−/− than in WT mice, and
that this abnormal wrapping increased in both groups during CPZ-induced de-
myelination. Some of these abnormalities were solved during remyelination in
WT CPZ but not in Lgals3−/− CPZ mice. Morphometric analyses showed that the
number of myelinated axons per field increased in both groups from demyelination
to remyelination, although more significantly in WT mice. Despite this increase,
WT CPZ remyelination values nearly reached those of WT (p = 0.3710); this did
Fig. 3 a Representative electron micrographs at 6000 (scale bar equals 0.5 µm) from
demyelinated and remyelinated axons. Each condition shows an inset with a highly magnified
image (20000) of a representative axon. Samples were taken in the zone highlighted in the black
box in the scheme. As compared to that of WT mice, myelin observed in Lgals3−/− mice was
loosely wrapped around the axons, an abnormality which was incremented during CPZ-induced
demyelination and not solved during remyelination. Black arrows indicate loosely wrapped axon
areas. b Table showing the percentage of correctly myelinated axons. Values represent the
mean ± SEM ns non-significant, *p < 0.05 and **p < 0.01 using two-way ANOVA followed by
Bonferroni post hoc tests. c, d Axon diameter frequency categorized by range (myelinated–
demyelinated) comparing WT CPZ with WT, and Lgals3−/− CPZ with Lgals3−/− after
remyelination. CPZ WT mice showed larger axons than WT, while Lgals3−/− mice exhibited
similar axon size as Lgals3−/− CPZ. In both cases, there is evidence for similar patterns of axon
size change between demyelination and remyelination. e Astrocytes immunolabeled with
anti-GFAP antibody in the CC showed astrocytic activation in response to CPZ treatment in
both types of animals during demyelination and its persistence in remyelination. Bottom right
image indicates the area evaluated (black box, 40). f Quantification of GFAP+ cells evaluated in
relationship to the WT condition (set at 100 %). The increased astroglial response was particularly
strong in Lgals3−/− mice in the last period of demyelination. During remyelination, the curve
slopes were similar in the CPZ condition. Percentages of GFAP+ cells were relative to each WT.
Both CPZ curves had a similar slope. g GFAP+ cells in the SVZ. Bottom right image indicates the
area evaluated (black box, 40). h Quantitative assessment of astrocytes in the SVZ. Lgals3−/−
CPZ mice had more GFAP+ cells than CPZ WT mice in demyelination, but not during
remyelination. i Western blot analysis of GFAP expression in the CC at the 8th week. The graph
shows GFAP levels relative to GAPDH. Values represent the mean ± SEM of five independent
experiments. ns non-significant, *p < 0.05, **p < 0.01 and ***p < 0.001 using two-way ANOVA
followed by Bonferroni post hoc tests
not occur for Lgals3−/− CPZ mice (p = 0.0328) (Fig. 3b). When data were analyzed
discriminating for axon diameter and myelination status, results in Lgals3−/− mice
showed an increase in the number of small axons at the expense of a decrease in the
number of large ones, both among myelinated and unmyelinated axons (Fig. 3d).
Astrocytic Activation Persists in Remyelinated Mice

Despite CPZ Removal and Was More Pronounced
in Lgals3−/− Mice
Astrocytes immunelabeled with anti-GFAP antibody in the CC showed there was

reactive astrogliosis in response to CPZ treatment in both groups during demyeli-
nation, with more pronounced levels in Lgals3−/− mice, in agreement with previous
results (Hoyos et al. 2014). This effect persisted during remyelination, and the
differences between animal types became more noticeable (Fig. 3e). The quantifi-
cation of GFAP+ cells, setting the WT condition at 100 %, showed an increase in
astroglial response, especially in Lgals3−/− and particularly at the critical point
(5 weeks) of demyelination (Fig. 3f). The analysis of GFAP+ cells in the SVZ
(Fig. 3g) revealed a larger amount in Lgals3−/− CPZ than in WT CPZ mice during
demyelination, a difference that remained statistically significant during remyeli-
nation (Fig. 3h). Support for these results was obtained from Western blot analyses
of GFAP expression at week 8 (Fig. 3i).
Gal-3 Could Be Critical for MMP-3 Expression
Immunohistochemical studies revealed a pronounced increase in the expression

levels of MMP-3 in WT CPZ at 5 weeks, the critical point of demyelination, and
after 2 weeks of remyelination. This increase was almost absent in Lgals3−/− mice,
which indicates that Gal-3 could be critical for MMP-3 expression. MMP-3 was
mainly detected in microglia, identified as Iba+ cells (Fig. 4a), and quantitative
support was obtained through the determination of MMP-3 IOD in the CC in the
different experimental situations (Fig. 4b).
Only WT Mice Exhibit Changes in the Microglial Phagocytic

Phenotype During Remyelination
The total number of microglia evaluated through Iba-1 immunelabeling decreased

during remyelination in both WT and Lgals3−/− mice (Fig. 5a and b). Ramification
values also decreased in both WT and Lgals3−/− mice (Fig. 5c).
However, the number of CD45+ cells during remyelination showed a significant
decrease only in WT mice. Similar results were obtained for TNF and TREM-2
expression, which displayed a significant decrease after remyelination only in WT
mice. In contrast, the number of CD11b+ cells decreased during remyelination in
both mouse types (Fig. 6a), in agreement with the results obtained for Iba+ cells.
There were no differences across experimental groups or conditions in neutrophil
migration in response to CPZ intoxication or Gal-3 depletion (Fig. 6b).
Fig. 4 a Representative sections showing MMP-3 and Iba-1 in CC (40). During demyelination,
there was an increase in MMP-3 protein (red) in the CC of WT mice, which was even greater
during remyelination. The increase in MMP-3 in Lgals3−/− mice represented 50 % of that found in
WT mice. During remyelination in WT mice and demyelination in Lgals3−/− mice, MMP-3
co-localized with microglia (Iba-1+ cells in green). b MMP-3 in WT and Lgals3−/− mice during
demyelination–remyelination
b Fig. 5 a Immunolabeled for Iba-1 in coronal brain slices during demyelination–remyelination in

WT and Lgals3−/− mice. b Iba-1 immunohistochemistry in the CC of WT and Lgals3−/− mice
during demyelination–remyelination. Quantification of Iba+ cells. c Filament plot for all
experimental conditions. Quantification of total ramification, ramification segments and terminal
points under the same conditions as mentioned above. Values represent the mean ± SEM of five
independent experiments. ns non-significant, *p < 0.05, **p < 0.01 and ***p < 0.001 using
two-way ANOVA followed by Bonferroni post hoc tests
Behavioral Performance After Remyelination Reached

Pre-CPZ-Intoxication Levels in Both Mouse Strains
After 2 weeks of recovery, both WT and Lgals3−/− mice significantly increased the
number of total arm entries (p < 0.05), the percentage of open arm entries
(p < 0.001) and the percentage of time spent in open arms (p < 0.001) (Fig. 6c).
These data suggest that, at this point in treatment, WT and Lgals3−/− mice present
lower levels of innate anxiety than their naïve counterparts. The WT recovery group
also showed an augmented number of counts in the locomotor activity test
(p < 0.001) (Fig. 6d), which reflects increased locomotion. On the other hand, WT
and Lgals3−/− recovering mice showed no significant changes in the number of total
arm entries or in the percentage of spontaneous alternations in the Y-maze test
(p < 0.05) (Fig. 6e).
Discussion
Following the description of Gal-3 participation in the demyelination process

(Hoyos et al. 2014), the present work was undertaken to gain insight into the role of
Gal-3 in the remyelination process using a CPZ-induced demyelination model in
C57BL/6 control and Lgals3−/− mice. CPZ emerges as one of the most suitable
models available to investigate remyelination for several reasons: first, it is simple
and easy to reproduce and entails low mortality (Kipp et al. 2009); second, the
absence of peripheral inflammatory cells within the demyelinated lesion, such as
lymphocytes or monocytes, leads to immune-independent demyelination/remyeli-
nation (McMahon et al. 2002; Remington et al. 2007; Ransohoff and Brown 2012);
third and last, nearly complete remyelination allows for the study of the mecha-
nisms underlying successful regeneration.
When demyelination is detected in this model, increased number of microglia
and astrocytes are observed within the lesion (Matsushima and Morell 2001), which
indicates that glial cells play a role both in demyelination and in remyelination.
Reactive glia are now recognized to mediate complex processes, including
Fig. 6 a Flow cytometry analysis of cell surface receptors and cytokine production by pre-sorted
microglial cells (CD11b+) from CC after remyelination. CD11b+ cells were purified by magnetic
micro beads conjugated to an antibody against CD11b, followed by immunolabeling with a
specific antibody against cell surface receptors and cytokines, and evaluated by flow cytometry.
Data were processed with Winmdi 2.8 software. Pre-immune sera were used as negative controls
for polyclonal antibodies. Isotype controls were used for specific monoclonal antibodies. At least 6
mice were analyzed per experimental condition at each time point. Values represent the
mean ± SEM. ns: non-significant, *p < 0.05, **p < 0.01 and ***p < 0.001 using two-way
ANOVA followed by Bonferroni post hoc tests. b Neutrophil evaluation was done using May
Grünwald Giemsa staining in samples of CC from WT and Lgals3−/− mice treated with CPZ.
Magnification 60. c–e Performance of WT and Lgals3−/− mice after 2-week remyelination in the
plus maze, locomotor activity and Y-maze tests. Results are expressed as the mean ± SEM of
(c) total arm entries, percentage of open arm entries and percentage of time spent in open arms
measured in the plus maze test; d locomotor activity counts; and e total arm entries and percentage
of spontaneous alternation measured in the Y-maze assay. Eight to fifteen animals were analyzed
per group. *p < 0.05, **p < 0.01 and ***p < 0.001 significantly different from WT mice and
+++
p < 0.001 significantly different from Lgals3−/− mice, using Newman-Keuls Multiple
Comparison Test after one-way ANOVA
beneficial and deleterious effects of brain injury and in neurodegeneration. Previous

studies have investigated the microglial phenotype during remyelination by per-
forming genome-wide gene expression analyses of microglia from the CC during
demyelination and remyelination in the CPZ mouse model (Olah et al. 2012; Voss
et al. 2012). They provide evidence for the existence of a microglial phenotype that
supports remyelination as early as the onset of demyelination and continuing
throughout remyelination. Transcriptomic analyses of the remyelination-supportive
microglial phenotype indicate that microglia maintain tissue homeostasis and pro-
mote regeneration.
As previously discussed in Chapters “Glial cells and Integrity of the Nervous
System”, “Microglia Function in the Normal Brain”, and “Purine Signaling and
Microglial Wrapping”, microglia release either neurotoxic or pro-recovery factors,
depending on whether they are differentiated toward an M1 or M2 phenotype. They
are involved in many types of inflammatory processes in the brain (Hanisch and
Kettenmann 2007; David and Kroner 2011) and have also been proposed to par-
ticipate in the initial stages of MS. Microglia also support cell survival during tissue
repair after injury to the CNS (David and Kroner 2011). Moreover, Gal-3 is
required for resident microglia activation and proliferation in response to ischemic
injury (Lalancette-Hébert et al. 2012). A pronounced increase in the phagocytic
capacity of microglial cells during CPZ-induced demyelination is associated mainly
with an upregulation of the phagocytic receptor TREM-2b in WT but not in
Lgals3−/− mice (Hoyos et al. 2014). These experiments have been now replicated
during remyelination, showing a significant decrease in the number of CD45+,
TNFa+ and TREM-2b+ cells in WT mice but no change in Lgals3−/− mice from
demyelination (5 weeks) to remyelination (7 weeks) points.
Our recently published results on demyelination (Hoyos et al. 2014) show that
OPCs generated in response to CPZ-induced demyelination in Lgals3−/− mice have
a decreased ability to differentiate, which could be due to the inhibitory effects of
impaired phagocytosis of myelin debris in Lgals3−/− microglia (Hoyos et al. 2014).
Moreover, this could also be explained by our previous findings that conditioned
media from Gal-3-expressing (but not Lgals3−/−) microglia promote OLG differ-
entiation (Pasquini et al. 2011). Previous papers have demonstrated that an inter-
ruption in OPC differentiation might be the reason for remyelination failure
(Franklin et al. 2008; Ulrich et al. 2008). Therefore, Lgals3−/− mice could show a
delay in remyelination due to a failure in Gal-3-induced OPC differentiation.
Moreover, as Lgals3−/− mouse remyelination occurs in an environment poorly
conditioned by microglia, myelin generated de novo is aberrant and appears loosely
wrapped around axons. This could explain the behavioral deficit observed in these
animals after remyelination, which will be discussed below.
Depending on various scenarios, astrocytes can either promote neuroplasticity or
secrete inhibitory matrix molecules that suppress axonal growth. Some authors have
postulated that astrocytes produce chemoattractants for OPCs, allowing them to
migrate from their resting positions toward the demyelination zone (Williams et al.
2007). This is probably the best explanation for the larger number of astrocytes in
remyelination than in demyelination in both animal strains. However, Lgals3−/−
mice reach higher levels of GFAP+ cells than WT littermates, which could reflect
more severe demyelination.
While demyelination renders a significant reduction in axon caliber and a loss of
small axons in Lgals3−/− mice after CPZ administration (Hoyos et al. 2014),
remyelination generates a greater recovery in WT mice than in Lgals3−/− mice in
small axons, which again indicates a failure in the remyelination process in the
absence of Gal-3. It is presumably the more abundant presence of small-caliber
axons that accounts for the incomplete remyelination observed in Lgals-3−/− mice.
It has been suggested that the reduction in axon caliber after initial demyelination is
largely reversible upon remyelination (Mason et al. 2001), but those axons that do
not remyelinate remain small in caliber. Small-caliber axons are probably initially
abundant in Lgals-3−/− mice and never become myelinated.
As already discussed in this chapter, the 21.5 kDa MBP isoform is the first to be
synthesized; it promotes not only proliferation but also branching of OPCs (Smith
et al. 2013). MBP isoforms including 18.2 and 14.0 kDa are involved in myelin
compaction and stabilization (Chernoff 1981). During demyelination, a dramatic
decrease in the 21.5 kDa MBP isoform is observed in the absence of Gal-3, which
explains the deficit in normal myelin formation (Pasquini et al. 2011). During
remyelination, Lgals3−/− mice do not seem to recover expression of the 21.5 and
18.2 kDa isoforms, which might explain abnormal remyelination.
In a variety of demyelinating diseases, there is a well-established role for MMPs,
a family of extracellular endopeptidases for tissue remodeling and regeneration
(Yong et al. 2001). MMPs cleave all components of the extracellular matrix and
thereby serve important homeostatic functions. During oligodendrogenesis and
remyelination, OPC can also release multiple factors to modulate neighboring cells
and their environment. Gal-3 is readily cleaved by MMPs, thus altering its own
carbohydrate-binding activity (Ochieng et al. 1994). We have demonstrated that
Gal-3 is cleaved by MMP-2 in OPC but not in differentiated OLG (Pasquini et al.
2011). In this situation, and as described in our previous work, MMP-3 is upreg-
ulated during CPZ treatment, while Gal-3 seems to be necessary to upregulate the
expression of MMP-3 and to promote microglial activation. As previously
demonstrated, MMP-3 could mediate mature OLG apoptosis and microglial acti-
vation (Kim and Joh 2006). These authors have also proved that the catalytically
active form of MMP-3 (actMMP-3) is released from apoptotic PC12 cells grown in
serum-deprived medium. ActMMP-3 leads to the production of microglial
inflammatory cytokines such as TNF through the ERK-NFjB signal transduction
pathway, which in turn exacerbates neural cell degeneration.
Behavioral observations from three different approaches (plus maze, locomotor
activity, and Y-maze tests) evidence changes in anxiety responses to the challenge
of novelty and height, motor performance, and spatial working memory activity.
We have previously demonstrated that Lgals3−/− mice exhibit decreased anxiety
consistent with abnormalities in their myelin structure (Pasquini et al. 2011) and
that CPZ treatment induces more pronounced demyelination in Lgals3-/- relative to
WT mice (Hoyos et al. 2014). In the current study, after the recovery period of
2 weeks, a decrease in anxiety persisted both for WT and Lgals3−/− mice, as
evidenced by augmented percentages of entries and time spent in open arms in the
plus maze test. Locomotor activity showed an increase in the locomotor activity
counts as well as in the number of total arm entries in the plus maze test.
Meanwhile, spatial working memory was reestablished, as compared to their
untreated counterparts. Similar behavioral performance was expected of recovering
mice and their naïve counterparts. However, the recovery group showed higher
open arms activity in the plus maze and augmented locomotion than untreated
animals. These data suggest that some capabilities are altered even when histo-
logical analyses evidence fiber remyelination. Previous studies have already found
that CPZ-fed mice display abnormal behavior during the demyelination process
with partial recovery of functions during the remyelination period, with enhanced
locomotor activity, improved spatial working memory, and decreased anxiety levels
(Stancic et al. 2012; Franco-Pons et al. 2007; Xu et al. 2009). Our results indicate
that behavioral deficits follow the course of demyelination–remyelination-induced
by CPZ administration, and that some of the changes persist and seem to be
irreversible even 2 weeks after CPZ withdrawal.
Remyelination is a key mechanism which restores myelin to normal conditions
after demyelination and which seems to be importantly regulated by Gal-3, a
molecule participating in OPC differentiation. Since Gal-3 is expressed in mi-
croglia, myelin restoration could be mediated by the Gal-3-induced M2 cell
polarization, or by Gal-3 acting directly on OPC differentiation. In conclusion, our
findings demonstrate that Gal-3 hierarchically governs the myelination process, at
least in the CPZ model, through the modulation of microglial cells, MMP activity,
and myelin architecture.
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Prenatal Systemic Hypoxia-Ischemia
and Oligodendroglia Loss in Cerebellum
Penha Cristina Barradas, Tiago Savignon, Alex C. Manhães,

Frank Tenório, Alan P. da Costa, Marta C. Cunha-Rodrigues
and Juliana Vaillant
Abstract Hypoxic-ischemic (HI) injury is an important cause of death and dis-

abilities. Despite all improvements in neonatal care, the number of children who
suffer some kind of injury during birth has remained stable in the last decade.
A great number of studies have shown alterations in neural cells and many animal
models have been proposed in the last 5 decades. Robinson et al. (2005) proposed
an HI model in which the uterine arteries are temporarily clamped on the 18th
gestation day. The findings were quite similar to the ones observed in postmortem
studies. The white matter is clearly damaged, and a great amount of astrogliosis
takes place both in the gray and white matters. Motor changes were also found but
no data regarding the cerebellum, an important structure related to motor perfor-
mance, was presented. Using this model, we have shown an increased level of
iNOS at P0 and microgliosis and astrogliosis at P9, and astrogliosis at P23 (up to
4 weeks from the insult). NO is important in migration, maturation, and synaptic
plasticity, but in exacerbated levels it may also contribute to cellular and tissue
damage. We have also evaluated oligodendroglia development in the cerebellum.
At P9 in HI animals, we found a decrease in the number of PDGFRa+ cells and an
apparent delay in myelination, suggesting a failure in oligodendroglial progenitors
migration/maturation and/or in the myelination process. These results point to an
injury in cerebellar development that might help to explain the motor problems in
HI.
P.C. Barradas (&) T. Savignon F. Tenório A.P. da Costa M.C. Cunha-Rodrigues

J. Vaillant
Depto. Farmacologia e Psicobiologia, Instituto de Biologia—UERJ,
Av. 28 de setembro, 87 fds 5° andar, Rio de Janeiro, RJ 20551-030, Brazil
A.C. Manhães
Depto. Ciências Fisiológicas; Instituto de Biologia Roberto Alcantara Gomes,
Universidade do Estado do Rio de Janeiro, Rio de Janeiro, RJ 20551-030, Brazil
T. Savignon
Depto. Farmacologia e Toxicologia, Instituto Nacional de Controle
de Qualidade em Saúde, Fundação Oswaldo Cruz,
Rio de Janeiro, RJ 21040-900, Brazil

DOI 10.1007/978-3-319-40764-7_16
334 P.C. Barradas et al.
Keywords Hypoxia-ischemia Nitric oxide synthase PDGFa receptor MBP

Development

CP Cerebral palsy
CREB cAMP response element-binding protein
ED1 Antibody that labels macrophage/microglia
GFAP Glial fibrillary acid protein
HI Hypoxia ischemia
NADPH-d Nicotinamide adenine dinucleotide phosphate reduced diaphorase
NADPH-d+ Nicotinamide adenine dinucleotide phosphate reduced diaphorase
positive
NM Non-manipulated
NO Nitric oxide
NOS Nitric oxide synthase
nNOS Neuronal nitric oxide synthase
PDGFRa Platelet derived growth factor receptor alpha
P0 Postnatal day 0, here considered as the day of birth
P2, 7, 9, 23 Postnatal day 2, 7, 9, and 23
SHAM Surgical control
SMV Superior Medullary Vellum
uANOVA Univariate analises of variance
WHO World health organization
General Considerations
Hypoxic-ischemic (HI) brain injury is an important cause of death and disabilities

around the world, both in developing and developed countries (Vannucci and
Vannucci 2005; Volpe 2009). According to WHO, about one million deaths occur
yearly due to birth issues (Lawn et al. 2005). Despite all efforts at neonatal care in
recent decades, the number of children who suffer injury during birth has remained
stable during the last decade (Nelson et al. 2003). After perinatal insults, infant
brains suffer oligodendrocyte loss, hypomyelination, astrogliosis (Marín-Padilla
1997), and perturbed cortical development (Marín-Padilla 1999). The mechanisms
underlying these pathological changes remain unclear.
Cerebral palsy (CP), a chronic debilitating disorder of impaired motor devel-
opment, is strongly associated with perinatal brain injury (Kuban and Leviton 1994;
Prenatal Systemic Hypoxia-Ischemia … 335
Volpe 2001, 2003). Various perinatal brain insults have been associated with CP,
including prematurity and chorioamnionitis (Perlman et al. 1996; Verma et al.
1997; Spinillo et al. 1998; Wu and Colford 2000; Terzidou and Bennett 2001).
Although full term infants can develop CP, it occurs more frequently in premature
infants (Cummins et al. 1993).
Because various insults at different gestational stages induce elevated levels of
cytokines and disrupt brain development, it has been proposed that aberrant cyto-
kine expression underlies perinatal brain injury (Adlinolfi 1993). The pathogenesis
of perinatal brain insults is, however, likely to involve numerous pathways asso-
ciated with cytokines and oxygen-free radical species (Haynes et al. 2003; Folkerth
et al. 2004), and their relative contributions have yet to be defined.
Perinatal brain injury invariably involves the gray and white matters, with the
balance between them depending on the stage of cerebral developmental and vessel
maturation. In order to study HI insult and its mechanisms of damage, several
animal models have been proposed. Each has focused on a particular developmental
stage, trying to mimic one of the many types of brain injury that occurs in humans
(Fig. 1).
Fig. 1 Timeline of brain development at the cellular level and the temporal relationship of rat and
mouse versus human brain development. Arrows point to ages that HI insult in rats/mouse in
prenatal life (light gray), at birth (medium gray) and postnatal life (dark gray) are most often
performed. Light gray boxes summarize the major effects observed in rodent HI models with the
numbers indicating representative references: 1—Tashima et al. (2001); 2—Grojean et al. (2003);
3—Loeliger et al. (2003); 4—Dieni and Rees (2003); 5—Robinson et al. (2005); 6—Olivier et al.
(2005)
The first model was proposed by Levine (1960) using adult rats with a perma-
nent ligation of the carotid artery. This model was particularly useful to study
stroke. Rice et al. (1981) adapted this model in postnatal day (P)7 rats with the
carotid ligation either permanent or temporary, creating one of the most used
models in HI field. This age was chosen because it is comparable to newborn
humans regarding several parameters, including cell proliferation rate, cell migra-
tion, and establishment of layering patterns in the cortex. This model has been
useful for understanding several mechanisms of HI injury. However, it excludes
close interaction between mother and fetus.
To include the relationship between mother and fetus, Wigglesworth proposed a
model of growth restriction in 1964, in which one uterine artery was permanently
ligated on embryonic day (E)17, inducing ischemia and probably hypoxia, yet the
purpose of the study was exactly to show ischemia. Pups from the ligated uterine
horn exhibited growth restriction at birth, in both rats and pigs (Wigglesworth 1964;
Minkowski et al. 1981; Morand et al. 1982; Chanez et al. 1993; Jensen et al. 1996;
Sadiq et al. 1999).
In a growth restriction model, Olivier et al. (2005) found damage to white matter
like that in humans who suffer perinatal hypoxia. The growth-restricted animals did
not recover weight, even in adulthood. Moreover, there were diffuse white matter
lesions, increased cell death, and macrophage invasion, indicating increased
inflammation. At P7, they observed a loss of pre-oligodendrocytes and deficient
myelination. Those characteristics resemble what is seen in preterm infants with
birth complications.
Another group in 2005 presented an HI model in which all four uterine arteries
were clamped for 15, 30, or 45 min on gestation day 18 (Robinson et al. 2005). The
results were similar to those of the growth restriction model. Additionally, only
45 min of HI mimicked the neuropathology of what is seen in humans
(Marín-Padilla 1997, 1999): white matter astrogliosis, oligodendrocyte death,
axonal injury, and altered cortical cerebral layering. Robinson et al. (2005) also
described increased proinflammatory cytokines both in amniotic fluid and frontal
lobe of the fetuses 4 and 24 hours after the insult. Motor performance also declined,
for locomotion diminished in the open field test and steps shortened in the stride
length test in adult animals.
The authors pointed out that this walking pattern is characteristic of children who
develop cerebral palsy and its spastic gait. This systemic rodent prenatal HI insult
accurately models human perinatal brain injury in several important ways,
including functional association of altered brain development with motor delay, and
consequently provides novel insights into the pathogenesis of human perinatal brain
insults. As the cerebellum has the major importance in motor learning, we wish to
obtain information concerning the effects of HI using a rodent systemic prenatal
model. After Robinson et al. (2005) found that 45 min is the time that mimics
human pathology, we ligated the four uterine arteries for this period.
Nitric Oxide Synthase Levels and Distribution Were

Impaired in a Prenatal Systemic HI Model
Enhancement of nitric oxide synthase (NOS) isoform expression has been reported in
CNS areas after HI events (Kaur et al. 2006; Vexler and Yenari 2009). NO over-
production contributes to excitotoxicity, resulting in cell death and axonal damage
(see Chapter “Glial Cells and Integrity of the Nervous System”). We measured the
levels of neuronal (nNOS) and inducible (iNOS) isoforms at P0 (day of birth, i.e., 5
days after the HI insult). There was no difference in the level of nNOS protein in the
cerebellum of HI animals compared to SHAM controls, as shown in Fig. 2a.
However, the level of iNOS was significantly increased in HI animals (Fig. 2b).
Glial cells have been suggested as the major source of this NO overproduction
(Kashiwagi et al. 2003). NADPH-d histochemistry labels the NOS family (all three
isoforms), the enzymes responsible for NO production. The number of NADPH-d+
cells is significantly increased in cerebellar white matter of young rats (Savignon
et al. 2012). At P9 there were no differences in the number of NADPH-d+ cells in
the cerebellar white matter comparing non-manipulated (NM), SHAM, and HI
animals. However, at P23, the number of NADPH-d+ cells decreased in NM and
SHAM animals, remaining significantly higher in HI animals (as discussed in Fig. 5
of Savignon et al. 2012).
We identified NADPH-d+ cells in the white matter using specific markers for
macrophage/microglia (ED1) or astrocytes (GFAP). At P9, both SHAM and HI
Fig. 2 Increase in iNOS following HI injury. Both nNOS and iNOS levels are shown in the rat
cerebellum at birth (P0) in SHAM and HI group. Data are represented as means ± SEM in
arbitrary units (AU), resulting from 3 independent experiments. a nNOS–SHAM = 14.9 ± 2.7;
HI = 13.4 ± 2.7, p = 0.7036. No significant difference was observed between groups (p > 0.05).
b iNOS–SHAM = 15.6 ± 4.5; HI = 52.6 ± 9.1, p = 0.0220. HI group presents a significant
increase in iNOS levels (p < 0.05)
animals presented NADPH-d+/ED1+ cells (Fig. 3a, b—arrows) and NADPH-d+/

GFAP+ cells (Fig. 3c, d—arrows). In both groups, the morphology of NADPH-d+/
ED1+ cells is typical of reactive microglia, i.e., small and rounded cells. At P23,
both groups still presented NADPH-d+/ED1+ cells in the white matter (Fig. 3e, f—
arrows), with the same morphology as in P9. However, at P23, HI animals still
presented NADPH-d+/GFAP+ cells similar to reactive astrocytes (Fig. 3h—ar-
rows), whereas SHAM animals did not present NADPH-d+/GFAP+ cells mor-
phologically similar to reactive astrocytes, but instead showed typical GFAP+
astroglia (Fig. 3g—arrowheads), indicating that the insult has long-term effects on
tissue (Savignon et al. 2012).
These results, mainly those found at P9, were not a complete surprise since the
surgery procedure and anesthesia may account for an inflammation component or
other damage. It is worth noting that microglia/astrocytes preferentially express the
iNOS isoform when reactive, as in cases of injury and inflammation, typifying what
is called microgliosis and astrogliosis (see Chapter “Glial Cells and Integrity of the
Nervous System”). Thus, we have shown that the cerebellar tissue presents an
environment hostile to other cells such as oligodendrocyte progenitors. It has been
shown in the last two decades that NO is important in migration, maturation, and
synaptic plasticity of a variety of cerebellar cells. However, it is also a contributing
factor to cellular and tissue damages if that production is greatly increased, as it
occurs during inflammation.
Oligodendroglia Loss in the Cerebellum
Neurons, oligodendrocytes, and particularly their progenitors are most affected by

HI (Back et al. 2002a, b). As mentioned in Chapter “Oligodendrocytes: Functioning
in a Delicate Balance Between High Metabolic Requirements and Oxidative
Damage”, oligodendroglia progenitors do not have a mature enzymatic system to
deal with the substantial free radicals delivered in HI events, particularly by
microglia (Thorburne and Juurlink 1996; Le Mellédo et al. 2004). NO produced by
glia expressing iNOS (You and Kaur 2000; Park et al. 2002) may also be
responsible for this vulnerability. It has been demonstrated that both neurons and
oligodendrocytes release considerable glutamate to the extracellular compartment,
and this together with increasing NO, may cause excitotoxicity and cell death (Back
et al. 2007). Activated microglia express glutamate receptors (Gottlieb and Matute
1997) and may be modulated by the excess extracellular glutamate, producing more
NO.
Oligodendrocytes are derived from various subpopulations of progenitors (see
Chapter “Glial Cells and Integrity of the Nervous System” for further reading on
oligodendrocyte development). In the subventricular layer one arises to populate
forebrain (cortex) and midbrain (thalamus and hypothalamus), while another in the
ceiling of the fourth ventricle populates hindbrain (cerebellum, pons, and brain
Fig. 3 NOS activity remains associated with reactive astrocyte end feet at blood vessels in P23 HI
cerebellum. Double labeling with NADPH-d histochemistry (dark-blue) and microglia or astroglia
immunoidentification (brown) in the vermis region of the cerebellar white matter (0.5 mm
mediolateral distance) during development. a–d P9; e–h P23. a, c, e and g (SHAM); b, d, f and
h (HI); a–b and e–f double labeled with anti-ED1 antibody; c, d and g, h double labeled with
anti-GFAP antibody. In both groups at P9, we can observe small, rounded NADPH-d+/ED1+ cells
(a and b) or NADPH-d+/GFAP+ cells (c and d), as indicated by arrows. In d, observe a blood
vessel, transversally cut, which presents NADPH-d staining, surrounded by GFAP+ astrocytic
endfeet (asterisk). At P23, observe small rounded NADPH-d+/ED1+ cells in both groups, as
indicated by arrows. HI animals display NADPH-d+/GFAP+ cells with typical reactive astrocyte
morphology (arrows). SHAM animals do not present NADPH-d+/GFAP+ cells resembling
reactive astrocytes. Arrowheads point to typical GFAP+ astrocytes, with no NADPH-d labeling.
Notice the presence of NADPH-d+ blood vessels (asterisks in g and h) that are surrounded by
GFAP+ astrocytic processes in HI animals (h) but not in SHAM animals (g). Calibration bar:
50 lm. Reproduced from Savignon et al. (2012)
stem). There is some disagreement regarding the timing of these events. In the
forebrain it is early and in the hippocampus and cerebellum it is quite late.
Reynolds and Wilkin (1988) showed the sequential changes in oligodendroglia
during development, beginning as nondifferentiated cells in the superior medullary
vellum (SMV) and the base of cerebellum, which then populate the whole organ.
Others described the phenotypic and antigenic changes that oligodendroglia
undergo during differentiation both in vitro as in vivo (Pfeiffer et al. 1993; Baumann
and Pham-Dinh 2001).
Oligodendroglial progenitors express alpha-receptor to platelet-derived growth
factor (PDGFRa) (Baumann and Pham-Dinh 2001). Data from our laboratory
showed that in both HI and control animals the density of PDGFRa+ progenitors at
P2 is about 20 cells/100 lm2 (unpublished data), escalating to about 50 cells per
field at P9, and returning at P23 to the same levels as at P2.
At P2, there were no differences in the number of PDGFRa+ cells in cerebellar
white matter in both groups (Fig. 4a, b). This was not a complete surprise, since
rodent cerebellum develops rapidly postnatally. At P9, there was a significant
increase in PDGFRa+ cells in both groups when compared to P2 (uANOVA;
F = 126.34, p < 0.001). However, HI animals showed a significant lower number
in this progenitor subpopulation compared to SHAM animals (Fig. 4c, d), indi-
cating that a prenatal HI event somehow affected the proliferation rate and/or
survival of oligodendroglial progenitors. At P23, in both groups a significant
reduction in PDGFRa+ counting was observed in both groups (Fig. 4e, f). This was
expected, since the rate of proliferation diminishes and the progenitors start to
differentiate, downregulate PDGFRa, and form myelin. Figure 4g depicts the cell
counting results for each group.
Myelin basic protein (MBP), a marker of mature oligodendrocyte and myelin
(see Chapters “Glial Cells and Integrity of the Nervous System” and
Requirements and Oxidative Damage”), was also impaired in the prenatal HI
systemic model. At P9 in SHAM animals, MBP+ fibers were observed close to the
calbindin-positive Purkinje cell layer (arrows in Fig. 5a), whereas in HI animals
those MBP+ fibers were clearly located in the main white matter tracts (Fig. 5b),
suggesting an apparent delay in myelination in the cerebellum. As development
proceeds, oligodendrocytes/myelin were found in all extents of the granular layer in
both groups. Yet, it appears that some failure occurred in the oligodendroglial
progenitors migration/maturation and/or in the myelination process, since we found
non-myelinated gaps in the granular layer (asterisks in Fig. 5d). This occurred only
in HI animals. This pattern was maintained in HI animals until adulthood (Fig. 5f).
Together, these results point to an injury in cerebellar oligodendroglia development
that might help to explain the motor problems observed in HI animals.
Fig. 4 Number of PDGFRa+ cells in the cerebellar white matter of SHAM and HI animals at P2,
P9 and P23. At P2, we have not observed differences in the number of PDGFRa+ cells in
cerebellar white matter (a, b). At P9 (c, d) there is a significant increase in the number of
PDGFRa+ cells in both groups when compared to P2 (uANOVA; F = 126.34, p < 0.001).
However, HI animals have a lower number of PDGFRa+ cells than SHAM at P9 (d, but better
shown in g). At P23, a significant reduction in PDGFRa+ counting was observed in both groups
(e, f) when compared to P9. g Depicts the cell counting for each group, with density measured as
number per 100 lm2. Calibration bar: 50 lm
Fig. 5 Myelination is delayed in HI animals. Myelin basic protein (MBP) is labeled in red and
calbindin, in Purkinje cells, in green. At P9, there is an apparent delay in myelination in the
cerebellum. In SHAM animals, some MBP+/Calbindin+ axons are close to Purkinje cell layer
(arrows in a), while in HI animals those axons are not (b). From P23 (c, d) until adulthood (e, f),
MBP+/Calbindin+ occupy all of the granular layer (arrows) in both SHAM and HI animals.
Calibration bar: 100 lm
Concluding Remarks
Multiple types of injury resulting from preterm birth in humans, including systemic
HI, converge to hinder brain cell survival, particularly for immature oligodendro-
cytes and cerebral neurons (Volpe 2009). Impaired brain cell survival and
differentiation continue for a prolonged period after the initial injury in animal
models (Robinson et al. 2005; Mazur et al. 2010). At the time of the HI insult and in
the days following, when the levels of cytokine and other inflammatory modulators
are still elevated (Robinson et al. 2005), several glial and neuronal progenitor
populations are entering the cerebellum parenchyma through the prospective
cerebellar white matter. These progenitors, especially of oligodendrocytes, are more
vulnerable to HI events because they lack the enzymatic complexes capable of
dealing with the great amount of free radicals produced during HI. NO forms free
radicals if produced in large amounts and is toxic to oligodendrocyte progenitors. In
addition, elevated NO may trigger N-methyl-D-aspartate (NMDA)-mediated intra-
cellular Ca++-influx and CREB-mediated transcription of apoptotic proteins such
as Bax, Bad, and Bcl-xl, causing neuronal death (Zubrow et al. 2002a, b; Mishra
et al. 2006).
Our results showed that in this systemic model of prenatal HI, oligodendroglial
differentiation in the cerebellum was impaired, with a reduction in the number of
PDGFRa-cells (oligodendrocyte progenitors) and mature oligodendroglial cells, as
demonstrated by reduced MBP immunostaning. This supports this model for use in
devising new therapeutic strategies for HI insults.
Acknowledgments We would like to thank Jorge Pereira das Neves for technical assistance and
Mariana Soares Magalhães for animal care.
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Index
A Development, 3, 5, 9, 12, 14, 69, 71, 74, 78,

Aconitase, 51, 54, 55 81, 334, 336, 338, 340
Adenosine, 153, 154, 158 Differentiation, 30, 32–35, 37–40
Adenosine triphosphate (ATP), 95, 96, 98, Disease, 39–41
100–103, 150, 152–154, 158
Aging, 206–211, 213, 215, 216 E
Alzheimer’s disease, 289, 299–301 Eat-me signals, 151, 158
Astrocyte, 3–9, 11–16, 48–51, 53–56, Ensheathment, 170
118–126, 128–133, 228–233, 235, 236, Exosomes, 192, 194–197
238–240 Experimental autoimmune encephalomyelitis,
Astrocyte dysfunction, 229, 231, 232, 234, 273
236, 237, 240
Astroglial signaling, 100 F
Axoplasm, 184, 191–194, 197 Fluoroacetate, 52, 54
Axotomy, 154, 156–158 Fluorocitrate, 53, 54
B G
Brainstem, 112, 113, 115, 117, 123–127, Galectin-3, 313–315, 219, 223, 226–330
129–131 Glia, 123, 126, 127
Glioblastoma, 247, 248
C Gliotransmitters, 95, 102, 104, 118, 120, 121,
CD39, 153, 154, 158 126, 128, 131–133
Central chemoreception, 113, 114, 117, 121, Glutamine synthetase, 51, 55, 56
122, 126, 127, 129, 133
Central nervous system, 69, 247, 249, 253–255 H
Connexins, 95, 98 Hypoxia-ischemia, 333–343
Cuprizone, 313
Cytokines, 69, 71, 74, 77–79, 209, 211, 212, I
214–216 Injury, 37, 39, 40
D L
Demyelinating diseases, 265, 267, 273, 289, Locus coeruleus nuclei, 112, 114
292, 293, 297, 298
Demyelination, 313–315, 319–321, 323, 326, M
328–330 MBP, 340, 342

DOI 10.1007/978-3-319-40764-7
348 Index
Microglia, 3, 11–15, 48, 52, 53, 206, 210–216, Oligodendrocytes, 29–32, 34, 35, 37–40, 289,
247–255, 265, 268–270, 313, 314, 317, 294, 299, 313, 314
318, 323, 326, 328, 330 Oxidative stress, 206–208
Microglial migration, 153, 154
Microtubular density, 189–191 P
MMPs, 313, 314, 329 Pannexins, 96, 102, 103
mRNA, 188, 192, 194, 195, 197 PDGFa receptor, 29, 32, 38, 40, 294, 317–320
Multiple sclerosis, 264, 267, 268 Phagocytosis, 152, 158, 159
Myelin, 9, 10, 16 Plasticity, 34
Myelination, 34, 35, 38, 39, 168–172, 315, Proliferation, 30, 32, 35, 37–40
322, 330 Purine receptors, 153, 158
Myelin damage, 232
R
N Raphe nuclei, 112, 114, 115, 127
Nerve injury, 198, 211 Reactive oxygen species (ROS), 171
Neurodegeneration, 231, 233 Remyelination, 314, 315, 319–321, 323, 326,
Neurodegenerative diseases, 206, 214 328–330
Neurodevelopmental diseases, 229, 240 Respiratory rhythm, 111, 113, 123–125,
Neuroimmunity, 214 128–130, 132
Neuroinflammation, 77, 213, 214 Retrotrapezoid nuclei, 111, 112, 125
Neuromyelitis optica, 267, 268
Neuronal activity, 34–38, 40, 41 S
Neuronal degeneration, 148 Schwann cells, 3, 9, 16
Neuron–glia crosstalk, 3 Systemic inflammation, 264, 269
NG-2 cells, 16
NG2-glia, 29–36, 37, 39–41 T
NG2-glia neuronal synapse, 27, 30 Therapy, 252–255
Nitric oxide synthase, 337 Tripartite synapses, 5
Nuclear receptors, 289, 292, 297, 299
W
O Wallerian degeneration, 191
Oligodendrocyte, 3, 9, 11, 16
Oligodendrocyte precursor cells (OPCs), 168,
171, 172, 174

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Advances in Experimental Medicine and Biology 949

Glial Cells in Health

ISSN 0065-2598 ISSN 2214-8019 (electronic)

© Springer International Publishing Switzerland 2016

Printed on acid-free paper

This Springer imprint is published by Springer Nature

Glial Cells and Integrity of the Nervous System . . . . . . . . . . . . . . . . . . . . 1

Part I Glial Cell Function in the Healthy Nervous System

Part II Glial Cell Dysfunction and Pathological Implications

Beatriz Almolda Unit of Histology, Department of Cell Biology, Physiology and

María José Contreras Grupo de Neurociencias y Muerte Celular, Facultad de

V. Gudi Department of Neurology, Hannover Medical School, Hannover,

Silvia Olivera-Bravo Cellular and Molecular Neurobiology, Instituto de

M. Stangel Department of Neurology, Hannover Medical School, Hannover,

Rommy von Bernhardi, Jaime Eugenín-von Bernhardi, Betsi Flores

Abstract Today, there is enormous progress in understanding the function of glial

R. von Bernhardi (&) B. Flores

© Springer International Publishing Switzerland 2016 1

The Concept of Neuron–Glia Crosstalk

Two major morphologies are recognized:

Astrocytes and the Tripartite Synapsis

Metabolic Support by Astrocytes

Astrocytes in Synaptogenesis and Synaptic Maintenance

Astrocytes regulate formation, maturation, maintenance, and stability of synapses.

Signaling in Astrocyte Networks and Gliotransmission

Glutamate metabotropic receptors in astrocytes activate intracellular signaling

As discussed in Chapters “Pharmacological Tools to Study the Role of

Oligodendrocytes and Schwann Cells

Myelination and Saltatory Nerve Conduction

Like astrocytes, oligodendrocytes are also interconnected by gap junctions formed

Myelination Impairment and Disease

As discussed in Chapters “Peripheral Inﬂammation and Demyelinating Diseases”,

As further discussed in Chapter “NG2–Glia, More Than Progenitor Cells”, NG2–

extracellular matrix, is produced by activity-dependent cleavage of NG-2 by the

When an insult to the nervous system is detected by microglia, they launch a

The precise nature of signals triggering microglial activation is not well

Davoust et al. 2008; Kawahara et al. 2009). Exposure to neurotransmitters, prox-

NO Production and Oxidative Stress

As previously mentioned, activated microglia can produce several inflammatory

Damage Versus Protection

Although research has mainly focused on the detrimental effects of microglia-

Acknowledgments Supported by Grants Fondo Nacional de Desarrollo Cientíﬁco y Tecnológico

through MEGF10 and MERTK pathways. Nature 504(7480):394–400. doi:10.1038/

PET in idiopathic Parkinson’s disease. Neurobiol Dis 21(2):404–412. doi:10.1016/j.nbd.2005.

Pellerin L, Bouzier-Sore AK, Aubert A, Serres S, Merle M, Costalat R, Magistretti PJ (2007)

Jaime Eugenín-von Bernhardi and Leda Dimou

J. Eugenín-von Bernhardi (&) L. Dimou (&)

© Springer International Publishing Switzerland 2016 27

NG2-glia in the Central Nervous System

Fig. 1 a Confocal microscopy image showing the multiprocess morphology of NG2-glia

Fig. 3 NG2-glia are heterogeneous in regard to their morphology. Magniﬁcation of confocal

Synapses Between NG2-glia and Neurons

In addition to the controversial possibility of generating action potentials, NG2-glia

typical postsynaptic receptors such as N-methyl-D-aspartate receptor (NMDAR)

and “Schwann Cell and Axon: An Interlaced Unit—From Action Potential to

circuits, therefore enhancing the computational properties of networks required to

NG2-glia Behavior and Dynamics Modulated