Cell Death and Differentiation (2007) 14, 1324–1335
& 2007 Nature Publishing Group All rights reserved 1350-9047/07 $30.00
www.nature.com/cdd
Review
Glia: the fulcrum of brain diseases
C Giaume1, F Kirchhoff2, C Matute3, A Reichenbach4 and A Verkhratsky*,5,6
Neuroglia represented by astrocytes, oligodendrocytes and microglial cells provide for numerous vital functions. Glial cells
shape the micro-architecture of the brain matter; they are involved in information transfer by virtue of numerous plasmalemmal
receptors and channels; they receive synaptic inputs; they are able to release ‘glio’transmitters and produce long-range
information exchange; finally they act as pluripotent neural precursors and some of them can even act as stem cells, which
provide for adult neurogenesis. Recent advances in gliology emphasised the role of glia in the progression and handling of the
insults to the nervous system. The brain pathology, is, to a very great extent, a pathology of glia, which, when falling to function
properly, determines the degree of neuronal death, the outcome and the scale of neurological deficit. Glial cells are central in
providing for brain homeostasis. As a result glia appears as a brain warden, and as such it is intrinsically endowed with two
opposite features: it protects the nervous tissue as long as it can, but it also can rapidly assume the guise of a natural killer,
trying to eliminate and seal the damaged area, to save the whole at the expense of the part.
Cell Death and Differentiation (2007) 14, 1324–1335; doi:10.1038/sj.cdd.4402144; published online 13 April 2007
Neuronal Doctrine Challenged: Glial Cells Shape Brain
Physiology and Pathology
‘What a piece of work is a man! how noble in reason!
how infinite in faculty! in form and moving how
express and admirable! in action how like an angel!
in apprehension how like a god! the beauty of the
world! the paragon of animals! y’
W. Shakespeare, The tragedy of Hamlet, Prince of Denmark,
Act 2, scene 2
The sudden emergence of an intellect, and therefore a
human being, which materialised only around a million years
ago, remains the main mystery for our self-understanding.
Similarly, we still do not know by which steps or transitions the
human intellect emerged from the animal kingdom and where
the fundamental difference between a man and an animal lies.
According to the neuronal doctrine, which governs modern
neuroscience since the beginning of the twentieth century,1,2
the neurone is regarded as a basic information processing unit
consisting of dendrites and axons with a unidirectional flow of
information from the receiving dendrites via the integrating cell
body to the terminal branches of the axon. Neuronal networks,
connected through synaptic contacts, are generally considered as the substrate of our intellect.
The number and size of neural cells increase with the size
of the body and of the brain of mammals. This increasing
quantity eventually has caused the generation of a new
quality, the intellect. Rather amazingly, however, there is a
relatively little difference in the morphology and physiology of
neurones between humans and beasts; similarly the density
of synaptic contacts in the brains of rodents and humans is
more or less constant at around 1100–1300 millions/mm3.3
On the contrary, evolution of the nervous system resulted
in great changes in the second type of neural cells, the
neuroglia.4 Indeed, phylogenetic advance in brain complexity
and capabilities coincided with a remarkable increase in the
number of glial cells: in the rodent cortex the glial to neurone
ratio is about 0.3 : 1, whereas in humans the same ratio is
several times higher being B1.65:1,5 while the total number of
glial cells in the human brain is B10 (or even more) times
larger than in lesser mammals. Astrocytes in higher primates
display a much larger complexity as compared, for example
with rodents.4 The linear dimensions of human protoplasmic
astroglial cells (which are the main type of glia in grey matter)
are about 2.75 times larger and their volume is about 27 times
greater than for the same cells in a mouse brain. Furthermore,
human protoplasmic astrocytes have about 40 main processes and these processes have immensely more complex
1
INSERM, U840 and Collège de France, Paris, France; 2Neurogenetics Max Planck Institute of Experimental Medicine, Hermann Rein Str. 3, 37075, Göttingen,
Germany; 3Departamento de Neurociencias, Universidad del Pais Vasco, Spain; 4Paul Flechsig Institute of Brain Research, Faculty of Medicine, University of Leipzig,
Jahnallee 59, 04109, Leipzig, Germany; 5Faculty of Life Sciences, The University of Manchester, Oxford Road, Manchester, M13 9PT, UK; 6Institute of Experimental
Medicine, ASCR, Videnska 1083, 142 20 Prague 4, Czech Republic
*Corresponding author: A Verkhratsky, Faculty of Life Sciences, The University of Manchester, 1.124 Stopford Building, Oxford Road, Manchester M13 9PT, UK.
Tel: þ 44 161 2755414; Fax: þ 44 161 2755463; E-mail: alex.verkhratsky@manchester.ac.uk
Keywords: glia; astrocyte; oligodendrocyte; microglia; reactive gliosis; brain pathology; brain damage and repair
Abbreviations: AMPA, a-amino-3-hydroxy-5-methylisoxazole-4-propionic acid; BDNF, brain-derived neurotrophic factor; bFGF, basic fibroblast growth factor; CNS,
central nervous system; CNTF, ciliary neurotrophic factor; Cx, connexin; EAAT-1 and EAAT-2, excitatory amino-acid transporters type 1 and 2; EAE, experimental
autoimmune encephalomyelitis; GABA, g-aminobutyric acid; GFAP, glial fibrillary acidic protein; IL-1b, interleukin 1b; iNOS, inducible form of nitric oxide synthase;
MCP-1, monocyte chemotactic protein-1; MS, multiple sclerosis; NMDA, N-methyl-D-aspartate; PVL, periventricular leukomalacia; PVR, proliferative vitreoretinopathy;
ROS, reactive oxygen species; RPE, retinal pigment epithelial; TNF-a, tumour necrosis factor-a; TrkB, tyrosine kinase B receptor; VEGF, vascular endothelial growth
factor.
Received 31.1.07; revised 08.3.07; accepted 09.3.07; Edited by P Nicotera; published online 13.4.07
Glia in neuropathology
C Giaume et al
1325
branching than mouse astrocytes (which bear only 3–4 main
processes). As a result, every human protoplasmic astrocyte
contacts and enwraps B2 million of synapses compared to
only B100 000 synapses covered by the processes of a
mouse astrocyte.4
In addition to these quantitative changes, central nervous
system (CNS) of Homo sapiens and other primates developed
specific types of astroglia, the interlaminar astrocytes and
polarised astrocytes,4,6,7 which are absent from the brain of
other species. The occurrence of interlaminar astrocytes is a
very recent achievement of primate evolution. They first
appear in the old world monkeys and are absent from new
world monkeys. Interlaminar astrocytes are thought to provide
for information transfer between different cortical layers. Their
somata lie mainly within layer 1 among the axonal fibres that
connect different regions of the brain. It is tempting to
speculate that the interlaminar processes receive signals that
are integrated at the soma level and afterwards transferred to
axonal fibre tracts. The glial cells, hence, may be much more
important in contributing to higher brain function, not to speak
of intellect, as was previously thought.
Past two decades brought upon us an incredible increase in
knowledge about appearance, physiological properties and
functions of glia (see for review for example,8–10 to name but a
few). We learned that these cells (and especially astroglial
cells – Figure 1) are as diverse as neurones; they shape the
micro-architecture of the brain matter; they are capable of
expressing the same receptors and channels as neurones do;
they receive synaptic inputs; they are organised as communicating networks; they are able to release ‘glio’transmitters
and produce long-range information exchange; finally they act
as pluripotent neural precursors and some of them are, most
likely, neural stem cells, which provide for adult neurogenesis. These advances in gliology constitute a tremendous
challenge to the neuronal doctrine, calling for a fundamental
reshaping of our perception of the brain organisation, which
undeniably will lead to an appearance of a more inclusive
theory of brain function.
Similarly, our perception of brain pathology, which, for a
long time was revolving around neuronal reactions, their
survival or death, has now turned into investigations which
very much emphasised the role of glia in the progression and
handling of the insults to the nervous system. The brain
pathology, is, to a very great extent, a pathology of glia, which,
when falling to function properly, determines the degree of
neuronal death, the outcome and the scale of neurological
deficit.
Indeed, glial cells are fundamental in determining neuronal
well-being and in providing all lines of defences to CNS. The
astroglia are forming neuronal-glial-vascular units in which
astrocytes forge the functional link between synaptic activity
and functional hyperaemia; simultaneously astrocytes feed
active neurones through the glucose-lactate shuttle.11,12
Astroglia rules over extracellular homeostasis in the brain
through controlling interstitial concentration of neurotransmitters (and most importantly the naturally toxic, yet the most
abundant neurotransmitter glutamate13), ions (K þ buffering,14) and regulating movements of water.15 In addition, brain
insults invariably trigger reactive astrogliosis, which reflects
the ancient and conserved astroglial defence reaction.16
The astrogliosis is fundamental for both limiting the areas of
damage (by scar formation through anisomorphic astrogliosis) and for the postinsult remodelling and recovery of neural
function (by isomorphic astrogliosis).
Similarly, the second macroglial cell type, the oligodendrocytes ensure the proper function of axons, by myelinating the
latter. Damage to oligodendrocytes triggers Wallerian degeneration and invariably results in axonal demise.17 Finally, the
microglia, which populates the whole of the brain parenchyma
and dwells in relatively independent territorial domains, is the
only system of specific immune and cellular defence, residing
beyond the blood–brain barrier.18 Malfunction of glia therefore
is fatal for the nervous system; all in all glial cells can survive
and operate in the presence of dead or dying neurones;
neurones, however, cannot survive in the absence of glia.
In this paper, we shall overview only some of many aspects
of glial control over damage and repair in the nervous tissue,
specifically concentrating on the astroglial gap junctions and
neuroprotection, on the role of Müller glial cells in retinopathies, on the dynamics of microglia and on the oligodendrocytes and white matter damage.
Glia and Neuropathology: General Perspectives
Figure 1 Confocal laser-scanning micrograph of a cortical astrocyte recorded
from a double-transgenic mouse in which astrocytes express EGFP and neurones
the red fluorescent protein HcRed1. Note the polarised shape of the astrocyte: one
endfoot is contacting a brain capillary, whereas thousands of distal processes are in
close contact to synapses
The brain, the most complex organ in our body, has to function
over a long time at the same time adapting to permanently
changing environmental challenges. As a result, the brain
circuitry has to be exceptionally plastic, and indeed many
brain areas (for example hippocampus or the visual cortex)
are prone to a constant remodelling. Other brain regions,
responsible for vital functions such as breathing, should
rigidly adhere to a conserved structure. This implies the
necessity of autoregulatory systems, which control all aspects
of brain development and function. To achieve this, the
Cell Death and Differentiation
Glia in neuropathology
C Giaume et al
1326
brain is isolated from the rest of the organism so that it can
control itself efficiently. This border is formed by the blood–
brain barrier. Astroglial endfeet plaster the blood vessels and
induce tight junctions among neighbouring endothelial cells of
the brain capillaries. Selective uptake and transport mechanisms in endothelial and astroglial membranes are essential for
most components of the blood stream to enter the brain.
Further, control over homeostasis in the brain parenchyma is
of a paramount importance and these are the glial cells that
act as creators and defenders of this homeostasis. As a result
glia appears as a brain warden, and as such it is intrinsically
endowed with two opposite features: it protects the nervous
tissue as long as it can, but it also can act as a natural killer,
trying to eliminate and seal the damaged area, to save the
whole at the expense of the part.
This two-sided role of glia is particularly exemplified upon
ischaemic brain insults.19 Disruption of the blood flow in the
brain causes considerable damage and death of neural cells.
Reduced oxygen supply (either hypoxia or anoxia) triggers
rapid depolarisation of neurones, and greatly compromises
their ability to maintain transmembrane ion gradients. This
is manifested in Na þ and Ca2 þ influx into the cells together
with a substantial K þ efflux; massive Ca2 þ influx initiates
glutamate release from neuronal terminals, thus further
amplifying the vicious circle by inducing ‘glutamate excitotoxicity’.20
Neurones and oligodendrocytes are the most vulnerable
and sensitive to ischaemic shock and glutamate excitotoxicity;
astrocytes are generally (but not always) more resilient. The
predominant mechanism of cell death, which follows the
stroke, is associated with excessive activation of ionotropic
glutamate receptors; particularly important is the long-lasting
opening of highly Ca2 þ permeable N-methyl-D-aspartate
(NMDA) receptors. The latter are expressed in majority of
neurones, in several types of astrocytes and in many
oligodendrocytes21; in all these cells overactivation of NMDA
receptors is pathologically relevant. The final result of
exocytotic signalling cascades is the cell overload with
Ca2 þ , which strains metabolic processes, impairs upon
mitochondrial function and activates numerous death-related
enzymes thus driving cells to fatal end through apoptotic or
necrotic route.22
The main brain defence against glutamate excitotoxicity is
formed by astrocytes. Astroglial cells, by virtue of numerous
transporters residing in their membrane, act as the main
sink for the glutamate in the CNS (see for example13 for
review). Astroglial protection against glutamate excitotoxicity
became very obvious from in vitro experiments: withdrawal
of astrocytes from neuronal cultures invariably produced a
very significant increase in the neuronal death triggered
by glutamate administration.13 On a more general
level, pharmacological or genetic downregulation of glial
glutamate transporters exacerbates brain damage. In
addition, astrocytes, which contain high concentrations of
main antioxidants glutathione and ascorbate, protect the brain
against reactive oxygen species, which are invariably
produced upon ischaemia. Finally, being the main site
for potassium buffering, astroglia removes the excess of K þ
from extracellular space, which may restrain neuronal
depolarisation.
Cell Death and Differentiation
The same mechanisms, however, underlie the darker side
of astroglial responses to ischaemic insults; when injury is too
severe astroglial cells may exacerbate damage of neural
tissue. First, astrocytes may act not only as a sink, but also as
a source of glutamate. Indeed, concentration of the latter in
astroglial cytoplasm may reach the level of several (up to 10)
mM. Depolarisation of astrocyte membrane together with an
increased extracellular Na þ concentration can reverse
glutamate transporter,23 thus producing glutamate efflux.
Further, glutamate may leave the astrocytes through hemichannels, which can be opened by lowering of extracellular
Ca2 þ and acidosis or even through P2X7 receptors, activated
by excessively high extracellular ATP; all these do happen
during ischaemia.24 Second, astrocytes may spread the death
signals through the brain parenchyma via gap junctions25 and/
or participate in developing spreading depression, which
determine the infarction progress through the penumbra.
Intercellular Communication in Glia and
Neuroprotection
Connexins in glia. In the brain, a typical, but not exclusive,
property of glial cells is prominent expression of connexins
(Cxs), the molecular constituents of gap junction channels
that allow direct intercellular communication between
adjacent cells.26 When expressed at the membrane, Cxs
are organised as hexamers, which, when associated headto-head between two neighboring cells, form a full gap
junction channel. Recent works have also demonstrated
that Cxs can operate as hemi-channels allowing
transplasmalemmal matters exchange.27 The central pore
defined by hemi- or full- Cx channels accounts for passage of
ions and small molecules with a cutoff selectivity of about
1–1.2 kDa. Cxs constitute a multigenic family of 20 or 21
members in rodents and humans, respectively, and so far at
least 11 different Cxs (Cx26, Cx29, Cx30, Cx32, Cx36, Cx37,
Cx40, Cx43, C 45, Cx46 and Cx47) have been detected in
the brain. Numerous studies have shown that biophysical
properties, compatibility of assembly between defined Cxs,
permeability and regulation of Cx channels depend on the
nature of their molecular constituents leading to the concept
of a ‘language’ of Cx.28 This concept, initially proposed for
gap junction channels, may now be extended to hemichannels as several ‘gliotransmitters’ can be released
through the latter and activate neighbouring glial cells and
neurones. In situ pattern of Cxs expression is distinct for
each brain cell type and all of them contain more than one Cx
suggesting that the properties and role of full- and hemichannels are cell specific.
Although the first evidence of gap junction-mediated
communication was demonstrated by electrophysiological
recordings of electrical coupling between excitable cells, the
permeability of Cx channels for small molecules is probably
more important for signalling between nonexcitable cells,
such as glia. Thus, addressing the question of the role of glial
Cxs requires the identification of signalling molecules exchanged through this intercellular pathway. There is now
converging evidence indicating that intercellular exchange
of signalling molecules occurs in cultured astrocytes and in
Glia in neuropathology
C Giaume et al
1327
glioma cell lines transfected with Cxs.29 The permeability of
astrocyte gap junctions for glucose and its metabolites,
including lactate, has been initially characterised by using
radiolabelled compounds. More recently, this has been
confirmed by using a fluorescent glucose molecule 2-(N-(7nitrobenz-2-oxa-1,3-diazol-4-Y1)amino)-2-deoxyglucose.30
Furthermore, Cx full- and hemi-channels are also instrumental
to the propagation of intercellular Ca2 þ waves thanks to their
permeability for InsP3 (intercellular route) and ATP (extracellular route).28
The level of Cx expression in most glial cells is high
compared to neurones, and this high expression persists
throughout adulthood. Morphological, biochemical and functional studies carried out in vitro as well as in situ indicate that
each glial cell type expresses a set of distinct Cxs, none of
them being specific for glia and that the strength of coupling
and the level of Cx expression depends on the glial subtype,
the developmental stage and the brain region.
Astrocytes. Cx43 is the main Cx detected in cultured
astrocytes.26 When astrocytes are isolated from Cx43
knockout mice, coupling is reduced to B5% of the control.
This residual coupling is provided by small amounts of other
Cxs, in particular Cx30, Cx40, Cx26, Cx46 and Cx45.
Similarly, expression of multiple Cxs mRNAs in astroglia
was revealed by single-cell RT–PCR performed on
astrocytes from hippocampal brain slices.31 Nevertheless,
Cx30 and Cx43 are considered the main Cxs expressed
in situ in astrocytes. Interestingly, Cx43 expression and
coupling efficiency vary in cultured astrocytes derived from
different brain regions. Similar heterogeneity was also found
between and within brain regions studied in acute slices.32
Moreover, Cx30 is only expressed in astrocytes of mature
grey matter, and the relative levels of Cx43 and Cx30 vary
according to the developmental stage and region studied.33
Although still debated,34 the presence of Cx26 was also
reported in sub-population of astrocytes.35
Oligodendrocytes are endowed with gap junctions found in
almost all areas of their plasma membranes, however, they
were not detected between successive layers of myelin.36 As
shown in culture and in brain slices, the coupling efficiency
between oligodendrocytes is low compared to astrocytes.
Expression of Cx32 and Cx47 has been shown in oligodendrocytes studied in vitro and in vivo.37 Distribution of Cx
sybtypes in adult brain sections suggests differences in their
subcellular localisation with Cx32 expression appearing first
coincidentally with the start of myelination.38 Expression of a
gene reporter indicates that Cx29 is also present in
oligodendrocytes in the white and grey matters.39
Microglial cells. Although nonactivated microglia lack Cx43
expression in culture it has been detected in adult rat
cerebral cortex. The expression of Cx43 is increased after
activation of microglial cells in vivo after a stab wound. In this
case, Cx43 is observed at the interfaces between activated
cells, which become dye-coupled through gap junctions.40
The expression of glial connexins is affected in brain
inflammation. Brain inflammation is a hallmark of many
brain diseases and it is characterised by a reactive gliosis
associated with phenotypic changes and proliferation of glial
cells (mainly astrocytes and microglia). These changes are
accompanied by modifications of Cx expression in
astrocytes, as complex changes in Cx43 expression and
gap junctional communication have been observed after
brain injuries and pathologies known to be associated with
reactive gliosis.41 During brain inflammation, microglial cells
and astrocytes synthesise a variety of inflammatory
mediators that regulate Cx43 expression and gap junctional
communication in astroglia. For instance, exposure of
cultured astrocytes to interleukin 1b (IL-1b) downregulates
their content of Cx43 at both mRNA and protein levels.42 This
effect is potentiated by another proinflammatory cytokine
tumour necrosis factor-a (TNF-a) as well as by b-amyloid.
These inhibitory effects are reproduced by co-culturing
astrocytes with activated microglia known to release
proinflammatory cytokines, indicating that tight interaction
occur between glial partners involved in reactive gliosis.43
The functional consequences of this inflammation-induced
Cx inhibition in astrocytes are not fully understood, however,
one possibility is that the decrease in Cx-mediated
communication may restrict the passage of active
molecules to neighbouring cells thus isolating the primary
lesion site. Therefore, reactive astrocytes with modified
intercellular communication should be considered as key
elements in a dynamically changing environment that is likely
to modify neuronal functions and survival.
Intercellular communication in astrocytes is either neuroprotective or deleterious. Given the high expression
of Cxs, the extent of gap junctional communication
among astrocytes and their role in spatial buffering of
ions (potassium, Ca2 þ ), long-range signalling and
exchange of small permeating molecules (glutamate, ATP,
glucose) within astroglial syncytium, a neuroprotective role
for astrocyte gap junctions has been hypothesised.
Alternatively, Cx-mediated intercellular communications
have been implicated in the propagation of cellular injury
between astrocytes. These contrasting opinions are based,
in part, on experimental brain ischaemia models and on the
clinical impact of such injuries.44 Several observations
support a neuroprotective role of astrocyte Cxs: (i) the
pharmacological inhibition of astrocyte gap junctions
enhances neuronal vulnerability either to glutamate
cytotoxicity or metabolic stress in neurone/glial co-cultures
as well as under experimental ischaemia using oxygen and
glucose depletion in hippocampal slice cultures; (ii) in vivo,
following middle cerebral artery occlusion, heterozygote
Cx43 þ / mice show a significantly increased stroke volume
compared to wild-type mice; and (iii) the use of targeted
deletion of Cx43, specifically in astrocytes, provides further
evidence that these proteins play a neuroprotective role in
ischaemic insults . On the other hand, evidence that gap
junctions enhance neuronal injury is also supported by
several observations. That is, (i) neuronal death caused by
oxygen and glucose depletion is decreased when Cx43
expression
is
blocked
by
specific
antisense
oligodeoxynucleotides in hippocampal slice cultures; (ii) the
stroke volume following occlusion of the medial cerebral
artery is reduced by gap junction inhibitors, and (iii) the
spreading depression caused by ischaemic insult propagates
via astrocyte gap junctions remaining open, a process that
Cell Death and Differentiation
Glia in neuropathology
C Giaume et al
1328
results in the expansion of the stroke volume. The role of
astroglial Cxs could also be mediated by hemi-channel
opening. Indeed, it has been recently reported that Cx hemichannels, which are normally closed became open under
experimental ischaemia induced by glucose and oxygen
deprivation.24
Up-to-now, a number of factors may account for the above
apparent contradictions.44 For instance, experimental studies
vary considerably with regard to the systems employed and
the way used to block hemi- or full- Cx channel-mediated
communication. In particular, chronology is likely an important
parameter to be considered. All in all, gap junctions as well as
hemi-channels may be beneficial at a defined step in the injury
cascade but deleterious when considered at another time.
Also, for a defined brain damage, the change in the level of Cx
expression is different in the core and at the periphery of the
damaged site, a process that likely depends on the nature of
the Cx involved (Cx43 versus Cx30). Finally, the contribution
of Cxs is certainly different when these proteins work as
hemi- or full-channels. Indeed, as each of these membrane
channels provides a pathway for intercellular communication,
it is not yet established that they target the same cellular
partner (neurones, astrocytes, oligodendrocytes and microglia) and that they allow the movement of the same signalling
molecules. These critical distinctions will become clear when
specific pharmacological tools discriminating between different Cxs, or between hemi- and full-channels will be available.
Müller Cells and Retinopathy
The retina, although being a highly specialised sensory organ,
is often used as a ‘simple’, veritable model of the brain. To
study the impact of glia in neurodegeneration, the mammalian
retina indeed offers numerous advantages. First, it contains a
peculiar type of macroglial cells, the so-called Müller (radial
glial) cells, either as the only type (in avascular retinae) or as
the dominant type of macroglia (in vascularised retinae,
astrocytes are additionally located in the innermost retinal
layers). Second, the well-layered structure of the retinal tissue
greatly facilitates both the identification and the quantification
of the neuronal cell type(s) undergoing degeneration. Third, all
neuronal compartments (somata, processes, synapses) and
non-neuronal elements of the retina and its environment
(blood vessels, vitreous body) form intimate contacts with the
branches of the Müller cells, each of which spans the entire
retinal thickness. This latter condition, together with their
biochemical and physiological properties, enables the Müller
cells in the healthy retina to perform a wealth of crucial
interactions with the neurones, to guarantee their developmental maturation, nutrition, normal function, and survival
(Figure 2a).45
However, this symbiosis between the Müller cells and their
‘assigned’ neurones,45 advantageous as it normally is, causes
dramatic problems for neuronal survival when the Müller cells
stop patronising neurones. Unfortunately, these are not rare
events; rather, a de-differentiation of Müller cells (‘reactive
Müller cell gliosis’) occurs in many acute and chronic retinal
injuries and diseases. Then, the neurones are endangered not
only by the loss of supportive glial functions, but also by
Cell Death and Differentiation
additional Müller cell reactions which often are directly
detrimental to them (Figure 2b).45
This ‘Janus-faced’ Müller cell impact on retinal neurones
can be better understood when one considers the essential
physiological features of these glial cells. The plasma
membrane of Müller cells is highly permeable to K þ as a
consequence of the high density of specialised K þ channels.
The high K þ permeability sets a very negative membrane
potential of these cells, which is close to the equilibrium
potential for K þ (about 80 mV), which, together with the
specific subcellular distribution of distinct K þ channel types, is
an essential precondition for virtually all neurone-supportive
functions of these cells. In particular, inwardly rectifying K þ
channels containing the Kir4.1 subunit are concentrated in
perivascular membrane sheets, and at the inner and outer
limiting membranes.46 These channels are responsible for the
export of excess K þ from retina into extraretinal ‘sinks’ such
as the blood vessels, the vitreous body and the subretinal
space. K þ ions are released into the perisynaptic extracellular
clefts by active neurones, from where they enter the Müller
cells via the channel complexes containing the Kir2.1
subunit46; this process of retinal K þ clearance has been
termed ‘K þ siphoning’.47 The K þ ions can leave the retina by
this mechanism only together with water molecules, for
osmotical reasons. This provides another essential Müller
cell function, namely the clearance of excess water which, for
instance, is generated by the aerobic metabolism of retinal
neurones (reviewed by Bringmann et al.48; see Figure 2a, i).
Another Müller cell function is called ‘neurotransmitter
recycling’ (Figure 2a, II). Müller cells possess high-affinity
uptake carriers for retinal neurotransmitters, including glutamate and g-aminobutyric acid (GABA), which remove excess
signalling molecules from the perisynaptic clefts. They also
express glutamine synthetase, which converts glutamate
into the nonactive molecule, glutamine. The latter is released
back for neuronal uptake and reconversion into glutamate
or GABA.49 It is noteworthy, that this enzymatic reaction is at
the same time the only possibility to remove ammonia from the
brain (glutamate þ ammonia-glutamine þ water). An important precondition for transmitter recycling is the highly
negative membrane potential of the Müller cells; depolarisation (caused for example by an inhibition or downregulation of
K þ channels) strongly impairs glial neurotransmitter uptake,
because the uptake carriers are electrogenic, and use the
membrane potential as a driving force.50
There are many more neurone-supportive functions of
mature Müller cells, including metabolic symbiosis by ‘feeding’
of neurones with lactate; clearance of CO2 by the glial
carbonic anhydrase, delivery of ROS scavengers such as
glutathione (Figure2a, III), and release of neurotrophic growth
factors such as basic fibroblast growth factor (bFGF) and
ciliary neurotrophic factor (CNTF), partially driven by stimulation of the brain-derived neurotrophic factor receptors of
tyrosine kinase B receptor type on Müller cells (Figure 2a,
IV).45 All these functions require an intact energy metabolism
of Müller cells, which is directly or indirectly related to
maintenance of negative membrane potential of the cells.
All the above-mentioned normal glia–neurone interactions
are supposed to be maintained, and probably even stimulated, in cases of mild and transient retinal injury. However,
Glia in neuropathology
C Giaume et al
1329
Figure 2 Survey of glia–neurone interactions in the normal and injured retina. (a) Role of Müller (glial) cells in maintaining K þ and water homeostasis (I), clearing
neurotransmitters (II) and protecting the retina against oxygen free radicals (III). Furthermore, Müller cells release neurotrophic factors (IV). (b) Failure of reactive ‘gliotic’ Müller
(glial) cells to maintain K þ homeostasis and to prevent the accumulation of water (I), to clear the retina from excess L-glutamate and ammonia (II), and to neutralise oxygen
free radicals is detrimental and may lead to permanent retinal damage (III). Moreover, reactive Müller cells even release nitric oxide (III) and detrimental (amounts of) cytokines
such as VEGF (causing leaky blood vessels) and TNF (contributing to neuronal cell death) (IV). Intracellular water accumulation causes glial cell swelling and oedema, which
exerts pressure upon retinal blood vessels and neurones (I). Abbreviations: A, amacrine cell; ATP, adenosine triphosphate; bFGF, basic fibroblast growth factor; BDNF, brainderived neurotrophic factor; C, cone photoreceptor; CAP, capillary; CB, cone bipolar cell; CNTF, ciliary neurotrophic factor; cyst, cysteine; EGF, epithelial growth factor; GABA,
g-aminobutyric acid; GC, ganglion cell; glut, glutamate; glu, glutamine; GS, glutamine synthetase; GSH, glutathione (reduced); GSH-synth, glutathione synthetase; GSSG,
glutathione (oxidised); HC, horizontal cell; iNOS, inducible form of nitric oxide synthase; MCP-1, monocyte chemotactic protein-1; NADPH, nicotinamide adenine dinucleotide
phosphate, reduced form; PEDF, pigment epithelium-derived factor; R, rod photoreceptor; RK, free radical molecule; RB, rod bipolar cell; TGFb, tumour growth factor-b; TNF,
tumour necrosis factor; VEGF, vascular endothelial growth factor
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more severe retinal damage inevitably triggers ‘unwelcome’
responses of Müller cells, manifested in reactive gliosis. This
process involves an early upregulation of the expression of
the intermediate filament protein, glial fibrillary acidic protein,
which can be visualised by immunohistochemistry. Further
progression of reactive gliosis is accompanied by a dedifferentiation of the cells; a key step consists in a downregulation
and redistribution of K þ channels in the glial cell plasma
membrane.45 Particularly, the Kir4.1 are no longer enriched in
the perivascular and vitreal endfoot membranes of Müller
cells,51 and the K þ conductance of the cell membrane at
potentials negative to –40 mV is strongly reduced or even
completely missing.51This is accompanied by a loss of the
negative resting membrane potential of the cells; such
reactive Müller cells display stochastically scattered membrane potentials up to levels as low as 20 mV.45,51
Thus, the reactive downregulation of K þ channels constitutes a switch from neuroprotective functioning of mature
glia to a ‘private live’ of dedifferentiated glial cells which now
abandon neurones to an increasing chaos of their waste
products. The neurones continue to release K þ and
glutamate neither of which can be buffered by the Müller cells
any longer (Figure 2b, I and II), owing to insufficient K þ
conductance and altered membrane potential (i.e., missing
driving force for glutamate uptake). This causes strong, longlasting depolarisations of the neurones, accompanied by
excessive Ca2 þ influx and, finally, excitotoxic neuronal death.
As a further complication, the insufficient K þ siphoning into
the blood vessels and other sinks coincides with an impaired
water export from the retina, resulting in glial cell swelling.45
This glial swelling and the subsequent cellular retinal oedema,
is further aggravated by a vascular endothelial growth factor
(VEGF)-induced leakiness of the retinal blood vessels (see
below) which cause a further drag of water into the retinal
tissue (and finally, into the glial cells). There are two conditions
which exacerbate the problem; first, K þ may still enter the
Müller cells because the inwardly rectifying Kir2.1 channels
remain available in membrane areas facing neuronal K þ
release and second, the aquaporin-4 water channels are still
present in the endfoot membranes of Müller cells. Together
this may even cause a reversal of the water fluxes at the
endfoot membranes, such that now water enters the Müller
cell cytoplasm from the blood vessels, rather than vice
versa.45 In any case, glial swelling and retinal oedema
constitute another danger for the survival of the neurones:
the swollen glial cells apply pressure on the blood vessels,
which decreases retinal supply of oxygen and nutrients, and
cause a further accumulation of neuronal waste products, and
deliver a pressure on the neurones themselves, thus causing
direct mechanical injury (Figure 2b, I). It should also be kept in
mind that the breakdown of neurotransmitter recycling is
accompanied by a failure of ammonia clearance, because
the glial glutamate uptake is no longer sufficient for a balanced
feeding of the glutamine synthetase reaction; increased
levels of ammonia contribute to neurotoxicity and neurodegeneration.
Moreover in the course of reactive gliosis, the Müller cells
become depleted of glutathione, resulting in impaired defence
against free radicals (Figure 2b, III). Rather, the inducible form
of nitric oxide synthase (iNOS) is now expressed by glial cells,
Cell Death and Differentiation
which causes an increased exposure of the neurones to
ROS.52 Finally, the pathological scenario involves a dramatic
change in the cytokine release pattern of the Müller cells
(Figure 2b, IV). Oedema/pressure-mediated reduction of
retinal blood flow and excitotoxic overstimulation of neuronal
metabolism contribute to retinal hypoxia which stimulates the
release of VEGF from Müller cells53 and retinal pigment
epithelial (RPE) cells.54 Increased VEGF levels make the
retinal vasculature leaky, which, in turn, leads to increased
efflux of water (i.e., to aggravated oedema) and to the release
of a variety of cytokines from vessels into the retinal tissue.
Some of these cytokines, such as epithelial growth factor
(EGF), are known to stimulate the release of TNF from Müller
cells. Finally, Müller cells stop releasing neuroprotective
factors such as bFGF and CNTF, but rather flood the
neurones with cell death-inducing factors such as TNF and
monocyte chemotactic protein-1.55
These pathological signalling pathways, further stimulated
by the release of ATP from injured neurones and activation of
ATP receptors on Müller cells (as well as by their depolarised
membrane potential, see above), eventually trigger the entry
of Müller cells into the cell cycle (Figure 2b, IV).45 Massive
Müller cell proliferation then causes the formation of glial
scars, the migration of dedifferentiated Müller cells out of the
retina, and the generation of cellular plates or ‘membranes’ on
both surfaces of the retina (Figure 3a). These cellular
membranes are constituted by several different cell types,
which all are capable of migration and proliferation, such as
RPE cells, fibroblasts and retinal glial cells. As they use to
grow into the vitreous body (and induce alterations there), this
phenomenon is called proliferative vitreoretinopathy (PVR).56
Proceeding PVR then leads to folding and detachment of
the retina (Figure 3b), excessive neurodegeneration and
blindness.
In summary, normal mature Müller cells display a set of
specific features that enable them to perform a wealth of
interactions with the retinal neurones. These interactions
support the survival and the proper functioning of nerve cells
in the healthy retina, and are stimulated in the early stages
of mild retinal injury, thus providing neuroprotective action.
Upon severe and/or rapidly progressing retinal injuries,
however, Müller cells undergo reactive gliosis; a key event
in this process is the downregulation and redistribution
of Kir4.1 channels, accompanied by a depolarisation of the
membrane potential. This leads to a blockade of the neuroprotective glia–neurone interactions, which in turn causes a
dramatic worsening of the survival conditions of the neurones.
Further, reactive Müller cells actively contribute to neuronal cell
death, by releasing toxic substances and cell death-mediating
cytokines; finally, excessive glial cell proliferation causes
retinal damage and detachment, which results in blindness.
Microglial motion: moving processes and brain
defence. Microglial cells are the immunocompetent cells
residing in the CNS, which, in essence, form the main brain
defence system, activated upon various kinds of injuries and
diseases.57 Microglia are of a myelomonocytic origin; the
microglial precursor cells invade the brain during early
embryonic development before closure of blood–brain
barrier.57 In the normal brain, microglial cells are present in
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Figure 3 Extensive proliferation and migration of Müller cells as a detrimental complication of reactive gliosis. (a) Transition between normal Müller cells in a healthy retina
(left side) and severe proliferative reactive gliosis in the injured retina with massive neurodegeneration (right side). (b) The outward migration of proliferating cells causes
mechanical forces that lead to retinal folding (arrows) and detachment, described as proliferative vitreoretinopathy (PVR). Ophthalmoscopic image of a rabbit retina with
experimentally induced PVR
the resting state, which is characterised by a small soma and
numerous very thin and highly branched processes (hence
these cells are also often called ‘ramified’).57. In the cortex,
each individual microglial cell is responsible for a clearly
defined territory of about 150 000 mm3; the processes of
resting cells are never in contact with each other.58 Brain
insults set into the motion the activation of microglia, which
is characterised by a complex pattern of biochemical and
morphological changes. The activated microglia, very
similarly to astrocytes, possess numerous mechanisms that
are simultaneously neuroprotective and neurodestructive,
depending on the severity of brain insult.
Up until recently resting, ‘ramified’ microglia have been
considered as rather inactive and dormant cells, which dwell
in the neuropil and wait for activation due to brain injuries of
various nature. Many in vitro studies identified a plethora of
substances, which cause microglia activation with morphological and functional transformations; these agents include
endogenous substances such as chemokines, cytokines or
metabolites such as IL1-b, IL-4, TNFa, fractalkine, complement fragments or ATP, but also ectopic substances such as
cell wall components of Gram-positive and -negative bacteria,
lipopolysaccharides or lipoteichoic acid.59–61
Most of microglial studies performed so far, however, used
acutely isolated brain preparations, which themselves can
induce cellular activation by releasing various active substances from damaged tissue. This disadvantage could now
be circumvented by employing two-photon laser-scanning
microscopy (2P-LSM) on genetically modified mice carrying
microglial markers, thus allowing noninvasive real-time
visualisation of microglia in the brain proper.58,62 For this
analysis, genetically modified mice were used in which
replacement of the chemokine receptor CX3CR1 gene (also
known as fractalkine receptor) by the enhanced green
fluorescent protein (EGFP) leads to microglia-specific labelling.63 Because the EGFP is expressed in the cytoplasm,
laser-scanning microscopy can reveal the complete cellular
structure.
Using this model fluorescently labelled cells can be
visualised transcranially through a thinned (B50 mm) window
made in the skull bone. Confocal imaging revealed the typical
shape of microglia: small rod-shaped somata with a radial
extension of numerous thin and highly ramified processes
(Figure 4). Microglial cells were found to be rather homogeneously distributed, suggestive of a territorial organisation.
On average about 6000–7000 microglial cells occupied every
mm3 of the brain volume. Interestingly, mice tolerated
anaesthesia and imaging for more than 10 h, which time
was quite sufficient for long-term analysis of microglial
motility. The real-time movies obtained with this technique
highlight the enormous dynamics of permanent membrane
extensions and process retractions.58 To penetrate the
neuropile, the tips of extending processes form bulbous
enlargements which spread into the neighbourhood at a
speed of about 1–2 mm per min. After such an extension
period, the processes were retracted and extended again into
another direction. Interestingly, the cellular somata stayed
very constant at their positions forming a three-dimensional
microglial network. As the extent of random process dynamics
stayed constant over several hours, it was concluded that it
serves a type of brain surveillance. This regular surveillance
pattern changes into a rapid response when the brain is
damaged. Using energy of light delivered through 2P-LSM,
micro-haemorrhages can be induced by focusing the scanning infrared laser onto a brain capillary for 30 s and
increasing its power threefold.58 As soon as components of
the blood arrive into the neuropil, microglial cells immediately
send their processes to the lesion site. Processes are also
being sent from rather distant regions (more than several
hundred micrometres apart). Process tips impinging on the
lesioned vessel broaden and form, together with processes
from other cells, a tight mesh that soon seals the capillary.
Similar targeted process extensions can be observed if single
cells such as neurones or astrocytes are ablated by the
infrared laser.62
The rapid movement of microglial processes is controlled
by metabotropic P2Y12 purinorepecptores, as in P2Y12
knockout mice localised microlesions do not induce rapid
process extensions of microglia.64 Therefore, early microglial
reactions to brain injury can be divided into at least three
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Figure 4 Time-lapse recording of microglia in the spinal cord of genetically modified mice in which EGFP is expressed from the CX3CR1 locus. (a–c) Snapshots of a
microglial cell taken at times indicated. Microglial processes are longitudinally orientated along axonal fiber tracts. Some small membrane protrusions (arrows) emanate from
the major processes within minutes, whereas others are retracted simultaneously. These terminal processes constantly survey their cellular neighbourhood. (d) Overlay of the
images shown on (a–c), colour coded in red, green and blue. The static portion of the cell therefore appears in white and the moving processes in different colours
phases: first, associated with ATP/P2Y12-mediated capability
to sense acute lesions and initiate secondary responses such
as process extension; second, which requires as yet unknown
signals to control growth process towards the lesion site and a
third phase which consists of the reorganisation of process
tips directly at the injured area.
The white matters: oligodendroglia death and brain
ischaemia. Oligodendrocytes are the major cell type of
white matter, which in humans constitutes about 50% of the
total brain volume. Oligodendroglia express glutamate
receptors and transporters, and are highly vulnerable to
excitotoxic and ischaemic insults (for recent reviews see
Alberdi et al.65) Excessive activation of a-amino-3-hydroxy-5methylisoxazole-4-propionic acid (AMPA), kainate or NMDA
receptors in oligodendrocytes leads to Ca2 þ overload and
cell death. A central event to this process is accumulation of
Ca2 þ within mitochondria, which leads to the depolarisation
of this organelle, increased production of oxygen free
radicals, and release of proapoptotic factors which activate
caspases (Figure 5).
Glutamate can also cause oligodendrocyte demise indirectly by inducing the release of toxic agents (such as TNF-a)
Cell Death and Differentiation
from microglia, which can potentiate glutamate oligotoxicity
via inhibition of glutamate uptake. Indeed, inhibition of the
expression and functioning of glutamate transporters in
axonal tracts is sufficient to induce oligodendroglial loss and
demyelination.66 Furthermore, glutamate at nontoxic concentrations can also induce oligodendrocyte death by sensitising
these cells to complement attack.67 Complement toxicity is
mediated by kainate, but not by AMPA, NMDA or metabotropic glutamate receptors and requires the formation of the
membrane attack complex which in turn increases membrane
conductance, induces Ca2 þ overload and mitochondrial
depolarisation as well as a rise in the level of reactive oxygen
species. Treatment with the antioxidant Trolox and inhibition
of poly(ADP-ribose) polymerase-1, but not of caspases,
protected oligodendrocytes against damage induced by
complement. This novel mechanism of glutamate-induced
toxicity to oligodendrocytes is also shared by neurones and
may be relevant to glutamate injury in acute and chronic
neurological disease with primary or secondary inflammation.
Loss of oligodendrocytes with subsequent damage to white
matter occurs in stroke, traumatic injury, neurodegenerative
diseases, multiple sclerosis (MS) as well as in psychiatric
diseases.17 Immature and adult oligodendrocytes are
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Figure 5 Signalling cascades triggered by activation of glutamate receptors induce oligodendrocyte death. Selective activation of AMPA receptors (AMPAR) and kainate
receptors (KAR) leads to Na þ and Ca2 þ influx through the receptor channel complex. Subsequent depolarisation activates voltage-gated Ca2 þ channels (VGCC) which
contributes to [Ca2 þ ]i increase. Ca2 þ overload induces rapid uptake by mitochondria, which results in attenuation of mitochondrial potential and an increase in the production
of reactive oxygen species (ROS). Cytochrome c (Cyt c) is released from depolarised mitochondria, interacts with apoptotic protease activating factor 1 (Apaf-1) and activates
caspases. Other proapoptotic factors include apoptosis-inducing factor (AIF) which activates poly(ADP-ribose)polymerase-1 (PARP-1). In oligodendrocytes, insults channelled
through Kai-R activate caspases 9 and 3, whereas those activating AMPA-R induces apoptosis by recruiting caspase 8, which truncates Bid, caspase 3 and PARP-1, or cause
necrosis. In addition, Ca2 þ influx triggered by Kai-R stimulation but not by AMPA-R activates calcineurin (CdP), which dephosphorylates Bad and facilitates apoptosis. Finally,
activation of NMDA receptors (NMDA-R) also initiates oligodendrocyte death which is entirely dependent on Ca2 þ influx; however, the molecular mechanisms activated by
these receptors are not known yet. Abbreviations: FADD, Fas-associated death domain; 14-3-3, phosphoserine-binding protein 14-3-3
particularly sensitive to transient oxygen and glucose deprivation. Both NMDA and AMPA/kainate receptors are activated
during ischaemia and their antagonists protect both oligodendrocytes and myelin.68 This feature is relevant to stroke as
well as to preterm and perinatal ischaemia. Thus, in vivo
models of stroke and cardiac arrest such as permanent middle
cerebral artery occlusion and brief transient global ischaemia
induce rapid oligodendroglial death.69 Few days after the
insult, there is an increase in the number of oligodendroglial
cells in areas bordering affected regions,70, as well as in the
number of immature oligodendrocytes surrounding the lateral
ventricles71 indicating that ischaemic damage to oligodendroglia can be compensated for, at least in part, by the
generation and migration of new oligodendrocytes.
Preterm and perinatal ischaemia can cause periventricular
leukomalacia (PVL), the main substrate for cerebral palsy,
which is characterised by diffuse injury of white matter
surrounding the lateral ventricles. White matter damage in
PVL is largely related to hypoxia-ischaemia and reperfusion in
the sick premature infant as a consequence of free radical
injury, cytokine toxicity and excitotoxicity. Injury to oligodendrocyte progenitors, caused in part by glutamate and the
subsequent derailment of Ca2 þ homeostasis, contributes to
the pathogenesis of myelination disturbances in this illness.72
In addition to this mechanism, glutamate-induced depletion
of glutathione and the subsequent oxidative stress in PVL
also contributes to damage to oligodendrocytes, which are
sensitive to oxidative stress in part because of their high lipid
and iron content. Notably, the vitamin K deficiency in preterm
infants is a risk factor for developing PVL, and in turn its
presence is protective against oxidative injury to immature
oligodendrocytes.73
Another illustrious example of white matter disease is MS,
in which the immune system attacks the white matter of the
brain and spinal cord, leading to disability and/or paralysis.
Myelin and oligodendrocytes are lost owing to the release
by immune cells of cytotoxic cytokines, autoantibodies and
toxic amounts of glutamate.74 In agreement with this idea,
experimental autoimmune encephalomyelitis (EAE), an animal model which exhibits the clinical and pathological features
Cell Death and Differentiation
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of MS, is alleviated by AMPA and kainate receptor antagonists. Remarkably, blockade of these receptors in combination with anti-inflammatory agents is effective even at an
advanced stage of unremitting EAE, as assessed by
increased oligodendrocyte survival and remyelination, and
corresponding decreased paralysis, inflammation, CNS
apoptosis and axonal damage.75
Glutamate levels are increased in acute MS lesions and
in normal-appearing white matter in MS patients.76 Potential
cellular sources contributing to enhanced glutamate levels in
cerebrospinal fluid include activated microglia, which can
release glutamate via the reversal of glutamate transporter
function, a process which is potentiated under pathological
conditions.74 In addition, oxidative stress may also contribute
to the increase in glutamate concentrations in the extracellular
space, because free radicals reduce the efficiency of
glutamate transporters.74 Other factors which may contribute
to perturbing glutamate homeostasis include altered activity
of the glutamate producing enzyme glutaminase in activated
macrophages/microglia in close proximity to dystrophic
axons,77 and altered expression of the glutamate transporters
excitatory amino-acid transporter type 1 (EAAT-1) and EAAT2 in oligodendrocytes as a consequence of enhanced
exposure to the proinflammatory cytokine TNFa.78 Overall,
these alterations likely lead to high extracellular glutamate
levels and an increased risk of oligodendrocyte excitotoxicity
in MS.
In summary, oligodendrocytes display great vulnerability to
excitotoxic insults mediated by glutamate receptors, a feature
which is relevant to acute and chronic diseases involving
white matter such as stroke and MS, respectively. The proper
functioning of glutamate uptake is critical to prevent glutamate-induced damage to oligodendrocytes, and positive
regulators of the expression of glutamate transporters have
a protective potential, as they contribute to ischaemic
tolerance after ischaemic preconditioning.79
Another set of molecular targets to prevent glutamate
insults to oligodendrocytes lie downstream of glutamate
receptor activation (see Figure 5). For instance, tetracyclines,
which attenuate mitochondrial damage subsequent to insults
including excitotoxicity, protect oligodendrocytes and white
matter, making these antibiotics promising candidates for the
treatment of acute and chronic diseases with oligodendrocyte
loss.80 On the other hand, drugs supporting the management
of Ca2 þ overload subsequent to the activation of glutamate
receptors may improve oligodendrocyte viability.
Conclusions
Increasing number of evidence indicates that glial cells shape
neural networks. Thus, it is time to reconsider their role in brain
functions and to ask whether they are the fulcrum in brain
pathology. Indeed, various types of glia are involved into
dysfunction and damage of the brain, and specific glial
functions may play a key role in the triggering or in the
progression of several brain pathologies. The identification of
molecular actors in glia such as junctional proteins, functional
purinergic and glutamatergic receptors or cell mobility should
certainly be considered when studying neurologic disorders.
Glial contribution to brain pathology in many cases takes two
Cell Death and Differentiation
faces, either protective or deleterious. Understanding of the
rules that govern this duality and its balance is yet nascent;
however, it is clear that the further definition of reactive gliosis,
which embraces different subclasses of glia will give important
clues in the near future. These clues will identify the role of glia
in neuroprotection, neural cells death and their repair, and
may result in developing new strategies of treating the insulted
brain.
Acknowledgements. CG research was supported by the CRPCEN;
the studies carried out in CM’s laboratory were supported by the Ministerio de
Educación y Ciencia, Ministerio de Sanidad y Consumo, Gobierno Vasco and
Universidad del Paı́s Vasco; FK was supported by grants from the Max Planck
Society and the DFG; AR were supported by the DFG; AV research was supported
by HIN and Alzheimer Research Trust (UK). The authors thank Dr. Thomas
Pannicke for inspiring discussions about this paper.
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Cell Death and Differentiation