Glutamate-Mediated Glial Injury: Mechanismsand Clinical ImportanceCARLOS MATUTE,* MAR�IA DOMERCQ, AND MAR�IA-VICTORIA S�ANCHEZ-G �OMEZDepartamento de Neurociencias, Universidad del Pa�ıs Vasco, Leioa, Vizcaya, Spain

KEY WORDSglutamate receptor; glutamate transporter; cell death; oligo-dendrocyte; pathology

ABSTRACTPrimary and/or secondary glial cell death can cause and/oraggravate human diseases of the central nervous system(CNS). Like neurons, glial cells are vulnerable to glutamateinsults. Astrocytes, microglia, and oligodendrocytes express awide variety of glutamate receptors and transporters thatmediate many of the deleterious effects of glutamate. Astro-cytes are responsible for most glutamate uptake in synapticand nonsynaptic areas and consequently, are the major regu-lators of glutamate homeostasis. Microglia in turn maysecrete cytokines, which can impair glutamate uptake andreduce the expression of glutamate transporters. Finally, oli-godendrocytes, the myelinating cells of the CNS, are verysensitive to excessive glutamate signaling, which can lead tothe apoptosis or necrosis of these cells. This review aims atsummarizing the mechanisms leading to glial cell death as aconsequence of alterations in glutamate signaling, and theirclinical relevance. A thorough understanding of these eventswill undoubtedly lead to better therapeutic strategies to treatCNS diseases affecting glia and in particular, those thatinvolve damage to white matter tracts. VVC 2005 Wiley-Liss, Inc.

INTRODUCTION

Glutamate is the principal excitatory neurotransmitterin the central nervous system (CNS), but it is also apotent neurotoxin that can kill nerve cells. Excessive glu-tamate signaling can lead to excitotoxicity, a phenomenonwhereby overactivation of glutamate receptors (GluRs)initiates cell demise. Excitotoxic cell death was firstdescribed during the late 1950s in retinal neurons (Lucasand Newhouse, 1957) and was found later to occur in vir-tually all neurons that express GluRs (Olney et al., 1969).Thereafter, glutamate excitotoxicity has been implicatedin acute injury to the CNS and in chronic neurodegenera-tive disorders (Choi, 1988; Lipton and Rosenberg, 1994;Lee et al., 1999). During the past few years, it has beenshown that glutamate can also be toxic to glial cells,including astrocytes (Haas and Erdo, 1991) and oligoden-drocytes (Yoshioka et al., 1995; Matute et al., 1997;McDonald et al., 1998). Moreover, glutamate can damagenerve cells by mechanisms that do not involve receptoractivation, but rather glutamate uptake (Oka et al, 1993).To understand how aberrant glutamate signaling cancause glial injury, we will first summarize current knowl-

edge of GluRs and glutamate transporters (GluTs) as wellas glutamate metabolism in glial cells. The mechanismsof glutamate toxicity in glia and its relevance to CNS dis-eases will then be discussed.

GLUTAMATE SIGNALING IN GLIAL CELLS

Glutamate signaling is carried out by GluRs andGluTs. Glutamate activates ionotropic receptors, whichgate membrane ion channels permeable to cations andmetabotropic receptors or seven-transmembrane (7TM)receptors, which are coupled to G proteins (for review, seeMichaelis, 1998; Dingledine et al., 1999; Cull-Candy andLeszkiewicz, 2004; Swanson et al., 2005; Mayer, 2005).Functional AMPA receptors are composed of GluR1-4subunits, whereas kainate receptors are composed ofGluR5-7 and KA1-2 subunits. Similarly, N-methyl-D-aspartate (NMDA) receptors consist of an NR1 subunit,together with NR2A-D subunits and NR3A-B (Dingledineet al., 1999; Cull-Candy and Leszkiewicz, 2004). AMPAreceptors are activated by AMPA and kainate whereaskainate receptors are also activated by kainate and arebest functionally isolated in the presence of GYKI53655,a selective AMPA receptor antagonist (Lerma, 2003). Inturn, metabotropic GluRs (mGluRs) can be classified asgroup I (mGluR1, mGluR5), group II (mGluR2, mGluR3),and group III (mGluR4, mGluR6-8) 7TM receptors(Swanson et al., 2005).

Functional GluRs are expressed by glial cells in gray aswell as in white matter (Verkhratsky and Steinh€auser,2000; Belachew and Gallo, 2004; Kettenmann andSteinh€auser, 2005). Ionotropic GluRs, mainly of theAMPA and kainate type, are expressed by astrocytes andoligodendrocytes and cultured microglia also possessesfunctional GluRs of these classes (Table 1). In particular,cells of the oligodendrocyte lineage express functionalAMPA/kainate type receptors throughout a wide range ofdevelopmental stages, although it is not clear yet whetherhuman mature oligodendrocytes express these receptors

Grant sponsor: Ministerio de Educaci�on y Ciencia, Ministerio de Sanidad y Con-sumo; Grant sponsor: Gobierno Vasco; Grant sponsor: Universidad del Pa�ıs Vasco.

*Correspondence to: Carlos Matute, Departamento de Neurociencias, Universi-dad del Pa�ıs Vasco, E-48940 Leioa, Vizcaya, Spain. E-mail: carlos.matute@ehu.es

Received 31 May 2005; Accepted 4 August 2005

DOI 10.1002/glia.20275

Published online 3 October 2005 in Wiley InterScience (www.interscience.wiley.com).

VVC 2005 Wiley-Liss, Inc.

GLIA 53:212–224 (2006)

(Wosik et al., 2004; P�erez-Cerd�a et al., 2005). The hetero-geneity of the native receptors expressed in glia is verylarge, since they are formed by homomeric and hetero-meric combinations of virtually all the AMPA and kainatereceptor subunits (Matthias et al, 2003). This complexityof this heterogeneity is further enhanced by the fact thatpatterns of expression of GluRs are spatially (regionally)and temporally distinct, thus providing a signaling sub-strate that can subserve a highly complex signaling sys-tem. In addition, recent data point to the expression ofNMDA receptors both in astrocytes and in oligodendro-cytes (Table 1; see also Kettenmann and Steinh€auser,2005). However, the presence of this family of receptors inglial cells appears to be very restricted, and the subunitsforming the receptors have not yet been characterized.Activation of ionotropic GluRs on glial cells induces Ca21

influx, which is coupled to a large variety of biologicalresponses, including the release of neurotransmitters andgrowth factors and modulation of neurotransmission (Vol-terra and Steinh€auser, 2004). Glutamate receptor signal-ing may thus play an active role in brain signaling andrepair. Overall, the receptors expressed by glial cells havethe same general properties as their neuronal counter-parts. However, they are edited to a lesser extent in thewhite matter (Matute et al., 1999) and are therefore morepermeable to Ca21 (Burnashev, 1996). In addition, theelectrophysiological properties of AMPA receptors in oli-godendroglial progenitors (OPCs) and in Bergmann gliastrongly suggest that they lack the edited GluR2 subunit,a feature that is relevant to neuron-to-glia communica-tion, since they provide a link between axonal activityand internal Ca21 levels in progenitors (Bergles et al.,2000), and AMPA receptor-mediated Ca21 influx is impor-tant in generating and maintaining the appropriate struc-tural and functional association between the neuronalelements of glutamatergic synapses in the cerebellum andBergmann glia (Iino et al, 2001). However, plastic changessubsequent to intense electrical activity may incorporatethe GluR2 subunit to preexisting receptors, as reported inneurons (Liu and Cull-Candy, 2000), which may exert aprotective switch to excitotoxity. It would be important tocheck whether glial cells have a similar regulatorymechanism. Interestingly, ischemia induces changes inthe expression of ionotropic GluRs in astrocytes as well asde novo expression in microglia (Gottlieb and Matute,1997; Krebs et al., 2003).

Glial cells also express group I, II, and III-type mGluRs(Kettenmann and Steinh€auser, 2005). Astrocytes expresspredominantly mGluR3 and mGluR5, which are coupledto adenylate cyclase and phospholipase C, respectively.Activation of mGluRs in astrocytes leads to Ca21-depen-dent, prostaglandin-mediated glutamate release (Bezziet al., 1998). Notably, the expression of these receptors inastrocytes is altered after traumatic injury, in amyo-trophic lateral sclerosis, in epilepsy, and in multiplesclerosis (MS) (Geurts et al., 2003; Kettenmann andSteinh€auser, 2005). In addition, oligodendrocytes alsoexpress receptors of all three groups of mGluRs but theirlevels are developmentally regulated and are very low inmature cells of this lineage (Deng et al., 2004). Finally,

microglia is endowed with group I, II, and III mGluR(Biber et al., 1999; Taylor et al., 2003, 2005).

Glutamate uptake from the extracellular space by spe-cific glutamate transporters (GluTs) is essential for theshaping of excitatory postsynaptic currents (Auger andAttwell, 2000) and for the prevention of excitotoxic deathdue to overstimulation of GluRs (Rothstein et al., 1996).At least five GluTs have been cloned (Danbolt, 2001;Huang and Bergles, 2004). Of these, glutamate transpor-ter 1 (GLT-1; EAAT2 in the modern nomenclature) exhi-bits the highest level of expression and is responsible formost glutamate transport (Danbolt, 2001). GluTs areexpressed by astrocytes and oligodendrocytes (Table 2).The main transporters expressed by astrocytes and oligo-dendrocytes are EAAT2 and the glutamate aspartatetransporter (GLAST; EAAT1 in the modern nomencla-ture). The neuronal transporter, termed excitatory aminoacid carrier 1 (EAAC1; EAAT3 in the modern nomencla-ture), is present in a subpopulation of adult oligodendro-cyte progenitor cells (Domercq et al., 1999) and also in asubpopulation of NG21 cells that do not belong to the oli-godendroglial lineage (Matthias et al., 2003). It thusappears that all macroglial cells differentially express thethree major GluTs present in the CNS. These transpor-ters maintain basal levels of extracellular glutamate inthe range of 1–2 lM and thus prevent overactivation ofGluRs under physiological conditions. Interestingly, acti-vated microglia and brain invading macrophages alsoexpress glutamate transporters after mechanical stimula-tion (van Landeghem et al., 2001) and HIV-induced CNSdamage (reviewed by Gras et al., 2003) (Table 2).

Glutamate is also a key metabolite that does not crossthe blood-brain barrier. Most of the glutamate present inthe brain is synthesized de novo by astrocytes (Hertzet al., 1999). These cells have somewhat lower levels ofcytosolic glutamate than neurons (2–3 mM vs. 5 mM)because they have glutamine synthase, an enzyme thatconverts glutamate into glutamine as part of the recy-cling of synaptically released glutamate (Hertz et al.,1999; Nedeergard et al., 2002). Glutamate released atnerve terminals is taken up by astrocytes via GluTs andis converted into glutamine or a-ketoglutarate. The gluta-mate–glutamine cycle is completed by shuttling of gluta-mine to neurons and its conversion into glutamate. Amajor source of extracellular glutamate is that which isinitially stored in synaptic vesicles. However, recent evi-dence shows that astrocytes have a vesicular compart-ment that is competent to exocytose glutamate (Bezziet al., 2004).

Alternatively, glutamate can be released from astrocytesby channel-mediated efflux, reversal of membrane gluta-mate transport subsequent to depolarization, or throughvolume-sensitive organic anion channels activated duringswelling (Nedergaard et al., 2002). Other as yet unknownregulated release mechanisms may also contribute to theextracellular concentration of glutamate. Together withglutamate leakage ensuing cellular damage, efflux of cyto-solic glutamate may account for the increased levels ofglutamate found in cerebrospinal fluid following strokeand traumatic injury (Nedergaard et al., 2002).

213GLUTAMATE-MEDIATED GLIAL INJURY

TABLE

1.Glutamate

Recep

tors

inGlialCells

oftheCNS

Cells

Recep

tortype

Preparation

Experim

entaltech

nique

Properties

Referen

ces

Astrocytes

AMPA/kainate

Rat/cerebral/cu

lture

Electrophysiolog

yDirectdep

olariza

tion

and

cation

influx

Bow

manandKim

elberg,1984;

Kettenmanet

al.,1984,

Son

theimer

etal.,1988

Bergmannglia

AMPA,

noGluR2

Rat/cerebelum/culture

Electrophysiolog

y,in

situ

hybridization

HighCa21permea

bility

Burn

ash

evet

al.,1992;

M€ uller

etal.,1992

Astrocytes

AMPA/kainate

Rat/hippocampus/cu

lture

Ca21im

aging

Ca21influxandCa21wave

gen

eration

CornellBellet

al.,1992

Astrocytes

GluR1-4

Mou

se/CA1region/acu

tely

isolatedcells

Electrophysiolog

y,single

cellRT-PCR

Preferentialcoex

pressionof

GluR1/G

luR2/G

luR3

Seifert

etal.,1997

Astrocytes

AMPA

Rabbit/retina/acu

teslices

Electrophysiolog

yglutamate

EC505

21lM

Clark

andMob

bs,

1992

Astrocytes

AMPA

Rat/op

ticnerve/acu

tepreparation

Ca21im

aging

Low

Ca21permea

bility

Krieg

lerandChiu,1993

Astrocytes

AMPA

Human/hippocampus/acu

tepreparation

Electrophysiolog

yLow

Ca21permea

bility

Seifert

etal.,1999

Astrocytes

GluR1-4

Ratandmou

se/

hippocampus/acu

tely

isolatedcellsandslices

Electrophysiolog

y,single

cellRT-PCR

Only

astrocyteslack

ing

glutamate

transp

orter

curren

tsresp

ondto

AMPA

Zhou

andKim

elberg,2001;

Matthiaset

al.,2003

Astrocytes

GluR5-7/

KA1-2

Bov

ineandrat/white

matter/tissu

eRT-PCR,im

munochem

istry

Expressionin

fibrous

astrocytes

Garc� ıa-B

arcinaand

Matute,1996

M€ uller

cells

NR1

Human/retina/culture

Electrophysiolog

y,im

munochem

istry

Dep

ressionof

inward

lyrectifyingK

1cu

rren

tsPuro

etal.,1996

Astrocytes

NMDA

Rat/hippocampus/acu

tely

isolatedcells

Electrophysiolog

y,im

munochem

istry

Functionalreceptors

inastrocytesafter

isch

emia

Sch

ipkeet

al.,2001;

Krebset

al.,2003

Astrocytes

GroupI

mGluR

Rat/brain/culture

Ca21im

aging,RT-PCR,

insitu

hybridization

Activation

ofmGluR5variants

inducesrelease

ofCa21from

intern

alstores

Pea

rceet

al.,1986;

Biber

etal.,1999

Astrocytes

GroupIand

IImGluR

Rat/brain/culture

Pharm

acology

Inhibitionof

CamP

form

ation

Prezeauet

al.,1994

Astrocytes

GroupImGluR

Rat/brain/culture

and

acu

teslices

Glutamate

release,

electrop

hysiolog

y,Ca21im

aging

Activation

oftypeImGluR

leadsto

Ca21dep

enden

t,prostaglandin-

med

iatedglutamate

release

Bezzi

etal.,1998

Astrocytes

GroupIand

IImGluR

Human/CNS/con

trols

andmultiple

sclerosis

Immunochem

istry

Increa

sedex

pressionof

mGluR1,

mGluR2/3,andmGluR5in

astrocytesin

multiple

sclerosis

Geu

rtset

al.,2003

OPCs

AMPA/kainate

Rat/op

ticnerve/acu

tely

isolatedcells

Electrophysiolog

yRespon

sesin

OPCsnot

inoligod

endrocytes

Barres

etal.,1990

OPCs

AMPA/kainate

Rat/op

ticnerve/cu

lture

Co2

1uptake

Influxof

Co2

1via

receptor

Fulton

etal.,1992

Oligod

endrocytes

AMPA/kainate

Mou

se/brain/culture

Electrophysiolog

y,Ca21im

aging

Ca21permea

ble

Borges

andKettenmann,1995;

Borges

etal.,1994

OPCs,

oligod

endrocytes,

andCG-4

cellline

GluR2-4,

GluR6-7,

KA1-2

Rat/cortex

/culture

Electrophysiolog

y,im

munochem

istry

Expressionof

heterom

eric

functional

AMPA

andkainate

receptors

Patnea

uet

al.,1994;

Puch

alskiet

al.,1994

OPCs

AMPA

Rat/cortex

/culture

Electrophysiolog

y,proliferation

assay

Inhibitionof

proliferation

andlinea

ge

progression

Galloet

al.,1996

Oligod

endrocytes

andCG-4

cellline

AMPA

and

kainate

Ratandrabbit/brain

and

opticnerve/cu

lture

and

invivoandCG-4

Tox

icityassays,

immunochem

istry

ExcitotoxicityinitiatedbyAMPA

and

kainate

receptors

Yoshiokaet

al.,1995;

Matute

etal.,1997;

McD

onald

etal.,1998

OPCs

AMPA

Ratand

mou

se/hippocampusand

cerebellum/acu

teslices

Electrophysiolog

y,im

munochem

istry

Pyramidalneu

ronsandclim

bing

fibersmakefunctionalsynapses

onOPCs

Bergleset

al.,2000;

Lin

etal.,2005

Oligod

endrocytes

AMPA

and

kainate

Humanbrain

andop

tic

nerve

Immunochem

istry

Absence

orpresence

ofAMPA

andkainate

receptors

Wosik

etal.,2004;

P� erez-C

erd� aet

al.,2005

OPCsand

oligod

endrocytes

AMPA

and

kainate

Ratandmou

se/brain

and

opticnerve/cu

lture

Tox

icityassays,

immunochem

istry,

oxidativestress

Overactivation

ofAMPA

andkainate

receptors

inducesOPCsand

oligod

endrocyte

dea

th

Liu

etal.,2002;

S� anch

ez-G

� omez

etal.,2003;

Leu

chtm

annet

al.,2003

Oligod

endrocytes

AMPA/kainate

Ratandhuman/

brain/culture

Tox

icityassays,

immunochem

istry,

electrop

hysiolog

y

Oligod

endrocytesare

insensitiveto

kainate

toxicity

Rosen

berget

al.,2003;

Wosik

etal.,2004

214 MATUTE ET AL.

GLIAL VULNERABILITY TO EXCESSIVEGLUTAMATE SIGNALING

Glutamate can damage nerve cells in at least two ways:by excitotoxicity, which is caused by sustained activation ofionotropic GluRs, and by receptor-independent mechanisms,which are secondary to glutamate uptake. In neurons,NMDA receptors, which are highly permeable to Ca21 anddistributed widely on CNS neurons, are the major initiatorsof excitotoxicity. However, activation of Ca21-permeableAMPA or kainate receptors can also trigger neuronal celldeath (reviewed in Weiss and Sensi, 1999) and antagonistsof these receptors are more efficient neuroprotectants thanNMDA receptor antagonists in some experimental neuro-degenerative paradigms (Gill and Lodge, 1997).

Numerous studies carried out over the past few yearshave shown that, in addition to neurons, glial cells can dieby excitotoxicity. The glial cell types that are most vulner-able to excitotoxicity are those of the oligodendrocyte line-age. However, there is evidence that sustained activationof ionotropic GluRs can also kill astrocytes and microglia.

Although astrocytes express functional GluRs, they aregenerally resistant to excitotoxic insults, and their vul-nerability to this cell death mechanism varies from oneregion to another. Thus, prolonged exposure of culturedhypothalamic astrocytes to high concentrations of gluta-mate and AMPA/kainate receptor agonists does not affecttheir viability (Prieto and Alonso, 1999). In contrast,similar insults are harmful to neocortical astrocytes, afeature that is greatly potentiated by blocking AMPAreceptor desensitization (David et al., 1996). Therefore,overactivation of AMPA receptors can be rapidly lethal toastrocytes, but the receptor desensitization mechanismnormally limits this toxicity. In addition, it has beenshown that brief glutamate exposure causes astrocyteswelling, whereas sustained incubation injures these cellsvia oxidative stress (Chen et al., 2000). Instead of an exci-tatory mechanism, glutamate-induced toxicity is me-diated predominantly by a reduction in glutathione con-tent (Fig. 1) and its effects are almost completely blockedby antioxidants and GluT inhibitors.

Microglial cells appear to be the glial cell type that is leastsusceptible to excitotoxicity. This is due to the fact thatmicroglial cells only express GluRs when they are reactive,as occurs in the post-ischemic brain (Gottlieb and Matute,1997) and in Alzheimer’s disease (Kingham et al., 1999).Thus, senile plaques in the Alzheimer’s brain are character-ized by activated microglia and immunoreactivity for thepeptide chromogranin A. Incubation of primary cultures ofrat brain-derived microglia with this peptide triggers nitricoxide production and enhanced microglial glutamate re-lease, a feature that is attenuated by inhibiting iNOS, micro-glial activation, free radical generation, and the cystine/glu-tamate antiport (Kingham et al., 1999). In turn, glutamaterelease causes microglial death, which is reduced by ionotro-pic GluR antagonists (Kingham et al., 1999).

The first evidence that oligodendrocytes are highly vul-nerable to glutamate was obtained in primary culturesmore than ten years ago (Oka et al., 1993). After a 24-hexposure to glutamate, oligodendroglial death was com-

TABLE

1.(con

tinued

).

Cells

Recep

tortype

Preparation

Experim

entaltech

nique

Properties

Referen

ces

OPCs

NR1

Rat/neu

rophypop

hysis/

culture

Electrophysiolog

y,Ca21

imaging,

immunocytoch

emistry

Recep

torblock

adedim

inishes

OPC

migration

Wanget

al.,1996

OPCsand

oligod

endrocytes

NMDA

Rat/whitematter/acu

teslices

Electrophysiolog

yDirectdep

olariza

tion

and

cation

influx

Ziaket

al.,1998;

Karadottiret

al.,2005

OPCsand

oligod

endrocytes

TypeI,

IIand

IIImGluR

Rat/forebrain/culture

Pharm

acology,

Ca21

uptake,

immunochem

istry,

toxicityassays

Expressionof

alltypes

ofmGluRsin

OPCs,

butnot

inoligod

endrocytes;

activation

oftypeImGluRs

protectsOPCsfrom

insu

lts

gen

eratingox

idativestress

Den

get

al.,2004

Microglia

GluR4andNR1

Rat/hippocampus/

isch

emia

Immunochem

istry

Expressionin

reactivemicroglia

GottliebandMatute,1997

Microglia

AMPA

and

kainate

Rat/cortex

/culture

Electrophysiolog

y,RT-PCR

Low

Ca21permea

bility,

GluR2-7,

KA1-2

Nod

aet

al.,2000

Microglia

GroupI

mGluR

Rat/brain/culture

Ca21im

aging,RT-PCR,in

situ

hybridization

Activation

ofmGluR5variants

inducesrelease

ofCa21

from

intern

alstores

Biber

etal.,1999

Microglia

GroupII

mGluR

Rat/brain/culture

Microglia/neu

ronco-cultures,

toxicityassays

MGluR2activation

triggersTNFa

release

from

microgliaand

neu

ronaldea

th

Tayloret

al.,2005

Microglia

GroupIII

mGluR

Rat/brain/culture

Microglia/neu

ronco-cultures,

viabilityassays

Activation

ofgroupIIImGluR

inducesneu

roprotection

Tayloret

al.,2003

215GLUTAMATE-MEDIATED GLIAL INJURY

TABLE

2.Glutamate

Tra

nsp

orters

inGlialCells

oftheCNS

Cells

Transp

orter

Preparation

Experim

entaltech

nique

Properties

Referen

ces

astrocytesand

Bergmannglia

EAAT1

rat/brain/tissu

eNorthern/insitu

hybridization

highex

pressionin

Bergmann

gliaandless

inthecerebru

mStorcket

al.,1992;Shash

idharan

andPlaitakis,1993

astrocytes

EAAT2

rat/brain

andsp

inalcord

/tissu

eim

munochem

istry

highex

pressionin

astrocytes

throughou

ttheCNS

Danboltet

al.,1992

astrocytes

EAAT2b

rat/brain

andsp

inalcord

/tissu

eim

munochem

istry

expressionin

astrocytesandneu

rons

Chen

etal.,2002

astrocytes

EAAT1and

EAAT2

ratandmou

se/hippocampus/

acu

tely

isolatedcellsandslices

electrop

hysiolog

yastrocyteslack

ingAMPA

receptors

displayglutamate

transp

ortcu

rren

tsZhou

andKim

elberg,2001;

Matthiaset

al.,2003

astrocytes

EAAT2

human/opticnerveand/

postm

ortem

tissue

RT-PCR,functionalassays,

immunochem

istry

expressionin

fibrousastrocytes

Vallejo-Illarramen

diet

al.,2005

oligod

endrocytes

EAAT1and

EAAT2

rat/op

ticnerve/cu

lture

andtissue

uptakeassays,

electrop

hysiolog

y,im

munochem

istry

oligod

endrocytesex

press

functional

glutamate

transp

orters

Dom

ercq

etal.,1999;

Dom

ercq

etal.,2005

oligod

endrocytes

EAAT1and

EAAT2

human/spinalcord

/culture

andtissue

uptakeandim

munochem

istry

uptakeandex

pressionis

neg

atively

regulatedbyTNFa

Werner

etal.,2001;

Pittet

al.2003

oligod

endrocytes

EAAT1

human/opticnerve/tissue

immunochem

istry

expressionin

interfascicular

oligod

endrocytes

Vallejo-Illarramen

diet

al.,2005

OPCs

EAAT3

ratandbov

ine/whitematter/tissu

ein

situ

hybridization

/im

munochem

istry

expressionof

EAAT3in

PDGFRa1

cellsof

theadult

corp

uscallosum

Dom

ercq

andMatute,1999;

Dom

ercq

etal.,1999;

KuglerandSch

mitt,

1999

activatedmicroglia

EAAT1

human/brain/tissu

eim

munochem

istry

expressionin

microgliaand

macrop

hages

inHIV

-infected

patien

tsandprion

disea

ses

Vallat-Decou

velaereet

al.,2003;

Chretien

etal.,2004

microglia

EAAT2

rat/brain/culture

uptake/im

munochem

istry

expressionof

functionalEAAT2

transp

orter

Nakajimaet

al.,2001

activatedmicroglia

EAAT2

rat/facialnucleu

s/axotom

yim

munochem

istry

Expresi� on

inactivatedmicroglia

surrou

ndingmoton

eurons

Lop

ez-R

edon

doet

al.,2000

earlyramified

microglia

EAAT1and

EAAT2

rat/brain/injury

immunochem

istry

expressiondenov

oin

microglia

atea

rlystages

postlesion

vanLandeg

hem

etal.,2001

NG21

cells

EAAT3

rat/hippocampus/acu

teslices

single

cell,RT-PCR,

electrop

hysiolog

yEAAT3transcripts

expressed

butnot

glialtransp

ortcu

rren

tsMatthiaset

al.,2003

216 MATUTE ET AL.

parable to that described in neurons. However, oligoden-droglial toxicity was not mediated by GluRs, as in neu-rons, but rather by a transporter-related mechanisminvolving the inhibition of cystine uptake, which resultsin glutathione depletion and cellular vulnerability totoxic free radicals (Oka et al., 1993; see also Fig. 1). Thistoxicity appears to occur predominantly in young cul-tures, since similar neurotoxic effects of glutamate havebeen observed in the early stages of neuronal cortical cul-tures (Choi et al., 1987; Schubert and Piasecki, 2001). Asimilar mechanism involving glutathione depletion andoxidative stress has also been proposed for astrocytes(Chen et al., 2000).

More recently, it was shown that prolonged activationof GluRs is toxic to cells of an oligodendroglial cell line(Yoshioka et al., 1996) and to oligodendrocytes in vitroand in vivo (Matute et al., 1997; McDonald et al., 1998; Liand Stys, 2000). Strikingly, oligodendrocytes, whichrarely express NMDA receptors, are highly vulnerable toglutamate excitotoxicity. Thus, short exposure to agonistsof AMPA and kainate receptors causes oligodendrocytedeath in vitro. This toxicity is directly related to Ca21

influx subsequent to receptor activation, and it is greatlyattenuated in the absence of Ca21 in the culture medium(S�anchez-G�omez and Matute, 1999).

Glutamate can also cause glial demise indirectly byinducing the release of toxic agents. In microglia, activa-tion of AMPA and kainate receptors results in the releaseof tumor necrosis factor-a (TNF-a), which can potentiateglutamate neurotoxicity and kill oligodendrocytes, des-troy myelin and damage axons (Merrill and Benveniste,1996). In turn, stimulation of the mGluR2 receptor onprimary rat microglia induces microglial activation andresults in a neurotoxic phenotype that secretes TNF-a(Taylor et al., 2005). This toxicity can be neutralized bythe activation of the mGluR3 receptor, which is also pre-sent in microglia (Taylor et al., 2005). Moreover, inflam-matory cytokines including TNF-a and interleukin-1b,which are commonly released by reactive microglia canimpair glutamate uptake and trigger excitotoxic oligoden-drocyte death (Takahashi et al., 2003). Indeed, inhibitionof the expression and functioning of GluTs in axonaltracts is sufficient to induce oligodendroglial loss anddemyelination, which demonstrates that the integrity of

Fig. 1. Signaling cascades triggered by glutamate-induced glialinjury. Selective activation of AMPA receptors (AMPA-R) and kainatereceptors (Kai-R) leads to Na1 and Ca21 influx through the receptorchannel complex. Subsequent depolarization activates voltage-gatedCa21 channels (VGCC) and reverse operation of the Na1/Ca21 exchan-ger (NCX), which contributes to [Ca21]i increase. Ca

21 overload inducesrapid uptake by mitochondria, which results in attenuation of mito-chondrial potential and an increase in the production of reactive oxygenspecies (ROS). The latter can also be generated by reversal of the gluta-mate-cystine exchanger, which results in cystine (CySS) efflux, andthereby glutathione (GSH) depletion. Cytochrome c (Cyt c) is released

from depolarized mitochondria, interacts with apoptotic protease acti-vating factor 1 (Apaf-1) and activates caspases. Other pro-apoptotic fac-tors include apoptosis-inducing factor (AIF), which activates poly(ADP-ribose)-1 (PARP-1). In oligodendrocytes, insults channeled through Kai-R activate caspases 9 and 3, whereas those activating AMPA-R induceapoptosis by recruiting caspase 8, which truncates Bid, caspase 3, andPARP-1, or cause necrosis. In addition, Ca21 influx triggered by Kai-Rstimulation, but not by AMPA-R activates calcineurin (CdP), whichdephosphorylates Bad and facilitates apoptosis. FADD, Fas-associateddeath domain; 14-3-3, phosphoserine-binding protein 14-3-3.

217GLUTAMATE-MEDIATED GLIAL INJURY

oligodendrocytes and white matter depends on properGluT function (Domercq et al., 2005).

MECHANISMS OF GLUTAMATE TOXICITY INGLIAL CELLS

Ionotropic GluRs mediating excitotoxicity are perme-able to Ca21. The degree of Ca21 permeability variesdepending on the subunit composition of the receptor.Excessive activation of AMPA, kainate, and NMDA recep-tors results in the influx of sodium and Ca21 ions throughthe channels formed by these receptors. Sodium influxcan in turn trigger a secondary increase in the concentra-tion of intracellular Ca21 through the activation of vol-tage-gated Ca21 channels and reverse operation of Na1/Ca21 exchange (Lee et al., 1999).

In oligodendrocytes, selective activation of AMPA andkainate receptors leads to Ca21 influx (Fig. 1), an effectthat is totally abolished by non-NMDA receptor antago-nists or by removing this cation from the culture medium.Blockade of voltage-gated Ca21 channels substantiallyreduces the amplitude of the Ca21 current triggered byAMPA receptor activation, but not that initiated by low-and high-affinity kainate receptors (Alberdi et al., 2002).In contrast, inhibition of the plasma membrane Na1/Ca21 exchanger only weakly attenuates the rise in thecytosolic Ca21 concentration ([Ca21]i) caused by activa-tion of kainate receptors. Surprisingly, excitotoxicity trig-gered by GluR overactivation is not reduced by Ca21

channel blockers or by inhibition of the Na1-Ca21

exchanger. This indicates that Ca21 influx via AMPA andkainate receptors alone is sufficient to initiate cell deathin oligodendrocytes, and it does not require the entry ofCa21 via other routes such as voltage-activated Ca21

channels or the plasma membrane Na1-Ca21 exchanger.Overall, these results are reminiscent of those that havebeen reported for neurons, indicating that rapid Ca21

influx through Ca21-permeable AMPA/kainate channelsmay result in mitochondrial Ca21 overload and in theproduction of oxygen radicals, which leads to neuronaldeath (Carriedo et al., 1998).

Several features may render oligodendrocytes vulner-able to overactivation of AMPA and kainate receptors.First, the subunit composition of the receptors endowsthem with higher Ca21 permeability. Thus, AMPA recep-tors in oligodendrocytes are homomeric and/or heteromericentities formed by the GluR3 and GluR4 subunits (Matuteet al., 2002), without GluR2, a configuration that permitsCa21 entry (Hollmann and Heineman, 1994). In addition, amajor constituent of kainate receptors in oligodendrocytesis the unedited version of GluR6, which again displayshigher Ca21 permeability (Burnashev, 1996). Second, oligo-dendrocytes do not express the major Ca21-binding pro-teins that are present in neurons and that contribute tomaintaining intracellular Ca21 homeostasis and prevent-ing cytosolic Ca21 overload (Baimbridge et al., 1992).

The types of excitotoxic cell death observed depend onthe intensity and duration of glutamate exposure andinvolve two temporally distinct phases of necrosis and

apoptosis, a feature that relies on mitochondrial physiology.Mitochondria accumulate much of the Ca21 that enters thecell during excitotoxic insult, and blockade of this processprevents cell death (Stout et al., 1998; Rego et al., 2001).Thus it seems that mitochondrial Ca21 accumulation is acritical event leading to the dysfunction of this organelle.Mitochondrial depolarization, increased production of oxy-gen free radicals, and release of proapoptotic factors thatactivate caspases (see Fig. 1) have all been reported to beconsequences of excitotoxic stimuli (Luetjens et al., 2000;Atlante et al., 2001). The relative contribution of theseevents determines whether ensuing cell death is via necro-sis or apoptosis (Ankarcrona et al., 1995). Consistent withthis, mitochondrial alterations subsequent to excitotoxicinsult in oligodendrocytes are reminiscent of those ob-served in neurons, in that they can trigger apoptotic andnecrotic death (S�anchez-G�omez et al., 2003). Thus, excito-toxic insults to these cells lead to massive Ca21 entry, mito-chondrial depolarization, and a rise in the level of reactiveoxygen species that correlates with a decrease in the levelsof reduced glutathione and ultimately causes caspase-dependent and -independent cell death.

Dysfunction of mitochondria leads to translocation ofcytochrome c to the cytoplasm, where it can activate cas-pases and trigger apoptosis. Alternatively, cytochrome crelease can also increase free radical production by themitochondrial electron transport chain at the levels ofcomplex I, III, and ubiquinone, causing nonspecific cellu-lar damage and necrotic-like morphology (Murphy et al.,1999). Depending on the levels of cytochrome c released bymitochondria, the amount of ATP may be insufficient tocomplete the apoptotic program. Consequently, cells candie by apoptosis or necrosis, as is indeed observed in exci-totoxic models (Bonfoco et al., 1995). Cell death by mildexcitotoxic insults can be prevented by the overexpressionof anti-apoptotic Bcl-2 and Bcl-xl (Lawrence et al., 1996;S�anchez-G�omez et al., 2003), whereas pro-apoptotic mem-bers such as Bad or Bax are involved in excitotoxin-induced mitochondrial dysfunction (Xiang et al., 1998).

CLINICAL RELEVANCE OF GLUTAMATE-MEDIATED GLIAL INJURY

Glial cells constitute the vast majority of CNS cellsand, as described above, are vulnerable to enhanced glu-tamate signals. In particular, glutamate toxicity in glialcells is implicated in most modes of white matter injurystudied so far (Stys, 2004). Since in humans white matterconstitutes about 50% of brain volume, glutamate-induced glial cell death is highly relevant to the patho-physiology of CNS diseases. In addition, primary and/orsecondary glutamate damage to glial cells in gray mattermay also contribute to the onset and progression of acuteand chronic neurodegenerative diseases.

Brain Ischemia

Glutamate excitotoxicity appears to be the predomi-nant mechanism underlying ischemic damage in the CNS

218 MATUTE ET AL.

(Lee et al., 1999). However, NMDA antagonists have notimproved the outcome in clinical trials of acute stroke(Lutsep and Clark, 2001), a failure that is likely due tothe lack of protection by these drugs of glial cells, whichcommonly do not express NMDA receptors. Indeed,AMPA and kainate receptor antagonists appear to bemore effective in reducing ischemic damage to both grayand white matter and have entered late-phase clinicaltrials (Akins and Atkinson, 2002).

In humans, most cases of focal ischemia and occlusionof major cerebral arteries damage both gray and whitematter. Energy deprivation causes neuronal death, axo-nal dysfunction, and loss of macroglia. Like neurons, oli-godendrocytes are very sensitive to transient oxygen andglucose deprivation (Goldberg and Ransom, 2003; Dewaret al., 2003; Stys, 2004). After 1 h under these conditions,the viability of oligodendrocytes in mixed glial cultures isseverely impaired, an effect that is attenuated by AMPA/kainate antagonists (McDonald et al., 1998). This indi-cates that glutamate released from astrocytes may initi-ate the excitotoxic cascade. In turn, immature oligoden-drocytes are even more sensitive to ischemic injury thantheir mature counterparts, and the death of these cellshas been reported to occur even in the absence of astro-cytes (Fern and M€oller, 2000). Thus, these cells mayrelease glutamate under ischemic conditions by reversefunctioning of their GluTs (Domercq et al., 1999, 2005).

Consistent with observations in vitro, permanent middlecerebral artery occlusion and brief transient global ische-mia induce rapid oligodendroglial death, and in certainbrain regions these cells are more vulnerable than neurons(Mandai et al., 1997; Petito et al., 1998). Interestingly, afew days after the insult, there is an increase in the num-ber of oligodendroglial cells in areas bordering affectedregions (Mandai et al., 1997), as well as in the number ofimmature oligodendrocytes surrounding the lateral ventri-cles (Gottlieb et al., 2000) indicating that ischemic damageto oligodendroglia can be compensated for, at least in part,by the generation of new oligodendrocytes.

A relatively prevalent and dramatic case of hypoxia-ischemia-related disease is periventricular leucomalacia,a condition characterized by diffuse cerebral white matterinjury as the predominant lesion. Injury to OPCs, causedin part by glutamate and the subsequent derailment ofCa21 homeostasis, contributes to the pathogenesis ofmyelination disturbances in this illness (Back and Riv-kees, 2004). In addition to this mechanism, glutamate-induced depletion of glutathione and the subsequent oxi-dative stress in PVL also contribute to hypoxia/ischemiadamage to oligodendrocytes (Haynes et al., 2003), whichare sensitive to oxidative stress in part because of theirhigh lipid and iron content. Notably, vitamin K, which isdeficient in preterm infants, is a risk factor for the devel-opment of PVL, and in turn is protective against oxida-tive injury to immature oligodendrocytes (Li et al., 2003).

In addition to oligodendrocytes, GFAP1 astrocyteinjury also occurs rapidly after hypoxia-ischemia in theneonatal striatum (Martin et al., 1997). Yet, the extent ofischemia-induced astrocyte death in the immature andadult brain remains uncertain, since protoplasmic astro-

cytes are poorly reactive to GFAP antibodies (Giffard andSwanson, 2005). Astrocyte death in response to hypoxiarequires external Ca21 and is blocked by inhibitors ofreversed Na1-Ca21 exchange, suggesting that injury wastriggered by a rise in [Ca21]i (Bondarenko et al., 2005).As in oligodendrocytes, hypoxia/ischemia induces mitho-chondrial depolarization in astrocytes (Smith et al.,2003), and their survival is improved by inhibitors of cas-pase-dependent cell death pathways, but also by blockingthe poly(ADP-ribose) polymerase-1 cell death pathway(Giffard and Swanson, 2005). However, it is not known ifdirect glutamate toxicity contributes substantially toastrocyte loss after ischemia.

Traumatic Injury

A cascade of events leading to what is termed ‘‘second-ary injury’’ determines the outcome of tissue damageafter an initial traumatic lesion to the spinal cord andother areas of the CNS, with most of the clinical disabilityarising from disruption of white matter tracts. Glutamateexcitotoxicity is a major contributor to secondary injurynot only via neuronal cell death but also via delayed post-traumatic white matter degeneration (Park et al., 2004).The mechanisms contributing to the latter include gluta-mate release by reversal of Na1-dependent glutamatetransport with subsequent activation of AMPA receptorsand oligodendrocyte death (Li et al., 1999). All threemajor Na1-dependent GluTs are expressed in variouscompartments of the white matter such as axon cylin-ders, astrocytes and oligodendrocytes and consequently,they could contribute to glutamate release (Li et al.,1999). Notably, NBQX, an AMPA/kainate receptor an-tagonist, reduces neurological deficits and tissue lossafter spinal cord injury (Rosenberg et al., 1999). In turn,glutamate applied to the spinal cord causes damage to oli-godendrocytes, astrocytes and myelin, all of whichexpress AMPA receptors (Li and Stys, 2000). Together,these data point to novel targets to treat traumatic injuryto the CNS and to AMPA and kainate antagonists aspotential drugs to reduce secondary damage subsequentto the initial lesion.

Neurodegenerative Disorders

Glial cells have been found to be damaged in Alzheimer’sdisease. The two histological hallmarks of this diseaseare the presence of aggregates of the amyloid b-peptide(Ab) in the form of plaques, and the presence of filamen-tous intracellular aggregates of the microtubule-asso-ciated protein tau, the so-called neurofibrillary tangles.Both features are often found in white matter (Nishimuraet al., 1995; Bronge, 2002), which suggests that whitematter damage contributes to the cognitive dysfunctionobserved in Alzheimer’s disease. Ab impairs glutamatetransport directly by lipid peroxidation of astrocytes andindirectly by inducing in microglia the release of TNF-�a,which is a negative regulator of GluT expression and

219GLUTAMATE-MEDIATED GLIAL INJURY

function (Mattson and Chan, 2003). In addition, Ab killsoligodendrocytes (Xu et al., 2001) and can increase theirvulnerability to being killed by glutamate (Pak et al.,2003). In turn, oligodendrocytes express presenilin 1(PS1) and oligodendrocytes from PS1 mutant knock-inmice exhibited increased vulnerability to excitotoxicitycompared to oligodendrocytes from wild-type mice (Paket al., 2003). Finally, when exposed to the demyelinatingagent cuprizone, PS1 mutant mice exhibit enhanceddamage in the white matter and learning and memorydeficits not seen in wild-type mice exposed to cuprizone(Pak et al., 2003).

Human immunodeficiency virus type 1 (HIV-1) infec-tion causes cognitive dysfunction in about 10–20% ofAIDS patients, a syndrome that may be due to excitotoxicneuronal damage (Kaul et al., 2005). Interestingly, HIVencephalitis is also characterized by myelin pallor, whichsuggests that oligodendrocyte excitotoxicity also occurs inthis condition as a consequence of an inhibition of gluta-mate uptake by eicosanoids and free radicals produced byHIV-infected brain mononuclear phagocytes (Lipton,1998) and/or of excessive glutamate release from astro-cytes (Bezzi et al., 2001). Indeed, the expression and func-tion of the glutamate transporter EAAT2 is reduced inhuman astrocytes exposed to the effects of HIV-1 and itsenvelope glycoprotein (Wang et al, 2003).

Multiple Sclerosis

In MS, the immune system attacks the white matter ofthe brain and spinal cord, leading to disability or paraly-sis, or both. Myelin and oligodendrocytes are lost due tothe release by immune cells of cytotoxic cytokines, auto-antibodies, and toxic amounts of glutamate (Matuteet al., 2001; Srinivasan et al. 2005). Excitotoxins such askainate infused onto the optic nerve cause glutamatereceptor-mediated MS-like lesions (Matute, 1998). Inturn, experimental autoimmune encephalomyelitis (EAE),an animal model that exhibits the clinical and pathologi-cal features of MS, is alleviated by AMPA and kainatereceptor antagonists (Smith et al., 2000; Pitt et al., 2000;Groom et al., 2003). Remarkably, blockade of these recep-tors in combination with anti-inflammatory agents iseffective even at an advanced stage of unremitting EAE,as assessed by increased oligodendrocyte survival andremyelination, and corresponding decreased paralysis,inflammation, CNS apoptosis and axonal damage (Kan-war et al., 2004).

The concentration of glutamate in cerebrospinal fluid(CSF) is higher in patients with acute rather than silentMS and in controls (Stover et al., 1997), and it is asso-ciated with the severity and course of the disease (Bar-khatova et al., 1998). Notably, glutamate levels areincreased in acute MS lesions and in normal-appearingwhite matter in MS patients (Srinivasan et al., 2005).Potential cellular sources contributing to enhanced gluta-mate levels in CSF include activated microglia, which canrelease glutamate via the reversal of GluT function, a pro-cess that is potentiated under pathological conditions

(Noda et al., 1999). In addition, oxidative stress may alsocontribute to the increase in glutamate concentrations inthe extracellular space, since free radicals reduce the effi-ciency of GluTs (Volterra et al., 1994). Other factors thatmay contribute to perturbing glutamate homeostasis in-clude altered activity of the glutamate producing enzymeglutaminase in activated macrophages/microglia in closeproximity to dystrophic axons (Werner et al., 2001), andreduced expression of the glutamate transporters EAAT-1and EAAT-2 in oligodendrocytes as a consequence of en-hanced exposure to the proinflammatory cytokine TNF-a(Pitt et al., 2003). Overall, these alterations likely lead tohigh extracellular glutamate levels and an increased riskof oligodendrocyte excitotoxicity in MS.

CONCLUSIONS AND FUTURE PROSPECTS

Glial cells are sensitive to glutamate-induced damage.Among them, oligodendrocytes display great vulnerabil-ity to overactivation of AMPA and kainate receptors. Thefailure of drugs aimed at preventing neuronal death todemonstrate significant improvement in the outcome ofacute and chronic neurodegeneration in humans is likelydue to the lack of protection by these drugs for the glialcells, which constitute the vast majority of cells withinthe CNS.

The proper functioning of glutamate uptake is criticalto prevent glutamate-induced damage to glia and drugsthat regulate the function and expression of GluTs havethe potential to attenuate glutamate insults to glial cells.Thus, reversal of GluTs can generate toxic extracellularglutamate levels during anoxia and trauma and signifi-cant protection can be achieved with specific inhibitors ofGluTs (Stys, 2004). Likewise, positive regulators of theexpression of GluTs also have a glioprotective potential,as they contribute to ischemic tolerance after ischemicpreconditioning (Romera et al., 2004). These includetumor growth factor-a (TGF-a) and epidermal growth fac-tor (EGF), which by signaling through EGF receptors andactivation of phosphoinositol-3-kinase and nuclear factor-jB, strongly enhance EAAT2 expression (Su et al., 2003).Remarkably, clinically used b-lactam antibiotics are alsopotent activators of GluT expression and thus hold greattherapeutic potential (Rothstein et al., 2005).

Another set of molecular targets to prevent glutamateinsults to glia lie downstream of GluR activation. Thus,the vulnerability of OPCs to glutamate excitotoxicity canbe attenuated with agonists of group I metabotropicGluRs by controlling oxidative stress generated down-stream of AMPA and kainate receptor activation (Denget al., 2004). Similarly, the clinically available anticonvul-sant topiramate prevents excitotoxicity in developing oli-godendrocytes (Follet et al., 2004). Together, these find-ings offer good prospects for future treatments for peri-ventricular leucomalacia, a condition in which there ismassive loss of immature oligodendrocytes.

In addition, the neuroprotective properties of tetracy-clines, which attenuate mitochondrial damage subse-quent to insults including excitotoxicity, have been

220 MATUTE ET AL.

expanded to oligodendrocytes and to white matter as awhole, making these antibiotics promising candidates forthe treatment of acute and chronic diseases with glial loss(Domercq and Matute, 2004). In contrast, drugs support-ing the management of Ca21 overload subsequent toGluR activation may improve glial cell viability. Notably,the major plasma membrane Ca21 extruding system, theNa1/Ca21 exchanger, is cleaved during brain ischemiaand in neuronal excitotoxic settings by calpains. Inhibi-tion of this process has been reported to enhance cell via-bility (Bano et al., 2005). Conceivably, similar interven-tions may prevent excitotoxic glial cell death.

In summary, knowledge about the mechanisms leadingto glutamate-mediated glial injury will open the way tonew pharmacological strategies for the treatment of CNSdisorders and in particular, those ailments in which gliaare primarily affected. Glioprotectants that prevent glu-tamate-induced glial cell death are about to emerge as anovel class of drugs with important clinical applications.Their use in conjunction with neuroprotectants is likelyto increase the effectiveness of current treatments forneurological dysfunction in acute and chronic neurode-generative diseases.

ACKNOWLEDGMENTS

The authors express our thanks to the agency ACTS(http://www.euskalnet.net/acts) for having improved theEnglish of this work.

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