human astrocytes in the diseased brain

Theyalsoregulatetheextracellularspacevolumeandmodulate themoment-to-momentsynapticplasticityAraqueetal.,2014; DalléracandRouach,2016.Manystudieshaveshowntheir con-tributiontoinformationp

Contents lists available atScienceDirect

Brain Research Bulletin

j o u r n a l h o m e p a g e :w w w e l s e v i e r c o m / l o c a t e / b r a i n r e s b u l l

Research report

Elena Dossi1, Flora Vasile1, Nathalie Rouach∗

Neuroglial Interactions in Cerebral Physiopathology, Center for Interdisciplinary Research in Biology, Collège de France, CNRS UMR 7241, INSERM U1050,

Labex Memolife, PSL Research University, Paris, France

a r t i c l e i n f o

Article history:

Received 31 October 2016

Received in revised form 8 February 2017

Accepted 9 February 2017

Available online xxx

Keywords:

Astrocytes

Humans

Brain

Pathology

a b s t r a c t

Astrocytesarekeyactiveelementsofthebrainthatcontributetoinformationprocessing.Theynotonly provideneuronswithmetabolicandstructuralsupport,butalsoregulateneurogenesisandbrainwiring Furthermore,astrocytesmodulatesynapticactivityandplasticityinpartbycontrollingtheextracellular spacevolume,aswellasionandneurotransmitterhomeostasis.Thesefindings,togetherwiththe discov-erythathumanastrocytesdisplaycontrastingcharacteristicswiththeirrodentcounterparts,pointtoa roleforastrocytesinhighercognitivefunctions.Dysfunctionofastrocytescantherebyinducemajor alter-ationsinneuronalfunctions,contributingtothepathogenesisofseveralbraindisorders.Inthisreview

wesummarizethecurrentknowledgeonthestructuralandfunctionalalterationsoccurringinastrocytes fromthehumanbraininpathologicalconditionssuchasepilepsy,primarytumours,Alzheimer’sdisease, majordepressivedisorderandDownsyndrome.Compellingevidencethusshowsthatdysregulationsof astrocytefunctionsandinterplaywithneuronscontributetothedevelopmentandprogressionofvarious neurologicaldiseases.Targetingastrocytesisthusapromisingalternativeapproachthatcouldcontribute

tothedevelopmentofnovelandeffectivetherapiestotreatbraindisorders

©2017TheAuthors.PublishedbyElsevierInc.ThisisanopenaccessarticleundertheCCBYlicense

(http://creativecommons.org/licenses/by/4.0/)

1 Introduction

Astrocytes areactive dynamic signalling players of the

cen-tralnervoussystem(CNS).Overthepast25yearsithasbecome

clear that astrocytes participate to a variety of essential

phys-iological processesin the healthybrain Indeed, farfrombeing

merelypassivecellsprovidingstructuralsupporttoneurons,

astro-cytesarenowviewedascrucialactiveanddynamicelementsof

thebraincircuitry:theyparticipateinformationandmaturation

of synapses, receptor trafficking, control of the homeostasis of

ionsandenergymetabolitesandclearanceofneurotransmitters

Theyalsoregulatetheextracellularspacevolumeandmodulate

themoment-to-momentsynapticplasticity(Araqueetal.,2014;

DalléracandRouach,2016).Manystudieshaveshowntheir

con-tributiontoinformationprocessingandmemoryformationinthe

Abbreviations: A␤, amyloid Beta; AQP, aquaporin; AD, Alzheimer’s disease; CNS,

central nervous system; Cx, connexin; DS, Down syndrome; GJ, gap junction; GFAP,

glial fibrillary acidic protein; GS, glutamine synthetase; HS, hippocampal sclerosis;

IP3R2, inositol triphosphate receptor 2; MDD, major depressive disorder; MTLE,

mesial temporal lobe epilepsy; mGluR5, metabotropic glutamate receptor 5.

∗ Corresponding author.

E-mail addresses: elena.dossi@college-de-france.fr (E Dossi),

(N Rouach).

1 E.D and F.V equally contributed to this work.

brain, thereby pointing to a role of astrocytes in higher inte-gratedbrainfunctions.Dynamicbidirectionalsignallingbetween astrocytesandneuronshasmainlybeenreportedin experimen-talanimalmodels.Recentdatahowevershowthatsuchreciprocal signallingalsooccursinthehumanbrain.Astrocytesfromhuman braintissueindeedexhibitCa2+-based“intrinsicexcitability”and canrespondtosynaptically-releasedneurotransmitters(Navarrete

etal.,2012).Furthermore,morphological,genomicandfunctional studieshaverevealedthathumanastrocytesdisplayspecific char-acteristicscomparedtotherodentcounterpart(Milleretal.,2010; Oberheimetal.,2006,2009;Zhangetal.,2016;Zhengetal.,2015) Humanastrocytes displayaremarkablemorphologicaldiversity accordingtocorticallayers,beinglargerandmorecomplexthan thoseofrodents;furthermore,theyexhibitahighexpressionof proteinsinvolvedinCa2+signallingandpropagateCa2+wavesat muchfastervelocitiesthantheirrodentcounterparts(Bazargani andAttwell,2016;Oberheimetal.,2009).Altogether,these find-ingssupporttheideathatinthehumanbrain,astrocytesmayplay

acrucialroleunderlyinghighercognitivefunctions.Alterationsin astrocytephysiologicalroleshavethusbeenhypothesizedto con-tributetocerebralpathology.Indeed,asearlyasinthe19thcentury, severalneuropathologistssuchasAlzheimer,FrommanandNissl, alreadyenvisionedaroleforgliainbraindiseases.Nonetheless, sincethebeginningofthe20thcenturytheconceptthat neurologi-caldiseasesresultprimarilyfromneuronaldysfunctiondominated

http://dx.doi.org/10.1016/j.brainresbull.2017.02.001

0361-9230/© 2017 The Authors Published by Elsevier Inc This is an open access article under the CC BY license ( http://creativecommons.org/licenses/by/4.0/ ).

toprominentadvancesintherapiesforbraindiseases.Such

dis-easesindeedstillremainthemostcomplicatedtounderstandand

treat.Growingevidencefromanalysisofpost-mortemorsurgically

resectedhumantissuesandfromanimalmodelsofCNSpathologies

indicatethatastroglialdysfunctionscontributetothe

pathogene-sisofseveralneurologicalandpsychiatricdisorders(Halassaetal.,

2007;RossiandVolterra,2009)

Inthisreviewwefocusonhuman-specificastroglialchanges

insomefrequent neurologicaldisorders,suchasepilepsy,brain

tumours,Alzheimer’sdisease,majordepressivedisorderandDown

syndrome

2 Astrogliosis as a hallmark of brain diseases

Acommonfeatureandpathological hallmark ofseveralCNS

diseases is reactive astrogliosis (Fig 1) It consists of a finely

gradedcontinuumofmolecular, cellularand functionalchanges

inastrocytesin responsetoCNSinjuries;thesealterations vary

accordingtotheseverityof thedisease(Andersonetal., 2014;

Eddleston and Mucke, 1993) and are regulated through

inter-andintracellularsignalling moleculesinacontext-specific

man-ner(Sofroniew,2009).Inmildormoderateastrogliosis,whichis

generallyassociatedwithmildtraumaorlocatedinareasata

cer-taindistancefromCNSlesions,astrocyticproliferationisalmost

absent Variable increased glial fibrillary acidic protein (GFAP)

expressionhasalsobeenobserved,togetherwithcellbody and

processhypertrophy,whichisnotalteringastrocyteorganization

intoindividualdistinctdomains(Wilhelmssonetal.,2006)

Fur-thermore,otherproteinsareup-regulatedinreactiveastrocytes,

suchascopper-zincsuperoxidedismutase,glutathioneperoxidase

ormetallothionein.Moderateastrogliosisalsoresultsinexpression

ofinduciblenitricoxidesynthaseandreleaseoftrophicfactorsand

cytokines,includingtumournecrosisfactors␣and␤,interleukins

andinterferons(ChenandSwanson,2003).Inmildormoderate

forms,reactiveastrogliosisexhibitsthepotentialfor resolution,

iftheinitialtriggeringinsultresolvesorisremoved;inthiscase,

cellsreturntoaconditionsimilartothatobservedinhealthytissue

(Sofroniew,2009).Onthecontrary,nearfocallesions,infectionsor

neurodegenerativeareasseverediffuseastrogliosisischaracterized

byenhancedastrocyticproliferation.Molecularfactorspromoting

proliferationofreactiveastrocytesarenotcompletely

character-ized,but a role for epidermal growthfactor, fibroblastgrowth

factor,endothelin1,ATP,lipopolysaccharideandnitricoxidehas

beenidentified(Gadeaetal.,2008;Levisonetal.,2000;Nearyand

Zimmermann,2009;SofroniewandVinters,2010).Thisenhanced

astrocyticproliferationcausesinterminglingand overlapping of

neighbouringastrocyticprocesses,whichdisruptsindividual

astro-cytedomains Insomecases,this potentastrocyticreactioncan

drivetheformationofacompactglialscar(Fig.1).Such scaris

characterizedbyastrocyteinteractionwithdifferentcelltypesand

ismainlyformedalongthebordersofseveretissuedamage,

necro-sis,tumours,chronicneurodegeneration,infectionorinflammatory

infiltration(Sofroniew,2009;SofroniewandVinters,2010).These

structuralchangesarelong-lastingandpersistaftertheresolution

ofthetriggeringinsult(Sofroniew,2009).Moreover,matureglial

scarsactasbarrierstoinflammatorycellstoprotectsurrounding

healthytissuefromnearbyareasofintenseinflammation.Reactive

astrocytescanalsoprotectCNScellsandtissuebyuptaking

exci-totoxicglutamate,producingglutathioneagainstoxidativestress,

degradingamyloid␤peptides,regulatingextracellularspace

vol-umeand ionbalance,facilitating blood brainbarrierrepair and

regulatingCNSinflammation.Nevertheless,growingevidencealso

shows that reactiveastrocytes cancontribute toor bethe

pri-mary sourceof CNS physiopathology Reactive astrocytes from

glialscarscanindeedsynthesizecollagenandsulphate proteogly-cans,whichpreventaxonregeneration(ChenandSwanson,2003)

Inaddition,alterationofthephysiologicalfunctionsofastrocytes resultingfromgeneticmutationscontributetobraindisorderssuch

asAlexander’sdiseaseandamyotrophiclateralsclerosis(Brenner

etal.,2001;Nagaietal.,2007).Theseoppositeeffectsofreactive astrocytesthuspointtoadualfunctionofastrogliosis(Sofroniew, 2009;SofroniewandVinters,2010)

3 Epilepsy

Epilepsy is one of the most prevalent neurological diseases affecting1%oftheworldpopulation(WorldHealthOrganisation,

2016,http://www.who.int/en/).Itischaracterizedbyrepetitively recurrentseizures,whichdisruptnormalbrainfunctionsandcan damagethebrainandworsenpre-existingneurologicaldeficits Contrarytothetraditionalviewassumingthatepilepticactivity

isgeneratedexclusivelyinandbyneurons,anastrocyticbasisfor epilepsyhasbeenproposed(Tianetal.,2005).Moreover, investi-gationsonspecimensfrommesialtemporallobeepilepsy(MTLE) patientshaveidentifiedchangesinastrocyticchannelsand recep-tors (Fig 2a–b), thus suggesting thatastrocyte dysfunctioncan participateinhyper-excitation,neurotoxicityandseizure spread-ing,inadditiontoestablishedneurogenicmechanisms

3.1 Epilepsy-associatedastrogliosis Reactiveastrogliosisispresentinalmostallformsofepilepsy, butitismostnotableinpresenceofhippocampalsclerosis(HS), whichisoftenassociatedwithMTLEandotherepilepsysyndromes (Thom,2014).Indeed,besidesaseverelossofprincipalneurons observedinCA1andCA3andgranulecelldispersion,HSis char-acterized by a chronic and fibrillary gliosis in CA1 and radial gliosisinthedentategyrus,wherethelengthofGFAP+ fibresis directlycorrelatedwiththedegreeofcelldispersioninthe den-tategyrus(Fahrneretal.,2007).Furthermore,inHS,togetherwith increasedconventionalGFAPexpression,anovelGFAPisoformhas beenidentified in smallmultinucleateCA1 and CA4astrocytes, GFAP-␥,whichisspeculatedtoregulateastrocytesizeandmotility (Martinianetal.,2009).WhetherHSisaprimarycauseofepilepsy

ortheresultof repeatedepilepticseizuresis stillcontroversial EveniftheprevailingviewtendstoconsiderHSasasecondary consequenceofepilepsy,experimentaldataonsurgicalsamples and autoptic tissuessuggest that HSaetiologyis multifactorial Febrileseizures,genetic susceptibility,alterationsof hippocam-paldevelopment,headinjuries,infectionsandinflammatoryand neurodevelopmentalfactorshaveindeedbeenidentifiedas pre-disposingelementstoHSdevelopment(SendrowskiandSobaniec, 2013;Thom,2014;Walker,2015)

3.2 KirchannelsandK+homeostasis

Itiswellknownthatastrocytesarekeyplayersinthe regula-tionofextracellularK+([K+]o),whichcantransientlyaccumulate duringprolongedneuronalactivityand causeneuronal depolar-izationandhyper-excitabilityifuncompensated(Heinemannand Lux,1977).[K+]o homeostaticcontrolisperformedbyK+ uptake andbyspatialK+buffering:whiletheformerismediatedbyglial and neuronal Na,K-ATPase orNa-K-Cl cotransporters,thelatter

isdrivenbythedifferencebetweentheglialsyncytiumnegative membranepotentialandthelocalK+equilibriumpotential.This resultsinredistribution ofK+fromsitesofhighneuronal activ-ity tosites of lower[K+]o through gapjunction(GJ)-connected astrocyticnetworks(Orkandetal.,1966;Walz,2000).This pecu-liarastrocyticpropertyismainlymediatedbyKir4.1K+channels,

Fig 1 Astrogliosis in pathological human brain.Top, schematic representation of different gradation of astrogliosis depending on the gravity of the insult Bottom, astrocyte morphology in normal tissue from a human autopsy specimen far from a lesion (left), and in presence of moderate (middle) and severe diffuse reactive astrogliosis (right) For each condition, a brightfield immunochemistry for GFAP counterstained with haematoxylin is shown on the left, and an enlarged view of the boxed areas on the right Scale bars: left, 50 ␮m; right, 20 ␮m [upper part modified from ( Sofroniew, 2009 ); bottom part from ( Sofroniew and Vinters, 2010 )].

whichareweakly-rectifyingchannelspossessingahighopen

prob-ability at rest and showingconductance increase at high[K+]o

(Butt andKalsi, 2006).Considering theirrole inK+ homeostasis

and sinceincreased levelsof[K+]o havebeen associatedtothe

pathophysiologyofepilepsy,Kirchannelshavebeeninvestigated

inexperimental(see(Cheveretal.,2010;Djukicetal.,2007))and

humanepilepsy.ByusingK+-sensitivemicroelectrodes,

measure-mentsofstimulus-induced[K+]o increaseshavebeenperformed

inhumanscleroticandnon-sclerotichippocampaltissues(HSand

non-HS)inpresenceofBa2+,ablockerofKirchannels(Kivietal.,

2000).TheserecordingshighlightedanimpairedK+bufferinginHS

slices:indeed,theyshowedthatBa2+wasabletoinduce[K+]o

accu-mulationinhippocampalslicesfromlesion-associatedTLEpatients

withnohistopathologicalhippocampalalterations;onthe

con-trary, it failed todo so in hippocampi from drug-resistantTLE

patientsshowingstrongneuronallossandgliosis(Gabrieletal.,

1998;Heinemannetal.,2000;Jauchetal.,2002;Kivietal.,2000)

Furthermore,patch-clamprecordingsandsinglecellRT-PCR

per-formedon humansclerotic hippocampusrevealeda significant

decrease(Hinterkeuseretal.,2000;Schröderetal.,2000)ora

com-pleteloss(BordeyandSontheimer,1998)ofKir4.1currents;this

hasalsobeenrecentlysupportedbyWesternblotand

immuno-histochemistryanalysisshowingadecreaseofKir4.1proteinlevels

inHStissuescomparedtonon-HSTLEpatientsorsudden-death

controls(Dasetal.,2012;Heuser etal.,2012).Thus,inHSTLE,

thereducedexpressionandfunctionalityofKir4.1channels

deter-mineanimpairedK+bufferingandenhanceseizuresusceptibility,

evenifitisstillcontroversialwhetherthisreductionisacauseora

consequenceofTLE

Interestingly,severalvariationsinhumanKir4.1gene(KCNJ10)

havebeen identifiedand associatedtodifferent seizure

pheno-types Indeed, Buono and colleagues found that patients with

refractoryMTLE,childhood absenceand juvenilemyoclonicand

idiopathicgeneralized epilepsy carried a missense mutation in

the C-terminal domainof KCNJ10 Such domain is involved in

ionic conductance, channel subunit dimerization and anchor-ingtotheplasma membrane(Buonoetal.,2004).Furthermore, othersinglenucleotidevariationsinnon-codingregulatoryKCNJ10 sequenceshavebeenfoundinTLEpatientswithfebrileseizures (Heuseretal.,2010).Moreover,patientswithEAST/SeSAME syn-drome,anautosomalrecessivemultiorgandisordercharacterized

by seizures, ataxia, sensorineural deafness, mental retardation and electrolyteimbalance,presentedloss-of-function mutations

inhighlyconservedaminoacidsequencesofKir4.1gene,which causesimpairmentin channelconductance and Ba2+-sensitivity (Bockenhaueretal.,2009;Reicholdetal.,2010;Scholletal.,2009; Williamsetal.,2010).Anothercohortofpatientsdisplayedinstead

a gain-of-functionmutationin KCNJ10affectingtheN-terminus andthefirsttransmembraneregionofthechannel:thisvariation causedanincreaseinKir4.1surfaceexpressionorconductanceand patientsdevelopedseizuresinassociationwithautismspectrum disordersandimpairedcognitiveabilities(Siccaetal.,2011) 3.3 Aquaporin-4(AQP4)

Ionhomeostasisinthebraindependsnotonlyonproper func-tioningofionchannels,butalsoonwatertransport.Theinfluxof waterbetweenthebloodandthebrainparenchymaistightly reg-ulatedbybloodvessels.Glialensheathmentofbloodvesselsalso contributestowaterinflux.Themovementofwateracrosscellular compartmentsismediatedbyaquaporins(AQPs),transmembrane proteinswhichactaswaterchannelsindifferentcellsandtissues

Inthebrain,thepredominantformisAQP4,whichislocalized,as Kir4.1channels,inastrocyticperivascularendfeetand perisynap-ticprocesses.AQP4participatesinthecontrolofextracellularfluid osmolarityandextracellularspacevolumebyregulatingwaterflow andK+buffering(Nagelhusetal.,2004).Itappearsthatwater move-mentisalteredinthehippocampusofHSTLEpatients,asithas beenshownthatitpresentsanincreasedT2signaldensityon mag-neticresonanceimagingandhigherapparentdiffusioncoefficients,

Fig 2 Alterations of astrocytes in CNS disorders.(a) Schematic representation of astrocyte-mediated regulation of synaptic activity in the healthy brain (b–f) Changes of astrocytic receptors, transporters, ion channels and intracellular proteins in epilepsy (b), brain tumours (c), Alzheimer’s disease (d), major depressive disorder(e)and Down syndrome (f) Increases of expression/levels are indicated in red, decreases in blue [AQP4: aquaporin 4; Cav: voltage-gated Ca 2+ channels; Cx43 and Cx30: connexins 43 and 30; GAT3: GABA transporter 3; GFAP: glial fibrillary acidic protein; GLAST and GLT1: glutamate transporters; Glu: glutamate; GS: glutamine synthetase; GJs: gap junctions; Kir4.1: inwardly-rectifying K + channels; mGluR5: metabotropic glutamate receptor 5; Nav: voltage-gated Na + channels; xCT: cysteine-glutamate system].

indicatingwateraccumulation(Bronenetal.,1991; Huggetal.,

1999;Wieshmannetal.,1999).ApotentialimpairmentofAQP4

functionhasbeenproposedinTLEpatienthippocampus:RT-PCR,

immunohistochemistryandgenechipanalysishaverevealedan

increasedexpressionofAQP4inHStissues;thiswasaccompanied

byreducedlevelsofdystrophin,aproteininvolvedinanchoring

AQP4tothemembraneinperivascularendfeet(Leeetal.,2004)

Theseresultsthussuggestedanalteredtraffickinganddistribution

ofAQP4onthemembranes.Quantitativeelectronmicroscopylater

confirmedthishypothesis:thelevelsofAQP4areindeedincreased

inMTLEcomparedtonon-MTLEhippocampi.However,AQP4

den-sityalongtheperivascularmembranedomainofastrocyteswas

reducedbyhalfinMTLECA1region,whilenodifferencewasfound

inAQP4densityonastrocytemembranesfacingneuropil(Eidetal.,

2005).Thesechangesweresecondarytoalteredperivascular

dys-trophinexpressioninscleroticareas(Eidetal.,2005;Leeetal.,

2004),whichcausedthelossofperivascularAQP4andresultedin

animpairedwaterfluxthroughastrocytes.Giventhatinpresence

ofhighneuronalactivityK+andwateraretakenupbythe astro-cytemembranefacingtheneuropil,transportedthroughastrocytic syncytiumandsiphonedintoblood orCSFthroughperivascular endfeetmembrane(PaulsonandNewman,1987),thisalteredflow

of water affected extracellular K+ buffering and contributed to epileptogenicity.SimilarchangesinAQP4 distributionhavealso beenfoundincorticalsamplesofepilepticfocalcortical dyspla-siatypeIIbpatients.AQP4wasindeedfoundtobeenrichedinthe neuropilandarounddysplasticneuronsandreducedatthe perivas-cularmembranesdue todisrupteddystrophincomplex (Medici

etal.,2011).TheinvolvementofAQP4inhumanepileptogenesis hasbeenfurtherconfirmedbya geneticstudywhich identified severalpolymorphismsofAQP4geneassociatedtoMTLEin com-binationwithKCNJ10singlenucleotidepolymorphisms(Heuser

etal.,2010).ThesedatasupportthatalterationsinwaterandK+

transportcontributetotheaetiologyofepilepsy

3.4 Connexin43andgapjunctions

The role of GJ-connected astrocytic networks in the

patho-physiology of epilepsy is still controversial, since they can act

bothasantiepileptic,byclearingandredistributingextracellular

K+,glutamateand GABA(Orkandet al.,1966;Walz,2000), and

proepileptic,byfuelingneuronswithglucoseanditsmetabolitesin

anactivity-dependentmanner(Rouachetal.,2008).Severalstudies

haveanalysedchangesinconnexin(Cx)expressionandcoupling

in human epileptic tissues: Naus and colleagues first reported

increasedCx43mRNAlevelsinthetemporalcortexofepileptic

patients(Nausetal.,1991).Thischangewasconfirmedbyother

groupsbothatthemRNAandproteinlevels(Aronicaetal.,2001;

Collignonetal.,2006;Fonsecaetal.,2002).However,unchanged

levelsofCx43havealsobeenreportedinhippocampaltissuesfrom

patientspresentingacomplexpartialseizuredisorderinthemedial

temporalcortexandhippocampus(Elisevichetal.,1997).Whether

expressionandfunctionofCx30,theotherastrocyticgapjunction

formingsubunit,arealteredinepilepsyremainsunclear.Studies

inrodentsindeedreportdifferentresults,whichmayberelated

tothediversityoftheepilepsymodels(Akbarpouretal.,2012;

Condorellietal.,2002;Söhletal.,2000),andnodataonhuman

epilepticbrainare yetavailable.Cx expressiondoesnot

neces-sarilyreflect activeandfunctional couplingbetweenastrocytes,

sincepost-translationalmodificationscanalterGJproperties,such

asconductance,openprobabilityandtrafficking.Fewfunctional

data on GJare available in human tissue An early study

per-formedinvitroonastrocyticculturesfromtissuesofMTLEpatients

reportedanenhancedcellcouplingbyusingthefluorescence

recov-ery afterphotobleaching technique(Lee etal., 1995).However,

recentworkshowedexvivoacompletelackofglialGJcoupling

in sclerotic hippocampaltissues from MTLE-HSpatients, and a

reducedcouplingduringtheepileptogenicphase inthekainate

mousemodelofTLE(Bedneretal.,2015).It isunclearwhether

culturedastrocytesmaintainthefunctionalpropertiestheydisplay

inthediseasedtissue.Alternatively,gapjunctioncouplingmaybe

differentiallyregulatedoverthecourseofepilepsy,asincreased

astroglialcouplingwasfoundfollowingstatusepilepticusinthe

kainatemouse(Takahashietal.,2010)

3.5 GlutamateandGABAhomeostasis

Neuronal activity leads to the release of excitatory and

inhibitoryneurotransmitters(mainlyglutamateandGABA)inthe

extracellularspace.Toensureappropriatesynapticresponsesand

themaintenanceofexcitation-inhibitionbalanceandtoavoid

neu-ronallossandseizures,properglutamateandGABAremovalfrom

thesynapticcleftbecomesessential.Astrocytesplayacrucialrole

inthisphenomenonthroughtheglutamine-glutamate-GABAcycle

(Eidetal.,2012):extracellularglutamateandGABAreleased

vesic-ularlyaretakenupbyastrocytesviaGLASTandGLT-1glutamate

transportersandGAT3GABAtransporter,respectively.Glutamine

synthetase(GS)thendirectlyconvertsglutamateintoglutamine,

whileGABAenterstheTCAcycleassuccinateandissubsequently

converted into alpha-ketoglutarate, glutamate, and glutamine

SpecializedN-transporterproteinsthenextrudeglutaminefrom

astrocytesinto theextracellularspace,while glutamatergicand

GABAergic neurons import glutamine through systemA

trans-porters,andconvertittoglutamateandGABA,respectively.Using

invivomicrodialysis,ithasbeenshownthatMTLEpatientspresent

five-fold higher extracellular glutamate levels in the epileptic

sclerotichippocampuscomparedtonon-HSandnon-epileptic

hip-pocampalformation,despiteneuronallossandatwo-foldincrease

inglialdensity(Cavusetal.,2005,2008).Thissuggestedthatan

impairedneurotransmitterhomeostasis couldparticipateinthe

pathophysiologyofepilepsy(CoulterandEid,2012).Indeed,

alter-ationsofglutamateandGABAtransportersaswellasGShavebeen identifiedinepilepticpatients

StudiesonglutamatetransportersinMTLEhavereported con-tradictoryresults.AninitialstudyfoundunchangedGLASTorGLT-1 expressionthroughinsituhybridizationandWesternblotanalysis

inTLEhippocampusandcortexcomparedtopost-mortemcontrols (Tessleretal.,1999).Similarresultshavealsobeenreportedintwo othermorerecentstudies(Bjørnsenetal.,2007;Eidetal.,2004) Conversely,ageneraldecreaseinGLT-1andGLASTexpression lev-elswasfoundinHSTLEpatientscomparedtonon-HSTLEsamples

asassessedbyimmunocytochemicalanalysis(Mathernetal.,1999; Properetal.,2002).Thissuggeststhatastrocyticglutamateuptake mayplayacrucialroleinpreventingepileptogenesis,evenifthe mechanisminvolvedremainsunknown

Glutamateexcessin TLEpatientsmaybeexplainednotonly

byadecreasedexpressioninglutamatetransporters,butalsoby impairedGSfunction.Indeed,ithasbeendemonstratedthatthere

isareductioninGSexpressionandfunctionalityinastrocytesof humansclerotichippocampi(Eidetal.,2004;vanderHeletal.,

2005):thisresultsinslowerratesofglutamate-glutaminecycling, accumulationofglutamateinastrocyticcytoplasmanddecreased synapticglutamateclearance(Petroffetal.,2002).Onthecontrary, subiculumastrocytes still expressGS,but theproteinisalmost absentinmostofthedistalastrocyticprocesses,comparedto non-epilepticcontrols(Eidetal.,2004).Furthermore,geneticmutations

inGSgene(GLUL)havebeenfoundinepilepticpatients:twoGLUL congenitalhomozygousmutationsoccurringatGSactivesiteshave beenidentifiedintwounrelatednewborns,oneofthemdisplaying severebrainmalformations,almostnoEEGactivityexceptshort thetaburstsandgeneralizedseizures(Haberetal.,2006;Häberle

etal.,2005).Noteworthy,athirdmutationhasrecentlybeenfound

inachildaffectedbyepilepticencephalopathyandpsychomotor retardation(Häberleetal.,2011)

AdeficiencyinGABAandGABA-mediatedinhibitionisthought

tocontributetoneuronalhyperexcitabilityinTLE.Aninvivo micro-dialysisstudyhasshownthattheepileptogenichippocampusof TLEpatientspresentedlowerextracellularGABAlevelsandhigher glutamateconcentrationsjustbeforetheonsetofseizures com-paredtothenon-epileptogenichippocampusofthesamepatient (DuringandSpencer,1993).Interestingly,ithasbeendemonstrated thatthiswasduetoanincreasedexpressionoftheastrocyticGABA transporter GAT3, which is normally only weakly expressedin humanhippocampalastrocytes(Leeet al.,2006):indeed,GAT3 wasmoreprominentlyexpressedincellsresemblingprotoplasmic astrocytes,locatedindentategyrusandhilusofsclerotic hippocam-palformations.ThisincreasedGAT3expressionmaythusexplain GABAreducedextracellularlevelsduringseizures,viaanincreased uptakebyastrocytes(DuringandSpencer,1993).Differentlyfrom TLE,absenceepilepsyischaracterizedbyanincreasedtonicGABAA receptor-mediatedinhibition in thalamocorticalneurons,which

isnecessary andsufficient forthegenerationofnon-convulsive seizures,typicalofthisformofepilepsy.ThisalterationofGABA levelsiscausedbyadysfunctionofGABAtransporterGAT-1,which

isexclusivelylocatedinastrocytesinthethalamusofbothhumans androdents(Crunellietal.,2011;Pirttimakietal.,2013)

Altogethertheseresultsindicatethatanimpairmentof gluta-mateandGABAextracellularlevels,duetoalteredexpressionof theirtransportersandreducedexpressionandfunctionalityofGS, canplayamajorroleinthepathophysiologyofepilepsy

3.6 Membranechannels Astrocytes of the sclerotic epileptic hippocampus exhibit unusualpropertiescomparedtoastrocytesofothernon-epileptic brainregions, duetoalterations of plasmamembrane channels andreceptors.Forinstance,ithasbeenshownthatastrocytesin

thehippocampusof HS-TLE patientspresent higher expression

ofmetabotropicglutamatergicreceptorsmGluR2/3,mGluR4and

mGluR8(TangandLee,2001)

Severalstudiesusingpatch-clamphavealsodemonstratedan

alteredexpressionofvoltage-gatedchannelsinastrocytesofthe

epileptichippocampus AdramaticincreaseinNa+current

den-sity was indeed found in cultures of MTLE astrocytes, which

displayedadepolarizedmembranepotentialandaction

potential-likeresponseswhenstimulatedwithcurrentinjections(O’Connor

etal.,1998).Similarresultswerealsoreportedinhumanacute

hip-pocampalslicesfromMTLEpatients(BordeyandSontheimer,1998;

BordeyandSpencer,2004):inthesetissuesastrocytespresented

complex, arborized and highly branched processes intensively

stainedforGFAP,andexpressedhighlevelsofTTX-sensitiveNa+

channels,allowing generation of slow action potentials A

sig-nificantup-regulation ofthe ␣1C subunitof voltage-gated Ca2+

channelswasalsoobservedasassessedbyimmunohistochemistry

onscleroticTLEhippocampi(Djamshidianetal.,2002);thisaltered

theproperties of L-typeCa2+ currents, suggestingan increased

astrocyticCa2+uptake

Allinall,investigationsofbraintissuesamplesfromepileptic

patientshaverevealedalterationsinexpression,localizationand

functionalityofseveralastrocyticproteins, suchasKir4.1

chan-nels,AQP4,Cx43,glutamateandGABAtransporters.Inaddition,an

enhancedexpressionofvoltage-gatedchannels,expressedatlow

levelsbyastrocytesinthehealthybrain,hasalsobeenobserved

in astrocytes from epilepticbrain specimens Accordingly,

dys-functional astrocytes can play a crucial role in the process of

epileptogenesisandcanthusbeconsideredasalternativetargets

todevelopnewantiepilepticdrugs

4 Brain tumours

Gliomas,representingthemajorityofprimarybraintumours,

mainlyoriginatefromglialcells.Theyareoneofthemost

aggres-siveneoplasias,sincetheycarryapoorprognosisdespiteaggressive

therapies due to their ability to infiltrate the brain and grow

They are classified according to the morphological properties

of the tumour-forming cells (Louis et al., 2007) In particular,

astrocyte-resemblingcellsareresponsibleforthedevelopmentof

astrocytomas,whichrepresentthemostcommonsubtypeofbrain

gliomas(Furnarietal.,2007).Severalstudieshaveidentified

astro-cyticmorphologicalandfunctionalchangesinastrocytoma,mainly

affectingextracellular glutamate levelsand membrane channel

functions(Fig.2c)

4.1 Structuralandmorphologicalchangesofastrocytes

Glioma cells share many characteristics with non-tumoural

astrocytes,butalsopresentseveralstructuralandmorphological

differences.Intissueofgliomapatients,anoverexpressionofGFAP

hasbeenidentifiedasbeingpositivelycorrelatedwithgliomasize,

butnotwiththedegreeofmalignancy(Herpersetal.,1986;Lee

etal.,2011a).Forthisreason,GFAPisusedasareliable

immuno-histochemicalmarkertostainsurgicallyresectedbraintumoursin

ordertoverifytheirastrocyticorigin(Vintersetal.,1998).Astrong

GFAPimmunoreactivityispresentalsoinreactiveastrocyteswithin

orsurroundingnon-glialtumours(Raoreetal.,2011).IngradeIto

IVastrocytomas,GFAPandvimentinareco-expressedbutin

differ-entcellularcompartments:indeed,whilevimentinislocatedcloser

tocellnucleus,GFAPispreferentiallydetectedincellularprocesses

(BordeyandSontheimer,1998;Cosgroveetal.,1989;Herpersetal.,

1986).BesidesGFAP,changes inothertypesofintermediate

fil-amentstructuralproteinshavebeenobserved:astrocytomacells

expressthevimentinbinding,300-kDaintermediatefilament

asso-ciatedprotein(IFAP–300kDa),whichisnormallypresentonlyin radialgliaand immatureastrocytesand absentinnormal adult brain(Yangetal.,1993,1994);furthermore,inastrocytomas,the proportionofkeratin-containingcells,whicharenormallydetected

intheneuroectoderm,isdirectlylinkedtothedegreeoftumour malignancy,thussupportingadedifferentiationoftumouralcells (Yangetal.,1994)

The modified expression of cytoskeletal elements may be responsible of the altered structure of astrocytic glioma cells: indeed, compared tonon-tumouralastrocytes, which present a stellateshapewith2–3majorandmanysmallerprocesses, low-gradepilocyticastrocytomacellsdisplayonly2–3thickprocesses (Bordey and Sontheimer,1998).Furthermore,astrocyticcells in low-gradegliomasdisplayminimaltomoderate nuclearatypia, scant cytoplasm and a high nucleus-to-cytoplasm ratio ( Burel-Vandenbosetal.,2011)

4.2 Extracellularglutamate Extracellularglutamateconcentrationiselevatedintumoural and peritumoural regions, especially close to tumours contain-ing necrotic areas in high-grade astrocytoma patients (Roslin

etal.,2003).Similarresultshavealsobeenobservedin oligoden-drogliomas,whichpresenthighlevelsofglutamateandglutamine

in the peritumoural area, as assessed by magnetic resonance spectroscopy (Rijpkema et al., 2003) This altered glutamate homeostasisexplainswhyduringthetimecourseofthedisease, 60–80%ofgliomapatientsexperienceseizures(Kurzwellyetal., 2010;Lynametal.,2007),whichoriginateclosetothetumourmass (Palludetal.,2013;Pattetal.,2000).Variousstudiesaimingat iden-tifyingthesourceofperitumouralglutamatereportedanimpaired expressionofglutamatetransportersongliomacells:braintissues fromglioblastomapatientsindeeddisplayastrongreductionin GLT-1levels,whileGLASTisnormallyexpressedbutis thought

tobemislocalizedincellnucleiratherthanattheplasma mem-brane(Lynametal.,2007;Savaskanetal.,2008;Yeetal.,1999) DecreasedGLT-1levelshavealsobeenobservedinhigh-grade com-paredtolow-gradeastrocytomasandnormalbrains(deGrootetal.,

2005).Furthermorethesechangesareaccompaniedbyanaltered expressionofthecysteine-glutamatesystem(xcsystem),aNa+ -independentexchangerthatcontrolstheintracellularglutathione levelsbyimportingonemoleculeofextracellularcysteine(required

inglutathionesynthesis)perreleasedglutamate.Gliomacelllines originatedfromtumoursandbrainspecimensfromglioblastoma patientsexpressthexcsystematsignificantlyhigherlevels com-paredtohumantissuesampleswithoutmalignanttransformation (Savaskan et al., 2008; Ye et al., 1999) Furthermore, a recent studyhasshownthat 50%of patient-derivedgliomashave ele-vatedexpressionofSLC7A11,thecatalyticsubunitofthexcsystem responsibleforxc-mediatedglutamaterelease(Robertetal.,2015) Interestingly,whenthesegliomacellsimplantedintracraniallyin micepropagated invivoasflanktumour xenolines,theycaused seizures,tumour-associatedexcitotoxicityandshortenedsurvival Altogethertheseresultsthussuggestthathighlevelsofthissystem contributetothereleaseofcytotoxicglutamatelevels,which pro-moteseizuresandactasanautocrine/paracrinesignalsustaining tumourgrowthandinvasion(Lyonsetal.,2007)

4.3 Gap-junctionsandmembraneionchannels Normalgrowthand metabolismofcells dependnotonly on theirorganellesandsubcellularstructures,butalsooncell-to-cell communication, in which GJs play a fundamental role Impair-ment of GJ-mediated intercellular communication may indeed resultinaberrantgrowthandtumourdevelopment(Omoriand Yamasaki,1998).Noteworthy,low-gradegliomaspresentastrong

peritumoural area; on the contrary,in surgicalspecimen from

high-grade astrocytoma patientsCx43 levels are reduced both

onmembranesandinthecytoplasm.Inaddition,onlythe

non-phosphorylated isoform of Cx43 was detected (Aronica et al.,

2001).Similarresultshavealsobeenobservedinprimary

astro-cyticcultures fromglioblastoma multiformpatients(Soroceanu

etal.,2001),wherereducedCx43expressionandGJ-mediated

cou-plingwerefound.Interestingly,thedecreaseinCx43expressionis

proportionaltotumourgradeandproliferativecapacity(Puetal.,

2004),andisnotduetoareducedgenetictranscription:gradeIII

andIVgliomasindeedpresentelevatedCx43mRNAbutlow

pro-teinslevels(Caltabianoetal.,2010), suggestinganalterationin

post-transcriptionalmechanismsingliomaastrocyticcells

BesidesGJchannels,astrocytictumouralcellspresentchanges

intheexpressionofNa+andK+channels:low-gradepilocytic

astro-cytomacellsindeeddisplayalmostexclusivelydelayedrectifying

K+currents,whilenotransientA-typeandinwardlyrectifyingK+

channelsweredetected.AlterationinK+channel-dependentcell

volume regulation resulted in a depolarizedmembrane

poten-tial and a round swollen cell body Furthermore, these cells

have increased TTX-sensitive Na+ currents, which enable them

togeneratespike-likeeventsaftercurrentinjections(Bordeyand

Sontheimer,1998).AlterationsinNa+channelexpressionhavealso

beenfoundinhigh-gradegliomas.PleomorphicGFAP+ cellsand

hypertrophicreactiveastrocytes adjacenttomultiform

glioblas-tomashowedstrongNav1.5expressionincellbodiesandprocesses,

comparedtoastrocytesinnormalwhitematter(Blacketal.,2010)

Malignant astrocytic gliomas, characterized by uncontrolled

cellularproliferationanddiffuseinfiltration,showanintense

resis-tance to apoptosis, which contributes to theineffectiveness of

traditional therapeutic approaches (such as surgical resection,

radiotherapyandchemotherapy).Astrocytesinthesecancers

dis-playmorphologicalchangesandincreasedGFAPexpression,two

phenotypesthathavebeendescribedinactivatedastrocytesaftera

CNSinjury.Moreover,GJcouplingisreduced:thisfavorsmalignant

transformationviaareductionofinhibitorysignalscontrollingcell

divisionandproliferationreceivedfromneighbouringcells.Glioma

astrocytesalsopresentimpairedextracellularglutamateregulation

andaberrantexpressionofvoltage-gatedchannels,contributing

toaberrantneuronalandastrocyticactivity.Inordertoconsider

thesedysregulatedastrocyticpathwaysaspotentialtherapeutical

targets,itisofcrucialimportancetounderstandhowgliomacells

modifyduringtumourprogressionandinteractwithneighbouring

normalandcancerouscellsinthetumourmicroenvironment

5 Alzheimer’s disease

Alzheimer’sdisease(AD)isthemostcommontypeofdementia

inelderly,accountingfor60–80%ofdementiapatients(Wortmann,

2012).It is characterizedbya subtle decline in episodic

mem-ory,appearingasa deficitinrecalling therecent past,followed

by a more global decline of cognitive abilities, such as loss of

long-termmemories,language,attentionandpersonalitychanges

(QuerfurthandLaFerla,2010).ADisidentifiedbytwo

histopatho-logicalhallmarks,extracellulardeposits(plaques)ofamyloid-beta

(A␤)proteinandintracellularneuronaltanglesformedby

abnor-mallyphosphorylatedtauprotein(QuerfurthandLaFerla,2010)

Thedistributionpattern of neurofibrillarytangles and neuronal

alterationsaregenerallyusedtodefinethestageofADprogression

(BraakandBraak,1991,1995).InstagesIandII,neuronal

alter-ationsareconfinedtothetransentorhinalregion,whileboththe

entorhinal/transentorhinalareasandhippocampusareinvolvedin

stagesIIIandIV.ThelaststagesofADprogression(VandVI)are

insteadmarkedbydevastatingneocorticaldistructionand

repre-sentthefullydevelopedAD(BraakandBraak,1995).Despitethe globaleconomicburdenofthisdisease,effectivetreatmentsarestill lackingandthecausesofthediseaseremainelusive.The patholog-icalpotentialofastrocytesinADhasbeeninitiallysuggestedin

1910byAloisAlzheimer,whofoundglialcellsinclosedassociation withdamagedneuronsandabundantlypopulatingsenileplaques (Verkhratskyetal.,2010).Thishasbeensubsequentlyconfirmed

bystudiesonhumantissuesandADanimalmodelsshowing astro-cytichypertrophy,particularlyinastrocytesassociatedwithsenile plaques(Nageleetal.,2004),aswellasglialchangesoften preced-ingplaqueandtangleformation(Rodríguez-Arellanoetal.,2016) (Fig.2d)

5.1 AstrogliosisinAD Reactiveastrogliosisisawell-knownhallmark ofAD,even if its role hasnot beenclearly understood yet.It is identified by

anincreasedexpression ofGFAP andhypertrophy ofastrocytes

inthevicinityofamyloidplaques.Post-mortemtissuesfromAD patientsindeeddisplayincreasedGFAPlevelsintemporal(Griffin

etal.,1989;Simpsonetal.,2010),occipital,parietalandfrontal lobes(Kashonetal.,2004).Moreover,in thecerebrospinalfluid

of ADpatients, higherlevelsof GFAPconcentrations have been measuredcomparedtoage-matchedcontrols(Jesseetal.,2009) Interestingly,somedegreeofcorrelationhasbeenfoundbetween GFAPexpressionandADprogression,withhigherGFAPlevelsat increasingBraakgroups(Simpsonetal.,2010)ordurationof clin-icalillness(Serrano-Pozoetal.,2013).However,this correlation remains uncertain,sinceanotherworkshowednodifferencein GFAP expression betweendemented and non-demented brains withinthesameBraak stage(Whartonet al.,2009).DuringAD progression,8 ofthe10differentGFAPisoformsdescribed (Hol and Pekny, 2015) areupregulated For instance,reactive astro-cytesindentategyrussubgranularzone,hilusandCA4areaofAD patientsdisplayaprominentexpressionofGFAP␦,butonlyCA1, CA3andsubiculumastrocytessurroundingplaquesshowedGFAP␦ upregulation withincreasingAD stage (Kamphuis etal., 2014) Additionally, the number of human-specificastrocyte subtypes expressingtheframe-shiftedGFAP variant,GFAP+1,is increased withADprogression,butonlyfewoftheseGFAP+1-expressingcells hasbeenidentifiedasassociatedtoplaques,withprocesses pro-trudingthroughthem(Kamphuisetal.,2014;Middeldorpetal.,

2009).AstrogliosisandGFAPupregulationarealsoaccompanied

bydysregulationintheexpressionofotherastrocyticcytoskeleton proteins.Forinstance,inthelateraltemporalcortexofadvanced

ADstages,thereisasignificantdecreaseintranscriptsencoding membersofthemyosinandkinesinfamilyandothercytoskeletal proteins,suchasactin␤,dyneinandintegrin␣.Moreover, tran-scriptsencoding tight junctionproteinsandadherensjunctions arealsoreducedduringADprogression(Simpsonetal.,2011).The effectofthealteredexpressionofthesegenesinastrocytesstill remainsunclear,butitmayaffectvariousintracellularsignalling pathways

5.2 InteractionsbetweenastrocytesandAˇ

InADbrainsatearlystagesofthepathology,activatedreactive astrocytes are predominantin the molecularlayerof the cere-bralcortexandclosetoamyloidplaquesinpyramidalcelllayers (WisniewskiandWegiel,1991).Inseveralbrainregionssuchas cortex, hippocampusand cerebellum, proliferatingprocesses of hypertrophicastrocytesnearesttoamyloiddepositscontactand surroundtheplaques;theypenetratemoreintonon-cored primi-tiveplaques,comparedtoclassiccompactcoredamyloiddeposits, thus merging with them and contributing to their fragmenta-tion,dispersionand theobservedvarietyof plaquemorphology

(Kato etal., 1998;Wisniewskiand Wegiel, 1991).Interestingly,

inthevisual cortexof ADbrainswithsevere pathology,

GFAP-immunoreactiveastrocytesandplaquesarearrangedinaspecific

laminardistribution: indeedgliosisis preferentiallylocalizedin

laminaeII,III,IVaandIVc,thelatterpresentingadiscrete

plaque-associatedglyotichorizontalbandattheloweredge(Beachand

McGeer,1988).Furthermore,amorerecentstudyhasshownthat

reactiveastrocytestogetherwithmicroglialcellsformspecific3D

reactiveglial nets around plaques in a plaque-specific way: at

A␤dense-coreplaques,astrocyticprocessesareintermingledwith

microglialcellbodieswhichenvelopthecoreA␤structure;while

atfibrillaryplaques,ahighernumberofglialcellsarerecruitedto

reactiveglialnetformationandbothmicroglialandastrocytic

pro-cessesinvadetheplaqueareaandinteractwithA␤protein(Bouvier

etal.,2016)

The stimuluscapable of inducing astrocyte reactivity in AD

brainsisstillunderdebate;however,studiesusingaggregatedA␤

proteinandtheintactcoreofA␤plaquesisolatedfromADbrain

tis-suehaveshownthatA␤cantriggeractivationofastrocytesinvitro,

causingGFAPup-regulationandmorphologicalchanges(DeWitt

etal.,1998).Furthermore,amyloidplaquescolocalizewithreactive

astrocytesintheabsenceofdystrophicneuritesinthehippocampus

ofmildADbrains,whiledecreasingoverthecourseofthepathology

(Pikeetal.,1995).Thissuggeststhatplaque-associatedastrocytosis

canactasacontributoryeventinADpathologyandthatonce

acti-vated,astrocytescanparticipateinA␤metabolism.A␤42indeed

accumulatesinthecytoplasmandprocessesofreactiveastrocytes

inthemolecularlayercompletelydevoidofamyloidplaquesinAD

brains.A␤42alsobuildsupinastrocytesassociatedwithplaquesin

pyramidalcelllayersoftheentorhinal,parietal,occipitaland

tem-poralcortex(Akiyamaetal.,1999;Funatoetal.,1998;Kurtetal.,

1999;Nageleetal.,2003;Thaletal.,1999,2000).Inthesecells,

non-fibrillarA␤42localizestosmalllysosomallipofuscin-likegranules,

organizedinclustersandmainlylocatedintheperinuclear

cyto-plasm(Funatoetal.,1998;Nageleetal.,2003;Thaletal.,1999;

Yamaguchietal.,1998).Moreover,ithasrecentlybeenreported

thatmorethan90%ofactivatedastrocytesinADbrainfrontal

cor-texcanaccumulateanewlydescribedclassofamyloidstructures

formedbyA␤,theannularprotofibrils.Thesefibrilsareabsentin

amyloidplaquesandinthebrainofage-matchedcontrols,andcan

inducereactiveoxygenspeciesgenerationandinactivationofGS

inthecell(Lasagna-ReevesandKayed,2011)

Theaccumulationof A␤42in astrocytesis directlylinkedto

theseverityoflocalADpathology.A␤42+ materialinastrocytes

isindeedproportionaltotheamountofA␤42containedin

sur-roundingneurons and tothe local density of amyloid plaques

withinthe pyramidalcell layer of theentorhinal cortex, while

theamountofA␤42inastrocytesofthemolecularlayerdevoid

ofplaqueswellcorrelateswiththeseverityofthepathology in

thesub-adjacentcorticallaminae(Nageleetal.,2003)

Interest-ingly,astrocyte-accumulatedA␤42isnotproducedbyastrocytes,

but hasa neuronal origin, deriving fromthe internalization of

degeneratingsynapsesanddendritesbyphagocytosis:astrocytic

A␤42colocalizeswithcholineacetyltransferaseand␣7nicotinic

acetylcholinereceptors,whichareneuron-specificproteins

accu-mulatinginastrocytesasaconsequenceoftheirdebris-clearing

activity(Nageleetal.,2004).Furthermore,withtheprogression

ofthedisease,A␤42-burdenedastrocytescanundergolysis and

formsmallsphericalGFAP+amyloidplaques,firstappearinginthe

subpialportionofthecorticalmolecularlayerclosetoastrocytes

containinglargeA␤42deposits(Nageleetal.,2003)

Theseresultsindicatethatnotonlyneurons,butalsoastrocytes

arecapableofgivingrisetoamyloidplaquesandcausing

morpho-logicalmodifications withintheseplaques;thisability canthus

account,atleastinpart,forthevarietyofplaquemorphology

iden-tifiedinADbrains.BlockingtheinitialaccumulationofA␤42in

neuronsrepresentsthemainearlytargettocontrolADpathology However,inviewoftheabilityofastrocytestointeractwithplaques andparticipateinA␤metabolism,limitingtheirrecruitmentcould alsocontributetolimitingordelayingADprogression

5.3 GlutamateandGABAhomeostasis Thereareseveralindicationsthatastrocyticglutamatergic func-tionisimpairedinAD.GLT-1immunoreactivityisreducedinthe frontalcortex of AD patients(Li et al.,1997; Tianet al., 2010) and thisreduction is inversely correlatedwithamyloid precur-sorproteinmRNAlevels,whilenochangehasbeenobservedfor GLASTexpression.GLT-1reducedexpressionisalsoaccompanied

bydecreasedglutamatergictransportactivityandincreasedmRNA levels,thusindicatinganimpairmentatpost-transcriptionallevel (Li et al.,1997).Similar resultshave alsobeenobtained inthe hippocampusandgyrusfrontalisofADpatientbrains.Amarked impairmentinGLT-1andGLASTexpressionatbothgeneand pro-teinlevelsoccuralreadyatearlystagesofthedisease,particularly

in thevicinityof amyloid plaques (Jacobet al., 2007) Further-more,in ADlateraltemporal cortex, GLT-1expressiontendsto decreasewithhigherBraakgroup.Thiscorrelationishoweverstill underdebate,sincecontradictoryresultshavebeenobtaineddue

tohighvariabilityofglutamatetransporterexpressionbetweenAD individuals(Beckstrømetal.,1999).Togetherwithreduced expres-sion,proteinsplicevariantscanalsoaccountforchangesinthe functionalityofglutamatetransporters.Interestingly,disease-and pathology-specificchangesinGLT-1splicevariantexpressionoccur

inautopticADbrains,whichcanaccountforthereducedastrocytic glutamateuptakeefficiencyinAD.Inparticular,areductioninthe functionalsplicevariantsb,whichisabletouptakeglutamate,has beenidentifiedinseveralbrainregions,togetherwithasignificant increaseofexon-skippingvariants,characterizedbyreduced trans-portcapacity(Scottetal.,2011).Altogether,theseresultssuggest

animpairmentofastrocyticglutamateclearancecapabilityinAD, whichmayleadtoneurotoxiceffectandcontributetotheincreased prevalenceofseizuresinADpatients(Scarmeasetal.,2009) Furthermore,thelevelsofGSarealsoalteredinADbrain: tem-poralneocortexastrocytesfromADbrainsamplesdisplayreduced

GS expression due to impairment of post-translational modifi-cations,which renderstheproteinhighlysensitivetooxidative lesioning(LePrinceetal.,1995).Incontrast,totalGSlevelsinthe CSFofADpatientsremainunchanged(Timmeretal.,2015) GABAergicdysfunctionalsoplaysaroleinAD,andseveralrecent studiespointtoabnormalitiesofGABAhomeostasisinreactiveAD astrocytes.AnaccumulationofGABAtogetherwithanincreasein GAD67andGAT3havebeenfoundinGFAP+astrocytesofdentate gyrus molecularlayer and inferior temporal cortex from post-mortemADpatients,butnotinage-relatedhealthysubjects(Mitew

etal.,2013;Wuetal.,2014).ThissuggestsanincreasedGABA pro-ductioninastrocytes.Consistentwiththishypothesis,astrocytic monoamineoxidase-B,theenzymeresponsibleforGABA produc-tion, is up-regulated in post-mortem brains of AD individuals ThishasalsobeenshowninADmousemodels,where accumulat-ingGABAisabnormallyreleasedfromreactiveastrocytesthrough bestrophin1 channels,which impairslearning and memory(Jo

etal.,2014)ThehighastrocyticGABAlevelidentifiedinhuman brainswithhighA␤loadmaythusbeusedasanovelbiomarkerand diagnostictoolforAD.Furthermore,sinceithasbeenshownthat GABAtransporterscanreversetheirtransportdirectionin pres-enceofexcessiveintracellularGABA(Leeetal.,2011b;Richerson andWu,2003),GAT3mayserveasanewdrugtarget

5.4 Astrocyte-specificproteinchanges

NotonlyareglutamateandGABAhomeostasisalteredinAD,

but alsootherastrocytic-specific functions,suchasGJ

commu-nicationandK+buffering.In1996Nagyandcolleaguesreported

forthefirsttimeanincreasedCx43expressionintemporal

corti-calareascontainingseveralamyloidplaquesinpost-mortemAD

humanbrains Furthermore,theyalsodemonstratedusing

elec-tronmicroscopythattheenhanced Cx43immunoreactivitywas

restricted to astrocytic GJs throughout the tissue, both within

and outside plaque-containing regions(Nagy et al.,1996).This

observationwaslaterconfirmed,asasimilarenrichmentofCx43

punctaoccurredinreactiveastrocyticprocessesinfiltrating

amy-loidplaques,togetherwithamilderincreaseofCx30(Koulakoff

etal.,2012).ADbraintissuesalsodisplayalteredKir4.1andAQP4

expression(Wilcocketal.,2009):astrongdecreaseinKir4.1and

AQP4mRNAlevelsoccurinthetemporalcortexofbrainsamples

fromADpatientswithmoderateandseverepathology

Further-moreAQP4 is alsomislocalized,and presentsa diffusestaining

patternwithpoorlydistinguishablebloodvesselsinsevere

pathol-ogycases(Wilcock etal.,2009).Asignificantincrease inAQP1,

normallyexpressedinthechoroidplexus,hasbeenidentifiedin

astrocytes of thefrontalcortex and temporal lobes in sporadic

andfamilialADcases,whereAQP1+ astrocyteswerefoundclose

toA␤42orA␤40deposits(Hoshietal.,2012;Pérezetal.,2007)

A deregulation of cellular Ca2+ levels hasbeen proposedto

contribute to the initialsteps of AD progression, by causing a

long-lastingoverloadinthecytoplasmandtheendoplasmic

retic-ulum, which triggers cell death Although this signalling was

consideredcompromisedmainlyinneurons,astrocytesarealso

affectedas demonstrated in rodent AD models and AD human

brainsamples.BasalCa2+levelsareindeedelevatedinthe

astro-cyticnetworkandCa2+transientsaremorefrequent,coordinated

acrosslongdistancesandindependentfromneuronalactivityin

thecortexofADmice(Kuchibhotlaetal.,2009).AnenhancedCa2+

responsemaybetheresultofanalteredexpressionofastrocytic

metabotropicglutamatereceptor5(mGluR5).Indeed,anincreased

mGluR5staininghasbeenidentifiedinhippocampalastrocytesof

ADpatients,inproximityofA␤plaques(Limetal.,2013)

Further-more,changesinS100␤levelshavebeenidentifiedinADpatients:

while S100␤is strongly overexpressedin AD hippocampaland

temporallobeastrocytescloselyassociatedwitheitherdiffuseor

neuriticA␤plaques,onlymoderateincreaseshavebeenfoundin

frontallobeandponsandunchangedlevelsinoccipitallobeand

cerebellum(Griffinetal.,1989;Marshaketal.,1992;VanEldik

and Griffin,1994).In thetemporal lobefromADhumanbrain,

S100␤-overexpressingastrocytesaremainlyassociatedwith

dif-fuseneuriticplaques,whilediffusenon-neuriticanddense-core

neuriticplaqueshavesmallnumbersofassociatedS100␤

astro-cytes.Astrocyteswereobservedincloseproximitytodense-core

non-neuriticplaquesonlyrarely(Mraketal.,1996).Thisindicates

thatactivatedastrocytesproducingS100␤arepresentalready

dur-ingtheearlieststages ofplaqueformation,and decreaseatthe

end-stageof plaqueprogression.Furthermore,a correlationhas

beenfound betweenneuritic plaque density and S100␤levels,

whicharesignificantlyincreasedinthebrainofADpatients

com-paredtoage-matchedcontrols(Mraketal.,1996).Thenumberof

activatedS100␤-overexpressingastrocytesassociatedwithsingle

neuriticplaquesandthedegreeofneuriticpathologyinthesame

plaquesarealsocorrelated(Mraketal.,1996;Shengetal.,1994,

1996).Remarkably,ADmicealsodisplayenhancedglutamatergic

gliotransmission,asindicatedbytheincreasedfrequencyinresting

conditionsoftheslowinwardcurrentsmediatedbyactivationof

NMDAreceptorsinneurons(Gómez-Gonzaloetal.,2017)

Further-more,astrocyticCa2+signalscanbeprotectiveduringtheinitial

phaseofAD,sincethedisruptionofinositoltriphosphate

recep-tors2(IP3R2)-mediatedCa2+signallinginastrocytesboostedthe progressionofA␤plaquedepositionandsynapticplasticity dys-function atvery early stages ofthe pathology (Gómez-Gonzalo

etal.,2017)

DuringADprogression,astrocytesdisplayacomplexpatternof dysfunctions,concerningcytoskeleton,celljunctions,gapjunction communication,intracellularsignallingmoleculesand neurotrans-mitter homeostasis These pathways are progressively affected withincreasingBraakstagesofthepathology,thussuggestinga continuousdeclineofastrocytefunctionsinAD.Further investi-gationsarenow requiredtoclarifywhetherandwhich ofthese changesplayanactiveroleinADdevelopment.Thiswillbecrucial

in designing newtherapies aimedat rescuingastrocytic physi-ologicalfunctionsthatmaylimitoreven preventAD-associated cognitivedecline

6 Major depressive disorder

Majordepressivedisorder(MDD),achronicrecurrentand debil-itating mental illness,is characterizedby depressedmood, loss

ofinterestandpleasure,weightchanges,sleepalterations,lossof energy,difficultiesofconcentrationandthoughtofdeathand sui-cide(AmericanPsychiatricAssociation,2013).Numerousstudies haverevealedthatMDDisadisorderwithprominent pathologi-calastrocyticalterations,whichaffectdensity,morphology,protein expressionandmembranechannelfunctionsofastrocytes(Fig.2e) However,astrocytic changes in MDDstrongly differ fromwhat

is observed in otherneurological and neurodegenerative disor-ders,suchasepilepsy,inflammationandAlzheimer’sdisease:while thesediseases present reactiveastrogliosis,glial scarformation andneuronalloss(Sofroniew,2009;SofroniewandVinters,2010), astrogliosisandprominentneuronalpathologyisnotpresentin MDD

6.1 Astrocytedensityandmorphology Many histopathological studies performed on post-mortem brain samples have unveiledprominent decreasesin astrocyte numberandpackingdensityinMDDsubjectscomparedto age-matchednon-psychiatriccontrols(Cotteretal.,2002,2001;Gittins andHarrison,2011;Ongüretal.,1998;Rajkowskaetal.,1999) Severalbrainregionsdisplayareducedastrocyticpopulation,such

as dorsolateralprefrontal(Cotter etal., 2002; Rajkowska etal.,

1999), orbitofrontal (Rajkowska etal., 1999), subgenual(Ongür

etal.,1998)andanteriorcingulatecortex(Cotteretal.,2001)and amygdala(Altshuleretal.,2010;Bowleyetal.,2002).However,an increaseinglialcelldensityhasalsobeenreportedin hippocam-palregionsanddentategyrusofMDDpatients(Stockmeieretal.,

2004),whilenochangehasbeenobservedintheorbitofrontal cor-texandinthesupragenualregionoftheanteriorcingulatecortex

inlate-lifedepressedpatients(Khundakaretal.,2011a,b)andin thehippocampus(Cobbetal.,2013).Changesinglialdensitymay thusdifferentiallyaffectspecificbrainregions.Interestingly,ithas beenshownthatthisalterationisage-dependent:ingreymatterof dorsolateralprefrontalcortexofyoungerdepressedpatients(<50 yearsold),thedensityofGFAP+astrocytesissignificantlyreduced comparedtocontrolsofsimilarage;incontrast,oldersubjectswith late-onsetdepressionpresentedincreasedastrocyticpopulationin thesamearea(Miguel-Hidalgo etal.,2000), probablyreflecting

acompensationtoneuronallossobservedinolderMDDpatients (Rajkowskaetal.,2005)

Inparalleltoalterationsofastrocytepackingdensity,thesizeof glialnucleiseemstobeaffectedinMDD.Astrocyteswithlarger nucleihave beenobserved inthedorsolateralprefrontalcortex (Rajkowska et al., 1999) and in the grey and white matter of

hadbiggercellbodiesandmoreramifiedprocessesindepressed

subjectscommittingsuicidecomparedtomatchedsudden-death

controls(Chana etal., 2003; Torres-Platas et al.,2011).On the

contrary,threeotherstudiesobservedunalteredglialsizeinthe

prefrontalandorbitofrontalcortexandinthehippocampus(Cotter

etal.,2002,2005;Stockmeieretal.,2004)

StudiesonGFAPexpressionadditionallyrevealedmarked

alter-ationsinMDDsubjects.Immunohistochemicalanalysisaimedat

quantifyingtheareacoveredbyGFAP+cellbodiesandprocesses

hasshownapredominantdecreaseofGFAPingreymatterofthe

prefrontalcortexinyoungdepressedsubjectscomparedtocontrols

(Miguel-Hidalgoetal.,2000),inwhitematterofanteriorcingulate

cortex(GittinsandHarrison,2011),inorbitofrontalcortex(

Miguel-Hidalgoetal.,2010),CA1andCA2hippocampalregions(Müller

etal.,2001),locuscoeruleus(Chandleyetal.,2013)andcerebellum

(Fatemietal.,2004).Moreover,GFAPdecreasehasalsobeen

con-firmedatmRNAandproteinlevels(Chandleyetal.,2013;Fatemi

etal.,2004;Johnston-Wilsonetal.,2000;Miguel-Hidalgoetal.,

2000;Websteretal.,2005)anditiscorrelatedwithageandonset

ofdepression:thelevelsofGFAPproteinareindeedsignificantly

lowerinlessthan60years-olddepressedpatientscomparedto

age-matchedcontrols,withnochangeobservedbetweenolderMDD

subjectsandtheircontrols(Sietal.,2004).Incontrast,anincreaseof

GFAPoccurredindorsolateralprefrontalcortexinlate-onsetMDD

patients(Davisetal.,2002;Miguel-Hidalgoetal.,2000)

6.2 GlutamateandGABAhomeostasis

Recentneuroimagingand post-mortemstudiesondepressed

subjectshaverevealedadysfunctionofastrocytic-mediated

reg-ulationofglutamatehomeostasis.MDDsubjectspresentedlower

levelsofglutamate,glutamineorcombinedglutamate/glutamine

(Glx),asassessedbymagneticresonancespectroscopy,inseveral

brainregionssuchasprefrontalareas(Hasleretal.,2007),frontal

lobe(Yildiz-YesilogluandAnkerst,2006),anteriorcingulate

cor-tex(Aueretal.,2000;Mirzaetal.,2004;Pfleidereretal.,2003)

andamygdala(Michaeletal.,2003),andinplasma(Altamuraetal.,

1995).Thissuggestsanimpairedglutamate/glutaminemetabolism

Thesechangesmayhowevernotappearhomogenouslyinallbrain

areasandinalldepressedpatients,sincetwootherstudiesreported

glutamate increases in occipital and frontalcortex (Hashimoto

et al., 2007; Sanacora et al., 2004), probably reflecting

region-specific alterations which can also depend on patient age and

chronicityofdepression

TheimpairedglutamatehomeostasisobservedinMDDpatients

mayberelatedtothereducedastrocyticpackingdensitydetected

frompost-mortemstudiesonMDDbrainsamples.Furthermore,

reduced expression of GLAST and GLT-1 occurin anterior

cin-gulate(Choudaryetal.,2005),dorsolateralprefrontal(Choudary

etal.,2005;Klempanetal.,2009)andorbitofrontalcortex(

Miguel-Hidalgoetal.,2010),andinlocuscoeruleus(Bernardetal.,2011;

Chandleyetal.,2013)andhippocampus(Medinaetal.,2016)of

subjectsdiagnosedwithMDD.Finally,differentcorticaland

sub-corticalregionsofdepressedsuicidevictims,suchasprefrontaland

premotorcortexandtheamygdala,alsodisplayreducedlevelsof

GS(Miguel-Hidalgoetal.,2010;Sequeiraetal.,2009).Interestingly,

ithasbeenshownthattheimpairmentinglutamate-relatedgene

expressionisspecifictoastrocyteinMDD,sinceitdoesnotoccurin

neuronsofMDDbrains(Bernardetal.,2011),thusunderlyingthe

astrocyticbasisofMDDpathology

In MDD not onlyglutamate regulation is impaired,but also

GABAhomeostasis.Indeed,reducedGABAlevelshavebeenfound

inthedorsomedial/dorsalanterolateralprefrontalcortex(Hasler

etal.,2007),andintheoccipitalcortex(Sanacoraetal.,2004)of

depressedbutnotremittedMDDpatients(Schüretal.,2016)

Fur-thermore,in thedorsolateralprefrontalcortex,GABAA receptor subunitsareup-regulated(Choudaryetal.,2005;Sequeiraetal.,

2009).Interestingly,it hasbeenshown that␣1and ␤3subunit expressionisselectivelyincreasedinsuicidecompleters(Choudary

etal.,2005)

6.3 Membranechannelsandproteins Several astrocytic-specific membrane channels have been demonstratedtobealteredinMDDsubjects.Adecreaseingene andproteinexpressionofCx43andCx30occursinthedorsolateral prefrontalcortexofsuicidecompleters(Ernstetal.,2011)andin thelocuscoeruleusofMDDpatients(Bernardetal.,2011).Cx43 levelsarealsoreducedintheorbitofrontalcortex(Miguel-Hidalgo

etal.,2014)andhippocampus(Medinaetal.,2016)

AstrocytesinMDDsubjectsdisplayalteredK+andwater homeo-stasis:Kir4.1channelsaredown-regulatedinthehippocampusof depressedpatients(Medinaetal.,2016);AQP4immunoreactivity andmRNAlevelsdecreaseinthegreymatteroforbitofrontal cor-tex,wherethecoverageofbloodvesselsbyastrocyticendfeetis reduced(Rajkowskaetal.,2013),inlocuscoeruleusandinthe hip-pocampusofMDDpatients,compared topsychiatrically-normal controlsubjects(Bernardetal.,2011;Medinaetal.,2016) Finally, another astrocytic marker, the Ca2+ binding protein S100␤,whichis predominantlyexpressedandsecreted bygrey matterastrocytesand involvedinseveralCa2+-dependent intra-cellular functions,is affectedin MDD pathology S100␤ mRNA levelsaredecreasedintheprefrontalcortexandhippocampusof depressedsuicide victims(Bernardetal., 2011;Klempan etal.,

2009),andthenumberofS100␤+astrocytesisstronglyreducedin CA1hippocampalregionofMDDpatients(Gosetal.,2013) Fur-thermore,in agreementwithMDD astrocytic pathology,S100␤ cerebrospinalfluidandserumlevelsareincreased(Grabeetal., 2001;Rothermundtetal.,2001;Schroeteretal.,2002,2008), prob-ablyreflecting leakageof S100␤fromastrocyticcytoplasminto extracellularcompartments

To summarize, key features of MDD include a reduction in astrocytepopulationandalterationsintheexpressionofseveral astrocyticmarkers,suchasGFAP,GJproteins,AQP4,Kir4.1 chan-nels, S100␤ and glutamate transporters MDD patients do not presentprominentastrogliosis,glialscarformationandneuronal degeneration,whichareconverselyobservedinotherbrain dis-orders,such as tumours,AD, amyotrophic lateralsclerosis and Huntington’sdisease.Astrocytescanthusbeconsideredasnovel targets for antidepressant medications Nonetheless, the recip-rocal communication between astrocytes and neuronsin MDD remainsstillunclear;moreover,itwouldbeinterestingto deter-minewhetheralterationsofastrocyticphysiologyandnumberare presentonlyduringepisodesofdepressionoralsoduringperiods

ofremission

7 Down syndrome

Downsyndrome(DS)isthemostcommonwell-known chro-mosomaldisorder,affectingoneinevery700babiesintheUnited States(Parkeretal.,2010).DSiscausedbythetrisomyof chro-mosome 21,and characterizedby mental retardation,language impairmentandotherphenotypicabnormalities,suchasslanting eyes,flatfacialfeatureandhypotonia.Furthermore,inadditionto developmentalfailure,DSischaracterizedbyAD-likepathology, withneuriticA␤plaqueswidelydevelopinginthehippocampus andenthorinalcortexof almostalladults withDS andin some

DSchildren (Hof etal.,1995;Hymanetal.,1995;Leverenz and Raskind,1998).Directlyrelatedtomentalretardation,anumber

ofneuropathologicalchangesfoundinDSCNShavebeenwidely