Research article

The potential role of glutamate transporters in the pathogenesis of normal tension glaucoma
Takayuki Harada,1,2,3 Chikako Harada,1,2 Kazuaki Nakamura,2 Hun-Meng A. Quah,1 Akinori Okumura,2 Kazuhiko Namekata,2 Tadashiro Saeki,4 Makoto Aihara,4 Hiroshi Yoshida,3 Akira Mitani,5 and Kohichi Tanaka1,6,7
of Molecular Neuroscience, School of Biomedical Science and Medical Research Institute, Tokyo Medical and Dental University, Tokyo, Japan. 2Department of Molecular Neurobiology, Tokyo Metropolitan Institute for Neuroscience, Fuchu, Japan. 3Department of Neuro-ophthalmology, Tokyo Metropolitan Neurological Hospital, Fuchu, Japan. 4Department of Ophthalmology, University of Tokyo School of Medicine, Tokyo, Japan. 5Human Health Science, Graduate School of Medicine, Kyoto University, Kyoto, Japan. 6Precursory Research for Embryonic Science and Technology, Japan Science and Technology Agency, Kawaguchi, Japan. 7Center of Excellence Program for Brain Integration and its Disorders, Tokyo Medical and Dental University, Tokyo, Japan.
1Laboratory

Glaucoma, a progressive optic neuropathy due to retinal ganglion cell (RGC) degeneration, is one of the leading causes of irreversible blindness. Although glaucoma is often associated with elevated intraocular pressure (IOP), IOP elevation is not detected in a significant subset of glaucomas, such as normal tension glaucoma (NTG). Moreover, in some glaucoma patients, significant IOP reduction does not prevent progression of the disease. Thus, understanding IOP-independent mechanisms of RGC loss is important. Here, we show that mice deficient in the glutamate transporters GLAST or EAAC1 demonstrate spontaneous RGC and optic nerve degeneration without elevated IOP. In GLAST-deficient mice, the glutathione level in Mller glia was decreased; administration of glutamate receptor blocker prevented RGC loss. In EAAC1-deficient mice, RGCs were more vulnerable to oxidative stress. These findings suggest that glutamate transporters are necessary both to prevent excitotoxic retinal damage and to synthesize glutathione, a major cellular antioxidant and tripeptide of glutamate, cysteine, and glycine. We believe these mice are the first animal models of NTG that offer a powerful system for investigating mechanisms of neurodegeneration in NTG and developing therapies directed at IOP-independent mechanisms of RGC loss.
Introduction Itisestimatedthatglaucomaaffectsnearly70millionindividualsworldwide,withatleast6.8millionbilaterallyblind(1).The diseaseischaracterizedbyaslowprogressivedegenerationof retinaloutputneurons(retinalganglioncells[RGCs])andtheir axons, which is usually associated with elevated intraocular pressure(IOP).Thecommonadult-onsetglaucomaisprimary open-angleglaucoma(POAG),whichisprobablycausedbya reductioninoutflowofaqueoushumorthroughthetrabecular outflowpathways(2).Normaltensionglaucoma(NTG),asubsetofPOAGthatindicatesstatisticallynormalIOP,alsoshows glaucomatousopticneuropathyandrelevantvisualfielddefect. SeveralpopulationstudieshavesuggestedthatNTGrepresents 20%90%ofallPOAG,withpercentagesseemingtovaryaccordingtorace(35).Interestingly,IOPstillseemstoplayarolein NTGbecauseasubstantialnumberofpatientswithNTGas wellasotherformsofPOAGbenefitfromloweringofIOP(6). Thus,NTGmaybecausedbythevulnerabilityofopticnerves tonormalrangeofIOP.However,itshouldbenotedthatsome ofNTGpatientsarestillprogressiveinspiteofsufficientIOP reduction.AlltheseobservationssuggestapossibilitythatfactorsnotdependentonIOPmaycontributetodiseaseprogress
Nonstandard abbreviations used:EAAC1,excitatoryaminoacidcarrier1;GCL, ganglioncelllayer;GLAST,glutamate/aspartatetransporter;GLT-1,glutamatetransporter1;GS,glutaminesynthetase;INL,innernuclearlayer;IOP,intraocularpressure; 2K,second-orderkernel;mfERG,multifocalelectroretinogram;NTG,normaltension glaucoma;ONL,outernuclearlayer;POAG,primaryopen-angleglaucoma;RGC, retinalganglioncell. Conflict of interest:Theauthorshavedeclaredthatnoconflictofinterestexists. Citation for this article:J. Clin. Invest.117:17631770(2007).doi:10.1172/JCI30178.

andthatelucidatingsuchfactorswouldbenecessarytounderstandthepathogenesisofglaucomaandguideeffortstoward improvedtherapeutics. Recentstudieshaveshownthatglaucomaisaffectedbymultiplegeneticandenvironmentalfactors(7,8),andthiscomplexity canmakeitdifficulttoreachdefinitiveconclusions (especiallyin humanstudies,inwhichmanyfactorscannotbecontrolled).Animalstudiescomplementhumanstudiesandcanprovideimportant insightsintogeneticetiologyandmolecularmechanismsforfurtherassessmentinpeople.Therearenowseveralpublishedmouse modelsofpressure-inducedRGCdeath.Theseincludeinherited andexperimentallyinducedmodels(9).However,noanimalmodel forNTGiscurrentlyknownandavailableforresearch. Besidesmoreextensivelyinvestigatedfactors,suchasreduced ocularbloodflow,ocularvasculardysregulation,andsystemic bloodpressurealterations,excessivestimulationoftheglutamatergicsystem,specificallytheNMDAsubtypes,hasbeenproposed tocontributetodeathofRGCsinglaucoma.Glutamatetransporteristheonlymechanismfortheremovalofglutamatefrom theextracellularfluidintheretina(10).Therefore,itishypothesizedthattheincreaseinglutamatemayresultfromafailureof theglutamatetransportersadjacenttoRGCs.IntheinnerplexiformlayerwheresynapsesonRGCsexist,thereare3transporters involvedinthistask:glutamatetransporter1(GLT-1),locatedin thebipolarcellterminals;excitatoryaminoacidcarrier1(EAAC1) in retinal neurons including RGCs; and glutamate/aspartate transporter(GLAST)inMllercells (11).However,thereisstill debateonwhetherexcitotoxicdamageisinvolvedinthepathophysiologyofglaucoma(12).Here,weutilizedmiceinwhich1 ofeachofthese3glutamatetransportershasbeenknockedout
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Figure 1
Expression of glutamate transporters in the retina. Immunohistochemical analysis of mouse retina doublestained with GLAST and GS, a specific marker for Mller glial cells (A); EAAC1 and calretinin, a specific marker for RGCs and amacrine cells (B); and GLT-1 and calretinin antibodies (C). Scale bar: 50 m.

andexaminedthelong-termeffectofretinalmorphologyduringpostnataldevelopmentonRGCsurvivalinvivo.Wefound thatGLAST-andEAAC1-knockoutmiceshowedspontaneously occurringRGCdeathandtypicalglaucomatousdamageofthe opticnervewithoutelevatedIOP.Toourknowledge,theseglutamatetransporterknockoutmicearethefirstanimalmodelsof NTG,whichofferapowerfulsystemfordeterminingmechanisms andevaluatingnewtreatmentsofNTG. Results Degeneration of RGCs in GLAST- and EAAC1-deficient mice. Immunohistochemicalstudiesdemonstratedthepresenceof3 subtypesofglutamatetransportersintheinnerplexiformlayer oftheratretina.GLASTisexpressedonMllercells,GLT-1on thebipolarcellterminals,andEAAC1onganglioncells(11).We

verifiedthattheselocalizationsarealsotrueforthemouse.As wepreviouslyreported(13),GLASTimmunoreactivityispresent throughouttheretinaanddouble-labeledbyglutaminesynthetase (GS),aspecificmarkerforMllerglialcells(Figure1A).EAAC1 andGLT-1areneural-typeglutamatetransporters;however,their distributionisdifferent.EAAC1ismainlylocalizedtotheinner retinallayeranddouble-labeledbycalretinin,aspecificmarkerfor RGCsandamacrinecells(Figure1B).Ontheotherhand,GLT-1 immunoreactivityispresentinasmallfractionofphotoreceptorsintheouternuclearlayer(ONL)inadditiontobipolarcells butnotinRGCs(Figure1C).Thefindingsareinagreementwith thosereportedbyRauenforratretina(11).TheretinaeofGLAST (14)andEAAC1(15)mutantmicehavenormalorganizationat birth (Figure2A).However,wenoticedsevereretinaldegenerationin8-month-oldGLAST/mice(seealsoFigure3,AandB),in

Figure 2
RGC degeneration in glutamate transporter mutant mice. (A) H&E staining of retinal sections during postnatal development. WT, GLAST+/, and GLAST/ mice are littermates. EAAC1+/ and EAAC1/ mice are littermates. (B) Quantification of RGC number in glutamate transporter mutant mice. The number of neurons in the GCL was counted in the retinal section from one ora serrata through the optic nerve to the other ora serrata at 0, 1, 2, 3, 5, 8, 16, and 32 weeks of age. Each plot represents the results of 3 to 6 independent experiments. Scale bar: 50 m (A). NBL, neuroblast layer.
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Figure 3
RGC degeneration in GLAST/ mice. H&E-stained sections show a decreased number of cells in the GCL in GLAST / mouse (arrowhead) (A and B). Retrogradely labeled RGCs in GLAST/ mouse were decreased compared with those in WT mouse (C and D). E and F are magnified images of C and D, respectively. Scale bar: 100 m (A, B, E, and F); 500 m (C and D).

whichcellnumberwasdecreasedintheganglioncelllayer(GCL) (53%6%;n=6,P<0.0001)andtheinnernuclearlayer(INL) (81%11%;n=6, P<0.05)butnotintheONL(97%5%;n=6, P=0.34).AsnearlyhalfofthecellsintherodentGCLaredisplaced amacrinecells,wedistinguishedRGCsfromdisplacedamacrine cellsbyretrogradelabeling(16).ThenumberofRGCspermm2 inretinaeof8-month-oldGLAST/mice(202248;n=18)was significantlyreducedcomparedwiththatinWTmice(4011106; n=18,P<0.0001)(Figure3,CF).WealsoexaminedRGCnumber bycountingthecellslocatedintheGCL.Becausequantification ofRGClossbyGCLcellcount(49%2%;n=6)wasconsistent withthatbyRGClabeling(50%1%;n=18,P=0.89)in8-montholdGLAST/mice,weexaminedtheGCLcellnumberfromP0to 8monthsinallmutantmice(Figure2B).SignificantRGClosswas alsoobservedinGLAST/miceafter2weeksofage,GLAST+/mice after5weeks,andEAAC1+/andEAAC1/miceafter8weeks.We wereunabletoexamine8-month-oldGLT-1/mice(17)because most of them died within 3 weeks; however, RGC number in 8-month-oldGLT-1+/micewasnormalcomparedwiththatinWT mice(101%2%;n=10;Figure2BandFigure4).Thus,GLT-1 mutantmicewerenotevaluatedfurther. Degeneration of optic nerve in GLAST- and EAAC1-deficient mice. Degenerationoftheopticnerveisoneofthehallmarksofglaucoma.ConsistentwithsevereRGCloss,opticnervecupping (Figure5B)andthinningoftheopticnerve(Figure5D)were apparentin8-month-oldGLAST/mutants.Quantitativeanalysis revealedthatopticnerveareainGLAST/mice(0.0740.002mm2; n=6)wassignificantlydecreasedcomparedwiththatinWTmice (0.0960.005mm2;n=6, P=0.0019).Toanalyzemorphologicalchangesintheopticnerve,semithintransversesectionswere cutandstainedwithtoluidineblue.Thedegeneratingaxonsin 5-week-oldGLAST/mutantshadabnormallydarkaxonalprofiles(Figure5F).Inaddition,comparedwithWTmice(Figure5E), thedensityofaxonsthroughtheopticnervewasclearlydeclined inGLAST/mice(Figure5F).Wenextexaminedtheiridocorneal angleinGLAST/mutantsandfoundthatitwaswellformed

withanobviousSchlemmcanalandanormalextentoftrabecularmeshwork(Figure5H).EAAC1/mutantsalsoshowedexcavationoftheopticnerveandnormaliridocornealanglemorphology(datanotshown). IOP measurement in GLAST- and EAAC1-deficient mice.GLASTand EAAC1mutantmiceshowedPOAG-likephenotype,whichsuggeststheinvolvementofincreasedIOP.Therefore,weexaminedthe IOPsofWT,EAAC1/,andGLAST/mice.IOPmeasurementwas carriedoutforbothyoung(4week)andadult(9to11month)mice around9pm,whenIOPishighestinmouseeyes(18).TheIOPs ofEAAC1/andGLAST/micewerenotsignificantlyincreased comparedwithWTatbothtimepoints(Figure5I).Theseresults suggestthatRGClossandglaucomatous-likechangesoftheoptic nerveinthesemutantsareIOP-independent. Impaired visual function in GLAST-deficient mice. To determine whethertheobserveddegenerationofRGCsandtheopticnerve isfunctionallysignificant,weanalyzedmultifocalelectroretinograms(mfERGs)ofGLAST/mouseretina.RGCscontributeto thehumanmfERGresponse,andthesecond-orderkernel(2K), whichappearstobeasensitiveindicatorofinnerretinaldysfunction(19),isimpairedinglaucomapatients(20).Theaveraged2K responsesof10miceareshowninFigure6A.Theresponsetopographydemonstratedthatthe2KcomponentderivedfromGLAST/ micetendedtobeshorter inallvisualfieldscomparedwiththat ofWTmice(Figure6B).Quantitativeanalysisrevealedthatthe2K amplitudewasreducedbyabouthalfinGLAST/micecompared withthatinWTmice(P<0.005)(Figure6C),suggestingthatRGC lossisfunctionallysignificantinGLAST/mice. Role of glutamate neurotoxicity in RGC degeneration in GLAST-deficient mice.TodeterminethecontributionofexcitotoxicitytoRGC deathinthesemodels,wefirstmeasuredtheintravitrealglutamate concentrationinWTandmutantmice.Glutamateconcentration wasnotsignificantlyincreasedinGLASTorEAAC1mutantmice (Figure7A).Becauseanincreaseinglutamateconcentrationinthe extracellularspacejustadjacenttoRGCsmightbeobscureddueto dilutionwithinthevitreous(21),failuretodetectglutamateelevationinthevitreousfluiddoesnotnecessarilyexcludeglutamate involvementinRGCdegenerationinthemutants.Toassessglu-

Figure 4
Normal retinal structure in GLT-1+/ mouse. H&E-stained sections show the absence of RGC degeneration in an 8-month-old GLT-1+/ mouse. Scale bar: 50 m.
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Figure 5
Optic nerve degeneration and normal IOP in glutamate transporter mutant mice. (A and B) Optic nerve atrophy in 8-month-old GLAST/ mice. In WT mice (A), the nasal and temporal nerve fibers (arrows) are beneath the internal limiting membrane (arrowhead) and enter a well-formed optic nerve (N). In GLAST/ mice (B), the nerve fiber layer has become thin and almost absent as it enters the nerve (bold arrows). Cupping extends to the posterior aspect of the inner retinal layer (arrowheads). (C and D) Thinning of optic nerve is apparent in GLAST/ (D) compared with WT (C) mice. (E and F) Staining of semithin sections with toluidine blue reveals the presence of abnormally dark axonal profiles (arrowheads) and decline of axons in GLAST/ (F) compared with WT (E) mice. (G and H) Aqueous humor drainage structures in WT (G) and GLAST/ (H) mice. Iridocorneal angle in GLAST/ mice is normal with an obvious Schlemms canal (arrow) and trabecular meshwork (asterisk) compared with those in WT mice. The angle recess between the cornea (C) and iris is wide open. (I) IOP of young (4 weeks old) and adult (911 months old) mice. Sample numbers are indicated in parentheses. Scale bar: 200 m (A, B, G, and H); 130 m (C and D); 9 m (E and F).

tamateinvolvementinRGCdegeneration,wenextexaminedthe effectoftheNMDA receptorantagonistmemantine(22)onRGC degenerationinGLASTmutants.Memantine(10mg/kginjected daily)preventedRGClosswhenappliedfromP7toP13(Figure7B), andtheRGCnumberinGLAST/micewasnormalatP14(Figure7C). Theprotectiveeffectofglutamatereceptorantagonistsuggests thatglutamateneurotoxicityispartlyinvolvedinRGClossin GLASTmutantmice.However,sinceglutamateconcentrationin thevitreousfluidwasnormal,otherfactors,inadditiontoglutamateneurotoxicity,mightcontributetoRGCdegenerationof GLAST-deficientmice. Role of oxidative stress in RGC degeneration of GLAST- and EAAC1deficient mice.Inadditiontoexcitotoxicity,oxidativestresshasalso beenproposedtocontributetoRGCdeathinglaucoma(23,24). InadultGLAST/andEAAC1/mice,lipidhydroperoxideswere increasedinretinae,suggestingtheinvolvementofoxidative stressinRGCloss(Figure8A).Previousstudiesdemonstrated thatglutathione,atripeptideofglutamate,cysteine,andglycine, hasacentralroleinprotectingRGCsagainstoxidativestressand thatglutamateuptakeisarate-limitingstepinglialglutathione synthesis(25,26).Becauseretinalglutathionewasspecifically distributedinMllercells(Figure8B),wehypothesizedthat oxidative stress may result from impaired glial glutathione synthesisinGLAST/mice.Inagreementwiththishypothesis,
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glutathionecontentintheretinaandinculturedMllercells wasdecreasedinGLAST/mice(Figure8C).Asimilarreductioninglutathionelevelwasrecentlyreportedintheplasmaof humanPOAGpatients(27).Toconfirmthatthereductionin MllercellglutathionelevelsmakesRGCsmorevulnerableto oxidativestress,weexaminedtheeffectofH2O2onWTRGCs inamixedculturewithMllercellsfromGLAST/orWTmice. WeusedaconcentrationofH2O2thatdoesnotinduceMller celldeath.RGClosswassignificantlyhigherwhenRGCswere mixedwithMllercellsfromGLAST/micethanwhenmixed withMllercellsfromWTmice(Figure8D).TheseresultsindicatethatGLASTdeficiencyleadstoglaucomatous-likeRGCloss in2ways:first,byexcessivestimulationofNMDAreceptorsand second,byreducingtheglutathionelevelsinMllercells,thus makingRGCsmorevulnerabletooxidativestress. UnlikeglialGLAST,neuronalEAAC1doesnotplayamajor roleinclearingglutamatefromtheextracellularspace(15,28). Instead,EAAC1cantransportcysteine,anobligateprecursorfor neuronalglutathionesynthesis,farmoreeffectivelythanGLAST (29).Aoyamaetal.recentlyreportedthatEAAC1deficiencyleads toimpairedneuronalglutathionemetabolismandage-dependent brainatrophy(29).InEAAC1/mutantmice,retinalglutathione content wasnotdecreased(Figure8C).Thesemeasurementsrepresentbulkglutathionecontentinretinalhomogenatesfrom

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Figure 6
Impaired multifocal electroretinogram in GLAST/ mice. (A) Averaged responses of 2K from 10 mice. The visual stimulus was applied to 7 different areas in the retina. The 7 individual traces demonstrate the average responses to the visual stimulus at the corresponding stimulus area. (B) Three-dimensional plots showing the amplitude variation across the arrays in A. (C) Quantitative analysis of 2K amplitude. The response amplitudes for each stimulus element were added and the result was divided by the total area of the visual stimulus. n = 10 per group; *P < 0.005. Values in B and C are given in nV per square degree (nV/deg2).

EAAC1/andWTmice.Thus,glutathionedeficiencyinRGCsof EAAC1/micemaynotbeapparentbecausealargeshareofretinal glutathioneislocalizedtoMllercells(26),whichdonotexpress EAAC1. To directly determine the vulnerability of EAAC1/ RGCstooxidants,primaryculturesofmutantandWTRGCswere treatedwithH2O2.CulturedRGCsfromEAAC1/miceweresusceptibletoH2O2comparedwiththosefromWTmice(Figure8E). ThesefindingssuggestthatEAAC1deficiencymakesRGCsmore vulnerabletooxidativestress. Discussion Here,weshowthatGLAST-andEAAC1-knockoutmiceshow progressiveRGClossandglaucomatousopticnervedegeneration withoutelevatedIOP.OurstudyraisesthepossibilitythatdysfunctionoftheglutamatetransportersGLAST(EAAT1inhumans)and EAAC1(EAAT3inhumans)playsaroleinRGCdeathinhuman NTG.ItisreportedthatEAAT1isdownregulatedinhumanglaucoma(30).Inaddition,adecreaseofEAAT1hasbeenshownin fibroblastsfrompatientswithAlzheimerdisease(31).Considering thehighfrequencyofPOAGinAlzheimerdiseasepatients(32), commonmechanisms,suchasGLASTdysfunction,mightcontributetothe2diseases.ThesefindingsindicatethatGLAST-and EAAC1-knockoutmiceareusefulasmodelsofNTG. BecauseGLASTisamajorglutamatetransporterinthemammalianretina,lossofGLASTisexpectedtorendertheretinahighly susceptibletoexcitotoxicdamage.However,wepreviouslyreported thattheGLAST/retinashowednosignsofRGCdegeneration althoughitwasmoresensitivetoprolongedischemiathannormal

retina(13).Inthepresentstudy,GLAST/miceshowedspontaneousRGClossandglaucomatousopticnervedegeneration.What factorsaccountforthisdiscrepancy?Onepossibilityisthegenetic backgroundofthemiceused.Inpreviousstudies,weusedGLAST/ miceonamixedC57BL/6129geneticbackground.Duringthe courseofbackcrossingtoaC57BL/6strain,GLAST/miceshowed spontaneousNTG-likeRGCdegeneration.Thisfindingcanallow ustoidentifystrain-specificmodifiergenesthatmightsuppress orenhanceRGCdegeneration.ItisintriguingthattheC57BL/6J micewithmutationsintheoptineurin(OPTN)gene,whichhave beenassociatedwithhumanPOAGandNTG,showextensiveRGC loss(33).Thus,theC57BL/6JstrainmayhaveapotentiallypathogenicalleleofOptn.Althoughmorestudiesareneededtodetermine whethertheNTG-likephenotypeinGLAST/miceonaC57BL/6J strainisrelatedtoOptn,suchstudieswillidentifynewcandidate molecules/pathwaysthatmaycontributetoRGCsurvival. TheresultsofthepresentstudysuggestthatglialGLASTand neuronalEAAC1playdifferentialrolesinpreventingRGCdegeneration.GLASTisessentialnotonlytokeeptheextracellular glutamateconcentrationbelowtheneurotoxiclevel,butalsoto maintaintheglutathionelevelsinMllercellsbytransporting glutamate,thesubstrateforglutathionesynthesis,intothecells. Incontrast,themainroleofEAAC1istotransportcysteineinto RGCsasaprecursorforneuronalglutathionesynthesis.Thus, GLASTdeficiencyleadstoRGCdegenerationcausedbybothexcitotoxicityandoxidativestresswhereasEAAC1deficiencyinduces RGClossmainlythroughoxidativestress.Inthepresentpaper,we demonstratethattheavailabilityofglutamateislimitingforthe maintenanceofnormalintracellularglutathionelevelinMller cells.Thisobservationdiffersfromfindingsinothertissues,where cysteinewasidentifiedastherate-limitingsubstance(34,35).How canthisbeexplained?PreviousstudiesdemonstratedthatMller cellspossessaveryfastglutamateturnover(36).Amajorpartof theavailableintracellularglutamateisusedforglutaminesynthesiscatalyzedbytheenzymeGS,whichisexclusivelylocatedinMllercells(37).Thismakestheintracellularglutamateconcentration ofMllercellsstronglydependentonglutamateuptakeviathe glutamatetransporterGLAST.Incontrast,thecysteineturnover ofMllercellsseemstoberatherslow(36).Therefore,therate-limitingfactorforglutathionelevelsinMllercellsisnotthecystine supplytothecellsbutrathertheprovisionofglutamate. Glutamateexcitotoxicityandoxidativestresshavebeenproposedtocontributetoretinaldamageinvariouseyediseases, includingretinalischemia,glaucoma,diabeticretinopathy,and age-relatedmaculardegeneration(3840).Therefore,thedesign ofcompoundscapableofactivatingglutamateuptakebyGLAST representsanovelstrategyforthemanagementofglaucomaand variousformsofretinopathy(41,42).WealsoshowedthatRGCs
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Figure 7
Effect of glutamate neurotoxicity on RGC degeneration. (A) Intravitreal glutamate concentration in glutamate transporter mutant mice. Sample numbers are indicated in parentheses. (B) H&Estained P14 retinal sections from WT and GLAST/ mice with or without treatment with memantine (10 mg/kg, i.p.) daily from P7 to P13. (C) Quantitative analysis of RGC number following memantine administration. The number of neurons in the GCL was counted in the retinal section from one ora serrata through the optic nerve to the other ora serrata. n = 6 per group. #P < 0.05; *P < 0.005. Scale bar: 50 m (B).

coculturedwithMllercellsfromGLAST/micearesusceptible tofreeradicalstimulation(Figure8D).Wepreviouslyreported thatneurotrophinsaltertheproductionofsometrophicfactors inMllercells,whichindirectlyleadstoneuralcellsurvivalduringphotoreceptordegeneration(43,44).Wesuggestthatsucha glia-neuronnetworkisfunctionalinvariousformsofneurodegenerativediseases,andthemessengersbetweengliaandneurons maybedifferentaccordingtothesituation.Inaddition,each singleMllercellextendsfromtheoutertotheinnersurfaceof theretina.ThisallowstheMllercellstoformelaborateintimate contactswiththesomataofalltypesofretinalneuronsaswellas withthefibersandsynapsesintheneuropileofthe2plexiform layers.TheirubiquitouspresencemakestheMllercellsasuitabletargetfordrugdeliveryandgenetherapyinretinaldegenerativediseases.Furthermore,recentstudieshaveshownthatMllercellscouldproliferateafterneurotoxicdamageandproduce bipolarcellsandrodphotoreceptorsintheadultmammalian retina(45,46).Therefore,Mllercellsmaybeanewtherapeutictargetforbothneuroprotectionandregenerationinretinal degenerativediseases(47,48). Methods
Mice. Experiments were performed using EAAC1 +/, EAAC1 / (15), GLT-1+/(17),GLAST +/,andGLAST/(14)micewithapprovalfrom theInstitutionalAnimalCareandUseCommitteeofTokyoMedical
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andDentalUniversityandtheTokyoMetropolitanInstituteforNeuroscience.AllthemiceusedinthisstudywerebackcrossedwithC57BL/6 morethan8times. Immunohistochemistry.Frozen12-mthickretinalsectionswereincubatedwith1of4setsofprimaryantibodymix.Primaryantibodiesused weremouseanti-GS(ChemiconInternational)andrabbitanti-GLAST(13) oranti-glutathione(SignatureImmunologics);andmouseanti-calretinin (ChemiconInternational)andrabbitanti-EAAC1(49)orantiGLT-1(13). FluorescenceimmunohistochemistrywasperformedusingCy2-conjugateddonkeyanti-mouseIgGandCy3-conjugateddonkeyanti-rabbitIgG (JacksonImmunoResearchLaboratoriesInc.).Sectionswereexaminedby fluorescencemicroscopy(Olympus). Histological and morphometric studies.Paraffinsections(7-mthick)of retinalspecimenswerecutthroughtheopticnerveandstainedwithH&E. RGCnumberandtheextentofretinaldegenerationwerequantifiedin3 ways.First,thecelldensityofeachlayerwasanalyzed(50).Second,inthe samesections,thenumberofneuronsintheGCLwascountedfromone oraserratathroughtheopticnervetotheotheroraserrata.Third,RGCs wereretrogradelylabeledfromthesuperiorcolliculuswithFluoro-Gold (Fluorochrome)(16).SevendaysafterFluoro-Goldapplication,eyeswereenucleatedandretinasweredetachedandpreparedasflattenedwholemounts in4%paraformaldehyde in0.1MPBSsolution.GCLwasexaminedinwholemountedretinaewithfluorescencemicroscopytodeterminetheRGCdensity.Fourstandardareas(0.04mm2)ofeachretinaatthepointof0.1mmfrom theopticdiscwererandomlychosen,labeledcellswerecountedbyobservers

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Figure 8
Increased oxidative stress in glutamate transporter mutant mice. (A) Lipid hydroperoxide concentration in whole retina of WT, EAAC1 /, and GLAST/ mice. Sample numbers are indicated in parentheses. (B) Immunohistochemical analysis of mouse retina double-stained with glutathione and GS. Glutathione-like immunoreactivity was observed in Mller glial cells (arrowheads). Scale bar: 20 m. (C) Mean glutathione concentration in whole retina and cultured Mller cells. Sample numbers are indicated in parentheses. (D) Lactate dehydrogenase (LDH) release from H2O2-treated RGCs cocultured with WT or GLAST/ Mller cells. n = 3 per group. (E) Lactate dehydrogenase release from H2O2-treated RGCs from WT or EAAC1/ mice. n = 4 per group. #P < 0.05; *P < 0.005.

blindedtotheidentityofthemice,andtheaveragenumberofRGCs/mm2 wascalculated.ThechangesinRGCnumberwereexpressedaspercentagesoftheWTcontroleyes.Insomeexperiments,memantine(10mg/kg; Merz)wasinjectedi.p.intoGLAST/miceandtheirlittermatesdailyfrom P7toP13.ThesemiceweresacrificedonP14andprocessedforRGCcount. Fortheanalysisoftheopticnerve,frozen10-mthicksectionswerecutat 2.5mmfromtheeyeballandstainedwithH&E.Quantitativeanalysisofthe opticnerveareawascarriedoutusingacomputerizedimageanalysisprogram (ScionImageBeta4.0.3;ScionCorporation).Fordetailedmorphologicalanalysis,opticnerveswerefixedin2%glutaraldehydeand2%paraformaldehyde in0.1Mphosphatebufferovernightat4C.Afterdissection,thepiecesof tissuewereplacedin1%osmiumtetroxide.Afterdehydration,thepieceswere embeddedinEPON(NisshinEM).Transversalsemithin(1m)sectionswere stainedwith0.2%toluidinebluein1.0%sodiumborate. IOP measurement.IOPwasdirectlymeasuredbyamicroneedlemethod inanesthetizedmiceasdescribedpreviously(18,51).Themicroneedle wasconnectedtoapressuretransducer,whichrelayeditssignaltoa bridgeamplifierandthentoananalog-to-digitalconverterandacomputer(WorldPrecisionInstruments).Themicroneedletipwasinserted throughthecorneaandthedatawereautomaticallycollectedonline intoacomputerdatabase.Tominimizevariation,datawerecollected duringatimewindow46minutesafterinjectionofanesthetic,duringwhichtimeIOPplateaued(51).Animalagewas4weeksor911 months,andbodyweightrangeswere1324gor2836gatthetime ofIOPmeasurement,respectively.Since24-hourIOPpatterninmouse eyesisbiphasicandIOPishighestaround9:00pm(18),weexamined IOPbetween8:00pmand11:00pm. mfERGs.Mice(1216weeksold)wereanesthetizedbyi.p.injectionofa mixtureofxylazine(10mg/kg)andketamine(25mg/kg).Pupilsweredilat

edwith0.5%phenylephrinehydrochlorideand0.5%tropicamide.mfERGs wererecordedusingaVERIS5.1system(Electro-DiagnosticImagingInc.). Thevisualstimulusconsistedof7hexagonalareasscaledwitheccentricity.Thestimulusarraywasdisplayedonahigh-resolutionblackandwhite monitordrivenataframerateof100Hz.The2K,whichisimpairedin patientswithglaucoma(20),wasanalyzed. Glutamate assay.Vitreoussamples(510l)weresurgicallyextractedfrom adultmice,andglutamatewasanalyzedbyserialenzymaticreactionsas previouslyreported(52). Cell culture.RGCs(16)andMllercells(44)werepreparedfromP7 mice.Insomeexperiments,WTRGCswerecoculturedwithMllercells fromGLAST/miceortheirWTlittermates.Theseculturecellswere stimulatedwith200MH2O2for16hours,andRGCdeathratewas analyzedusingalactatedehydrogenasecytotoxictestkit(Wako)aspreviouslyreported(16). Lipid peroxidation and glutathione assay.TheconcentrationsoflipidperoxidesandglutathioneweremeasuredusingBioxytechLPO-560(Oxis HealthProductsInc.)andGlutathioneassaykit(CaymanChemical)per themanufacturersprotocols. Statistics.Forstatisticalcomparisonof2samples,weuseda2-tailedStudents ttestorMann-WhitneyUtest.Inothercases,wealsoused1-factor ANOVAandTukey-Kramertest.DataarepresentedasthemeanSEM. P<0.05wasregardedasstatisticallysignificant.

Acknowledgments WethankX.Guo,K.Ajiki,andM.Ichikawafortechnicalassistance.ThisstudywassupportedinpartbygrantsfromtheMinistryofEducation,Culture,Sports,ScienceandTechnologyof Japan,theTokyoBiochemicalResearchFoundation,theNaito

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research article
Foundation,andtheUeharaMemorialFoundation(toT.Harada). C.HaradawassupportedbyafellowshipfromJapanSocietyfor thePromotionofScienceforYoungScientists. ReceivedforpublicationAugust28,2006,andacceptedinrevised formApril24,2007.
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