abnormalities in glutamate metabolism and excitotoxicity in the

Hindawi Publishing CorporationScientificaVolume 2013 Article ID 528940 13 pageshttpdxdoiorg1011552013528940

Review ArticleAbnormalities in Glutamate Metabolism and Excitotoxicity inthe Retinal Diseases

Makoto Ishikawa

Department of Ophthalmology Akita Graduate University Faculty of Medicine 1-1-1 Hondo Akita 010-8543 Japan

Correspondence should be addressed to Makoto Ishikawa makomedakita-uacjp

Received 10 October 2013 Accepted 17 November 2013

Academic Editors M Hamann M Milanese M J Seiler and L Tremolizzo

Copyright copy 2013 Makoto Ishikawa This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

In the physiological condition glutamate acts as an excitatory neurotransmitter in the retina However excessive glutamate can betoxic to retinal neurons by overstimulation of the glutamate receptors Glutamate excess is primarily attributed to perturbation inthe homeostasis of the glutamate metabolism Major pathway of glutamate metabolism consists of glutamate uptake by glutamatetransporters followed by enzymatic conversion of glutamate to nontoxic glutamine by glutamine synthetase Glutamatemetabolismrequires energy supply and the energy loss inhibits the functions of both glutamate transporters and glutamine synthetase In thisreview we describe the present knowledge concerning the retinal glutamate metabolism under the physiological and pathologicalconditions

1 Introduction

Glutamate is the most prevalent neurotransmitter in thevisual pathway including the retina [1ndash3] After releasefrom the presynaptic neural terminal glutamate binds tothe postsynaptic glutamate receptors inducing the influxof Na+ and Ca2+ resulting in membrane depolarizationWhen glutamate is in excess it can become toxic to retinalneurons by overstimulation of the glutamate receptors [4ndash7] In as early as 1957 Lucas and Newhouse [4] reportedthat subcutaneous administration of sodium L-glutamateinduced necrosis in the inner retina of albino mice withina few hours of injection indicating that high concentrationsof glutamate cause retinal cell death Therefore efficientremoval of glutamate from the extracellular space is criticalformaintenance of retinal function and preventing the retinalneurons against glutamate toxicity (Figure 1)

Glutamate is metabolized by two major processes itsuptake and the subsequent enzymatic degradation Der-ouiche and Rauen (1995) [8] have shown that glutamate isdegraded into nontoxic glutamine by glutamine synthetasefollowing uptake by major glutamate transporter GLASTinto Muller cells A prerequisite for an effective glutamate-glutamine cycle in glial cells is the regulated coordinationbetween glutamate uptake and glutamate degradation [9]

Although glutamate is considered as a potent exotoxinexogenous glutamate is only weakly toxic to the retinawhen glutamate transporters on Muller glial cells are opera-tional [10]When glutamate transporter is pharmacologicallyblocked inner retinal neurons are exposed by a higheramount of endogenous glutamate resulting in severe exci-totoxic degeneration [10] These observations suggest thatglutamate is neurotoxic when the uptake system is impairedrather than when the release is excessive

The first part of this article surveys physiology of glu-tamate metabolism with particular interest in glutamatetransporters (GLASTGLT-1 EAAC-1) glutamine synthetaseand energy supply The second part describes excitotoxicretinal degeneration induced by abnormalities of glutamatemetabolism in the retinal ischemia glaucoma and diabeticretinopathy

2 Glutamate Metabolism inthe Physiological Condition

21 Glutamate Uptake by Glutamate Transporters All neu-ronal and glial cells in the retina express high-affinity glu-tamate transporters [9] At least five glutamate transportershave been cloned GLAST (EAAT1) GLT-1 (EAAT2) [11 12]

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Glutamate

GlutamineGlutaminesynthetaseSynaptic

cleft

Glutamatetransporter

Glutamate

Photoreceptors

Mueller cell

Bipolar cell

Non

-NM

DA-

R

NM

DA-

R

Met

abol

ic-R

Synaptic vesicles

Figure 1 Glutamatergic synaptic transduction and uptake andmetabolism of glutamate in the Muller cell The photoreceptorcell synthesizes glutamate which is continuously released duringdarkness Glutamate released from the synaptic terminal reaches tothe postsynaptic receptors of bipolar cell dendrites then is promptlytaken up from the synaptic cleft The glutamate transporters are thepredominant mechanism for uptake of glutamate in the Muller cellandmaintain the proper concentration of this potentially excitotoxicamino acid Glutamate transported into the Muller cell is degradedto glutamine by glutamine synthetase

EAAC-1 (EAAT3) [13] EAAT4 [14] and EAAT5 [15] In theretina four out of the five knownEAATs have been describedImmunohistochemical analysis localize the distributions ofthese glutamate transporters respectively [16 17] GLAST isdistributed in the Muller glial cells GLT-1 and EAAT5 arecolocalized in the photoreceptor cells and the bipolar cellsEAAC-1 (EAAT3) is localized in the horizontal cells ganglioncells and some amacrine cells

Since glutamate is a potent neurotoxin the functionalrole of glutamate transporters is critical to prevent the retinalexcitotoxicity Izumi et al [10] reported the pharmacologicalblockade of glutamate transporters using TBOA (DL-threo--benzyloxyaspartate) [18ndash20] TBOA is an inhibitor of alltypes of glutamate transporters and does not evoke currentsin neurons or glial cells Izumi et al [10] used TBOA aloneand in combination with exogenous glutamate to examinethe role of glial glutamate transporters in excitotoxic retinaldegeneration In the presence of TBOA the potency ofglutamate as a neurotoxin is greatly enhanced SurprisinglyTBOA alone is neurotoxic and the toxicity is inhibited by acombination of ionotropic NMDA and non-NMDA receptorantagonists suggesting that the damage is excitotoxicityFurthermore TBOA-induced neuronal damage is inhibitedby the glutamate release inhibitor riluzole suggesting that the

damage is mediated by endogenous glutamate rather thanresulted from a direct action of TBOAThese results stronglysuggest that the neurotoxic actions of TBOA result fromblocking glutamate transport and pathological conditionsor treatments that impair glial glutamate transport greatlyaugment the toxic effects of endogenous glutamate

Recently antisense knockout techniques or the con-struction of transgenic and gene knockout mouse modelsrepresent the successful approach to achieving suppressionor elimination of a genetic message of specific glutamatetransporters In this section we describe the characterizationof these three glutamate transporter precisely based on theexperimental results mainly obtained by gene engineering

211 GLAST GLAST is the prominent glutamate transporterin the retina andmainly expressed in theMuller cells [1 8 21ndash24] In the mouse Muller cells glutamate removal by GLASTis calculated as 50 among the retinal glutamate trans-porters [25] Glutamate uptake via GLAST is accompaniedby cotransport of three Na+ and one H+ and the counter-transport of one K+ at each glutamate transport Sodiumions are extruded at least in part by Na+K+-adenosinetriphosphate (ATP) ase in a reaction that consumes ATP

To elucidate the role of GLAST in the regulation of retinalfunctionHarada et al [26] developedGLAST-deficientmiceand revealed reduction of the scotopic ERG b-wave indicat-ing the reduction of glutamate-mediated neurotransmissionactivity in the retina After induction of the retinal ischemiaby increasing intraocular pressure above systolic pressurefor 60min more severe excitotoxic degeneration is foundin GLAST-deficient mice than in wild-type indicating thatGLAST is neuroprotective against ischemia [27]

Barnett and Pow [28] injected intravitreally antisenseoligonucleotides to GLAST into rat eyes Although a markedreduction of GLAST activity was detected the retinas dis-played no evidence of excitotoxic neuronal degeneration andthe distribution of glutamate was unaffected by antisensetreatment Significant inhibition in GLAST function wasapparent 5 days after injection of antisense oligonucleotideand was sustained for at least 20 days The observed lackof neuronal degeneration suggests that reduced glutamateuptake into the Muller cells does not cause excitotoxic tissuedamage

HoweverHarada et al [29] examined the long-term effect(32 weeks) of GLAST deficiency on the retinal morphologyduring postnatal development in vivo and revealed spon-taneous loss of retinal ganglion cell (RGC) and optic nervedegeneration without elevated intraocular pressure (IOP) InGLAST-deficient mice administration of glutamate receptorantagonist prevented RGC loss These findings suggest thatGLAST is necessary to prevent retinal excitotoxicity

Taken together the glutamate removal by glutamatetransportes is prerequisite for the maintenance of normalretinal transmission and preventive against excitotoxicity

212 GLT-1 Unlike in the central nervous system [30] it isconsidered thatGLT-1 does not play a predominant role in thephysiological glutamate transmission in the retina because

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antisense GLT-1-knockout mice exhibit almost normal reti-nal function [24 30] However other researchers reportedthat treatment with antisense oligonucleotides against GLT-1 increased vitreal glutamate levels leading to ganglion celldeath in the rat retina [31] and the retinal damage inducedby ischemia was exacerbated in GLT-1 deficient mice [26]Harada et al [29] tried to examine the long-term effect ofGLT-1 deficiency on the retinal morphology during postnataldevelopment in vivo However almost all of GLT-1minusminusmicedied within 3 weeks There seems to be no difference in RGCnumber between GLT-1+minusmice and wild-type mice

Despite these contradictory findings the involvement ofGLT-1 of the retina in the homeostasis of glutamate cannot beexcluded

213 Splice Variants of Glutamate Transporters Splice vari-ant is a result of alternative splicing of pre-mRNA wherethe exons are reattached in a different manner to producedifferent mRNAs [32] Alternative splicing occurs in 95 ofmultiexonic genes [33] Splice variants are translated fromalternatively splicedmRNAs that contain diversities in aminoacid sequence or biological properties

(a) GLAST The presence of three types of splice variantsof GLAST (GLAST1a GLAST1b and GLAST1c) has beenreported GLAST1a and GLAST1b lack exon 3 and 9 respec-tively [34 35] GLAST1a preferentially located in the endfeetof the Muller cells The localization of GLAST1a is differentfrom normally expressed GLAST which is entirely expressedin the Muller cell body [34] However the clarification ofthe precise role of GLAST1a in the retina is still remainedGLAST1b is expressed at low levels in neurons in the normalbrain while expression levels rise dramatically in neuronsafter a hypoxic insult indicating that expression is regulatedto maintain the homeostasis of the glutamate concentration[36] GLAST1c lacks both exon 5 and 6 and coded fora 430 amino acid protein [37] GLAST1c is present inmultiple species and is widely expressed by astroglial cellsand oligodendrocytes in the brains of various mammalianspecies as well as in the optic nerve and the retinas of humanSimilarities between GLAST1c and a functional prototypictransporter may support the speculation that GLAST1c is afunctional glutamate transporter and possibly represents aprimitive form of GLAST However the precise localizationof GLAST1c in the retina has not been identified

(b) GLT-1 GLT-1 (EAAT2) exists in several distinct formsincluding the originally described form (GLT-1a) along withGLT-1b (also called GLT1v) and GLT1c [38] GLT-1a appearsto be associated mainly with a population of amacrine cellswhereas GLT-1b is associated with cone photoreceptorssubpopulations of bipolar cells and astrocytes [39 40] GLT-1c is normally only expressed by the photoreceptors in themammalian retina [38] In the rat GLT1c is colocalized withGLT1b in cone photoreceptors GLT1c expression is develop-mentally regulated only appearing at around postnatal day7 in the rat retina when photoreceptors first exhibit a darkcurrent [41] In the normal eyes of humans and rats GLT-1c was expressed only in photoreceptors In glaucoma there

was an apparent increase in expression of GLT-1c in retinalganglion cells including occasional displaced ganglion cellsAlthough the precise role of GLT-1c still remains to beelucidated upregulation of GLT-1c might be an attempt toadapt to the pathological condition such as glaucoma

Based on these findings transcriptional regulation andmRNA splicing causing differential expression of GLAST orGLT-1 may affect glutamate transport and may provide ancomplicated mechanism for glutamate uptake

(c) EAAC-1 EAAT3 is also known as excitatory aminoacid carrier 1 (EAAC-1) EAAC-1 localizes in the synapticlayers (the outer and inner plexiform layers) horizontal cellssubgroups of amacrine cell and the ganglion cell In additionimmunoreactivity reveals the presence of EAAC-1-positiveamacrine cells distant from the synaptic sites Consideringthe location EAAC-1 may regulate glutamate uptake indifferentmanner both near and well away from synaptic sitesHowever knockdownof the expression ofGLASTorGLT-1 inrats using antisense oligonucleotides increased the extracel-lular glutamate concentration whereas EAAC-1 knockdownmice showed no increase in extracellular glutamate [42]These findings indicate that glutamate uptake is not a majorrole of EAAC1 EAAC-1 can transport cysteine significantlyhigher than GLAST or GLT-1 [43] and contribute to generateglutathione Partial knock-down of EAAC-1 decreases theneuronal glutathione contents and increases oxidant levels[44]These results indicate that EAAC-1 is responsible for themetabolism of glutathione which plays a critical role as anantioxidant

22 Glutamate Degradation by Glutamine Synthetase Glu-tamine synthetase is the only enzyme to synthesize glutamineand plays an important role in glutamate detoxification [45]Glutamine synthetase seems critical for the cell survivalbecause the deficiency in the glutamine synthetase geneinduces early embryonic death [46]

In the retina glutamine synthetase is specifically localizedin the Muller cells [47] and catalyzes the ATP-dependentcondensation of glutamate [48] In salamander retina glu-tamine synthesis stimulates glial glycolysis to match energydemands [49] Additionally glutamine synthetase activity ismost prominent in the presence of high levels of ammoniaand is more limited in the presence of low levels of ammonia[49] Immunohistochemistry revealed that inhibition of glu-tamine synthetase by dl-methioninel-sulfoximine (MSO)caused a dramatic increase in the glutamate level in theMuller cells and subsequently the rapid loss of the glutamatecontent of the photoreceptor cells bipolar cells and ganglioncells [8] Barnett et al [50] injected MSO intraocularly inWistar rats and revealed prompt suppression of the sco-topic ERG b-wave indicating the reduction of glutamatergicneurotransmission activity in the retina These results alsoindicate that inhibition of glutamine synthetase may rapidlyimpair the retinal response to light

23 Importance of Energy Supply in Glutamate MetabolismGlutamate uptake can be influenced by changes in cellular

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energy levels [51 52] ATP is the usable form of chem-ical energy for the central nervous system including theretina ATP is provided by two different sources oxidativemetabolism and glycolysis The generation of ATP throughoxidative metabolism (oxidative ATP) is selectively blockedby oxygen deprivation The generation of ATP through gly-colysis (glycolytic ATP) is not blocked by glucose deprivationas long as glycogen stores are available for use Iodoacetate(IA) inhibits both oxidative and glycolytic ATP generation

Oxygen deprivation produces little morphologicalchange and does not induce glutamate-induced excit-otoxicity In contrast inhibition of glycolysis by IA producedsevere neuronal damage The neuronal damage producedby IA was inhibited by pyruvate a substrate that sustainsoxidative energy pathways In the presence of IA pluspyruvate glutamate became neurotoxic at low concentrationsthrough activation of non-NMDA receptors [53] Theseresults indicate that glycolytic energy metabolism plays acritical role in sustaining ionic balances that one requiredfor Muller cell glutamate uptake and glial uptake helps toprevent glutamate-mediated excitotoxicity

Even in the presence of normal energy metabolism how-ever ATP levels will be depressed when ATP consumptionexceeds production Thus excessive neuronal activity andenergy demands may acutely reduce ATP levels Energyfailure increases vulnerabilities of retinal neurons to excito-toxicity because the critical steps in glutamate metabolismare ATP-dependent In this section we give an overview ofthe relations between the energy supply and the glutamatemetabolism

231 Glutamate Transporters Although glutamate uptake byglutamate transporters does not require ATP consumptionGlutamate transporte is the Na+-dependent glutamate trans-porters which utilize ionic gradients of Na+ K+ and H+ todrive glutamate transport against the concentration gradientSince the ionic gradients aremaintained by the sodiumpumpthey are dependent upon ATP production When ATP levelsdrop increased extracellular K+ and reduced extracellularNa+ result in a neurotoxic release of glutamate and areattributed to reversed operation of glutamate transporters[54] Reversal of the glutamate transporter is considered tofacilitate the increase of extracellular glutamate concentrationto excitotoxic levels These findings also indicate that theglutamate transporter may transport glutamate in eitherdirection depending on the ionic gradient across the plasmamembrane

232 Glutamine Synthetase Degradation of glutamate byglutamine synthetase also consumes ATP molecule as in thefollowing reaction

Glutamate + NH4+ + ATP

997888rarr glutamine + ADP + Pi +H+(1)

in the presence of manganese or magnesium Thereforethe suppression of intracellular ATP causes a decrease inglutamine synthetase activity

GLAST and glutamine synthetase are the primary roleplayers that transport glutamate into theMuller cell and con-vert it into glutamine Jablonski et al [55] reported the geneticregulation of both genes Slc1a3 and Glul using an array of 75recombinant inbred strains of mice Slc1a3 and Glul encodeGLAST and glutamine synthetase respectively Interestinglydespite their independent regulation gene ontology analysisof tightly correlated genes reveals that the enriched andstatistically significant molecular function categories of bothdirected acyclic graphs have substantial overlap indicatingthat the shared functions of the correlates of Slc1a3 andGlul include production and usage of adenosine triphosphate(ATP) These results indicate that ATP depletion may induceexcitotoxicity via downreguration of Slc1a3 and Glul Consis-tently it has been reported that energy deprivation decreasesglutamate uptake within 2-3min [56]

In addition to the suppression on the glutamate trans-porters and glutamine synthetase ATPdepletion also inducesthe failures in membranous Na+K+ pump maintaining theresting membrane potential or in Ca2+ extrusion Intra-cellular calcium increase leads to mitochondrial impair-ment further accelerating ATP depletion [57] RecentlyNguyen et al propose a vicious cycle involving excitotoxicityoxidative stress and mitochondrial dynamics Oxidativestress produced by mitochondrial impairment leads to theupregulation of the NMDA receptors and exaggerates theexcitotoxicity [58]

3 Abnormalities of Glutamate Metabolism

Excitotoxicity has been considered to be involved in severalocular pathologies including ischemia induced by retinal orchoroidal vessel occlusion glaucoma and diabetic retinopa-thy [1ndash7] Inhibition of retinal degeneration afforded byadministration with glutamate receptor antagonists supportsthis hypothesis [1 3 8 9 59 60]

Excitotoxic cell death does not always result from excessof glutamate Extracellular levels of glutamate achieved dur-ing retinal ischemia [61ndash63] may not be sufficient to induceneuronal damage under normal conditions [64] This sug-gests that clearance of glutamate is important in preventingretinal excitotoxicity in response to glutamate In support ofthis it has been shown that retinas of GLAST deficiencymiceare extremely sensitive to the ischemic insults [26]

In this section we describe the abnormalities of glutamatemechanisms in retinal ischemia glaucoma and diabeticretinopathy respectively

31 Retinal Ischemia In general ischemia means the patho-logical conditions with a restriction in blood flow causing aninadequate supply of oxygen and glucose needed for cellularmetabolism In consequence the prolonged retinal ischemiainduces irreversible morphological and functional changesFor example the experimental obstruction of the centralretinal artery of old atherosclerotic hypertensive rhesusmonkeys induced no remarkable changes within 97minwhile the longer the arterial obstruction the more extensivethe retinal damage [65]

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Acute retinal ischemia induces irreversible damages asthe consequence of ATP depletion [66ndash68] ATP depletionlowers the function of Na+-K+-ATP pump resulting in influxof Na+ Clminus and water which can cause hypo-osmoticswelling of cell organelles and their dysfunction [69] Reper-fusion after ischemia has been known to induce more exag-gerated retinal degeneration The cessation of oxygen andnutrients supply during ischemia increases the susceptibilityof the retina for inflammation [70] or the oxidative stressinduced by the restoration of blood circulation Excitotoxicityis considered as one of the essential elements to trigger theabove-mentioned ischemiareperfusion degeneration [71]

311 Glutamate Transporters In the retinal ischemia theexpressional and functional changes of glutamate trans-porters have been reported Barnett et al [72] have reportedthat GLAST functions are reduced after retinal ischemiawas induced by the central retinal artery occlusion for60min while the GLAST expression appears to be normalDecrease in glutamate uptake by GLAST may induce diffu-sion of glutamate into vitreous cavity or anterior chambersIn consistence it is reported that glutamate concentrationsignificantly increased in aqueous humor sample obtainedfrom patients with retinal artery obstruction [73]

The ischemiareperfusion model revealed a markedincrease in GLAST mRNA expression in the inner retina[74] By contrast Barnett and Grozdanic [75] reported thatthe GLAST function recovers rapidly upon reperfusion andsuggest that the loss of transporter function might be dueto acute metabolic changes induced by the ischemia [76ndash78] rather than rapid downregulation of transporter geneexpression

Recently Russo et al [79] evaluated the expression ofGLAST and GLT-1 in a rat ischemiareperfusion modelThey reported that there were no remarkable changes inGLAST expression while modulation of GLT-1 expressionwas observed in the isolated retinal synaptosomes Theseresults support a role for GLT-1 in glutamate accumulationobserved in the retina following an ischemic event

Despite of discrepancies these findings suggest thatglutamate transporters play an important role in the regula-tion of extracellular glutamate concentration under ischemicconditions

312 Glutamine Synthetase As glutamine synthetase con-sumes ATP to convert glutamate to glutamine [80] it seemsplausible that ATP suppression in the ischemic retina [21]promptly induces the impairment of delegation of glutamateresulting in the elevation of the extracellular concentrationof glutamate However glutamine synthetase activity wasrelatively well preserved during 60 minutes of simulatedacute retinal ischemia induced by deprivation of both oxygenand glucose using ex vivo rat retinal preparation [21] Suchresults indicate that the retina can efficiently generate ATPfrom glycolysis despite of the deprivation of oxygen andglucose [81] In ischemiareperfusion rat eyes it actually takes

several days to induce a significant depression of glutaminesynthetase after the onset of the retinal ischemia [82ndash84]

The response of the retina to a postischemic reperfusionphase seems to depend upon the intensity of the ischemicstress The other researcher reported that the expression inglutamine synthetase inMuller cells increased at 6 hours afterischemia reached its peak at 24 hours and decreased on day14 compared with normal level in the ischemiareperfusionmodel [85]

It is considered that glutamine synthetase activity maybe upregulated for a short period after onset of ischemia toprotect retinal neurons against excitotoxicity As the rates ofglycolysis decrease according to the depletion of glycogenstorage an intracellular ATP level falls causing inhibition ofglutamine synthetase In addition it is plausible that ischemiainduced upregulation of protein kinase C delta resulting inthe downregulation of glutamine synthetase [86]

32 Glaucoma Glaucoma is characterized by progressiveand accelerated loss of retinal ganglion cells and theiraxons The prominent pathological finding in glaucoma isthe apoptotic cell death of the RGC [87] However thepathogenesis of apoptotic RGC death in glaucoma has notbeen clarified It is hypothesized that glutamate-mediatedexcitotoxicity may contribute to pathogenesis of glaucoma[88] Although an elevation of glutamate concentration inthe vitreous humor was reported [89 90] the followingstudies could not reproduce these results [91ndash94] Howeveran elevation in the glutamate level in the vitreous humor isnot necessary to induce excitotoxicity in the experimentalanimals or humans with glaucoma [95 96] It is becauseglutamate increase is likely to occur only in localized areas ofthe retina or optic nerve at any one time during glaucomatousneurodegeneration If this is true abnormalities in the gluta-matemetabolism result in excitotoxic damage to theRGCandcontribute to the pathophysiology of glaucoma

321 Glutamate Transporters Glutamate transporters arecritical for maintaining optimal extracellular concentrationsof glutamate [4 5 59 97 98] Since glutamate transport isthe only mechanism for removing glutamate from the extra-cellular fluid it is hypothesized that functional impairment ofglutamate transportersmay play amajor role in excitotoxicityand contribute to the pathogenesis of glaucoma [99 100]Changes of GLAST GLT-1 and its splice variant and ECCA-1have been reported in glaucomaGLAST GLAST is themajor glutamate transporter expressedin retinal Muller cells [48] However the expressionalchanges of GLAST in glaucoma are still controversial Somestudies have shown that GLAST expression diminishes [99ndash101] or remains stable [102] in experimental glaucomawhereas others have reported an increased expression [103]In this section I will overview the role of GLAST in thepressure-dependent and the pressure-independent glaucomamodels

(a) Pressure-Dependent Glaucomatous Changes with GLASTIt is generally recognized that elevated intraocular pressure

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Bubble

Bubble

Bubble

Manometer

Eyecup preparations

Valve

10(mmHg)

75

Buffer columnheight

135 cm

1012 cm

Gas darr

darr Antagonist

Figure 2 Outline of the present experiments Eyecups preparations were sunken to the bottom of a glass cylinder with different heights Eachglass cylinder was filled with incubation buffer at 30∘C for 24 hoursThe buffer was bubbled with 95O

2-5 CO

2Hydrostatic pressure at the

bottom of the cylinder was calculated to be 10mmHg and 75mmHg when a CSF was added to a height of 135 cm and 1012 cm respectivelyGlutamate receptor antagonists or agonists were added to the buffer during some experiments

is the most significant risk factor for accelerated ganglioncell death in glaucoma However the mechanism of cellulardamage caused by elevated IOP is still unknown

It has been reported that GLAST expression is reducedin experimental glaucoma models of rat [100] and mouse[101] as well as in glaucoma patients [99] By contrastthere is another report that GLAST expression increasedtime-dependently in a rat glaucoma model [103] Thesediscrepancies might be mainly caused by the differences inthe antibodies and glaucoma model

We recently developed a rat ex vivo hydropressure model(Figure 2) to examine the expressional changes of GLASTinduced by elevated pressure (75mmHg) for 24 hours [104]Such acute high pressures can induce retinal ischemia clini-cally and in in vivo glaucoma models [105ndash107] The ex vivohydrostatic pressure model takes advantages to exclude theeffects of ischemia and could investigate direct the effectsof pressure-induced retinal injury on glutamate metabolism(Figure 3) In this acute model Western blot and real-timeRT-PCR analyses revealed that 75mmHg pressure inhibitedGLAST expression [104]

(b) Pressure-Independent Glaucomatous Changes withGLAST The population-based study revealed that thenormal-tension glaucoma is the most prevalent form ofglaucoma in Japan Moreover in some glaucoma patientssignificant IOP reduction does not prevent the progressionof the disease Harada et al [29] show that GLAST deficientmice demonstrate spontaneous ganglion cell death and opticnerve degenerationwithout elevated IOP InGLAST-deficientmice administration of glutamate receptor blocker preventedRGC loss indicating that GLAST is necessary to preventexcitotoxic retinal damage Additionally GLAST maintainsthe glutathione levels in Muller cells by transportingglutamate the substrate for glutathione synthesis into thecells Glutathione has strong antioxidative properties Takentogether GLAST deficiency leads to RGC degenerationcaused by both excitotoxicity and oxidative stress

GLT-1 GLT-1 is one of the major glutamate transportersalong with GLAST and is found only in cones and varioustypes of bipolar cells The characteristic localization of GLT-1 on bipolar cells in the vicinity of the ganglion cell implies

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ILM

IPL

INL

OPL

ONL

10mmHg 75mmHg(a) (b)

(c)

(d)

Figure 3 Immunofluorescent localization of glial fibrillary acidicprotein by confocal fluorescent microscopy (a) GFAP expression isrecognized as FITC fluorescence and localized in the Muller cellsendfeet of (arrow) in a normal retina (10mmHg) (b) Apparentfluorescent reaction is recognized in the Muller cell body (arrow-heads) and the Muller cells endfeet (arrow) at 75mmHg (c) and(d) Tangential view of the GFAP expression at the level of theinner nuclear layer (c) and the outer nuclear layer (d) GFAP-stained Muller cell bodies were recognized as (rhodamine-stained)reddish dots among the DAPI- (410158406-diamidino-2-phenylindoledihydrochloride) stained nuclei Note the obliquely running Mullercell process (arrows) Figures (a) to (d) are in the same magnifica-tion Figures (a) to (d) are in the same magnification Bar = 20 120583m

that GLT-1 may regulate glutamate concentration aroundganglion cell synapse [100] It is because the specific subtypeof ganglion cells is known to degenerate during glaucoma[108]

GLT-1 is reported to be down-regulated in glaucomatouseyes in rats [100] and mice [101] However Park et al [102]reported that GLT-1 was expressed in cone photoreceptorsand some cone bipolar cells and the levels of expressionwere significantly increased in in vivo rat glaucoma modelIn contrast GLAST expression which occurred in Mullercells the main retinal glial cells remained stable during theexperimental period These results suggest that integrity ofGLT-1may be a prerequisite for themaintenance of glutamatehomeostasis in the retina undergoing glaucoma [109]

Additionally one of the splice variants of GLT-1 GLT-1cshowed clear response against the IOP elevation In normaleyes of humans and rats GLT-1c was expressed only inphotoreceptors In glaucoma there was additional prefer-ential expression of GLT-1c in retinal ganglion cells [36]The induction of GLT-1c expression by retinal ganglion cellsmay indicate that the perturbation in glutamate homeostasisis evident in glaucoma and that such anomalies selectivelyinfluence retinal ganglion cells These results suggest thatthe expression of GLT-1c may represent an attempt byretinal ganglion cells to protect themselves against elevatedlevels of glutamate GLT-1c may be a useful indicator ofthe extent of stress of the retinal ganglion cells and thus atool for examining outcomes of potential therapeutic andexperimental interventions

EAAC-1 Harada et al [29] utilized EAAC1-deficient miceand examined the long-term effect of retinal morphologyduring postnatal development on RGC survival in vivo Theyfound that EAAC-1-knockout mice showed spontaneouslyoccurring RGC death and typical glaucomatous damage ofthe optic nerve without elevated IOP The main role ofEAAC-1 is to transport cysteine into RGCs as a precursor forneuronal glutathione synthesis [47 110ndash112] Thus EAAC-1deficiency induces RGC loss mainly through oxidative stress

322 Glutamine Synthetase It remains controversial wheth-er pressure elevation changes the expression and activities ofglutamine synthetase Although increases in the expression ofglutamine synthetase were reported after pressure elevation[21 113 114] decreases in activity and expression of glutaminesynthetase have been also reported [115 116] Shen et al [113]reported that high concentration of vitreal glutamate inducedupregulation of glutamine synthetase Such upregulation wasblocked if IOP was acutely elevated for 24 hours but wasrestored if IOP remained elevated for 1 week

These findings suggest that moderate elevation of IOPcauses only short-term functional changes of glutamatemetabolism by retinal Muller cells However it is not knownto what extent endogenous extracellular glutamate can regu-late glutamine synthetase expression in normal eyes or in eyeswith glaucoma

In primary glaucoma in dogs [116] it is reported thatdecreases in glutamine synthetase immunoreactivity wereassociatedwith the damaged regions of the retinaThese find-ings may indicate that the decrease in glutamine synthetasepotentiates ischemia-induced early glutamate redistributionand neuronal damage in canine primary glaucoma

Ishikawa et al examined the enzyme activity [104] andexpressional changes of GLAST [117] induced by elevatedpressure (75mmHg) for 24 hours using a rat ex vivohydropressure model In this acute model elevated pressuresuppresses activity and expression of glutamine synthetaseIn addition it is revealed that depressed GLAST expressionresults in downregulation of glutamine synthetase activityThese results may indicate that during pressure-loadingimpairment of GLAST takes place first and results in down-regulation of glutamine synthetase activity as a second effect

323 Energy Deprivation It has been reported that theRGC loses the retrograde axonal transport by mechanicalcauses resulting in apoptotic or necrotic cell death in theexperimental glaucoma model induced by perilimbal andepiscleral vein photocauterization [118] Axonal transportis the prominent energy-consuming process to transportcell organelles neurotrophic factors and other substances[119] Ju et al [120] reported that IOP elevation directlydamaged mitochondria in the optic nerve head axons inthe glaucomatous DBA2J mice Mitochondrial impairmentinduces cellular ATP reduction resulting in disturbance ofaxonal transport and influence the viability of the retinalganglion cells via retrograde axonal degeneration [121 122]Excitotoxicity is also considered to be closely associated with

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ATP-depletion induced by mitochondrial dysfunction [123ndash128] These results indicate the important features about thepotential risk of energy deprivation in the glaucoma

33 Diabetic Retinopathy Diabetic retinopathy is the mostcommon complication of the diabetes and a leading cause ofvisual impairment during the working-age in the industrial-ized countries [129] Diabetic retinopathy has been classicallyconsidered as a microcirculatory disease of the retina How-ever it has been recently reported that retinal degenerationprecedes the impairment of the microcirculation In theearly stage of the diabetic retinopathy elevated levels ofglutamate oxidative stress the overexpression of the renin-angiotensin system and the upregulation of receptor foradvanced glycation end-products play an essential role [130]These results mean that main features of retinal degenerationare already present in the retinas of diabetes without anymicrocirculatory abnormalities In the later stage retinalexcitotoxicity participates in vascular endothelial growthfactor (VEGF-) inducedmicrocirculatory abnormalities suchas disruption of blood-retinal barrier or an increase in thevascular permeability

The relationship between the excitotoxicity and theinduction of VEGF is one of the most interesting path-ways linking neurodegeneration with vascular impairmentNMDA receptors exert a tonic inhibition of VEGF secretionin cultures of rat purified Muller cells thus indicating that inhealthy retina glutamatergic stimulation could have a pro-tective role [131] In addition it has been demonstrated thatthe elevation of VEGF expression and blood-retinal barrier(BRB) breakdown in streptozotocine-induced diabetic ratsare blocked by the NMDA receptor channel blocker and theuncompetitive antagonist memantine [132]

These results suggest that hyperglycemia induces anincrease in extracellular glutamate and the subsequent over-activation of NMDA receptors mediates VEGF productionBRB breakdown and RGC damage observed in diabeticretinopathy In this regard it has recently been reportedthat the attenuation of retinal NMDA receptor activity bybrimonidine (an alpha-2 adrenergic receptor agonist) resultsin amarkeddecrease in vitreoretinalVEGF and the inhibitionof BRB breakdown in diabetic rats [132]

331 Glutamate Transporters Increased concentration ofglutamate in vitreous body of diabetic retinopathy patientshas been reported [133] Early in the course of diabeticretinopathy the function of the glutamate transporter inMuller cells is reported to decrease by a mechanism that islikely to involve oxidation [134]

As the activity of glutamate transporter can be rapidlyrestored it seems possible that targeting this molecule fortherapeutic intervention may restore glutamate homeostasisand ameliorate sight-threatening complications of diabeticretinopathy [135]

Recently Lau et al [136] reported decreases in the tran-script levels of genes related to glutamate neurotransmissionand transport as diabetes progresses in the rat retinaDiabetescaused significant decrease in the transcriptional expression

of glutamate transporter SLC1A3 gene encoding GLASTprotein leading to the decreased removal of glutamate fromthe extracellular space suggesting that diabetes impairs theglutamate transporter function of Muller cells Consistentlyfaint GLAST immunoreactivity was restricted to the innerretina with the diabetic retinopathy compared with thethroughout staining of the normal retina [137]

332 Glutamine Synthetase In diabetic rats an increase inglutamine synthetase and a decrease in glutamate transporterare reported [138]SeriesAnalysis ofGene Expression (SAGE)analysis of the diabetic retina revealed a 456 reduction intranscript levels of glutamine synthetase in streptozotocin-induced diabetic rats compared with normal rats [139] RT-PCR and colorimetric enzyme activity assays revealed sig-nificant decreases in glutamine synthetase mRNA expressionand the enzyme activity as early as the first month ofdiabetes development with a progressive decrease in GSmRNA level and enzyme activity over a 12-month periodNorthern blot analysis indicated a linear correlation betweenthe reduction in glutamate synthetase expression and thetime course of diabetic retinopathy which was validatedby real-time RT-PCR These results implicate glutaminesynthetase as a possible biomarker for evaluating the severityof developed diabetic retinopathy over the time course ofdiabetes progression Immunohistochemistry revealed thatantiglutamine synthetase labeling was prominent in the outerand inner plexiform layer as well as the ganglion cell layer inthe normal control while the signal was diminished in thediabetic retina compared to control retina [137] By contrastSilva et al reported that Muller cells exposed to high-glucosemedium produced higher levels of glutamine synthetase butreduced levels of glutamate transporter [138]

4 Concluding Comments

Glutamate uptake and its enzymatic degradation are thecritical steps to maintain the homeostasis of extracellularglutamate concentration in the retina Down-regulation ofthese process induces excitotoxic neuronal degeneration inretinal diseases such as ischemia glaucoma and diabeticretinopathy The abnormalities in glutamate metabolism arealso caused by energy failure due to mitochondrial dysfunc-tion As glutamine synthetase consumes ATP to convert glu-tamate to glutamine ATP depletion induces the impairmentof delegation of glutamate resulting in the elevation of theextracellular concentration of glutamate Although glutamatetransporters do not require ATP consumption they are theNa+-dependent glutamate transporters which utilize ionicgradients of Na+ K+ and H+ to drive glutamate transportagainst the concentration gradient Since the ionic gradientsare maintained by the sodium pump they are dependentupon ATP production When ATP levels drop both gluta-mate uptake and degradation are remarkably inhibited andthe risk to induce excitotoxicity significantly increases

Retinal ischemia induces irreversible excitotoxic degen-eration as the consequence of ATP depletion Reperfusionafter ischemia induces exaggerated retinal degeneration

Scientifica 9

In ischemiareperfusion model GLAST function decreasesin the early phase followed by prompt functional recoveryGlutamine synthetase activity is upregulated for a shortperiod after onset of ischemia to protect retinal neuronsagainst excitotoxicity As the rates of glycolysis decreaseaccording to the depletion of glycogen storage glutaminesynthetase activity decreases In the experimental glaucomamodel elevated IOP suppresses GLAST expression first andresults in the downregulation of glutamine synthetase activityas a second effect IOP elevation also damagedmitochondriainducing cellular ATP depletion resulting in ganglion celldeath In the diabetic rat the decrease in the expression ofglutamate transporters and glutamine synthetase is reportedHyperglycemia also induces an glutamate excess and thesubsequent overactivation of NMDA receptors mediatesVEGF production and RGC damage

These results indicate the importance of maintainingthe homeostasis of glutamate metabolism to prevent theexcitotoxicity in retinal diseases such as the retinal ischemiaglaucoma and diabetic retinopathy

Acknowledgments

The author thanks Professor Takeshi Yoshitomi (Departmentof Ophthalmology Akita Graduate University School ofMedicine) and Professor Yukitoshi Izumi (Department ofPsychiatry Washington University School of Medicine) fortheir instructions and suggestions This work was supportedby JSPS KAKENHI Grant no 24592666 to MI

References

[1] W B Thoreson and P Witkovsky ldquoGlutamate receptors andcircuits in the vertebrate retinardquo Progress in Retinal and EyeResearch vol 18 no 6 pp 765ndash810 1999

[2] S C Massey and R F Miller ldquoExcitatory amino acid receptorsof rod- and cone-driven horizontal cells in the rabbit retinardquoJournal of Neurophysiology vol 57 no 3 pp 645ndash659 1987

[3] S C Massey and R F Miller ldquoN-Methyl-D-aspartate receptorsof ganglion cells in rabbit retinardquo Journal of Neurophysiologyvol 63 no 1 pp 16ndash30 1990

[4] D R Lucas and J P Newhouse ldquoThe toxic effect of sodiuml-glutamate on the inner layers of the retinardquo Archives ofOphthalmol vol 58 no 2 pp 193ndash201 1957

[5] J W Olney ldquoThe toxic effects of glutamate and related com-pounds in the retina and the brainrdquo Retina vol 2 no 4 pp341ndash359 1982

[6] D R Sisk and T Kuwabara ldquoHistologic changes in theinner retina of albino rats following intravitreal injection ofmonosodium L-glutamaterdquo Graefersquos Archive for Clinical andExperimental Ophthalmology vol 223 no 5 pp 250ndash258 1985

[7] C K Vorwerk S A Lipton D Zurakowski B T Hyman BA Sabel and E B Dreyer ldquoChronic low-dose glutamate istoxic to retinal ganglion cells toxicity blocked by memantinerdquoInvestigativeOphthalmology andVisual Science vol 37 no 8 pp1618ndash1624 1996

[8] A Derouiche and T Rauen ldquoCoincidence of L-glutamateL-aspartate transporter (GLAST) and glutamine synthetase (GS)immunoreactions in retinal glia evidence for coupling of

GLAST and GS in transmitter clearancerdquo Journal of Neuro-science Research vol 42 no 1 pp 131ndash143 1995

[9] T Rauen andMWieszligner ldquoFine tuning of glutamate uptake anddegradation in glial cells common transcriptional regulation ofGLAST1 and GSrdquoNeurochemistry International vol 37 no 2-3pp 179ndash189 2000

[10] Y Izumi K Shimamoto A M Benz S B Hammerman J WOlney and C F Zorumski ldquoGlutamate transporters and retinalexcitotoxicityrdquo Glia vol 39 no 1 pp 58ndash68 2002

[11] T Storck S Schulte K Hofmann and W Stoffel ldquoStructureexpression and functional analysis of a Na+-dependent gluta-mateaspartate transporter from rat brainrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 89 no 22 pp 10955ndash10959 1992

[12] G Pines Y Zhang and B I Kanner ldquoGlutamate 404 is involvedin the substrate discrimination of GLT-1 a (Na+ + K+)-coupledglutamate transporter from rat brainrdquo Journal of BiologicalChemistry vol 270 no 29 pp 17093ndash17097 1995

[13] Y Kanai and M A Hediger ldquoPrimary structure and func-tional characterization of a high-affinity glutamate transporterrdquoNature vol 360 no 6403 pp 467ndash471 1992

[14] W A Fairman R J Vandenberg J L Arriza M P Kavanaughand S G Amara ldquoAn excitatory amino-acid transporter withproperties of a ligand-gated chloride channelrdquo Nature vol 375no 6532 pp 599ndash603 1995

[15] J L Arriza S Eliasof M P Kavanaugh and S G Amara ldquoExci-tatory amino acid transporter 5 a retinal glutamate transportercoupled to a chloride conductancerdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 94 no8 pp 4155ndash4160 1997

[16] A Bringmann T Pannicke J Grosche et al ldquoMuller cells inthe healthy and diseased retinardquo Progress in Retinal and EyeResearch vol 25 no 4 pp 397ndash424 2006

[17] Y Izumi C O Kirby-Sharkey A M Benz et al ldquoSwelling ofMuller cells induced by AP3 and glutamate transport substratesin rat retinardquo Glia vol 17 no 4 pp 285ndash293 1996

[18] K Shimamoto B LeBrun Y Yasuda-Kamatani et al ldquoDL-threo-120573-benzyloxyaspartate a potent blocker of excitatoryamino acid transportersrdquo Molecular Pharmacology vol 53 no2 pp 195ndash201 1998

[19] K Shimamoto Y Shigeri Y Yasuda-Kamatani B LebrunN Yumoto and T Nakajima ldquoSyntheses of optically pure 120573-hydroxyaspartate derivatives as glutamate transporter block-ersrdquo Bioorganic and Medicinal Chemistry Letters vol 10 no 21pp 2407ndash2410 2000

[20] Y Shigeri K Shimamoto Y Yasuda-Kamatani et al ldquoEffects ofthreo-120573-hydroxyaspartate derivatives on excitatory amino acidtransporters (EAAT4 and EAAT5)rdquo Journal of Neurochemistryvol 79 no 2 pp 297ndash302 2001

[21] Y Izumi S B Hammerman C O Kirby A M Benz JW Olney and C F Zorumski ldquoInvolvement of glutamate inischemic neurodegeneration in isolated retinardquo Visual Neuro-science vol 20 no 2 pp 97ndash107 2003

[22] Y Otori S Shimada K Tanaka I Ishimoto Y Tano and MTohyama ldquoMarked increase in glutamate-aspartate transporter(GLASTGluT-1) mRNA following transient retinal ischemiardquoMolecular Brain Research vol 27 no 2 pp 310ndash314 1994

[23] T Rauen J D Rothstein and H Wassle ldquoDifferential expres-sion of three glutamate transporter subtypes in the rat retinardquoCell and Tissue Research vol 286 no 3 pp 325ndash336 1996

10 Scientifica

[24] K P Lehre S Davanger and N C Danbolt ldquoLocalization ofthe glutamate transporter protein GLAST in rat retinardquo BrainResearch vol 744 no 1 pp 129ndash137 1997

[25] V P Sarthy L Pignataro T Pannicke et al ldquoGlutamate trans-port by retinal Muller cells in glutamateaspartate transporter-knockout micerdquo Glia vol 49 no 2 pp 184ndash196 2005

[26] T Harada C Harada M Watanabe et al ldquoFunctions of thetwo glutamate transporters GLAST and GLT-1 in the retinardquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 95 no 8 pp 4663ndash4666 1998

[27] J D RothsteinM Dykes-Hoberg C A Pardo et al ldquoKnockoutof glutamate transporters reveals a major role for astroglialtransport in excitotoxicity and clearance of glutamaterdquo Neuronvol 16 no 3 pp 675ndash686 1996

[28] N L Barnett and D V Pow ldquoAntisense knockdown of GLASTa glial glutamate transporter compromises retinal functionrdquoInvestigative Ophthalmology and Visual Science vol 41 no 2pp 585ndash591 2000

[29] T Harada C Harada K Nakamura et al ldquoThe potential role ofglutamate transporters in the pathogenesis of normal tensionglaucomardquo Journal of Clinical Investigation vol 117 no 7 pp1763ndash1770 2007

[30] N J Maragakis J Dietrich V Wong et al ldquoGlutamate Trans-porter Expression and Function in Human Glial ProgenitorsrdquoGlia vol 45 no 2 pp 133ndash143 2004

[31] C K Vorwerk R Naskar F Schuettauf et al ldquoDepression ofretinal glutamate transporter function leads to elevated intrav-itreal glutamate levels and ganglion cell deathrdquo InvestigativeOphthalmology and Visual Science vol 41 no 11 pp 3615ndash36212000

[32] D L Black ldquoMechanisms of alternative pre-messenger RNAsplicingrdquo Annual Review of Biochemistry vol 72 pp 291ndash3362003

[33] Q Pan O Shai L J Lee B J Frey and B J Blencowe ldquoDeepsurveying of alternative splicing complexity in the human tran-scriptome by high-throughput sequencingrdquo Nature Geneticsvol 40 no 12 pp 1413ndash1415 2008

[34] L T Macnab S M Williams and D V Pow ldquoExpression of theexon 3 skipping form of GLAST GLAST1a in brain and retinardquoNeuroReport vol 17 no 18 pp 1867ndash1870 2006

[35] L TMacnab andDV Pow ldquoCentral nervous system expressionof the exon 9 skipping form of the glutamate transporterGLASTrdquo NeuroReport vol 18 no 8 pp 741ndash745 2007

[36] R K P Sullivan EWoldeMussie L Macnab G Ruiz and D VPow ldquoEvoked expression of the glutamate transporter GLT-1c inretinal ganglion cells in human glaucoma and in a rat modelrdquoInvestigativeOphthalmology andVisual Science vol 47 no 9 pp3853ndash3859 2006

[37] A Lee A R Anderson S J Beasley N L Barnett P Poronnikand D V Pow ldquoA new splice variant of the glutamate-aspartatetransporter cloning and immunolocalization of GLAST1c inrat pig and human brainsrdquo Journal of Chemical Neuroanatomyvol 43 no 1 pp 52ndash63 2012

[38] T Rauen M Wieszligner R Sullivan A Lee and D V Pow ldquoAnew GLT1 splice variant cloning and immunolocalization ofGLT1c in the mammalian retina and brainrdquo NeurochemistryInternational vol 45 no 7 pp 1095ndash1106 2004

[39] P Reye R Sullivan E L Fletcher and D V Pow ldquoDistributionof two splice variants of the glutamate transporter GLT1 in theretinas of humans monkeys rabbits rats cats and chickensrdquoJournal of Comparative Neurology vol 445 no 1 pp 1ndash12 2002

[40] P Reye R Sullivan and D V Pow ldquoDistribution of two splicevariants of the glutamate transporter GLT-1 in the developingrat retinardquo Journal of Comparative Neurology vol 447 no 4 pp323ndash330 2002

[41] S Holmseth H A Scott K Real et al ldquoThe concentrations anddistributions of three C-terminal variants of the GLT1 (EAAT2slc1a2) glutamate transporter protein in rat brain tissue suggestdifferential regulationrdquo Neuroscience vol 162 no 4 pp 1055ndash1071 2009

[42] L Had-Aissouni ldquoToward a new role for plasma membranesodium-dependent glutamate transporters of astrocytes main-tenance of antioxidant defenses beyond extracellular glutamateclearancerdquo Amino Acids vol 42 no 1 pp 181ndash197 2012

[43] N Zerangue and M P Kavanaugh ldquoInteraction of L-cysteinewith a human excitatory amino acid transporterrdquo Journal ofPhysiology vol 493 no 2 pp 419ndash423 1996

[44] K Aoyama W S Sang A M Hamby et al ldquoNeuronalglutathione deficiency and age-dependent neurodegenerationin the EAAC1 deficient mouserdquo Nature Neuroscience vol 9 no1 pp 119ndash126 2006

[45] H Lie-Venema T B Hakvoort F J van Hemert A F Moor-man and W H Lamers ldquoRegulation of the spatiotemporalpattern of expression of the glutamine synthetase generdquoProgressin Nucleic Acid Research andMolecular Biology vol 61 pp 243ndash308 1998

[46] Y He T B M Hakvoort J L M Vermeulen et al ldquoGlutaminesynthetase deficiency in murine astrocytes results in neonataldeathrdquo Glia vol 58 no 6 pp 741ndash754 2010

[47] R E Riepe and M D Norenburg ldquoMuller cell localisation ofglutamine synthetase in rat retinardquo Nature vol 268 no 5621pp 654ndash655 1977

[48] A Bringmann T Pannicke B Biedermann et al ldquoRole ofretinal glial cells in neurotransmitter uptake and metabolismrdquoNeurochemistry International vol 54 no 3-4 pp 143ndash160 2009

[49] S Poitry C Poitry-Yamate J Ueberfeld P R MacLeish andMTsacopoulos ldquoMechanisms of glutamate metabolic signaling inretinal glial (Muller) cellsrdquo Journal of Neuroscience vol 20 no5 pp 1809ndash1821 2000

[50] N L Barnett D V Pow and S R Robinson ldquoInhibition ofMuller cell glutamine synthetase rapidly impairs the retinalresponse to lightrdquo Glia vol 30 no 1 pp 64ndash73 2000

[51] DNicholls andD Attwell ldquoThe release and uptake of excitatoryamino acidsrdquo Trends in Pharmacological Sciences vol 11 no 11pp 462ndash468 1990

[52] I A Silver and M Erecinska ldquoEnergetic demands of theNa+K+ATPase in mammalian astrocytesrdquo Glia vol 21 pp 35ndash45 1997

[53] Y Izumi C O Kirby A M Benz J W Olney and C F Zorum-ski ldquoMuller cell swelling glutamate uptake and excitotoxicneurodegeneration in the isolated rat retinardquo Glia vol 25 no4 pp 379ndash389 1999

[54] D J Rossi T Oshima and D Attwell ldquoGlutamate release insevere brain ischaemia is mainly by reversed uptakerdquo Naturevol 403 no 6767 pp 316ndash321 2000

[55] M M Jablonski N E Freeman W E Orr et al ldquoGeneticpathways regulating glutamate levels in retinal muller cellsrdquoNeurochemical Research vol 36 no 4 pp 594ndash603 2011

[56] D Jabaudon M Scanziani B H Gahwiler and U GerberldquoAcute decrease in net glutamate uptake during energy depri-vationrdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 97 no 10 pp 5610ndash5615 2000

Scientifica 11

[57] A Novelli J A Reilly P G Lysko and R C HenneberryldquoGlutamate becomes neurotoxic via the N-methyl-D-aspartatereceptor when intracellular energy levels are reducedrdquo BrainResearch vol 451 no 1-2 pp 205ndash212 1988

[58] D Nguyen M V Alavi K-Y Kim et al ldquoA new viciouscycle involving glutamate excitotoxicity oxidative stress andmitochondrial dynamicsrdquo Cell Death and Disease vol 2 no 12article e240 2011

[59] T Rauen W R Taylor K Kuhlbrodt and M Wiessner ldquoHigh-affinity glutamate transporters in the rat retina a major roleof the glial glutamate transporter GLAST-1 in transmitterclearancerdquo Cell and Tissue Research vol 291 no 1 pp 19ndash311998

[60] T Rauen ldquoDiversity of glutamate transporter expression andfunction in the mammalian retinardquo Amino Acids vol 19 no 1pp 53ndash62 2000

[61] P Louzada-Junior J J DiasW F Santos J J Lachat H F Brad-ford and J Coutinho-Netto ldquoGlutamate release in experimentalischaemia of the retina an approach using microdialysisrdquoJournal of Neurochemistry vol 59 no 1 pp 358ndash363 1992

[62] M J Neal J R Cunningham PHHutson and J Hogg ldquoEffectsof ischaemia on neurotransmitter release from the isolatedretinardquo Journal of Neurochemistry vol 62 no 3 pp 1025ndash10331994

[63] AMullerM Villain and C Bonne ldquoThe release of amino acidsfrom ischemic retinardquo Experimental Eye Research vol 64 no 2pp 291ndash293 1997

[64] D S Casper R L Trelstad and L Reif-Lehrer ldquoGlutamate-induced cellular injury in isolated chick embryo retina mullercell localization of initial effectsrdquo Journal of Comparative Neu-rology vol 209 no 1 pp 79ndash90 1982

[65] S S Hayreh M B Zimmerman A Kimura and A SanonldquoCentral retinal artery occlusion Retinal survival timerdquo Exper-imental Eye Research vol 78 no 3 pp 723ndash736 2004

[66] H Weiss ldquoThe vitreous body as an energy depot in intraocularischemiardquo Bericht uber die Zusammenkunft Deutsche Ophthal-mologische Gesellschaft vol 71 pp 416ndash418 1972

[67] D Kaskel H Valenzuela O Hockwin and M Muntau ldquoEnzy-mologic and histologic investigations in normal and pressureischemic retina of rabbitsrdquo Albrecht von Graefes Archiv furKlinische und Experimentelle Ophthalmologie vol 200 no 1 pp71ndash78 1976

[68] PWassilewaOHocksin and I Korte ldquoGlycogen concentrationchanges in retina vitreous body and other eye tissues caused bydisturbances of blood circulationrdquo Albrecht von Graefes Archivfur Klinische und Experimentelle Ophthalmologie vol 199 no 2pp 115ndash120 1976

[69] G D Zeevalk and W J Nicklas ldquoActivity at the GABAtransporter contributes to acute cellular swelling produced bymetabolic impairment in retinardquoVision Research vol 37 no 24pp 3463ndash3470 1997

[70] S Berger S I Savitz S Nijhawan et al ldquoDeleterious roleof TNF-120572 in retinal ischemia-reperfusion injuryrdquo InvestigativeOphthalmology and Visual Science vol 49 no 8 pp 3605ndash36102008

[71] N N Osborne R J Casson J P M Wood G ChidlowM Graham and J Melena ldquoRetinal ischemia mechanisms ofdamage and potential therapeutic strategiesrdquo Progress in Retinaland Eye Research vol 23 no 1 pp 91ndash147 2004

[72] N L Barnett D V Pow and N D Bull ldquoDifferential perturba-tion of neuronal and glial glutamate transport systems in retinal

ischaemiardquoNeurochemistry International vol 39 no 4 pp 291ndash299 2001

[73] Y Wakabayashi T Yagihashi J Kezuka D Muramatsu MUsui and T Iwasaki ldquoGlutamate levels in aqueous humor ofpatients with retinal artery occlusionrdquo Retina vol 26 no 4 pp432ndash436 2006

[74] A Bringmann O Uckermann T Pannicke I Iandiev AReichenbach and P Wiedemann ldquoNeuronal versus glial cellswelling in the ischaemic retinardquo Acta Ophthalmologica Scan-dinavica vol 83 no 5 pp 528ndash538 2005

[75] N L Barnett and S D Grozdanic ldquoGlutamate transporterlocalization does not correspond to the temporary functionalrecovery and late degeneration after acute ocular ischemia inratsrdquo Experimental Eye Research vol 79 no 4 pp 513ndash5242004

[76] R A Swanson J Liu J W Miller et al ldquoNeuronal regulation ofglutamate transporter subtype expression in astrocytesrdquo Journalof Neuroscience vol 17 no 3 pp 932ndash940 1997

[77] M Conradt andW Stoffel ldquoInhibition of the high-affinity brainglutamate transporter GLAST-1 via direct phosphorylationrdquoJournal of Neurochemistry vol 68 no 3 pp 1244ndash1251 1997

[78] N D Bull and N L Barnett ldquoAntagonists of protein kinase Cinhibit rat retinal glutamate transport activity in siturdquo Journalof Neurochemistry vol 81 no 3 pp 472ndash480 2002

[79] R Russo F Cavaliere G P Varano et al ldquoImpairment ofneuronal glutamate uptake and modulation of the glutamatetransporter GLT-1 induced by retinal ischemiardquo PLoS ONE vol8 no 8 Article ID e69250 2013

[80] E Kosenko M Llansola C Montoliu et al ldquoGlutamine syn-thetase activity and glutamine content in brain modulationby NMDA receptors and nitric oxiderdquo Neurochemistry Interna-tional vol 43 no 4-5 pp 493ndash499 2003

[81] J Stone J Maslim K Valter-Kocsi et al ldquoMechanisms of pho-toreceptor death and survival in mammalian retinardquo Progress inRetinal and Eye Research vol 18 no 6 pp 689ndash735 1999

[82] P G Hirrlinger E Ulbricht I Iandiev A Reichenbach andT Pannicke ldquoAlterations in protein expression and membraneproperties during Muller cell gliosis in a murine model oftransient retinal ischemiardquo Neuroscience Letters vol 472 no 1pp 73ndash78 2010

[83] D Dorfman D C Fernandez M Chianelli M Miranda ML Aranda and R E Rosenstein ldquoPost-ischemic environmentalenrichment protects the retina from ischemic damage in adultratsrdquo Experimental Neurology vol 240 pp 146ndash156 2013

[84] D C Fernandez M S Chianelli and R E RosensteinldquoInvolvement of glutamate in retinal protection againstischemiareperfusion damage induced by post-conditioningrdquoJournal of Neurochemistry vol 111 no 2 pp 488ndash498 2009

[85] X Wang X Mo D Li et al ldquoNeuroprotective effect oftranscorneal electrical stimulation on ischemic damage in therat retinardquo Experimental Eye Research vol 93 no 5 pp 753ndash760 2011

[86] M Hlavackova K Kozichova J Neckar et al ldquoUp-regulationand redistribution of protein kinase C-120575 in chronically hypoxicheartrdquoMolecular Cell Biochemistry vol 345 no 1-2 pp 271ndash2822010

[87] H A Quigley R W Nickells L A Kerrigan M E PeaseD J Thibault and D J Zack ldquoRetinal ganglion cell death inexperimental glaucoma and after axotomy occurs by apoptosisrdquoInvestigative Ophthalmology and Visual Science vol 36 no 5pp 774ndash786 1995

12 Scientifica

[88] R J Casson ldquoPossible role of excitotoxicity in the pathogenesisof glaucomardquoClinical and Experimental Ophthalmology vol 34no 1 pp 54ndash63 2006

[89] D E Brooks G A Garcia E B Dreyer D Zurakowski and RE Franco-Bourland ldquoVitreous body glutamate concentration indogs with glaucomardquo American Journal of Veterinary Researchvol 58 no 8 pp 864ndash867 1997

[90] E B Dreyer D Zurakowski R A Schumer S M Podosand S A Lipton ldquoElevated glutamate levels in the vitreousbody of humans and monkeys with glaucomardquo Archives ofOphthalmology vol 114 no 3 pp 299ndash305 1996

[91] L Carter-Dawson M L J Crawford R S Harwerth et alldquoVitreal glutamate concentration inmonkeyswith experimentalglaucomardquo Investigative Ophthalmology and Visual Science vol43 no 8 pp 2633ndash2637 2002

[92] R A Honkanen S Baruah M B Zimmerman et al ldquoVitreousamino acid concentrations in patients with glaucoma undergo-ing vitrectomyrdquo Archives of Ophthalmology vol 121 no 2 pp183ndash188 2003

[93] H Levkovitch-Verbin K R G Martin H A Quigley L ABaumrindM E Pease andDValenta ldquoMeasurement of aminoacid levels in the vitreous humor of rats after chronic intraoc-ular pressure elevation or optic nerve transectionrdquo Journal ofGlaucoma vol 11 no 5 pp 396ndash405 2002

[94] S Wamsley B T Gabelt D B Dahl et al ldquoVitreous glutamateconcentration and axon loss in monkeys with experimentalglaucomardquoArchives of Ophthalmology vol 123 no 1 pp 64ndash702005

[95] A J Lotery ldquoGlutamate excitotoxicity in glaucoma truth orfictionrdquo Eye vol 19 no 4 pp 369ndash370 2005

[96] N N Osborne G Chidlow and J P M Wood ldquoGlutamateexcitotoxicity in glaucoma truth or fiction By AJ Loteryrdquo Eyevol 20 no 12 pp 1392ndash1394 2006

[97] Y Kanai and M A Hediger ldquoThe glutamateneutral aminoacid transporter family SLC1 molecular physiological andpharmacological aspectsrdquo Pflugers Archiv European Journal ofPhysiology vol 447 no 5 pp 469ndash479 2004

[98] Y Izumi C O Kirby A M Benz J W Olney and CF Zorumski ldquoSwelling of Muller cells induced by AP3 andglutamate transport substrates in rat retinardquo Glia vol 17 no 4pp 285ndash293 1996

[99] R Naskar C K Vorwerk and E B Dreyer ldquoConcurrentdownregulation of a glutamate transporter and receptor inglaucomardquo Investigative Ophthalmology and Visual Science vol41 no 7 pp 1940ndash1944 2000

[100] K RGMartinH Levkovitch-VerbinDValenta L BaumrindM E Pease and H A Quigley ldquoRetinal glutamate trans-porter changes in experimental glaucoma and after optic nervetransection in the ratrdquo Investigative Ophthalmology and VisualScience vol 43 no 7 pp 2236ndash2243 2002

[101] F Schuettauf S Thaler S Bolz et al ldquoAlterations of aminoacids and glutamate transport in the DBA2J mouse retinapossible clues to degenerationrdquo Graefersquos Archive for Clinical andExperimental Ophthalmology vol 245 no 8 pp 1157ndash1168 2007

[102] CK Park J Cha S C Park et al ldquoDifferential expression of twoglutamate transporters GLAST and GLT-1 in an experimentalrat model of glaucomardquo Experimental Brain Research vol 197no 2 pp 101ndash109 2009

[103] E Woldemussie MWijono and G Ruiz ldquoMuller cell responseto laser-induced increase in intraocular pressure in ratsrdquo Gliavol 47 no 2 pp 109ndash119 2004

[104] M Ishikawa T Yoshitomi C F Zorumski and Y IzumildquoEffects of acutely elevated hydrostatic pressure in a rat ex vivoretinal preparationrdquo Investigative Ophthalmology and VisualScience vol 51 no 12 pp 6414ndash6423 2010

[105] S W Park K-Y Kim J D Lindsey et al ldquoA selective inhibitorof Drp1Mdivi-1 increases retinal ganglion cell survival in acuteischemic mouse retinardquo Investigative Ophthalmology and VisualScience vol 52 no 5 pp 2837ndash2843 2011

[106] S Yoneda E TanakaWGoto TOta andHHara ldquoTopiramatereduces excitotoxic and ischemic injury in the rat retinardquo BrainResearch vol 967 no 1-2 pp 257ndash266 2003

[107] N A Whitlock N Agarwal J-X Ma and C E CrossonldquoHsp27 upregulation by HIF-1 signaling offers protectionagainst retinal ischemia in ratsrdquo Investigative Ophthalmologyand Visual Science vol 46 no 3 pp 1092ndash1098 2005

[108] HAQuigley ldquoNeuronal death in glaucomardquoProgress in Retinaland Eye Research vol 18 no 1 pp 39ndash57 1999

[109] M Persson and L Ronnback ldquoMicroglial self-defencemediatedthrough GLT-1 and glutathionerdquo Amino Acids vol 42 no 1 pp207ndash219 2012

[110] R Dringen B Pfeiffer and B Hamprecht ldquoSynthesis of theantioxidant glutathione in neurons supply by astrocytes ofCysGly as precursor for neuronal glutathionerdquo Journal ofNeuroscience vol 19 no 2 pp 562ndash569 1999

[111] GWu Y-Z Fang S Yang J R Lupton andN D Turner ldquoGlu-tathione metabolism and its implications for healthrdquo Journal ofNutrition vol 134 no 3 pp 489ndash492 2004

[112] W Reichelt J Stabel-Burow T Pannicke H Weichert and UHeinemann ldquoThe glutathione level of retinal Muller glial cellsis dependent on the high-affinity sodium-dependent uptake ofglutamaterdquo Neuroscience vol 77 no 4 pp 1213ndash1224 1997

[113] F Shen B Chen J Danias et al ldquoGlutamate-induced glutaminesynthetase expression in retinal Muller cells after short-termocular hypertension in the ratrdquo Investigative Ophthalmology andVisual Science vol 45 no 9 pp 3107ndash3112 2004

[114] S Zhang H Wang Q Lu et al ldquoDetection of early neurondegeneration and accompanying glial responses in the visualpathway in a ratmodel of acute intraocular hypertensionrdquoBrainResearch vol 1303 pp 131ndash143 2009

[115] M C Moreno P Sande H A Marcos N de Zavalıa M I KSarmiento and R E Rosenstein ldquoEffect of glaucoma on theretinal glutamateglutamine cycle activityrdquo The FASEB Journalvol 19 no 9 pp 1161ndash1162 2005

[116] C-T Chen K Alyahya J R Gionfriddo R R Dubielzig and JE Madl ldquoLoss of glutamine synthetase immunoreactivity fromthe retina in canine primary glaucomardquoVeterinaryOphthalmol-ogy vol 11 no 3 pp 150ndash157 2008

[117] M Ishikawa T Yoshitomi C F Zorumski and Y IzumildquoDownregulation of glutamine synthetase via GLAST suppres-sion induces retinal axonal swelling in a rat ex vivo hydrostaticpressure modelrdquo Investigative Ophthalmology amp Visual Sciencevol 52 no 9 pp 6604ndash6616 2011

[118] M Vidal-Sanz M Salinas-Navarro F M Nadal-Nicolas et alldquoUnderstanding glaucomatous damage anatomical and func-tional data from ocular hypertensive rodent retinasrdquo Progress inRetinal and Eye Research vol 31 no 1 pp 1ndash27 2012

[119] P J Hollenbeck and W M Saxton ldquoThe axonal transport ofmitochondriardquo Journal of Cell Science vol 118 no 23 pp 5411ndash5419 2005

[120] W-K Ju J D Lindsey M Angert A Patel and R N Wein-reb ldquoGlutamate receptor activation triggers OPA1 release and

Scientifica 13

induces apoptotic cell death in ischemic rat retinardquo MolecularVision vol 14 pp 2629ndash2638 2008

[121] W-K Ju Q Liu K-Y Kim et al ldquoElevated hydrostatic pressuretriggers mitochondrial fission and decreases cellular ATP indifferentiated RGC-5 cellsrdquo Investigative Ophthalmology andVisual Science vol 48 no 5 pp 2145ndash2151 2007

[122] W-K Ju K-Y Kim J D Lindsey et al ldquoElevated hydrostaticpressure triggers release of OPA1 and cytochrome C andinduces apoptotic cell death in differentiated RGC-5 cellsrdquoMolecular Vision vol 15 pp 120ndash134 2009

[123] D G Nicholls and M W Ward ldquoMitochondrial membranepotential and neuronal glutamate excitotoxicity mortality andmillivoltsrdquo Trends in Neurosciences vol 23 no 4 pp 166ndash1742000

[124] W-K Ju K-Y Kim M Angert et al ldquoMemantine blocksmitochondrial OPA1 and cytochrome c release and subsequentapoptotic cell death in glaucomatous retinardquo Investigative Oph-thalmology and Visual Science vol 50 no 2 pp 707ndash716 2009

[125] M M Y Fan and L A Raymond ldquoN-Methyl-d-aspartate(NMDA) receptor function and excitotoxicity in Huntingtonrsquosdiseaserdquo Progress in Neurobiology vol 81 no 5-6 pp 272ndash2932007

[126] M F Beal ldquoAging energy and oxidative stress in neurodegen-erative diseasesrdquoAnnals of Neurology vol 38 no 3 pp 357ndash3661995

[127] R C Henneberry A Novelli J A Cox and P G LyskoldquoNeurotoxicity at theN-methyl-D-aspartate receptor in energy-compromised neurons An hypothesis for cell death in agingand diseaserdquo Annals of the New York Academy of Sciences vol568 pp 225ndash233 1989

[128] T Nakamura P Cieplak D-H Cho A Godzik and S ALipton ldquoS-Nitrosylation of Drp1 links excessive mitochondrialfission to neuronal injury in neurodegenerationrdquo Mitochon-drion vol 10 no 5 pp 573ndash578 2010

[129] J W Y Yau S L Rogers R Kawasaki et al ldquoGlobal prevalenceand major risk factors of diabetic retinopathyrdquo Diabetes Carevol 35 no 3 pp 556ndash564 2012

[130] C Hernandez and R Simo ldquoNeuroprotection in diabeticretinopathyrdquo Current Diabetes Reports vol 12 no 4 pp 329ndash337 2012

[131] A R Cervantes-Villagrana J Garcia-Roman C Gonzlez-Espinosa and M Lamas ldquoPharmacological inhibition of n-methyl d-aspartate receptor promotes secretion of vascularendothelial growth factor in muller cells effects of hyper-glycemia and hypoxiardquo Current Eye Research vol 35 no 8 pp733ndash741 2010

[132] J Kusari S X Zhou E Padillo K G Clarke and D W GilldquoInhibition of vitreoretinal VEGF elevation and blood-retinalbarrier breakdown in streptozotocin-induced diabetic rats bybrimonidinerdquo Investigative Ophthalmology and Visual Sciencevol 51 no 2 pp 1044ndash1051 2010

[133] J Ambati K V Chalam D K Chawala et al ldquoElevated 120574-aminobutyric acid glutamate and vascular endothelial growthfactor levels in the vitreous of patients with proliferative diabeticretinopathyrdquoArchives of Ophthalmology vol 115 no 9 pp 1161ndash1166 1997

[134] Q Li and D G Puro ldquoDiabetes-induced dysfunction of theglutamate transporter in retinal Muller cellsrdquo InvestigativeOphthalmology and Visual Science vol 43 no 9 pp 3109ndash31162002

[135] D G Puro ldquoDiabetes-induced dysfunction of retinal Mullercellsrdquo Transactions of the American Ophthalmological Societyvol 100 pp 339ndash352 2002

[136] J C Lau R A Kroes J R Moskal and R A LinsenmeierldquoDiabetes changes expression of genes related to glutamateneurotransmission and transport in the Long-Evans rat retinardquoMolecular Vision vol 19 pp 1538ndash1553 2013

[137] K Zeng H Xu M Mi et al ldquoDietary taurine supplementationprevents glial alterations in retina of diabetic ratsrdquo Neurochem-ical Research vol 34 no 2 pp 244ndash254 2009

[138] K C Silva M A Rosales D E Hamassaki et al ldquoGreentea is neuroprotective in diabetic retinopathyrdquo InvestigativeOphthalmology amp Visual Science vol 54 no 2 pp 1325ndash13362013

[139] Y Xu-hui Z HongW Yu-hong L Li-juan T Yan and L PingldquoTime-dependent reduction of glutamine synthetase in retinaof diabetic ratsrdquo Experimental Eye Research vol 89 no 6 pp967ndash971 2009

Submit your manuscripts athttpwwwhindawicom

Neurology Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Research and TreatmentSchizophrenia

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2 Scientifica

Glutamate

GlutamineGlutaminesynthetaseSynaptic

cleft

Glutamatetransporter

Glutamate

Photoreceptors

Mueller cell

Bipolar cell

Non

-NM

DA-

R

NM

DA-

R

Met

abol

ic-R

Synaptic vesicles

Figure 1 Glutamatergic synaptic transduction and uptake andmetabolism of glutamate in the Muller cell The photoreceptorcell synthesizes glutamate which is continuously released duringdarkness Glutamate released from the synaptic terminal reaches tothe postsynaptic receptors of bipolar cell dendrites then is promptlytaken up from the synaptic cleft The glutamate transporters are thepredominant mechanism for uptake of glutamate in the Muller cellandmaintain the proper concentration of this potentially excitotoxicamino acid Glutamate transported into the Muller cell is degradedto glutamine by glutamine synthetase

EAAC-1 (EAAT3) [13] EAAT4 [14] and EAAT5 [15] In theretina four out of the five knownEAATs have been describedImmunohistochemical analysis localize the distributions ofthese glutamate transporters respectively [16 17] GLAST isdistributed in the Muller glial cells GLT-1 and EAAT5 arecolocalized in the photoreceptor cells and the bipolar cellsEAAC-1 (EAAT3) is localized in the horizontal cells ganglioncells and some amacrine cells

Since glutamate is a potent neurotoxin the functionalrole of glutamate transporters is critical to prevent the retinalexcitotoxicity Izumi et al [10] reported the pharmacologicalblockade of glutamate transporters using TBOA (DL-threo--benzyloxyaspartate) [18ndash20] TBOA is an inhibitor of alltypes of glutamate transporters and does not evoke currentsin neurons or glial cells Izumi et al [10] used TBOA aloneand in combination with exogenous glutamate to examinethe role of glial glutamate transporters in excitotoxic retinaldegeneration In the presence of TBOA the potency ofglutamate as a neurotoxin is greatly enhanced SurprisinglyTBOA alone is neurotoxic and the toxicity is inhibited by acombination of ionotropic NMDA and non-NMDA receptorantagonists suggesting that the damage is excitotoxicityFurthermore TBOA-induced neuronal damage is inhibitedby the glutamate release inhibitor riluzole suggesting that the

damage is mediated by endogenous glutamate rather thanresulted from a direct action of TBOAThese results stronglysuggest that the neurotoxic actions of TBOA result fromblocking glutamate transport and pathological conditionsor treatments that impair glial glutamate transport greatlyaugment the toxic effects of endogenous glutamate

Recently antisense knockout techniques or the con-struction of transgenic and gene knockout mouse modelsrepresent the successful approach to achieving suppressionor elimination of a genetic message of specific glutamatetransporters In this section we describe the characterizationof these three glutamate transporter precisely based on theexperimental results mainly obtained by gene engineering

211 GLAST GLAST is the prominent glutamate transporterin the retina andmainly expressed in theMuller cells [1 8 21ndash24] In the mouse Muller cells glutamate removal by GLASTis calculated as 50 among the retinal glutamate trans-porters [25] Glutamate uptake via GLAST is accompaniedby cotransport of three Na+ and one H+ and the counter-transport of one K+ at each glutamate transport Sodiumions are extruded at least in part by Na+K+-adenosinetriphosphate (ATP) ase in a reaction that consumes ATP

To elucidate the role of GLAST in the regulation of retinalfunctionHarada et al [26] developedGLAST-deficientmiceand revealed reduction of the scotopic ERG b-wave indicat-ing the reduction of glutamate-mediated neurotransmissionactivity in the retina After induction of the retinal ischemiaby increasing intraocular pressure above systolic pressurefor 60min more severe excitotoxic degeneration is foundin GLAST-deficient mice than in wild-type indicating thatGLAST is neuroprotective against ischemia [27]

Barnett and Pow [28] injected intravitreally antisenseoligonucleotides to GLAST into rat eyes Although a markedreduction of GLAST activity was detected the retinas dis-played no evidence of excitotoxic neuronal degeneration andthe distribution of glutamate was unaffected by antisensetreatment Significant inhibition in GLAST function wasapparent 5 days after injection of antisense oligonucleotideand was sustained for at least 20 days The observed lackof neuronal degeneration suggests that reduced glutamateuptake into the Muller cells does not cause excitotoxic tissuedamage

HoweverHarada et al [29] examined the long-term effect(32 weeks) of GLAST deficiency on the retinal morphologyduring postnatal development in vivo and revealed spon-taneous loss of retinal ganglion cell (RGC) and optic nervedegeneration without elevated intraocular pressure (IOP) InGLAST-deficient mice administration of glutamate receptorantagonist prevented RGC loss These findings suggest thatGLAST is necessary to prevent retinal excitotoxicity

Taken together the glutamate removal by glutamatetransportes is prerequisite for the maintenance of normalretinal transmission and preventive against excitotoxicity

212 GLT-1 Unlike in the central nervous system [30] it isconsidered thatGLT-1 does not play a predominant role in thephysiological glutamate transmission in the retina because

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6 Scientifica

Bubble

Bubble

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Manometer

Eyecup preparations

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10(mmHg)

75

Buffer columnheight

135 cm

1012 cm

Gas darr

darr Antagonist


2-5 CO








Scientifica 7

ILM

IPL

INL

OPL

ONL


(c)

(d)











8 Scientifica













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Acknowledgments


References

























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12 Scientifica


































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Neural Plasticity










Psychiatry Journal









Journal of


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4 Scientifica
















Scientifica 5













6 Scientifica

Bubble

Bubble

Bubble

Manometer

Eyecup preparations

Valve

10(mmHg)

75

Buffer columnheight

135 cm

1012 cm

Gas darr

darr Antagonist


2-5 CO








Scientifica 7

ILM

IPL

INL

OPL

ONL


(c)

(d)











8 Scientifica













Scientifica 9



Acknowledgments


References

























10 Scientifica


































Scientifica 11

































12 Scientifica


































Scientifica 13

































Neural Plasticity










Psychiatry Journal









Journal of


4 Scientifica
















Scientifica 5













6 Scientifica

Bubble

Bubble

Bubble

Manometer

Eyecup preparations

Valve

10(mmHg)

75

Buffer columnheight

135 cm

1012 cm

Gas darr

darr Antagonist


2-5 CO








Scientifica 7

ILM

IPL

INL

OPL

ONL


(c)

(d)











8 Scientifica













Scientifica 9



Acknowledgments


References

























10 Scientifica


































Scientifica 11

































12 Scientifica


































Scientifica 13

































Neural Plasticity










Psychiatry Journal









Journal of


Scientifica 5













6 Scientifica

Bubble

Bubble

Bubble

Manometer

Eyecup preparations

Valve

10(mmHg)

75

Buffer columnheight

135 cm

1012 cm

Gas darr

darr Antagonist


2-5 CO








Scientifica 7

ILM

IPL

INL

OPL

ONL


(c)

(d)











8 Scientifica













Scientifica 9



Acknowledgments


References

























10 Scientifica


































Scientifica 11

































12 Scientifica


































Scientifica 13

































Neural Plasticity










Psychiatry Journal









Journal of


6 Scientifica

Bubble

Bubble

Bubble

Manometer

Eyecup preparations

Valve

10(mmHg)

75

Buffer columnheight

135 cm

1012 cm

Gas darr

darr Antagonist


2-5 CO








Scientifica 7

ILM

IPL

INL

OPL

ONL


(c)

(d)











8 Scientifica













Scientifica 9



Acknowledgments


References

























10 Scientifica


































Scientifica 11

































12 Scientifica


































Scientifica 13

































Neural Plasticity










Psychiatry Journal









Journal of


Scientifica 7

ILM

IPL

INL

OPL

ONL


(c)

(d)











8 Scientifica













Scientifica 9



Acknowledgments


References

























10 Scientifica


































Scientifica 11

































12 Scientifica


































Scientifica 13

































Neural Plasticity










Psychiatry Journal









Journal of


8 Scientifica













Scientifica 9



Acknowledgments


References

























10 Scientifica


































Scientifica 11

































12 Scientifica


































Scientifica 13

































Neural Plasticity










Psychiatry Journal









Journal of


Scientifica 9



Acknowledgments


References

























10 Scientifica


































Scientifica 11

































12 Scientifica


































Scientifica 13

































Neural Plasticity










Psychiatry Journal









Journal of


10 Scientifica


































Scientifica 11

































12 Scientifica


































Scientifica 13

































Neural Plasticity










Psychiatry Journal









Journal of


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12 Scientifica


































Scientifica 13

































Neural Plasticity










Psychiatry Journal









Journal of


12 Scientifica


































Scientifica 13

































Neural Plasticity










Psychiatry Journal









Journal of


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Neural Plasticity










Psychiatry Journal









Journal of














Neural Plasticity










Psychiatry Journal









Journal of


abnormalities in glutamate metabolism and excitotoxicity in the

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