Neuronal glutathione deficiency and age-dependentneurodegeneration in the EAAC1 deficient mouse

Koji Aoyama1,2, Sang Won Suh1,2, Aaron M Hamby1,2, Jialing Liu2,3, Wai Yee Chan1,2, Yongmei Chen1,2 &Raymond A Swanson1,2

Uptake of the neurotransmitter glutamate is effected primarily by transporters expressed on astrocytes, and downregulation of

these transporters leads to seizures and neuronal death. Neurons also express a glutamate transporter, termed excitatory amino

acid carrier–1 (EAAC1), but the physiological function of this transporter remains uncertain. Here we report that genetically

EAAC1-null (Slc1a1–/–) mice have reduced neuronal glutathione levels and, with aging, develop brain atrophy and behavioral

changes. EAAC1 can also rapidly transport cysteine, an obligate precursor for neuronal glutathione synthesis. Neurons in

the hippocampal slices of EAAC1–/– mice were found to have reduced glutathione content, increased oxidant levels and

increased susceptibility to oxidant injury. These changes were reversed by treating the EAAC1–/– mice with N-acetylcysteine,

a membrane-permeable cysteine precursor. These findings suggest that EAAC1 is the primary route for neuronal cysteine

uptake and that EAAC1 deficiency thereby leads to impaired neuronal glutathione metabolism, oxidative stress and

age-dependent neurodegeneration.

Sodium-dependent excitatory amino acid transporters (EAATs) regu-late extracellular glutamate concentrations in the central nervoussystem. Five EAATs have been identified, termed glutamate-aspartatetransporter (GLAST or EAAT1), glutamate transporter 1 (GLT-1 orEAAT2), EAAC1 (EAAT3), EAAT4 and EAAT5 (ref. 1). GLAST andGLT-1 are localized primarily to astrocytes, whereas EAAC1, EAAT4 andEAAT5 are localized primarily to neurons1–4. EAAT4 and EAAT5 arerestricted to cerebellar Purkinje cells and retina, respectively, but EAAC1is widely expressed in neurons throughout the nervous system3,4.

The function of EAAC1 in the brain has not been established. Unlikethe astrocyte glutamate transporters, EAAC1 does not play a major rolein clearing glutamate from the extracellular space5–7. Also unlike theastrocyte glutamate transporters, which are clustered near glutamater-gic synapses, EAAC1 is localized diffusely over cell bodies and pro-cesses2,3,8, suggesting a function other than the re-uptake ofsynaptically released glutamate. An exception to this pattern is pre-synaptic GABAergic terminals, where EAAC1 uptake of glutamatecontributes to re-synthesis of GABA9. An additional distinguishingfeature of EAAC1 is that it can bind and transport cysteine far moreeffectively than the astrocyte glutamate transporters can10,11.

Cysteine is normally the rate-limiting substrate for the synthesis ofglutathione, the principal cellular thiol antioxidant12–14. Most cell typesacquire cysteine in the form of cystine, by hetero-exchange withglutamate (system Xc

–)12, but cell culture studies suggest that neuronslose this capacity during development13,15,16. Consistent with this,studies of the intact mature brain show an absence of Xc

– expression

in neurons17 and extremely low (o 300 nM) cystine concentrations inbrain extracellular fluid18,19. Neuronal glutathione synthesis is sup-ported by astrocytes through an indirect route involving astrocyteglutathione release, its cleavage to cysteinylglycine and the subsequentrelease of free cysteine by an ectopeptidase located on the neuronal cellsurface13,20. The route of cysteine uptake into neurons has not beenascertained, but cell culture studies suggest EAAC1 as a candidateneuronal cysteine transporter because pharmacological inhibitors ofEAAC1 prevent neuronal glutathione synthesis in the presence ofextracellular cysteine14,21,22. Cysteine is transported by EAAC1 at arate comparable to that of glutamate and with an affinity roughlytenfold greater than that of the astrocyte transporters GLAST andGLT-1 (ref. 10).

Glutathione is important for the metabolism of hydrogen peroxide(H2O2), nitric oxide and other reactive oxygen species and for themaintenance of reduced thiol groups on proteins23. Pharmacologicallyinduced glutathione deficiency causes neurodegeneration24, and low-ered glutathione content is found in neurodegenerative disordersassociated with oxidative stress25, suggesting that impairment inneuronal cysteine uptake could lead to neurodegeneration. Here wereport that mice deficient in EAAC1 are deficient in neuronal thiolcontent and develop age-dependent behavioral abnormalities and brainatrophy. Neurons in hippocampal slices from EAAC1–/– mice showedincreased vulnerability to oxidants (but not to glutamate) and reducedcapacity to metabolize reactive oxygen species. Neuronal thiol contentand resistance to oxidant stress were normalized in the EAAC1–/– mice

Received 13 September; accepted 28 October; published online 27 November 2005; doi:10.1038/nn1609

1Department of Neurology, University of California San Francisco, San Francisco, California 94143, USA. 2Veterans Affairs Medical Center, 4150 Clement Street,San Francisco, California 94121, USA. 3Department of Neurosurgery, University of California San Francisco, San Francisco, California 94143, USA. Correspondenceshould be addressed to R.A.S. (ray@itsa.ucsf.edu).

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by the administration of N-acetylcysteine (NAC), a membrane-permeable cysteine precursor that does not require active transport.These findings suggest that EAAC1 functions as a neuronal cysteinetransporter and that dysfunction of this system leads to impairedglutathione homeostasis and neurodegeneration.

RESULTS

We determined mouse genotype by polymerase chain reaction (PCR)and confirmed genotype results by western blotting and immunostain-ing for EAAC1 protein expression (Fig. 1). Western blots showedEAAC1 immunoreactivity at the predicted molecular weight(B63 kDa)2,26 in the brains of wild-type mice (Fig. 1b) and noimmunoreactivity in the brains of EAAC1–/– mice. Similarly, immu-nostaining of hippocampal sections showed that EAAC1 was expressedon neuronal cell membranes of wild-type but not EAAC1–/– mice(Fig. 1c). Previous studies have found no change in the expression ofthe major astrocyte glutamate transporters GLT-1 and GLAST inresponse to EAAC1 gene deficiency6 or downregulation7, but expres-sion of these transporters has not been examined in the aged EAAC1–/–

mouse brain. Here, western blots for GLT-1 and GLAST showed nodifference between the wild-type mice and EAAC1–/– mice at either7 weeks (data not shown) or 11 months of age (Fig. 1d), as determinedby densitometry.

Behavioral abnormalities in the EAAC1–/– mice

In maintaining the EAAC1–/– mouse colonies, it became apparent thatthe older mice showed increased aggressiveness and impaired self-grooming compared to age-matched wild-type mice. We studied theirbehavioral changes further with the Morris water maze test27. Theperformance of the wild-type and EAAC1–/– mice was similar at7 weeks of age, showing progressively shortened target latency withrepeated trials on both the visible platform and the hidden platformtasks. By contrast, 11-month-old EAAC1–/– mice did not improve oneither task with repeated trials (Fig. 2a). This impairment was not dueto gross visual disturbances, because the aged EAAC1–/– mice reachednormally for nearby small surfaces when suspended by the tail.Spontaneous locomotor activity was also not significantly altered inthe aged EAAC1–/– mice (data not shown). Spontaneous swim velocitywas slower in the aged EAAC1–/– mice (Fig. 2b), but not slow enough toaccount for the failure to shorten the target latency with repeated trials.Notably, the aged EAAC1–/– mice differed from the other groups in thattheir spontaneous swim speed was well below their maximal swimspeed. Together, these observations suggest cognitive or motivationalimpairment in the aged EAAC1–/– mice.

Brain atrophy and oxidative stress in EAAC1–/– mice

A comparison of coronal sections from wild-type and EAAC1–/– mousebrains showed age-dependent cortical thinning and ventricular enlar-gement in the EAAC1–/– mice. Wild-type mice showed a small increasein ventricular size between the ages of 7 weeks and 11 months (Fig. 3);by contrast, EAAC1–/– mice showed slightly larger ventricle size thanthe wild-type mice at 7 weeks and much larger ventricle size at11 months. Accordingly, measures of the hippocampal CA1 cell layerand the corpus callosum both showed reduced size in the agedEAAC1–/– mice (Fig. 3).

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b Figure 1 Genotyping and glutamate transporter expression. (a) PCR analysis

of genomic DNA shows loss of the EAAC1 band and presence of the NEO

cassette in the outbred EAAC1–/– mouse. (b) Western blots show the major

EAAC1 band at 63 kDa in the wild-type brain and no immunoreactivity in the

EAAC1–/– mouse brain. (c) Immunostaining of the hippocampal CA1 region

shows EAAC1 expression localized to neuronal cell membranes in the wild-

type brain and no signal from the EAAC1–/– mouse brain. Scale bar, 40 mm.

(d) Western blots for brain GLT-1 and GLAST expression in 11-month-oldwild-type and EAAC1–/– mice.

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Wild type EAAC1–/–Figure 2 Performance on the Morris water maze test. (a) At age 7 weeks,

time to reach platform (latency) and rate of latency change was similar in the

wild-type and EAAC1–/– mice during both visible and hidden platform

sessions. For both the visible and hidden platform tasks, the 11-month-old

EAAC1–/– mice showed profound impairment (P o 0.01) as compared to the

7-week- and 11-month-old wild-type mice and the 7-week-old EAAC1–/– mice(n ¼ 10). The dashed and dotted lines indicate the mean rate of change

over the designated testing intervals. (b) Spontaneous swim velocity was

moderately reduced in the 11-month-old EAAC1–/– mice relative to the

7-week-old EAAC1–/– and wild-type mice and the 11-month-old wild-type

mice (P o 0.01, n ¼ 10).

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The age-dependent brain atrophy in the EAAC1–/– mice was accom-panied by markers of oxidative stress. Immunoreactivity for nitro-tyrosine and 4-hydroxy-2-nonenal (HNE), which are formed byoxidant interactions with proteins and lipids, respectively, wasincreased in the aged EAAC1–/– mouse brains. The increase wasprominent in the hippocampal cell fields and the cerebral cortex(Fig. 4). Both nitrotyrosine and HNE localized to neurons in theaged EAAC1–/– mouse brains (Fig. 4). These markers were not detectedin the corpus callosum or other white matter tracts (SupplementaryFig. 1 online).

Increased vulnerability of EAAC1–/– neurons to oxidants

We prepared hippocampal slices from young (6–8 week) wild-type andEAAC1–/– mice to study possible mechanisms by which EAAC1 genedeficiency could lead to the observed age-dependent changes. BecauseEAAC1 can function as both a glutamate and a cysteine transporter10,11,we used hippocampal slices to compare the vulnerability of the wild-type and EAAC1–/– mouse brains to glutamate and oxidant exposures.Neurons in slices from the EAAC1–/– mice did not show increasedvulnerability to glutamate over a range of bath glutamate concentra-tions (Fig. 5). This result is consistent with earlier studies reporting a

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Figure 3 Brain atrophy in the EAAC1–/– mice. (a,b) Coronal brain sections show cortical thinning and ventricular enlargement in the older EAAC1–/– mice. The

top rows are at the level of the anterior commissure and the bottom rows are 2.7 mm posterior to the anterior commissure. Scale bar, 2 mm in a; 1 mm in b.

(c) Ventricle size is larger in the EAAC1–/– mice at age 7 weeks, and the difference is further increased at age 11 months (n ¼ 6–14). (d) Hematoxylin-eosin

staining of the CA1 hippocampal cell field; scale bar, 40 mm. (e) Fluoro-myelin staining of the corpus callosum (green) in coronal section; scale bar, 100 mm.

Nuclear counterstaining (red) of the pyramidal cell layer caught obliquely on these sections is included for scale and orientation. (f) Quantified measures of the

CA1 cell layer and corpus callosum (n ¼ 3–6). *P o 0.05; **P o 0.01. Error bars denote s.e.m.

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Figure 4 Oxidative stress in neurons of EAAC1–/– mouse brain. (a,b) Immunostaining for (a) nitrotyrosine and (b) HNE showed increased immunoreactivity in

neurons of cerebral cortex and hippocampal CA1 and CA3 cell fields in 11-month-old EAAC1–/– mice. Scale bar, 40 mm. (c,d) Immunostaining for nitrotyrosine

and HNE, colocalized with the neuronal marker microtubule-associated protein-2 (MAP-2). Representative of three brains in each group.

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negligible role for neuronal glutamate transporters in regulating extra-cellular glutamate concentrations1,9. By contrast, the neurons inEAAC1–/– mice were several times more sensitive to H2O2 and to3-morpholinosydnonimine (SIN-1), which generates superoxide, nitricoxide, peroxynitrite and related oxygen species28.

Cysteine uptake is a rate-limiting step in neuronal synthesis ofglutathione21,23,29, and glutathione has a central role in the metabolismof both peroxide and nitrosyl oxidants23,30,31. SIN-1 greatly increasedneuronal nitrotyrosine immunoreactivity in brain slices fromEAAC1–/– mice under conditions that produced a negligible increasein slices from the wild-type mice (Fig. 6a,b), suggesting an impairedcapacity for the scavenging of nitrosyl radicals in the EAAC1–/– mousebrain31. We performed parallel studies with hippocampal slices fromwild-type mice that had been treated with buthionine sulfoximine(BSO) to reduce brain glutathione content32. Slices from these mice

also showed increased nitrotyrosine formation (Fig. 6a,b), supportingthe possibility that EAAC1 gene deficiency leads to impaired neuronalglutathione synthesis. To further assess this possibility, hippocampalslices from EAAC1–/–, wild-type and BSO-treated wild-type mice wereevaluated with 5-(and 6-)carboxy-2¢,7¢-dichlorodihydrofluoresceindiacetate (DCF), which is oxidized to a fluorescent compound byoxygen species derived from H2O2 or peroxynitrite33. We observed amodest increase in neuronal DCF fluorescence in the wild-type slicesduring incubation with H2O2 or SIN-1 and much larger increases (10-to 40-fold) in the slices from EAAC1–/– mice and BSO-treated wild-typemice (Fig. 6c,d). The effect of BSO pretreatment was slightly less thanthe effect of the EAAC1–/– genotype.

We measured bulk glutathione content in brain homogenates fromEAAC1–/–, wild-type and BSO-treated wild-type mice (Fig. 6e). Thesemeasurements are likely to underestimate the degree of glutathionedeficiency in the EAAC1–/– neurons because a large share of brainglutathione is localized to astrocytes, which do not express EAAC1(ref. 23). Similarly, because the decrease in glutathione in the BSO-treated mice reflects impaired glutathione synthesis in both neuronsand astrocytes, the comparable glutathione reductions observed in theEAAC1–/– mouse brains and BSO-treated mouse brains may indicate amuch greater neuronal glutathione depletion in the EAAC1–/– mice.Glutathione measurements from the livers of wild-type and EAAC1–/–

mice gave comparable values (mean ± s.e.m.)—1.60 ± 0.12 and 1.50 ±0.06 mmol per mg of protein, respectively (n ¼ 4)—suggesting that thereduced glutathione levels in the EAAC1–/– mouse brains results fromlocal rather than systemic effects of EAAC1 gene deficiency.

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Figure 5 Increased vulnerability of neurons in EAAC1–/– mice to oxidative

stress. (a) Neuron death in hippocampal slice preparations was identified by

PI fluorescence. PI fluorescence was evaluated at 0 or 4 h under control

conditions, or 3.5 h after a 30-min incubation with glutamate, hydrogen

peroxide or SIN-1; scale bar, 40 mm. (b) There was a severalfold increased

sensitivity to SIN-1 and H2O2 in the slices from EAAC1–/– mice, but no

increased sensitivity to glutamate. **P o 0.01; n ¼ 3–5. Error bars

represent s.e.m.

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Figure 6 Reduced scavenging of reactive oxygen species in neurons of EAAC1–/– mice.

(a) Hippocampal slices were prepared from EAAC1–/– mice, wild-type mice or wild-type mice

that had been treated with BSO to reduce brain glutathione content. The slices were evaluated

for nitrotyrosine immunoreactivity after incubation with SIN-1; scale bar, 40 mm. (b) SIN-1

produced a small increase in nitrotyrosine immunoreactivity in the wild-type brain slices

(P o 0.01) and a much larger increase in slices from EAAC1–/– mice and BSO-treated wild-

type mice (**P o 0.01, n ¼ 4). (c) The presence of reactive oxygen species was evaluatedwith DCF after 30-min incubations with H2O2 or SIN-1; bar, 40 mm. (d) Both of the oxidants

produced a small increase in the neuronal DCF signal in the wild-type hippocampal slices

(P o 0.01), and both oxidants produced much larger increases in slices from EAAC1–/– mice

and BSO-treated wild-type mice. (P o 0.01, n ¼ 4). (e) Brain glutathione content was reduced

in the EAAC1–/– mouse brains and in wild-type mice treated with BSO (**P o 0.01, n ¼ 4–8).

Error bars denote s.e.m.

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Oxidant effects on neurons are unaffected by bicuculline

The uptake of glutamate by EAAC1 provides a substrate for GABAformation in GABAergic neurons9, raising the possibility that EAAC1deficiency could promote oxidant stress in neurons by reducingGABAergic tone. GABA itself has no significant antioxidant properties,but reduced activation of GABAA receptors could, in principle,indirectly amplify oxidant effects on neurons by increasing neuronaldepolarization, NMDA receptor activation and glutamate release34,35.The comparable neurotoxicity of glutamate in brain slices from wild-type and EAAC1–/– mice (Fig. 5) suggests that this effect, if present,must be small; but to directly test this possibility, we examined theeffect of the GABAA receptor antagonist (+)-bicuculline on theneuronal response to oxidants in the brain slice preparation. Weobserved no effect of 20 mM bicuculline36 on DCF fluorescence inwild-type slices after incubation with SIN-1 or H2O2 (Fig. 7a–d).Bicuculline also had no effect on neuronal survival after incubationwith SIN-1 or H2O2 (Fig. 7e–h).

NAC normalizes neuronal glutathione in EAAC1–/– mice

To determine directly whether EAAC1 is involved in neuronal glu-tathione homeostasis, we used fluorescently tagged C5 maleimide toquantify reactive thiol content (of which glutathione is the principalcomponent) in hippocampal slices from wild-type and EAAC1–/– mice.As expected, the C5 maleimide fluorescence was markedly reduced inneurons from the hippocampal slices of EAAC1–/– mice (Fig. 8a). NACcan passively cross lipid membranes and thereby provide cysteine tocells that lack cysteine transport37,38. The C5 maleimide signal was

normalized in EAAC1–/– mice given NAC 5 h before brain harvest(Fig. 8a). To confirm that this thiol signal was due to glutathione ratherthan to NAC or cysteine, we also prepared slices from EAAC1–/– micethat were given BSO along with NAC to prevent de novo synthesis ofglutathione. The effect of NAC was blocked in these mice (Fig. 8a),confirming that the C5 maleimide signal is primarily attributable toglutathione. Biochemical measures of glutathione further confirmedthat the NAC-induced increase in brain glutathione content wasblocked by BSO and that the BSO treatment did not deplete pre-existing glutathione stores over this time interval (Fig. 8b). Asexpected, neuronal death after oxidant exposure was decreased in slicesfrom NAC-treated EAAC1–/– mice relative to untreated EAAC1–/– mice(Figs. 5 and 8), and this decrease was negated by the coadministrationof BSO (Fig. 8c,d).

DISCUSSION

The original description of the EAAC1–/– mouse reported reducedspontaneous activity but no gross neurodegeneration6. Our studies,using descendants of these mice, similarly showed no gross neuro-degeneration at young ages, but did show brain atrophy and pro-nounced behavioral abnormalities by 11 months of age. These changeswere accompanied by histochemical markers of neuronal oxidativestress, and experiments with acutely prepared hippocampal slicesconfirmed an impaired neuronal resistance to reactive oxygen species.The capacity of EAAC1 to function as a cysteine transporter raised thepossibility that these abnormalities could be caused by impairedneuronal glutathione metabolism, and we confirmed decreased reactive

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Figure 7 Bicuculline does not potentiate the oxidant effects of H2O2 or SIN-1. (a–h) The presence of reactive oxygen species in wild-type hippocampal

cultures was evaluated with DCF after 30-min incubations with H2O2 (a,c) or SIN-1 (b,d) in the presence and absence of 20 mM bicuculline. Neuron death

in wild-type hippocampal slice preparations was assessed by PI fluorescence after incubation with H2O2 (e,g) or SIN-1 (f,h) in the presence and absence of

20 mM bicuculline. Scale bar, 40 mm; n ¼ 4 under each condition. Error bars denote s.e.m.

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thiol content in hippocampal neurons of the EAAC1–/– mice. Theadministration of the cell-permeable cysteine precursor NAC correctedboth the neuronal thiol content and neuronal oxidant-scavengingcapacity. The effects of NAC on oxidant-scavenging capacity are dueprimarily to its role as a substrate for glutathione synthesis39,40; weconfirmed this here by showing that the effects of NAC are negated bythe simultaneous administration of BSO.

Immunostaining showed nitrotyrosine and HNE accumulation inneurons in the EAAC1–/– mouse brain, consistent with the localizationof EAAC1 selectively to neurons. The measured loss of neurons in thehippocampal CA1 cell layer was small, however, relative to theincreased ventricular volume. This discrepancy may be explained inpart by associated axonal loss, as demonstrated by the narrowing of thecorpus callosum in the aged EAAC1–/– mice. In addition, there may besecondary loss of glial elements contributing to brain atrophy.

EAAC1 is unique among the cysteine transporters in that itstransport of substrates is coupled to the transport of three Na+ ions,thereby enabling uptake against a steep concentration gradient41. Thepresent findings show that EAAC1 is quantitatively important forneuronal glutathione homeostasis but do not exclude alternative routesof neuronal cysteine uptake that may partially compensate for EAAC1deficiency. In fact, a complete loss of cysteine uptake would not becompatible with prolonged neuronal survival.

EAAC1 is also expressed in several extraneural tissues6. In the kidney,EAAC1 is expressed by renal tubule cells, in which it serves as a majorroute of glutamate and aspartate re-uptake from the urine. EAAC1–/–

mice show dicarboxylic aminoaciduria6, and it is possible that thismetabolic defect could influence neuronal glutathione metabolismindirectly. However, the rapid normalization of neuronal glutathionewith NAC identifies cysteine uptake as the limiting step for glutathionesynthesis in EAAC1–/– mice. Similarly, the normal glutathione contentmeasured in the liver of the EAAC1–/– mouse argues against a systemicdeficiency of sulfur amino acids.

A notable feature of the EAAC1–/– mouse is that the brain atrophyand behavioral abnormalities are markedly age dependent, despite thefact that brain slices obtained from young mice showed large biochem-ical abnormalities when compared to slices from wild-type mice. Thedelayed onset of gross atrophy and behavioral changes may be due to

other interacting factors associated with aging or, alternatively, to theaccumulated effect of a prolonged impairment in neuronal glutathionehomeostasis. In either case, the parallel between the delayed onset ofdisease in these mice and the delayed onset of most human neurode-generative disorders suggests that common mechanisms could beinvolved. There have been case reports of neurological developmentaldelay associated with dicarboxylic aminoaciduria42, but a humangenetic EAAC1 deficiency has not been identified. EAAC1 expressionis, however, regulated by several factors, including protein kinase C43,cholesterol44, presenilin26 and others. It will be of interest to learnwhether the magnitude of these regulatory influences is sufficient toalter neuronal glutathione metabolism.

METHODSStudies were performed in accordance with a protocol approved by the Animal

Use Committee at the San Francisco Veterans Affairs Medical Center. Reagents

were obtained from Sigma-Aldrich except where noted.

EAAC1–/– mice. The EAAC1–/– mice were descendants of the strain established

in a previous study6, in which exon 1 is disrupted by a neomycin resistance

(NEO) cassette. These mice were outbred to wild-type CD-1 mice for six or

more generations before these studies. Littermate and age-matched wild-type

CD-1 mice were used as controls.

Genotyping. Genotypes were confirmed by PCR of tail DNA, using oligonu-

cleotide primers pairs for EAAC1 exon 1 and for the inserted NEO cassette:

5¢-CCGCCACGCAAAACCACCGTGCTCGGTCCC-3¢ (EAAC1.1), 5¢-CTAG

TACCACGGCGGCCACGGTTGAGAGCA-3¢ (EAAC1.2), 5¢-CATTCGACCAC

CAAGCGAAAC-3¢ (NEO.1) and 5¢-CAGCAATGTCACGGGTAGCCAAC-3¢(NEO.2). The amplified products were separated by 1.5% agarose gel electro-

phoresis and visualized by ethidium bromide staining.

Western blotting. Western blotting was performed as described pre-

viously45. Rabbit antibodies to EAAC1, GLAST or GLT-1 (Alpha Diagnostic

International) were used at 0.5 mg ml–1, and mouse monoclonal antibody to

b-actin (Sigma) was used at a 1:1,000 dilution. The antibodies were visualized

by chemiluminescence after incubation with horseradish peroxidase–

conjugated antibody to rabbit IgG. Optical densities of the protein bands

were measured using the NIH ImageJ software program. The protein band

densities were normalized in each case to the density of the b-actin band from

the same sample.

NAC2.0

1.5

1.0

0.5

0

Glu

tath

ione

(µm

ol p

er m

g pr

otei

n)

0

Time (h)

20 ** **

**

EAAC1–/–

+ NAC15

10

5

0

0 h

cont

rol

4 h

cont

rol

4 h

SIN-1

500

µM

4 h

H 2O 2

200

µM

Flu

ores

cenc

e in

tens

ity(a

rbitr

ary

units

)

4 6

BSONAC + BSO

EAAC1–/–

+ NAC + BSO

Wild type EAAC1–/–

EAAC1–/– + NACEAAC1–/–

+ NAC + BSO

NAC

EAAC1–/–

NAC + BSO NAC

EAAC1–/–

NAC + BSO

0 hcontrol

4 hcontrol

4 h H2O2200 µM

4 h SIN-1500 µM

a b

d

c

Figure 8 Neuronal glutathione deficiency and vulnerability to oxidant stress in EAAC1–/– mice are both

reversed by NAC. (a) Reactive thiols were evaluated in brain slices using C5-maleimide fluorescence. There

was reduced fluorescence in the neurons in EAAC1–/– slices relative to those in the wild-type slices. This

signal was increased in slices from mice treated with the cell-permeant cysteine precursor NAC, but not in

slices from mice treated with BSO together with NAC to prevent glutathione synthesis. n ¼ 3 in each

group. (b) Biochemical determination of glutathione in EAAC1–/– mouse brains after treatment with NAC or

NAC plus BSO confirmed that the effect of NAC on glutathione levels is attenuated by the concomitant

administration of BSO. *P o 0.05 versus control, n ¼ 3–7. (c,d) PI staining in hippocampal slices

prepared from NAC-treated EAAC1–/– mice showed increased neuronal resistance to H2O2 or SIN-1compared to untreated EAAC1–/– mice (Fig. 5). This effect of NAC was blocked by the coadministration of

BSO. **P o 0.01, n ¼ 3. Error bars denote s.e.m.

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Immunostaining. Brain sections were prepared and immunostained as

described45. The primary antibodies were 5 mg ml–1 rabbit antibody to EAAC1

(Alpha Diagnostic International); 1:500 dilution rabbit antibody to nitro-

tyrosine (Chemicon); and 1:500 dilution rabbit antibody to HNE (Alpha

Diagnostic International). After washing, the slices ware incubated with a

1:500 dilution of Alexa Fluor 488–conjugated goat anti-rabbit IgG (Molecular

Probes). Confocal photomicrographs were acquired using 10-nm optical

thickness sections.

Brain morphology measurements. Ventricle area was measured on three

consecutive 30-mm coronal slices taken at the level of the anterior commissure,

and on an additional three slices taken 2.7 mm posterior to the anterior

commissure. Width of the CA1 cell layer in each mouse was averaged from

measurements from three consecutive hematoxylin-eosin–stained slices. Width

of the corpus callosum was measured on three consecutive slices stained with

lipophilic dye fluoromyelin (Molecular Probes) and counterstained with

propidium iodide (PI). Slice images were imported to Photoshop software

for the image analysis, and measurements from the three consecutive slices were

averaged for each data point ‘n’.

Behavioral studies. A total of 40 mice were studied, in two groups of 20. The

first group consisted of 10 wild-type mice and 10 EAAC1–/– mice of age 11–12

months. The second group consisted of 10 wild-type mice and 10 EAAC1–/–

mice of age 6–8 weeks. To minimize the effects of social influences on behavior,

mice were housed individually in the testing room beginning 3 d before testing.

As a first step, spontaneous open field activity was assessed to establish any

locomotor differences. On each of three consecutive days, open field activity

was recorded for 10 min after an initial 1-min adaptation period. Subsequently,

spatial learning and memory were evaluated by the Morris water maze task, as

previously detailed46. The mice were trained first to locate a visible platform

(days 1 and 2), and then to locate a hidden platform (days 3 through 5). The

mice received two training sessions per day, each consisting of three 1-min trials

with a 10-min intertrial interval. Swim speed and time to reach the platform

(latency) was recorded with an EthoVision video tracking system (Noldus

Information Technology). Mice that did not reach the platform within 60 s

were manually placed on the platform and assigned a latency interval of 60 s.

Hippocampal slice preparation. Brains from wild-type and EAAC1–/– mice,

6–8 weeks of age, were vibratome-sectioned into 300-mm coronal slices. The

slices were placed in ice-cold artificial cerebrospinal fluid (aCSF) containing

130 mM NaCl, 3.5 mM KCl, 1.25 mM NaH2PO4, 2 mM MgSO4, 2 mM CaCl2,

20 mM NaHCO3 and 10 mM glucose at pH 7.2 while equilibrated with 95%

oxygen and 5% CO2. The aCSF osmolality was 290 ± 15 mOsm kg–1 as

determined by Wescor vapor pressure osmometer. All studies were done using

brain slices from wild-type and EAAC1–/– mice treated in parallel. Experiments

were initiated by transferring the slices to a circulating bath of aCSF at 30 1C

that was continuously bubbled with 95% oxygen and 5% CO2. Glutamate and

oxidants were added for 30-min incubation intervals. Slices were then trans-

ferred to fresh aCSF and maintained at 22 1C until harvested for cell death or

immunostaining 3.5 h later. Where designated, 20 mM (+)-bicuculline (Tocris)

was incubated with the slices beginning 30 min before the H2O2 or SIN-1

exposures to block GABAA receptor function36,47.

Cell death determinations. Cell death in the brain slices was assessed as

described previously48, with minor changes. Slices were incubated for 3 min

with 5 mM PI, washed, fixed in 4% paraformaldehyde and stored in the dark at

4 1C. Digitized images were prepared with a confocal fluorescent microscope

within 24 h of staining. The PI signal intensity was measured in the CA1 cell

body layer of each slice using a ‘region of interest’ mask of constant size. The

background signal was measured from the stratum radiatum of each slice,

which contained no PI fluorescence, and subtracted from the value obtained in

the CA1 region. Values from three slices per brain were averaged for each ‘n’.

Measurement of reactive oxygen species in hippocampal slices. Hippocampal

slices were pre-incubated in 10 mM 5-(and 6-)carboxy-2¢,7¢-dichlorodihydro-

fluorescein diacetate (DCF; Molecular Probes) for 30 min to allow intracellular

loading. After washing with aCSF, slices were exposed to H2O2 or SIN-1 for

30 min. The slices were immediately photographed to quantify DCF fluores-

cence or fixed in 4% paraformaldehyde for nitrotyrosine immunostaining. DCF

fluorescence and nitrotyrosine immunofluorescence was quantified by the same

method as used for PI fluorescence.

Manipulation and measurement of glutathione level. BSO was injected at a

dose of 660 mg kg–1 i.p. twice a day for 4 d to reduce brain levels of glutathione

in wild-type mice32. NAC was administered as a single 150 mg kg–1 intra-

peritoneal injection 4–6 h before brain harvest to elevate neuronal glutathione

levels in EAAC1–/– mice12,39,49. In some mice, the NAC injection was accom-

panied by BSO (1320 mg kg–1), to prevent de novo glutathione formation.

Biochemical glutathione determinations were performed by the NADPH-

dependent glutathione reductase method, as described previously21. In situ

evaluation of reactive thiols, of which glutathione is the dominant species, was

performed using a fluorescent maleimide derivative50. After incubations under

the designated conditions, hippocampal slices were washed with aCSF, fixed in

4% paraformaldehyde, and incubated overnight at 4 1C with 2.5 mM Alexa

Fluor 488 C5 maleimide (Molecular Probes) in phosphate-buffered saline

containing 2% goat serum, 0.2% Triton X-100 and 0.1% bovine serum

albumin. The slices were photographed with a confocal microscope and the

images quantified as described for PI fluorescence.

Statistics. Data are expressed as means ± s.e.m. The behavioral studies were

assessed using repeated-measures analysis of variance (ANOVA). The brain

morphology and biochemical measurements were assessed by ANOVA and the

Bonferroni test for multiple group comparisons. The fluorescent indicators

used for cell death, immunostaining and thiol determinations were evaluated

with the Kruskal-Wallis test followed by Dunn’s test for multiple comparisons

between the wild-type and EAAC1–/– preparations.

Note: Supplementary information is available on the Nature Neuroscience website.

ACKNOWLEDGMENTSWe thank D. Burns for technical assistance and M. Yenari and S. Massa forcritical reading of the manuscript. This work was supported by grants fromthe US National Institutes of Health and the Department of Veterans Affairs.

COMPETING INTERESTS STATEMENTThe authors declare that they have no competing financial interests.

Published online at http://www.nature.com/natureneuroscience/

Reprints and permissions information is available online at http://npg.nature.com/

reprintsandpermissions/

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