Oxidative stress on EAAC1 is involved in MPTP-induced glutathione depletion and motor dysfunction

Oxidative stress on EAAC1 is involved in MPTP-inducedglutathione depletion and motor dysfunction

Koji Aoyama, Nobuko Matsumura, Masahiko Watabe and Toshio NakakiDepartment of Pharmacology, Teikyo University, School of Medicine, 2-11-1 Kaga, Itabashi, Tokyo 173-8501, Japan

Keywords: amino acid transporters, n-acetylcysteine, oxidative stress, Parkinson’s disease

Abstract

Excitatory amino acid carrier 1 (EAAC1) is a glutamate transporter expressed on mature neurons in the CNS, and is the primary routefor uptake of the neuronal cysteine needed to produce glutathione (GSH). Parkinson’s disease (PD) is a neurodegenerative disorderpathogenically related to oxidative stress and shows GSH depletion in the substantia nigra (SN). Herein, we report that 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated mice, an experimental model of PD, showed reduced motor activity, reduced GSHcontents, EAAC1 translocation to the membrane and increased levels of nitrated EAAC1. These changes were reversed by pre-administration of n-acetylcysteine (NAC), a membrane-permeable cysteine precursor. Pretreatment with 7-nitroindazole, a specificneuronal nitric oxide synthase inhibitor, also prevented both GSH depletion and nitrotyrosine formation induced by MPTP.Pretreatment with hydrogen peroxide, l-aspartic acid b-hydroxamate or 1-methyl-4-phenylpyridinium reduced the subsequentcysteine increase in midbrain slice cultures. Studies with chloromethylfluorescein diacetate, a GSH marker, demonstrateddopaminergic neurons in the SN to have increased GSH levels after NAC treatment. These findings suggest that oxidative stressinduced by MPTP may reduce neuronal cysteine uptake, via EAAC1 dysfunction, leading to impaired GSH synthesis, and that NACwould exert a protective effect against MPTP neurotoxicity by maintaining GSH levels in dopaminergic neurons.

Introduction

Parkinson’s disease (PD) is a progressive, late-onset disorder resultingfrom dopaminergic neurodegeneration in the substantia nigra (SN).Although the precise pathogenesis of PD is still unclear, oxidativestress plays an important role in the underlying mechanism (Halliwell,1992). In patients with PD, the SN shows high levels of oxidativeby-products (Dexter et al., 1989; Yoritaka et al., 1996; Alam et al.,1997) and iron (Dexter et al., 1987), which can react with hydrogenperoxide (H2O2) via the Fenton reaction to form hydroxyl radicals(Youdim et al., 1989) and low glutathione (GSH) levels (Perry &Yong, 1986; Sian et al., 1994).GSH plays a critical role in protecting cells from oxidative stress

and xenobiotics, as well as maintaining the thiol redox state. However,brain GSH declines with ageing (Maher, 2005), and GSH depletionenhances oxidative stress leading to neuronal degeneration (Schulzet al., 2000; Bharath et al., 2002). Although patients with PDexclusively show GSH loss in the SN, the precise mechanism has notyet been clarified.GSH is a tripeptide composed of glutamate, cysteine and glycine.

Cysteine is the rate-limiting substrate for GSH synthesis in neurons(Dringen et al., 1999). In primary neuron culture, approximately 90% oftotal cysteine uptake is mediated by sodium-dependent systems, mainlyexcitatory amino acid transporters (EAATs), also known as systemXAG- (Shanker et al., 2001; Chen & Swanson, 2003; Himi et al.,2003b). There are five EAATs, termed GLAST, GLT-1, EAAC1,EAAT4 and EAAT5 (Danbolt, 2001). GLAST and GLT-1 are localizedprimarily to astrocytes; EAAC1, EAAT4 and EAAT5 to neurons.EAAT4 and EAAT5 are restricted to cerebellar Purkinje cells and the

retina, respectively, whereas EAAC1 is widely expressed in the CNS(Maragakis & Rothstein, 2004). Knockdown expression of GLAST orGLT-1 in rats using antisense oligonucleotides increased the extra-cellular glutamate concentration, whereas EAAC1 knockdown had noeffect on extracellular glutamate (Rothstein et al., 1996). Astrocyteglutamate transporters are limited to glutaminergic synapses, whereasEAAC1 is detected diffusely over cell bodies and processes (Rothsteinet al., 1994). These findings suggest that clearing extracellular glutamateis not a major role of EAAC1. EAAC1 can also transport cysteine at arate comparable to that of glutamate, with an affinity 10–20-fold higherthan that of GLAST or GLT-1 (Zerangue & Kavanaugh, 1996).A recent study demonstrated age-dependent neurodegeneration withdecreased GSH content, increased oxidant levels and increasedsusceptibility to oxidative stress in EAAC1-deficient mice (Aoyamaet al., 2006). Notably, these EAAC1-deficient mice also showed anage-dependent decrease in neuronal number in the SN (Chan et al.,2005; Berman et al., 2007). However, to our knowledge there havebeen no studies examining EAAC1 in any of the PD models.1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) is known as

an exogenous neurotoxin, which induces mitochondrial dysfunctionleading to increased oxidative stress, dopamine depletion in thestriatum and parkinsonism (Langston et al., 1983).Herein, we report that oxidative stress may reduce neuronal cysteine

uptake via EAAC1 leading to impaired GSH synthesis in the MPTPmouse model of PD.

Materials and methods

Animals

C57BL ⁄ 6 male mice, 4–5 and 8–10 weeks old, were used for sliceculture and MPTP experiments, respectively. All mice were kept in a

Correspondence: Dr T. Nakaki, as above.E-mail: [email protected]

Received 20 June 2007, revised 15 October 2007, accepted 6 November 2007

European Journal of Neuroscience, Vol. 27, pp. 20–30, 2008 doi:10.1111/j.1460-9568.2007.05979.x

ª The Authors (2007). Journal Compilation ª Federation of European Neuroscience Societies and Blackwell Publishing Ltd

temperature-controlled room at 23 �C under a 12 h light : dark cyclewith food and water available ad libitum. The protocols were approvedby the Animal Ethics Committee of Teikyo University School ofMedicine.

MPTP intoxication

TheMPTPgroup (n ¼ 10) received intraperitoneal injections ofMPTP-HCl (25 mg ⁄ kg ⁄ day, Sigma Aldrich, Tokyo, Japan) for 5 days(cumulative MPTP dose of 125 mg ⁄ kg). The control group (n ¼ 10)received the same volume of saline intraperitoneally. The n-acetylcys-teine (NAC) ⁄ MPTP group (n ¼ 10) received NAC (150 mg ⁄ kg ⁄ day,Sigma Aldrich) intraperitoneally at 3 h before the MPTP injection(25 mg ⁄ kg ⁄ day) for 5 days. The NAC group (n ¼ 8) received NAC(150 mg ⁄ kg ⁄ day) intraperitoneally for 5 days. Mice (n ¼ 7) alsoreceived intraperitoneal injections of 7-nitroindazole (7-NI, 50 mg ⁄ kg,Sigma Aldrich) 30 min before each MPTP injection (25 mg ⁄ kg ⁄ day)for 5 days.

Rotarod test

Motor activities of mice were measured using the KN-75 Rotarodsystem (Natsume, Tokyo, Japan). This system has individualcompartments for each mouse and an automatic revolving rod(diameter 9 cm). Animals were placed daily on a stationary rod for5 min and then trained to stay on it as the rod rotated at 10 r.p.m. for30 s (training session). Trial sessions were undertaken on Days 1, 3, 5and 7. The trials were started at a speed of 15 r.p.m., the minimumneeded to keep the mice walking on the rod, in order to establishmaximal performance at 4 min. The mean time on the rod wascalculated for three consecutive trials. The trial sessions were skippedin the event of failure to achieve the training session goal on any givenday.

Tissue preparation

The mouse brains were removed on Day 7 or on Day 21 for samplepreparation. Mice were anesthetized with diethyl ether and perfusedwith cold saline. The brain was removed from the skull and fixed for24 h in 4% paraformaldehyde followed by 30% sucrose for immu-nohistochemistry. For GSH assay and Western blotting, midbrainswere homogenized with 5% sulfosalicylic acid or fraction buffer[0.25 m sucrose ⁄ 50 mm Tris-HCl (pH 7.2) ⁄ 1 mm EDTA ⁄ 1 mm dith-iothreitol (DTT) ⁄ 3.3 mm CaCl2 ⁄ 5 mm MgCl2 ⁄ 10 mm KCl ⁄ 1 mm

phenylmethylsulfonyl fluoride (PMSF) ⁄ 5 lg ⁄ mL leupeptin, pepstatinand aprotinin], respectively. For immunoprecipitation, samples werehomogenized with RIPA buffer [50 mm Tris-HCl (pH 7.2) ⁄ 150 mm

NaCl ⁄ 1% NP-40 ⁄ 0.25% sodium deoxycholate ⁄ 1 mm EDTA ⁄ 1 mm

PMSF ⁄ 1 mm NaF ⁄ 1 mm Na3VO4 ⁄ 5 lg ⁄ mL of leupeptin, pepstatinand aprotinin].

Preparation of plasma membrane fraction

The mouse midbrain was gently homogenized using a Dounce-typehomogenizer, in cold suspension buffer [250 mm sucrose ⁄ 50 mm

Tris-HCl (pH 7.2) ⁄ 3.3 mm CaCl2 ⁄ 5 mm MgCl2 ⁄ 10 mm KCl ⁄ 1 mm

DTT ⁄ 1 mm EDTA ⁄ 1 mm PMSF ⁄ 5 lg ⁄ mL of leupeptin, pepstatinand aprotinin]. The lysate was centrifuged at 1200 g for 10 min at4 �C, and the supernatant was then centrifuged again at 8000 g for10 min at 4 �C. The supernatant was further centrifuged at 105 000 g

for 60 min at 4 �C. The precipitant was used as a sample for Westernblotting.

Total GSH assay

Total GSH [reduced GSH plus glutathione disulfide (GSSG)] wasmeasured by the NADPH-dependent GSH reductase method, aspreviously described (Tietze, 1969). Midbrain samples were homog-enized with ice-cold 5% sulfosalicylic acid to precipitate cellularmacromolecules and extract GSH from both cells and tissues. Aftercentrifugation at 1200 g for 15 min, the supernatant solution was usedfor measurements. Brain solutions were diluted with phosphate-buffered saline (PBS) containing 1 mm EDTA and adjusted to pH 7.0.A reaction mixture containing 1 mm EDTA ⁄ 0.3 mm DTNB ⁄ 0.4 mm

NADPH ⁄ 2 IU ⁄ mL GSH reductase was added to the same volume ofdiluted brain solution and GSH standard solutions. Total absorbance at405 nm was measured and calibrated to GSH standards. Values werenormalized to protein content determined by the bicinchoninic acidmethod (Smith et al., 1985).

Immunohistochemistry

Coronal 50-lm sections were cut and placed in PBS containing 2%goat serum ⁄ 0.2% Triton X-100 ⁄ 0.1% bovine serum albumin for30 min at room temperature. After washing with PBS, the slices wereincubated overnight at 4 �C with 5 lg ⁄ mL rabbit antibody to EAAC1(Alpha Diagnostics, San Antonio, TX, USA) and 2 lg ⁄ mL mouseanti-nitrotyrosine antibody (Upstate, Lake Placid, NY, USA) or5 lg ⁄ mL mouse antibody to tyrosine hydroxylase (TH; AffinityBioReagents, Golden, CO, USA). After washing with PBS, the sliceswere incubated for 2 h with a 1 : 1000 dilution of Alexa Fluor 488-conjugated goat anti-rabbit IgG (Molecular Probes, Eugene, OR,USA) and ⁄ or a 1 : 1000 dilution of Alexa Fluor 594-conjugated goatanti-mouse IgG (Molecular Probes). The sections were mounted usinga ProLong Antifade Kit (Molecular Probes) and photographed with aBio-Rad inverted laser-scanning fluorescent microscope MRC-1024using Laser Sharp 2000 software (Bio-Rad, Tokyo, Japan).

Western blot

Sample lysates were mixed with a threefold concentrated loadingbuffer [150 mm Tris-HCl (pH 6.8), 30% glycerol, 3% sodium dodecylsulfate, 320 mm sucrose, 1 mm EGTA, 5 mm NaN3, 15%b-mercaptoethanol, 3 mm DTT and 0.0075% bromophenol blue].The mixture was then boiled for 3 min. Equal amounts of total cellularprotein were separated by 7.5% sodium dodecyl sulfate–polyacryl-amide gel electrophoresis (SDS–PAGE) and transferred to PVDFmembranes. Membranes were incubated overnight at 4 �C with0.5 lg ⁄ mL rabbit anti-EAAC1 antibody (Alpha Diagnostics) or0.5 lg ⁄ mL mouse anti-nitrotyrosine antibody (Upstate) in 1% bovineserum albumin ⁄ 0.5% Tween-20 ⁄ 20 mm Tris-buffered saline ⁄ 5%milk, pH 7.4, with 0.05% sodium azide. After washing, themembranes were incubated with a 1 : 10 000 dilution of horseradishperoxidase-conjugated anti-rabbit or mouse IgG antibody (Amersham,Buckinghamshire, UK) for 2 h at room temperature. The membraneswere again washed, treated with the chemiluminescent substrate ECLPlus Western Blotting Detection Reagents (Amersham), followed byimmediate X-ray film exposure. To adjust for protein loading,membranes were also immunostained with a 1 : 5000 dilution ofrabbit anti-actin antibody (Sigma Aldrich) and then with a 1 : 10 000

Oxidative stress on EAAC1 in MPTP mice 21

ª The Authors (2007). Journal Compilation ª Federation of European Neuroscience Societies and Blackwell Publishing LtdEuropean Journal of Neuroscience, 27, 20–30

dilution of horseradish peroxidase-conjugated anti-rabbit IgG antibody(Amersham).

Coomassie Brilliant Blue staining

After 7.5% SDS–PAGE, the gel was stained with 0.25% CoomassieBrilliant Blue R-250 (Sigma Aldrich) according to the manufacturer’sinstructions.

Immunoprecipitation

Lysates in RIPA buffer were preincubated with 20 lL protein Aagarose (Oncogene, Boston, MA, USA) for 3 h on ice to remove non-specific binding proteins. After centrifugation at 1200 g for 15 min,the supernatant was incubated with 5 lg rabbit anti-EAAC1 antibodyovernight at 4 �C and then with 75 lL protein A agarose for 3 h onice. Pellets were precipitated by centrifugation at 1200 g for 15 minand washed three times with RIPA buffer. After boiling for 3 min todissociate the immune complexes, the sample was again centrifuged at1200 g for 15 min and the supernatant was used for Western blots.Optical densities of protein bands were measured using the NIHimageJ software program. The nitrotyrosine band densities werenormalized in each case to the density of the EAAC1 band from thesame sample. The normalized band densities were then expressed as apercentage of control values running on the same immunoblot.

Slice culture preparations

After decapitation, the brain was cut into 300-lm slices in gassed(95% oxygen ⁄ 5% CO2) ice-cold artificial cerebrospinal fluid (aCSF)containing (in mm): NaCl, 130; KCl, 3.5; NaH2PO4, 1.25; MgSO4, 2;CaCl2, 2; NaHCO3, 20; glucose, 10; pH 7.4. All of the experimentswere initiated by transferring midbrain slices to tubes each containingaCSF at 30 �C that was continuously bubbled with 95% oxygen ⁄ 5%CO2. Cysteine uptake measurements were initiated by adding theslices to tubes each containing 100 lm cysteine. Each tube alsocontained 100 lm DTT to prevent cysteine oxidization (Chen &Swanson, 2003). In the H2O2 experiments, the slices were preincu-bated with different concentrations of H2O2 for 30 min. The sliceswere washed and then transferred to new tubes containing 100 lm

cysteine ⁄ DTT in aCSF, followed by another 15-min incubation. In theexperiments using l-aspartic acid b-hydroxamate (LAbHA, SigmaAldrich) or dihydrokainic acid (DHK, Tocris, Ellisville, MO, USA),the slices were preincubated for 30 min with different concentrationsof these compounds and then coincubated with 100 lm cysteine ⁄ DTTfor 15 min. In the 1-methyl-4-phenylpyridinium (MPP+, SigmaAldrich) experiment, the slices were preincubated with 5 mm MPP+

for 30 min, and then washed and incubated with 100 lm cyste-ine ⁄ DTT for 15 min. In the experiment with NAC, the slices wereincubated for 30 min with 5 mm NAC and then incubated with NAC-free medium for another 30 min. After incubation, the slices werewashed three times with aCSF containing 100 lm DTT on ice andfrozen at )80 �C until high-performance liquid chromatography(HPLC) analysis.

Cell culture experiment

Human dopaminergic SH-SY5Y cells were grown in Dulbecco’smodified Eagle’s medium supplemented with 10% fetal calf serum at37 �C under 5% CO2 in air. SH-SY5Y cells were treated with 5 mm

MPP+ for 3 h. After washing with PBS, cells were incubated with

OPTI-MEM (Gibco, Glasgow, Scotland, UK) containing 10 lm

cysteine and 100 lm DTT for 5 min. After collecting the medium,cysteine amounts in the medium were measured using an HPLC-fluorescence detection system. Cysteine concentrations were calcu-lated by the peak area standardized with known amounts of cysteine.

HPLC-fluorescence detection

Brain tissues were homogenized with a 10-fold volume of 5%trichloroacetic acid containing 5 mm Na2EDTA and centrifuged at1200 g for 15 min. The supernatants were used for measurements.Tissue cysteine was detected with 4-fluoro-7-sulfamoylbenzofurazan(ABD-F, Dojindo, Kumamoto, Japan), a fluorogenic labeling reagentfor thiols, according to the published protocol (Toyo’oka et al., 1989).An LC-9A liquid chromatograph system (Shimadzu, Kyoto, Japan)was used for detection. An analytical column, Inertsil ODS-2(150 · 4.6 mm ID 5 lm; GL Sciences, Tokyo, Japan) was fixed at40 �C and connected through a corresponding guard column(10 · 4.0 mm ID 5 lm; GL Sciences). A stepwise gradient elutionwas programmed with solvents A (50 mm potassium biphthalate atpH 4.0) and B (8% acetonitrile in solvent A). The mobile phase washeld at 80% solvent A and 20% B for 6 min followed by a 10-minprogram held at 100% solvent B. The flow rate of the eluate was1.0 mL ⁄ min. All samples were injected into the column with an AutoInjector (Shimadzu). An RF-530 fluorescence spectromonitor (Shi-madzu) was used with excitation and emission at 380 nm and 510 nm,respectively. The signals from the detector were recorded on aChromatopac C-R4A (Shimadzu). Tissue cysteine concentrations werecalculated by the peak area standardized with known amounts ofcysteine.

Tissue fluorescence GSH detection

Green-fluorescent chloromethylfluorescein diacetate (CMFDA,Molecular Probes) passes freely through cell membranes and istransformed into a cell-impermeant fluorescent dye after reacting withGSH (Barhoumi et al., 1993). Midbrain slices were exposed to 5 mm

H2O2, 5 mm MPP+ or 5 mm NAC for 30 min, and then transferred tofresh aCSF containing 5 lm CMFDA. Slices were then incubated at30 �C for 30 min and transferred to fresh aCSF for another 30 minincubation. The slices were fixed in 4% paraformaldehyde and thenstored in 30% sucrose for immunohistochemistry as described above.

Statistical analysis

Data are expressed as means ± SEM. Statistical significance wasdetermined using either Student’s t-test for two group comparisons, oranova with the Bonferroni ⁄ Dunn test for multiple group compari-sons. A P-value of < 0.05 was considered significant.

Results

Rotarod motor performance

To show behavioral MPTP toxicity and the protective effect of NAC,we measured motor activities using the Rotarod test. Trial sessionperformances were variable unless a training session had beensuccessfully conducted on the same day. Two trials were eliminatedfrom the data because of unsuccessful training sessions. Four of 38mice (saline group, 1; MPTP group, 2; NAC ⁄ MPTP group, 1) diedduring the test period. The times on the rod were compared among

22 K. Aoyama et al.


groups on the same day. There were no statistically significantdifferences prior to treatments among the groups. After Day 3, MPTP-treated mice showed poor performances on the rod as compared withthe saline-treated mice (Fig. 1). NAC ⁄ MPTP-treated mice alsoshowed decreased times on the rod, but the difference was notsignificant as compared with saline-treated mice on Day 3, thoughthey had recovered by Day 5. On Day 7, the MPTP group showedsignificantly decreased times on the rod as compared with the othergroups (P < 0.05), whereas there were no differences among thesaline, NAC and NAC ⁄ MPTP groups.

Enzymatic total GSH assay

To examine total GSH (reduced GSH + GSSG) concentrations in themice treated with saline, MPTP, NAC ⁄ MPTP and 7-NI ⁄ MPTP, weperformed enzymatic GSH assays using mouse midbrains. Mice wereinjected for 5 days as described in the Materials and methods section.Mouse brains were removed 2 days after the last injection. Withouttreatment, the total GSH level in the mouse midbrain (0.61 ±0.04 lmol ⁄ mg protein, n ¼ 9, mean ± SEM) was significantly lowerthan that in the cortex (0.79 ± 0.07 lmol ⁄ mg protein, n ¼ 7,P < 0.05). MPTP administration decreased the midbrain GSH levelby approximately 28% as compared with that of saline-injected controls(Fig. 2). Systemic administration of NAC can increase GSH in the CNS(Pocernich et al., 2000). In this study, NAC pretreatment prevented themidbrain GSH decrease induced by MPTP. Furthermore, pretreatmentwith 7-NI, a specific neuronal nitric oxide synthase (nNOS) inhibitor,also preserved midbrain GSH levels in MPTP-treated mice. This resultis in agreement with a previous report demonstrating nNOS-deficientmice to be resistant to MPTP (Przedborski et al., 1996).

Immunohistochemistry

Human pigmented dopaminergic neurons reportedly contain EAAT3(EAAC1) at high levels (Plaitakis & Shashidharan, 2000). Our dataalso showed EAAC1 immunoreactivity in TH-positive neurons in themouse SN (Fig. 3A). MPTP treatment caused a redistribution of

EAAC1 to the cell surface, whereas with NAC plus MPTPco-treatment there was no translocation. An oxidative stress marker,nitrotyrosine, was increased in the SN of MPTP-treated mice(Fig. 3B). The signal was partially co-localized with that of EAAC1on the cell surface. Co-treatment with NAC plus MPTP prevented theincrease in nitrotyrosine. 7-NI also prevented the nitrotyrosineincrease, while having no influence on the EAAC1 translocation tothe cell surface induced by MPTP (Fig. 3B).

Western blot ⁄ immunoprecipitation

Amounts of EAAC1 protein were unchanged in midbrain wholelysates (Fig. 4A), while cell membrane EAAC1 expression wassignificantly increased in MPTP-treated mice (Fig. 4B and C). Toinvestigate whether EAAC1 is subjected to oxidative stress by MPTPtreatment, midbrain samples were immunoprecipitated with EAAC1antibody and then used for Western blots with nitrotyrosine antibody.Figure 4D shows a representative Western blot result demonstrating anincrease in the amount of nitrotyrosine in EAAC1 protein with MPTPtreatment. Densitometric analysis of the band adjusted by that ofEAAC1 showed that MPTP treatment increased the nitrotyrosine levelin EAAC1 and that NAC co-treatment blunted this increase to astatistically significant degree (Fig. 4E).

Cysteine content in slice culture

To investigate the influence of pre-incubations with H2O2, EAATinhibitors or MPP+, we examined slice culture tissues containingcysteine by the fluorescence HPLC method. ABD-F is a fluorogeniclabeling reagent for thiols and is detected by HPLC (Fig. 5A;Toyo’oka et al., 1989). Cysteine contents in slice culture tissues were

Fig. 1. Mean time of overall rod performances. Mice were treated with25 mg ⁄ kg ⁄ day 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) with(n ¼ 10) or without (n ¼ 10) 150 mg ⁄ kg ⁄ day n-acetylcysteine (NAC), orwith saline (n ¼ 10) or 150 mg ⁄ kg ⁄ day NAC (n ¼ 8) alone for 5 days. Micegiven MPTP showed significant motor dysfunction, with loss of the ability tokeep walking on the rod. NAC ⁄ MPTP treatment had significantly restored themotor functions of mice by Day 7. Only NAC had no effect on rod time ascompared with saline. *P < 0.05, **P < 0.01 vs Day 1. #P < 0.05 vs the othergroup on the same day.

Fig. 2. Total midbrain GSH assay. Mice were treated with saline,25 mg ⁄ kg ⁄ day 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) or25 mg ⁄ kg ⁄ day MPTP plus 150 mg ⁄ kg ⁄ day n-acetylcysteine (NAC) for5 days. Total midbrain GSH (reduced GSH plus GSSG) contents weresignificantly reduced in MPTP-treated mice, but preserved in 7-nitroindazole(7-NI), and in NAC plus MPTP-treated mice. Mouse brains were extracted onDay 7. n ¼ 6–9. *P < 0.05, **P < 0.01.



Fig. 3. Effect of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) on excitatory amino acid carrier 1 (EAAC1) distribution in dopaminergic cells.(A) Immunostaining for EAAC1 (green) is colocalized with TH (red) in the SN. MPTP treatment induces EAAC1 translocation to the cell surface, whereasco-treatment with n-acetylcysteine (NAC) and MPTP does not. (B) Immunostaining for EAAC1 (green) and nitrotyrosine (red) in the SN. MPTP treatmentincreases immunoreactivity for nitrotyrosine in EAAC1-positive cells. EAAC1 is colocalized with nitrotyrosine on the cell surface in the MPTP-treated mouse butnot in the NAC ⁄ MPTP or 7-nitroindazole (7-NI) ⁄ MPTP-treated mice. Mouse brains were extracted and fixed on Day 7. Results are representative of three brains ineach group. Scale bar: 5 lm.

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increased by approximately sixfold as compared with extracellularfluid (100 lm) at 30 min after the start of incubation (Fig. 5B). Thiscysteine increase was significantly blunted by pre-incubation withH2O2, MPP+ or LAbHA, a relatively selective EAAC1 inhibitor, butnot DHK, a selective GLT-1 inhibitor (Arriza et al., 1994; Fig. 6A).

Cysteine uptake in dopaminergic cellsSH-SY5Y, a human dopaminergic neuroblastoma cell line, expressesEAAC1 (Sala et al., 2005). We measured medium-containing cysteineloss, which is considered to represent cysteine uptake into SH-SY5Ycells, with and without MPP+ pre-treatment. Three-hour pre-treatment

Fig. 4. Western blot analysis of excitatory amino acid carrier 1 (EAAC1). (A) Total proteins extracted from midbrains were probed with EAAC1 antibody.1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) treatment had no effect on the total EAAC1 amount on Day 7, or on Day 21. (B) Midbrain tissues werefractionated on Day 7 as described in Materials and methods. EAAC1 expression on the plasma membrane is increased after MPTP treatment, whereasn-acetylcysteine (NAC) ⁄ MPTP treatment does not induce translocation (upper panel). Samples (3 lg ⁄ lane) were loaded onto each well. Coomassie Brilliant Blue(CBB) staining, shown in the lower panel, indicates that equal amounts of total protein have been loaded. (C) The optical band density of EAAC1 is expressed as apercentage of the control (saline) group value on the same membrane. n ¼ 4 independent experiments. *P < 0.05 vs saline group. (D) Midbrain samplesimmunoprecipitated with EAAC1 antibody were probed with nitrotyrosine antibody. (E) The optical band density of nitrotyrosine is normalized by that of EAAC1and expressed as a percentage of the control (saline) group value on the same membrane. The amount of EAAC1 containing nitrotyrosine is increased after MPTPtreatment. NAC co-treatment reduces the nitration of EAAC1 in MPTP-treated mice. n ¼ 3 independent experiments. **P < 0.01 vs saline group. #P < 0.05 vsMPTP group.



with 5 mm MPP+ significantly inhibited the 5-min uptake of cysteineinto SH-SY5Y cells (Fig. 6B).

Fluorescence GSH detection in slice culture

CMFDA is a membrane-permeant dye labeling GSH via reaction withglutathione-S-transferase. CMFDA can pass through the cell mem-brane and is transformed into a cell-impermeant fluorescent dye-thioether adduct. In this experiment, H2O2 or MPP+ treatmentdecreased, while NAC treatment increased GSH levels in dopaminer-gic cells in the SN (Fig. 7A). The effect of NAC on the GSH level inthe midbrain was also confirmed by HPLC (Fig. 7B). NACadministration increased cysteine and GSH levels in midbrain slices.These data support the efficacy of NAC, which raises GSH levels indopaminergic neurons in vivo, against MPTP toxicity.

Discussion

Neurons rely mainly on extracellular cysteine for GSH synthesis(Dringen et al., 1999), because neurons have no means of direct GSHuptake. Extracellular supplies of the other amino acids, glutamate andglycine, do not increase GSH synthesis (Almeida et al., 1998; Dringenet al., 1999), as intracellular concentrations are already sufficient(Dringen, 2000). Cystine is an oxidized form of two cysteines with adisulfide linkage and is utilized as a substrate for GSH synthesis insome cell types (Bannai & Kitamura, 1980). However, mature neuronsutilize cysteine but not cystine for GSH synthesis (Sagara et al., 1993;Kranich et al., 1996). Because cystine is not an EAAC1 substrate(Kanai & Hediger, 1992) and mature neurons do not have cystinetransporters, i.e. system xc–, which is expressed in regions facing theCSF, a role in redox buffering of the cysteine ⁄ cystine balance in theCSF has been suggested (Sato et al., 2002). Recently, mice lacking

Fig. 5. Cysteine measurement using HPLC-fluorescence detection system. (A) Chromatogram of cysteine and glutathione (GSH) solutions with knownconcentrations as standards (left panel) and midbrain slices at different time points after incubation with 100 lm cysteine (right panel). (B) Tissue cysteineconcentrations were evaluated employing comparisons with standard cysteine solutions and were normalized according to the wet tissue weight. Cysteine levels inmidbrain slices rise significantly as the incubation time increases. n ¼ 4 independent experiments. **P < 0.01 vs the control (0 min).

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this cystine transporter were reported to show no changes in brainGSH contents (Sato et al., 2005). Therefore, the availability ofcysteine, but not other amino acids, is critically important for neuronalGSH synthesis.

Previous reports demonstrated that glutamate transporters arevulnerable to oxidative stress and that glutamate uptake is inhibitedby preincubation with peroxynitrite or H2O2 in vitro (Trotti et al.,1998). However, little is known about the influence of oxidative stresson the capacity of EAAC1 to function as a cysteine transporter.Previous studies have suggested that glutamate uptake is regulated bythe redox state of sulfydryl groups on cysteine residues of EAAC1 andthat oxidation of the ‘redox site’ by H2O2 decreases glutamate uptake(Trotti et al., 1997a, b). Our data from acute slice culture experiments

demonstrate that preincubation with H2O2 reduces subsequent cyste-ine uptake in the midbrain. Similarly, the marked reduction of cysteineuptake observed in the presence of LAbHA, but not DHK, suggeststhat EAAC1 is the primary cysteine transporter in the midbrain, as hasbeen demonstrated in the hippocampus (Aoyama et al., 2006). Thesefindings suggest that oxidative stress may impair neuronal GSHsynthesis via EAAC1 dysfunction in the midbrain.Peroxynitrite is a potent oxidant generated by the reaction between

superoxide anion and nitric oxide (Kuhn et al., 2004), and plays amajor role in MPTP neurotoxicity (Schulz et al., 1995). MPTPneurotoxicity is dependent on its metabolism to MPP+, which canspecifically enter dopaminergic neurons via the dopamine transporter(Javitch et al., 1985) to inhibit complex I of the mitochondrialrespiratory chain, and thus leads to reactive oxygen species productionand ATP depletion (Tipton & Singer, 1993). Dopamine transporter-deficient mice preserved nearly normal striatal dopamine levels whenexposed to neurotoxic doses of MPTP (Gainetdinov et al., 1997),while dopamine transporter over-expressing mice showed enhancedMPTP neurotoxicity (Donovan et al., 1999). These indicate thatMPTP is a neurotoxin specific to dopaminergic neurons in vivo. In thisstudy, we showed pretreatment with MPP+ to decrease subsequentcysteine uptake in midbrain slices and SH-SY5Y cells, and also toreduce GSH levels in dopaminergic neurons of the SN. These resultsindicate that MPTP neurotoxicity would be enhanced by inhibitingneuronal cysteine uptake leading to impaired GSH synthesis.A previous report demonstrated MPTP neurotoxicity to be attenu-

ated in nNOS-deficient mice (Przedborski et al., 1996). Therefore, it isimportant to elucidate the mechanism underlying peroxynitrite-med-iated neurotoxicity in the MPTP model. Nitrotyrosine is a permanentmarker of peroxynitrite attack on proteins (Beckman, 1994) and isfound in post mortem PD brain samples (Good et al., 1998). In theMPTP model, tyrosine nitration inactivates TH, a key dopaminesynthesis enzyme, and is found in a-synuclein, a major component ofLewy bodies (Kuhn et al., 2004). Peroxynitrite can oxidize cysteineresidues and ⁄ or nitrate tyrosine residues on glutamate transporters, andthereby impair their function (Trotti et al., 1997b, 1998). To date, nodirect evidence of EAAC1 nitration has been obtained. In this study, wefound an increased amount of nitrotyrosine on EAAC1 and colocal-ization at the plasma membrane in the SN of MPTP-treated mice.Pretreatment with 7-NI, a selective inhibitor of nNOS, prevented thenitrotyrosine formation and GSH depletion induced by MPTP. Theseresults may explain the decrease in GSH, which occurs with EAAC1dysfunction in the midbrains of MPTP-treated mice. A 30% decline intotal GSH appears to be rather large for a partial impairment ofneuronal EAAC1 by oxidative stress. Because GSH contents may varyamong TH-positive neurons, TH-negative neurons and glial cells, theapparently large decline in total GSH might suggest a predominantGSH distribution in TH-positive neurons in the midbrain.A previous study demonstrated that MPTP administration decreased

the striatal GLT-1 level after 21 days (Holmer et al., 2005). In ourstudy, total EAAC1 amounts were unchanged, while amounts ofEAAC1, though nitrated, on the plasma membrane were increased inthe midbrains of MPTP-treated mice. Redistribution of EAAC1 to themembrane surface has been demonstrated to be regulated by proteinkinase C, particularly protein kinase C subtype a (Davis et al., 1998).Indeed, protein kinase C activation induced by peroxynitrite has beenidentified in fibroblasts (Bapat et al., 2001), pulmonary arteryendothelial cells (Phelps et al., 1995) and the myocardium (Pagliaroet al., 2001). Interestingly, in this study, treatment with 7-NI, whichblocks peroxynitrite formation, did not influence the EAAC1redistribution to the membrane surface induced by MPTP. Thisredistribution might be induced by signals other than peroxynitrite.

Fig. 6. Cysteine measurement by HPLC. (A) Midbrain slices were exposedto H2O2, the EAAC1 inhibitor l-aspartic acid b-hydroxamate (LAbHA), theGLT-1 selective inhibitor dihydrokainic acid (DHK) or 1-methyl-4-phenylpy-ridinium (MPP+) solution for 30 min before the subsequent 15-min incubationwith 100 lm cysteine ⁄ aCSF solution. Pre-incubation with H2O2 dose-depen-dently blocked subsequent cysteine increases in the slices. A high dose(1.2 mm) of LAbHA or 5 mm MPP+ preincubation also significantly reducedthe subsequent cysteine increase, while DHK did not. n ¼ 4 or moreindependent experiments. *P < 0.05, **P < 0.01 vs the control. ##P < 0.01vs no treatment. (B) SH-SY5Y, a human dopaminergic neuroblastoma cellline, was exposed to a 10 lm cysteine solution for 5 min to measureextracellular cysteine loss, which is considered to represent cysteine uptake intothe cells, with or without MPP+ pretreatment. Three-hour pretreatment with5 mm MPP+ significantly inhibited the subsequent cysteine uptake. n ¼ 8.**P < 0.01 vs the control.



Fig. 7. Effect of n-acetylcysteine (NAC) on GSH synthesis in slice culture. (A) H2O2 or 1-methyl-4-phenylpyridinium (MPP+) treatment (5 mm) for 30 minreduced chloromethylfluorescein diacetate (CMFDA) fluorescence, a marker for intracellular GSH, on TH-positive neurons in the SN, while NAC treatment (5 mm)for 30 min increased this fluorescence. Results are representative of three brains in each group. Scale bar: 5 lm. (B) Incubation with 5 mm NAC for 30 minincreased cysteine and GSH contents, which were measured by HPLC, in midbrain slices. n ¼ 5. **P < 0.01 vs the control.

28 K. Aoyama et al.


Further study is needed to elucidate the mechanism of the traffickingsystem in this model.

NAC acts as a precursor for GSH synthesis by supplying cysteine(De Vries & De Flora, 1993) and activates the GSH cycle (De Floraet al., 1991). NAC enters the cell readily (Mazor et al., 1996) and isthen deacetylated to form l-cysteine regardless of whether EAAC1 ispresent (Himi et al., 2003a; Aoyama et al., 2006). NAC exerts a directchemical effect as an antioxidant, although with less potency than thatof GSH (Hussain et al., 1996). Systemic administration of NAC candeliver cysteine to the brain and raise GSH levels in the CNS(Pocernich et al., 2000). Therefore, NAC would exert its protectiveeffects against oxidative stress mainly by serving as a substrate forGSH synthesis. Our slice culture results showed NAC to act as aneffective precursor for GSH synthesis in dopaminergic neurons. Thereare no reports demonstrating GSH-related protective effects of NACagainst MPTP neurotoxicity using behavioral, biochemical or histo-chemical analysis in vivo, although one study demonstrated NACtreatment to restore the striatal dopamine level in MPTP-treated mice(Perry et al., 1985). Our present results demonstrate that NAC pre-administration ameliorates motor dysfunction in addition to restoringGSH levels in MPTP-treated mice. We also found the nitrotyrosinelevel on EAAC1 to be reduced in the midbrains of NAC ⁄ MPTP-treated mice as compared with MPTP-treated mice. Although whetherNAC would be clinically beneficial in PD is as yet unknown, its lowtoxicity and ease of administration warrant further investigation of thiscompound.

Acknowledgement

This work was supported in part by a Grant-in-Aid for Young Scientists (B)from the Japan Society for the Promotion of Science.

Abbreviations

ABD-F, 4-fluoro-7-sulfamoylbenzofurazan; aCSF, artificial cerebrospinal fluid;CMFDA, chloromethylfluorescein diacetate; DHK, dihydrokainic acid; DTT,dithiothreitol; EAAC1, excitatory amino acid carrier 1; EAAT, excitatory aminoacid transporter; GSH, glutathione; GSSG, glutathione disulfide; HPLC, high-performance liquid chromatography; LAbHA, l-aspartic acid b-hydroxamate;MPP+, 1-methyl-4-phenylpyridinium; MPTP, 1-methyl-4-phenyl-1,2,3,6-tetra-hydropyridine; NAC, n-acetylcysteine; 7-NI, 7-nitroindazole; nNOS, neuronalnitric oxide synthase; PBS, phosphate-buffered saline; PD, Parkinson’s disease;PMSF, phenylmethylsulfonyl fluoride; SDS–PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis; SN, substantia nigra; TH, tyrosinehydroxylase.

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Oxidative stress on EAAC1 is involved in MPTP-induced glutathione depletion and motor dysfunction