GST� expression mediates dopaminergic neuronsensitivity in experimental parkinsonismMichelle Smeyne*, Justin Boyd*, Kennie Raviie Shepherd*, Yun Jiao*, Brooks Barnes Pond*, Matthew Hatler*,Roland Wolf†, Colin Henderson†, and Richard Jay Smeyne*‡

*Department of Developmental Neurobiology, St. Jude Children’s Research Hospital, 332 North Lauderdale Street, Memphis, TN 38105; and†Cancer Research UK, Molecular Pharmacology Unit, Ninewells Hospital and Medical School, Biomedical Research Centre, Level 5,Dundee DD1 9SY, Scotland, United Kingdom

Communicated by Richard L. Sidman, Harvard Institutes of Medicine, Boston, MA, December 13, 2006 (received for review March 27, 2006)

The cause of 95% of Parkinson’s disease (PD) cases is unknown. Itis hypothesized that PD arises from an interaction of free-radical-generating agents with an underlying genetic susceptibility tothese compounds. Here we use the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine model of parkinsonism to examine the role ofa dual function protein, GST�, in dopaminergic neuron death.GST� is the only GST family member expressed in substantia nigraneurons. GST� reduction by pharmacological blockade, RNA inhi-bition, and gene targeting increases sensitivity to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, suggesting that differential ex-pression of GST� contributes to the sensitivity to xenobiotics in thesubstantia nigra and may influence the pathogenesis of reactiveoxygen species-induced neurological disorders including PD.

glutathione � oxidative stress � Parkinson’s disease � substantia nigra �detoxification

Inhibition of free radicals and the damage they produce iscritical for cell survival, particularly in neurons. Glutathione

S-transferases (GSTs) are a class of inducible, multifunctional,detoxifying enzymes that catalyze the reduction of hydrophobicelectrophiles, like those generated by pesticides and chemother-apeutic compounds (1, 2). In mammals, six classes of cytosolicGSTs have been identified, although only three have beendescribed in the CNS (�, �, and �) (3, 4). Of these three GSTisoenzymes, GST� has also been shown to inhibit JNK-activatedsignaling, blocking the phosphorylation of cJUN and apoptosisof the cell (5). Epidemiology and experimental models indicatethat several neurological disorders, including idiopathic Parkin-son’s disease (PD), arise from the combination of a geneticsusceptibility and environmental exposure to compounds thatgenerate oxidative stress (6). Although several familial parkin-sonian syndromes have been identified (including those thatinduce cell death by the generation of free radicals), the causeof idiopathic PD remains elusive. Several models of parkinson-ism have been developed, including the use of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Systemic adminis-tration of MPTP causes a loss of substantia nigra (SN)dopaminergic neurons by mitochondrial inhibition, protein ni-trosylation, and release of intracellular dopamine. Each of theseprocesses results in the secondary elevation of reactive oxygenspecies in humans, nonhuman primates, and mice (7).

Brains of PD patients show numerous indicators of oxidativestress, including decreased levels of glutathione (GSH), in-creased lipid peroxidation, presence of dopamine quinones,DNA damage, and increases in JNK-mediated activation ofc-Jun (8, 9). Neurons in the SN pars compacta (SNpc) arethought to be particularly sensitive to oxidative stress becausethey contain elevated levels of Fe3�, �-synuclein, and dopamine(10). In the murine MPTP model of parkinsonism, MAO-B inglial cells converts MPTP to 1-methyl-4-phenylpyridinium(MPP�), which enters dopaminergic neurons through dopaminetransporters and blocks complex I respiration (7). The subse-quent energy depletion results in a cascade of effects, producing

free radicals and neuron death. Previously it was shown thatthere is naturally occurring strain-dependent sensitivity toMPTP (11). Here we examine the expression of GST isoenzymesin the MPTP-sensitive C57BL/6 mouse strain and the MPTP-resistant Swiss–Webster (SW) mouse strain in an early responseto xenobiotic exposure.

ResultsGST Expression in the Basal Ganglia. Basal level and MPTP-inducedexpression of GST was examined in two regions of the brainaffected in PD, the SN and striatum, and one region not affected,the cerebral cortex of adult C57BL/6 and SW mice. GST� andGST� were expressed in each of these brain regions, whereasGST� was not observed in any of the regions (Fig. 1a). GST�mRNA distribution was diffuse and uniform in the SN whereasGST� followed a more distinct pattern of expression (Fig. 1b).Western blot analysis using class-specific anti-GST antibodiesdemonstrated that GST� and GST� were present in all threeregions of the CNS examined. Consistent with the RT-PCRresults, GST� was not detected (Fig. 1c). GST� was expressedin the dopaminergic neurons of the SNpc as well as in astrocytes[Fig. 1e and supporting information (SI) Fig. 5a], whereas GST�was not found in the dopaminergic neurons of the SNpc but wasapparent in the astrocytes surrounding the neurons (Fig. 1d andSI Fig. 5b). GST� protein was also abundantly expressed intyrosine hydroxylase (TH)-positive neurons of normal humanSN obtained from postmortem tissue (Fig. 1f ). Thus, of the threeknown GST isoforms present in the CNS, only GST� was presentin the population of dopaminergic neurons that are lost in PD.

Strain Differences in GST� Expression Are Detected After Adminis-tration of MPTP. Previously we have shown that the levels of MPP�,determined by HPLC, were similar in the SN and striatum ofC57BL/6 and SW strains after administration of MPTP (12).Because GST� is a detoxifying enzyme present in the SN, MPTPwas administered to C57BL/6 and SW mice to assess whether levelsof GST� protein changed after toxic challenge. Immediately afterfour injections of MPTP (20 mg/kg) spaced 2 h apart, GST� proteinin C57BL/6 SN was virtually absent, whereas MPTP-resistant SWmice maintained a significantly higher, although reduced, level of

Author contributions: M.S., J.B., and K.R.S. contributed equally to this work; M.S., K.R.S.,and R.J.S. designed research; M.S., J.B., K.R.S., Y.J., B.B.P., and M.H. performed research;R.W. and C.H. contributed new reagents/analytic tools; M.S., J.B., and Y.J. analyzed data;and M.S. and R.J.S. wrote the paper.

The authors declare no conflict of interest.

Freely available online through the PNAS open access option.

Abbreviations: PD, Parkinson’s disease; MPTP, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyri-dine; GSH, glutathione; SN, substantia nigra; SNpc, SN pars compacta; MPP�, 1-methyl-4-phenylpyridinium; EA, ethacrynic acid; 3,4-DBA, 3,4-dihydroxybenzoic acid; SW, Swiss–Webster; TH, tyrosine hydroxylase.

‡To whom correspondence should be addressed. E-mail: richard.smeyne@stjude.org.

This article contains supporting information online at www.pnas.org/cgi/content/full/0610978104/DC1.

© 2007 by The National Academy of Sciences of the USA

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the enzyme (P � 0.05) (Fig. 2 a and b). In addition, at this time pointGST� protein levels were significantly different from control SN ineach strain (C57BL/6, P � 0.01; SW, P � 0.05). Twelve hours afterMPTP, GST� expression in the SN of both strains recovered tobasal levels (Fig. 2a). The loss of GST� was specific to the SN,because GST� expression did not change in regions of the brainwhere MPTP-induced cell loss did not occur (SI Fig. 6). Thereduction of GST� is not due to free radical-induced oligomeriza-tion (13), because Western blot analysis did not reveal the presenceof any high-molecular-weight bands. Also, no strain differenceswere detected in GSH levels in SN or striatum (SI Fig. 7 a and b).The recovery of GST� protein 12 h after MPTP in C57BL/6 miceappears to be primarily in nondopaminergic cells. In contrast, SWdopaminergic neurons, as well as other cell types in the MPTP-resistant SW mouse, never completely lost expression of GST� andrecovered to basal levels as early as 12 h after MPTP (Fig. 2b).

Phosphorylated cJUN expression is a hallmark of cell deathinduction, and GST� has been shown to inhibit JNK-mediatedphosphorylation of cJUN (5). After MPTP, phosphorylatedcJUN was elevated in the C57BL/6 SN but not in SW SN (Fig.2c). The timing of the increase in cJUN expression coincides withthe loss of GST� expression. Therefore, maintenance of sub-stantial levels of the detoxifying enzyme GST� in SW dopami-

nergic neurons may also confer resistance to MPTP exposure byinhibition of apoptosis.

Inhibition of GST� Alters Sensitivity to MPP� in Vitro. SN primarycultures treated with MPP� resulted in strain-dependent dopa-minergic cell death (14). GST� protein was expressed in dopa-minergic neurons as well as astrocytes in vitro (Fig. 3a). SNcultures generated from SW mice were treated with ethacrynicacid (EA), an inhibitor of GSTs (15), to determine whether thisinfluences MPP�-induced dopaminergic cell death. SW dopa-minergic neurons exposed to EA before MPP� had a 30 � 4%loss of dopaminergic neurons whereas no loss was observed withMPP� alone (P � 0.005) (Fig. 3b). RNAi was used to test theeffects of specific inhibition of GST� on the survival of dopa-minergic neurons isolated from SW mice. The TH-positive cellloss in SW SN cultures transfected with siRNA targeted to GST�and treated with MPP� was compared with nontransfected

Fig. 1. Expression of GST in SW and C57BL/6 brain. (a) RT-PCR of GST�, GST�,and GST� mRNA in cerebral cortex (Ctx), striatum (Str), and SN. (b Upper) Insitu hybridization of GST� and GST� in C57BL/6 brain in region of SN (arrows).(b Lower) Enlargement of boxed region showing SN (arrows). (c) Immunoblotof GST�, GST�, and GST� in SN, striatum, cerebral cortex, and liver betweenmouse strains. (d and e) GST� and GST� expression in the SN. TH identifiesdopaminergic neurons. ( f) GST� and TH in normal-aged human SN. [ Scale bar:f (applies to d–f), 25 �m.]

Fig. 2. Cellular response to MPTP in C57BL/6 and SW SN. (a) Immunoblot ofGST� in C57BL/6 (C) and SW (S) SN after saline (control), four doses of 20 mg/kgMPTP, and four doses of 20 mg/kg MPTP plus 12 h. GAPDH was used as loadcontrol. Relative quantitation is shown in Right (*, P � 0.01; #, P � 0.05). (b)Immunofluorescent labeling of GST� (green) in the SN of C57BL/6 (C57) andSW (same conditions as in a) mice. Each Inset shows a magnified imagecontained within the field of view. (c) Immunofluorescent labeling of basallevels of phospho-c-Jun (p-cJUN, green) in C57BL/6 and SW (SN neurons areidentified by TH, red) after administration of 80 mg/kg MPTP. [Scale bars: b(applies to b and c), 70 �m; b Inset (applies to all Insets), 25 �m.]

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cultures treated with MPP� from the same culture cohort.Quantitative PCR of cultures treated with GST� siRNA andMPP� showed a 41 � 10% mean decrease in GST� mRNAexpression with a 49 � 9% loss of dopaminergic neurons. Whentransfected with GST� siRNA alone, there was a 20 � 6%decrease in GST� expression and a 26 � 11% loss of dopami-nergic neurons. Nontransfected SW cultures treated with MPP�

showed 0 � 0% dopaminergic neuron loss (P � 0.005) (Fig. 3c).Statistical comparisons showed no significant difference in do-pamine cell death between cultures treated with GST� siRNAalone and those not treated. Transfection with scrambled (neg-ative control) siRNA or vehicle alone resulted in a 0–7%decrease in GST� mRNA in five of six culture cohorts and nodopaminergic neuron loss. We hypothesize that the nonsignifi-cant loss of SW dopaminergic neurons measured after inhibitionof GST� alone is due to the inherent oxidative conditions in cellculture (16).

Genetic Deletion of GST� Increases the Sensitivity of SNpc Dopami-nergic Neurons to MPTP. To further examine the role of GST� ininduction of experimental parkinsonism, we backcrossed theGST�-null mouse (17) onto the MPTP-resistant SW mouse (SIFig. 8). Seven days after MPTP administration, GST� (�/�)and (�/�) mice were killed with wild-type littermates, and SNdopaminergic neuron loss was assessed. Wild-type and GST�(�/�) littermates displayed similar resistance to MPTP toxicity(Fig. 4 a, b, and d). GST� (�/�) mice, however, showed a 37 �3% MPTP-mediated dopamine cell loss (Fig. 4 c and d). This lossis comparable to that seen in the MPTP-sensitive C57BL/6 strain(18). In addition, reduced levels of dopamine, 3,4-dihydroxy-phenylacetic acid, and homovanillic acid were also seen in thestriatum of GST� (�/�) mice compared with untreatedGST�(�/�) mice 7 days after MPTP treatment (Fig. 4 e–g).

Loss of GST� did not affect dopamine cell number in the SN,indicating that the decrease in cell number after MPTP resultedfrom the combination of toxic challenge and loss of GST�.Intracellular levels of MPP� in the SN of SW mice increasedslightly after each MPTP injection, whereas in GST� (�/�)mice the rise in MPP� levels was significant after the secondinjection and remained elevated until after the fourth injection(Fig. 4h). Measurement of 3,4-dihydroxybenzoic acid (3,4-DBA)in SN, an indicator of free radical formation, increased 300% 6 hafter MPTP administration in GST� (�/�) mice compared withthat seen in GST� (�/�) mice (Fig. 4i).

DiscussionGSTs are phase II detoxifying enzymes that catalyze the con-jugation of electrophiles with GSH and reduce free radicals.Free radical compounds conjugated with GSH bind to the Nterminus of GSTs and are excreted from the brain (4, 19). GSH,a tripeptide synthesized from glutamate, cysteine, and glycine(20), is an abundant antioxidant in mammalian cells. Totalinhibition of GSH results in increases in free radical formationand sensitivity to a number of oxidative stressors (21), whereasdecreased levels of GSH alone are not sufficient to causedopaminergic degeneration (22).

Numerous studies have demonstrated the importance of GSH(23), GSH peroxidase (24), and enzymes involved in GSH synthesis(25) in neuroprotection after oxidative stress. GSTs and GSH likelyboth play an essential role in maintaining a balanced milieu whenthe cell is exposed to excess reactive oxygen species (ROS).Decreased levels or function of detoxifying enzymes such as GSTsor GSH peroxidase may lead to increased dependence on theantioxidant properties of GSH, thereby lowering the level of GSHwhen the cell is exposed to a significant free radical challenge (SIFig. 7c). It has been suggested that the rate of spontaneous reactionof GSH with some electrophiles is too low to protect cells (1), anddirect measurement of electrophile reduction by GSH conjugatedto GST� was 600% more efficient than GSH alone (26). Expressionpatterns in this study show that GST� is the only GST expressed inthe neurons of the SNpc, and this may contribute to the sensitivityof this population of neurons to MPTP as well as other parkinso-nian toxins such as paraquat (27) and rotenone (28). In support ofthis, we observe that there is an increase in intracellular MPP� andfree radical formation in the absence of GST� protein. Onepossible explanation for this is that MPDP and MPP� act asnoncompetitive inhibitors of GSTs in addition to inhibiting theGSH peroxidase II activity of GSTs (29), and by inhibiting dopa-mine transporter function (30). The MPP� increase seen in theGST� (�/�) mice after the second injection may be due to theinhibition of dopamine transporter function by free intracellularMPP� in the dopaminergic neurons that cannot effectively reduceor clear ROS, resulting in a loss of sequestration of MPP� byVMAT (31).

In addition to binding free radicals on the N terminus, the Cterminus of GST� binds to JNK, inhibiting the phosphorylationof cJun and blocking the cascade of apoptosis (5, 32). Thebifunctionality of this enzyme may also provide a key to under-standing why different modes of cell death, i.e., apoptosis versusnecrosis, have been observed in PD (33). Because GST� andGST� are the only proteins of this class of enzyme present in theSN, they are good candidates for mediating the detoxification ofROS and, if defective, may lead to free radical overload anddeath of dopaminergic neurons. GST� is present in dopaminer-gic neurons and astrocytes in the SN of SW (MPTP-resistant)and C57BL/6 (MPTP-sensitive) mice, whereas GST� is presentonly in astrocytes. It is interesting to note that, although SW SNhas twice the number of astrocytes as C57BL/6 SN (12), inhi-bition or lack of GST� alone in mice on a SW background stillresults in dopaminergic neuron death similar to that seen inC57BL/6 SN. Further investigation of the role of astrocytes and

Fig. 3. GST inhibition increases dopaminergic neuron death in an MPTP-resistant strain. (a) SN neuron, in vitro, immunostained for TH (red) and GST�

(green). The coexpression of the two fluorophores is seen as yellow. (b) EA (50nM) treatment before MPP� (50 nM for 2 days) resulted in 30 � 4% TH-positivecell loss compared with 0% with MPP� or 16 � 12% with EA alone (F � 5.36,P � 0.05). (c) GST� inhibition (50 nM) with MPP� (50 nM for 2 days) resultedin 49 � 9% dopamine cell loss, compared with 0% loss in nontransfectedcultures treated with MPP� (F � 5.23, P � 0.005) and 26 � 11% loss with GST�

siRNA inhibition alone (not significant). (Scale bar: 25 �m.)

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GST� in dopaminergic neuron protection during oxidative stressshould clarify this issue.

In humans, polymorphisms in GST� have been loosely cor-related with increased risk of idiopathic PD after exposure topesticides (34, 35), and polymorphisms in a drosophila GSTenzyme have been shown to enhance dopaminergic neuron lossin a parkin mutant (36). GST� mRNA and protein levels havealso been reported to be lower in CNS regions affected byamyotrophic lateral sclerosis than in control subjects or regionsunaffected by the disease (37). The majority of PD cases have anunknown etiology, although it is accepted that there is aninteraction of genetic susceptibility with exposure to a variety ofenvironmental factors (38). In addition to these environmentalcauses, at least seven genes in familial PD have been identified(39) that when mutated fail to modulate the ubiquitin-proteasome system, resulting in increased generation of freeradicals, inflammatory changes, and neuronal necrosis (40) ordirect activation of apoptotic pathways in dopaminergic neurons(41). Although it is likely that there are a multitude of individualmechanisms that lead to dopaminergic neuron loss, oxidative

stress and the accumulation of free radicals are common toidiopathic and familial forms of parkinsonism. We hypothesizethat cellular GST� levels may be a useful target for riskassessment or therapeutic intervention of PD.

MethodsAll animals used in this study were treated in accordance withNational Institutes of Health guidelines, and methods wereapproved by the St. Jude Children’s Research Hospital AnimalCare and Use Committee.

In Situ Hybridization and Immunohistochemistry. Fixed frozen brainswere sectioned in series at 15 �m. Adjacent series were used tolocalize GST�, GST�, and GST�. For in situ hybridization, slideswere labeled with 33P-conjugated riboprobes designed in VectorNTI (Invitrogen, Carlsbad, CA) with T3 and T7 polymeraserecognition sequencing flanking the sense (forward) and antisense(reverse) primers, respectively (42). Sequences used for probes wereas follows: GST� (NM010356), forward, 5�-ATTAACCCTCAC-TAAAGGGACAAGATTATCTCGTTGGCAA-3�; GST�, re-

Fig. 4. MPTP induced neuronal loss in the SN of GST� (�/�), GST� (�/�), and GST� (�/�) mice. (a–c) Representative midbrain sections showing SNimmunostained for TH and counterstained for Nissl in GST� (�/�), GST� (�/�), and GST� (�/�) mice 7 days after MPTP. (d) Stereological analysis of the numberof dopaminergic neurons within the SN of GST� (�/�), GST� (�/�), and GST� (�/�) mice 7 days after MPTP. (e–g) HPLC measurement of dopamine (e),3,4-dihydroxyphenylacetic acid (f), and homovanillic acid (g) in striatum from GST� (�/�), control, and MPTP-treated mice. (h) HPLC measurement of MPP� fromSN of SW and GST� (�/�) mice 15 min after four individual i.p. injections (spaced at 2-h intervals) of MPTP (F � 16.73, P � 0.001; *, P � 0.05, SW compared withGST� (�/�) SN). (i) Measurement of hydroxyl radical formation in the SN of SW and GST� (�/�) mice at baseline and 15 min after three injections of MPTP (F �25.8, P � 0.0001).

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verse, 5�-TAATACGACTCACTATAGGGGCTTCAATGTGA-TGCAAAATT-3�; GST� (NM008183), forward, 5�-ATTAAC-CCTCACTAAAGGGATACCTTGCCCGAAAGCACAA-3�;GST�, reverse, 5�-TAATACGACTCACTATAGGGGGAAG-GCGTCCAGGCACTTGC-3�; GST� (NM013541), forward, 5�-ATTAACCCTCACTAAAGGGAGATATGGTGAAGGA-ATGATGGGGT-3�; GST�, reverse, 5�-TAATACGACTCAC-TATAGGGGGATCTTGGGCCGGGCACTGA-3�.

Sections containing the SNpc from three separate brains wereimmunolabeled with monoclonal anti-GST� (610719; BD Bio-sciences, San Jose, CA) and polyclonal anti-TH (Pel-Freez,Rogers, AR), polyclonal anti-GST� (NCL-GST�M2; Novocas-tra, Newcastle upon Tyne, U.K.) and monoclonal anti-TH (1299;Sigma, St. Louis, MO), or polyclonal anti-GST� (NCL-GST�;Novocastra). GST isoforms were visualized by using FITC-conjugated secondary antibodies, and TH was visualized byusing RITC-conjugated secondary antibodies (MolecularProbes, Carlsbad, CA). Sections were visualized by using acomputer-assisted Leica TCS SP confocal microscope.

RT-PCR and Western Blot Analysis. Mice were killed by cervicaldislocation followed by rapid brain removal, dissection intocomponent parts, and snap-freezing for RT-PCR and Westernblot analysis (n � 4 per strain per condition) 15–30 min afterdifferent doses of MPTP: two doses of 20 mg/kg MPTP, fourdoses of 20 mg/kg MPTP, and four doses of 20 mg/kg plus 12 h.For RT-PCR, total mRNA was isolated by using the RNeasy Kit(Qiagen, Valencia, CA) as per the manufacturer’s directions.One hundred nanograms of RNA was reverse-transcribed byusing the SuperScript II Kit (Invitrogen), and GST�, GST�, andGST� were cDNA-amplified by using the primers describedabove. For Western blot analysis, tissue was homogenized inRIPA lysis buffer with a protease inhibitor mixture. Proteinconcentration was determined by using a protein assay kit(Bio-Rad, Hercules, CA). Thirty micrograms of total protein wasresolved on a 4–12% SDS gradient PAGE and electrotrans-ferred to polyvinylidene membrane (Millipore, Billerica, MA).GST�, GST�, and GST� protein levels were determined byusing rabbit anti-GST A1-1 (1:1,000, GS 62; Oxford BiomedicalResearch, Oxford, MI), rabbit anti-GST M1-1 (1:1,000, GS 67;Oxford Biomedical Research), and mouse anti-GST-pi (1:1,000,610718; BD Biosciences). Phospho-JNK levels were determinedby using an anti-phospho-JNK polyclonal antibody (1:500; Bio-Source, Carlsbad, CA), and phospho-c-Jun (Ser-73) was deter-mined by using a polyclonal antibody (1:500; Cell SignalingTechnology, Danvers, MA). Secondary antibodies conjugatedwith HRP (1:10,000; Pierce, Rockford, IL) were visualized byusing Supersignal West Dura chemiluminescence (Pierce). Sub-sequent to this procedure, membranes were stripped and re-probed by using mouse anti-GAPDH (1:5,000, ab8245; Abcam,Cambridge, MA). Chemiluminescence was repeated to visualizeprotein bands. Western blots were digitally scanned and analyzedwith NIH Image (National Institutes of Health, Bethesda, MD)for quantification per the manufacturer’s directions (http://rsb.info.nih.gov/nih-image). Adjusted scores for the target pro-teins were divided by the adjusted scores for GAPDH to obtainthe normalized scores (as percentage GAPDH).

GST� Inhibition. SN from postnatal day 0 to postnatal day 3 ND4SW mice were cultured as previously reported (14). At 7 DIV,cells were treated with 50 nM EA (Sigma) (pH 7.3) 30 min beforetreatment with 50 nM MPP�. EA and MPP� treatment wererepeated on the same cultures 24 h later. At 5 days after MPP�

treatment, cultures were fixed (4% formalin/1� TBS) andimmunostained for TH (1:250). The number of TH-positive cellswere counted as previously reported (14). RNAi experimentswere performed as above, but at 7 DIV cultures were transfectedwith predesigned GST� siRNA (50 nM; Ambion, Austin, TX)

and treated 4 and 24 h after transfection with 50 nM MPP�.Transfection efficiency of siRNA was calculated by countingCy3-labeled GST� siRNA in all cells and compared with thetotal number of DAPI-labeled cells. A labeling efficiency of 42%was empirically determined (expected Cy-3 label of siRNA is45–55% as per Ambion). Cells from the same cohort were fixedfor TH immunocytochemistry or harvested for RNA (Qiagen)48–72 h after transfection. Immunostained SN cultures werecounted and compared as above, and quantitative PCR of GST�was performed by using an Applied Biosystems Model 7900 withforward primer 5�-GTGCCCGGCCCAAGAT-3�, reverseprimer 5�-TTGATGGGACGGTTCACATG-3�, and FAM/Tamra-labeled probe 5�-6FAM-AAGGCCTTTCTGTCCTC-CCCGGA-TAMRA-3�. To normalize RNA levels, GST� levelswere compared with 18S levels (Applied Biosystems) within thesame RNA sample. Mouse brain total RNA (Ambion) wascompared as a positive control. Sequences used for RNAi andquantitative PCR were within the ORF for mouse GST� (43),evaluated by BLAST search, and were found to align specificallyto Mus musculus GST�. GST� mRNA levels are reported aspercentage of expression relative to nontransfected cells fromthe same culture cohort. Four to six independent culture cohortswere analyzed in each experiment.

Analysis of SNpc Number. C57BL/6, ND4 SW (Harlan), and GST�(�/�) mice backcrossed onto SW for seven generations wereused in this study. At this number of backcrosses, 80% of thegenome is fixed in any individual animal (44), and all of themicrosatellite markers we use to distinguish C57BL/6 and SW(18) were fixed for SW. For studies using GST (�/�) and (�/�)mice, wild-type littermates were used as controls. Three- to4-month-old mice of each genotype (n � 4) were given four i.p.injections of 20 mg/kg MPTP (Sigma) or saline at 2-h intervals(total dose, 80 mg/kg MPTP). For examination of cell number,mice were processed and analyzed as previously reported (12) byusing the optical fractionator (45) on a Bioquant Image AnalysisSystem (R & M Biometrics, Nashville, TN).

Measurement of MPP� Levels. C57BL/6, SW, and GST� (�/�)mice, 3–6 months in age, received four i.p. injections of 20 mg/kgMPTP-HCl. Mice were killed 15 min after each injection or 7days after the last injection. Striatum and SN were dissected onice and homogenized in 10 volumes of ice-cold 0.1 M perchloricacid. The homogenate was centrifuged at 12,000 � g for 15 minat 4°C, and the supernatants were filtered in 0.2� filters for 2 minat 8,000 � g. One hundred microliters of supernatant wasinjected into a HPLC equipped with a Nucleosil 100-5 C18 (4 �125 mm, 5 �m) column (Agilent Technologies, Santa Clara, CA)and a UV detector. The mobile phase consisted of 50 �Msulfuric acid, 10% acetonitrile, 90% water, and 75 mM trieth-ylamine adjusted to a final of pH 2.3 (run under isocraticconditions at 1.0 ml/min). The UV detector was set to 295 nmfor MPP� detection, as previously described (46). The concen-trations of MPP� were determined by comparison with knownamounts of MPP� iodide dissolved in 0.1 M perchloric acid andextrapolation from a standard curve.

Determination of Hydroxyl Free Radicals. The generation of hy-droxyl free radicals in the SN was determined by using the4-hydroxybenzoic acid method with slight modifications (47).Briefly, C57BL/6 and SW mice, 3–6 months in age, received onei.p. injection of 4-hydroxybenzoic acid (400 mg/kg) 1 h beforereceiving three i.p. injections of 20 mg/kg MPTP-HCl separatedby 2-h intervals. Mice were killed 15 min after the third injectionof MPTP, and SN were dissected and homogenized in 10volumes of 0.1 M perchloric acid and centrifuged for 12 min at10,000 � g at 4°C. The supernatant was passed through a 0.2-�mfilter and stored at �70°C. 4-Hydroxybenzoic acid reacts with

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hydroxyl radicals to produce 3,4-DBA; thus 3,4-DBA was ana-lyzed by using HPLC combined with EC detection under iso-cratic conditions. The guard cell was set at �350, with ascreening electrode set at �150 mv and working electrode set at�700 mv. The mobile phase consisted of 50 mM NaH2PO4 and0.05 mM octane sulfonic acid, 25 �M EDTA, and 2% acetoni-trile, adjusted to pH 2.7 with phosphoric acid. The mobile phasewas delivered at a flow rate of 0.6 ml/min onto a DHBA column.The levels of 3,4-DBA were determined by injecting knownconcentrations of 3,4-DBA and extrapolating from a standardcurve.

Dopamine and Metabolite Analysis. C57BL/6 mice, SW mice, andGST� (�/�) mice, 3–6 months in age, received four i.p.injections of 20 mg/kg MPTP-HCl at 2-h intervals and werekilled after 7 days. The brains of mice were rapidly removed andplaced on an ice-cooled plate for dissection of the striatum.Tissue was weighed and homogenized in 10 volumes of ice-cooled 0.1 M perchloric acid and centrifuged for 12 min at10,000 � g at 4°C. The supernatant was passed through a 0.2-�mfilter and stored at �70°C. Dopamine, 3,4-dihydroxyphenylac-etic acid, and homovanillic acid were analyzed by using reverse-phase ion-pairing HPLC combined with EC detection under

isocratic conditions. The guard cell was set at �350 mV, with ascreening electrode set at �150 mV and working electrode setat �220 mV. The mobile phase consisted of 75 mM sodiumdihydrogen phosphate monohydrate, 1.7 mM 1-octane sulfonicacid sodium salt, 0.73 mM triethylamine, 25 �M EDTA, and 10%acetonitrile/90% water. The pH was adjusted to 3.0 with phos-phoric acid. The mobile phase was delivered at a flow rate of 0.6ml/min onto the reversed-phase column MD-150 (3 � 150 mm;3 �m). Twenty-microliter aliquots were injected by an autoin-jector with a cooling module set at 4°C. The levels of dopamine,3,4-dihydroxyphenylacetic acid, and homovanillic acid were de-termined by injecting known concentrations of each protein andextrapolating from a standard curve.

Statistical Analysis. Differences in cell number and measurementsof protein levels using densitometric analysis were determined byusing a two-way ANOVA and Fisher’s post hoc analysis todetermine whether statistical differences were present. All sta-tistics were performed by using Statview (SAS Institute,Cary, NC).

Funding was provided by National Institutes of Health Grant NS39006(to R.J.S.) and the American Lebanese Syrian Associated Charities.

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