Proteome analysis of substantia nigra and striatal tissue in the mouse MPTP model of Parkinson's disease

RESEARCH ARTICLE

Proteome analysis of substantia nigra and striatal tissue

in the mouse MPTP model of Parkinson’s disease

Xin Zhao1, Quan Li1, Lei Zhao1 and XiaoPing Pu1, 2

1 Department of Molecular and Cellular Pharmacology, School of Pharmaceutical Sciences,Peking University, Beijing, P. R. China

2 National Key Research laboratory of Natural and Biomimetic Drugs, Peking University, Beijing, P. R. China

The dopaminergic neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) replicatesmany of the pathological hallmarks of Parkinson’s disease (PD) in mice via selective destructionof dopamine neurons of the substantia nigra and striatum. Although MPTP has been widelyused to study downstream effects following the degeneration of dopaminergic neurons, theunderlying mechanisms of MPTP action remain poorly understood. To determine the under-lying mechanisms of MPTP action at the protein level, a 2-DE-based proteomics approach wasused to evaluate the changes in protein expression in substantia nigra and striatal tissue inC57BL/6 mice after MPTP administration. We identified nine proteins that were markedlyaltered and are likely to be involved in mitochondrial function, heat shock protein activity, andwhich contribute enzyme activities for energy metabolism and protein degradation.

Received: January 27, 2007Revised: June 7, 2007

Accepted: June 11, 2007

Keywords:

MPTP / Parkinson’s disease / Substantia nigra and striatum

Proteomics Clin. Appl. 2007, 1, 1559–1569 1559

1 Introduction

Parkinson’s disease (PD) is characterized by preferentialdopaminergic neurodegeneration in the substantia nigrapars compacta (SNpc) with a subsequent loss of striataldopamine and the presence of Lewy bodies in theremaining nigral neurons. Although the mechanismsunderlying the development of both genetic and sporadicPD remain unknown, mitochondrial and proteasomaldysfunction and oxidative stress are widely recognized asmajor contributors [1, 2]. A pivotal role for these pathwaysis further supported by the fact that all chemicallyinduced Parkinsonian models, including 1-methyl-4-phe-

nyl-1,2,3,6-tetrahydropyridine (MPTP) [3], rotenone [4],and more recently epoxomicin [5], induce their effects viamitochondrial and proteasomal dysfunction, as well asthrough increased oxidative stress. Nevertheless, despitedecades of research, attempts to conclusively identify themolecules involved in dopaminergic neurodegenerationhas yielded limited success. In some forms of familialPD, mutations in the genes encoding a-synuclein, parkin,and ubiquitin C-terminal hydrolase L1 (UCH-L1) areassociated with protein accumulation and Lewy body for-mation in the substantia nigra, locus coeruleus, and cer-ebral cortex [6–8]. In sporadic PD, the levels of oxidativelydamaged proteins and protein aggregation are elevated inthe substantia nigra, and are likely to be secondary to themitochondrial dysfunction observed in these patients [9–11]. In addition, recent studies have revealed structuraland functional defects in 26/20S proteasomes in the sub-stantia nigra in sporadic PD [12–14].

Mouse model of PD produced by systemically adminis-tering the neurotoxin MPTP and rat model of hemi-parkinsonism obtained by intranigral injection of the neuro-toxin 6-hydroxydopamine (6-OHDA) were two most widelyused animal models for PD [15]. In our study, we chose the

Correspondence: Dr. Xiao-Ping Pu, Department of Molecular andCellular Pharmacology, School of Pharmaceutical Sciences, Pek-ing University, Beijing 100083, P. R. ChinaE-mail: [email protected]: 186-10-82802431

Abbreviations: MPP1, 1-methyl-4-phenylpyridinium ion; MPTP, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; PD, Parkinson’s dis-ease; SNpc, substantia nigra pars compacta; UPS, ubiquitin-pro-teasome system

DOI 10.1002/prca.200700077

© 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.clinical.proteomics-journal.com

1560 X. Zhao et al. Proteomics Clin. Appl. 2007, 1, 1559–1569

MPTP-induced mouse model as the animal model for PD todo the research. Once in the brain, MPTP is converted bymonoamine oxidase B within nondopaminergic cells, pre-dominantly glial cells, to an active metabolite 1-methyl-4-phenylpyridinium ion (MPP1) which enters dopaminergicneurons through the dopamine transporter. When MPP1

ions accumulate in substantia nigra pars compacta neurons,they inhibit complex I of the mitochondrial electron trans-port chain, ultimately leading to cell death [16]. Thus, in thepresent study, the MPTP mouse model was selected to per-form a proteome analysis of substantia nigra and striatal tis-sue in an animal Parkinson’s disease model.

Current research on the molecular basis of PD hasused high-throughput screening techniques to detectaltered neuronal gene expression in the rat and mousebrain following exposure to dopamine neurotoxins [17, 18].However, changes in mRNA levels do not always correlatewith similar changes in protein levels or activity [19].Studies evaluating protein expression changes in PDmodels are therefore crucial. The most common methodof proteome analysis is a comparative 2-DE separation ofproteins from two different biological states, followed byMS mapping or MS/MS to identify the differentiallyexpressed proteins. 2-DE separation exhibits remarkableresolving power and ease of use for quantitative analysis,which increases the sensitivity, reproducibility, andthroughput of proteome analysis [20–22]. Here, we used a2-DE-based proteomics approach to examine changes inprotein expression in the substantia nigra and striatum inthe mouse upon MPTP exposure. Several protein func-tional groups were found to be differentially regulated inthe MPTP model group, including mitochondrial proteins,heat shock proteins (HSP), and enzymes involved inenergy metabolism and protein degradation. The identifi-cation and characterization of these proteins will not onlyshed more light on the pathogenesis of PD, but shouldalso yield potential therapeutic targets that can prevent PDonset or halt its clinical progression.

2 Materials and methods

2.1 Chemicals and reagents

MPTP, 3,30-diaminobenzidine tetrahydrochloride (DAB),urea, CHAPS, DTT, and iodoacetamide were supplied bySigma (St. Louis, MO, USA). Tyrosine hydroxylase (TH) goatpolyclonal antibody was purchased from Chemicon Interna-tional (Temecula, CA, USA). Histostain™-SP Kits were pur-chased from Zymed Laboratories (San Francisco, CA, USA).Acrylamide was obtained from Merck (Darmstadt, Ger-many). The other reagents for the polyacrylamide gel prepa-ration, IPG strips, and carrier ampholytes (Resolyte 3–10)were purchased from BioRad (Hercules, CA, USA). Theprotease inhibitor cocktail set (Merck) contained 4-(2-amino-ethyl) benzenesulfonyl fluoride (AEBSF), trans-epoxy-

succinyl-L-leucylamido (4-guanidino) butane (E-64), EDTA,leupeptin, and aprotinin, and was used according to themanufacturer’s instructions.

2.2 Animals and treatments

Adult male C57BL/6 mice weighing 20–25 g at the begin-ning of this experiment were purchased from the Laborato-ry Animal Center of Peking University Health ScienceCenter (Beijing, China). Purchased mice met the approvalof the local animal committee with confirmation number:SCXK (Jing) 2002-0001. Animals were housed in groups of3–5 under standard conditions (temperature 22 6 27C,relative humidity 55 6 5%, 12-h light/dark cycle) with foodand water available ad libitum. In the present study, allexperiments were performed under the guidelines of theExperimental Laboratory Animal Committee of PekingUniversity Health Science Center and were in strict accor-dance with the principles and guidelines of the NationalInstitutes of Health Guide for the Care and Use of Labora-tory Animals.

After being allowed to acclimatize in our facilities for 1wk, mice were randomly divided into the following twogroups, containing 19 mice per group: an MPTP modelgroup and a vehicle control group that received an equal vol-ume of normal saline. Mice were treated with a conventionalMPTP paradigm. MPTP (30 mg/kg, dissolved in saline) wasintraperitoneally injected once a day for five consecutivedays. Control animals were injected with saline under thesame regimen [23, 24]. Behavioral experiments, includingspontaneous motor activity and rotarod evaluation, wereconducted 1 and 2 h after the final MPTP injection.

2.3 Spontaneous motor activity test

To evaluate spontaneous motor activity, we used an activitymonitor (Experimental Factory of Chinese Academy of Med-ical Sciences, Beijing, China) consisting of four Plexiglascylinders (23 cm630 cm, diameter6height), each equippedwith three infrared beams and an automated counting sys-tem. Ten mice were randomly selected from each group. Themouse was placed in the cylinder and, following a 3 minhabituation period, its overall activity level was assessed bycounting the number of infrared beam crossings in the pho-tocell apparatus per 5 min period [25].

2.4 Rotarod test

To assess sensorimotor coordination, the mice were eval-uated in the rotarod task [26, 27]. The rotarod unit(Experimental Factory of Peking University Health ScienceCenter, Beijng, P. R. China) consists of a rotating spindle(diameter 7.3 cm) and five individual compartments to testfive mice at a time. After training twice daily over twosuccessive days (speed 12 rpm on the first day and 18 rpmon the second day), the rotation speed was increased to



25 rpm on the third day in a test session. The time eachmouse remained on the rotating bar was recorded overthree trials for each mouse. Each trial was separated by a5-min interval and each trial had a maximum length of60 s. Data are presented as the mean time on the rotatingbar over the three test trials.

2.5 TH immunohistochemistry

Five days after the last MPTP or saline injection, four micefrom each group were anaesthetized with sodium pento-barbital (50 mg/kg) and the heads were perfusion-fixed with10% buffered formalin following a normal saline flush. Thebrains were removed 1 h after perfusion fixation and wereimmersed in the same fixative for a minimum of 2 wk untilthey were embedded in paraffin. Paraffin sections from thesubstantia nigra (10 mm thick) were mounted and immuno-histochemically stained with TH using a polyclonal anti-THantibody followed by a Histostain-SP kit. After a 5-min rinsein 10 mM PBS (pH 7.4), the sections were blocked for 1 h inPBS containing 10% normal goat serum. Sections were thenincubated overnight at 47C in a solution of PBS plus 0.3%Triton X-100 containing the anti-TH primary antibody (1:200).After a 15-min rinse in fresh 10 mM PBS, a biotinylated sec-ondary antibody was applied for 2 h, followed by incubationwith an avidin–biotin peroxidase complex for 30 min at roomtemperature. Immunoreactivity was visualized with 0.05%DAB. Sections were dehydrated through an alcohol series,cleared in xylene, and coverslipped. All sections were coun-terstained with bisbenzimide (10 mg/mL) for 5 min andwashed with 0.02% Triton X-100 in 10 mM PBS. Photos weretaken of sections from the same position in each brain with anOlympus digital camera attached to a microscope with106objective. The number of surviving dopaminergic neu-rons was determined manually by counting the TH-markedsoma under bright-field illumination in the right SNpc usinga 106objective. Cell counts were determined using threeanatomically matched sections from each of the animals(n = 4 per group) and the obtained value was divided by threeto yield an average cell count per section. The data were ana-lyzed statistically using a one-way analysis of variance(ANOVA), followed by posthoc t-tests where appropriate.

2.6 Sample preparation

C57BL/6 mice were anaesthetized and sacrificed by exsan-guination. Striatal and substantia nigra tissue were dissectedfrom control and MPTP treated mice. Samples (0.3 g tissuefrom five mice) were homogenized using an ultrasonichomogenizer in 1.5 mL lysis buffer (7 M urea, 40 mM Tris,pH 7.4, 65 mM DTT, 2% CHAPS) containing a protease in-hibitor cocktail set, and incubated at 47C for 1h. Homoge-nized samples were then centrifuged at 15 000 rpm for 1 h at47C. The supernatants were collected as crude extracts. Tri-plicates each consisting of the pooled combined brainregions of five mice were used in 2-DE.

2.7 2-DE

Supernatants of the homogenized samples were cleanedusing a 2-D clean-up kit (Amersham Biosciences). The totalprotein concentration of the two samples was determinedusing a bicinochoninic acid (BCA) protein assay reagent kit(Pierce, Rockford, IL, USA) and then adjusted to the sameconcentration (7 mg/mL). 2-DE was performed essentially asreported previously [28]. Samples of protein (800 mg) wereapplied to 17 cm immobilized pH 3–10 nonlinear gradientstrips by in-gel rehydration (12 h at 50 V). Focusing was per-formed at 207C according to the following schedule: 0.5 h 0–250 V linear, 1 h 250–1000 V linear, 5 h 1000 V–10 000 Vlinear, 6 h 10 000 V. The second-dimensional separation wasperformed on 12.5% SDS-polyacrylamide gels. The gels wererun at 50 mA per gel in a Protean II xi system. After proteinfixation for 2 h in 50% methanol containing 10% acetic acid,the gels were silver stained. Molecular weights were deter-mined by running standard protein markers (Institute ofBiochemistry, Shanghai), covering a range of 10–100 kDa. pIvalues were provided by the supplier of the IPG strips. Silver-stained gels were scanned in an Image Scanner and elec-tronic images of the gels were recorded using Photoshop(Adobe) software. Protein spots were outlined automatically,gel were matched and spot quantitation were analyzed usingPDQuest 2-D Gel analysis software (Version 7.1; BioRad).The Student’s t-test was applied to compare the spot patternsin gels derived from individual control (15 mice, pooling 5mice samples, n = 3) and MPTP-treated animals (15 mice,pooling 5 mice samples, n = 3). Significant spots that showedat least 1.5-fold difference in intensity between the groupswere selected for protein identification.

2.8 Protein identification and database search

Protein spots of interest were excised from the gels and sub-jected to in situ digestion with TPCK-trypsin according topreviously described procedures [29, 30], with some mod-ifications. In brief, the gel particles were rinsed with 50%ACN v/v for removal of salts and SDS, followed by reductionof cysteines with dithiotreitol, and subsequent carbamido-methylation of free cysteines with iodoacetamide. In situtryptic degradation was performed overnight at 377C, fol-lowed by two subsequent extractions of peptides. The pooledextracts were lyophilized and finally reconstituted in 20 mL of0.1% TFA v/v prior to MALDI-MS analysis. Samples wereconcentrated, purified and prepared for MALDI-MS analysis.The mass spectrometer used in the present work was a Voy-ger Biospectrometer™ workstation (ABI, USA). The instru-ment is equipped with a nitrogen laser (337 nm, 3 ns outputpulse) and a high current detector. All data were obtained inthe reflection mode with an acceleration voltage of 20 kV.Each mass spectrum was generated from the accumulateddata of 50 laser shots. A saturated solution of CHCA in 50%ACN was used as the matrix. Spectra were calibrated usingmatrix and tryptic autodigestion ion peaks as an internal



standard. Protein identification was achieved using theMASCOT search engine (http://www.matrixscience.com)query for the entire theoretical mouse peptide masses in theNCBI and Swiss-Prot protein databases, based on theassumption that peptides are monoisotopic, oxidized atmethionine residues and carbamidomethylated at cysteineresidues. Up to one missed trypsin cleavage was allowed, al-though most matches did not contain any missed cleavages.A mass tolerance of 150 ppm was allowed for matchingpeptide mass values. Probability-based MOWSE scores werecalculated by the software by comparison of search resultsagainst estimated random match population and werereported as 2106log10 (p), where p is the absolute prob-ability.

2.9 Western blot analysis

To confirm the differentially expressed proteins in MPTPtreated mice, striatal and substantia nigra tissue were dis-sected from controls and MPTP treated mice. Tissues werehomogenized in lysis buffer (comprising the following:80 mM Tris-HCl (pH 7.4), 150 mM NaCl, 2 mM Na2EDTA,0.1% SDS w/v, 0.4 mM DTT) containing a protease inhibitorcocktail set (Merck), and the homogenates were placed on icefor 1 h. Homogenates were clarified by centrifugation at15 0006g for 10 min and the total protein content of the twosamples was determined using a BCA protein assay reagentkit. Equal amounts (20 mg) of protein extracts were subjectedto 12.5% SDS-PAGE analysis and transferred to a PVDFmembrane (Millipore) using the Mini Trans-Blot Cell System(BioRad) for 50 min at 250 mA. The membranes wereblocked with 5% nonfat dry milk in TBST (20 mM Tris-HClpH 7.4, 0.15 M NaCl, 0.05% Tween 20) for 1 h at room tem-perature under agitation. Then, anti-Cpn 10 (1:2000, Stress-gen), anti-GST M1 (mu) (1:1000, Upstate), and anti-uMtCK(1:500, Santa Cruz) antibodies were incubated with mem-branes. HRP-conjugated goat anti-rabbit antibody (SantaCruz) diluted 1:5000 was used as the secondary antibody. Toensure equal protein loading, membranes were stripped andimmunostained for actin using an anti-b-actin antibody(1:1000, Santa Cruz) as the primary antibody. Relative bandintensities were determined by Gel-Pro Analyzer software(Media Cybernetics, USA).

3 Results

3.1 MPTP-induced motor behavior in mice

MPTP is widely used to model nigrostriatal dopaminergicneuron degeneration in mice and nonhuman primates, al-though the dosing paradigms vary widely. We used a classicmethod [23, 24] as described above (30 mg/kg/day, i.p. for5 days). Because of our interest in protein expression chang-es occurring in nigrostriatal dopamine systems after MPTPintoxication, we used the 2-DE-based proteomics approach

and Western blotting to analyze differentially expressed pro-teins in striatal and substantia nigra tissue.

Figures 1A and B show results for the spontaneousmotor activity and rotarod tests, respectively. MPTP admin-istration (MPTP model group) resulted in a significantreduction in spontaneous motor activity relative to vehicle-treated mice (vehicle control group). For the rotarod test,MPTP-treated mice remained on the bar for a reducedamount of time compared to vehicle-treated mice (p,0.01 forboth comparisons).

Figure 1. Behavioral testing in MPTP-treated mice. (A) The num-ber of total movements recorded in the spontaneous locomotiontest over a 5 min period. Relative to the vehicle control group(295.2 6 21.9), locomotion counts were drastically reduced byMPTP treatment (63.1 6 7.3). (B) The latency to fall on therotarod. Relative to the vehicle control group (57.2 6 5.6 s), theduration of time on the rotating rod was significantly decreasedfollowing MPTP treatment (21.3 6 2.7 s). Values are presented asmean 6 SEM (n = 10). **p,0.01 compared with the vehicle con-trol group.

3.2 TH immunohistochemistry

TH is the rate-limiting enzyme in dopamine biosynthesis.Immunostaining of the substantia nigra using an anti-THantibody followed by a Histostain-SP kit demonstrated



MPTP-induced loss of TH-positive dopaminergic neurons inthe substantia nigra (Figs. 2A (b), 2B, p,0.05). In controlmice, the cytoplasm and fibers of dopaminergic neurons

Figure 2. Immunohistochemical staining of dopaminergic neu-rons with an anti-tyrosine hydroxylase antibody in the substantianigra in the mouse MPTP model. Photomicrographs were takenat a magnification of 2006. (A) (a) Vehicle control group: numer-ous substantia nigra cells are observed. The cytoplasm is richlystained and the cellular processes are evident. (b) MPTP modelgroup (30 mg/kg, 1x/day for 5 days): few immunoreactive posi-tive cells are observed and the cell processes are not visible in themajority of cells. (B) Cell counts in the SNpc of vehicle and MPTP-treated mice. Values represent mean 6 SEM of four mice pergroup. **p,0.01 compared with the vehicle control group.

were intensely stained and the cellular processes were evi-dent (Fig. 2A (a)). By contrast, MPTP treatment resulted in amarked loss of DA-containing SNpc neurons, as few TH-positive cells were observed and the cellular processes wereabsent for most cells (Fig. 2A (b)).

Consistent with the above cellular morphological obser-vations, the average cell count in the vehicle control groupwas 59 6 8 DA neurons per section, whereas the mean in theMPTP model group was 17 6 7 per section (p,0.05, n = 4)(Fig. 2B). These results suggest that we successfully inducedDA cell loss in the mouse MPTP model for PD.

3.3 2-DE of control and MPTP treatment samples and

identification of differentially expressed proteins

Proteins extracted from the striatal and substantia nigra tis-sue of both the control and MPTP treated mice were ana-lyzed in parallel by 2-DE using silver staining. The gel spotpatterns of these two specimens are summarized in Fig. 3.The images were analyzed using PDQuest software (BioRad)and each specimen was found to contain a total of about 500spots. A total of 345 spots from the MPTP treated specimenscould be matched to the control specimens with a match rateof 67%. Of the protein spots with varying staining inten-sities, nine spots were selected for further analysis from thesilver-stained gels. The relative abundance of these nine

Figure 3. The raw 2-D images of the substantia nigra and stria-tum extracts of control (A) and MPTP-treated (B) mice, visualizedby silver stain. The extracts were separated using IPG gel (pH 3–10, 17 cm) in the first phase and 12.5% polyacryamide gel in SDS-PAGE. Sampled protein in one gel was 800 mg.



selected proteins (spots 1–9) in the two gels was analyzed byPDQuest software (Table 1). The quantitative changes inselected proteins as described by the p value compared withcontrol group and the ratio MPTP/control are shown inTables 1 and 2. Enlarged regions of the respective gels areshown in Fig. 4. Moreover, the protein patterns betweenthese samples were highly reproducible.

The nine spots were excised from control and MPTPgels, digested with trypsin and analyzed with MALDI-MS.Peptide masses were used to query the NCBI and Swiss-Protsequence databases, and the nine spots were all significantwith MOWSE scores greater than 63. The results, compris-ing of the percentage of sequence coverage, the number ofmatching peptides, the accession names, variations,descriptions, the theoretical pI, and MW, are included inTable 2. There were five up-regulated protein spots and fourdown-regulated. The five proteins with an increase in

Table 1. Quantitative change of proteins from substantia nigraand striatum in response to MPTP administration

Spotno.

Control MPTP p-value

Spotdensity

CV (%) Spotdensity

CV (%)

1 1486 19.12 800 31.17 0.0052 859 31.88 163 26.43 0.0353 183 48.87 421 43.22 0.0484 886 16.24 248 42.92 0.0015 78 21.43 222 9.39 0.0066 55 24.11 171 37.78 0.0547 183 15.64 369 7.74 0.0028 364 18.38 995 20.05 0.0149 350 23.62 49 13.88 0.023

Spot number (spot no.) corresponds to the same number shownin Fig. 4. Spot density is the average OD of the same spot fromthree independent samples belonging to the same experimentalgroup. CV shows the variation of the OD of the spots across thesethree samples.

Figure 4. Enlarged regions of the respective gels are shown inthis figure for nine differentially expressed proteins. The proteins(no. 1–9) indicated by the arrows exhibited robust differences inexpression between the vehicle control group and MPTP modelgroup. The graphs show the relative abundance of the proteinindicated across the two gels (C is the control group; M is theMPTP-treated group). The scales of the graphs are in arbitraryunits.

expression included biliverdin reductase (BVR) B (spot 3),polyubiquitin (spot 5), Ckmt1 protein (spot 6), proteasome(prosome, macropain) subunit, alpha type 2 (spot 7), and es1protein (spot 8). The four proteins with a decrease in expres-sion were HSP 1 (spot 2), GST mu 1 (spot 4), ubiquitin-con-jugating enzyme E2M (spot 9), and peptidylprolyl isomeraseA (spot 1). Figure 5 reports the peptide mass fingerprint andidentification of GST mu 1 (spot 4) to provide an example ofthe peptide mass fingerprints of the nine proteins.

3.4 Western blot

To further confirm the protein abundance changes observedin 2-DE, a Western blot was performed. As shown in Fig. 6,HSP 1 and GST mu 1 were down-regulated in the MPTPmodel group, whereas the Ckmt1 protein was up-regulated.The trends observed for these three proteins by Westernblotting were consistent with the results from 2-DE.

4 Discussion

MPTP is a recognized environmental cause of PD that wasoriginally identified in epidemiologic studies of drug ab-users. This neurotoxin causes selective destruction of thedopaminergic neurons of the nigrostriatal pathway inhumans and other primates as well as in mice [31, 32]. In thepresent study, behavioral experiments revealed that MPTPinduces behavioral deficits in spontaneous locomotion andmotor coordination in mice. By using tyrosine hydroxylaseimmunohistochemistry, we were able to demonstrate thatMPTP causes a loss of tyrosine hydroxylase-positive neuronsin the mouse SNpc, indicating that we had successfullyinduced PD-like symptoms/pathology in the MPTP model.

Proteomics tools are broadly applied in human disease,and the techniques are frequently used for the discovery ofdifferentially expressed proteins. Here, we used the 2-DE andMS techniques to identify changes in protein expression inMPTP treated mice relative to vehicle treated mice, in orderto identify differentially regulated proteins and to furtherclarify the role of these altered proteins in the genesis of PD.

Recently, studies have identified various proteins thatmay be involved in pathogenesis of PD using proteomicstechnology [33–35]. Here, we have identified nine proteinswhich were differentially expressed in the MPTP and controlsamples. Three of them belong to the ubiquitin-proteasomesystem (UPS), including the proteasome (prosome, macro-pain) subunit, alpha type 2 (spot 7), ubiquitin-conjugatingenzyme E2M (spot 9), and polyubiquitin (spot 5). HSP 1(spot 2), Ckmt1 protein (spot 6), and es1 protein (spot 8) areinvolved in mitochondrial functioning. The other three pro-teins are BVR B (spot 3), GST mu 1 (spot 4) and peptidyl-prolyl isomerase A (spot 1).

Polyubiquitin (spot 5) belongs to the HSP gene family[36]. In the present study, polyubiquitin was up-regulated inthe MPTP model group. This may be due to an increase in



Figure 5. MS and identification of spot 4. (A) The peptide mass fingerprint of spot 4 from mouse substantia nigra and striatum analyzed withMALDI-MS. (B) Identification of spot 4 as GST mu 1 with 65% amino acid sequence coverage by matched peptides using MASCOTsoftware.Matched peptides were shown in bold. (C) Using the data, the MASCOT program searched the protein in the NCBI protein database.



Table 2. Identification of proteins that showed quantitative change in response to MPTP administration

Spotno.

Protein identitiy Accessionname

No. ofpeptidesmatched

%Sequencecoverage

MW(kDa)

pI RatioMPTP/control

Description

1 Peptidylprolyl isomerase A gi)6679439 7 49 18.131 7.74 0.54 Peptidylprolyl isomerase2 Heat shock protein 1 gi)19353434 5 47 10.956 7.93 0.19 Chaperonin, play fundamental roles in

the folding, assembly, andtranslocation of other proteins

3 BVR B gi)21450325 7 50 22.297 6.49 2.30 Serine/threonine kinase4 GST mu 1 gi)28386202 18 65 26.067 7.71 0.28 Play an important role in the defense

mechanism of the organism5 Similar to polyubiquitin gi)94370512 5 64 8.723 6.56 2.85 UPS6 Ckmt1 protein gi)19683937 9 25 47.373 8.39 3.11 Ubiquitous mitochondrial creatine

kinase: a member of creatinekinases

7 Proteasome (prosome, macropain)subunit, alpha type 2

gi)20070847 8 47 26.024 6.92 2.01 A subunit of 20S proteasome

8 es1 protein gi)20070420 5 34 28.415 9.00 2.74 May be serving a basic function inmitochondria

9 Ubiquitin-conjugating enzyme E2M gi)4507791 6 18 21.172 7.57 0.14 Ubiquitin-conjugating enzyme

Spot number corresponds to the same number shown in Fig. 4. MW is the theoretical molecular mass calculated from the amino acidsequence. pI is the theoretical pI calculated from the amino acid sequence of the predicted mature protein. Ratio MPTP/Control representsthe ratio of spot intensity of the MPTP-treated against the control group.

Figure 6. Expression of HSP1 (spot 2), GSTM1 (spot 4), andCkmt1 (spot 6) in control and MTPT-treated mice were analyzedvia Western blot. HSP1 and GSTM1 were down-regulated in theMPTP model group whereas the Ckmt1 protein was up-regu-lated.

proteasome activity following environmental stress and sub-sequent protein damage. This in turn might lead to reducedubiquitination and therefore impaired clearance of abnormalproteins in PD. Ubiquitin-conjugating enzyme E2M (spot 9)is a member of the ubiquitin-conjugating enzyme family.The proteasome (prosome, macropain) subunit, alpha type 2(spot 7) is a subunit of the 20S proteasome. Together, thesefindings indicate robust alterations in the UPS followingMPTP treatment, which has not been completely understoodto date.

Our results are consistent with previous studies whichimplicate the UPS in onset of PD. The UPS is one of thepathways involved in the detoxification and targeting ofdamaged proteins for degradation [37]. The current findingssupport the contention that proteasome dysfunction may beinvolved in the pathogenesis of PD [38–41].

The Ckmt1 protein (spot 6) is a crucial enzyme in cellenergy metabolism [42]. The active metabolite of MPTP, the

MPP1 is an inhibitor of complex I of the mitochondrial elec-tron transport chain. This ion accumulates in dopaminergicneurons and results in mitochondrial dysfunction. One ofthe deleterious effects is a decrease in ATP production,resulting in an energy shortage in dopaminergic neurons[43]. Therefore, increased expression of Ckmt1 protein mayindicate an attempt to compensate for the decrease in ATPsynthesis.

HSP1 (also named HSP10, spot 2) together with HSP60,is involved in the folding of imported mitochondrial proteins[44–46]. There have been no reports to date concerning therelationship between HSP10 and PD, yet previous studieshave established a relationship between other HSP, such asHSP70, and PD [47–49]. In the present work, HSP10 wasdown-regulated in the MPTP model group, although theimplication of this remains unclear. Similarly, the role of thees1 protein (spot 8) that is up-regulated in the MPTP modelgroup in PD is also not clear. Recent studies have indicatedthat human es1 protein belongs to the DJ-1/PfpI family.Based on the assumption that structural similarities reflect afunctional relationship, we may speculate that human es1protein serves a basic role in mitochondrial function [50, 51].The altered expression levels of the HSP10 and es1 proteinsmay indicate mitochondrial dysfunction or alterations inother, as-yet unidentified cellular processes. Mitochondrialdysfunction and concomitant free radical production resultin increased oxidative stress and are likely to have a primaryrole in the pathogenesis of PD [52]. Ckmt1, HSP10, and es1protein are all involved in mitochondrial activity, and our



results indicate that these three proteins may be involved incases of PD induced by environmental toxins. However, thedetailed mechanism of action by these proteins requires fur-ther study.

The two other proteins found to be differentiallyexpressed in the MPTP and control samples are twoenzymes involved in energy metabolism. GST mu 1(GSTM1, spot 4), a GST, plays an important role inintracellular detoxification pathways. In addition, maleswith a deletion of the GSTM1 gene were found to bemore susceptible to PD [53, 54]. In our study, theexpression of GSTM1 was down-regulated in the MPTPmodel group. While the underlying reasons for thedecreased expression remain unknown, such a decreasewould be expected to result in a decrease in the cellulardetoxification capabilities, thus increasing the suscepti-bility to PD. The second enzyme, BVR (spot 3), is a ser-ine/threonine kinase that catalyzes the reduction of bili-verdin, the heme oxygenase (HO) activity product, tobilirubin [55, 56]. To date, there are no reports concern-ing the relationship between BVR-B and PD. However,the increase in BVR-B expression in the present studymay indicate an increase in bilirubin, which has anti-oxidant properties and may protect cells under oxidativestress conditions.

The last protein to be differentially expressed in ourresults is peptidylprolyl isomerase A (also named cyclophi-lin A, spot 1), which is the receptor for cyclosporin A andwhich accelerates protein folding by catalyzing the cis–transisomerization of proline imidic peptide bonds in oligopep-tides [57, 58]. Peptidylprolyl isomerase A is down-regulatedin the MPTP model group, although its role in PD remainsunclear.

Among the nine differentially expressed proteins identi-fied in this study, we chose three for further evaluation viaWestern blot analysis: HSP 10, GSTM1, and Ckmt1. Eachprotein was found to have commercially available antibodies.We found that HSP 10 and GSTM1 were down-regulated inthe MPTP model group whereas the Ckmt1 protein was up-regulated. These semi-quantitative observations were con-sistent with the results of 2-DE studies.

To the best of our knowledge, the current studydemonstrates for the first time a multitude of steady stateprotein level changes following MPTP administration.Many of the identified proteins derive from different cas-cades and pathways and have never previously been asso-ciated with MPTP administration. Deregulation of specificproteins may indicate deranged function, and our findingsencourage the functional proteomics study and the sub-sequent effects of protein up- or down-regulation. Thebiological significance of altered protein levels may simplyreflect the pathophysiological phenomena of MPTPadministration. Further studies will show whether proteinderangements are specific for MPTP or whether the pro-tein changes observed will turn out to be specific PD bio-markers.

This work was supported by a grant from the National Pro-gram for Key Basic Research Projects (no.2004CB518902) andthe National Natural Science Foundation of China(no.30472164).

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Proteome analysis of substantia nigra and striatal tissue in the mouse MPTP model of Parkinson's disease