Proteomic Analysis of Expression and Protein Interactions in a 6-Hydroxydopamine-Induced Rat Brain Lesion Model

Neurochemistry International 57 (2010) 16–32

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Neurochemistry International

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Proteomic analysis of expression and protein interactions in a 6-hydroxydopamine-induced rat brain lesion model

Bokyung Park a, Junyoung Yang a, Nuri Yun a, Kwang-Min Choe a, Byung K. Jin b, Young J. Oh a,* a Department of Biology, College of Life Science and Biotechnology, Yonsei University, 134 Shinchon-Dong, Seodaemoon-Gu, Seoul 120-749, Republic of Korea b School of Medicine Kyung-Hee University, Seoul 120-749, Republic of Korea

ARTICLE INFO ABSTRACT

Article history: Parkinson’s disease (PD) is the second most common neurodegenerative disorder caused by selective Received 6 August 2009 degeneration of the dopaminergic (DA) neurons in the substantia nigra pars compacta (SNc). Although Received in revised form 28 February 2010 mitochondrial abnormality, oxidative stress and proteasomal dysfunction are recognized as major Accepted 1 April 2010 contributors to the progression of PD, there is a limited understanding of the key molecular events that Available online 18 April 2010 provoke degeneration of DA neurons. Using a proteomic approach, we attempted to identify profiles of proteins with altered expression levels in rats following unilateral stereotaxic injection of 6- Keywords: hydroxydopamine into the SNc. Protein expression profiles of these proteins in the substantia nigra 6-Hydroxydopamine and the striatum were made using two-dimensional gel electrophoresis in conjunction with a mass Neuronal cell death Parkinson’s disease spectrometry. More than 70 identified proteins displayed significant differences in their temporal and Proteomics spatial expression pattern between experimental and vehicle-operated control groups. Based on the identity of the proteins, we further searched for potential binding partners using biological databases available on the web and constructed a protein interaction network. Among several interconnected proteins in the network, we verified the interaction between prohibitin and the NADH-ubiquinone oxidoreductase 30 kDa subunit (NDUFS3 subunit; a mitochondrial complex I subunit) by co- immunoprecipitation. We also confirmed, using immunohistochemical localization, that both prohibitin and the NDUFS3 subunit were increased in the dying DA neurons, suggesting its potential role in regulating mitochondrial function in dying DA neurons. Furthermore, knockdown of prohibitin accelerated 6-hydroxydopamine-induced cell death in SH-SY5Y cells. Our results raise the possibility that interconnected proteins in the network may positively or negatively impact the progression of DA neuronal death. ß 2010 Elsevier Ltd. All rights reserved.

Parkinson’s disease (PD) is a typical neurodegenerative disorder esis of PD is not fully understood, several important factors have accompanying such clinical manifestation as rigidity, resting been proposed and both types of PD seem to share certain common tremor, bradykinesia and postural instability. The pathological mechanisms in the progression of DA neuronal death. hallmarks of PD are selective degeneration of dopaminergic (DA) Important molecules and signaling pathways underlying DA neurons located in the substantia nigra pars compacta (SNc), and neuronal death have been examined using experimental models of the formation of Lewy bodies and dystrophic Lewy neurites PD. Gene-based models including a-synuclein, parkin, PINK1, (Giasson and Lee, 2003). There are two forms of PD, sporadic LRRK2 and DJ-1 have recently been used to evaluate consequences accounting for approximately 95% of the cases and the remaining of functional disruption by several forms of mutations (Moore 5% being inherited. Therefore, both environmental and genetic et al., 2005; Klein and Schlossmacher, 2006). Given the biochemical factors are believed to play important roles in PD pathogenesis changes reminiscent of those occurring in patients with PD, by (Broussolle and Thobois, 2002). Although the molecular pathogen- contrast, studies using toxin-based models induced by 6-hydroxydopamine (6-OHDA), 1-methyl-4-phenyl-1,2,3,6-tetahydropyr- idine (MPTP) and rotenone have been widely used to identify Abbreviations: 2-DE, two-dimensional gel electrophoresis; 6-OHDA, 6-hydroxydo- multiple molecular targets and the associated molecular mechan- pamine; DA, dopaminergic; GFAP, glial ﬁbrillary acidic protein; NDUFS3 subunit, isms underlying DA neuronal death (Beal, 2001). For example, NADH-ubiquinone oxidoreductase 30 kDa subunit; PD, Parkinson’s disease; SD rat, stereotaxic injection of 6-OHDA into the substantia nigra (SN) or Sprague–Dawley rat; SN, substantia nigra; SNc, substantia nigra pars compacta; striatum (STR), or administration of MPTP to monkeys and mice SNr, substantia nigra pars reticulata; STR, striatum; TH, tyrosine hydroxylase. * Corresponding author. Tel.: +82 2 2123 2662; fax: +82 2 312 5657. has been used to establish animal models of PD that accompany E-mail address: [email protected] (Y.J. Oh). relatively selective degeneration of DA neurons in the SNc (Javoy

0197-0186/$ – see front matter ß 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.neuint.2010.04.005 B. Park et al. / Neurochemistry International 57 (2010) 16–32 17 et al., 1976; Irwin et al., 1993; Ovadia et al., 1995). Using these 1.2. Sample preparation and 2-DE animal models, as well as cell culture models of PD, most of the At 5, 10 and 20 d after surgery, six rats in each group were decapitated, and the current hypotheses regarding potential pathophysiology of DA defined areas of SN and STR were consistently dissected out from the brains placed neuronal degeneration have been proposed (Dauer and Przed- on ice. Subsequent experiments for 2-DE were basically performed as previously borski, 2003). These include overproduction of reactive oxygen described with minor modification (Park et al., 2004a,b; Lee et al., 2008). The dissected tissues were solubilized in a 2-DE buffer containing a protease inhibitor species, abnormal mitochondrial function, generation of abnormal cocktail (Roche, Mannheim, Germany) using a tip-probe attached to the Sonic protein aggregates and inflammation (Fahn and Cohen, 1992; Dismembrator (Fisher Scientific, USA). Protein content was determined by two- Schapira, 1997; Beal, 2003; Abou-Sleiman et al., 2006). Regardless dimensional Quantikit (Amersham Bioscience, Uppsala, Sweden). Isoelectric of the recent progress in our understanding about the etiology and focusing (IEF) in the first dimension was performed by an in-gel rehydration pathogenesis of PD, we still lack understanding of the key method using 24 cm IPG strips (Amersham Biosciences) with a pH range between 3 and 10 NL (non-linear). Briefly, samples (1 mg protein per each gel) were diluted molecular events that provoke degeneration of DA neurons. with a total volume of 450 ml lysis buffer containing 0.5% IPG buffer (pH 3–10 NL, Currently, several approaches using proteome analyses have Amersham Biosciences), rehydrated overnight and focused for 96 kVh. The second been reported in neurodegenerative disorders including PD dimension, SDS-PAGE was performed using 10–16% gradient polyacrylamide gels (Choudhary and Grant, 2004; Fountoulakis and Kossida, 2006; without stacking gels for 16 h at 18 mA/gel and stopped just before the dye front ran off the gels. Gels were then stained with 0.1% Coomassie Brilliant Blue G-250 Licker et al., 2009). Both fluid and tissue samples obtained from (Sigma) for 24 h. The stained gels were scanned using a densitometer (Powerlook experimental models of PD or from patients with PD have been 2100XL, UMAX, USA), and the gel images were analyzed by the ProteomeWeaver subjected to proteomic analyses. For example, protein expression software system ver. 2.1 (Definiens, Germany). The protein spots in the different has been compared in the postmortem SN of PD patients and age- gels were defined as up-regulated or down-regulated by comparing the intensity of matched controls (Basso et al., 2004; Jin et al., 2006; Werner et al., the protein spots in 6-OHDA-injected and the matching vehicle-injected samples. Significance of difference between two groups was determined by Student’s t-test. 2008). Simultaneous measurement of relative changes in the proteome has been performed with samples of cerebrospinal fluid 1.3. In-gel trypsin digestion and mass spectrometric analysis and human plasma (El-Agnaf et al., 2003; Abdi et al., 2006; Waragai et al., 2006; Zhang et al., 2008). Quantitative proteomic analyses Protein spots of interest were manually cut into approximately 1 mm cubes from have been conducted for established experimental animal and cell the gels and destained by washing with a 50% acetonitrile (ACN) in 25 mM ammonium bicarbonate buffer, pH 8.0. The destained gel slices were completely culture models of PD after treatment with MPTP (Jin et al., 2005; dried for 30 min under vacuum in a SpeedVac centrifuge (BioTron, Korea). Gel slices Diedrich et al., 2008), 6-OHDA (Lee et al., 2003; De Iuliis et al., were rehydrated and digested at 37 8C for 17 h with 10 mg/ml porcine trypsin in 2005; Nakamura et al., 2006; Lee et al., 2008), L-DOPA (Valastro 25 mM ammonium bicarbonate buffer, pH 8.0. Digestion was stopped by addition of et al., 2007), dopamine quinone (Van Laar et al., 2008)or 15 ml of 50% ACN, 0.1% trifluoroacetic acid. After in-gel trypsin digestion, the extracted tryptic peptides were purified with a POROS R2 column (Applied lipopolysaccharide (McLaughlin et al., 2006). Possible changes in Biosystems, Foster City, CA). The purified peptide mixtures were combined with a- the brain proteome of parkin knockout mice have been studied by cyano-4-hydroxycinnamic acid matrix (1:1, v/v) and spotted on stainless steel running 2-dimensional gel electrophoresis (2-DE; Palacino et al., MALDI sample plates (Applied Biosystems, Framingham, MA). The peptide mixtures 2004; Periquet et al., 2005). were acquired in reflectron mode (20 keV accelerating voltage) with 500 ns delayed In the present study, we specifically aimed to further identify extraction, averaging 1000 laser shots per spectrum using 4700 Proteomics Analyzer (Applied Biosystems). Trypsin autolysis reference ions (842.51 and the temporal and spatial expression pattern of proteins in both the 2211.10 Da peptides) were used to internally calibrate each spectrum to a mass SN and STR after a unilateral injection of 6-OHDA into the rat SNc accuracy within 20 ppm. The spectra were analyzed by GPS Explorer TM software using 2-DE in conjunction with a mass spectrometry. We identified ver. 3.5 (Applied Biosystems) using Mascot server ver. 1.9.05 (Matrix Science, London, more than 70 proteins showing altered expression levels in both UK). The peptide peaks with a signal to noise ratio greater than 10–15 and a mass between m/z 800 and 4000 were searched against NCBInr (2007.04.30, 4886660 SN and STR. By specifically attempting to construct a network out sequences, 1688057783 residues; Taxonomy, Rattus, 40234 sequences) or SwissProt of the identified proteins, we found several hub proteins that (2007.04.17, 264492 sequences, 96880444 residues; Taxonomy, Rattus, 5959 potentially interact with several other important proteins that may sequences) database after contaminants were excluded. Carbamidomethyl cysteine be associated with the progression of neurodegeneration. We also was set as fixed modification and oxidized methionines and deamidation of asparagine provided evidence to show binding of a mitochondrial chaperone and glutamine as variable modifications. When protein appears in database under different names and accession numbers, we first checked the names through other protein with component of the mitochondrial complex I, their Database including NCBI (http://www.ncbi.nlm.nih.gov) or the Human Protein increased staining intensity in dying DA neurons and potential Reference Database (HPRD, http://www.hprd.org) on the web to confirm whether protective role for the mitochondrial chaperone protein, raising the those different names indicate the same protein. Several other criteria including the possibility that interconnected proteins in the network may protein score, C.I. % and statistic significance were also taken into our consideration. In addition, selection of the protein was also assisted by a comparison of the theoretical impact the progression of DA neuronal death. values of molecular weight and pI acquired from the Database and values appeared on the 2-DE gel itself. 1. Experimental procedures

1.1. Animals and surgical procedures 1.4. Construction of a protein interaction network

All animal experiments were conducted in accordance with the guidelines set by To gather information on the differentially expressed proteins and to find their Yonsei University. Male Sprague–Dawley rats (SD; 9-week-old, 280–300 g; Orient, binding partners, we searched through the open databases on the web including the Korea) were housed in a temperature controlled chamber and maintained at National Center for Biotechnology Information (NCBI, http://www.ncbi.nlm.nih.- 22 2 8C with a 12 h light–dark cycle and free access to food and water. Rats were first gov; Pruitt et al., 2000), SWISS-PROT (http://www.expasy.org; Boeckmann et al., distributed into two groups at random: unilaterally 6-OHDA-injected groups and 2003), the Database of Interacting Proteins (DIP, http://dip.doe-mbi.ucla.edu; vehicle-operated (ascorbate-saline vehicle-only injected) groups. For the rotational Xenarios et al., 2000) or the Human Protein Reference Database (HPRD, http:// behavioral test and 2-DE, 3 or 6 rats were assigned for each group, respectively. The www.hprd.org/; Peri et al., 2003). The output results from the databases were stereotaxic procedures and further processing were performed as previously described manually curated using Excel and Access software (Microsoft) to file up the list of by us with modifications (Choi et al., 2003; Lee et al., 2008). Briefly, rats in each group annotated protein information. Based on the output information, we constructed a were anaesthetized with chloral hydrate (400 mg/kg, i.p.), and 5 ml of 6-OHDA (5 mg/ protein linkage map by connecting those proteins categorized as signal transduc- ml in 0.2 mg/mL ascorbate-saline; Sigma, St. Louis, MO) or vehicle-only was tion and cell communication as a basic frame of their interaction partners. ipsilaterally administered into the SNc (AP, 5.3 mm posterior to bregma; L, 2.3 mm from the midline; D, 7.6 mm from the skull surface). After surgery, rats were 1.5. Co-immunoprecipitation and Western blot analysis maintained in a chamber until use. For the rotational behavior test, rats in each group were given D-amphetamine (5 mg/kg, i.p.) and their rotational behavior tested for The dissected tissues of SN or STR (5–10 d after lesion) were solubilized using a 90 min on the 10th and 30th day after surgery as previously described (Ungerstedt and tip-probe attached to Sonic Dismembrator in a cold RIPA buffer (50 mM Tris–HCl, Arbuthnott, 1970; Schwarting and Huston, 1996). The number of rotations in each pH 7.4, 150 mM NaCl, 1% NP-40, 0.25% sodium deoxycholate, 1 mM EGTA) group was counted on both the ipsilateral and contralateral direction. containing a protease inhibitor cocktail. The tissue lysates were centrifuged at 18 B. Park et al. / Neurochemistry International 57 (2010) 16–32

12,000 g,48C for 10 min and total protein content was measured using Bio-Rad 1.6. Immunohistochemistry protein assay kit. An equal amount of soluble proteins was incubated with protein G- or A-agarose (Santa Cruz Biotechnology, Santa Cruz, CA) and mouse monoclonal SD rats were transcardially perfused through the ascending aorta with 50 ml of anti-NDUFS3 subunit antibody (1:100; Abcam, Cambridge, UK) or mouse isotonic saline, followed by 200–250 ml of ice-cold 4% paraformaldehyde in 0.1 M monoclonal anti-prohibitin antibody (1:100; Calbiochem, Darmstadt, Germany) phosphate buffer, pH 7.4. The excised brains were post-fixed overnight in the same at 4 8C for overnight on a rotator. After incubation, the agarose pellets were carefully fixation solution and then transferred to 20% sucrose in 0.1 M phosphate buffered resuspended and washed in a RIPA buffer, and proteins were eluted from the beads saline (PBS). Six series of 40-mm-thick coronal sections were cut on a freezing by addition of 2 SDS-PAGE loading buffer. Eluted proteins were separated on a microtome and stored 4 8C until ready to use. For immunohistochemical 12.5% SDS-PAGE and processed for western blot analysis with anti-NDUFS3 subunit localization of TH, glial fibrillary acidic protein (GFAP), prohibitin, NDUFS3 subunit, antibody (1:2000) or anti-prohibitin antibody (1:1000). For measuring levels of or a combination, tissue sections from the SN were first washed with PBS and tyrosine hydroxylase (TH; a rate-limiting enzyme of dopamine biosynthesis), rabbit incubated in 0.2% Triton X-100/3% bovine serum albumin (BSA)/PBS for 30 min. polyclonal anti-TH (1:1000; Pel-Freez, Rogers, AR) was used as a primary antibody. After permeabilization, tissues sections were incubated in 3% BSA/PBS overnight at Rabbit polyclonal anti-actin antibody (1:3000; Sigma) or mouse monoclonal anti- 4 8C with the indicated primary antibodies. These included rabbit polyclonal anti- glyceraldehyde-3-phosphate dehydrogenase (GAPDH; 1:3000; Chemicon, Billerica, TH (1:500; Pel-Freez), rabbit polyclonal anti-GFAP (1: 500; Sigma), mouse MA) was used as a loading control. Peroxidase-conjugated secondary antibodies monoclonal anti-prohibitin (1:200) and mouse monoclonal anti-NDUFS3 subunit (1:2000; Amersham Biosciences) were used as appropriate. Bands were visualized (1:200). Tissue sections were subsequently washed with PBS and incubated with an by enhanced chemiluminescence (Amersham Biosciences). appropriate secondary antibody. These included Alexa Fluor 488 goat anti-rabbit

Fig. 1. Characterization of rat model of PD following unilateral stereotaxic injection of 6-OHDA into the SNc. (A) Immunohistochemical localization of TH was conducted in tissue sections obtained from the STR (dotted area) and SN (dotted area) on 10th or 30th day after unilateral injection of 6-OHDA into the SNc of SD rats. (B) Western blot analysis of TH was carried out in tissue lysates harvested from the ipsilateral side of SN or STR of vehicle- or 6-OHDA-lesioned rats at the indicated time periods. Duplicated blots were probed with polyclonal anti-actin antibody to confirm equal loading of proteins in each lane. (C) D-amphetamine-induced rotational behavior test conducted in 6- OHDA-lesioned rats at the indicated time periods. The number of rotations of ipsilateral (filled bars) and contralateral direction (shaded bars) between vehicle or 6-OHDA- injection group was measured for 90 min. Each bar represents the mean SEM (n = 3). *Significance of difference was determined by Student’s t-test (p < 0.001). B. Park et al. / Neurochemistry International 57 (2010) 16–32 19

IgG antibody and Alexa Fluor 568 goat anti-mouse IgG antibody (1:200, Molecular OHDA, indicating the magnitude of nigrostriatal lesion in our Probes, Eugene, OR). Nuclei were stained with a DNA intercalator, Hoechst 33258 model system (Fig. 1C). (Calbiochem-Novabiochem AG, Laufelfingen, Switzerland). After being washed with PBS, tissue sections were mounted onto slides with Vectashield mounting medium (Vector Laboratories, Burlingame, CA) for further microscopic examina- 2.2. 2-DE tion. An Axio imager D1 transmitted light microscope (Carl Zeiss, Zena, Germany) or a laser scanning confocal microscope (Bio-Rad LS2000) were used to collect the At the indicated time periods (5, 10 and 20 d) after stereotaxic images. injection of 6-OHDA or vehicle only, tissue obtained from the ipsilateral side of the SN and STR of the two groups were processed 1.7. Cell culture and gene silencing for a 2-DE-based proteomic analysis. Six rats were assigned for The SHSY-5Y cells were cultivated in Dulbecco’s modified Eagle’s medium each group (a total of 36 rats), and each dissected tissue from the supplemented with 10% heat-inactivated fetal bovine serum in an atmosphere of SN and STR was subjected to 2-DE (a total of 72 gels). Gel images 10% CO2. For post-transcriptional gene silencing, cells were transiently transfected that were equally grouped depending on tissue origin and time with 100 nM small interfering RNA (siRNA) of prohibitin or control siRNA as recommended by the manufacturer (Qiagen, Valencia, CA). Knock-down level of periods following the lesion were further edited into a final single prohibitin was confirmed by western blot analysis using mouse monoclonal anti- image, respectively. Fig. 2A and B shows the typical 2-DE master prohibitin (1:1000). Cell viability following treatment with 6-OHDA was measured gel images obtained from the SN or STR of the vehicle-injected by the MTT reduction assay (Choi et al., 1999). control group, respectively. Although there seemed to be no apparent difference in the overall distribution pattern of protein 2. Results spots obtained from the SN and STR, the basal amount of expression levels of proteins were different (e.g., neurofilaments, 2.1. Characterization of 6-OHDA-lesioned rat model 14-3-3 proteins), and the location of variant forms differed (e.g., annexin III). By comparing 2-DE gel images obtained from the 6- The goal of the present study was to collect information on OHDA- or vehicle-injected samples at a given days of post- proteins and the changes in their expression levels that may be injection, all of the differentially expressed protein spots by at least intimately associated with the process of dopaminergic neuronal 1.5-fold with a p-value of less than 0.1 were numbered on Fig. 2 cell death using the 6-OHDA-lesioned rat model. We first and subsequently identified by mass spectrometry. Information on characterized our rat model system by measuring expression these proteins from the SN and STR are described in Table 1A and B, levels of TH and amphetamine-induced rotational behavior after respectively. For all proteins listed in Table 1, the selected protein unilateral stereotaxic injection of 6-OHDA into the SNc. Immuno- scores depicted as C.I.% were close to 97–100%. When more than histochemistry showed that TH-positive neurons in the SNc and one candidate protein with similarly close calls were acquired from their axonal projections in the STR were largely damaged (Fig. 1A). the search through NCBInr and SwissProt Database, we basically TH-positive neurons in the ventral tegmental area were spared. As tried to verify each protein spot by performing repeated PMF shown in Fig. 1B, the expression level of TH in the SN and STR was analysis, searching against different databases, considering the significantly decreased 5 d after 6-OHDA injection and continu- protein score depicted as C.I.%, and referring to their location on 2- ously diminished in a time-dependent manner. In good correlation DE gel itself. Since alteration of the various isoforms of 14-3-3 were with the present immunohistochemical data for TH and our detected both in the SN and STR, in addition, we critically previous study demonstrating a diminished number of Nissl- examined and compared the PMF spectral data obtained from two positive neurons in SNc (Lee et al., 2008), measurement of the brain tissues to decide which protein spot on the 2-DE gel most amphetamine-induced rotational behavior demonstrated an truly represents the specific isoform of 14-3-3. Therefore, only the ipsilateral turning response in rats unilaterally injected with 6- proteins that were best fitted at all significance levels were listed in

Fig. 2. Typical 2-DE gel images of the SN and the STR. SD rats were stereotaxically injected with vehicle or 6-OHDA into the SNc of the brains, and the tissue samples from the SN and STR were collected. The protein mixture from each sample was analyzed on pH 3–10 NL strips followed by 10–16% gradient SDS-PAGE. Following analysis of spot intensity between vehicle- and 6-OHDA-treated groups, the altered protein spots were indicated by the arrows and the numbers on 2-DE gels of the SN (A) and STR (B). Identiﬁcation were done by protein mass spectrometry and listed in Table 1. 20

Table 1 Summary of the proteins identiﬁed by proteomic analysis of the altered proteins in the SN (A) and STR (B) after unilateral injection of 6-OHDA.

(14-3-3 protein tau) 16–32 (2010) 57 International Neurochemistry / al. et Park B. 5 14-3-3 protein zeta/delta Down (10 d) 1433Z-RAT 27,928 4.73 65 47 18 53 68.6 99.91774287 (Protein kinase C inhibitor protein 1) (KCIP-1) 6 3-Mercaptopyruvate Down (5 d), gij20304123 33,209 5.88 65 52 13 51 97.7 100 sulfurtransferase (MST) Up (20 d) 7 40S ribosomal protein SA Up (20 d) RSSA_RAT 32,803 4.8 65 50 15 42 103.0 100 (p40) (34/67 kDa laminin receptor) 8 4-Aminobutyrate aminotransferase, Down (10 d) gij13591900 57,173 8.46 65 36 29 60 111.0 100 mitochondrial precursor (GABA transaminase) 9 4-Aminobutyrate aminotransferase, Down (10 d) gij13591900 57,173 8.46 65 37 28 52 109.0 100 mitochondrial precursor (GABA transaminase) 10 4-Aminobutyrate aminotransferase, Down (10 d) GABT_RAT 57,173 8.15 65 40 25 56 117.0 100 mitochondrial precursor (GABA transaminase) 11 Acyl-CoA hydrolase Down (10 d) gij6981462 37,936 7.16 65 50 15 31 73.9 99.83609468 12 Adenosine kinase Down (5 d) ADK_RAT 40,456 5.72 65 46 19 31 75.6 99.98310831 13 Adenylyl cyclase-associated Down (10 d) CAP1_RAT 51,905 7.16 65 44 21 48 106.0 100 protein 1 (CAP 1) 14 Alpha-synuclein Up (20 d) SYUA_RAT 14,506 4.74 65 56 9 45 61.1 99.53743418 15 Annexin A3 (Annexin III) (Lipocortin III) Up (10, 20 d) ANXA3_RAT 36,573 5.96 65 45 20 58 145.0 100 (Placental anticoagulant protein III) (PAP-III) 16 Annexin A3 (Annexin III) (Lipocortin III) Up (10 d) gij122065130 36,573 5.96 65 43 22 57 121.0 100 (Placental anticoagulant protein III) (PAP-III) 17 Annexin A5 (Annexin V) (Lipocortin V) Up (20 d) ANXA5_RAT 35,779 4.93 65 38 27 71 171.0 100 (Endonexin III) (Calphobindin I) (CBP-I) 18 Annexin A5 (Annexin V) (Lipocortin V) Down (20 d) ANXA5_RAT 35,780 4.93 65 47 18 71 175 100 (Endonexin III) (Calphobindin I) (CBP-I) 19 Annexin A5 (Annexin V) (Lipocortin V) Down (10 d), ANXA5_RAT 35,780 4.93 65 38 27 67 164 100 (Endonexin III) (Calphobindin I) (CBP-I) Up (20 d) 20 Apolipoprotein E precursor (Apo-E) Down (10 d) APOE_RAT 35,789 5.23 65 45 20 47 82.5 99.99664901 21 Complement component 1 Q Down (10 d) C1QBP_RAT 31,326 4.77 65 44 21 60 55.6 98.35875454 subcomponent-binding protein, mitochondrial precursor 22 Eukaryotic translation initiation Down (10 d) IF2A_RAT 36,375 5.02 65 40 25 49 126.0 100 factor 2, subunit 1 alpha 23 Eukaryotic translation initiation factor 2, Down (5 d) gij9506571 36,375 5.02 65 44 21 48 107.0 100 subunit 1 alpha 24 Exocyst complex component 6 (Exocyst Down (10 d) EXOC6_RAT 93,872 5.74 65 42 23 25 59.3 99.29987842 complex component Sec15A) (Sec15-lake 1) (rSec15) 25 Ferritin heavy chain (Ferritin H subunit) Up (5 d) FRIH_RAT 21,287 5.85 65 57 8 37 56.1 98.53723844 26 Glia maturation factor beta (GMF-beta) Up (20 d) GMFB_RAT 16,900 5.32 65 54 11 50 71.1 99.95239275 27 Protein disulfide isomerase A3 precursor Down (5 d) gij8393322 57,018 5.88 65 33 32 59 228.0 100 (Glucose regulated protein, 58 kDa) (Disulfide isomerase ER-60) 28 Glutathione S-transferase P (GST 7-7) Down (10 d) GSTP1_RAT 23,656 6.89 65 44 21 65 97.3 100 (Chain 7) (GST class-pi) 29 Glutathione S-transferase, mu type 3 Down (10 d) gij13592152 25,838 6.84 65 42 23 61 116.0 100 (GST-Yb3) (Glutathione S-transferase Yb-3) 30 Guanine deaminase Up (20 d) gij7533042 51,449 5.48 65 51 14 43 129.0 100 31 Guanine deaminase Down (5 d) gij7533042 51,449 5.48 65 42 23 41 126.0 100 32 Hemoglobin subunit beta-1 (Hemoglobin Down (20 d) HBBI_RAT 16,085 7.88 65 51 14 74 106.0 100 beta-1 chain) (Beta-1-globin) 33 L-lactate dehydrogenase B chain Up (20 d) LDHB_RAT 36,879 5.7 65 42 23 54 120 100 (LDH subunit H) 34 NADH dehydrogenase [ubiquinone] 1 Down (5 d) NDUAA_RAT 40,753 7.64 65 45 20 56 125.0 100 .Pr ta./Nuohmsr nentoa 7(00 16–32 (2010) 57 International Neurochemistry / al. et Park B. alpha subcomplex subunit 10, mitochondrial precursor 35 NADH-ubiquinone oxidoreductase 24 kDa Down (5 d) gij205628 26,859 6 65 50 15 54 61.2 96.94793952 subunit, mitochondrial precursor 36 Neurofilament, light polypeptide Down (5, 10, 20 d) gij13929098 61,356 4.63 65 42 23 37 86.4 99.99078293 37 Neurofilament, light polypeptide Down (5, 10, 20 d) gij13929098 61,355 4.63 65 38 27 42 137.0 100 38 OTU domain, ubiquitin aldehyde Down (10 d) gij19527388 31,250 4.85 65 55 10 49 98.5 100 binding 1 (Otubain 1) 39 Phosphatidylethanolamine-binding Down (5 d) PEBPI_RAT 20,904 5.48 65 54 11 65 62.4 99.64708281 protein 1 (Raf kinase inhibitor protein) 40 Prohibitin Up (20 d) PHB_RAT 29,860 5.57 65 50 15 59 94.3 100 41 Protamine-2 (Sperm protamine-P2) Up (20 d) PRM2_RATFU 13,835 11.86 65 62 3 36 57.0 98.81102319 (Sperm histone P2) (Basic nuclear protein HPS2) 42 Proteasome activator complex subunit 2 Up (20 d) PSME2_RAT 27,072 5.52 65 54 11 442 66.5 99.86269923 (Proteasome activator 28 beta subunit) 43 Protein disulfide isomerase A3 precursor Down (10 d) gij1352384 57,044 5.88 65 33 32 52 211.0 100 (Glucose regulated protein, 58 kDa) (Disulfide isomerase ER-60) 44 Protein phosphatase 1, catalytic subunit, Down (5 d) gij4506005 37,975 5.84 65 50 15 43 75.3 99.88126113 beta isoform 1 (PP 1B) 45 Reticulocalbin-2 precursor (Calcium-binding Down (10 d) RCN2_RAT 37,410 4.27 65 46 19 54 104.0 100 protein ERC-55) 46 Serine/threonine-protein phosphatase 2A Up (10 d) PPP5_RAT 57,518 5.84 65 44 21 42 72.5 99.96551161 regulatory B (PR53) (PP2A, subunit B) 47 Similar to NADH dehydrogenase (ubiquinone) Down (5, 20 d) gij27661165 24,419 5.87 65 51 14 50 64.6 98.60493847 FE–S protein 8 48 Similar to Serine/threonine-protein phosphatase Down (5 d) gij109467991 36,884 5.88 65 49 16 57 98.8 100 2A regulatory subunit B (PR53) (PP2A, subunit B) 49 Transketolase (TK) Down (10 d) TKT_RAT 38,355 7.23 65 35 30 51 161.0 100 50 Transketolase (TK) Down (10 d) TKT_RAT 68,355 7.23 65 38 27 48 129.0 100 51 Tyrosine 3-monooxygenase (Tyrosine Down (10 d) TY3H_RAT 56,337 5.74 65 49 16 50 98.3 100 3-hydroxylase) (TH) 52 Ubiquitin-conjugating enzyme E2N Up (5 d) UBE2N_RAT 17,171 6.13 65 54 11 72 75.0 99.98115599 (Ubiquitin-protein ligase N) (Bendless protein) 53 Vimentin Down (5 d), gij14389299 53,758 5.06 65 44 21 38 128.0 100 Up (20 d) 21 22

Table 1 (Continued )

No. a Protein name Changed pattern b Accession Protein Protein No. of mass No. of mass No. of mass Sequence Protein Protein number c MWd pId searched unmatched matched coverage % score e score C.I. % f (B) STR 1 14-3-3 protein beta/alpha (Protein kinase Up (10 d) 1433B_RAT 28,152.85 4.81 65 51 14 52 58.59999847 99.17742873 C inhibitor protein 1) (KCIP-1) 2 14-3-3 protein gamma Down (10 d) 1433G_RAT 28,458.93 4.8 65 46 19 49 74.8 99.98026791 3 14-3-3 protein gamma Up (10 d) 1433G_RAT 28,284.91 4.8 65 45 20 45 136.0 100 4 14-3-3 protein theta (14-3-3 protein tau) Down (10 d) 1433T_RAT 28,050.81 4.69 65 47 18 40 70.5 99.94689036 5 14-3-3 protein zeta /delta (Protein kinase C Up (10 d), 1433Z_RAT 27,927.76 4.73 65 51 14 41 52.9 96.94386009 inhibitor protein 1) (KCIP-1) Down (10 d) 6 26S protease regulatory subunit 7 (Proteasome Down (5 d) PRS7_RAT 48,950.11 5.59 65 40 25 46 167.0 100 26S subunit ATPase 2) (Protein MSS1)

7 2-oxoisovalerate dehydrogenase subunit alpha, Down (5 d) gij129032 50,132.95 7.68 65 44 21 44 153.0 100 16–32 (2010) 57 International Neurochemistry / al. et Park B. mitochondrial precursor 8 3(2), 5-bisphosphate nucleotidase Down (5 d) gij25282455 331,353.05 5.58 65 54 11 41 86.9 99.99178527 9 Aconitase 2, mitochondrial Up (20 d) gij40538860 85,379.97 7.87 65 42 23 36 156.0 100 10 Adenylate kinase isozyme 1 Down (20 d) gij8918488 21,646.18 7.71 65 50 15 68 84.1 99.98434715 11 Alpha-enolase (2-phospho-D-glycerate hydro-lyase) Down (5 d) gij56757324 47,446.28 6.16 65 41 24 55 104.0 100 (Non-neural enolase) (NNE) (Enolase 1) 12 Alpha-synuclein Down (5 d) SYUA_RAT 14,506.2 4.74 65 57 8 45 93.5 100 13 ARP3 actin-related protein 3 homolog Down (5 d) gij23956222 47,791.01 5.61 65 43 22 42 110 100 14 Biliverdin reductase A Down (10 d) BIEA_RAT 33,718.53 5.82 65 51 14 50 69.9 99.93724146 15 Calretinin (CR) Down (10, 20 d) CALB2_RAT 31,500.5 4.94 65 47 18 54 72.5 99.96649008 16 Dimethylarginine dimethylaminohydrolase 2 Down (5 d) gij47087079 30,017.39 5.66 65 52 13 45 104 100 17 Glia maturation factor beta (GMF-beta) Down (5 d) GNAL_RAT 44,742.46 6.23 65 49 16 41 85.19999695 99.99814786 18 Glia1 fibrillary acidic protein delta Up (10 d) gij5030428 48,810.24 5.72 65 35 30 63 158.0 100 19 Protein disulfide isomerase A3 precursor Down (20 d) gij8393322 55,553.72 5.88 65 37 28 56 228.0 100 (Glucose regulated protein, 58 kDa) (Disulfide isomerase ER-60) 20 Glucose-6-phosphate dehydrogenase Down (5 d) gij8393381 59,801.88 5.97 65 33 32 54 200.0 100 21 Glutamate oxaloacetate transaminase 1 Down (20 d) gij122065118 46,399.5 6.73 65 42 23 62 216.0 100 (Aspartate aminotransferase, cytoplasmic) (Transaminase A) 22 Glutamine synthetase Down (5 d) GLNA_RAT 42,994.37 6.64 65 49 16 33 71.1 99.95374342 (Glutamate-ammonia ligase) (GS) 23 Glutathione S-transferase P (GST 7-7) (Chatin 7) Up (20 d) GSTP1_RAT 23,656.09 6.89 65 50 15 60 90.4 100 (GST class-pi) 24 Glyoxalase I (Lactoylglutathione lyase) Down (5 d) LGUL_RAT 20,980.35 5.12 65 51 14 57 83.8 99.99751588 (Methylglyoxalase) (Aldoketomutase) 25 Heat-shock protein beta-1 (HspB1) (Heat shock Up (10 d) HSPB1_RAT 22,936.62 6.12 65 53 12 53 117.0 100 27 kDa protein) (HSP 27) 26 Imprinted and ancient (IMPACT) Up (20 d) gij58866042 36,320.21 5.07 65 50 15 34 69 99.49348395 27 Karyopherin (importin) beta 1 Down (20 d) gij8393610 98,396.12 4.66 65 40 25 28 99.3 100 28 NADH dehydrogenase [ubiquinone] 1 alpha Down (5 d) NDUAA_RAT 40,467.64 7.64 65 32 13 48 112 100 subcomplex subunit 10, mitochondrial precursor 29 N-ethylmaleimide sensitive fusion protein Down (20 d) gij13489067 82,599.96 6.55 65 34 31 38 192.0 100 30 N-ethylmaleimide sensitive fusion protein Down (20 d) gij13489067 82,599.96 6.55 65 31 34 42 235.0 100 31 Phosphoserine aminotransferase Down (20 d) gij29692074 40,910.74 8.09 65 43 22 45 99.5 100 32 Platelet-activating factor acetylhydrolase, Up (20 d) gij7305363 46,640.13 6.97 65 46 19 46 118.0 100 isoform 1b, beta 1 subunit 33 Prohibition Down (5 d) gij6679299 29,801.9 5.57 65 56 9 51 125.0 100 34 Protein tyrosine phosphatase, non-receptor type 5 Up (5 d) gij94400885 42,976.99 5 65 45 20 46 126.0 100 35 Pyruvate dehydrogenase [lipoamide] kinase Down (5 d) PDK2_RAT 46,308.15 6.13 65 44 21 51 77.4 99.98915641 isozyme 2, mitochondrial precursor 36 Rab GDP dissociation inhibitor alpha (Rab GDI alpha) Down (20 d) GDIA_RAT 50,504.15 4 65 35 30 56 214.0 100 37 Rab GDP dissociation inhibitor beta Down (5 d) gij40254781 50,504.69 5.93 65 37 28 48 197.0 100 (GDP dissociation inhibitor 2) 38 Rab1B protein Down (5 d) gij226486 22,350.21 5.55 65 55 10 58 64.5 98.57244381 39 Serine/threonine kinase receptor associated protein Up (20 d) gij58865512 38,722.03 4.99 65 50 15 47 106.0 100 40 Seine/threonine-protein phosphatase PP1-beta Down (5 d) PP1B_RAT 37,974.7 5.84 65 53 12 36 57.5 98.90938124 catalytic subunit (Protein phosphatase 1) (PP 1B) 41 Similar to Alpha-Centractin (Centractin) Down (5 d) gij109460227 54,974.59 8.27 65 41 24 50 123.0 100 (Centrosome-associated acting homolog) 42 Similar to NADH dehydrogenase Down (5 d) gij27702072 30,207.62 7.07 65 47 18 52 203.0 100 (ubiquinone) FE–S protein 3 43 Similar to NADH dehydrogenase Down (5 d) gij27702072 30,207.62 7.07 65 46 19 61 214.0 100 (ubiquinone) FE–S protein 3 44 Similar to NADH dehydrogenase Up (20 d) gij27661165 23,954.73 5.87 65 51 14 44 87.1 99.99215499 (ubiquinone) FE–S protein 8 45 Similar to purine-nucleoside phosphorylase Down (5 d) gij34869683 32,571.07 6.45 65 43 22 50 67.30000305 99.25080839 46 Triosephosphate isomerase Up (20 d) TPIS_RAT 27,353.8 6.89 65 49 16 54 95.9 100 (TIM) (Triosephosphate isomerase) 47 Tyrosine 3-monoxygenase Down (5, 10, 20 d) TY3H_RAT 56,337.36 5.74 65 44 21 58 151.0 100 .Pr ta./Nuohmsr nentoa 7(00 16–32 (2010) 57 International Neurochemistry / al. et Park B. (Tyrosine 3-hydroxylase) (TH) 48 Tyrosine 3-monoxygenase Down (5, 10, 20 d) gij6981652 55,931.32 5.74 65 55 10 32 104.0 100 (Tyrosine 3-hydroxylase) (TH) 49 Tyrosine 3-monoxygenase Down (5, 10, 20 d) TY3H_RAT 55,931.32 5.74 65 50 15 44 118.0 100 (Tyrosine 3-hydroxylase) (TH) 50 Tyrosine 3-monoxygenase Down (5, 10, 20 d) TY3H_RAT 56,330.47 5.74 65 50 15 44 96.4 100 (Tyrosine 3-hydroxylase) (TH) 51 Ubiquinol-cytochrome c reductase, Up (20 d) gij57114330 29,717.26 9.04 65 51 14 38 90.59999847 99.99649576 Rieske iron-sulfur polypeptide 1 52 Ubiquitin carboxyl-terminal hydrolase isozyme L3 Down (5 d) UCHL3_RAT 26,107.03 5.01 65 49 16 56 120 100 53 Ubiquitin-conjugating enzyme E2N Down (5 d) gij16758810 17,112.96 6.13 65 53 12 79 118.0 100 a Number(Ubiquitin-protein represents the protein ligase N) spot (Bendless of the 2-DE protein) master gel shown in Fig. 2. b Changed pattern represents spot volume of proteins that were either down or up-regulated. Day after 6-OHDA injection showing significant change in volume was indicated in parenthesis. c Accession number in SwissProt and NCBInr databases. d Theoretical M r (Da) and pI. e A non-probabilistic protein score, derived from the ions scores, and the number of peptide matches. f Ion score confidence interval calculated as % (PMF data) using GPS Explorer TM software ver. 3.5 (Applied Biosystems). The closer the confidence interval percentage value is to 100%, the more likely the protein is correctly identified. 23 24 Table 2 Functionally categorized lists of the proteins showing differential expression levels after unilateral injection of 6-OHDA. Proteins are listed by alphabetical order within each functional category.

Biological process Molecule function Gene name Identiﬁed proteins

Cell growth and/or maintenance Protein serine/threonine PPP1CB Protein phosphatase 1, catalytic phosphatase activity subunit, beta isoform (PP IB) Structural constituent of ACTR1A Alpha-centractin (Centractin) cytoskeleton (Actin-related protein 1A) (Centrosome-associated actin homolog) Structural constituent of ACTR3 ARP3 actin-related protein 3 cytoskeleton homolog (ACTR3) (Actin-related protein 3) (ARP3) Structural constituent of VIM Vimentin cytoskeleton Structural molecule activity GFAP Glial ﬁbrillary acidic protein delta Structural molecule activity NEFL Neuroﬁlament, light polypeptide Unknown CAP1 Adenylyl cyclase-associated protein (CAP) homolog MCH1

Metabolism; energy pathways ATPase activity NSF N-ethylmaleimide sensitive fusion protein .Pr ta./Nuohmsr nentoa 7(00 16–32 (2010) 57 International Neurochemistry / al. et Park B. Catalytic activity ACO2 Aconitase 2 (Citrate hydrolyase aconitase) (Mitochondrial aconitase) Catalytic activity BCKDHA 2-Oxoisovalerate dehydrogenase subunit alpha, mitochondrial precursor (BCKAD) (MSUD1) Catalytic activity EXO1 Alpha-enolase (2-phospho-D-glycerate hydro-lyase) (Non-neural enolase) (NNE) (Enolase 1) Catalytic activity G6PD Glucose-6-phosphate dehydrogenase Catalytic activity LDHB L-lactate dehydrogenase B chain (LDH subunit H) Catalytic activity NDUFS3 NADH dehydrogenase (ubiquinone) Fe–S protein 3 Catalytic activity PDK2 Pyruvate dehydrogenase [lipoamide] kinase isozyme 2, mitochondrial precursor (PDK2) Catalytic activity TH Tyrosine 3-monooxygenase (Tyrosine 3-hydroxylase) (TH) Catalytic activity AK1 Adenylate kinase isozyme 1 Catalytic activity UQCKFS1 Ubiquinol-cytochrome c reductase, Rieske iron-sulfur polypeptide 1 (RISP) Glutathione transferase GSTP1 Glutathione S-transferase P (GST 7-7) (Chain 7) activity (GST class-pi) Glutathione transferase GSTM3 Glutathione S-transferase, mu type 3 (GST Yb3) activity (Glutathione S-transferase Yb-3) Glutathione transferase GLO1 Glyoxalase I (Lactoylglutathione lyase) (Methylglyoxalase) activity (Aldoketomutase) Hydrolase activity Cte1 Acyl-CoA hydrolase Hydrolase activity DDAH2 Dimethylarginine dimethylaminohydrolase 2 (Dimethylargininase 2) Isomerase activity Tpi1 Triosephosphate isomerase (TIM) (Triosephosphate isomerase) Oxidoreductase activity Blvra, Blvr Biliverdin reductase A Oxidoreductase activity NDUFA10 NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 10, mitochondrial precursor Oxidoreductase activity NDUFS8 NADH dehydrogenase:ubiquinone Fe–S protein 8 Oxidoreductase activity NDUFV2 NADH-ubiquinone oxidoreductase 24 kDa subunit, mitochondrial precursor Phosphorylase activity NP Purine-nucleoside phosphorylase (PNP) (Nucleoside phosphorylase) Sulfotransferase activity Mpst 3-Mercaptopyruvate sulfurtransferase (MST) Transaminase activity ABAT 4-Aminobulyrate aminotransferase (Gamma Aminobutyrate Transaminase) (GABA transaminase) Transaminase activity GLUL; GLNS Glutamine synthetase (Glutamate ammonia ligase) (GS) Transaminase activity GOT1 Glutamate oxaloacetate transaminase 1 (Aspartate aminotransferase, cytoplasmic) (Transaminase A) Transaminase activity PSAT1 Phosphoserine aminotransferase Transketolase activity TKT Transketolase (TK)

Immune response Complement receptor C1QBP Complement component 1 Q subcomponent-binding activity protein, mitochondrial precursor (GClq-R protein) (HABP1)

Protein metabolism Chaperone activity HSPB1 Heat-shock protein beta-1 (HspB1) (Heat shock 27 kDa protein) (HSP 27) Chaperone activity SNCA Alpha synuclein Isomerase activity PDIA3 Protein disulfide isomerase A3 precursor (Glucose regulated protein, 58 kDa) (Disulfide isomerase ER-60) Translation regulator EIF2S1 Eukaryotic translation initiation factor 2, subunit 1 alpha activity Ubiquitin-specific protease OTUB1 OTU domain, ubiquitin aldehyde binding 1 (Otubain 1) activity Ubiquitin-specific protease PSMC2 26S protease regulatory subunit 7 (Proteasome 26S subunit activity ATPase 2) (Protein MSS1) Ubiquitin-specific protease PSME2 Proteasome activator complex subunit 2 (Proteasome activity activator 28 beta subunit)

Ubiquitin-speciﬁc protease Ube2n Ubiquitin-conjugating enzyme E2N (Ubiquitin-protein 16–32 (2010) 57 International Neurochemistry / al. et Park B. activity ligase N) (Bendless protein) Ubiquitin-speciﬁc protease UCHL3 Ubiquitin carboxyl-terminal hydrolase isozyme L3 (UCHL3) activity (Ubiquitin thiolesterase L3)

Regulation of nucleobase, nucleoside. nucleotide Catalytic activity ADK Adenosine kinase and nucleic acid metabolism DNA binding PRM2 Protamine-2 (Sperm protamine-P2) (Sperm histone P2) (Basic nuclear protein HPS2)

Signal transduction; cell communication Calcium ion binding ANXA3 Annexin A3 (Annexin III) (Lipocortin III) (Placental anticoagulant protein III) (PAP-III) Calcium ion binding ANXA5 Annexin A5 (Annexin V) (Lipocortin V) (Endonexin II) (Calphobindin I) Calcium ion binding Calb2 Calretinin (CR) Calcium ion binding RCN2 Reticulocalbin 2 precursor (Calcium-binding protein ERC-55) Growth factor activity GMFB Glia maturation factor beta (GMF-beta) GTPase activity RAB1B Rab1B protein GTPase activator activity GDI1 Rab GDP dissociation inhibitor alpha (Rab GDI alpha) (GDI-1) GTPase activity GNAL Guanine nucleotide-binding protein G(olf) subunit alpha Heterotrimeric G-protein GNAO1 Guanine deaminase GTPase activity Hydrolase activity PAFAH1B2 Platelet-activating factor acetylhydrolase, isoform 1b, beta1 subunit Hydrolase activity, acting on ester bonds Bpnt1 3(2),5-Bisphosphate nucleotidase 1 Protease inhibitor activity PEBP1 Phosphatidylethanolamine-binding protein 1 (Raf kinase inhibitor protein) Protein serine/threonine PPP2CB Serine/threonine-protein phosphatase 2A regulatory phosphatase activity subunit B (PR53) (PP2A, subunit B) Protein serine/threonine PPP5C Serine/threonine-protein phosphatase 5 (PP5) phosphatase activity (Protein phosphatase T) Protein tyrosine phosphatase PTPN5 Protein tyrosine phosphatase, non-receptor type 5 (PTPN) activity Receptor binding STRAP Serine/threonine kinase receptor associated protein (MAWD) (UNRIP) Receptor signaling complex Ywhab 14-3-3 protein beta/alpha (Protein kinase C inhibitor scaffold activity protein 1) (KCIP-1) Receptor signaling complex Ywhae 14-3-3 protein epsilon (Mitochondrial import stimulation scaffold activity factor L subunit) (MSF L) Receptor signaling complex Ywhag 14-3-3 protein gamma 25 scaffold activity Receptor signaling complex Ywhaq 14-3-3 protein theta (14-3-3 protein tau) scaffold activity 26 B. Park et al. / Neurochemistry International 57 (2010) 16–32

Table 1. Reflecting the degree of lesion in the nigrostrial DA projection shown in Fig. 1, the level of TH in both the SN and STR was found to be decreased. On the other hand, cytoskeletal and its associated proteins including neurofilament light proteins, transketolase and vimentin were down-regulated in the SN but not in the STR. Proteins associated with Lewy body such as a-synuclein and 14-3-3 proteins were found to be differentially expressed in the SN and STR. Several of the other differentially expressed proteins in our study including ubiquinol-cytochrome c reductase, hemoglobin b-chain, glial fibrillary acidic protein and ferrintin heavy chain were the same as those found in the human substantia nigra specimens from PD patients (Basso et al., 2004), indirectly supporting the validity of the rat 6-OHDA-lesion model to search for potential target proteins associated with the progression of DA neuronal death in patients with PD. The vast majority of the proteins spots with different expression levels in both the SN and STR in our study were previously unidentified. laminin receptor) protein 1) (KCITP-1) component Sec15A) (Sec15-like 1) (rSec15) inhibitor 2) (Rab GDI beta) 2.3. Functional classification and a protein interaction network

To classify the differentially expressed proteins in our study into several functional categories, we first gathered information on these proteins by searching through the open databases including the NCBI, SWISS-PROT or the HPRD. As shown in Table 2, the differentially expressed proteins were distributed into such biological processes as cell growth and/or maintenance, metabolism/energy pathways, immune response, protein metabolism, Ywhaz, Msfs1 14-3-3 protein zeta/delta (Protein kinase C inhibitor EXOC6 Exocyst complex component 6 (Exocyst complex PHB Prohibitin GDI2 Rab GDP dissociation inhibitor beta (GDP dissociation regulation of nucleobase/nucleic acid metabolism, signal transduction/cell communication, and transport. The majority of the proteins identified in our study were associated with various metabolic activities and signal transduction pathways including calcium-, receptor-, or scaffold activity-mediated signaling pathways. We then attempted to determine relationships among the proteins with altered levels of expression that were categorized into several biological functions. To evaluate the potential interacting protein profiles, we searched for potential interacting proteins through the HPRD or the DIP. Based on information gathered from these databases, we first constructed a main frame linkage map by connecting the identified proteins in our study that were categorized into the signal transduction/cell communication (marked in red ovals) and other functional categories (marked in blue ovals; Fig. 3). Based on the search using the databases mentioned above, proteins that were not found in this study (marked in black) were also incorporated into a further expanded protein network to evaluate whether and how these newly Receptor signaling complex scaffold activity activity Ribosomal subunit Rpsa, Lamr1 40S ribosomal protein SA (p40) (34/61 kDa Receptor signaling complex scaffold activity Auxiliary transport protein activity Storage proteinTransporter activityTransporter activityTransporter activityincorporated APOE FTMT HBB KPNB1 proteins might Apolipoprotein E Ferritin precursorbe heavy (Apo-E) chain (Ferritin Hemoglobin Karyopherin H beta (importin) subunit) interconnected chain beta 1 with the identified proteins in the progression of DA neuronal cell death. Fig. 3 shows the potential linkage map and interrelationships of the differentially expressed and newly incorporated proteins with their protein partners. Intriguingly, not only proteins categorized as signal transduction/cell communication but proteins in other categories were conjugated and interrelated with each other through their binding partners. To find the key players that influence other proteins within the constructed protein network, we focused on the hub proteins that have many binding partners. Among several proteins that may act as hub proteins, for example, proteins including isoforms of 14-3-3, raf kinase inhibitor and prohibitin were found to have altered expression levels during DA neuronal death in our study. Since mitochondrial abnormality has been proposed to be involved in ) the progression of DA neuronal death, prohibitin and its binding partner, NDUFS3 subunit attracted our immediate attention. Change in the expression level of prohibitin is associated with Continued various types of disorders including aging and degenerative diseases (Nijtmans et al., 2002; Mishra et al., 2005). Among many Biological process Molecule function Gene name Identified proteins Transport Auxiliary transport protein

Table 2 ( previously described functions of prohibitin, it has been well B. Park et al. / Neurochemistry International 57 (2010) 16–32 27

Fig. 3. Schematic diagram of the predicted protein interaction network. We constructed a protein interaction network by connecting the binding partners of the proteins showing differential expression categorized in the signal transduction and cell communication (marked in red ovals) and other functional categories (marked in blue ovals) as described in Table 2. Proteins marked in black ovals were incorporated from the databases. (For interpretation of the references to color in this ﬁgure legend, the reader is referred to the web version of the article.) established that prohibitin acts as a chaperone involving the 2.4. Interaction between prohibitin and the NDUFS3 subunit stabilization of mitochondrial proteins including subunits of respiratory enzymes (Nijtmans et al., 2000, 2002). Considering To validate the interaction between prohibitin and the NDUFS3 previous studies suggesting that a changes in prohibitin levels subunit, we performed co-immunoprecipitation assays. Tissue might be associated with the accumulation of mitochondrial lysates harvested from the SN and STR of post-lesioned rats were oxygen radicals-mediated damage (Camougrand and Rigoulet, immunoprecipitated with anti-NDUFS3 subunit or with anti- 2001; Ahn et al., 2006; Theiss et al., 2007), we attempted to further prohibitin antibody followed by western blot analysis with the validate its interaction with the NDUFS3 subunit and their appropriate antibodies. As shown in Fig. 4, it was conﬁrmed that immunostaining intensity in dying DA neurons. prohibitin binds the NDUFS3 subunit in both the SN and STR. To

Fig. 4. Co-immunoprecipitation of prohibitin and the NDUFS3 subunit in tissue lysates. Tissue lysates obtained from the SN and STR of post-operated rats were incubated with protein G-agarose resin and anti-NDUFS3 subunit (A) or anti-prohibitin (B) antibody for co-immunoprecipitation analysis followed by immunoblotting with anti-prohibitin (A) or anti-NDUFS3 (B), respectively. Total tissue lysates were also analyzed by western blot using anti-prohibitin (A) or anti-NDUFS3 subunit (B) antibody. Blot images shown here are representative of at least three separate experiments with protein G-agarose resin. Similar results were obtained with protein A-agarose resin. Double arrows indicate the heavy chains of antibodies. 28 B. Park et al. / Neurochemistry International 57 (2010) 16–32 examine the cell-type specific immunostaining pattern of these prohibitin compared to those in vehicle-injected group. Immuno- proteins, nigral sections obtained from rats 5 d post the 6-OHDA histochemical colocalization of prohibitin and an astrocytic marker lesion were subjected to immunohistochemistry. At this time protein, GFAP indicated that increased staining intensity of point, the whole sectional views indicated that TH-positive prohibitin was not detected in GFAP-positive cells (Fig. 6B), neurons and bundles were apparently damaged in the ipsilateral suggesting 6-OHDA-lesion-induced upregulation of prohibitin side of 6-OHDA-lesioned SNc as compared to those in the may primarily occur in dying DA neuronal cells. Similarly, we contralateral side of 6-OHDA-lesioned or the ipsilateral side of examined under a higher magnification the staining intensity of vehicle-injected SNc (Fig. 5; also refer to Lee et al., 2008). In these the NDUFS3 subunit in the nigral sections 5 d post 6-OHDA lesion. preparations, immunostaining intensity of prohibitin was largely As shown in Fig. 7A, increased staining intensity of the NDUFS3 increased in the ipsilateral side of the 6-OHDA-lesioned SNc and subunit was detected in TH-positive dying neurons in the SNc of 6- the area above. No discernible increase of prohibitin was detected OHDA-lesioned side. Among 88 TH-positive neurons counted in 6- in the substantia nigra pars reticulata (SNr). Increased expression OHDA-injected group, approximately 65.6% of cells showed of the NDUFS3 subunit was also detected in the ipsilateral side of increased staining intensity for NDUFS3 subunit compared to SNc of the 6-OHDA-lesioned rats. Unlike the expression pattern of those in vehicle-injected group. Moderate but noticeable increase prohibitin, higher levels of the NDUFS3 subunit were also detected of staining intensity of the NDUFS3 subunit was also detected in in the SNr (Fig. 5). To determine cell types that express higher GFAP-positive neurons in the SNc of the 6-OHDA-lesioned side levels of prohibitin and the NDUFS3 subunit, confocal microscopic (Fig. 7B). examination at a higher magnification was performed. Results showed that increased staining intensity of prohibitin was most 2.5. Acceleration of 6-OHDA-induced cell death in siRNA for obvious in dying TH-positive neuronal cell body with fragmented prohibitin-transfected cells neurites of the SNc (Fig. 6A and B; i images). Among 108 TH- positive neurons counted in 6-OHDA-injected group, approxi- 2-DE followed by an immunoblot analysis demonstrated that mately 81.5% of cells showed increased staining intensity for expression levels of prohibitin was increased in the nigral tissue

Fig. 5. Immunohistochemical staining patterns of prohibitin and the NDUSF3 subunit. SD rats stereotaxically injected with vehicle or 6-OHDA were sacriﬁced 5 d after the injection. To examine immunohistochemical localization of prohibitin or NDUFS3, the nigral sections were immunostained with anti-prohibitin or anti-NDUSF3 antibody in combination with anti-tyrosine hydroxylase (TH). Tissue sections were further incubated with appropriate ﬂuorescence-conjugated secondary antibodies as described in Section 1. B. Park et al. / Neurochemistry International 57 (2010) 16–32 29

Fig. 6. Cellular localization of prohibitin in the SN. Basically, tissue sections were obtained from the SN and processed for immunofluorescent localization of prohibitin as described in Fig. 5. To examine cellular localization of prohibitin, nigral sections were immunostained with anti-prohibitin antibody in combination with anti-TH (A) or anti- GFAP (B) antibody, respectively. Tissue sections were further incubated with appropriate fluorescence-conjugated secondary antibodies. For localization of nuclei, tissue sections were also incubated with a DNA intercalator, Hoechst 33258. The images were visualized with a confocal microscope. A and B images shown here indicate enlarged views obtained from the SNc of ipsilateral side injected with vehicle only (a, d, g, and j); and contralateral side (b, e, h, and k) or ipsilateral side (c, f, i, and l) injected with 6- OHDA following triple localization of nuclei, prohibitin, and TH (A) or GFAP (B). Merged images are shown in rows j, k and l. White arrows in (i) of (A and B) indicate TH- positive neuronal cells. Scale bars, 10 mm. lysates of 5 d post 6-OHDA lesion (Fig. 8A). To evaluate how regions following a unilateral injection of 6-OHDA. Secondly, we expression level of prohibitin is potentially associated with studied temporal changes in the expression levels of proteins in neurodegeneration, SHSY5Y neuronal cells were transiently these hemiparkinsonian rats. Tissue samples were obtained at 3 transfected with prohibitin siRNA for post-transcriptional gene different time points (5, 10 and 20 d) and subjected to proteomic silencing. As determined by western blot analysis (Fig. 8B), analyses. Third, we compared protein spots obtained from the approximately 70% of knockdown of prohibitin was achieved ipsilateral side of rats injected with 6-OHDA and vehicle-only to 72 h post-transfection. Following treatment with 100 mM 6-OHDA rule out that the proteins may be altered by an injection per se. for various time periods, the rate of cell death determined by the Finally, we mapped relationships between proteins with differing MTT reduction assay was accelerated at all time periods in expression levels in order to understand the interconnection of prohibitin siRNA knock-down cell line (Fig. 8C). various important proteins in the course of DA neuronal cell death. Based on scanning images of the 2-DE gels in our study, there 3. Discussion seemed to be no qualitative differences in the overall pattern of the displayed protein spots obtained from the control SN and STR Proteomics technologies have been used to investigate the samples. However, as described in the results section, the basal molecular targets associated with the pathophysiology of several levels of certain protein spots in each tissue were different. neurodegenerative and psychiatric disorders (Fountoulakis and Therefore, we directly compared 2-DE gel images of 6-OHDA- Kossida, 2006; Licker et al., 2009). However, there are only a few injected SN and STR separately to their equivalent vehicle control proteomics analyses on postmortem brains or cerebrospinal fluid gels. By doing so, we identified more than 70 proteins with of patients with PD, or neurotoxin-based animal models of PD. quantitative expression differences in both tissues. Among these Furthermore, extensive attempt to identify the multiple targets has identified proteins, some overlapped with proteins already found not been made using 6-OHDA-injected animal model. Therefore, in previous proteomic studies using postmortem specimens from the purpose of our study was to further identify bulk protein PD patients, parkin-deficient mice or 6-OHDA-injected models of targets that change their expression levels following a unilateral PD (e.g., neurofilament light protein, a-enolase, glutamine stereotaxic injection of 6-OHDA into the SNc using a gel-based 2- synthetase, NADH-ubiquinone oxidoreductase subunit, 14-3-3 DE analysis. We chose the intranigral injection model because of a isoforms, hemoglobin b-chain and ferritin heavy chain). However, previous report suggesting that the pattern of DA neuronal cell loss the vast majority of the proteins spots with different expression in this model is quite similar to an advanced stage of the human levels in both the SN and STR in our study were previously pathological situation of PD (Deumens et al., 2002). Several other unidentified. At present, it is not clear whether these newly important features were also considered in our study. First, we identified proteins alone or in combination are associated with the attempted to analyze the proteome from both the SN and STR potential pathophysiology of DA neuronal death. In any case, many 30 B. Park et al. / Neurochemistry International 57 (2010) 16–32

Fig. 7. Cellular localization of NDUFS3 subunit in the SN. Tissue sections were obtained from the SN and processed for immunofluorescent localization of NDUFS3 subunit as described in Fig. 5. A and B confocal images shown here indicate enlarged views obtained from the SNc of ipsilateral side injected with vehicle only (a, d, g, and j); and contralateral side (b, e, h, and k) or ipsilateral side (c, f, i, and l) injected with 6-OHDA following triple localization of nuclei, NDUFS3 subunit, and TH (A) or GFAP (B). Merged images are shown in row j, k and l. White arrows in (i) of (A) indicate TH-positive neuronal cells. Scale bars, 10 mm. of the newly identified proteins in our rat PD model are involved in there is accumulating evidence indicating that mitochondrial metabolism and signal transduction pathways. We also found damage, particularly to the respiratory chain complex I (NADH: proteins that belong to such biological processes as cell growth, ubiquinone oxidoreductase) and decrease in complex I activity, immune response and transport. Interestingly, proteins such as 14- underlies the pathology of neurological disorders including PD 3-3 isoforms, alpha-enolase, glutamine synthetase, NADH-ubiqui- (Schapira et al., 1990, 1998; Betarbet et al., 2000; Sherer et al., none oxidoreductase subunits, triosephosphate isomerase 1, 2002), we further studied prohibitin that is connected to various neurofilament light chain and vimentin found to have differen- protein partners including mitochondrial complex I subunits, in tially expressed levels in our model have also been reported to be particular with the NDUFS3 subunit. Previous studies have involved in other CNS diseases (e.g., Alzheimer’s disease, Down’s suggested that change in the expression level of prohibitin is syndrome, Creutzfeldt-Jakob’s disease, Schizophrenia; Fountoula- associated with senescence and may be a potential target for new kis and Kossida, 2006). This raises the possibility that there may be therapeutics (Nijtmans et al., 2002; Mishra et al., 2005). Further- an overall consistency with changes in protein levels belonging to more, it has been demonstrated that a significant change of important functional categories in various neurodegenerative and prohibitin is found in the substantia nigra and the frontal cortex in psychiatric diseases. PD, suggesting that disease-specific changes in the expression of In general, proteins do not function as isolated entities within a mitochondria-related proteins may be closely associated with the cell. Rather, their biological functions result from an outcome of progression of the diseases (Ferrer et al., 2007). A previous study relatively simple protein-protein interactions or of larger protein has also shown that prohibitin might act as a chaperone to protect complexes in the context of a network (Xenarios et al., 2000; Peri complex I subunits before assembly (Bourges et al., 2004). With et al., 2003). Once a protein network is constructed, one can make these reports, our data suggesting binding of prohibitin and the assumptions with regards to whether and how altered protein NDUFS3 subunit seems to raise the possibility that upregulation of expression affects the functional outcome of their interactions and prohibitin in the SN following 6-OHDA injection may be related consequently the kinetics of DA neuronal death. Using the with its known chaperone activity involving stabilization of information of the proteins with altered expression levels in our mitochondrial proteins (Merkwirth and Langer, 2009; Artal-Sanz study and of available databases, we attempted to construct a and Tavernarakis, 2010). Our data obtained from gene silencing for protein network to find the key interacting molecules that may prohibitin showed that cells are more sensitive to 6-OHDA- play important roles in DA neuronal death. Although it has to be induced cell death. These results are in line with the recent report unequivocally established in subsequent studies, our attempt to by Liu et al. (2009) demonstrating that oxidative stress can construct an interaction map raises the possibility of not only one- increase the prohibition content in the mitochondria and over- to-one protein interactions but also hub proteins that have many expression of prohibitin protects against oxidative stress-induced binding partners. Among several proteins that may act as hub cell death. Although it remains largely elusive as to which proteins, for example, proteins including isoforms of 14-3-3, raf mechanism(s) is closely associated with increased levels of kinase inhibitor and prohibitin were found to have altered prohibitin in our 6-OHDA-lesioned brain model, this phenomenon expression levels during DA neuronal death in our study. Since may suggest that upregulation of prohibitin following 6-OHDA B. Park et al. / Neurochemistry International 57 (2010) 16–32 31

drial chaperone protein and complex I subunit. Because prohibitin acts as a hub protein that has many other binding partners (e.g., p53 and RAF) as well as a close relationship with other proteins (e.g., mitochondrial complex I proteins), it is plausible that complex I activity may be regulated by far more complicated processes than expected. It is plausible that both direct and indirect contributions of the proteins in the network may be involved in the progression of DA neuronal death. Our present study expands on the notion that multiple analyses of proteomes would be an efﬁcient way to provide the extended framework of protein events associated with DA neuronal degeneration and to reveal potential drug targets or diagnostic markers for PD. Further systematic studies using the identiﬁed targets and the protein network may elucidate the important death cascades of DA neuronal cells intimately associated with PD.

Acknowledgments

We thank Dr. Young-Ki Paik at Yonsei University for his critical reading of our manuscript. This work was supported by a grant from the Ministry of Science and Technology through BRC, and in part by the KOSEF (SRC, R11-2008-036-00000-0), KRF-C00204 and WCU (R33-2008-000-10014-0).

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