Mitochondrial Dysfunction and Oxidative Damage in parkin-deficient Mice

Abstract

Loss-of-function mutations in parkin are the predominant cause of familial Parkinson's disease. We previously reported that parkin-/- mice exhibit nigrostriatal deficits in the absence of nigral degeneration. Parkin has been shown to function as an E3 ubiquitin ligase. Loss of parkin function, therefore, has been hypothesized to cause nigral degeneration via an aberrant accumulation of its substrates. Here we employed a proteomic approach to determine whether loss of parkin function results in alterations in abundance and/or modification of proteins in the ventral midbrain of parkin-/- mice. Two-dimensional gel electrophoresis followed by mass spectrometry revealed decreased abundance of a number of proteins involved in mitochondrial function or oxidative stress. Consistent with reductions in several subunits of complexes I and IV, functional assays showed reductions in respiratory capacity of striatal mitochondria isolated from parkin-/- mice. Electron microscopic analysis revealed no gross morphological abnormalities in striatal mitochondria of parkin-/- mice. In addition, parkin-/- mice showed a delayed rate of weight gain, suggesting broader metabolic abnormalities. Accompanying these deficits in mitochondrial function, parkin-/- mice also exhibited decreased levels of proteins involved in protection from oxidative stress. Consistent with these findings, parkin-/- mice showed decreased serum antioxidant capacity and increased protein and lipid peroxidation. The combination of proteomic, genetic, and physiological analyses reveal an essential role for parkin in the regulation of mitochondrial function and provide the first direct evidence of mitochondrial dysfunction and oxidative damage in the absence of nigral degeneration in a genetic mouse model of Parkinson's disease. Loss-of-function mutations in parkin are the predominant cause of familial Parkinson's disease. We previously reported that parkin-/- mice exhibit nigrostriatal deficits in the absence of nigral degeneration. Parkin has been shown to function as an E3 ubiquitin ligase. Loss of parkin function, therefore, has been hypothesized to cause nigral degeneration via an aberrant accumulation of its substrates. Here we employed a proteomic approach to determine whether loss of parkin function results in alterations in abundance and/or modification of proteins in the ventral midbrain of parkin-/- mice. Two-dimensional gel electrophoresis followed by mass spectrometry revealed decreased abundance of a number of proteins involved in mitochondrial function or oxidative stress. Consistent with reductions in several subunits of complexes I and IV, functional assays showed reductions in respiratory capacity of striatal mitochondria isolated from parkin-/- mice. Electron microscopic analysis revealed no gross morphological abnormalities in striatal mitochondria of parkin-/- mice. In addition, parkin-/- mice showed a delayed rate of weight gain, suggesting broader metabolic abnormalities. Accompanying these deficits in mitochondrial function, parkin-/- mice also exhibited decreased levels of proteins involved in protection from oxidative stress. Consistent with these findings, parkin-/- mice showed decreased serum antioxidant capacity and increased protein and lipid peroxidation. The combination of proteomic, genetic, and physiological analyses reveal an essential role for parkin in the regulation of mitochondrial function and provide the first direct evidence of mitochondrial dysfunction and oxidative damage in the absence of nigral degeneration in a genetic mouse model of Parkinson's disease. Parkinson's disease (PD) 1The abbreviations used are: PD, Parkinson's disease; ROS, reactive oxygen species; 4HNE, 4-hydroxynonenal; DA, dopamine; DAT, dopamine transporter; MS, mass spectrometry; pI, isoelectric point; TMPD, N,N,N′,N′-tetramethylphenylenediamine; FCCP, carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazine; PRDX, peroxiredoxin. 1The abbreviations used are: PD, Parkinson's disease; ROS, reactive oxygen species; 4HNE, 4-hydroxynonenal; DA, dopamine; DAT, dopamine transporter; MS, mass spectrometry; pI, isoelectric point; TMPD, N,N,N′,N′-tetramethylphenylenediamine; FCCP, carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazine; PRDX, peroxiredoxin. is the second most prevalent neurodegenerative disease. Clinical manifestations of PD include postural instability, bradykinesia, resting tremor, and rigidity. Neuropathologically, the disease is characterized by the selective degeneration of the dopaminergic neurons in the substantia nigra (1Olanow C.W. Tatton W.G. Annu. Rev. Neurosci. 1999; 22: 123-144Google Scholar). The etiology of PD is still unknown, although clinical and experimental evidence implicate the involvement of mitochondrial dysfunction (2Beal M.F. Ann. N. Y. Acad. Sci. 2003; 991: 120-131Google Scholar, 3Kosel S. Hofhaus G. Maassen A. Vieregge P. Graeber M.B. Biol. Chem. 1999; 380: 865-870Google Scholar) and oxidative stress (4Jenner P. Olanow C.W. Neurology. 1996; 47: 5161-5170Google Scholar, 5Zhang Y. Dawson V.L. Dawson T.M. Neurobiol. Dis. 2000; 7: 240-250Google Scholar). Analysis of mitochondria isolated from idiopathic PD patients showed inhibited capacity of NADH-ubiquinone reductase, complex I of the mitochondrial electron transport chain, and increased production of reactive oxygen species (ROS) (6Schapira A.H. Biochim. Biophys. Acta. 1998; 1366: 225-233Google Scholar). Similar changes have been seen in autopsy cases of patients with presymptomatic PD, suggesting that mitochondrial dysfunction and oxidative stress precede clinical manifestations (7Dexter D.T. Sian J. Rose S. Hindmarsh J.G. Mann V.M. Cooper J.M. Wells F.R. Daniel S.E. Lees A.J. Schapira A.H.V. Jenner P. Marsden C.D. Ann. Neurol. 1994; 35: 38-44Google Scholar). Exposure to selective neurotoxins, including paraquat and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), has been linked to either increased risk of PD or chemically induced parkinsonism (8Langston J.W. Ballard P. Tetrud J.W. Irwin I. Science. 1983; 219: 979-980Google Scholar, 9Koller W.C. Neurology. 1986; 361147Google Scholar). These compounds have been shown experimentally to decrease mitochondrial function and selectively inhibit the activity of complex I (10Singer T.P. Ramsay R.R. McKeown K. Trevor A. Castagnoli Jr., N.E. Toxicology. 1988; 49: 17-23Google Scholar). In vitro chemical inhibition of complex I results in reduced oxidative phosphorylation and increased mitochondrial generation of ROS, similar to what was observed in mitochondria from PD patients (11Sherer T.B. Betarbet R. Stout A.K. Lund S. Baptista M. Panov A.V. Cookson M.R. Greenamyre J.T. J. Neurosci. 2002; 22: 7006-7015Google Scholar, 12Sousa S.C. Maciel E.N. Vercesi A.E. Castilho R.F. FEBS Lett. 2003; 543: 179-183Google Scholar, 13Schmuck G. Rohrdanz E. Tran-Thi Q.H. Kahl R. Schluter G. Neurotox. Res. 2002; 4: 1-13Google Scholar). Pathological examinations of PD brains have revealed increases in protein and lipid byproducts of ROS, including protein carbonyls and 4-hydroxynonenal (4HNE) (14Alam Z.I. Daniel S.E. Lees A.J. Marsden D.C. Jenner P. Halliwell B. J. Neurochem. 1997; 69: 1326-1329Google Scholar, 15Yoritaka A. Hattori N. Uchida K. Tanaka M. Stadtman E.R. Mizuno Y. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 2696-2701Google Scholar). Furthermore, 4HNE forms adducts with and inhibits the activities of the D1 dopamine (DA) receptor and the DA transporter (DAT), suggesting that lipid peroxides may contribute to the disruption of DA signaling (16Shin Y. White B.H. Uh M. Sidhu A. Brain Res. 2003; 968: 102-113Google Scholar, 17Morel P. Tallineau C. Pontcharraud R. Piriou A. Huguet F. Neurochem. Int. 1998; 33: 531-540Google Scholar). Cultured dopaminergic neurons have been shown to exhibit enhanced sensitivity to paraquat and MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) as well as ROS (18Chun H.S. Gibson G.E. DeGiorgio L.A. Zhang H. Kidd V.J. Son J.H. J. Neurochem. 2001; 76: 1010-1021Google Scholar). These findings suggest that mitochondrial dysfunction and accompanying ROS generation could be a common mechanism for the selective loss of substantia nigra neurons and the nigrostriatal DA signal in PD (19Dawson T.M. Dawson V.L. Science. 2003; 302: 819-822Google Scholar). In addition to the more prevalent, idiopathic form, a subset of PD patients exhibits familial inheritance patterns. Large numbers and varieties of autosomal recessively inherited mutations in parkin are the predominant cause of familial PD (20Vaughan J.R. Davis M.B. Wood N.W. Ann. Hum. Genet. 2001; 65: 111-126Google Scholar). Initially described as juvenile-onset, atypical parkinsonism lacking Lewy bodies, subsequently identified cases are often clinically and pathologically indistinguishable from early onset familial or sporadic PD, including the presence of Lewy bodies in a single case (21Kitada T. Asakawa S. Hattori N. Yamamura Y. Minoshima S. Yokochi M. Mizuno Y. Shimizu N. Nature. 1998; 392: 605-608Google Scholar, 22Farrer M. Chan P. Chen R. Tan L. Lincoln S. Hernandez D. Forno L. Gwinn-Hardy K. Petrucelli L. Hussey J. Singleton A. Tanner C. Hardy J. Langston J.W. Ann. Neurol. 2001; 50: 293-300Google Scholar, 23Hayashi S. Wakabayashi K. Ishikawa A. Nagai H. Saito M. Maruyama M. Takahashi T. Ozawa T. Tsuji S. Takahashi H. Movement Disorders. 2000; 15: 884-888Google Scholar). We have recently reported that the loss of parkin function in mice results in nigrostriatal dysfunction, as evidenced by increased extracellular dopamine concentration in the striatum, reduced synaptic excitability in the striatal neurons, and behavioral deficits in paradigms that are sensitive to alterations in the nigrostriatal pathway (24Goldberg M.S. Fleming S.M. Palacino J.J. Cepeda C. Lam H.A. Bhatnagar A. Meloni E.G. Wu N. Ackerson L.C. Klapstein G.J. Gajendiran M. Roth B.L. Chesselet M.F. Maidment N.T. Levine M.S. Shen J. J. Biol. Chem. 2003; 278: 43628-43635Google Scholar). Despite measurable differences in nigrostriatal function in parkin-/- mice (24Goldberg M.S. Fleming S.M. Palacino J.J. Cepeda C. Lam H.A. Bhatnagar A. Meloni E.G. Wu N. Ackerson L.C. Klapstein G.J. Gajendiran M. Roth B.L. Chesselet M.F. Maidment N.T. Levine M.S. Shen J. J. Biol. Chem. 2003; 278: 43628-43635Google Scholar), no reduction in the number of dopaminergic neurons was observed in two independently generated parkin-/- mice (24Goldberg M.S. Fleming S.M. Palacino J.J. Cepeda C. Lam H.A. Bhatnagar A. Meloni E.G. Wu N. Ackerson L.C. Klapstein G.J. Gajendiran M. Roth B.L. Chesselet M.F. Maidment N.T. Levine M.S. Shen J. J. Biol. Chem. 2003; 278: 43628-43635Google Scholar, 25Itier J.M. Ibanez P. Mena M.A. Abbas N. Cohen-Salmon C. Bohme G.A. Laville M. Pratt J. Corti O. Pradier L. Ret G. Joubert C. Periquet M. Araujo F. Negroni J. Casarejos M.J. Canals S. Solano R. Serrano A. Gallego E. Sanchez M. Denefle P. Benavides J. Tremp G. Rooney T.A. Brice A. De Yebenes J.G. Hum. Mol. Genet. 2003; 12: 2277-2291Google Scholar). Parkin has been reported as an E3 ubiquitin-protein ligase (26Shimura H. Hattori N. Kubo S. Mizuno Y. Asakawa S. Minoshima S. Shimizu N. Iwai K. Chiba T. Tanaka K. Suzuki T. Nat. Genet. 2000; 25: 302-305Google Scholar). Previous reports described several substrates for parkin-mediated ubiquitinylation (27Cookson M.R. Neuromolecular Med. 2003; 3: 1-13Google Scholar). It has been suggested that the loss of parkin function results in aberrant accumulation of substrate proteins including PAEL receptor, synphlin-1, and CDC-rel1. Accumulation of one or more of these proteins has been postulated to confer toxicity upon dopaminergic neurons in the substantia nigra (28Xu J. Kao S.Y. Lee F.J. Song W. Jin L.W. Yankner B.A. Nat. Med. 2002; 8: 600-606Google Scholar). However, steady-state levels of these substrates are unchanged in parkin-/- brains (24Goldberg M.S. Fleming S.M. Palacino J.J. Cepeda C. Lam H.A. Bhatnagar A. Meloni E.G. Wu N. Ackerson L.C. Klapstein G.J. Gajendiran M. Roth B.L. Chesselet M.F. Maidment N.T. Levine M.S. Shen J. J. Biol. Chem. 2003; 278: 43628-43635Google Scholar). 2J. J. Palacino and J. Shen, unpublished results. 2J. J. Palacino and J. Shen, unpublished results. Recent evidence has also suggested a role for parkin in the protection of monoaminergic neurons against proteasomal dysfunction, α-synuclein overexpression-mediated cell death (29Petrucelli L. O'Farrell C. Lockhart P.J. Baptista M. Kehoe K. Vink L. Choi P. Wolozin B. Farrer M. Hardy J. Cookson M.R. Neuron. 2002; 36: 1007-1019Google Scholar), and kainic acid-induced toxicity (30Staropoli J.F. McDermott C. Martinat C. Schulman B. Demireva E. Abeliovich A. Neuron. 2003; 37: 735-749Google Scholar). It was shown that parkin is localized in mitochondria and inhibits mitochondria-dependent cell death (31Darios F. Corti O. Lucking C.B. Hampe C. Muriel M.P. Abbas N. Gu W.J. Hirsch E.C. Rooney T. Ruberg M. Brice A. Hum. Mol. Genet. 2003; 12: 517-526Google Scholar). Other studies demonstrate that overexpression of mutant parkin elevates cellular markers of oxidative stress, whereas overexpression of wild-type parkin results in reduced levels of these markers (32Hyun D.H. Lee M. Hattori N. Kubo S. Mizuno Y. Halliwell B. Jenner P. J. Biol. Chem. 2002; 277: 28572-28577Google Scholar). These observations are consistent with findings from parkin-null flies, which exhibit altered mitochondrial morphology and degeneration of tissues such as wing flight muscles and spermatocytes (33Greene J.C. Whitworth A.J. Kuo I. Andrews L.A. Feany M.B. Pallanck L.J. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 4078-4083Google Scholar). These results raised the possibility that parkin may be involved in mitochondrial function. Based on these observations, we hypothesized that lack of parkin function may cause impairment of mitochondrial function in parkin-/- mice. To determine whether a lack of parkin causes changes in protein abundance and/or modification, we conducted a nonbiased proteomic analysis of the ventral midbrain of parkin-/- and wild-type mice. Using a well established method for two-dimensional analysis of brain lysates (34Klose J. Nock C. Herrmann M. Stuhler K. Marcus K. Bluggel M. Krause E. Schalkwyk L.C. Rastan S. Brown S.D. Bussow K. Himmelbauer H. Lehrach H. Nat. Genet. 2002; 30: 385-393Google Scholar), we were able to detect ∼8000 discrete protein spots from extracts of the ventral midbrain of parkin-/- and wild-type mice. Comparative analysis of 10 pairs of wild-type and parkin-/- brain samples revealed reproducible, quantitative changes of fifteen protein spots by silver staining. Subsequent mass spectrometric (MS) analysis revealed that these 15 spots represented 14 distinct proteins, 13 of which exhibited decreases in abundance in brains of parkin-/- mice and 1 additional protein which exhibited altered electrophoretic mobility, consistent with differential post-translational modification. Eight of these proteins were involved in either oxidative phosphorylation or antioxidant activities. Consistent with these findings, parkin-/- mice exhibited decreases in oxidative phosphorylation, weight gain, and antioxidant capacity as well as increased ROS-mediated tissue damage, suggesting an essential role for parkin in regulating normal respiratory function of mitochondria as well as in the protection of cells from oxidative stress. Mice—Mice bearing a germline disruption of exon 3 of the parkin gene were generated as previously described (24Goldberg M.S. Fleming S.M. Palacino J.J. Cepeda C. Lam H.A. Bhatnagar A. Meloni E.G. Wu N. Ackerson L.C. Klapstein G.J. Gajendiran M. Roth B.L. Chesselet M.F. Maidment N.T. Levine M.S. Shen J. J. Biol. Chem. 2003; 278: 43628-43635Google Scholar). Mice used for all studies except the proteomic analysis were in the hybrid background of C57BL/6 and 129/Sv. Mice used for proteomic studies were the 129/Sv inbred strain. Two-dimensional Gel Electrophoresis and Mass Spectrometry—Protein samples for two-dimensional gel electrophoresis were prepared from the dissected ventral midbrain (including the substantia nigra) of each of the 10 pairs of parkin-/- and wild-type mice as previously described (35Klose J. Methods Mol. Biol. 1999; 112: 67-85Google Scholar) with the following modifications. The solutions used for extractions were100 mm phosphate buffer, pH 7.1 (0.2 m KCl, 20% w/v glycerol, and 4% w/v 3-[(3-chloramidopropyl) dimethylammonio]-1-propanesulfonate) (A), protease inhibitor solution I (1 Complete™ tablet (Roche Applied Science) dissolved in 2 ml of buffer A) (B), and protease inhibitor solution II (1.4 μm pepstatin A and 1 mm phenylmethylsulfonyl fluoride in ethanol) (C). The frozen tissue was transferred into a mortar placed in a liquid nitrogen bath. An aliquot of 1.25 parts v/w of A, 0.08 parts v/w of protease inhibitor I, and 0.02 parts v/w of protease inhibitor II were added to the tissue and ground to a fine powder. The resulting powder was filled into a 2-ml microtube, quickly thawed, supplied with 0.034 parts of glass beads, and then sonicated in an ice-cold water bath 6 times for 10 s with intervals of 1 min 50 s. The homogenate was stirred 30 min in the presence of 0.025 parts v/w of Benzonase (Merck). 6.5 m urea, 2 m thiourea, and 70 mm dithiothreitol solution were added, and stirring was continued for an additional 30 min. The protein extract was supplied with 0.1 parts v/w of ampholyte mixture Servalyte pH 2–4 (Serva, Heidelberg, Germany) and stored at -80 °C or analyzed immediately. Proteins were separated by large two-dimensional gels as described previously (34Klose J. Nock C. Herrmann M. Stuhler K. Marcus K. Bluggel M. Krause E. Schalkwyk L.C. Rastan S. Brown S.D. Bussow K. Himmelbauer H. Lehrach H. Nat. Genet. 2002; 30: 385-393Google Scholar, 35Klose J. Methods Mol. Biol. 1999; 112: 67-85Google Scholar). Briefly, the gel format was 40 cm (isoelectric focusing, prepared with carrier ampholyte mixture covering pH 3–10) × 30 cm (SDS-PAGE, 15%) × 0.75 mm. The amount of the protein sample applied to the gel was 5 μl (60 μg/μl). For sample comparisons brain extracts from each pair of parkin-/- and control mice were run and stained in parallel. High sensitivity silver staining was used to visualize proteins (35Klose J. Methods Mol. Biol. 1999; 112: 67-85Google Scholar). Two-dimensional gels were evaluated visually pairwise, and changes of spots were considered with respect to variation in the presence or absence, quantity, and spot position. Protein spots found to be reproducibly altered in parkin-/- patterns versus wild type were evaluated with the Proteomweaver imaging software Version 2.1 (Definiens, Munich, Germany). Although the mice we used are in a homogenous genetic background (129/Sv inbred strain), we still observed individual variations. Protein alterations confirmed in more than six pairs of mice were scored. All 10 parkin-/- mice investigated were affected at least in 7 of 14 proteins, and 5 mice were affected in more than 12 proteins. Data were analyzed by Student's t test. For protein identification using MS, 18-μl (60 μg/μl) samples were electrophoresed on 1.5-mm gels and stained with MS-compatible silver stain or colloidal Coomassie Brilliant Blue G-250. Protein spots of interest were excised from gels and subjected to in-gel trypsin digestion without reduction or alkylation. Tryptic fragments were analyzed by a combination of matrix-assisted laser desorption ionization time-of-flight and liquid chromatography/electrospray ionization ion trap MS. The mass spectra were analyzed using Protein Prospector (MS-Fit) and Sequest Version 3.1 software. Mitochondrial Respiration—Mice were euthanized by CO2 inhalation, and tissues were rapidly dissected on ice. Brains were removed, and striata were isolated as described previously (24Goldberg M.S. Fleming S.M. Palacino J.J. Cepeda C. Lam H.A. Bhatnagar A. Meloni E.G. Wu N. Ackerson L.C. Klapstein G.J. Gajendiran M. Roth B.L. Chesselet M.F. Maidment N.T. Levine M.S. Shen J. J. Biol. Chem. 2003; 278: 43628-43635Google Scholar). Striata from 2 mice of each genotype were pooled for mitochondrial isolation. Tissue samples were homogenized in 10 ml of buffer A (320 mm sucrose, 5 mm Tris, 2 mm EGTA, pH 7.4, at 4 °C) with 5 strokes of a Teflon Dounce. Samples were centrifuged for 3 min at 2000 × g to remove nuclei and tissue particles. Supernatants were collected and centrifuged for 10 min at 12,000 × g to pellet mitochondria and synaptosomes. The crude pellet was resuspended in 10 ml of buffer A with the addition of 0.02% w/v of digitonin to disrupt synaptosomal membranes and release trapped mitochondria. The resuspended pellet was centrifuged for 10 min at 12,000 × g to pellet mitochondria, which was resuspended in 100 μl of buffer A, and protein content was determined by BCA assay (Pierce). Mitochondria were resuspended at a final concentration of 0.4 mg/ml protein in 0.5 ml of buffer B (120 mm KCl, 3 mm HEPES, 1 mm EGTA, 5 mm KH2PO4, pH 7.2) with 1% w/v of bovine serum albumin and assayed for respiration using an excess of 8 mm glutamate, 8 mm malate (complex I), 4 mm succinate (complex II), or 0.4 mm N,N,N′,N′-tetramethylphenylenediamine (TMPD)/1 mm ascorbate (complex III/IV) as electron donors. ADP was added in limiting amounts (14 μm), and state 3 respiration was measured. After depletion of ADP, state 4 respiration was measured. After determination of coupled respiration, 400 nm carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP) was added to the reaction chamber, and respiration was measured in the absence of a proton gradient. Mitochondrial respiration was determined using a platinum electrode with a 1-ml buffer chamber (DM-10, Rank Bros., Ltd., UK). Due to intra-day variations in respiration rates, oxygen consumption values are represented as a fraction of the wild-type state 3 respiration for succinate. Data were analyzed by unpaired Student's t test. Electron Microscopy—Wild-type and parkin-/- mice were euthanized by CO2 inhalation and transcardially perfused with 20 ml of phosphate-buffered saline followed by 10 ml of fresh 2.5% glutaraldehyde plus 2.5% formaldehyde in 100 mm cacodylate buffer. Brains were removed and post-fixed in the above fixative for an additional 18 h at 4 °C. Brains were washed in phosphate-buffered saline and cut into 1-mm thick coronal sections, and striata were dissected and processed for standard electron microscopy by epon embedding with osmium tetroxide fixation and uranyl acetate counterstaining. Mitochondrial number and morphology were determined in images from 2–5 different fields from 2 mice per genotype by an investigator blind to the genotype. Body Weight Measurements—Body weights were measured in parkin-/- and wild-type mice at regular intervals beginning 10 days after weaning. Weights were analyzed by two-way analysis of variance followed by Bonferroni post-hoc analysis. Data for adult male mice were collected in conjunction with behavioral analysis and were analyzed by unpaired Student's t test. Serum Antioxidant Capacity Assay—Mice were fasted overnight to minimize variability due to dietary uptake. Mice were euthanized by CO2 inhalation, blood was collected in heparinized tubes, and serum was isolated by centrifugation at 1200 rpm for 10 min. Serum total antioxidant capacity was measured by the conversion of Cu2+ to Cu1+ using a colorimetric assay and is represented as μm uric acid equivalents and carried out as per manufacturer's instructions (Total Antioxidant Potential, Oxis Research). Data were analyzed by unpaired Student's t test. Protein Carbonyl Assay—Brains were homogenized in 50 mm Tris, 150 mm NaCl, and 1% v/v Triton X-100, pH 7.5, and insoluble material was removed by centrifugation. Supernatants were assayed for protein content (BCA, Pierce) and 20 μg of protein was assayed for protein carbonyls as per the manufacturer's instructions (OxyBlot, Chemicon). Briefly, proteins were diluted into a final concentration of 6% SDS and reacted with 2,4-dinitrophenylhydrazine for 15 min. After the reaction, samples were neutralized, electrophoresed on NuPage gels (Invitrogen), transferred to nitrocellulose (Protran BA-83, Schleicher & Schuell), and Western-blotted using an antibody specific to the dinitrophenylhydrazone-derivatized residues on oxidatively damaged proteins. Blots were stripped and subsequently reprobed for actin (AC-15, Abcam, Cambridge, MA) to confirm equivalent protein loading. Immunohistochemistry—Brains were removed, fixed in neutral-buffered formalin for 2 h at room temperature, paraffin-embedded, and sectioned as previously described (24Goldberg M.S. Fleming S.M. Palacino J.J. Cepeda C. Lam H.A. Bhatnagar A. Meloni E.G. Wu N. Ackerson L.C. Klapstein G.J. Gajendiran M. Roth B.L. Chesselet M.F. Maidment N.T. Levine M.S. Shen J. J. Biol. Chem. 2003; 278: 43628-43635Google Scholar). Tissue sections were blocked with 10% goat serum, incubated with an antibody to Michael adducts of 4HNE (#393207, 1:200, Calbiochem) and then with an avidin-conjugated secondary. Immunoreactivity was visualized with diaminobenzidine. Proteomic Analysis of parkin-/- Mice—Because parkin is an E3 ubiquitin ligase (26Shimura H. Hattori N. Kubo S. Mizuno Y. Asakawa S. Minoshima S. Shimizu N. Iwai K. Chiba T. Tanaka K. Suzuki T. Nat. Genet. 2000; 25: 302-305Google Scholar), we anticipated that loss of parkin would result in accumulation of its substrates, which may in turn cause nigrostriatal dysfunction and nigral degeneration. To identify the proteomic difference between parkin-/- and wild-type mice, we used large-gel two-dimensional electrophoresis (34Klose J. Nock C. Herrmann M. Stuhler K. Marcus K. Bluggel M. Krause E. Schalkwyk L.C. Rastan S. Brown S.D. Bussow K. Himmelbauer H. Lehrach H. Nat. Genet. 2002; 30: 385-393Google Scholar) to separate proteins in the ventral midbrain of each of the 10 pairs of parkin-/- and wild-type mice at ∼8 months of age. Proteins were resolved in the first dimension by their isoelectric point (pI) on a 40-cm tube gel using carrier ampholytes and subsequently resolved in the second dimension by their molecular weight on 40 × 30-cm SDS-PAGE gels. After silver staining, we detected reproducible, specific changes in 15 of ∼8000 discrete spots between the genotypes (Fig. 1). Contrary to our expectations, the staining intensity of all but one of these 15 protein spots was decreased in parkin-/- mice. Isolation of protein spots from these gels followed by trypsin digestion and subsequent matrix-assisted laser desorption ionization and electrospray ionization MS provided the identification of these proteins (Table I). Fourteen of the 15 spots represented distinct proteins, whereas one protein was detected twice in 2 adjacent spots (A5 and A6) with varying pI, suggesting a post-translational modification. Changes in protein spot intensity that were confirmed in more than six pairs of parkin-/- and wild-type mice were scored (Table I).Table IProtein alterations in parkin -/- miceProteinSpot numberAccession numberaSWISS-PROT/TrEMBL accession numberFrequency of alterationbNumber of pairs of mice showing consistent alterations in parkin -/- miceQuantitative changescPercentage changes shown as the mean ± S.E. All alterations are statistically significant by Student's t test (p < 0.05)%Proteins involved in mitochondrial OXPHOS Pyruvate dehydrogenase E1α1B6P354877-57.2 ± 15.4 NADH-ubiquinone oxidoreductase 24-kDa subunitA5Q9D6J67+41.4 ± 10.9 NADH-ubiquinone oxidoreductase 24-kDa subunitA6Q9D6J67-27.8 ± 11.4 NADH-ubiquinone oxidoreductase 30-kDa subunitA7Q9DCT28-9.1 ± 2.4 Cytochrome c oxidase, subunit VbA2P120757-48.7 ± 11.7Proteins involved in oxidative stress Peroxiredoxin 2A3Q611718-26.8 ± 7.4 Peroxiredoxin 6A9O087096-28.6 ± 8.8 Peroxiredoxin 1B2P357008-27 ± 5.1 Lactoylglutathione lyaseA4Q9CPU09-20.0 ± 4.7Other Proteins Profilin IIA1Q9JJV27-24.2 ± 8.0 Hypothetical protein dJ37E16.5 (novel protein similar to nitrophenylphosphatases from various organisms)A8Q9UGY28-22.0 ± 8.1 Vacuolar protein sorting-29B1Q9QZ886-13.0 ± 1.9 α-Crystallin, chain BB3P239277-27.6 ± 6.5 Heterogeneous nuclear ribonucleoprotein A1B4P493129-14.6 ± 4.6 Lasp-1B5Q617927-49.3 ± 13.2a SWISS-PROT/TrEMBL accession numberb Number of pairs of mice showing consistent alterations in parkin -/- micec Percentage changes shown as the mean ± S.E. All alterations are statistically significant by Student's t test (p < 0.05) Open table in a new tab The majority of the proteins altered in parkin-/- mice are functionally implicated in either mitochondrial respiration (subunits of pyruvate dehydrogenase and mitochondrial complexes I and IV) or oxidative stress (peroxiredoxin (PRDX) 1, 2, and 6 and lactolylglutathione lyase), indicating a connection between loss of parkin expression and mitochondrial and/or antioxidant deficiencies. The 24-kDa subunit of complex I was shifted to a more acidic pI in the parkin-/- brain (Fig. 1E, spots A5 and A6), indicating the protein had undergone a differential post-translational modification in the parkin-/- mouse brain. Based on the nature and degree of the pI shift from spot A6 to A5, possible modifications included oxidation or nitration of the protein. These modifications have been shown previously to appear in cells and tissues after exposure to oxidative stress (36MacMillan-Crow L.A. Crow J.P. Thompson J.A. Biochemistry. 1998; 37: 1613-1622Google Scholar, 37Viner R.I. Williams T.D. Schoneich C. Biochemistry. 1999; 38: 12408-12415Google Scholar, 38Yang K.S. Kang S.W. Woo H.A. Hwang S.C. Chae H.Z. Kim K. Rhee S.G. J. Biol. Chem. 2002; 277: 38029-38036Google Scholar). The alteration of both 24- and 30-kDa subunits suggested a general impairment of complex I in parkin-/- mice. Furthermore, spot B1 (subunit Vb of complex IV) was also down-regulated in parkin-/- mice, suggesting additional alterations in complex IV of the mitochondrial respirat