Mitochondrial Dysfunction and Oxidative Damage in parkin-deficient Mice

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). were resuspended at a concentration of protein in ml of buffer mm KCl, 3 mm 1 mm EGTA, 5 mm pH with w/v of serum and for using an of mm mm 4 mm or mm mm as electron was added in and 3 was of 4 was of carbonyl cyanide was added to the and was in the absence of a Mitochondrial was determined using a with a buffer to in oxygen are represented as a of the wild-type 3 for Data were analyzed by Student's t test. Electron and parkin-/- mice were euthanized by CO2 and with ml of followed by 10 ml of in 100 mm Brains were and in the for an additional at 4 Brains were in and into and striata were dissected and for electron by with and Mitochondrial number and morphology were determined in from from 2 mice genotype by an to the were in parkin-/- and wild-type mice at intervals 10 were analyzed by analysis of followed by Data for mice were collected in with behavioral analysis and were analyzed by Student's t test. were to to Mice were euthanized by CO2 inhalation, was collected in and serum was isolated by at for 10 min. antioxidant capacity was by the of to using a assay and is represented as μm and as Data were analyzed by Student's t test. Protein were homogenized in 50 mm Tris, mm and pH and was by Supernatants were for protein content and of protein was for protein carbonyls as the Briefly, proteins were into a concentration of and with for 15 min. the samples were electrophoresed on gels transferred to and using an to the on proteins. were and subsequently for to protein were removed, in for 2 at and 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 were with with an to adducts of 4HNE and then with an was with Analysis of parkin-/- 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 that loss of parkin in accumulation of its which may in cause nigrostriatal dysfunction and nigral degeneration. To the proteomic parkin-/- and wild-type we used 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 proteins in the ventral midbrain of each of the 10 pairs of parkin-/- and wild-type mice at of Proteins were in the first by isoelectric on a gel using carrier and subsequently in the second by weight on 40 × silver we reproducible, changes in 15 of ∼8000 discrete spots the to the staining of all one of these 15 protein spots was decreased in parkin-/- mice. of protein spots from these gels followed by trypsin digestion and matrix-assisted laser desorption ionization and ionization the identification of these proteins of the 15 spots represented distinct proteins, whereas one protein was in 2 spots and with pI, suggesting a post-translational modification. in protein spot that were confirmed in more than six pairs of parkin-/- and wild-type mice were alterations in parkin of of pairs of mice consistent alterations in parkin changes shown as the S.E. All alterations are by Student's t involved in mitochondrial NADH-ubiquinone NADH-ubiquinone NADH-ubiquinone involved in oxidative stress Proteins protein protein similar to from protein 6.5 of pairs of mice consistent alterations in parkin changes shown as the S.E. All alterations are by Student's t in a The of the proteins altered in parkin-/- mice are in either mitochondrial of and mitochondrial complexes I and or oxidative stress and 6 and a loss of parkin and mitochondrial and/or antioxidant The of complex I was to a more in the parkin-/- brain spots and the protein a differential post-translational modification in the parkin-/- mouse Based on the and of the from spot to or of the These have been shown previously to in cells and tissues to oxidative stress L.A. 1998; 37: Scholar, C. 1999; Scholar, H.A. S.C. K. J. Biol. Chem. 2002; 277: Scholar). The of and subunits suggested a impairment of complex I in parkin-/- mice. Furthermore, spot of complex was also in parkin-/- suggesting additional alterations in complex of the mitochondrial