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BIBTEX RIS

Novel Monoclonal Antibodies Demonstrate Biochemical Variation of Brain Parkin with Age

2003; Elsevier BV; Volume: 278; Issue: 48 Linguagem: Inglês

10.1074/jbc.m306889200

ISSN

1083-351X

Autores

Aaron C. Pawlyk, Benoit I. Giasson, Deepak M. Sampathu, Francisco A. Perez, Kah‐Leong Lim, Valina L. Dawson, Ted M. Dawson, Richard D. Palmiter, John Q. Trojanowski, Virginia M.‐Y. Lee,

Tópico(s)

Genetics and Neurodevelopmental Disorders

Resumo

Autosomal recessive juvenile parkinsonism is a movement disorder associated with the degeneration of dopaminergic neurons in substantia nigra pars compacta. The loss of functional parkin caused by parkin gene mutations is the most common single cause of juvenile parkinsonism. Parkin has been shown to aid in protecting cells from endoplasmic reticulum and oxidative stressors presumably due to ubiquitin ligase activity of parkin that targets proteins for proteasomal degradation. However, studies on parkin have been impeded because of limited reagents specific for this protein. Here we report the generation and characterization of a panel of parkin-specific monoclonal antibodies. Biochemical analyses indicate that parkin is present only in the high salt-extractable fraction of mouse brain, whereas it is present in both the high salt-extractable and RIPA-resistant, SDS-extractable fraction in young human brain. Parkin is present at decreased levels in the high salt-extractable fraction and at increased levels in the SDS-extractable fraction from aged human brain. This shift in the extractability of parkin upon aging is seen in humans but not in mice, demonstrating species-specific differences in the biochemical characteristics of murine versus human parkin. Finally, by using these highly specific anti-parkin monoclonal antibodies, it was not possible to detect parkin in α-synuclein-containing lesions in α-synucleinopathies, thereby challenging prior inferences about the role of parkin in movement disorders other than autosomal recessive juvenile parkinsonism. Autosomal recessive juvenile parkinsonism is a movement disorder associated with the degeneration of dopaminergic neurons in substantia nigra pars compacta. The loss of functional parkin caused by parkin gene mutations is the most common single cause of juvenile parkinsonism. Parkin has been shown to aid in protecting cells from endoplasmic reticulum and oxidative stressors presumably due to ubiquitin ligase activity of parkin that targets proteins for proteasomal degradation. However, studies on parkin have been impeded because of limited reagents specific for this protein. Here we report the generation and characterization of a panel of parkin-specific monoclonal antibodies. Biochemical analyses indicate that parkin is present only in the high salt-extractable fraction of mouse brain, whereas it is present in both the high salt-extractable and RIPA-resistant, SDS-extractable fraction in young human brain. Parkin is present at decreased levels in the high salt-extractable fraction and at increased levels in the SDS-extractable fraction from aged human brain. This shift in the extractability of parkin upon aging is seen in humans but not in mice, demonstrating species-specific differences in the biochemical characteristics of murine versus human parkin. Finally, by using these highly specific anti-parkin monoclonal antibodies, it was not possible to detect parkin in α-synuclein-containing lesions in α-synucleinopathies, thereby challenging prior inferences about the role of parkin in movement disorders other than autosomal recessive juvenile parkinsonism. Autosomal recessive juvenile parkinsonism (AR-JP) 1The abbreviations used are: AR-JPautosomal recessive juvenile parkinsonismELISAenzyme-linked immunosorbent assayDLBdementia with Lewy bodiesGCIglial cytoplasmic inclusionHShigh salt bufferHSThigh salt Triton bufferLSlow salt buffermAbmonoclonal antibodyMSAmultiple system atrophyPBSphosphate-buffered salinePDParkinson's diseasePMIpost-mortem intervalPMSFphenylmethanesulfonyl fluorideRIPAradioactive immunoprecipitation assay bufferIBRin-between RING fingerCHOChinese hamster ovaryDAPI4′,6-diamidino-2-phenylindole. is an especially insidious form of parkinsonism that can strike as early as the 1st decade of life. A major locus for this disease was mapped to chromosome 6q, and the gene was subsequently identified and termed parkin (1Kitada T. Asakawa S. Hattori N. Matsumine H. Yamamura Y. Minoshima S. Yokochi M. Mizuno Y. Shimizu N. Nature. 1998; 392: 605-608Crossref PubMed Scopus (4323) Google Scholar). Human parkin is a 465-amino acid protein with a predicted molecular mass of 52 kDa that contains an N-terminal ubiquitin-like domain, a linker region, and a C-terminal TRIAD domain consisting of two RING fingers on either side of an in-between RING (IBR) finger region (2van der Reijden B.A. Erpelinck-Verschueren C.A. Lowenberg B. Jansen J.H. Protein Sci. 1999; 8: 1557-1561Crossref PubMed Scopus (64) Google Scholar). Deletions and insertions of one or more exons resulting in premature translation termination are some of the most common mutations in parkin, but numerous missense point mutations in parkin have also been shown to be causal of AR-JP (3West A. Periquet M. Lincoln S. Lucking C.B. Nicholl D. Bonifati V. Rawal N. Gasser T. Lohmann E. Deleuze J.F. Maraganore D. Levey A. Wood N. Durr A. Hardy J. Brice A. Farrer M. Am. J. Med. Genet. 2002; 114: 584-591Crossref PubMed Scopus (192) Google Scholar). Since the identification of parkin, many studies focused on elucidating the function of this protein, and it has been shown it can function as a ubiquitin-protein isopeptide ligase and that its overexpression in vivo enhances the ubiquitinylation of synphilin-1, Pael-R, and CDCRel-1. Immunoprecipitated parkin has been reported to catalyze the ubiquination of these substrates in vitro as well as O-glycosylated α-synuclein p22 and cyclin E (4Imai Y. Soda M. Hatakeyama S. Akagi T. Hashikawa T. Nakayama K.I. Takahashi R. Mol. Cell. 2002; 10: 55-67Abstract Full Text Full Text PDF PubMed Scopus (435) Google Scholar, 5Shimura H. Schlossmacher M.G. Hattori N. Frosch M.P. Trockenbacher A. Schneider R. Mizuno Y. Kosik K.S. Selkoe D.J. 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Kitada T. Matsumine H. Asakawa S. Minoshima S. Yamamura Y. Shimizu N. Mizuno Y. Ann. Neurol. 1999; 45: 668-672Crossref PubMed Scopus (259) Google Scholar, 22Zarate-Lagunes M. Gu W.J. Blanchard V. Francois C. Muriel M.P. Mouatt-Prigent A. Bonici B. Parent A. Hartmann A. Yelnik J. Boehme G.A. Pradier L. Moussaoui S. Faucheux B. Raisman-Vozari R. Agid Y. Brice A. Hirsch E.C. J. Comp. Neurol. 2001; 432: 184-196Crossref PubMed Scopus (47) Google Scholar) have reported that parkin is extractable from cultured cells as well as from human and rodent brain by using low salt buffers with or without the addition of mild detergents. Parkin has also been found in Lewy body-enriched preparations from human brain (20Schlossmacher M.G. Frosch M.P. Gai W.P. Medina M. Sharma N. Forno L. Ochiishi T. Shimura H. Sharon R. Hattori N. Langston J.W. Mizuno Y. Hyman B.T. Selkoe D.J. Kosik K.S. Am. J. Pathol. 2002; 160: 1655-1667Abstract Full Text Full Text PDF PubMed Scopus (278) Google Scholar). However, there is a discrepancy among these reports on the number and size of human versus rodent parkin isoforms as well as discrepancies in the subcellular distribution of parkin (12Horowitz J.M. Vernace V.A. Myers J. Stachowiak M.K. Hanlon D.W. Fraley G.S. Torres G. J. Chem. Neuroanat. 2001; 21: 75-93Crossref PubMed Scopus (38) Google Scholar, 13Huynh D.P. Scoles D.R. Ho T.H. Del Bigio M.R. Pulst S.M. Ann. Neurol. 2000; 48: 737-744Crossref PubMed Scopus (69) Google Scholar, 15Kubo S.I. Kitami T. Noda S. Shimura H. Uchiyama Y. Asakawa S. Minoshima S. Shimizu N. Mizuno Y. Hattori N. J. Neurochem. 2001; 78: 42-54Crossref PubMed Scopus (117) Google Scholar, 21Shimura H. Hattori N. Kubo S. Yoshikawa M. Kitada T. Matsumine H. Asakawa S. Minoshima S. Yamamura Y. Shimizu N. Mizuno Y. Ann. Neurol. 1999; 45: 668-672Crossref PubMed Scopus (259) Google Scholar, 23Hase A. Yamada H. Arai K. Sunada Y. Shimizu T. Matsumura K. Brain Res. 2002; 930: 143-149Crossref PubMed Scopus (12) Google Scholar). To clarify some of these issues, sensitive and specific antibodies to parkin were generated. A panel of antiparkin monoclonal antibodies (mAb) that recognize different domains of parkin was produced, and their specificity in murine and human brain was demonstrated. Detailed analyses of the biochemical properties of parkin in mouse and human brain tissue by using serial fractionation with buffers of increasing protein extraction strength showed unusual properties of parkin in the human brain. Finally, by using these highly specific parkin mAbs in conjunction with biochemical and immunostaining methods, we were unable to detect parkin in α-synuclein containing lesions in patients with α-synucleinopathies thereby calling into question prior studies about the role of parkin in movement disorders other than AR-JP. The full-length human parkin cDNA was cloned into the baculovirus expression vector pFASTBAC-HTb and expressed in Sf9 cells (Invitrogen). The resulting His6-tagged parkin protein was purified on a column packed with nickel-nitrilotriacetic acid resin (Invitrogen). In brief, Sf9/parkin cells were lysed in a buffer containing 50 mm Tris-HCl, pH 8.5, 0.1 m KCl, 5 mm 2-mercaptoethanol, 1 mm PMSF, and 1% Nonidet P-40 and then centrifuged at 10,000 × g for 15 min at 4 °C. The supernatant was applied to a nickel-nitrilotriacetic acid column pre-equilibrated in equilibration buffer (20 mm Tris-HCl, pH 8.5, 0.5 m KCl, 20 mm imidazole, 5 mm 2-mercaptoethanol, 1 mm PMSF, 10% glycerol). The column was washed with 10 column volumes of the same buffer followed by 2 column volumes of wash buffer (20 mm Tris-HCl, pH 8.5, 1 m KCl, 5 mm 2-mercaptoethanol, 1 mm PMSF, and 10% glycerol) and then 2 column volumes of equilibration buffer. His6-parkin was eluted with 20 mm Tris-HCl, pH 8.5, 0.1 m KCl, 100 mm imidazole, 5 mm 2-mercaptoethanol, 1 mm PMSF, and 10% glycerol (see Fig. 1A). The rabbit polyclonal antibodies CS2132 and AB5112 were purchased from Cell Signaling Technology (Beverly, MA) and Chemicon International, Inc. (Temecula, CA), respectively. Murine anti-human parkin mAbs were generated by immunization of mice with recombinant human parkin as described (30Giasson B.I. Jakes R. Goedert M. Duda J.E. Leight S. Trojanowski J.Q. Lee V.M.-Y. J. Neurosci. Res. 2000; 59: 528-533Crossref PubMed Scopus (194) Google Scholar). Fusion was conducted by using spleen lymphocytes from immunized Balb/c mice and SP2 cells to produce hybridomas. Resulting hybridoma supernatants were screened by enzyme-linked immunosorbent assay (ELISA) using plates coated with parkin. The epitopes of the anti-parkin mAbs were mapped by Western blotting using proteins expressed by Gene-Porter 2 (Gene Therapy Systems, San Diego, CA)-mediated transfection of QBI293 cells with pcDNA3.1-myc/His (Invitrogen) harboring subcloned domains of parkin. To generate these constructs, PCR fragments spanning the coding region for each domain (ubiquitin-like domain, 1–77; linker region, 78–220; first RING finger, 221–305; IBR region, 306–398; second RING finger, 399–465) were digested and ligated into the host vector. The predicted sequence of each construct was confirmed by DNA sequencing. Antibodies were isotyped by antigen-captured ELISA by using goat anti-mouse antibodies to each immunoglobulin subtype (Sigma). Proteins were resolved on 10 and 15% SDS-polyacrylamide gels for parkin and α-synuclein, respectively, and electrophoretically transferred to nitrocellulose membranes (Schleicher & Schuell) as described previously (29Forman M.S. Schmidt M.L. Kasturi S. Perl D.P. Lee V.M.-Y. Trojanowski J.Q. Am. J. Pathol. 2002; 160: 1725-1731Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar, 30Giasson B.I. Jakes R. Goedert M. Duda J.E. Leight S. Trojanowski J.Q. Lee V.M.-Y. J. Neurosci. Res. 2000; 59: 528-533Crossref PubMed Scopus (194) Google Scholar). Western blotting was conducted by following published protocols by using either goat anti-mouse (Jackson Immuno-Research Laboratories, West Grove, PA) or goat anti-rabbit (Santa Cruz Biotechnology, Santa Cruz, CA) conjugated to horseradish peroxidase as secondary antibodies (29Forman M.S. Schmidt M.L. Kasturi S. Perl D.P. Lee V.M.-Y. Trojanowski J.Q. Am. J. Pathol. 2002; 160: 1725-1731Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar, 30Giasson B.I. Jakes R. Goedert M. Duda J.E. Leight S. Trojanowski J.Q. Lee V.M.-Y. J. Neurosci. Res. 2000; 59: 528-533Crossref PubMed Scopus (194) Google Scholar). Immuno-positive protein bands were detected with Renaissance Enhanced Luminol Reagents (PerkinElmer Life Sciences) and X-Omat Blue XB-1 film (Eastman Kodak Co.). Wild type Chinese hamster ovary (CHO) cells and CHO cells stably expressing human parkin (CHO-PAR4) were maintained in α-minimal essential medium (Invitrogen), 10% fetal bovine serum (Invitrogen), 100 units/ml penicillin, and 100 units/ml streptomycin. HeLa, Neuro-2A, and HEK-293 cells were maintained in Dulbecco's modification of Eagle's medium (Cellgro by Mediatech, Herndon, VA), 10% fetal bovine serum, 100 units/ml penicillin, and 100 units/ml streptomycin. SH-SY5Y cells were maintained in RPMI medium 1640 (Invitrogen) with l-glutamine, 10% fetal bovine serum, 100 units/ml penicillin, and 100 units/ml streptomycin. Cells were grown to ∼80% confluency, lysed in 2% SDS, 50 mm Tris, pH 7.6, and heated to 100 °C for 5 min. Protein concentrations of cell lysates were determined by bicinchoninic acid assay (Pierce) prior to SDS-PAGE and Western blotting. Parkin-null mice were generated by genomic deletion of exon 2 of parkin, removing most of the coding region for the ubiquitin-like domain. 2F. A. Perez and R. D. Palmiter, manuscript in preparation. The parkin-null mice were maintained on a C57Bl6/J × Sv129S hybrid background. Young wild type and parkin null mice used in these studies were 4–12 weeks old, and aged wild type mice were >22 months old. Whole mouse brains were dissected for biochemical fractionation studies. The brain tissue samples from human subjects that were used for biochemical analyses are summarized in Table I. Two extraction methods (I and II) were used for these tissues as summarized below.Table ISummary of subjects used in this study Young (Y) and aged (A) cases are described according to their pathologically confirmed diagnoses, age at death (Age), sex and post-mortem interval (PMI). The abbreviations are as follows: NML, normal; SCHZ, schizophrenia; DLB, dementia with Lewy bodies; MSA, multiple system atrophy.CaseDiagnosisAgeSexPMIyearshY-1NML14M10Y-2NML23M8Y-3NML22M4A-1SCHZ86F7.5A-2NML92F5A-3SCHZ83F7.5A-4SCHZ70F17.5A-5NML69M11A-6SCHZ76F10A-7NML60M14A-8NML74F6A-9NML74F3.5A-10NML43M30.5A-11DLB90F5A-12DLB75F6A-13DLB79M20.5A-14NML75M17A-15MSA65M43A-16MSA57F8A-17MSA54M25 Open table in a new tab Method I—Murine or human tissue samples were homogenized in 2 ml/g of high salt (HS) buffer (50 mm Tris, pH 7.5, 2 mm EDTA, 750 mm NaCl) and sedimented at 125,000 × g for 30 min at 4 °C. Supernatants from the initial fractionation were saved as the HS fraction, and the pellets were washed and re-extracted sequentially with buffers of increasing protein extraction strengths. For each buffer, two cycles of extraction and washes were conducted using 2 ml/g of HS + 1% Triton X-100 (HST) followed by 2 ml/g of RIPA (150 mm NaCl, 0.1% SDS, 0.5% sodium deoxycholate, 1% Nonidet P-40, 50 mm Tris, pH 8.0). Supernatants were saved as the HST and RIPA fractions, respectively. Floatation and removal of contaminating myelin using HS + 20% sucrose was performed prior to the RIPA extraction. A final extraction using 2 ml/g of 2% SDS in 50 mm Tris, pH 7.6, was conducted on the pellet and sedimented at 125,000 × g for 30 min at 22 °C. The supernatant was saved as the SDS fraction. Method II—Brain tissues from patients with α-synucleinopathies and control patients were homogenized in 10 ml/g low salt (LS) buffer (10 mm Tris, pH 7.5, 5 mm EDTA, 1 mm dithiothreitol, and 10% sucrose) and sedimented at 25,000 × g for 30 min at 4 °C. Supernatants were saved as the LS fraction, and pellets were washed by re-extraction in LS buffer. Resulting pellets were subjected to two sequential extractions in 10 ml/g Triton X (TX) buffer (LS + 1% Triton X-100 + 0.5 m NaCl) and sedimented at 180,000 × g for 30 min at 4 °C. Supernatants from the first of these extractions were saved as the TX fraction. Pellets were then homogenized in 10 ml/g Sarkosyl buffer (LS + 1% N-lauroylsarcosine + 0.5 m NaCl) and incubated at 22 °C on a shaker for 1 h prior to sedimentation at 180,000 × g for 30 min at 22 °C. Supernatants were saved as the Sarkosyl extraction buffer fraction. Remaining pellets were extracted in 2.5 ml/g SDS buffer (2% SDS, 50 mm Tris, pH 7.6) prior to centrifugation at 25,000 × g for 30 min at 22 °C. Supernatants were saved as the SDS fraction. All extraction buffers contained a mixture of protease inhibitors (1 μg/ml each of leupeptin, pepstatin A, N-p-tosyl-l-phenylalanine chloromethyl ketone, Nα-p-tosyl-l-lysine chloromethyl ketone, soybean trypsin inhibitor, and 0.5 mm PMSF). For experiments using quantitative amounts of protein, total protein concentration was determined with the BCA protein assay kit (Pierce) using bovine serum albumin as a standard. SDS sample buffer (10 mm Tris, pH 6.8, 1 mm EDTA, 40 mm dithiothreitol, 1% SDS, 10% sucrose) was added to samples of HS, HST, RIPA, LS, TX, and Sarkosyl extraction buffer fractions; sample buffer without SDS was added to the SDS samples. All samples were heated for 5 min at 100 °C prior to SDS-PAGE. PRK8-coupled dextran beads were generated using the Carbo-Link column kit (Pierce) following the manufacturer's protocol. Proteins were extracted from human frontal cortex tissue by two extractions with HST, and myelin was floated and removed as described above. The resulting pellet was homogenized in 8 m urea in 50 mm Tris, pH 7.6, with protease inhibitors. Following sedimentation at 125,000 × g, the supernatant was diluted to 2 m urea and incubated by gentle rocking with PRK8-coupled beads for 4 h. The incubation and all subsequent steps were performed at 4 °C. The beads were washed 3 times with 2 m urea, 1% Triton X-100 in 50 mm Tris, pH 7.6, and bound parkin was then eluted with Pierce ImmunoElution Buffer by gentle rocking for 1 h. SDS sample buffer was added to an aliquot of the eluant. Samples were not boiled to limit carbamoylation of protein in the presence of urea. The harvesting, fixation, and further processing of the tissue specimens used in this study were conducted as described previously (24Duda J.E. Giasson B.I. Gur T.L. Montine T.J. Robertson D. Biaggioni I. Hurtig H.I. Stern M.B. Gollomp S.M. Grossman M. Lee V.M.-Y. Trojanowski J.Q. J. Neuropathol. Exp. Neurol. 2000; 59: 830-841Crossref PubMed Scopus (136) Google Scholar, 25Schmidt M.L. Murray J. Lee V.M.-Y. Hill W.D. Wertkin A. Trojanowski J.Q. Am. J. Pathol. 1991; 139: 53-65PubMed Google Scholar). Briefly, tissue blocks of cingulate cortex from DLB or cerebellum from MSA brains were fixed with 70% ethanol, 150 mm NaCl or neutral buffered formalin and infiltrated with paraffin. The diagnostic assessment of all DLB and MSA cases (both of which are α-synucleinopathies characterized by α-synuclein inclusions) was performed in accordance with published guidelines (26Gilman S. Low P.A. Quinn N. Albanese A. Ben Shlomo Y. Fowler C.J. Kaufmann H. Klockgether T. Lang A.E. Lantos P.L. Litvan I. Mathias C.J. Oliver E. Robertson D. Schatz I. Wenning G.K. J. Neurol. Sci. 1999; 163: 94-98Abstract Full Text Full Text PDF PubMed Scopus (975) Google Scholar, 37McKeith I.G. Galasko D. Kosaka K. Perry E.K. Dickson D.W. Hansen L.A. Salmon D.P. Lowe J. Mirra S.S. Byrne E.J. Lennox G. Quinn N.P. Edwardson J.A. Ince P.G. Bergeron C. Burns A. Miller B.L. Lovestone S. Collerton D. Jansen E.N. Ballard C. de Vos R.A. Wilcock G.K. Jellinger K.A. Perry R.H. Neurology. 1996; 47: 1113-1124Crossref PubMed Scopus (3676) Google Scholar). Whole mouse brains (wild type or parkin null) were fixed with 70% ethanol, 150 mm NaCl and paraffin-embedded. Immunohistochemistry was carried out using the avidin-biotin complex detection system (Vector Laboratories, Burlingame, CA) and 3,3′-diaminobenzidine as described (24Duda J.E. Giasson B.I. Gur T.L. Montine T.J. Robertson D. Biaggioni I. Hurtig H.I. Stern M.B. Gollomp S.M. Grossman M. Lee V.M.-Y. Trojanowski J.Q. J. Neuropathol. Exp. Neurol. 2000; 59: 830-841Crossref PubMed Scopus (136) Google Scholar, 25Schmidt M.L. Murray J. Lee V.M.-Y. Hill W.D. Wertkin A. Trojanowski J.Q. Am. J. Pathol. 1991; 139: 53-65PubMed Google Scholar). The anti-synuclein mouse monoclonal antibody Syn303 (28Duda J.E. Giasson B.I. Mabon M.E. Lee V.M.-Y. Trojanowski J.Q. Ann. Neurol. 2002; 52: 205-210Crossref PubMed Scopus (282) Google Scholar) was used to detect α-synuclein inclusions by immunohistochemistry as described previously (27Duda J.E. Giasson B.I. Chen Q. Gur T.L. Hurtig H.I. Stern M.B. Gollomp S.M. Ischiropoulos H. Lee V.M.-Y. Trojanowski J.Q. Am. J. Pathol. 2000; 157: 1439-1445Abstract Full Text Full Text PDF PubMed Scopus (241) Google Scholar, 28Duda J.E. Giasson B.I. Mabon M.E. Lee V.M.-Y. Trojanowski J.Q. Ann. Neurol. 2002; 52: 205-210Crossref PubMed Scopus (282) Google Scholar, 29Forman M.S. Schmidt M.L. Kasturi S. Perl D.P. Lee V.M.-Y. Trojanowski J.Q. Am. J. Pathol. 2002; 160: 1725-1731Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar, 30Giasson B.I. Jakes R. Goedert M. Duda J.E. Leight S. Trojanowski J.Q. Lee V.M.-Y. J. Neurosci. Res. 2000; 59: 528-533Crossref PubMed Scopus (194) Google Scholar). Sections were also lightly counter-stained with hematoxylin. In order to try to stain α-synuclein pathological inclusions in human diseased cases with parkin antibodies, a variety of fixation and retrieval methods were applied. These include the following: 1) formalin- or ethanol-fixed, paraffin-infiltrated tissue followed by antigen retrieval with urea, microwave irradiation, or formic acid; 2) formalin- or ethanol-fixed floating sections; 3) frozen sections post-fixed with ethanol or formalin; and 4) fresh frozen un-fixed sections. Double labeling indirect immunofluorescence analyses for brain sections were performed as described previously (29Forman M.S. Schmidt M.L. Kasturi S. Perl D.P. Lee V.M.-Y. Trojanowski J.Q. Am. J. Pathol. 2002; 160: 1725-1731Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). The rabbit anti-α-syn antibody SNL4 (30Giasson B.I. Jakes R. Goedert M. Duda J.E. Leight S. Trojanowski J.Q. Lee V.M.-Y. J. Neurosci. Res. 2000; 59: 528-533Crossref PubMed Scopus (194) Google Scholar) and several of the parkin mAbs were used to examine possible co-localization of α-synuclein and parkin in α-synuclein inclusions by immunofluorescence. For tissue culture experiments, cells were grown on glass coverslips, transfected as described above, and fixed for 10 min with 4% paraformaldehyde in PBS followed by the permeabilization with 0.2% Triton X-100 in PBS for 10 min. Alexa Fluor 488-(green) and Alexa Fluor 594 (red)-conjugated secondary antibodies (Molecular Probes, Eugene, OR) were used to detect immunostaining, and slides were coverslipped with Vectashield 4′,6-diamidino-2-phenylindole (DAPI) mounting medium (Vector Laboratories, Burlingame, CA). CHO cells were maintained in α-minimal essential medium, 10% fetal bovine serum (Invitrogen), and 100 units/ml penicillin, 100 units/ml streptomycin. Cells were transfected with the plasmid parkin/pcDNA 3.1 expressing full-length un-tagged parkin using LipofectAMINE reagents (Invitrogen) according to the manufacturer's instructions. Cells stably transfected with parkin/pcDNA 3.1 were selected and maintained with 200 μg/ml geneticin (Invitrogen). Following harvesting with trypsin, cells were washed with PBS, fixed with 70% ethanol, 150 mm NaCl, compacted into a pellet by centrifugation at 13,000 × g, and paraffin-embedded. Following sectioning, immunocytochemistry or immunofluorescence staining was performed as described above. To produce antibodies specific to parkin, a battery of murine mAbs was raised against recombinant human parkin as described under "Experimental Procedures." We identified 60 hybridomas with strong immunoreactivity for the recombinant protein by ELISA. To confirm the specificity of these parkin mAbs, we conducted Western blot analyses using brain extracts from wild type and parkin-null mice as well as extracts from the human brain. Surprisingly, out of our panel of 60 mAbs, only four anti-parkin antibodies (PRK8, PRK28, PRK106, and PRK109) were able to detect protein bands with apparent molecular masses corresponding to that of parkin while at the same time showing no cross-reactivity with proteins in extracts from parkin null mice (Fig. 1 and Table II). These four parkin mAbs detected ∼50- and ∼44-kDa bands in mouse brain extracts but not in extracts from parkin-null mice; representative Western blots are shown in Fig. 1B. A similar doublet of ∼50- and ∼46-kDa immunobands also was detected in human brain extracts with PRK8, PRK28, PRK35, PRK106, PRK109, and a commercially available antibody, CS2132 (Figs. 1B and 2). A very minor cross-reacting band at ∼142 kDa was detected in both wild type and parkin-null mouse brain extracts by some of the parkin mAbs, but this ∼142-kDa band is the major species recognized by CS2132 (Fig. 1B). The other commercially purchased anti-parkin antibody AB5112 did not recognize authentic parkin since this antibody detected a number of protein bands in mouse brain extracts that were also present in brain extracts from parkin-null mice (Fig. 1B).Table IICharacterization of selected anti-parkin antibodiesAntibodyDomainaUbl, ubiquitin-like domain; Md, linker domain; IBR, in-between RINGs domain; R2, second RI

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