Prolyl-isomerase Pin1 Accumulates in Lewy Bodies of Parkinson Disease and Facilitates Formation of α-Synuclein Inclusions
2005; Elsevier BV; Volume: 281; Issue: 7 Linguagem: Inglês
10.1074/jbc.m507026200
ISSN1083-351X
AutoresAkihide Ryo, Takashi Togo, Toshiki Nakai, Akiko Hirai, Mayuko Nishi, Akira Yamaguchi, Kyoko Suzuki, Yoshio Hirayasu, Hideki Kobayashi, Kilian Perrem, Yih‐Cherng Liou, Ichiro Aoki,
Tópico(s)Parkinson's Disease Mechanisms and Treatments
ResumoParkinson disease (PD) is a relatively common neurodegenerative disorder that is characterized by the loss of dopaminergic neurons and by the formation of Lewy bodies (LBs), which are cytoplasmic inclusions containing aggregates of α-synuclein. Although certain post-translational modifications of α-synuclein and its related proteins are implicated in the genesis of LBs, the specific molecular mechanisms that both regulate these processes and initiate subsequent inclusion body formation are not yet well understood. We demonstrate in our current study, however, that the prolyl-isomerase Pin1 localizes to the LBs in PD brain tissue and thereby enhances the formation of α-synuclein immunoreactive inclusions. Immunohistochemical analysis of brain tissue from PD patients revealed that Pin1 localizes to 50–60% of the LBs that show an intense halo pattern resembling that of α-synuclein. By utilizing a cellular model of α-synuclein aggregation, we also demonstrate that, whereas Pin1 overexpression facilitates the formation of α-synuclein inclusions, dominant-negative Pin1 expression significantly suppresses this process. Consistent with these observations, Pin1 overexpression enhances the protein half-life and insolubility of α-synuclein. Finally, we show that Pin1 binds synphilin-1, an α-synuclein partner, via its Ser-211-Pro and Ser-215-Pro motifs, and enhances its interaction with α-synuclein, thus likely facilitating the formation of α-synuclein inclusions. These results indicate that Pin1-mediated prolyl-isomerization plays a pivotal role in a post-translational modification pathway for α-synuclein aggregation and in the resultant Lewy body formations in PD. Parkinson disease (PD) is a relatively common neurodegenerative disorder that is characterized by the loss of dopaminergic neurons and by the formation of Lewy bodies (LBs), which are cytoplasmic inclusions containing aggregates of α-synuclein. Although certain post-translational modifications of α-synuclein and its related proteins are implicated in the genesis of LBs, the specific molecular mechanisms that both regulate these processes and initiate subsequent inclusion body formation are not yet well understood. We demonstrate in our current study, however, that the prolyl-isomerase Pin1 localizes to the LBs in PD brain tissue and thereby enhances the formation of α-synuclein immunoreactive inclusions. Immunohistochemical analysis of brain tissue from PD patients revealed that Pin1 localizes to 50–60% of the LBs that show an intense halo pattern resembling that of α-synuclein. By utilizing a cellular model of α-synuclein aggregation, we also demonstrate that, whereas Pin1 overexpression facilitates the formation of α-synuclein inclusions, dominant-negative Pin1 expression significantly suppresses this process. Consistent with these observations, Pin1 overexpression enhances the protein half-life and insolubility of α-synuclein. Finally, we show that Pin1 binds synphilin-1, an α-synuclein partner, via its Ser-211-Pro and Ser-215-Pro motifs, and enhances its interaction with α-synuclein, thus likely facilitating the formation of α-synuclein inclusions. These results indicate that Pin1-mediated prolyl-isomerization plays a pivotal role in a post-translational modification pathway for α-synuclein aggregation and in the resultant Lewy body formations in PD. Parkinson disease (PD) 2The abbreviations used are: PD, Parkinson disease; LB, Lewy body; dn, dominant-negative; DRB, 5,6-dichrolo-1-β-d-ribofuranosylbenzimidazole; CKII, casein kinase II; PBS, phosphate-buffered saline; GST, glutathione S-transferase; HA, hemagglutinin; GFP, green fluorescent protein; siRNA, small interference RNA; RNAi, RNA interference. is one of the most common neurodegenerative disorders and is characterized by the loss of dopaminergic neurons in the substantia nigra and by the presence of cytoplasmic inclusions known as Lewy bodies (LBs) in surviving neurons (1.Dunnett S.B. Bjorklund A. Nature. 1999; 399: A32-A39Crossref PubMed Scopus (516) Google Scholar, 2.Lotharius J. Brundin P. Nat. Rev. Neurosci. 2002; 3: 932-942Crossref PubMed Scopus (943) Google Scholar). LBs have classically been considered as a pathological hallmark of PD, consisting of many components, including α-synuclein, which is one of the major constituents (3.Spillantini M.G. Schmidt M.L. Lee V.M. Trojanowski J.Q. Jakes R. Goedert M. Nature. 1997; 388: 839-840Crossref PubMed Scopus (6178) Google Scholar, 4.Baba M. Nakajo S. Tu P.H. Tomita T. Nakaya K. Lee V.M. Trojanowski J.Q. Iwatsubo T. Am. J. Pathol. 1998; 152: 879-884PubMed Google Scholar). The first indication of a pathogenic role for α-synuclein in PD came from the results of linkage analysis of mutations in its gene in autosomal dominant forms of the disease (5.Polymeropoulos M.H. Lavedan C. Leroy E. Ide S.E. Dehejia A. Dutra A. Pike B. Root H. Rubenstein J. Boyer R. Stenroos E.S. Chandrasekharappa S. Athanassiadou A. Papapetropoulos T. Johnson W.G. Lazzarini A.M. Duvoisin R.C. Di Iorio G. Golbe L.I. Nussbaum R.L. Science. 1997; 276: 2045-2047Crossref PubMed Scopus (6667) Google Scholar, 6.Kruger R. Kuhn W. Muller T. Woitalla D. Graeber M. Kosel S. Przuntek H. Epplen J.T. Schols L. Riess O. Nat. Genet. 1998; 18: 106-108Crossref PubMed Scopus (3321) Google Scholar). α-Synuclein is an unfolded protein in its native state, but in a pathological state it can be induced to form either α-helical or β-sheet structures that result in the formation of insoluble α-synuclein aggregates (7.Recchia A. Debetto P. Negro A. Guidolin D. Skaper S.D. Giusti P. FASEB J. 2004; 18: 617-626Crossref PubMed Scopus (245) Google Scholar, 8.Lundvig D. Lindersson E. Jensen P.H. Brain Res. Mol. Brain Res. 2005; 134: 3-17Crossref PubMed Scopus (72) Google Scholar). The aggregation of α-synuclein can be modified by a range of factors, both in vitro and in vivo, including environmental regulators of pH, temperature, ionic strength, and oxidative stress and by intrinsic intracellular pathways (8.Lundvig D. Lindersson E. Jensen P.H. Brain Res. Mol. Brain Res. 2005; 134: 3-17Crossref PubMed Scopus (72) Google Scholar). The latter of these regulatory networks includes several α-synuclein-binding proteins such as synphilin-1 (9.Engelender S. Kaminsky Z. Guo X. Sharp A.H. Amaravi R.K. Kleiderlein J.J. Margolis R.L. Troncoso J.C. Lanahan A.A. Worley P.F. Dawson V.L. Dawson T.M. Ross C.A. Nat. Genet. 1999; 22: 110-114Crossref PubMed Scopus (440) Google Scholar) and post-translational modifications of related molecules, such as phosphorylation and ubiquitination (10.Giasson B.I. Lee V.M. Cell. 2003; 114: 1-8Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar, 11.Okochi M. Walter J. Koyama A. Nakajo S. Baba M. Iwatsubo T. Meijer L. Kahle P.J. Haass C. J. Biol. Chem. 2000; 275: 390-397Abstract Full Text Full Text PDF PubMed Scopus (424) Google Scholar, 12.Fujiwara H. Hasegawa M. Dohmae N. Kawashima A. Masliah E. Goldberg M.S. Shen J. Takio K. Iwatsubo T. Nat. Cell Biol. 2002; 4: 160-164Crossref PubMed Scopus (159) Google Scholar). Synphilin-1 was identified as a protein that interacts with α-synuclein and has been shown to be a substrate of the PD-associated ubiquitin ligases, Parkin, SIAH, and dorfin (9.Engelender S. Kaminsky Z. Guo X. Sharp A.H. Amaravi R.K. Kleiderlein J.J. Margolis R.L. Troncoso J.C. Lanahan A.A. Worley P.F. Dawson V.L. Dawson T.M. Ross C.A. Nat. Genet. 1999; 22: 110-114Crossref PubMed Scopus (440) Google Scholar, 13.Chung K.K. Zhang Y. Lim K.L. Tanaka Y. Huang H. Gao J. Ross C.A. Dawson V.L. Dawson T.M. Nat. Med. 2001; 7: 1144-1150Crossref PubMed Scopus (660) Google Scholar, 14.Liani E. Eyal A. Avraham E. Shemer R. Szargel R. Berg D. Bornemann A. Riess O. Ross C.A. Rott R. Engelender S. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 5500-5505Crossref PubMed Scopus (161) Google Scholar, 15.Nagano Y. Yamashita H. Takahashi T. Kishida S. Nakamura T. Iseki E. Hattori N. Mizuno Y. Kikuchi A. Matsumoto M. J. Biol. Chem. 2003; 278: 51504-51514Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar, 16.Ito T. Niwa J. Hishikawa N. Ishigaki S. Doyu M. Sobue G. J. Biol. Chem. 2003; 278: 29106-29114Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). Recent studies indicate that synphilin-1 ubiquitination via these ubiquitin ligases is important for the formation of LBs (13.Chung K.K. Zhang Y. Lim K.L. Tanaka Y. Huang H. Gao J. Ross C.A. Dawson V.L. Dawson T.M. Nat. Med. 2001; 7: 1144-1150Crossref PubMed Scopus (660) Google Scholar, 14.Liani E. Eyal A. Avraham E. Shemer R. Szargel R. Berg D. Bornemann A. Riess O. Ross C.A. Rott R. Engelender S. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 5500-5505Crossref PubMed Scopus (161) Google Scholar, 17.Lim K.L. Chew K.C. Tan J.M. Wang C. Chung K.K. Zhang Y. Tanaka Y. Smith W. Engelender S. Ross C.A. Dawson V.L. Dawson T.M. J. Neurosci. 2005; 25: 2002-2009Crossref PubMed Scopus (448) Google Scholar). In fact, the co-expression of the α-synuclein and synphilin-1 proteins results in the formation of cytoplasmic inclusions in a subset of cultured cells (9.Engelender S. Kaminsky Z. Guo X. Sharp A.H. Amaravi R.K. Kleiderlein J.J. Margolis R.L. Troncoso J.C. Lanahan A.A. Worley P.F. Dawson V.L. Dawson T.M. Ross C.A. Nat. Genet. 1999; 22: 110-114Crossref PubMed Scopus (440) Google Scholar, 18.O'Farrell C. Murphy D.D. Petrucelli L. Singleton A.B. Hussey J. Farrer M. Hardy J. Dickson D.W. Cookson M.R. Brain Res. Mol. Brain Res. 2001; 97: 94-102Crossref PubMed Scopus (59) Google Scholar). However, it is currently not well understood how these two molecules function cooperatively during inclusion body formation or how their functional interaction is regulated. Phosphorylation-dependent prolyl-isomerization is a recently characterized post-translational modification mechanism that regulates the function and properties of specific proteins (19.Lu K.P. Trends Biochem. Sci. 2004; 29: 200-209Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar, 20.Joseph J.D. Yeh E.S. Swenson K.I. Means A.R. Winkler K.E. Prog. Cell Cycle Res. 2003; 5: 477-487PubMed Google Scholar). These modifications are catalyzed by a peptidyl prolyl-isomerase, Pin1, which specifically binds phosphorylated serine or threonine residues immediately preceding proline (pSer/Thr-Pro motifs) in a subset of proteins, and promotes the cis/trans isomerization of the peptide bond (19.Lu K.P. Trends Biochem. Sci. 2004; 29: 200-209Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar, 20.Joseph J.D. Yeh E.S. Swenson K.I. Means A.R. Winkler K.E. Prog. Cell Cycle Res. 2003; 5: 477-487PubMed Google Scholar). Such conformational changes have been shown to have profound effects on the function of Pin1 substrates as they can modulate catalytic activity, phosphorylation status, protein-protein interactions, subcellular localization, and protein stability (21.Ryo A. Nakamura M. Wulf G. Liou Y.C. Lu K.P. Nat. Cell Biol. 2001; 3: 793-801Crossref PubMed Scopus (422) Google Scholar, 22.Ryo A. Suizu F. Yoshida Y. Perrem K. Liou Y.C. Wulf G. Rottapel R. Yamaoka S. Lu K.P. Mol. Cell. 2003; 12: 1413-1426Abstract Full Text Full Text PDF PubMed Scopus (558) Google Scholar). Consequently, Pin1 has been shown to be involved in the regulation of many cellular events, including proliferation, differentiation, and cell death, and has been reported to be associated with several human diseases, including cancer and Alzheimer disease (AD) (19.Lu K.P. Trends Biochem. Sci. 2004; 29: 200-209Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar, 23.Ryo A. Liou Y.C. Lu K.P. Wulf G. J. Cell Sci. 2003; 116: 773-783Crossref PubMed Scopus (171) Google Scholar, 24.Liou Y.C. Sun A. Ryo A. Zhou X.Z. Yu Z.X. Huang H.K. Uchida T. Bronson R. Bing G. Li X. Hunter T. Lu K.P. Nature. 2003; 424: 556-561Crossref PubMed Scopus (382) Google Scholar). In AD brain tissue, Pin1 accumulates in pathological neurofibrillary tangles, thereby specifically interacting with phosphorylated tau proteins on their Thr-231-Pro motifs (24.Liou Y.C. Sun A. Ryo A. Zhou X.Z. Yu Z.X. Huang H.K. Uchida T. Bronson R. Bing G. Li X. Hunter T. Lu K.P. Nature. 2003; 424: 556-561Crossref PubMed Scopus (382) Google Scholar, 25.Lu P.J. Wulf G. Zhou X.Z. Davies P. Lu K.P. Nature. 1999; 399: 784-788Crossref PubMed Scopus (634) Google Scholar, 26.Zhou X.Z. Kops O. Werner A. Lu P.J. Shen M. Stoller G. Kullertz G. Stark M. Fischer G. Lu K.P. Mol. Cell. 2000; 6: 873-883Abstract Full Text Full Text PDF PubMed Scopus (479) Google Scholar). Significantly, by catalyzing the cis/trans-isomerization of tau, Pin1 restores the ability of phosphorylated tau to bind microtubules and promotes tau dephosphorylation by the PP2A phosphatase (25.Lu P.J. Wulf G. Zhou X.Z. Davies P. Lu K.P. Nature. 1999; 399: 784-788Crossref PubMed Scopus (634) Google Scholar, 26.Zhou X.Z. Kops O. Werner A. Lu P.J. Shen M. Stoller G. Kullertz G. Stark M. Fischer G. Lu K.P. Mol. Cell. 2000; 6: 873-883Abstract Full Text Full Text PDF PubMed Scopus (479) Google Scholar). However, although the crucial role of Pin1 in the pathogenesis of AD has begun to be characterized, its involvement in other neurodegenerative diseases is unknown. In our current study, we provide evidence that Pin1 plays a role in the aggregation and degradation of α-synuclein. We show that Pin1 accumulates in the LBs of PD tissue and co-localizes with α-synuclein in a cell culture model of α-synuclein inclusions. Furthermore, the overexpression of Pin1 enhances the formation of α-synuclein inclusions, which is accompanied by their increased half-life and insolubility. We also demonstrate that Pin1 interacts with synphilin-1 in a phosphorylation-dependent manner and regulates its interaction with α-synuclein. These results together indicate that Pin1-mediated prolyl-isomerization may be involved in the formation of LBs and, consequently, in the pathogenesis of PD. Immunohistochemistry—The sampling and usage of all brain tissues in this study was performed under the guidelines of the committee in Yokohama City University for the use of clinical samples, protocol no. 04-008. A total of six autopsied brains from patients with PD and five normal controls were examined in this study. Tissue sections were placed on slides, deparaffinized in xylene, hydrated in 100 and 75% ethanol, and then washed with PBS. The tissue slides were pretreated with 99% formic acid for 3 min. After extensive washing with PBS, the slides were then treated with PBS containing 5% goat serum and 0.1% Triton X-100 for blocking and then incubated with either anti-Pin1 polyclonal antibodies (1:500) (21.Ryo A. Nakamura M. Wulf G. Liou Y.C. Lu K.P. Nat. Cell Biol. 2001; 3: 793-801Crossref PubMed Scopus (422) Google Scholar) or anti-α-synuclein polyclonal antibodies (1:100) (27.Suzuki K. Iseki E. Katsuse O. Yamaguchi A. Katsuyama K. Aoki I. Yamanaka S. Kosaka K. Neuroreport. 2003; 14: 551-554Crossref PubMed Scopus (45) Google Scholar) at 4 °C in humidified chamber for 12 h. After washing with PBS, the slides were then incubated with biotinylated secondary antibody for 2 h. Immunohistochemical analysis was then performed using Vectastain ABC kit and DAB-staining solution (Vector Laboratories, Burlingame, CA). Protein Degradation Assay—293T cells were transfected with α-synuclein, with Xpress-LacZ used as a control. Cycloheximide (100 μg/ml) was added to the media 24 h after transfection, and cells were harvested at different time points. Total cell lysates in SDS sample buffer were sonicated and then analyzed by immunoblotting with either anti-Xpress (Invitrogen) or anti-α-synuclein antibodies (27.Suzuki K. Iseki E. Katsuse O. Yamaguchi A. Katsuyama K. Aoki I. Yamanaka S. Kosaka K. Neuroreport. 2003; 14: 551-554Crossref PubMed Scopus (45) Google Scholar). The blots were scanned and semiquantified using National Institutes of Health Image 1.6.2 software (22.Ryo A. Suizu F. Yoshida Y. Perrem K. Liou Y.C. Wulf G. Rottapel R. Yamaoka S. Lu K.P. Mol. Cell. 2003; 12: 1413-1426Abstract Full Text Full Text PDF PubMed Scopus (558) Google Scholar). GST Pull-down Assay, Immunoprecipitation, and Immunoblotting Analyses—293T and COS-1 cells were lysed with GST pull-down buffer (50 mm HEPES (pH 7.4), 150 mm NaCl, 10% glycerol, 1% Triton X-100, 1.5 mm MgCl2, 1 mm EGTA, 100 mm NaF, 1 mm Na3VO4, 1 mm dithiothreitol, 0.5 μg/ml leupeptin, 1.0 μg/ml pepstatin, and 0.2 mm phenylmethylsulfonyl fluoride) and incubated with 30 μl of glutathione-agarose beads containing either GST-Pin1 or GST at 4 °C for 2 h. The precipitated proteins were then washed three times with lysis buffer and subjected to SDS-PAGE. For immunoprecipitation, cells were harvested 24 h after transfection and lysed with Nonidet P-40 lysis buffer (10 mm Tris HCl (pH 7.5), 100 mm NaCl, 0.5% Nonidet P-40, 1 mm Na3VO4, 100 mm NaF, 0.5 μg/ml leupeptin, 1.0 μg/ml pepstatin, and 0.2 mm phenylmethylsulfonyl fluoride). Cell lysates were incubated for 1 h with Protein A/G-Sepharose/non-immunized IgG complexes. Supernatant fractions were recovered and immunoprecipitated with 5 μg of anti-HA or anti-Xpress antibody and 30 μl of Protein A/G-Sepharose. After washing three times with lysis buffer, pellets were analyzed on SDS-PAGE gels and subjected to immunoblot analysis. For GST-pull-down assay with in vitro translated proteins, pcDNA-synphilin-1 and pcDNA-synphilin-1(Δ207–216) were translated in vitro with the TnT-coupled transcription/translation kit (Promega). They were then incubated in Xenopus extracts, as described previously (21.Ryo A. Nakamura M. Wulf G. Liou Y.C. Lu K.P. Nat. Cell Biol. 2001; 3: 793-801Crossref PubMed Scopus (422) Google Scholar). Proteins translated in vitro were incubated with 20 μl of glutathione-agarose beads containing GST-Pin1 or GST at 4 °C for 2 h, as described previously (21.Ryo A. Nakamura M. Wulf G. Liou Y.C. Lu K.P. Nat. Cell Biol. 2001; 3: 793-801Crossref PubMed Scopus (422) Google Scholar). The precipitated proteins were washed with GST-lysis buffer and subjected to SDS-PAGE. Construction of Synphilin-1 and α-Synuclein Vectors—The cloning of full-length synphilin-1 cDNA was performed as described previously (28.Iseki E. Takayama N. Furukawa Y. Marui W. Nakai T. Miura S. Ueda K. Kosaka K. Neurosci. Lett. 2002; 326: 211-215Crossref PubMed Scopus (18) Google Scholar). Synphilin-1 cDNA was further subcloned into the pcDNA-HisMax vector (Invitrogen) to generate N-terminal Xpress-tagged synphilin-1. α-Synuclein cDNA was amplified by PCR from a human brain cDNA library and inserted into either the pcDNA or pcDNA-HA expression vector (Invitrogen) to generate untagged or C-terminal HA-tagged α-synuclein, respectively. Immunofluorescent Analysis—Exponentially growing 293T cells on coverslips were transfected with pcDNA-α-synuclein and pcDNA-synphilin-1 using Effectene reagent (Qiagen), according to the manufacturer's instructions. 24 h after transfection, 10 μm MG-132 was added to the culture medium for the final 12 h of culturing. Cells were then fixed with 3% formaldehyde and treated with PBS, containing 5% goat serum and 0.1% Triton X-100 for blocking, and then incubated with anti-α-synuclein polyclonal antibodies at room temperature in a humidified chamber for 2 h (27.Suzuki K. Iseki E. Katsuse O. Yamaguchi A. Katsuyama K. Aoki I. Yamanaka S. Kosaka K. Neuroreport. 2003; 14: 551-554Crossref PubMed Scopus (45) Google Scholar). After washing with PBS, slides were incubated with anti-rabbit-Alexa568 secondary antibody (Molecular Probes) for 1 h followed by immunofluorescent analysis using fluorescent laser microscopy (Olympus, Tokyo, Japan). More than 200 GFP-positive cells per slide were scored for inclusion body formation. Phospho-peptides Pull-down Assay—1 μg of Biotin-labeled phosphorylated peptides was captured with 30 μl of streptavidin magnetic beads (New England Biolabs) and then mixed with 2 μg of recombinant GST or GST-Pin1 in GST-lysis buffer. After a 1-h incubation at 4 °C, the beads were washed three times with GST-lysis buffer and then incubated with 2× sample buffer with boiling for 5 min. The supernatant fractions were then subjected to immunoblotting analysis with anti-Pin1 antibody. Construction of Synphilin-1 siRNA—Synphilin-1 siRNAs were created as described previously (29.Ryo A. Uemura H. Ishiguro H. Saitoh T. Yamaguchi A. Perrem K. Kubota Y. Lu K.P. Aoki I. Clin. Cancer Res. 2005; 11: 7523-7531Crossref PubMed Scopus (104) Google Scholar) with the following sequences: synph-RNAiA (GATTCTGGATGTGCCTTAT); synph-RNAiB (CTCACCTCCTCTGGTTAAA); and control-RNAi (TCGTATGTTGTGTGGAATT). Pin1 Accumulates in the Lewy Bodies of Parkinson Disease—The pathogenesis of PD has been implicated in the formation of LBs, accompanied by specific post-translational modifications of related molecules (2.Lotharius J. Brundin P. Nat. Rev. Neurosci. 2002; 3: 932-942Crossref PubMed Scopus (943) Google Scholar, 10.Giasson B.I. Lee V.M. Cell. 2003; 114: 1-8Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar). Because the prolyl-isomerase Pin1 regulates a broad range of post-translational modification processes (19.Lu K.P. Trends Biochem. Sci. 2004; 29: 200-209Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar, 23.Ryo A. Liou Y.C. Lu K.P. Wulf G. J. Cell Sci. 2003; 116: 773-783Crossref PubMed Scopus (171) Google Scholar), we hypothesized that it may play some role also in the pathogenesis of PD. To test this hypothesis, we first attempted immunohistochemical analyses of PD brain tissue from affected patients with both anti-Pin1 and anti-α-synuclein antibodies. We subsequently found that the anti-Pin1 antibody positively stained the LBs in the midbrains of each of the PD patients and that ∼50–60% of these LBs were detectable by this immunostaining (Fig. 1A). In addition, Pin1 was observed to be localized more intensely in the halo of the majority of the LBs, whereas the neuronal cytoplasm was very weakly stained (Fig. 1, B and C). Anti-α-synuclein antibody labeling also detected LBs in all of our PD patients and intense halo or punctate staining was observed (Fig. 1, B and C), as described previously (3.Spillantini M.G. Schmidt M.L. Lee V.M. Trojanowski J.Q. Jakes R. Goedert M. Nature. 1997; 388: 839-840Crossref PubMed Scopus (6178) Google Scholar, 4.Baba M. Nakajo S. Tu P.H. Tomita T. Nakaya K. Lee V.M. Trojanowski J.Q. Iwatsubo T. Am. J. Pathol. 1998; 152: 879-884PubMed Google Scholar). However in control brain cells, no LB-like inclusions were detected with either antibody (data not shown). These results indicate that Pin1 is a component of Lewy bodies and may affect their formation. Pin1 Binds and Co-localizes with α-Synuclein in Intracellular Inclusions—Our observations, that Pin1 localizes to LBs and that its staining pattern resembles that of α-synuclein, suggested the possibility that these two proteins interact in these structures. We therefore examined the interaction between Pin1 and α-synuclein in cultured cells. 293T cells were transfected with HA-tagged α-synuclein, and lysates from these cells were immunoprecipitated with an anti-HA antibody. The resulting immunoblotting analysis revealed that endogenous Pin1 does indeed bind to α-synuclein (Fig. 2A). To further confirm this specific interaction in vitro, GST-pull-down experiments were performed. Lysates of 293T cells that had been transfected with α-synuclein were incubated with either GST or GST-Pin1. α-Synuclein was subsequently detected in GST-Pin1-bound agarose beads, but not the control GST beads (Fig. 2B). This further indicated that a specific interaction occurs between Pin1 and α-synuclein. Pin1 has been shown to specifically bind motifs containing a phosphorylated serine or threonine preceding proline (Ser/Thr-Pro). Because α-synuclein has no such motif, we speculated that Pin1 binding to α-synuclein could be indirect. To address this question, we performed an in vitro binding assay. Recombinant His-tagged α-synuclein or tau proteins were treated with Xenopus mitotic extracts and then subjected to GST-pull-down analysis as performed previously (21.Ryo A. Nakamura M. Wulf G. Liou Y.C. Lu K.P. Nat. Cell Biol. 2001; 3: 793-801Crossref PubMed Scopus (422) Google Scholar). As shown in Fig. 2C, although both proteins were well phosphorylated with Xenopus extracts, only tau, but not α-synuclein was pulled down with GST-Pin1. These data therefore show that Pin1 associates with α-synuclein in cell lysates but that it cannot bind to α-synuclein in experiments using purified recombinant proteins. This suggests that there is an indirect interaction between these two proteins. It has been previously shown that the co-transfection of α-synuclein and synphilin-1 results in the formation of intracellular inclusions in cultured cells (9.Engelender S. Kaminsky Z. Guo X. Sharp A.H. Amaravi R.K. Kleiderlein J.J. Margolis R.L. Troncoso J.C. Lanahan A.A. Worley P.F. Dawson V.L. Dawson T.M. Ross C.A. Nat. Genet. 1999; 22: 110-114Crossref PubMed Scopus (440) Google Scholar, 14.Liani E. Eyal A. Avraham E. Shemer R. Szargel R. Berg D. Bornemann A. Riess O. Ross C.A. Rott R. Engelender S. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 5500-5505Crossref PubMed Scopus (161) Google Scholar). We utilized this finding to investigate whether Pin1 co-localizes with α-synuclein in these inclusions. 293T cells were co-transfected with α-synuclein, synphilin-1, and GFP-Pin1, followed by immunocytochemical analysis. Pin1 was again found to co-localize with α-synuclein in intracellular inclusions that showed either halo or spot patterns (Fig. 2, D and E), as described previously (9.Engelender S. Kaminsky Z. Guo X. Sharp A.H. Amaravi R.K. Kleiderlein J.J. Margolis R.L. Troncoso J.C. Lanahan A.A. Worley P.F. Dawson V.L. Dawson T.M. Ross C.A. Nat. Genet. 1999; 22: 110-114Crossref PubMed Scopus (440) Google Scholar, 14.Liani E. Eyal A. Avraham E. Shemer R. Szargel R. Berg D. Bornemann A. Riess O. Ross C.A. Rott R. Engelender S. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 5500-5505Crossref PubMed Scopus (161) Google Scholar). However, Pin1 overexpression alone did not lead to the formation of either aggregates or inclusions with other proteins (supplemental Fig. S1). These results further indicate that Pin1 indirectly interacts with α-synuclein in intracellular inclusions. Pin1 Facilitates the Formation of α-Synuclein Immunoreactive Inclusions—We next addressed whether Pin1 overexpression affects the formation of α-synuclein inclusions. It has been shown in earlier studies that the co-expression of α-synuclein and its binding partner synphilin-1 in mammalian cells results in the formation of cytoplasmic inclusions composed of α-synuclein (9.Engelender S. Kaminsky Z. Guo X. Sharp A.H. Amaravi R.K. Kleiderlein J.J. Margolis R.L. Troncoso J.C. Lanahan A.A. Worley P.F. Dawson V.L. Dawson T.M. Ross C.A. Nat. Genet. 1999; 22: 110-114Crossref PubMed Scopus (440) Google Scholar, 14.Liani E. Eyal A. Avraham E. Shemer R. Szargel R. Berg D. Bornemann A. Riess O. Ross C.A. Rott R. Engelender S. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 5500-5505Crossref PubMed Scopus (161) Google Scholar). We expanded upon this finding to investigate whether Pin1 has any effects on inclusion body formation. 293T cells were transfected with α-synuclein and synphilin-1, together with either GFP, GFP-Pin1, or a GFP-dominant negative (dn)-Pin1, and examined for α-synuclein immunopositive inclusions. The co-expression of GFP-Pin1 in this experiment resulted in a significant increase in the formation of α-synuclein immunoreactive inclusions and in the numbers of inclusion positive cells, when compared with control GFP-transfected cells (Fig. 3, A and C). In contrast, when a dominant-negative Pin1 construct was co-expressed in parallel experiments, there were significant decreases in the number of cells containing α-synuclein inclusions (Fig. 3, A and C). These phenomena were also confirmed by an additional experiment with non-tagged Pin1 constructs (data not shown). However, transfection of GFP, GFP-Pin1, or GFP-dnPin1 without co-transfection of α-synuclein and synphilin-1 did not result in the inclusion formations described by previous studies (supplemental Fig. S1). Likewise, hematoxylin and eosin staining also revealed in parallel experiments that GFP-Pin1 enhances, whereas GFP-dnPin1 decreases, the number of cells with eosinophilic cytosolic inclusions (Fig. 3B). These results suggested that a specific functional interaction exists between Pin1 and α-synuclein in the formation of α-synuclein inclusions. To address whether either the binding or the catalytic activity of Pin1 is required for inclusion body formation, we performed an experiment using either a WW-domain (binding domain) mutant (W34A) or peptidyl-prolyl-cis/trans-isomerase domain (catalytic domain) mutant (K63A) of Pin1. The subsequent inclusion body formation analysis revealed that both of these Pin1 mutants failed to enhance the formation of α-synuclein inclusions (Fig. 3D), indicating that both domains are required for inclusion body formation. Taken together, our results indicate that Pin1 plays a role in the formation of intracellular α-synuclein inclusions. Pin1 Enhances the Protein Half-life and Insolubility of α-Synuclein—Our data showing that Pin1 interacts with α-synuclein and promotes α-synuclein inclusion formation prompted us to examine the possible function of Pin1 in the post-translational modification of the α-synuclein protein. We initially examined both the stability and the solubility of α-synuclein, because these two properties are important for aggregate formation. 293T cells were transfected with α-synuclein, together with either a GFP vector control or GFP-Pin1 construct, followed by treatment with cycloheximide to inhibit protein synthesis. We then harvested the cells at different time points and examined α-synuclein protein levels by Western blot analysis. Interest
Referência(s)