Assembly of Lysine 63-linked Ubiquitin Conjugates by Phosphorylated α-Synuclein Implies Lewy Body Biogenesis
2007; Elsevier BV; Volume: 282; Issue: 19 Linguagem: Inglês
10.1074/jbc.m700422200
ISSN1083-351X
AutoresChao Liu, Erkang Fei, Nali Jia, Hongfeng Wang, Rui-Song Tao, Atsushi Iwata, Nobuyuki Nukina, Jiang‐Ning Zhou, Guanghui Wang,
Tópico(s)Nuclear Receptors and Signaling
Resumoα-Synuclein (α-syn) and ubiquitin (Ub) are major protein components deposited in Lewy bodies (LBs) and Lewy neurites, which are pathologic hallmarks of idiopathic Parkinson disease (PD). Almost 90% of α-syn in LBs is phosphorylated at serine 129 (Ser129). However, the role of Ser129-phosphorylated α-syn in the biogenesis of LBs remains unclear. Here, we show that compared with coexpression of wild type (WT)α-syn and Ub, coexpression of phospho-mimic mutant α-syn (S129D) and Ub in neuro2a cells results in an increase of Ub-conjugates and the formation of ubiquitinated inclusions. Furthermore, S129D α-syn fails to increase the Ub-conjugates and form ubiquitinated inclusions in the presence of a K63R mutant Ub. In addition, as compared with WT α-syn, S129D α-syn increased cytoplasmic and neuritic aggregates of itself in neuro2a cells treated with H2O2 and serum deprivation. These results suggest that the contribution of Ser129-phosphorylated α-syn to the Lys63-linked Ub-conjugates and aggregation of itself may be involved in the biogenesis of LBs in Parkinson disease and other related synucleinopathies. α-Synuclein (α-syn) and ubiquitin (Ub) are major protein components deposited in Lewy bodies (LBs) and Lewy neurites, which are pathologic hallmarks of idiopathic Parkinson disease (PD). Almost 90% of α-syn in LBs is phosphorylated at serine 129 (Ser129). However, the role of Ser129-phosphorylated α-syn in the biogenesis of LBs remains unclear. Here, we show that compared with coexpression of wild type (WT)α-syn and Ub, coexpression of phospho-mimic mutant α-syn (S129D) and Ub in neuro2a cells results in an increase of Ub-conjugates and the formation of ubiquitinated inclusions. Furthermore, S129D α-syn fails to increase the Ub-conjugates and form ubiquitinated inclusions in the presence of a K63R mutant Ub. In addition, as compared with WT α-syn, S129D α-syn increased cytoplasmic and neuritic aggregates of itself in neuro2a cells treated with H2O2 and serum deprivation. These results suggest that the contribution of Ser129-phosphorylated α-syn to the Lys63-linked Ub-conjugates and aggregation of itself may be involved in the biogenesis of LBs in Parkinson disease and other related synucleinopathies. Parkinson disease (PD) 2The abbreviations used are: PD, Parkinson disease; LB, Lewy body; Ub, ubiquitin; α-syn, α-synuclein; β-syn, β-synuclein; WT, wild type; EGFP, enhanced green fluorescent protein; UPS, ubiquitin proteasome system; PBS, phosphate-buffered saline; DAPI, 4′,6-diamidino-2-phenylindole; DMEM, Dulbecco's modified Eagle's medium; GFP, green fluorescent protein. is the most common neurodegenerative movement disorder (1Dawson T.M. Dawson V.L. Science. 2003; 302: 819-822Crossref PubMed Scopus (1408) Google Scholar). Pathologically, it is characterized by loss of dopamine neurons and the presence of cytoplasmic inclusions (Lewy bodies, LBs) in surviving neurons in the substantia nigra pars compacta, accompanied by the presence of dystrophic neurites (Lewy neurites) (1Dawson T.M. Dawson V.L. Science. 2003; 302: 819-822Crossref PubMed Scopus (1408) Google Scholar, 2Nussbaum R.L. Ellis C.E. N. Engl. J. Med. 2003; 348: 1356-1364Crossref PubMed Scopus (1034) Google Scholar). Although many studies have focused on the LBs, the mechanism that underlies LB biogenesis is poorly understood (3Olanow C.W. Perl D.P. DeMartino G.N. McNaught K.S. Lancet Neurol. 2004; 3: 496-503Abstract Full Text Full Text PDF PubMed Scopus (250) Google Scholar, 4Shults C.W. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 1661-1668Crossref PubMed Scopus (363) Google Scholar). α-SYN was identified as the first "PD-gene" (5Gasser T. J. Neurol. 2001; 248: 833-840Crossref PubMed Scopus (128) Google Scholar). Three PD related mutations in the α-SYN gene contribute to the rare familial forms of PD (6Kruger 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 (3344) Google Scholar, 7Polymeropoulos 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 (6734) Google Scholar, 8Zarranz J.J. Alegre J. Gomez-Esteban J.C. Lezcano E. Ros R. Ampuero I. Vidal L. Hoenicka J. Rodriguez O. Atares B. Llorens V. Tortosa E. Gomez del Ser T. Munoz D.G. de Yebenes J.G. Ann. Neurol. 2004; 55: 164-173Crossref PubMed Scopus (2192) Google Scholar). The deposition of α-syn has been found in PD, dementia with LBs (DLB) (9Spillantini M.G. Crowther R.A. Jakes R. Hasegawa M. Goedert M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6469-6473Crossref PubMed Scopus (2443) Google Scholar), multiple system atrophy (10Wakabayashi K. Yoshimoto M. Tsuji S. Takahashi H. Neurosci. Lett. 1998; 249: 180-182Crossref PubMed Scopus (534) Google Scholar), and juvenile onset neuroaxonal dystrophy (11Wakabayashi K. Yoshimoto M. Fukushima T. Koide R. Horikawa Y. Morita T. Takahashi H. Neuropathol. Appl. Neurobiol. 1999; 25: 363-368Crossref PubMed Scopus (67) Google Scholar). These diseases are so called synucleinopathies, suggesting that α-syn plays a common role in certain neurodegenerative diseases (12Jellinger K.A. Mov. Disord. 2003; 18: S2-S12Crossref PubMed Scopus (240) Google Scholar). In synucleinopathic brains and transgenic animal models, α-syn is selectively and extensively phosphorylated at serine 129 (13Fujiwara 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 (162) Google Scholar, 14Yamada M. Iwatsubo T. Mizuno Y. Mochizuki H. J. Neurochem. 2004; 91: 451-461Crossref PubMed Scopus (220) Google Scholar, 15Anderson J.P. Walker D.E. Goldstein J.M. de Laat R. Banducci K. Caccavello R.J. Barbour R. Huang J. Kling K. Lee M. Diep L. Keim P.S. Shen X. Chataway T. Schlossmacher M.G. Seubert P. Schenk D. Sinha S. Gai W.P. Chilcote T.J. J. Biol. Chem. 2006; 281: 29739-29752Abstract Full Text Full Text PDF PubMed Scopus (926) Google Scholar, 16Takahashi M. Kanuka H. Fujiwara H. Koyama A. Hasegawa M. Miura M. Iwatsubo T. Neurosci. Lett. 2003; 336: 155-158Crossref PubMed Scopus (118) Google Scholar). Phosphorylation of α-syn at Ser129 is critical for α-syn neurotoxicity (17Chen L. Feany M.B. Nat. Neurosci. 2005; 8: 657-663Crossref PubMed Scopus (529) Google Scholar) and furthermore, targets α-syn for ubiquitination (15Anderson J.P. Walker D.E. Goldstein J.M. de Laat R. Banducci K. Caccavello R.J. Barbour R. Huang J. Kling K. Lee M. Diep L. Keim P.S. Shen X. Chataway T. Schlossmacher M.G. Seubert P. Schenk D. Sinha S. Gai W.P. Chilcote T.J. J. Biol. Chem. 2006; 281: 29739-29752Abstract Full Text Full Text PDF PubMed Scopus (926) Google Scholar, 18Hasegawa M. Fujiwara H. Nonaka T. Wakabayashi K. Takahashi H. Lee V.M. Trojanowski J.Q. Mann D. Iwatsubo T. J. Biol. Chem. 2002; 277: 49071-49076Abstract Full Text Full Text PDF PubMed Scopus (348) Google Scholar). Ubiquitination is a process whereby a small protein called ubiquitin (Ub) conjugates to its target protein. Monoubiquitination occurs through the isopeptide bond between the C-terminal glycine (Gly-76) residue on Ub and the ɛ-amino group of the lysine (Lys) side chain in the target protein by multisteps that are activated sequentially by E1 (Ub-activating), E2 (Ub-conjugating), and E3 (Ub-ligase) enzymes (19Hershko A. Ciechanover A. Annu. Rev. Biochem. 1998; 67: 425-479Crossref PubMed Scopus (6959) Google Scholar). The addition of one or more Ub moieties to the Ub on the target protein results in di- or polyubiquitination. Ub, one of the major components in LBs, is an essentially required component in the ubiquitin-proteasome system (UPS) (20Lowe J. Blanchard A. Morrell K. Lennox G. Reynolds L. Billett M. Landon M. Mayer R.J. J. Pathol. 1988; 155: 9-15Crossref PubMed Scopus (470) Google Scholar, 21Sakamoto M. Uchihara T. Hayashi M. Nakamura A. Kikuchi E. Mizutani T. Mizusawa H. Hirai S. Exp. Neurol. 2002; 177: 88-94Crossref PubMed Scopus (39) Google Scholar). UPS is an intracellular proteolytic system that degrades its targeted proteins (22Weissman A.M. Nat. Rev. Mol. Cell. Biol. 2001; 2: 169-178Crossref PubMed Scopus (1262) Google Scholar, 23Layfield R. Cavey J.R. Lowe J. Aging Res. Rev. 2003; 2: 343-356Crossref PubMed Scopus (92) Google Scholar). To date, many studies suggest that malfunction of protein degradation linked to UPS plays an important role in the pathogenesis of PD (23Layfield R. Cavey J.R. Lowe J. Aging Res. Rev. 2003; 2: 343-356Crossref PubMed Scopus (92) Google Scholar, 24Snyder H. Wolozin B. J. Mol. Neurosci. 2004; 24: 425-442Crossref PubMed Scopus (46) Google Scholar). A protein tagged by a chain of four or more Ubs, instead of a single Ub, is targeted for proteasomal degradation (25Thrower J.S. Hoffman L. Rechsteiner M. Pickart C.M. EMBO J. 2000; 19: 94-102Crossref PubMed Scopus (1321) Google Scholar). Specifically, Lys48-linked poly-Ub chains instead of Lys63-linked poly-Ub chains are considered to be a proteasomal degradation signal (22Weissman A.M. Nat. Rev. Mol. Cell. Biol. 2001; 2: 169-178Crossref PubMed Scopus (1262) Google Scholar, 23Layfield R. Cavey J.R. Lowe J. Aging Res. Rev. 2003; 2: 343-356Crossref PubMed Scopus (92) Google Scholar). In vivo, the aggregation of α-syn is enhanced by Lys63-linked ubiquitination (26Liu Y. Fallon L. Lashuel H.A. Liu Z. Lansbury Jr., P.T. Cell. 2002; 111: 209-218Abstract Full Text Full Text PDF PubMed Scopus (699) Google Scholar). Also, both wild type α-syn and mutant α-syn, incubated with rabbit reticulocyte Fraction IIA in vitro, increase Lys63-linked Ub-conjugate formation with the mutants being more effective (27Doss-Pepe E.W. Chen L. Madura K. J. Biol. Chem. 2005; 280: 16619-16624Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). The increased Ub-conjugates are detectable by anti-Ub antibodies, but not by anti-α-syn antibody, indicating that α-syn may function in regulating assembly of Ub-Lys63 chains (27Doss-Pepe E.W. Chen L. Madura K. J. Biol. Chem. 2005; 280: 16619-16624Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). In this study, we investigate the possible role of phosphorylation of α-syn at Ser129 in the biogenesis of LBs using a cellular model. Coexpression of S129D α-syn, on which serine 129 was converted to aspartate to mimic phosphorylated α-syn, and Ub resulted in an increase in Ub-conjugates and cytoplasmic ubiquitinated aggregates compared with the coexpression of WT α-syn with Ub. We also observed that coexpression of S129D α-syn and Ub caused the formation of ubiquitinated inclusions in neuro2a cells. Furthermore, in neuro2a cells cotransfected with K63R mutant Ub, S129D α-syn failed to increase the Ub-conjugates or form ubiquitinated inclusions. Compared with WT α-syn, S129D α-syn increased the ubiquitination of itself when coexpressed with Ub. Additionally, S129D α-syn formed more aggregates in neuro2a cells treated with H2O2 and serum deprivation than WT α-syn. Our data may provide insight into a molecular mechanism that links phosphorylation of α-syn and Lys63-linked Ub-conjugates to LB biogenesis. Plasmid Construction—The WT α-SYN cDNA was constructed by subcloning the PCR product amplified using primers 5′-GAAGATCTCCATGGATGTATTCATG-3′ and 5′-GCGTCGACAAGGCTTCAGGTTCGTAG-3′ from previous pCI vector (28Iwata A. Maruyama M. Kanazawa I. Nukina N. J. Biol. Chem. 2001; 276: 45320-45329Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar) containing the wild type α-SYN gene. These products were inserted into the BglII/SalI sites of the pEGFP-N1 vector (Clontech) and BamHI/XhoI sites of pCDNA3.1/V5-HisB vector (Invitrogen) to construct pEGFPN1-α-SYN (WT) and pCDNA3.1/V5-HisB-α-SYN (WT). Full-length β-SYNUCLEIN cDNA was created by PCR using primers 5′-GAAGATCTGGATGGACGTGTTCATG-3′ and 5′-GCGTCGACAACGCCTCTGGCTCATAC-3′ with a human fetal brain cDNA library (Clontech) as a template and inserted into pEGFP-N1 vector (Clontech) via the BglII/SalI sites. UB cDNA was obtained by reverse transcriptase-PCR using primers 5′-CTGGATCCACATGCAGATCTTCGTG-3′ and 5′-CATGAATTCTTACCCACCTCTGAGACG-3′ with total RNA extracted from HeLa cells and inserted in-frame into pGEX-5X-1 (Amersham Biosciences) at BamHI/EcoRI sites. pcDNA4/HisA-UB was created by excising the UB gene at BamHI/EcoRI sites from pGEX-5X-1-UB vector and inserting into pcDNA4-HisA vector (Invitrogen) at BamHI/EcoRI sites. p3XFlag-myc-CMV-24-UB was created by excising the fragments at KpnI/XbaI sites from pcDNA4HisA-UB and inserting into the p3XFlag-myc-CMV-24 vector (Sigma) at KpnI/XbaI sites. The following point mutations of the gene were introduced using site-directed mutagenesis (MutanBEST kit, TAKARA): 1) α-syn phosphomimic mutant (S129D) was created using primers 5′-GATGAGGAAGGGTATCAAGACTAC-3′ and 5′-AGGCATTTCATAAGCCTCATTGTC-3′; 2) α-syn phospho-dead mutant (S129A) was created using primers 5′-GCTGAGGAAGGGTATCAAGACTAC-3′ and 5′-AGGCATTTCATAAGCCTCATTGTC-3′; 3) Ub missense mutation K48R, using primers 5′-AGACAGCTGGAAGATGGACGCACC-3′ and 5′-CCCAGCAAAGATCAACCTCTGCTG-3′; 4) Ub missense mutation K63R, using primers 5′-AGAGAGTCCACCCTGCACCTGGTG-3′ and 5′-CTGGATGTTGTAGTCAGACAGGGT-3′. The fidelity of all constructs was confirmed by sequencing. Cell Culture and Transfection—Neuro2a cells cultured overnight in Dulbecco's modified Eagle's medium (DMEM) (Invitrogen) containing 10% newborn calf serum (Invitrogen) were washed with Opti-MEM and then transiently transfected with expressing plasmids using Lipofectamine™ 2000 reagent (Invitrogen) in Opti-MEM without serum. The same volume of DMEM containing 10% newborn calf serum was added to the culture media 6 h after transfection. Two days later, the transfected cells were observed by an inverted microscope (Olympus, IX71, Japan) or subjected to fluorescent observation, immunoblot, or coimmunoprecipitation analysis. Immunocytochemistry—Transfected cells grown in chamber slides were washed with PBS, fixed in PBS with 4% paraformaldehyde for 5 min, and then treated with 0.25% Triton X-100 for 10 min. After blocking in 4% BSA/PBS for 1 h, the cells were incubated with anti-FLAG antibody (1:5000, Sigma) for 1 h at room temperature. After washing with PBS, the cells were incubated with rhodamine-conjugated donkey anti-mouse antibodies (1:200, Santa Cruz Biotechnology) for 1 h at room temperature. Then the cells were washed with PBS and incubated with DAPI (Sigma) for 3 min at room temperature. Finally, the cells were observed using an inverted fluorescence microscope. Coimmunoprecipitation—Transfected cells were treated with 10 μm MG132 (Calbiochem, La Jolla, CA) for 12 h, 48 h after transfection. The cells were harvested and sonicated in modified TSPI buffer (50 mm Tris-HCl (pH 7.5), 150 mm sodium chloride, 1 mm EDTA, 1 μg/ml of aprotinin, 10 μg/ml of leupeptin, 0.5 μm Pefabloc SC, 10 μg/ml of pepstatin, and 1 mm phenylmethylsulfonyl fluoride) containing 1% Nonidet P-40. Cellular debris was removed by centrifugation at 16,000 × g for 30 min at 4 °C. The supernatants were preincubated with protein G-Sepharose (Roche) for 2 h at 4 °C and then incubated with monoclonal anti-GFP antibody (Roche) for 1 h at 4 °C. After incubation, protein G-Sepharose was used for precipitation. The beads were washed with TSPI buffer four times and then eluted with SDS sample buffer for immunoblot analysis. Immunoblot Analysis—Proteins were separated by SDS-PAGE and then transferred onto polyvinylidene difluoride membrane (Millipore Corporation, Bedford, MA). The following primary antibodies were used: monoclonal anti-GFP anti-body (1:5000, Roche), monoclonal anti-V5 antibody (1:500, Invitrogen), monoclonal anti-FLAG antibody (1:5000, Sigma), and monoclonal anti-α-tubulin antibody (1:200, Santa Cruz Biotechnology). A sheep anti-mouse IgG-horseradish peroxidase antibody (1:5000, Amersham Biosciences) was used as the secondary antibody. The proteins were visualized using an ECL detection kit (Amersham Biosciences). Fractionation Experiments—For Nonidet P-40 soluble or insoluble fractionation experiments, cells were lysed in a modified TSPI buffer. After sonication, cells were centrifuged at 16,000 × g for 30 min at 4 °C. Nonidet P-40-insoluble pellets were dissolved in a buffer containing 1% SDS, 1% Nonidet P-40. The soluble and insoluble fractions (dissolved by 1% SDS) were subjected to immunoblot analysis using the antibodies previously described. Aggregation Induction—Cells were transfected with various constructs. At 24 h after transfection, the DMEM containing 10% newborn calf serum was replaced with newborn calf serum-free DMEM containing 400 μm H2O2. Cells were cultured for a further 72 h, then washed with PBS, fixed in PBS with 4% paraformaldehyde for 5 min, and then treated with 0.25% Triton X-100 for 10 min at room temperature. After being washed with PBS, cells were incubated with DAPI (Sigma) for 3 min at 4 °C. After incubation, cells were washed with PBS and observed using an inverted fluorescence microscope. Characterization of WT α-Syn, S129D α-Syn, or β-Syn Expressed in Neuro2a Cells—It has been reported that almost 90% of α-syn in LBs is phosphorylated at Ser129 (13Fujiwara 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 (162) Google Scholar). To investigate the possible role of Ser129-phosphorylated α-syn in LB biogenesis, we created a mutant form of α-syn in which serine (S) 129 was converted to aspartate (D) to mimic phosphorylated α-syn (S129D) (Fig. 1A). To use enhanced green fluorescent protein (EGFP) as a reporter, we generated plasmids expressing EGFP-tagged WT α-syn, S129D α-syn or β-syn (Fig. 1A). To exclude the possible effects of EGFP on the α-syn conformation, we also created plasmids expressing V5-tagged WT α-syn or S129D α-syn (Fig. 1A, bottom). Neuro2a cells were transfected with EGFP, EGFP-tagged WT α-syn, S129D α-syn or β-syn. Cells overexpressing these EGFP-tagged proteins displayed a diffusive distribution in cytoplasm, nuclei, and neurites (Fig. 1, B-E). Forty-eight hours after transfection, cells were harvested and the total cell lysates were subjected to immunoblot analysis using anti-GFP antibody. Expression of all the constructs was confirmed by immunoblot analysis (Fig. 1J). Aggregation of these fusion proteins was not observed in transfected neuro2a cells. Transfection with V5-lacZ, V5-tagged WT, or S129D α-syn was also performed. Forty-eight hours after transfection, cells were harvested and the soluble and insoluble fractions dissolved with 1% SDS were subjected to immunoblot analysis using anti-V5 antibody (Fig. 1K). Aggregation of these fusion proteins was not observed in transfected neuro2a cells. Coexpression of S129D α-Syn and Ub Increases Ub-conjugates and Induces LB-like Structure Formation—To investigate whether the phosphorylation of α-syn at serine 129, which has been linked to the pathogenesis of PD, has effects on UPS, we examined the ubiquitinated proteins in neuro2a cells cotransfected with FLAG-Ub along with EGFP, EGFP-tagged WT α-syn or S129D α-syn. We also examined cells cotransfected with FLAG-Ub along with V5-lacZ, V5-tagged WT α-syn, or S129D α-syn. Forty-eight hours after transfection, the cells were analyzed by immunoblotting or immunofluorescence. Immunoblotting analysis revealed the smear bands of anti-FLAG immunoreactivity that represented the Ub-conjugates. In Nonidet P-40-soluble fractions, coexpression of WT or S129D α-syn with Ub resulted in more Ub-conjugates, especially in the high molecular weight, than coexpression of Ub and EGFP or V5-lacZ (Fig. 2, A and B). In Nonidet P-40-insoluble fractions, S129D α-syn resulted in more Ub-conjugates than WT α-syn (Fig. 2, A and B). As a comparison, the S129A α-syn mutant, where Ser129 was converted to alanine (A) to abolish phosphorylation at this site, was used. Coexpression of S129A α-syn and FLAG-Ub did not contribute to the Ub-conjugate formation compared with coexpression of FLAG-Ub and WT or S129D α-syn (supplemental Fig. S1). However, when using anti-GFP or anti-α-syn antibody, no low migrating bands, compatible with the ubiquitinated α-syn species, were detected in cell lysates (data not shown). The transfected cells were visualized by excitation of EGFP and immunochemistry analysis using the anti-FLAG antibody. As shown in Fig. 2, C-K, the number of ubiquitinated aggregates per Ub-positive cell was counted and subjected to data analysis. About 50% of Ub-positive cells, in the presence of EGFP and Ub, formed ubiquitinated aggregates. In the presence of WT α-syn and Ub, ubiquitinated aggregates were found in more than 60% of Ub-positive cells. Whereas in the presence of S129D α-syn and Ub, cells with ubiquitinated aggregates increased significantly to ∼80% of Ub-positive cells (Fig. 2L). The quantitative data showed that coexpression of S129D α-syn and Ub significantly increased the number of cells with three or more ubiquitinated aggregates as compared with coexpression of Ub and EGFP or WT α-syn (Fig. 2M). These results are consistent with our immunoblotting results suggesting that phosphorylated α-syn contributes to the accumulation of ubiquitinated proteins. Characterization of LB-like Structure Biogenesis Induced by Coexpression of S129D α-Syn and Ub—Neuro2a cells were cotransfected with EGFP-tagged S129D α-syn and FLAG-Ub. Forty-eight hours after transfection, EGFP-tagged S129D α-syn was visualized by excitation of EGFP and FLAG-Ub was visualized by immunocytochemistry analysis using anti-FLAG anti-body. There are different patterns of α-syn intracytoplasmic aggregation in cotransfected cells (Fig. 3, A-T). Some cells contained diffusive or "cloud-like" α-syn fluorescence (Fig. 3, A-D). Occasionally, this diffuse α-syn fluorescence showed a more intense aggregation or a greater tendency to stain an outer rim, whereas Ub was more likely to stain the core (Fig. 3, E-H). Colocalization of α-syn with Ub was found in both large and small aggregates scattered in the cytoplasm (Fig. 3, I-L). Two patterns of ubiquitinated inclusions were observed in neuro2a cells: one shows amorphous inclusion with Ub immunoreactivity and α-syn fluorescence (Fig. 3, M-P); another shows well defined colocalization of α-syn with Ub (Fig. 3, Q-T). Effects of S129D α-Syn on Ub-conjugates and LB-like Structure Formation Depend on Lysine 63 on Ub—Ubiquitination of specific proteins that target themselves to proteasomal degradation is believed to be dependent on the conjugation of the poly-Ub chain, which is linked through specific lysines on Ub (22Weissman A.M. Nat. Rev. Mol. Cell. Biol. 2001; 2: 169-178Crossref PubMed Scopus (1262) Google Scholar). The most prominent sites are the degradation related Lys48 and degradation unrelated Lys63 on Ub. To further investigate whether Lys48 or Lys63 on Ub is involved in the Ub-conjugate formation and why these conjugates escape from proteasomal degradation, we created two mutant forms of Ub on which lysine 48 or 63 was converted to arginine. We examined the ubiquitinated proteins in neuro2a cells cotransfected with K48R or K63R mutant FLAG-Ub together with EGFP, EGFP-tagged WT α-syn, or S129D α-syn for 48 h. Immunoblotting analysis using anti-FLAG antibody showed that coexpression of WT or S129D α-syn with the K48R mutant Ub both resulted in more poly-Ub-conjugates, especially in the high molecular weight, as compared with coexpression of EGFP and K48R mutant Ub (Fig. 4A). Furthermore, in Nonidet P-40 insoluble fractions, S129D α-syn resulted in more Ub-conjugates than WT α-syn. These results are similar to our observations in cells that were cotransfected with WT or S129D α-syn and wild type Ub (Fig. 2A). Coexpression of WT or S129D α-syn with K63R mutant Ub did not contribute to poly-Ub-conjugate formation in either Non-idet P-40-soluble or -insoluble fractions any more than coexpression of EGFP with K63R mutant Ub (Fig. 4B). To further ascertain whether Lys63 on Ub is involved in LB-like structure formation, neuro2a cells were cotransfected with K63R mutant FLAG-Ub along with EGFP, EGFP-tagged WT α-syn, or S129D α-syn. Forty-eight hours after transfection, the cells were visualized by excitation of EGFP and by immunochemistry analysis using anti-FLAG antibody. We found that both WT and S129D α-syn failed to increase the ubiquitinated aggregates or form ubiquitinated inclusions (Fig. 5, A-I). Similar results were observed in neuro2a cells cotransfected with K48R or K63R mutant FLAG-Ub along with V5-lacZ, V5-tagged WT α-syn, or S129D α-syn (Fig. 4, C and D). These results suggest that the contribution of S129D-phosphorylated α-syn to the Ub-conjugates and ubiquitinated inclusions depends on the Lys63 site not the Lys48 site on Ub.FIGURE 5Contribution of S129D α-syn to ubiquitinated inclusions depends upon Lys63 on Ub. A-I, neuro2a cells were cotransfected with K63R mutant FLAG-Ub along with EGFP, EGFP-tagged WT α-syn, or S129D α-syn. Forty-eight hours after transfection, cells were subjected to fluorescent observation. EGFP and EGFP-tagged proteins (A, D, and G, green) were visualized by excitation of EGFP. Immunocytochemistry analysis for FLAG-Ub (B, E, and H, red) was carried out with anti-FLAG antibody. The nuclei were stained with DAPI (C, F, and I, blue). Bar, 10 μm. These experiments were repeated three times with similar results.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Phosphorylation of α-Syn at Ser129 Increases Self-modification by Ub—Abundant deposition of α-syn and Ub in LBs may indicate an important role for α-syn-Ub linkage in the pathogenesis of LBs (9Spillantini M.G. Crowther R.A. Jakes R. Hasegawa M. Goedert M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6469-6473Crossref PubMed Scopus (2443) Google Scholar). However, using immunoprecipitation of GFP-tagged proteins followed by Western blotting with anti-FLAG antibody, we did not observe ubiquitinated α-syn in the soluble samples used in Figs. 2A and 4A (data not shown). To further explore a possible role for α-syn-Ub linkage in LB biogenesis, we examined the ubiquitination of α-syn within cells cotransfected with WT FLAG-Ub along with EGFP-tagged WT α-syn or S129D α-syn under the treatment of MG132. Neuro2a cells coexpressing β-syn and Ub were used as a control. Forty-eight hours after transfection, the cells were subsequently treated with MG132 (10 μm) for 12 h. After treatment, cells were harvested and a coimmunoprecipitation assay was performed using anti-GFP antibody. The immunoprecipitants were subjected to immunoblot analysis using anti-FLAG antibody. As shown in Fig. 6, low migrating smeared bands compatible with ubiquitinated α-syn were observed. Smeared bands compatible with ubiquitinated β-syn were not detected (Fig. 6). Based on the apparent molecular mass of α-syn-EGFP (≈47 kDa) and FLAG-Ub (≈18 kDa), the high molecular mass species accounted for as mono-, di-, or tri-FLAG-Ub modified α-syn appear predominantly at the phosphomimic mutant (indicated by arrows in Fig. 6). These results support our earlier findings in this study that α-syn is co-localized with Ub in some of the aggregates. Considering the large amount of Ub-conjugates formed by the expression of S129D α-syn (Fig. 2, A and B), these results also suggest that phosphorylation of α-syn at Ser129 may facilitate the ubiquitination of other proteins as well as itself. Expression of S129D α-Syn Increases α-Syn Aggregate Formation under Oxidative Stress and Serum Deprivation—Lewy neurites, also known as dystrophic neurites, are one of the pathological characteristics of synucleinopathies. We therefore cultured neuro2a cells expressing EGFP, EGFP-tagged WT, or S129D α-syn in serum-free DMEM 24 h after transfection. Seventy-two hours later, the cells were observed using an inverted system microscope (Olympus, IX71, Japan). We observed that some of the surviving intact cells developed long neurites that are similar with the dystrophic neurites in morphous. It has been proposed that oxidative stress is associated with the pathogenesis of PD. A previous study suggests that oxidative stress induces the formation of α-syn inclusions (29Smith W.W. Margolis R.L. Li X. Troncoso J.C. Lee M.K. Dawson V.L. Dawson T.M. Iwatsubo T. Ross C.A. J. Neurosci. 2005; 25: 5544-5552Crossref PubMed Scopus (212) Google Scholar). To explore whether phosphorylation of α-syn affects self-aggregation under oxidative stress, neuro2a cells were transfected with EGFP, EGFP-tagged WT, or S129D α-syn for 24 h, followed by deprivation of serum and treatment with or without exposure to oxidative stress. Seventy-two hours later, the cells were observed using an inverted system microscope. The surviving cells with intact nuclei were selected for data analysis. We found that neuro2a cells overexpressing EGFP, EGFP-tagged WT, or S129D α-syn formed a few aggregates in the absence of serum (Fig. 7, A-D) with no statistic significance among the cells by quantitative data (Fig. 7J). However, oxidative stress induced the cytoplasmic and neuritic aggregates of α-syn in surviving neuro2a cells (Fig. 7, E-I) with cells overexpressing S129D α-syn forming approximately two times more α-syn aggregates than cells overexpressing WT α-syn (Fig. 7J). These results suggest that Ser129-phosphorylated α-syn may be more prone to aggregation under oxidative stress. Typically, LBs are found in dopaminergic neurons in the substantia nigra pars compacta, but they are also observed in noradrenergic neurons in other brain regions and the periphe
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