Wild Type α-Synuclein Is Degraded by Chaperone-mediated Autophagy and Macroautophagy in Neuronal Cells
2008; Elsevier BV; Volume: 283; Issue: 35 Linguagem: Inglês
10.1074/jbc.m801992200
ISSN1083-351X
AutoresTereza Vogiatzi, Maria Xilouri, Kostas Vekrellis, Leonidas Stefanis,
Tópico(s)Nerve injury and regeneration
Resumoα-Synuclein (ASYN) is crucial in Parkinson disease (PD) pathogenesis. Increased levels of wild type (WT) ASYN expression are sufficient to cause PD in humans. The manner of post-transcriptional regulation of ASYN levels is controversial. Previously, we had shown that WT ASYN can be degraded by chaperone-mediated autophagy (CMA) in isolated liver lysosomes. Whether this occurs in a cellular and, in particular, in a neuronal cell context is unclear. Using a mutant ASYN form that lacks the CMA recognition motif and RNA interference against the rate-limiting step in the CMA pathway, Lamp2a, we show here that CMA is indeed involved in WT ASYN degradation in PC12 and SH-SY5Y cells, and in primary cortical and midbrain neurons. However, the extent of involvement varies between cell types, potentially because of differences in compensatory mechanisms. CMA inhibition leads to an accumulation of soluble high molecular weight and detergent-insoluble species of ASYN, suggesting that CMA dysfunction may play a role in the generation of such aberrant species in PD. ASYN and Lamp2a are developmentally regulated in parallel in cortical neuron cultures and in vivo in the central nervous system, and they physically interact as indicated by co-immunoprecipitation. In contrast to previous reports, inhibition of macroautophagy, but not the proteasome, also leads to WT ASYN accumulation, suggesting that this lysosomal pathway is also involved in normal ASYN turnover. These results indicate that CMA and macroautophagy are important pathways for WT ASYN degradation in neurons and underline the importance of CMA as degradation machinery in the nervous system. α-Synuclein (ASYN) is crucial in Parkinson disease (PD) pathogenesis. Increased levels of wild type (WT) ASYN expression are sufficient to cause PD in humans. The manner of post-transcriptional regulation of ASYN levels is controversial. Previously, we had shown that WT ASYN can be degraded by chaperone-mediated autophagy (CMA) in isolated liver lysosomes. Whether this occurs in a cellular and, in particular, in a neuronal cell context is unclear. Using a mutant ASYN form that lacks the CMA recognition motif and RNA interference against the rate-limiting step in the CMA pathway, Lamp2a, we show here that CMA is indeed involved in WT ASYN degradation in PC12 and SH-SY5Y cells, and in primary cortical and midbrain neurons. However, the extent of involvement varies between cell types, potentially because of differences in compensatory mechanisms. CMA inhibition leads to an accumulation of soluble high molecular weight and detergent-insoluble species of ASYN, suggesting that CMA dysfunction may play a role in the generation of such aberrant species in PD. ASYN and Lamp2a are developmentally regulated in parallel in cortical neuron cultures and in vivo in the central nervous system, and they physically interact as indicated by co-immunoprecipitation. In contrast to previous reports, inhibition of macroautophagy, but not the proteasome, also leads to WT ASYN accumulation, suggesting that this lysosomal pathway is also involved in normal ASYN turnover. These results indicate that CMA and macroautophagy are important pathways for WT ASYN degradation in neurons and underline the importance of CMA as degradation machinery in the nervous system. α-Synuclein (ASYN) 3The abbreviations used are:ASYNα-synucleinCMAchaperone-mediated autophagydoxdoxycyclineLamplysososome-associated membrane proteinRNAiRNA interferenceWTwild type3-MA3-methyladenineEGFPenhanced green fluorescent proteinPBSphosphate-buffered salineBisTris2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diolFBSfetal bovine serumRTreverse transcriptionsiRNAsmall interfering RNACNScentral nervous systemANOVAanalysis of variancescrscrambledGAPDHglyceraldehyde-3-phosphate dehydrogenaseshRNAsmall hairpin RNAm.o.i.multiplicity of infectionepxepoxomicinERKextracellular signal-regulated kinaseAbantibodyTHtyrosine hydroxylase. 3The abbreviations used are:ASYNα-synucleinCMAchaperone-mediated autophagydoxdoxycyclineLamplysososome-associated membrane proteinRNAiRNA interferenceWTwild type3-MA3-methyladenineEGFPenhanced green fluorescent proteinPBSphosphate-buffered salineBisTris2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diolFBSfetal bovine serumRTreverse transcriptionsiRNAsmall interfering RNACNScentral nervous systemANOVAanalysis of variancescrscrambledGAPDHglyceraldehyde-3-phosphate dehydrogenaseshRNAsmall hairpin RNAm.o.i.multiplicity of infectionepxepoxomicinERKextracellular signal-regulated kinaseAbantibodyTHtyrosine hydroxylase. is central in Parkinson disease (PD) pathogenesis. Point mutations in the gene encoding ASYN, as well as multiplications of the gene locus, are identified in rare cases of familial PD (1Kruger 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 (3235) Google Scholar, 2Polymeropoulos 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 (6441) Google Scholar, 3Singleton A. Gwinn-Hardy K. Lancet. 2004; 364: 1105-1107Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). Genetic polymorphic variants within the ASYN locus that may be associated with increased production of ASYN, confer an increased risk for sporadic PD (4Maraganore D.M. de Andrade M. Elbaz A. Farrer M.J. Ioannidis J.P. Kruger R. Rocca W.A. Schneider N.K. Lesnick T.G. Lincoln S.J. Hulihan M.M. Aasly J.O. Ashizawa T. Chartier-Harlin M.C. Checkoway H. Ferrarese C. Hadjigeorgiou G. Hattori N. Kawakami H. Lambert J.C. Lynch T. Mellick G.D. Papapetropoulos S. Parsian A. Quattrone A. Riess O. Tan E.K. Van Broeckhoven C. J. Am. Med. Assoc. 2006; 296: 661-670Crossref PubMed Scopus (424) Google Scholar). These data suggest that modulation of wild type (WT) ASYN levels is critical for PD pathogenesis. Control of protein levels is in part achieved by differential degradation that modulates cellular protein half-life. The subject of ASYN degradation is controversial. α-synuclein chaperone-mediated autophagy doxycycline lysososome-associated membrane protein RNA interference wild type 3-methyladenine enhanced green fluorescent protein phosphate-buffered saline 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol fetal bovine serum reverse transcription small interfering RNA central nervous system analysis of variance scrambled glyceraldehyde-3-phosphate dehydrogenase small hairpin RNA multiplicity of infection epoxomicin extracellular signal-regulated kinase antibody tyrosine hydroxylase. α-synuclein chaperone-mediated autophagy doxycycline lysososome-associated membrane protein RNA interference wild type 3-methyladenine enhanced green fluorescent protein phosphate-buffered saline 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol fetal bovine serum reverse transcription small interfering RNA central nervous system analysis of variance scrambled glyceraldehyde-3-phosphate dehydrogenase small hairpin RNA multiplicity of infection epoxomicin extracellular signal-regulated kinase antibody tyrosine hydroxylase. Initial studies showed that ASYN accumulated in cells upon proteasomal inhibition, suggesting that the proteasome was responsible for ASYN degradation (5Bennett M.C. Bishop J.F. Leng Y. Chock P.B. Chase T.N. Mouradian M.M. J. Biol. Chem. 1999; 274: 33855-33858Abstract Full Text Full Text PDF PubMed Scopus (382) Google Scholar, 6Imai Y. Soda M. Takahashi R. J. Biol. Chem. 2000; 275: 35661-35664Abstract Full Text Full Text PDF PubMed Scopus (645) Google Scholar). This has also been shown in more recent work (7Webb J.L. Ravikumar B. Atkins J. Skepper J.N. Rubinsztein D.C. J. Biol. Chem. 2003; 278: 25009-25013Abstract Full Text Full Text PDF PubMed Scopus (1122) Google Scholar, 8Tofaris G.K. Layfield R. Spillantini M.G. FEBS Lett. 2001; 509: 22-26Crossref PubMed Scopus (324) Google Scholar). Other studies however, including our own, failed to detect accumulation of endogenous or overexpressed ASYN with proteasomal inhibition (9Ancolio K. Alves da Costa C. Ueda K. Checler F. Neurosci. Lett. 2000; 285: 79-82Crossref PubMed Scopus (103) Google Scholar, 10Rideout H.J. Larsen K.E. Sulzer D. Stefanis L. J. Neurochem. 2001; 78: 899-908Crossref PubMed Scopus (251) Google Scholar, 11Rideout H.J. Stefanis L. Mol. Cell. Neurosci. 2002; 21: 223-238Crossref PubMed Scopus (114) Google Scholar, 12Paxinou E. Chen Q. Weisse M. Giasson B.I. Norris E.H. Rueter S.M. Trojanowski J.Q. Lee V.M. Ischiropoulos H. J. Neurosci. 2001; 21: 8053-8061Crossref PubMed Google Scholar). In contrast, ASYN appeared to accumulate with general lysosomal inhibition (12Paxinou E. Chen Q. Weisse M. Giasson B.I. Norris E.H. Rueter S.M. Trojanowski J.Q. Lee V.M. Ischiropoulos H. J. Neurosci. 2001; 21: 8053-8061Crossref PubMed Google Scholar, 13Cuervo A.M. Stefanis L. Fredenburg R. Lansbury P.T. Sulzer D. Science. 2004; 305: 1292-1295Crossref PubMed Scopus (1511) Google Scholar). In lysosomes, degradation of cytoplasmic components is achieved through distinct types of autophagic pathways as follows: chaperone-mediated autophagy (CMA), microautophagy, and macroautophagy (14Levine B. Klionsky D.J. Dev. Cell. 2004; 6: 463-477Abstract Full Text Full Text PDF PubMed Scopus (3103) Google Scholar, 15Cuervo A.M. Trends Cell Biol. 2004; 14: 70-77Abstract Full Text Full Text PDF PubMed Scopus (699) Google Scholar). CMA involves the selective targeting of proteins containing a KFERQ peptide motif to lysosomes. This requires binding to the lysosomal receptor, Lamp2a, the rate-limiting step in CMA (16Cuervo A.M. Dice J.F. Science. 1996; 273: 501-503Crossref PubMed Scopus (679) Google Scholar, 17Majeski A.E. Dice J.F. Int. J. Biochem. Cell Biol. 2004; 36: 2435-2444Crossref PubMed Scopus (301) Google Scholar, 18Massey A. Kiffin R. Cuervo A.M. Int. J. Biochem. Cell Biol. 2004; 36: 2420-2434Crossref PubMed Scopus (149) Google Scholar). Microautophagy involves the pinocytosis of small quantities of cytosol directly by lysosomes (18Massey A. Kiffin R. Cuervo A.M. Int. J. Biochem. Cell Biol. 2004; 36: 2420-2434Crossref PubMed Scopus (149) Google Scholar, 19Muller O. Sattler T. Flotenmeyer M. Schwarz H. Plattner H. Mayer A. J. Cell Biol. 2000; 151: 519-528Crossref PubMed Scopus (136) Google Scholar). Macroautophagy involves the sequestration of cytosolic regions into autophagosomes that deliver their contents to late endosomal and lysosomal compartments for degradation (20Shintani T. Klionsky D.J. Science. 2004; 306: 990-995Crossref PubMed Scopus (2110) Google Scholar). Direct assessment of the contribution of macroautophagy to WT ASYN degradation was performed in two studies using the selective macroautophagy inhibitor 3-methyladenine (3-MA). In both cases (7Webb J.L. Ravikumar B. Atkins J. Skepper J.N. Rubinsztein D.C. J. Biol. Chem. 2003; 278: 25009-25013Abstract Full Text Full Text PDF PubMed Scopus (1122) Google Scholar, 8Tofaris G.K. Layfield R. Spillantini M.G. FEBS Lett. 2001; 509: 22-26Crossref PubMed Scopus (324) Google Scholar), 3-MA application failed to enhance ASYN levels, suggesting the lack of involvement of macroautophagy in normal ASYN turnover. ASYN contains the pentapeptide motif KFERQ that could target it to the CMA pathway. Analysis in an in vitro system of purified liver lysosomes confirmed that ASYN can be degraded by CMA. Coupled with cellular data, which indicated that rat ASYN is degraded in ventral midbrain cultures by a lysosomal pathway, we had proposed that CMA may be the major pathway used for WT ASYN degradation (13Cuervo A.M. Stefanis L. Fredenburg R. Lansbury P.T. Sulzer D. Science. 2004; 305: 1292-1295Crossref PubMed Scopus (1511) Google Scholar). However, there is no direct proof that CMA is responsible for ASYN degradation in cells, and in particular neuronal cells; rather this hypothesis is based on the in vitro data with purified liver lysosomes and the exclusion of other degradation pathways. Therefore, molecular techniques targeted specifically toward CMA are needed to prove or disprove the hypothesis that CMA represents a major route for WT ASYN degradation in cellular systems. Ideally, such experiments should be performed in neuronal cells, and particularly those that are most relevant to PD. This is all the more prescient, as the rate-limiting step in CMA, the levels of Lamp2a, have been reported to be very low in the CNS (21Konecki D.S. Foetisch K. Zimmer K.P. Schlotter M. Lichter-Konecki U. Biochem. Biophys. Res. Commun. 1995; 215: 757-767Crossref PubMed Scopus (84) Google Scholar, 22Furuta K. Yang X.L. Chen J.S. Hamilton S.R. August J.T. Arch. Biochem. Biophys. 1999; 365: 75-82Crossref PubMed Scopus (46) Google Scholar). Accordingly, we have undertaken the present study to ascertain whether CMA is indeed responsible for WT ASYN degradation in neuronal cells. Because of the controversy surrounding the issues of proteasomal and macroautophagy-dependent degradation of WT ASYN, we have also examined the contribution of these pathways in various neuronal cell culture systems. Animals—Wistar rats and wild type control or double transgenic C57BI/C3H mice expressing human A53T ASYN under the control of the prion promoter were used. The generation and phenotype of these mice have been described previously (23Giasson B.I. Duda J.E. Quinn S.M. Zhang B. Trojanowski J.Q. Lee V.M. Neuron. 2002; 34: 521-533Abstract Full Text Full Text PDF PubMed Scopus (887) Google Scholar). The mice were purchased from The Jackson Laboratories (Bar Harbor, ME) and were housed in the animal facility of the Biomedical Research Foundation of the Academy of Athens in a room with a controlled light-dark cycle (12 h light-12 h dark) and free access to food and water. For immunoprecipitation and Western immunoblotting experiments, the animals were sacrificed by cervical dislocation; brains were harvested, dissected on ice to obtain the region of interest, and immediately frozen. All animals were processed in a similar manner. Tissue was stored at –80 °C until further use. Genotyping was performed by quantitative Southern dot blot analysis with a 32P-labeled oligonucleotide-primed ASYN DNA probe as described previously (23Giasson B.I. Duda J.E. Quinn S.M. Zhang B. Trojanowski J.Q. Lee V.M. Neuron. 2002; 34: 521-533Abstract Full Text Full Text PDF PubMed Scopus (887) Google Scholar). All efforts were made to minimize animal suffering and to reduce the number of the animals used, according to the European Communities Council Directive (86/609/EEC) guidelines for the care and use of laboratory animals. All animal experiments were approved by the Institutional Animal Care and Use Committee of Biomedical Research Foundation of the Academy of Athens. Generation of Stable Cell Lines and Transfections—We generated the ΔDQ mutation in the ASYN open reading frame by substituting the amino acids DQ at positions 98–99 with AA, using PCR-based site-directed mutagenesis, as described previously (13Cuervo A.M. Stefanis L. Fredenburg R. Lansbury P.T. Sulzer D. Science. 2004; 305: 1292-1295Crossref PubMed Scopus (1511) Google Scholar). Human ASYN in pcDNA3 was used as template (24Stefanis L. Larsen K.E. Rideout H.J. Sulzer D. Greene L.A. J. Neurosci. 2001; 21: 9549-9560Crossref PubMed Google Scholar). Naive SH-SY5Y cells were transfected overnight with the Tet-Off vector (10 μg) (Clontech) using the Lipofectamine 2000 reagent (Invitrogen). Selection was performed with 500 μg/ml G418 (Calbiochem). Growth medium was changed every 2 days. G418-resistant colonies were picked. Inducibility of each clone was determined by transient transfection of a pTRE-LUC vector, in the presence or absence of doxycycline (dox, 2 μg/ml) TET-approved medium (Clontech). Two clones were finally chosen for tight regulation. Maintenance of the clones was in 250 μg/ml G418. One of the clones was further used for generation of stable pTRE-ASYN expression as described below. Mutant ΔDQ/WT ASYN was generated by PCR from human WT ASYN. PCR products were then cloned into a TOPO-pCRII vector (Invitrogen). WT, ΔDQ/WT ASYN were subcloned into the HindIII and XbaI sites of the pTRE-tight vector (Clontech) and transfected along with the pTK-hygromycin vector (Clontech) using Lipofectamine 2000, following the manufacturer's recommendations. Selection was with 200 μg/ml G418 and 25 μg/ml (for PC12 cells) or 50 μg/ml (for SH-SY5Y cells) hygromycin B (Roche Diagnostics). Single colonies were isolated and maintained in medium with/without dox (2 μg/ml for PC12 or 3 μg/ml for SH-SY5Y cells) for 4 days. The clones were tested for ASYN expression by immunocytochemistry and Western blot analysis. Resistant clones were picked, and ASYN inducibility was examined by immunofluorescence in the presence or absence of dox (2 μg/ml added for 4 days) in Tet-Off approved medium using the monoclonal antibody Syn 211 sc-12767 (Santa Cruz Biotechnology, Santa Cruz, CA). Clones with the tightest regulation were further confirmed by Western immunoblotting with the same antibody. Cell Culture—PC12 cells were cultured in RPMI 1640 medium (Invitrogen) with 10% horse serum (Biowest, Nuaillé, France) and 5% fetal bovine serum (FBS, Biowest, France) on rat tail collagen-coated plates. SH-SY5Y cells were cultured in RPMI 1640 medium with 10% FBS. Stable cell lines were co-cultured with 200 μg/ml G418 and 25 μg/ml (for PC12 cells) or 50 μg/ml (for SH-SY5Y cells) hygromycin B. For pharmacological studies, 3-methyladenine (3-MA, Sigma), NH4Cl (Sigma), epoxomicin (epx, Sigma), and dox (Clontech) were added at indicated times and concentrations. Serum deprivation in SH-SY5Y cells was in RPMI 1640 medium + 0.5% FBS. All plasticware was from Greiner (Greiner, Bio One GmbH, Germany). RNAi—A 21-nucleotide, small interfering RNA was designed against the rat Lamp2a mRNA (GenBank™ accession number NM_017068) according to the criteria of Elbashir et al. (25Elbashir S.M. Harborth J. Lendeckel W. Yalcin A. Weber K. Tuschl T. Nature. 2001; 411: 494-498Crossref PubMed Scopus (8018) Google Scholar) and Reynolds et al. (26Reynolds A. Leake D. Boese Q. Scaringe S. Marshall W.S. Khvorova A. Nat. Biotechnol. 2004; 22: 326-330Crossref PubMed Scopus (1612) Google Scholar). The nucleotide sequence targeting rat Lamp2a was 5′-AAGCGCCATCATACTGGATAT-3′ (L1) and was subjected to a BLAST search to verify specificity. As a control, we used a scrambled (scr) siRNA containing the 21-nucleotide sequence 5′-AATTTAGCCGATACTGCCTAG-3′. Briefly, PC12 cells were grown in 12-well dishes, and siRNAs (L1 and scr) at a concentration of 25 nm were delivered with Lipofectamine 2000 (Invitrogen) following the manufacturer's instructions. Six hours later, the medium was removed and replaced with normal culture medium. Lamp2a down-regulation was assessed 24 and 48 h post-transfection. Primary Neuronal Cultures—Cultures of rat (embryonic day 18, E18) or mouse (E16) cortical neurons were prepared as described previously (27Stefanis L. Park D.S. Friedman W.J. Greene L.A. J. Neurosci. 1999; 19: 6235-6247Crossref PubMed Google Scholar, 28Dietrich P. Rideout H.J. Wang Q. Stefanis L. Mol. Cell. Neurosci. 2003; 24: 430-441Crossref PubMed Scopus (37) Google Scholar). Dissociated cells were plated onto poly-d-lysine-coated 6-well or 12-well dishes at a density of ∼150,000–200,000/cm2. Cells were maintained in Neurobasal medium (Invitrogen), with B27 serum-free supplements (Invitrogen), l-glutamine (0.5 mm), and penicillin/streptomycin (1%). More than 98% of the cells cultured under these conditions represent post-mitotic neurons (11Rideout H.J. Stefanis L. Mol. Cell. Neurosci. 2002; 21: 223-238Crossref PubMed Scopus (114) Google Scholar). Rat midbrain cultures derived from postnatal day 1 were prepared using standard procedures (13Cuervo A.M. Stefanis L. Fredenburg R. Lansbury P.T. Sulzer D. Science. 2004; 305: 1292-1295Crossref PubMed Scopus (1511) Google Scholar) with modifications. Briefly, material dissected form the ventral portion of the midbrain was cleaned free of meningeal tissue, minced, and enzymatically dissociated in a mixture of trypsin/DNase. Dissociated cells were plated at a density of ∼200,000 cells per cm2 on poly-d-lysine-coated plates. The neurons were maintained in neurobasal A medium with B27 serum-free supplements. Intracellular Protein Degradation—Total protein degradation in cultured cells (PC12 cells, cortical neurons) was measured by pulse-chase experiments (13Cuervo A.M. Stefanis L. Fredenburg R. Lansbury P.T. Sulzer D. Science. 2004; 305: 1292-1295Crossref PubMed Scopus (1511) Google Scholar, 29Franklin J.L. Johnson E.M. J. Cell Biol. 1998; 142: 1313-1324Crossref PubMed Scopus (43) Google Scholar) with modifications. Briefly, confluent PC12 cells or cortical neurons (day 7 in culture) were labeled with [3H]leucine (2 μCi/ml) (leucine, L-3,4,5; PerkinElmer Life Sciences) at 37 °C for 48 or 24 h respectively. The cultures were then extensively washed with medium and returned in complete growth medium containing 2 mm of unlabeled leucine for 6 h. This medium containing mainly short lived proteins was removed and replaced with fresh medium containing cold leucine, and/or the general lysosomal inhibitor NH4Cl (30Hart P.D. Young M.R. J. Exp. Med. 1991; 174: 881-889Crossref PubMed Scopus (123) Google Scholar), or the inhibitor of macroautophagy 3-MA (31Seglen P.O. Gordon P.B. Proc. Natl. Acad. Sci. U. S. A. 1982; 79: 1889-1892Crossref PubMed Scopus (1159) Google Scholar) at indicated concentrations. Aliquots of the medium were taken at different times (14 h for PC12 cells and 12 and 24 h after labeling for cortical neurons), and proteins in the medium were precipitated with 20% trichloroacetic acid for 20 min on ice and centrifuged (10,000 × g, 10 min, 4 °C). Radioactivity in the supernatant (representing degraded proteins) and pellet (representing undegraded proteins) was measured in a liquid scintillation counter (Wallac T414, PerkinElmer Life Sciences). At the last time point, cells were lysed with 0.1% NaOH. Proteolysis was expressed as the percentage of the initial total acid-precipitable radioactivity (protein) in the cell lysates transformed to acid-soluble radioactivity (amino acids and small peptides) in the medium during the incubation. Total radioactivity incorporated in cellular proteins was determined in triplicate samples as the amount of acid-precipitable radioactivity in labeled cells. Measurement of Half-lives of WT (Endogenous and Over-expressed) and ΔDQ/WT Synucleins—85% confluent cell cultures were grown in methionine/cysteine-deprived RPMI 1640 medium (Sigma) for 10 min and then labeled with [35S]methionine/cysteine mixture (0.2 mCi/ml) (Express Labeling Mix, PerkinElmer Life Sciences) for 2 h. For cortical neurons the radiolabeling was performed for 24 h in Neurobasal medium without previous amino acid starvation. After extensive washing with medium, cells were maintained in complete or serum-deprived medium (0.5% FBS) and at the indicated times lysed in RIPA buffer (150 mm NaCl, 50 mm Tris, pH 7.6, 0.1% SDS, 1% Triton X-100, 2 mm EDTA, and 0.1% deoxycholate) with protease inhibitors (complete mini, Roche Diagnostics) and subjected to immunoprecipitation with an antibody against ASYN. The antibodies used were the sc-7011 (C-20) rabbit polyclonal antibody (Santa Cruz Biotechnology) for PC12 and SH-SY5Y cells and the monoclonal Syn 1 antibody (BD Biosciences) for cortical cultures. Protein G+-agarose beads were from Santa Cruz Biotechnology. Immunoprecipitates were resolved by SDS-PAGE (12%), and the gels were dried and then exposed on a PhosphorImager Screen and quantified using Gel Analyzer version 1.0 software (Biosure, Greece). Western Immunoblotting—PC12 cells, SH-SY5Y cells, and primary neurons were washed twice in cold PBS and then harvested in lysis buffer (150 mm NaCl, 50 mm Tris, pH 7.6, 0.1% SDS, 1% Triton X-100, 2 mm EDTA) with protease inhibitors. Lysates were centrifuged at 10,000 × g for 10 min at 4 °C. The detergent-insoluble pellets were washed twice in PBS and resuspended in 2× Laemmli buffer. Protein concentrations in soluble fractions were determined using the Bradford or Lowry methods (Bio-Rad). Proteins were resolved on 12% SDS-polyacrylamide gels or 4–12% BisTris NuPAGE gels (Invitrogen) and transferred onto nitrocellulose membranes. Blots were probed with antibodies directed against the following: 1) ASYN, monoclonal Syn 1 (1:1000; BD Biosciences), polyclonal C20 (1:1000; Santa Cruz Biotechnology); 2) polyclonal Lamp2a (Igp96), (1:1000; Zymed Laboratories Inc.); 3) monoclonal Lamp1 (1:1000; Santa Cruz Biotechnology); 4) polyclonal ERK (loading control; 1:5000; Santa Cruz Biotechnology); 5) monoclonal GAPDH (1:1000; Chemicon); 6) monoclonal ubiquitin (1:750; Chemicon); 7) monoclonal GFP (1:500, Santa Cruz Biotechnology); 8) monoclonal β-actin (1:20,000; Sigma); and 9) polyclonal TH (1:1000, Chemicon). Blots were probed with horseradish peroxidase-conjugated secondary antibodies (Jackson ImmunoResearch), visualized with Western Lightning® (PerkinElmer Life Sciences), and exposed to Super RX film (Fuji Film). After scanning the images, the intensity of each immunoreactive band was estimated by densitometric quantification using the Gel Analyzer version 1.0 software. Immunocytochemistry—Cortical or ventral midbrain neurons grown on 24-well plates were fixed in freshly prepared 3.7% formaldehyde for 45 min. Blocking was with 10% normal goat serum, 0.4% Triton X-100 for 1 h at room temperature. Mouse anti-ASYN (1:200; BD Biosciences) and rabbit anti-tyrosine hydroxylase (TH, 1:500; Calbiochem) antibodies were applied overnight at 4 °C. Fluorescent secondary antibodies (mouse Cy3, 1:250; rabbit Cy2, 1:150, Jackson ImmunoResearch) were added for 1 h. The fluorescent marker Hoechst 33258 (1 μm; Sigma) was used to assess cell nuclei. RT-PCR—Total RNA was extracted from cortical neurons 48 and 72 h after infection with the lentiviruses bearing the L1 or the scrambled siRNAs using TRIzol (Invitrogen), and cDNA was generated with the reverse transcription system (Invitrogen), according to the manufacturer's instructions. RT-PCR was performed using the cDNA as template. All primer pairs were optimized to be in the log -exponential phase of amplification. The following primers were used: 1) ASYN-forward, ttctgcggaagcctagagag, and ASYN-reverse, tcctccaacatttgtcacttgc (product size = 253 bp); 2) β-actin-forward, tcaccatggatgatgatatcgcc, and β-actin-reverse, ccacacgcagctcattgtagaagg (product size = 282 bp); all primers were from Clough and Stefanis (32Clough R.L. Stefanis L. FASEB J. 2007; 21: 596-607Crossref PubMed Scopus (42) Google Scholar). Products were subsequently resolved on 1% agarose gels and stained with ethidium bromide, and the signal intensity was quantified using Gel Analyzer version 1.0 software. Design of siRNAs and Cloning of Small Hairpin RNAs—A stem-loop structure incorporating the 21-nucleotide targeting rat Lamp2a sequence was created based on Rubinson et al. (33Rubinson D.A. Dillon C.P. Kwiatkowski A.V. Sievers C. Yang L. Kopinja J. Rooney D.L. Zhang M. Ihrig M.M. McManus M.T. Gertler F.B. Scott M.L. Van Parijs L. Nat. Genet. 2003; 33: 401-406Crossref PubMed Scopus (1336) Google Scholar), so that small hairpin RNA (shRNA) could be produced from the lentiviral vector PLL3.7 gift from Dr. Dimitrios Thanos (Laboratory for Molecular Biology, Biomedical Research Foundation of the Academy of Athens). Complementary oligonucleotides encoding the shRNAs were synthesized, annealed, and cloned into pLL3.7 vector by ligation into HpaI- and XhoI-digested vector (L1 vector). The pLL3.7 vector carries loxP sites, a cytomegalovirus promoter driving expression of EGFP, and the mouse U6 promoter with downstream restriction sites (HpaI and XhoI) to allow efficient introduction of oligonucleotides encoding shRNAs. Lentivirus Production—The lentiviruses were generated by co-transfection of human embryonic kidney (HEK) 293T cells with three plasmids using the calcium phosphate method (34Dull T. Zufferey R. Kelly M. Mandel R.J. Nguyen M. Trono D. Naldini L. J. Virol. 1998; 72: 8463-8471Crossref PubMed Google Scholar). We cultured HEK 293T cells in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% FBS and 1% penicillin/streptomycin. For transfection, 107 cells were plated into 150-cm2 flasks. The next day, the 293T cells were transfected with 50 μg of vector DNA (PLL3.7-L1, PLL3.7-scr), 17.5 μgof pMDG2 (env), and 32.5 μg of R8 91 (gag/pol) plasmids, using calcium-phosphate precipitation (all the plasmids for lentiviral vector packaging were kindly provided by Dr. Dimitrios Thanos). After 16 h, the medium was removed, and the cells were washed twice with PBS and returned to the normal culture medium. Medium containing recombinant lentivectors was collected at 24, 48, and 72 h post-transfection and centrifuged (400 × g, 10 min, 4 °C) to remove cellular debris. After filtration through 0.45-μm filter unit (Millipore), the supernatant from each time point was centrifuged at 75,000 × g or 90 min at 4 °C in Sorvall Discovery TH641 swing bucket rotor. The supernatant was discarded, and the virus (pellet) was resuspended in 50 μl/tube of PBS supplemented with 0.5% bovine serum albumin, aliquoted, and stored at –80 °C. Lentiviral titers for the viruses collected each day were determined by seeding HeLa cells in 12-well plates at 5 × 104 cells per well, 3–4 h before infection with serial dilutions of the concentrated viral stock. After incubation for 2 days, the medium was removed, and the EGFP-expressing cells were identified using a fluorescence-activated cell sorter. Titers ranged from 3 to 6 × 107 infectious units (IU/m
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