title: Crosstalk between the proteasome system and autophagy in the clearance of α-synuclein.
abstract: OBJECTIVE
  A growing body of evidence suggests that α-synuclein accumulation may play an important role in the pathogenesis of Parkinson's disease. The aim of this study was to investigate the roles of the proteasome and autophagy pathways in the clearance of wild-type and mutant α-synuclein in PC12 cells.
  
  
  METHODS
  PC12 cells overexpressing either wild-type or A30P mutant α-synuclein were treated with the proteasome inhibitor epoxomicin, the macroautophagy inhibitor 3-MA and the macroautophagy activator rapamycin alone or in combination. The cell viability was assessed using MTT assay. Immunofluorescence and Western blot analysis were used to detect the level of α-synuclein, LAMP-2A, E1 activase, and E2 ligase in the cells. Chymotrypsin-like proteasomal activity was measured using a commercial kit.
  
  
  RESULTS
  When the proteasome and macroautophagy in the wild-type and mutant cells were inhibited with epoxomicin and 3-MA, respectively, the cell viability was significantly decreased, and the α-synuclein level was increased. Both epoxomicin and 3-MA activated the chaperone-mediated autophagy (CMA) by increasing the level of the CMA-limiting enzyme LAMP-2A. Furthermore, 3-MA or epoxomicin significantly decreased chymotrypsin-like proteasomal activity. 3-MA or epoxomicin did not change E1 activase expression in either mutant or wild-type cells, but increased E2 ligase expression, especially when used together. Macroautophagy inducer rapamycin increased the cell viability and reduced epoxomicin-induced α-synuclein accumulation. Interestingly, CMA was also activated by rapamycin.
  
  
  CONCLUSIONS
  Our results demonstrate the existence of complex crosstalk between different forms of autophagy and between autophagy and the proteasome pathway in the clearance of α-synuclein in PC12 cells.
authors.affiliation: Department of Neurology, Second Affiliated Hospital of Soochow University, Suzhou 215004, China.
authors.forename: Fang
authors.initials: F
authors.lastname: Yang
authors.forename: Ya-ping
authors.initials: YP
authors.lastname: Yang
authors.forename: Cheng-jie
authors.initials: CJ
authors.lastname: Mao
authors.forename: Ling
authors.initials: L
authors.lastname: Liu
authors.forename: Hui-fen
authors.initials: HF
authors.lastname: Zheng
authors.forename: Li-fang
authors.initials: LF
authors.lastname: Hu
authors.forename: Chun-feng
authors.initials: CF
authors.lastname: Liu
mesh_terms: D000225:Adenine / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D001343:Autophagy / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D002918:Chymotrypsin / Q000378:metabolism; D006801:Humans; D009842:Oligopeptides / Q000494:pharmacology; D016716:PC12 Cells; D010300:Parkinson Disease / Q000378:metabolism; D017354:Point Mutation; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism*; D061988:Proteasome Inhibitors / Q000494:pharmacology; D051381:Rats; D051844:alpha-Synuclein / Q000235:genetics / Q000378:metabolism*
chemical_list: D009842:Oligopeptides; D061988:Proteasome Inhibitors; D051844:alpha-Synuclein; C025946:3-methyladenine; D002918:Chymotrypsin; D046988:Proteasome Endopeptidase Complex; D000225:Adenine; C078846:epoxomicin