title: Parkin represses 6-hydroxydopamine-induced apoptosis via stabilizing scaffold protein p62 in PC12 cells.
abstract: OBJECTIVE
  Parkin has been shown to exert protective effects against 6-hydroxydopamine (6-OHDA)-induced neurotoxicity in different models of Parkinson disease. In the present study we investigated the molecular mechanisms underlying the neuroprotective action of parkin in vitro.
  
  
  METHODS
  HEK293, HeLa and PC12 cells were transfected with parkin, parkin mutants, p62 or si-p62. Protein expression and ubiquitination were assessed using immunoblot analysis. Immunoprecipitation assay was performed to identify the interaction between parkin and scaffold protein p62. PC12 and SH-SY5Y cells were treated with 6-OHDA (200 μmol/L), and cell apoptosis was detected using PI and Hoechst staining.
  
  
  RESULTS
  In HEK293 cells co-transfected with parkin and p62, parkin was co-immunoprecipitated with p62, and parkin overexpression increased p62 protein levels. In parkin-deficient HeLa cells, transfection with wild-type pakin, but not with ligase activity-deficient pakin mutants, significantly increased p62 levels, suggesting that parkin stabilized p62 through its E3 ligase activity. Transfection with parkin or p62 significantly repressed ERK1/2 phosphorylation in HeLa cells, but transfection with parkin did not repress ERK1/2 phosphorylation in p62-knockdown HeLa cells, suggesting that p62 was involved in parkin-induced inhibition on ERK1/2 phosphorylation. Overexpression of parkin or p62 significantly repressed 6-OHDA-induced ERK1/2 phosphorylation in PC12 cells, and parkin overexpression inhibited 6-OHDA-induced apoptosis in PC12 and SH-SY5Y cells.
  
  
  CONCLUSIONS
  Parkin protects PC12 cells against 6-OHDA-induced apoptosis via ubiquitinating and stabilizing scaffold protein p62, and repressing ERK1/2 activation.
authors.affiliation: Laboratory of Molecular Neuropathology, Jiangsu Key Laboratory of Translational Research and Therapy for Neuro-Psycho-Diseases and College of Pharmaceutical Sciences, Soochow University, Suzhou 215021, China.
authors.forename: Xiao-ou
authors.initials: XO
authors.lastname: Hou
authors.affiliation: Laboratory of Molecular Neuropathology, Key Laboratory of Brain Function and Diseases and School of Life Sciences, University of Science & Technology of China, Chinese Academy of Sciences, Hefei 230027, China.
authors.forename: Jian-min
authors.initials: JM
authors.lastname: Si
authors.affiliation: Laboratory of Molecular Neuropathology, Jiangsu Key Laboratory of Translational Research and Therapy for Neuro-Psycho-Diseases and College of Pharmaceutical Sciences, Soochow University, Suzhou 215021, China.
authors.forename: Hai-gang
authors.initials: HG
authors.lastname: Ren
authors.affiliation: Laboratory of Molecular Neuropathology, Jiangsu Key Laboratory of Translational Research and Therapy for Neuro-Psycho-Diseases and College of Pharmaceutical Sciences, Soochow University, Suzhou 215021, China.
authors.forename: Dong
authors.initials: D
authors.lastname: Chen
authors.affiliation: Laboratory of Molecular Neuropathology, Jiangsu Key Laboratory of Translational Research and Therapy for Neuro-Psycho-Diseases and College of Pharmaceutical Sciences, Soochow University, Suzhou 215021, China.
authors.forename: Hong-feng
authors.initials: HF
authors.lastname: Wang
authors.affiliation: Laboratory of Molecular Neuropathology, Jiangsu Key Laboratory of Translational Research and Therapy for Neuro-Psycho-Diseases and College of Pharmaceutical Sciences, Soochow University, Suzhou 215021, China.
authors.forename: Zheng
authors.initials: Z
authors.lastname: Ying
authors.affiliation: Laboratory of Molecular Neuropathology, Jiangsu Key Laboratory of Translational Research and Therapy for Neuro-Psycho-Diseases and College of Pharmaceutical Sciences, Soochow University, Suzhou 215021, China.
authors.forename: Qing-song
authors.initials: QS
authors.lastname: Hu
authors.affiliation: Laboratory of Molecular Neuropathology, Jiangsu Key Laboratory of Translational Research and Therapy for Neuro-Psycho-Diseases and College of Pharmaceutical Sciences, Soochow University, Suzhou 215021, China.
authors.forename: Feng
authors.initials: F
authors.lastname: Gao
authors.affiliation: Laboratory of Molecular Neuropathology, Jiangsu Key Laboratory of Translational Research and Therapy for Neuro-Psycho-Diseases and College of Pharmaceutical Sciences, Soochow University, Suzhou 215021, China.
authors.forename: Guang-hui
authors.initials: GH
authors.lastname: Wang
mesh_terms: D000818:Animals; D017209:Apoptosis; D057809:HEK293 Cells; D006367:HeLa Cells; D006360:Heat-Shock Proteins / Q000378:metabolism*; D006801:Humans; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism; D016627:Oxidopamine / Q000378:metabolism*; D016716:PC12 Cells; D051381:Rats; D000071456:Sequestosome-1 Protein; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism*; D054875:Ubiquitination
chemical_list: D006360:Heat-Shock Proteins; D000071456:Sequestosome-1 Protein; C491079:Sqstm1 protein, rat; D016627:Oxidopamine; D044767:Ubiquitin-Protein Ligases; C111567:parkin protein; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3