Parkin represses 6-hydroxydopamine-induced apoptosis via stabilizing scaffold protein p62 in PC12 cells |
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Authors: | Xiao-ou Hou Jian-min Si Hai-gang Ren Dong Chen Hong-feng Wang Zheng Ying Qing-song Hu Feng Gao Guang-hui Wang |
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Affiliation: | 1.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;2.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 |
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Abstract: | Aim: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.Conclusion:Parkin protects PC12 cells against 6-OHDA-induced apoptosis via ubiquitinating and stabilizing scaffold protein p62, and repressing ERK1/2 activation. |
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Keywords: | Parkinson disease parkin p62 ERK1/2 6-OHDA PC12 cells |
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