title: Proteasome inhibitor-induced autophagy in PC12 cells overexpressing A53T mutant α-synuclein.
abstract: The aim of the present study was to examine the effects of proteasome inhibitor (PI)‑induced autophagy on PC12 cells overexpressing A53T mutant α‑synuclein (α‑syn) by detecting alterations in the levels of microtubule‑associated protein 1A/1B light chain (LC3)+ autophagosomes and the lysotracker‑positive autolysosomes using immunofluorescence, the expression of LC3‑II using western blot analysis and the morphology of PC12 cells using transmission electron microscopy. It was found that the addition of MG132 (500 nmol/l) significantly increased the number of autophagosomes and autolysosomes and upregulated the expression of LC3‑II. The autophagy inhibitor 3‑methyladenine (3‑MA) completely inhibited the autophagy induced by MG132 (500 nmol/l). The autophagy enhancer trehalose significantly increased the number of autophagosomes and autolysosomes and improved the protein level of LC3‑II induced by MG132. To examine the effect of PI‑induced autophagy on the degradation of A53T mutant α‑syn, the expression of α‑syn was detected by western blot analysis. It was revealed that MG132 increased the expression of A53T α‑syn and trehalose counteracted the increase of A53T α‑syn induced by MG132. Combined inhibition of 3‑MA and PI significantly increased the accumulation of A53T α‑syn as compared with treatment using either single agent. In addition, combination of MG132 (500 nmol/l) with trehalose (50 mmol/l) or 3‑MA (2 mmol/l) markedly decreased the cell viability as compared with treatment using either single agent individually as demonstrated using a 3‑(4,5‑dimethylthiazol‑2‑yl)‑2,5‑diphenyltetrazolium bromide assay. These results suggest that the PI, MG132, could induce autophagy in PC12 cells overexpressing A53T mutant α‑syn and this autophagy could be completely inhibited by 3‑MA, indicating that PI‑induced autophagy is mediated by the upregulation of the macroautophagy class III PI3K pathway. PI‑induced autophagy may act as a compensatory degradation system for degradation of A53T α‑syn when the ubiquitin‑proteasome system is impaired. Autophagy activation may directly contribute to the survival of PC12 cells treated with proteasome inhibitors. The present study may assist in illuminating the association between PI and autophagy in the pathogenesis of Parkinson's disease.
authors.affiliation: Department of Neurology, Shanghai Changzheng Hospital, Second Military Medical University, Shanghai 200003, P.R. China.
authors.forename: Danmei
authors.initials: D
authors.lastname: Lan
authors.affiliation: Department of Neurology, Shanghai Changzheng Hospital, Second Military Medical University, Shanghai 200003, P.R. China.
authors.forename: Wenzhao
authors.initials: W
authors.lastname: Wang
authors.affiliation: Department of Neurology, Shanghai Changzheng Hospital, Second Military Medical University, Shanghai 200003, P.R. China.
authors.forename: Jianhua
authors.initials: J
authors.lastname: Zhuang
authors.affiliation: Department of Neurology, Shanghai Changzheng Hospital, Second Military Medical University, Shanghai 200003, P.R. China.
authors.forename: Zhongxin
authors.initials: Z
authors.lastname: Zhao
mesh_terms: D000225:Adenine / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D001343:Autophagy / Q000187:drug effects* / Q000235:genetics*; D016923:Cell Death / Q000187:drug effects / Q000235:genetics; D015870:Gene Expression; D007976:Leupeptins / Q000494:pharmacology; D009154:Mutation; D016716:PC12 Cells; D061988:Proteasome Inhibitors / Q000494:pharmacology*; D051381:Rats; D058570:TOR Serine-Threonine Kinases / Q000378:metabolism; D051844:alpha-Synuclein / Q000235:genetics* / Q000378:metabolism
chemical_list: D007976:Leupeptins; D061988:Proteasome Inhibitors; D051844:alpha-Synuclein; C025946:3-methyladenine; D058570:TOR Serine-Threonine Kinases; D000225:Adenine; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde