Proteasome inhibitor-induced autophagy in PC12 cells overexpressing A53T mutant α-synuclein

Lan,Danmei; Wang,Wenzhao; Zhuang,Jianhua; Zhao,Zhongxin

doi:10.3892/mmr.2014.3011

Proteasome inhibitor-induced autophagy in PC12 cells overexpressing A53T mutant α-synuclein

Authors:
- Danmei Lan
- Wenzhao Wang
- Jianhua Zhuang
- Zhongxin Zhao
View Affiliations

Affiliations: Department of Neurology, Shanghai Changzheng Hospital, Second Military Medical University, Shanghai 200003, P.R. China
Published online on: November 27, 2014 https://doi.org/10.3892/mmr.2014.3011
Pages: 1655-1660
Copyright: © Lan et al. This is an open access article distributed under the terms of Creative Commons Attribution License [CC BY_NC 3.0].

Metrics: Total Views: 0 (Spandidos Publications: | PMC Statistics: )

Metrics: Total PDF Downloads: 0 (Spandidos Publications: | PMC Statistics: )

Cited By (CrossRef): 0 citations Loading Articles...

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.

View Figures

View References

1	Polymeropoulos MH, Lavedan C, Leroy E, et al: Mutation in the alpha-synuclein gene identified in families with Parkinson’s disease. Science. 276:2045–2047. 1997. View Article : Google Scholar : PubMed/NCBI
2	Kruger R, Kuhn W, Muller T, et al: Ala30Pro mutation in the gene encoding alpha-synuclein in Parkinson’s disease. Nat Genet. 18:106–108. 1998. View Article : Google Scholar
3	Masliah E, Rockenstein E, Veinbergs I, et al: Dopaminergic loss and inclusion body formation in alpha-synuclein mice: implications for neurodegenerative disorders. Science. 287:1265–1269. 2000. View Article : Google Scholar : PubMed/NCBI
4	Feany MB and Bender WW: A Drosophila model of Parkinson’s disease. Nature. 404:394–398. 2000. View Article : Google Scholar : PubMed/NCBI
5	Webb JL, Ravikumar B, Atkins J, Skepper JN and Rubinsztein DC: Alpha-Synuclein is degraded by both autophagy and the proteasome. J Biol Chem. 278:25009–25013. 2003. View Article : Google Scholar : PubMed/NCBI
6	McNaught KS and Jenner P: Proteasomal function is impaired in substantia nigra in Parkinson’s disease. Neurosci Lett. 297:191–194. 2001. View Article : Google Scholar : PubMed/NCBI
7	Moore DJ, Dawson VL and Dawson TM: Role for the ubiquitin-proteasome system in Parkinson’s disease and other neurodegenerative brain amyloidoses. Neuromolecular Med. 4:95–108. 2003. View Article : Google Scholar
8	McNaught KS, Belizaire R, Isacson O, Jenner P and Olanow CW: Altered proteasomal function in sporadic Parkinson’s disease. Exp Neurol. 179:38–46. 2003. View Article : Google Scholar
9	McNaught KS, Bjorklund LM, Belizaire R, Isacson O, Jenner P and Olanow CW: Proteasome inhibition causes nigral degeneration with inclusion bodies in rats. Neuroreport. 13:1437–1441. 2002. View Article : Google Scholar : PubMed/NCBI
10	McNaught KS, Perl DP, Brownell AL and Olanow CW: Systemic exposure to proteasome inhibitors causes a progressive model of Parkinson’s disease. Ann Neurol. 56:149–162. 2004. View Article : Google Scholar : PubMed/NCBI
11	McNaught KS and Olanow CW: Proteasome inhibitor-induced model of Parkinson’s disease. Ann Neurol. 60:243–247. 2006. View Article : Google Scholar : PubMed/NCBI
12	Komatsu M, Waguri S, Chiba T, et al: Loss of autophagy in the central nervous system causes neurodegeneration in mice. Nature. 441:880–884. 2006. View Article : Google Scholar : PubMed/NCBI
13	Alvarez-Erviti L, Rodriguez-Oroz MC, Cooper JM, et al: Chaperone-mediated autophagy markers in Parkinson disease brains. Arch Neurol. 67:1464–1472. 2010.PubMed/NCBI
14	Yang Q and Mao Z: Parkinson disease: a role for autophagy? Neuroscientist. 16:335–341. 2010. View Article : Google Scholar : PubMed/NCBI
15	Conway KA, Harper JD and Lansbury PT: Accelerated in vitro fibril formation by a mutant alpha-synuclein linked to early-onset Parkinson disease. Nat Med. 4:1318–1320. 1998. View Article : Google Scholar : PubMed/NCBI
16	Komatsu M, Waguri S, Ueno T, et al: Impairment of starvation-induced and constitutive autophagy in Atg7-deficient mice. J Cell Biol. 169:425–434. 2005. View Article : Google Scholar : PubMed/NCBI
17	Ding WX, Ni HM, Gao W, et al: Linking of autophagy to ubiquitin-proteasome system is important for the regulation of endoplasmic reticulum stress and cell viability. Am J Pathol. 171:513–524. 2007. View Article : Google Scholar : PubMed/NCBI
18	Fels DR, Ye J, Segan AT, et al: Preferential cytotoxicity of bortezomib toward hypoxic tumor cells via overactivation of endoplasmic reticulum stress pathways. Cancer Res. 68:9323–9330. 2008. View Article : Google Scholar : PubMed/NCBI
19	Pandey UB, Nie Z, Batlevi Y, et al: HDAC6 rescues neurodegeneration and provides an essential link between autophagy and the UPS. Nature. 447:859–863. 2007. View Article : Google Scholar : PubMed/NCBI
20	Zhu K, Dunner K Jr and McConkey DJ: Proteasome inhibitors activate autophagy as a cytoprotective response in human prostate cancer cells. Oncogene. 29:451–462. 2010. View Article : Google Scholar :
21	Ebrahimi-Fakhari D, Cantuti-Castelvetri I, Fan Z, et al: Distinct roles in vivo for the ubiquitin-proteasome system and the autophagy-lysosomal pathway in the degradation of alpha-synuclein. J Neurosci. 31:14508–14520. 2011. View Article : Google Scholar : PubMed/NCBI
22	Kadowaki M and Karim MR: Cytosolic LC3 ratio as a quantitative index of macroautophagy. Methods Enzymol. 452:199–213. 2009. View Article : Google Scholar : PubMed/NCBI
23	Mizushima N and Yoshimori T: How to interpret LC3 immunoblotting. Autophagy. 3:542–545. 2007. View Article : Google Scholar : PubMed/NCBI
24	Tanida I, Ueno T and Kominami E: LC3 and autophagy. Methods Mol Biol. 445:77–88. 2008. View Article : Google Scholar : PubMed/NCBI
25	Dingle JT and Barrett AJ: Uptake of biologically active substances by lysosomes. Proc R Soc Lond B Biol Sci. 173:85–93. 1969. View Article : Google Scholar : PubMed/NCBI
26	Sarkar S, Davies JE, Huang Z, Tunnacliffe A and Rubinsztein DC: Trehalose, a novel mTOR-independent autophagy enhancer, accelerates the clearance of mutant huntingtin and alpha-synuclein. J Biol Chem. 282:5641–5652. 2007. View Article : Google Scholar
27	Lan DM, Liu FT, Zhao J, et al: Effect of trehalose on PC12 cells overexpressing wild-type or A53T mutant alpha-synuclein. Neurochem Res. 37:2025–2032
28	Levine B and Yuan J: Autophagy in cell death: an innocent convict? J Clin Invest. 115:2679–2688. 2005. View Article : Google Scholar : PubMed/NCBI
29	Casarejos MJ, Solano RM, Gomez A, Perucho J, de Yebenes JG and Mena MA: The accumulation of neurotoxic proteins, induced by proteasome inhibition, is reverted by trehalose, an enhancer of autophagy, in human neuroblastoma cells. Neurochem Int. 58:512–520. 2011. View Article : Google Scholar : PubMed/NCBI
30	Pan T, Kondo S, Zhu W, Xie W, Jankovic J and Le W: Neuroprotection of rapamycin in lactacystin-induced neurodegeneration via autophagy enhancement. Neurobiol Dis. 32:16–25. 2008. View Article : Google Scholar : PubMed/NCBI