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.

Figure 1

3-MA inhibits MG132-induced macroautophagy and the processing of LC3 in PC12 cells overexpressing A53T α-synuclein. (A) Treatment with 500 nmol/l MG132 was demonstrated to induce the formation of LC3 and Lysotracker RED-positive autolysosomes (white arrows, original magnification, ×400), (B) increase the expression level of LC3-II and (C) affect the morphology of autolysosomes (black arrows) as observed by TEM; these effects could be inhibited completely by 3-MA. (A and C) No evident autophagosomes were detected in 3-MA pre-treated cells, even after 24 h treatment with 500 nmol/l MG132. ^**P<0.01 vs. control group. 3-MA, 3-methyladenine; LC3, microtubule-associated protein 1A/1B light chain.

Figure 2

Tre enhances MG132-induced macroautophagy. (A) Inhibition of the ubiquitin-proteasome system significantly activated macroautophagy, and 50 mmol/l Tre further increased the number of LC3⁺ and Lysotracker-positive autolysosomes (white arrows) induced by MG132. Original magnification, ×400. (B) In A53T α-synuclein-overexpressing PC12 cells, 500 nmol/l MG132 significantly increased LC3-II expression (P<0.01). Treatment with 50 mmol/l Tre further upregulated MG132-induced LC3-II expression. ^**P<0.01 vs. control group, ^##P<0.01 vs. MG132 group. LC3, microtubule-associated protein 1A/1B light chain; Tre, trehalose.

Figure 3

Effects of autophagy enhancers or inhibitors on MG132-induced accumulation of α-synuclein. The A53T α-syn-overexpressing PC12 cells were treated with MG132 (500 nmol/l), Tre (50 mmol/l, tre), MG132 (500 nmol/l) and Tre (50 mmol/l), 3-MA (2 mmol/l), MG132 (500 nmol/l) and 3-MA (2 mmol/l). The expression of α-syn was analyzed by immunoblotting. ^**P<0.01 vs. control group, ^##P<0.01 vs. MG132 group. α-syn, α-synuclein; Tre, trehalose; 3-MA, 3-methyladenine.

Figure 4

Effects of autophagy enhancers or inhibitors on proteasome inhibitor-induced cell death. The cell viability was measured using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and the results are expressed as the percentage of control. A combination of MG132 (500 nmol/l) with Tre (50 mmol/l) or 3-MA (2 mmol/l) markedly decreased the cell viability compared with treatment using either single agent. ^**P<0.01 vs. control group, ^##P<0.01 vs. MG132 group. 3-MA, 3-methyladenine; Tre, trehalose.