Bibenzyl compound 20c protects against endoplasmic reticulum stress in tunicamycin-treated PC12 cells in vitro

Abstract

Aim: Accumulation of α-synuclein (α-syn) in the brain is a characteristic of Parkinson's disease (PD). In this study, we investigated whether treatment with tunicamycin, an endoplasmic reticulum (ER) stress inducer, led to the accumulation of α-syn in PC12 cells, and where α-syn protein was accumulated, and finally, whether bibenzyl compound 20c, a novel compound isolated from Gastrodia elata (Tian ma), could alleviate the accumulation of α-syn and ER stress activation in tunicamycin-treated PC12 cells. Methods: PC12 cells were treated with tunicamycin for different time (6 h, 12 h, 24 h, 48 h). Cell viability was determined by a MTT assay. Subcellular fractions of ER and mitochondria were extracted with the Tissue Endoplasmic reticulum Isolation Kit. The levels of α-syn protein and ER-stress-associated downstream chaperones were detected using Western blots and immunofluorescence. Results: Treatment of PC12 cells with tunicamycin (0.5-10 μg/mL) dose-dependently increased the accumulation of α-syn monomer (19 kDa) and oligomer (55 kDa), and decreased the cell viability. Accumulation of the two forms of α-syn was observed in both the ER and mitochondria with increasing treatment time. Co-treatment with 20c (10 -5 mol/L) significantly increased the viability of tunicamycintreated cells, reduced the level of α-syn protein and suppressed ER stress activation in the cells, evidenced by the reductions in phosphorylation of eIF2α and expression of spliced ATF6 and XBP1. Conclusion: Tunicamycin treatment caused accumulation of α-syn monomer and oligomer in PC12 cells. Bibenzyl compound 20c reduces the accumulation of α-syn and inhibits the activation of ER stress, which protected PC12 cells against the toxicity induced by tunicamycin.Keywords: Gastrodia elata; bibenzyl compound 20c; PC12 cells; tunicamycin; α-synuclein; ER stress; Parkinson's disease; neuroprotection Acta Pharmacologica Sinica (2016Sinica ( ) 37: 1525Sinica ( -1533 doi: 10.1038/aps.2016 proteins in the ER [6,7] . The activation of the PERK pathway promotes the phosphorylation of eIF2α and the expression of transcription factor ATF4, which increases the expression of ER chaperones to reduce protein entry into the ER. During ER stress, ATF6 translocates to the Golgi and releases transcription factors to migrate into the nucleus and regulate gene expression. When IRE1 is activated, it catalyzes the splicing of the mRNA encoding the X-box-binding protein 1 (XBP1) to produce XBP1 and regulate target genes [8] . Although the mechanisms of PD pathogenesis remain unclear, many studies have indicated that the accumulation of α-syn may be a neurotoxic factor [9,10] . However, the mechanisms underlying α-syn accumulation and how α-syn accumulation contributes to neurodegeneration remain poorly understood. Studies have indicated that sodium butyrate, which is an ER stress inducer, can induce an increase in the oligomeric forms of α-syn in 3D5 cells, and this effect was blocked by cotreatment with the ER stress inhibitor salubrinal, which suggested that ER stress co...

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