The Regulation And Underlying Mechanisms Of Hydrogen Sulfide On Autophagy And α-synuclein Degradation In PC12 Cells

Objective:To study the possible effects and underlying mechanism of H2 S on autophagy and α-synuclein degradation in PC12 cells.Methods:The neurotoxins rotenone was used to establish in vitro PD model in PC12 cells. Na HS and GYY4137 served as hydrogen sulfide(H2S) fast-releasing hydrogen sulfide donor and the slow-releasing donors, respectively. Cell viability was determined by MTT assay. Western blot was used to detect the level of autophagy substrate SQSTM1/p62, microtubule-associated protein 1 light chain 3(LC3), Beclin1, m TOR, p70S6 K, AMPK, α-synuclein; NP40 was used to separate the soluble and insoluble fractions of α-synuclein; reverse-transcription PCR(RT-PCR) was used to determine the m RNA level of α-synuclein; PC12 cells were transiently transfected with GFP-LC3 for visualizations of the number and distribution of LC3 dots; AMPK S-sulfhydration was detected by Biotin Switch Technique(BST).Results:100 μM Na HS treatment significantly upregulated LC3 II expression and decreased p62 protein level, and 50 μM GYY4137 had similar effects on LC3 and p62; 100 μM Na HS and 50 μM GYY4137 treatment for 24 h significantly increased the number of LC3 positive dots compared to control group, without significant effect on cell viability.(2) Neither Na HS nor GYY4137 changed the level of Beclin1 protein; nevertheless, both 100 μM Na HS and 50 μM GYY4137 down-regulated the phosphorylation of m TOR and p70S6 K. Furthermore, Na HS and GYY4137 increased p-AMPK and S-sulfhydration level.(3) 100 n M rotenone increased the level of α-synuclein especially NP40-insoluble fraction, and Na HS pretreatment significantly reduced rotenone-induced α-synuclein accumulation. RT-PCR study showed Na HS and rotenone had no effect on α-synuclein m RNA level respectively.(4) Na HS pretreatment evidently reversed rotenone-induced p62 increase and LC3 decrease, which implied that hydrogen sulfide may reduce intracellular α-synuclein accumulation via facilitating its autopahgic degradation.Conclusion:The findings demonstrate hydrogen sulfide may increase AMPK phosphorylation via S-sulfhydration and thus inhibit m TOR/p70S6 K activity, which plays a positive role in upregulating autohagy and decreasing intracellular α-synuclein accumulation.