Effects Of Autophagy On The α-synuclein Metabolism And The Relevance Of HDAC6 In Rotenone-induced Injury Of SH-SY5Y Cells

AIM:To study the role of autophagy in Rotenone-induced injury of SH-SY5Ycells, and to preliminary investigate the relationship among autophagy,α-synuclein and HDAC6; and the pathogenesis of Parkinson'mechanism.Method: In this study ,we have established cellular models of Parkinson'disease , which using rotenone treatment with human neuroblastoma cell line SH-SY5Y. To determinnation the effect of proliferation inhibition of Rotenone on the SH-SY5Y cell using WST-1; the cells'morphological changes were observed using optical microscopy; the relevance of autophagic protein LC3 as well as HDAC6,α-synuclein expression were determined with immunofluorescence(IF) and Western blot; the effects of autophagy specific inhiboitor 3-methyadenine (3-MA), autophagy activator rapamycin onα-synuclein accumulation and the LC3 and HDAC6 expression changes were also determined with immunofluorescence(IF).Results: The results of WST-1 showed that 100nM Rotenone without proliferation inhibition effect on the SH-SY5Ycells;but optical microscopy detect the morphological change; However , the SH-SY5Y cell growth is inhibited obviously with pre-join the autophay specific inhibitor 3-methyadenine(3-MA ); optical microscopy showed that cells appear to die and its antenna can not be restored. Immunofluorescence showed rotenone inducedα-synuclein increase, aggregation and aggeromes formation; and Immunofluorescence also demonstrated that the increases of LC3 and HDAC6 ,also form the inclusion bodies which were similar to aggresomes. Western blot showed a dynamic change ofα-synuclein and LC3. Double immunofluorescence showedα-synuclein and HDAC6 co-expression; LC3 andα-synuclein , LC3 and HDAC6 also exists the phenomenon of co-expression. Inhibition of autophagy by 3-MA significantly aggravatedα-synuclein aggreation and accumulation. At the same time, HDAC6 expression decreased and inhibited in some extent.α-synuclein showed a relatively high level with time detected by western blot; the activator of autophagy, IF and western blot showed that expression ofα-synuclein decreased; and HDAC6 expression increased in advance of activate autophagy by rapamycin. Rapamycin maybe increase HDAC6 expression , while accelerating the autophagyic degradation ofα-synuclein.Conclusion: Rotenone can improve the level of SH-SY5Y cell autophagy and increase expression ofα-synuclein and form Lewy bodies; HDAC6 andα-synuclein co-expression demestrate that HDAC6 also is an important component of Lewy bodies ;autophagy is the main way for clearance ofα-synuclein; activation autophagy maybe accelerateα-synuclein metabolism. HDAC6 response mechanism maybe dependent on autophagy.