HDAC6-mediated Autophagy In α-synuclein Degradation And Its Regulatory Mechanisms

PartⅠHDAC6 regulates aggresome-autophagy degradation pathway of alpha-synuclein in response to MPP~+-induced stressObjective: Increasing evidence suggests that the ubiquitin-binding histone deacetylase-6 (HDAC6) plays an important role in the clearance of misfolded proteins by autophagy. Here, we treated PC-12 cells overexpressing human mutant (A53T) alpha-synuclein (α-SN) and SH-SY5Y cells with MPP~+, and aimed to study wherther HDAC6 regulates aggresome-autophagy degradation pathway ofα-SN in response to MPP~+-induced stressMethods: In this study,α-SNA53T~+PC12 and SH-SY5Y cells were exposed to MPP~+ (1.5mM) for 12h. The cells were observed by immunoblotting in HDAC6 protein levels, and by confocal immunofluorescence were observed the co-localization of HDAC6 andγ-tubulin/LAMP-1. RNA interference or Tubacin were processed to inhibit the HDAC6, and using immunoblotting and immunofluorescence confocal technique to observe HDAC6,α-SN and LC3 protein levels and its localization in the cell; and further observation by extracting the nucleus protein to determine the subcellular localization ofα-SN.Results: It was found that HDAC6 expression significantly increased and mainly colocalized withα-SN in the perinuclear region to form aggresome-like bodies in response to MPP~+-induced stress. HDAC6 deficiency blocked the formation of aggresome-like bodies and interfered with the autophagy in response to MPP~+-induced stress. Moreover, misfoldedα-SN accumulated into the nuclei, resulting in its reduced clearance. Conclusion: Taken together, HDAC6 participated in the degradation of MPP~+-induced misfoldedα-SN aggregates by regulating the aggresome-autophagy pathway. Understanding the mechanism may disclose potential therapeutic targets for synucleinopathies such as Parkinson disease.PartⅡHDAC6 regulates chaperone-mediated autophagic clearance ofα-synuclein in response to MPP~+Objective: To study wherther HDAC6 regulates chaperone-mediated autophagic clearance ofα-SN in response to MPP~+,the PC12 cells overexpressing human wild-type (WT) or mutant (A30P) alpha-synuclein (α-SN) were exposed to MPP~+.Methods: siRNA technology was used to interfere the HDAC6 ofα-SNWT~+PC12 andα-SNA30P~+PC12 cells, and by western blot identification of interference efficiency; siRNA interference and / or MPP~+ were processed toα-SNWT~+PC12 andα-SNA30P~+PC12 cells. By western blot we observed the protein levels ofα-SN, LAMP-2a and HSC70, and through the confocal immunofluorescence technique to observe the co-location of LAMP-2a and LAMP-1 inα-SNWT~+PC12 cells. At the same time, we use of ELISA to observe the changes of RNase A inα-SNWT~+PC12 cells, and by immunoprecipitation further observed the role of HDAC6 on the acetylation of HSP90 and the interaction of HSP90 with LAMP-2a and HSC70.Results: It was found that the activity of chaperone-mediated autophagy (CMA) was increased and abnormal accumulated wild-typeα-SN was degraded in compensation, whilst mutantα-SN failed to be degraded by CMA. Inhibition of HDAC6 by RNAi resulted in different changing profiles of LAMP-2a and HSC70 in response to MPP~+, a slight decline in the former whereas an increase in the latter inα-SNA30P~+PC12 cells. Further studies revealed that the inhibition of HDAC6 interfered with the interaction of HSP90 with LAMP-2a and HSC70 by regulating the level of acetylated HSP90, resulting in the obstacles of chaperone-mediatedα-SN autophagic degradation pathway.Conclusions: These results demonstrate for the first time that by regulating HSP90 acetylation, HDAC6 disrupts the CMA-mediated removal of misfoldedα-SN aggregates induced by MPP~+. The findings may provide some clues to the potential therapeutic targets for synucleinopathies such as Parkinson disease.PartⅢThe role of HDAC6 on the cell injury resulted by MPP~+-inducedα-synuclein aggregatesObjective: To study the role of HDAC6 on the cell injury resulted by MPP~+-induced alpha-synuclein (α-SN) aggregates, we treated PC-12 cells overexpressing human mutant (WT/A30P/A53T)α-SN and SH-SY5Y cells with MPP~+.Methods: At 12h after drug treatment, the cell viability was measured by means of MTT methods and LDH release assay. The apoptosis ratio was assessed by flow cytometry. The hallmarks of apoptosis and autophagy were assessed with transmission electron microscopy.Results: (1) Exposed to MPP~+ for 12 hours , the cell viability decreased, and lack of HDAC6 leads to the cell viability of overexpression of WT, A30P, A53Tα-SN PC12 further reduced; (2) Exposed to MPP~+ for 12 hours, cell injury increases, and lack of HDAC6 leads to the cell injury of over-expression WT, A30P, A53Tα-SN PC12 further increase; (3) Exposed to MPP~+ for 12 hours, the cells in early apoptosis significantly increase, inhibition of HDAC6 leads to the early apoptosis of overexpression of WT, A30P, A53Tα-SN PC12 cells further increase; (4) Live cell morphology and cell shape changes are induced by MPP~+, and the lack of HDAC6 leads to overexpression of WT, A30P, A53T α-SN PC12 cells accumulated, particularly in the cells overexpressing A53Tα-SN. (5) The cell autophagy increase in the ultrastructure of the cells treated with MPP~+; inhibition of HDAC6 leads to intracellular autophagic vesicles reduce, and different kinds of cells apoptosis changes were observed in theα-SNWT~+PC12 andα-SNA53T~+PC12 cellsConclusion: HDAC6 is a major factor to resist the MPP~+-induced toxicity stress. By regulating the degradation ofα-SN aggregates HDAC6 resists the MPP~+-induced toxicity stress.