【作者】 苏敏；

【导师】 刘春风；

【作者基本信息】 苏州大学 ， 神经病学， 2011， 博士

【摘要】 第一部分HDAC6介导了MPP~+诱导的α-synuclein聚集体的aggresome-大自噬降解途径目的:利用MPP~+处理过表达人类突变型(A53T)α-synuclein的PC12细胞株(α-SNA53T~+PC12)和SH-SY5Y细胞株,构建帕金森病细胞模型,探讨HDAC6对MPP~+诱导的α-synuclein聚集体的aggresome-大自噬降解途径的调控。方法:用1.5mM的MPP~+处理α-SNA53T~+PC12和SH-SY5Y细胞,通过免疫印迹法观察细胞内HDAC6蛋白水平的变化,并利用免疫荧光共聚焦技术观察HDAC6与γ-tubulin和LAMP-1的共定位情况;分别用自噬抑制剂3-MA、siRNA干扰HDAC6及HDAC6特异性抑制剂Tubacin和/或MPP~+共处理α-SNA53T~+PC12细胞后,利用免疫印迹法和免疫荧光共聚焦技术观察HDAC6、α-synuclein和LC3蛋白水平及其在细胞内定位的改变;并进一步通过提取细胞核蛋白观察HDAC6对α-synuclein亚细胞定位的影响。结果:(1)MPP~+诱导细胞内HDAC6表达明显增加并且定位发生改变,其与γ-tubulin、LAMP-1和α-synuclein共定位于细胞核周围形成aggresome样小体。(2)MPP~+诱导α-synuclein聚集体与LC3共定位于细胞核周围及细胞突起,抑制自噬导致α-synuclein增多。(3)敲减或抑制HDAC6导致MPP~+诱导的α-synuclein聚集体进一步在细胞内蓄积,且不能在核周形成aggresome样小体,并大量聚集于细胞核内。(4)进一步观察发现敲减或抑制HDAC6阻止了MPP~+诱导下LC3-Ⅱ蛋白表达的上调以及LC3荧光信号的增强。结论:HDAC6通过调控aggresome-大自噬途径参与了MPP~+诱导的异常聚集的α-synuclein的降解,缺乏HDAC6阻碍了MPP~+诱导的α-synuclein聚集体形成aggresome及自噬活性的反应性上调,导致大量α-synuclein蓄积于细胞核内。第二部分HDAC6调控了分子伴侣介导的自噬途径对MPP~+诱导的α-synuclein聚集体的降解目的:利用MPP~+处理过表达人类野生型(WT)和突变型(A30P)α-synuclein的PC12细胞株(α-SNWT~+PC12和α-SNA30P~+PC12),构建帕金森病细胞模型,探讨HDAC6对MPP~+诱导的α-synuclein聚集体的分子伴侣介导的自噬降解途径(CMA)的调控。方法:利用siRNA技术干扰α-SNWT~+PC12和α-SNA30P~+PC12细胞的HDAC6,并通过免疫印迹法鉴定干扰效率;用siRNA干扰和/或MPP~+共处理α-SNWT~+PC12和α-SNA30P~+PC12细胞后,利用免疫印迹法观察α-synuclein、LAMP-2a和HSC70蛋白水平的变化;并通过免疫荧光共聚焦技术观察α-SNWT~+PC12细胞内LAMP-2a和LAMP-1的共定位;同时,采用ELISA方法观察α-SNWT~+PC12细胞内RNase A的变化;并进一步通过免疫共沉淀方法观察HDAC6对α-SNWT~+PC12细胞内HSP90乙酰化及其与LAMP-2a和HSC70相互作用的影响。结果:(1)RNA干扰48小时,两株细胞中的HDAC6表达均明显降低,大约在60%左右;(2)MPP~+处理后突变型α-synuclein蛋白水平增加,野生型α-synuclein增加不明显,而缺乏HDAC6导致两株细胞中的α-synuclein均明显增加;(3)MPP~+诱导两株细胞LAMP-2a和HSC70表达增加,缺乏HDAC6致使野生型细胞内LAMP-2a进一步增加,而突变型LAMP-2a表达轻度下降,HSC70表达代偿性增加;(4)MPP~+减少了α-SNWT~+PC12细胞内的RNase A,缺乏HDAC6导致细胞内RNAse A明显增多;(5)MPP~+诱导α-SNWT~+PC12细胞的LAMP-2a荧光信号增强,缺乏HDAC6致使LAMP-2a荧光信号进一步增加,且LAMP-1荧光信号也相应增强;(6)MPP~+诱导α-SNWT~+PC12细胞内HSP90乙酰化水平下降,其与HSC70和LAMP-2a的相互作用增加,而缺乏HDAC6导致HSP90乙酰化水平增加,其与HSC70和LAMP-2a的结合下降。结论:MPP~+诱导了CMA活性的上调,且上调的CMA可代偿性降解MPP~+诱导的野生型α-synuclein;HDAC6参与了MPP~+诱导的CMA活性的反应性上调,并通过调节HSP90的乙酰化影响野生型α-synuclein的CMA降解途径。第三部分HDAC6对MPP~+诱导的α-synuclein聚集体所致的细胞损伤的拮抗作用目的:利用MPP~+处理PC12细胞株(PC12、α-SNWT~+PC12、α-SNA30P~+PC12和α-SNA53T~+PC12)和SH-SY5Y细胞株,构建帕金森病细胞模型,探讨HDAC6对MPP~+诱导的α-synuclein聚集体所致的细胞损伤的拮抗作用。方法:以siRNA干扰或Tubacin抑制HDAC6,并与MPP~+相结合的方法共处理各株细胞达12小时,然后通过MTT法检测细胞活力,LDH法评定细胞损伤,并通过流式细胞仪检测各组细胞凋亡率,光镜观察活细胞一般形态学变化,同时采用透射电镜方法观察细胞超微结构的改变。结果:(1)MPP~+处理12小时后各株细胞活力均降低,敲减HDAC6导致过表达WT、A30P、A53Tα-synuclein PC12细胞活力进一步降低;(2)暴露于MPP~+处理12小时致使各株细胞LDH释放量均增加,敲减HDAC6导致过表达WT、A30P、A53Tα-synuclein PC12细胞损伤进一步增加;(3)MPP~+作用12小时后细胞的早期凋亡显著增高,抑制HDAC6导致过表达WT、A30P、A53Tα-synuclein PC12细胞的早期凋亡进一步增加;(4)活细胞形态学观察MPP~+诱导细胞形态改变,缺乏HDAC6导致过表达WT、A30P、A53Tα-synuclein PC12细胞明显积聚,尤其是过表达A53T的PC12细胞;(5)细胞超微结构显示MPP~+处理后细胞内自噬结构增多,抑制HDAC6细胞内自噬囊泡减少,在过表达WT或A53Tα-synuclein PC12细胞内可见各种细胞凋亡改变。结论:HDAC6是一种细胞内主要的应对MPP~+毒性应激反应的调节因子,其极可能通过对易聚集α-synuclein清除过程的调控来抵抗MPP~+毒性,从而在一定程度上发挥保护功能。更多还原

【Abstract】 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.更多还原