SIRT3通过调节线粒体自噬保护PD模型的机制研究

Parkinson’s disease is the second most common neurodegenerative disease affecting approximately five million people worldwide. Aging is the most important factor of PD. The pathology of this disease is complex and not entirely understood. Evidence suggests that PD is related with α-synuclein accumulation, mitochondrial dysfunction and oxidative stress. SIRT3 belongs to the Sirtuins family, which associates with aging closely. SIRT3 could reduce the reactive oxygen species so as to ameliorate the damge of oxidative stress. In our previous study, we found that SIRT3 is protective to rotenone-induced PD cell model through up-regulation of autophagy. The familial PD is reported to be related with PINK1 and Parkin mutation, which play important roles in mitophagy. In order to clarify the relationship between SIRT3 and mitophagy and PD injury, we plan to use rotenone to construct PD cell and mouse model. Using siRNA to interfere the expression of SIRT3 and constructing vector to overexpress SIRT3 and transgenic animal to overexpress SIRT3. Detecting the expression of SIRT3, α-synuclein accumulation, mitophadiral dysfunction, oxidative stress, mitophagy and SIRT3-p53-Parkin、SIRT3-FOXOs-Parkin、SIRT3-PGC-1α-Parkin pathway by using techniques like trsmission electron mictoscope, fluorescence probe, western blot, immunofluorescence staining, fow cytometry and immunoprecipitation. By this, we could demonstrate the correlation between SIRT3 and mitophagy and the modulation machenism between SIRT3 and mitophagy. This work would give us a new target for PD protection.

本课题组前期研究发现SIRT3可通过诱导自噬促进α-synuclein降解和损伤线粒体清除，从而对PD细胞模型起保护作用。文献报道家族性PD相关突变基因Parkin、PINK1均与线粒体自噬相关。而SIRT3对线粒体自噬的调节关系及其具体机制尚不清楚。本课题拟建立鱼藤酮诱导PD细胞及动物模型，利用透射电镜、荧光探针标记技术、免疫荧光染色、Western blot、流式细胞术、动物行为学检测等多种实验手段，采用基因转染和RNAi基因沉默技术及转基因动物，明确SIRT3是否通过上调线粒体自噬对PD产生保护作用。并用Western blot、免疫共沉淀技术、免疫荧光染色等方法检测SIRT3是否通过SIRT3-p53-Parkin、SIRT3-FOXOs-Parkin、SIRT3-PGC-1α-Parkin通路上调线粒体自噬。SIRT3对PD的保护作用及其机制研究，将为PD防治提供科学依据。

帕金森病（PD）是我国第二大神经系统退行性疾病，对个人、家庭及社会造成沉重负担。目前PD的治疗手段多为基于增加多巴胺的替代治疗，只能暂时缓解临床症状，且PD诊断的误诊率高，做出诊断时间晚。因此，寻找有效的神经保护靶点及寻找有效地PD早期发病生物标志物刻不容缓。本研究分为细胞试验及动物实验。利用透射电镜、荧光探针标记技术、免疫荧光染色、Western blot、流式细胞术、动物行为学检测等多种实验手段，采用基因转染和RNAi基因沉默技术及转基因动物，明确SIRT3是否通过上调线粒体自噬对PD产生保护作用。并用Western blot、免疫共沉淀技术、免疫荧光染色等方法检测SIRT3是否通过SIRT3-p53-Parkin、SIRT3-FOXOs-Parkin、SIRT3-PGC-1α-Parkin通路上调线粒体自噬。此外，通过SERS技术检测溶液中重要神经递质多巴胺的水平。研究意义：1.已发现sirt3可上调大自噬及其作用通路，进一步探索sirt3与线粒体自噬的关系及作用机制，为发现新的对PD有神经保护作用的靶点提供策略。2. PD发病机制是黑质纹状体内多巴胺能神经元变性缺失，通过SERS技术检测溶液中多巴胺分子浓度，为进一步检测PD相关分子标志物提供有力的检测手段。