帕金森病中CDK5磷酸化依赖的C9orf72泛素化降解介导alpha-synuclein清除障碍和神经元死亡的机制研究

Parkinson disease (PD) is characterized by the loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc), and by the presence of Lewy bodies (LBs) in this region, but the mechanism is still unclear. In the current study, we used quantitative proteomics to detect the protein expression and phosphorylation, ubiquitination levels in the SNpc of A30P*A53T α-synuclein transgenic mouse model of PD. We found that the expression of Chromosome 9 open reading frame 72 (C9orf72) decreased, phosphorylation and ubiquitination levels increased. C9orf72 has been demonstrated to be associated with the pathogenesis of amyotrophic lateral sclerosis and frontotemporal dementia, but its role in the pathogenesis of PD has not been reported. This project intends to explore the molecular mechanism of C9orf72 involvement in autophagy dysfunction and neuron death in PD. Firstly, the phosphorylation and ubiquitination levels of C9orf72 protein were detected in PD clinical specimens, cell and animal models. The molecular mechanism of C9orf72 degradation in neurons was analyzed. Secondly, We assumed that C9orf72 could phosphorylated by CDK5 at S9, consequently promote the ubiquitination and degradation of C9orf72. Thirdly, we investigated whether C9orf72 degradation is involved in the disorder of α-synuclein autophagy clearance and neuron death in PD. Finally, we verified whether interference of CDK5 phosphorylation of C9orf72 could alleviate autophagy dysfunction, reduce neuronal death and improve movement performance in the PD model, in order to provide a new idea and drug development target for PD research.

帕金森病（PD）是以α-synuclein清除障碍形成的路易小体和黑质多巴胺能神经元进行性死亡为病理学特征的疾病，但其分子机制尚不清楚。本项目应用定量蛋白组学检测早期PD转基因小鼠黑质蛋白表达及磷酸化与泛素化修饰水平，发现与自噬相关的C9orf72蛋白表达下降，修饰水平升高，C9orf72已被证实与渐冻症和额颞叶痴呆发病相关，但其在PD发病中的作用尚无报道。本项目拟深入探究C9orf72参与PD中自噬障碍和神经元死亡的分子机制：首先在PD临床标本和细胞/动物模型中检测C9orf72蛋白磷酸化和泛素化水平及降解情况和降解通路；然后研究PD时CDK5通过磷酸化C9orf72的S9位点介导其泛素化降解；继而探讨C9orf72降解参与α-synuclein自噬清除障碍和神经元死亡的机制；最后验证特异干预CDK5磷酸化C9orf72能否减轻PD中神经元死亡和运动障碍，为预防和早期治疗PD提供新靶点。

帕金森病（PD）是以α-synuclein清除障碍形成的路易小体和黑质多巴胺能神经元进行性死亡为病理学特征的疾病，但其分子机制尚不清楚。前期我们应用定量蛋白质组学对PD转基因小鼠黑质组织的蛋白质表达变化进行全面观察，并通过文献检索选择与自噬密切相关的C9orf72作为靶蛋白进行深入探究，C9orf72已被证实与渐冻症和额颞叶痴呆发病相关，但其在PD发病中的作用尚无报道。本项目遵循转化医学理念，通过组学检测—筛选靶点—阐明机制—干预治疗的研究思路，在分子—细胞—组织—动物等多个水平对C9orf72靶蛋白在PD发病过程中的关键作用及其分子机理进行深入的研究和探讨：我们在多种PD的细胞和动物模型检测发现，C9orf72蛋白的表达水平显著降低，mRNA表达没有改变，磷酸化水平增加，其表达减少主要通过泛素-蛋白酶体通路降解；继而发现神经系统关键激酶CDK5可直接磷酸化C9orf72蛋白的S9位点，C9orf72蛋白的S9位点的磷酸化可促进C9orf72蛋白的降解；敲减C9orf72，可加重PD小鼠黑质神经元α-synuclein聚集、自噬清除障碍、神经元丢失及运动功能障碍；过表达C9orf72可以减轻PD时神经元的α-synuclein聚集、自噬功能障碍；最后发现特异性干扰CDK5磷酸C9orf72的S9位点，可减轻PD时C9orf72蛋白降解介导的α-synuclein聚集、自噬功能障碍及神经元的死亡和运动功能障碍。本研究开发的穿膜小分子多肽，可特异性干扰神经元内CDK5磷酸化C9orf72的特定位点，不影响CDK5磷酸化细胞内其它底物，从而避免对其正常生理功能的影响和副作用，具有一定的临床应用价值。本研究极大地丰富和加深了对PD发病机制的认识和理解，对预防和早期治疗PD具有重要的临床意义，为帕金森病黑质多巴胺能神经元死亡的机制研究提供新的研究思路和治疗靶点。