利用等遗传背景对照研究Parkin基因突变通过微管致神经元变性的机制

Parkinson’s disease (PD) is characterized by the progressive loss of dopamine-generating cells in the substantia nigra. Mutation of Parkin gene is one common cause of familial form, idiopathic form of Parkinson's disease, especially for the early-onset type; however the mechanism is unclear. Recently, increasing studies show that microtubule cytoskeleton represents a point of convergence in the genetic and environmental factors, plays an important role in the pathogenesis of PD, but its specific mechanism remains unkown. On the other hand, due to the lack of isogenic control, it is hard to differentiate the minor but disease-related phenotype changes from the variations under different genetic background. Previously, we have successfully rescued the deletion exon of Parkin gene in the induced pluripotent stem cells (iPSCs) based on homologous recombination mediated by gene editing tools (CRISPR/Cas9 and TALENs); and confirmed that mutations of Parkin gene influence the development of DA neurons. This project will generate new Parkin gene mutation PD patients and normal controls isogenic iPSCs model (repair mutation of parkin gene, mutate the normal parkin gene) mediated by CRISPR/Cas9 technology based on the previous results mentioned above. Our project will deeply study the mechnism behind the pathogenesis of Parkin gene regulates the development of dopamine neurons through microtubule cytoskeleton and may provide new understanding to the cause and treatment of Parkinson’s disease.

帕金森病(PD)是以中脑黑质多巴胺能(DA)神经元丢失为主要特征的神经变性疾病;Parkin是家族性、特发性及早发型PD的常见致病基因,目前其突变致病机制不明。近年来越来越多的研究证明微管细胞骨架正作为遗传与环境因素的汇聚点在PD发病中起着重要作用，然其具体作用机制仍不清楚；同时因缺乏遗传上匹配（isogenic）的对照，要区分某些细微但与PD相关的表型改变是一件困难的事情。前期我们通过同源重组技术在PD患者特异性iPSCs上对Parkin基因缺失外显子成功进行了修复，并发现Parkin基因突变影响DA神经元突起的生长。因此本课题拟在前期研究基础上利用CRISPR/Cas9技术修复或突变Parkin基因，建立新的Parkin基因突变PD患者及正常对照的isogenic iPSC细胞组模型，深入探索Parkin基因通过微管调控DA神经元生长及其突变的致病机制，以期为PD发病和治疗提供新思路。

帕金森病（PD）是最常见的神经退行性疾病之一，目前病因不明；Parkin基因是最常见的早发型及家族性、散发性PD的致病基因，但致病机制不清。本项目主要研究内容是建立Parkin基因突变PD患者特异性及正常对照的Parkin基因isogenic对照iPSCs细胞模型，并定向分化为中脑多巴胺（DA）能神经元来进行PD相关表型的探索，以及探索Parkin基因突变对中脑DA能神经元生长发育及对微管的影响等。.取得的重要研究结果及科学意义：1）成功建立了Parkin基因突变特异性PD患者和正常对照的Parkin基因同遗传背景isogenic对照iPSCs细胞模型，为进一步精准研究Parkin基因突变的致病机制提供了平台；2）在对建立Parkin基因isogenic对照iPSCs细胞模型定向分化为中脑腹侧被盖区（VTA）DA能神经元（A10区）验证之前的表型外，更创新性定向分化成经典PD病理相关的中脑黑质致密度（SNpc）DA能神经元（A9区），为探索Parkin基因的功能提供了更接近与PD主要病理的特异性SNpc DA能神经元；3）首次发现PD患者特异性iPSCs细胞分化的中脑SNpc DA能神经元有自发性自主同步起搏放电autonomic pacemaking现象，而Parkin基因突变减低了SNpc DA能神经元的autonomic pacemaking频率，可能因导致线粒体氧化应激的改变而成为SNpc DA能神经元易感的原因，为Parkin基因突变致PD的分子机制阐明了新的方向；4）通过数据库分析，首次发现APOE基因变异可影响PD运动症状进展，为PD分子分型及预后预测提供了新的证据； 5）对早发型和晚发型PD患者外周免疫状态分析研究发现，多种免疫细胞表达在早发型和晚发型PD中有改变，为PD的免疫机制提供了线索；6）在影像研究中，发现了PD发生与类淋巴系统的障碍相关，可能为PD的诊治提供新的线索。

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