The role of endogenous PINK1 and Parkin mutations in human dopaminergic neurons

帕金森病（Parkinson disease，PD）是第二常见的神经退行性疾病，其特征是黑质多巴胺能神经元的丢失。类似于“特发性”PD的临床综合征已被报告为复发性遗传性Parkin和PINK 1连锁PD。我们在本申请中的总体假设是，脆弱神经元中线粒体功能的损伤在PD的发病机制中起关键作用，并且线粒体功能的恢复将导致神经保护。由于编码蛋白的功能主要在非神经元系统中或通过敲除方法进行研究，因此必须在适当的人源性和生物学相关细胞模型中研究内源性突变的作用。本研究的目的是通过使用一种新的分化策略来产生和表征iPS细胞衍生的多巴胺能神经元的纯群体。将检查Parkin和PINK 1突变神经元的毒性和线粒体功能，并与对照进行比较。为了更直接地测试所观察到的表型是否与致病性突变相关，我们打算检查拯救策略。我们将表达PGC-1α，一种线粒体生物发生的重要调节因子，并使用锌指技术进行遗传拯救，该技术允许选择性修复内源性突变。

Parkinson disease (PD) is the second most common neurodegenerative disorder, characterized by the loss of dopaminergic neurons in the substantia nigra. A clinical syndrome resembling ‘idiopathic’ PD has been reported for recessively inherited Parkin- and PINK1-linked PD. Our overall hypothesis in this application is that impairment of mitochondrial function in vulnerable neurons plays a key role in the pathogenesis of PD and that restoration of mitochondrial function will result in neuroprotection. As the function of the encoded proteins has been mainly investigated in non-neuronal systems or through knockdown approaches, there is an imperative to examine the role of endogenous mutations in appropriate human-derived and biologically relevant cell models. The objective of this study is to generate and characterize pure populations of iPS cell-derived dopaminergic neurons by using a novel differentiation strategy. Parkin and PINK1 mutant neurons will be examined for toxicity and mitochondrial function and compared to controls. In order to more directly test whether the observed phenotypes are related to pathogenic mutations, we intend to examine rescue strategies. We will express PGC-1α, an important regulator of mitochondrial biogenesis, and employ genetic rescue using zinc finger technology that allows for selective repair of endogenous mutations.