PINK1激酶对Bad的磷酸化调控在非人灵长类神经变性中的作用

PINK1突变可因基因功能缺失导致青少年型帕金森病，但具体分子机制尚不清楚。大量体外研究发现PINK1在线粒体自噬中有重要功能，但Pink1敲除小鼠无神经死亡特征且线粒体自噬无明显变化，使得PINK1在体内作用缺乏深入研究。我们前期发现CRISPR/Cas9介导的猴大脑PINK1敲除可造成严重神经变性，且PINK1蛋白在猴大脑中表达丰富，其缺失可显著下调促凋亡蛋白Bad的磷酸化水平但Pink1敲除小鼠却无此变化。因此提出假说：PINK1对Bad的磷酸化调控在猴神经细胞存活中至关重要。本研究拟利用已建立的PINK1敲除猴模型，通过生化技术及组学方法探究PINK1调控Bad磷酸化的机理；拟通过过表达模拟磷酸化Bad对PINK1敲除猴治疗，利用核磁共振成像、行为学及病理学等方法评估猴肢体运动障碍及神经变性的症状是否得到改善。该研究可揭示PINK1突变造成神经变性的分子机理及为治疗PD提供新思路。

Mutations in the human PINK1 gene were found to result in juvenile-onset Parkinson disease (PD) by loss of function, but the specific molecular mechanism remains unclear. Based on the in vitro findings that PINK1 plays a primary role in mitophagy, a number of Pink1 knockout (KO) mouse models have been established for investigating PD pathogenesis. However, these Pink1 KO mouse models have not recapitulated selective and overt neurodegeneration seen in PD or validated the important in vitro findings for the function of PINK1 in mitophagy. Our recent studies of CRISPR-Cas9-targeted PINK1 monkeys revealed that loss of PINK1 in the monkey brains can lead to dramatic neurodegeneration. Our preliminary findings also show that PINK1 is abundant in the monkey brain and that PINK1 deficiency can reduce the phosphorylation of Bad, which can trigger apoptosis in neuronal cells. While there is no significant changes of the phosphorylation of Bad in Pink1 KO mouse brain. This indicates that the phosphorylation of Bad by PINK1 kinase may play a primary role in neuronal survival of monkey brain. The current application proposes the further investigation of how PINK1 kinase is involved in neurodegeneration in the monkey brain with a focus on Bad phosphorylation. We will also perform stereotaxic injection of AAV vector expressing phosphorylation mimic BAD and use MRI, behavioral and pathological analysis to examine whether it can improve the motor dysfunction and neuronal degeneration of PINK1 mutant monkey models. The studies aim to provide new insights into the pathogenesis of PD due to the mutations in the PINK1 gene and the therapy for Parkinson disease.