Analyze mouse and cell culture models for PINK1 related Parkinson's disease

描述（由申请人提供）：线粒体功能障碍和氧化应激被认为与许多神经退行性疾病有关，包括帕金森病（PD）。线粒体功能障碍与帕金森病发病最直接的因果关系证据来自新发现的PINK1，一种家族性隐性早发帕金森病的疾病基因。PINK1蛋白是一种核基因组编码蛋白，具有线粒体靶向信号肽和保守的激酶结构域，但其正常功能和在发病机制中的作用仍有待阐明。PINK1- pd患者的突变导致PINK1蛋白被截断或很可能在功能上失活。假设：我们的总体假设是PINK1- pd病理是由线粒体功能破坏引起的，因为突变的PINK1蛋白不能磷酸化其正常底物。为了验证这一假设，我们产生了遗传PINK1-/-敲除小鼠和其他有价值的试剂，我们提出以下具体目标。具体目的1：我们将分析PINK1-/-小鼠是否在黑质和其他脑区出现进行性运动缺陷和神经元病理。特异性目的2：随后，但不依赖于目的1的结果，我们将在PINK1-/-小鼠和PINK1-/-多巴胺能细胞系中研究线粒体功能障碍。特异性目的3：随后，但不依赖于目的1的结果，我们将鉴定和确认PINK1激酶的底物。意义：总的来说，PINK1敲除小鼠和多巴胺能PINK1-/-细胞系的产生和表征，PINK1突变引起的线粒体功能障碍的研究，PINK1激酶底物的鉴定，对于了解PINK1- pd的分子机制，确定潜在的治疗靶点，以及药物筛选和测试至关重要。

DESCRIPTION (provided by applicant): Mitochondria dysfunction and oxidative stress have been suggested to be associated with many neurodegenerative diseases, including Parkinson's Disease (PD). The most direct CAUSALITY evidence of mitochondria dysfunction to PD pathogenesis arises from the newly identified PINK1, a disease gene of familial, recessive early-onset PD. PINK1 protein is a nuclear genome encoded protein with a mitochondria targeting signal peptide and a conserved kinase domain, however, its normal functions and roles in pathogenesis remain to be elucidated. The mutations in PINK1-PD patients are causing the PINK1 proteins to be truncated or most likely functionally inactive. HYPOTHESIS: Our overall hypothesis is that PINK1-PD pathology is caused by disruption of mitochondria functions because the mutant PINK1 protein fails to phosphorylate its normal substrates. To test this hypothesis, we have generated genetic PINK1-/- knockout mice and other valuable reagents, and we propose the following specific aims. SPECIFIC AIM 1: we will analyze whether PINK1-/- mice develop progressive motor deficits and neuronal pathology in substantia nigra and other brain regions. SPECIFIC AIM 2: Subsequently, but not dependent on the Aim 1 outcome, we will investigate mitochondria dysfunction in PINK1 -/- mice and in PINK1-/- dopaminergic cell lines. SPECIFIC AIM 3: Subsequently, but not dependent on the Aim1 outcome, we will identify and confirm the substrates of PINK1 kinase. SIGNIFICANCE: Collectively, the generation and characterization of the PINK1 knockout mice as well as the dopaminergic PINK1-/- cell lines, the investigation of the mitochondria dysfunction caused by PINK1 mutations, and the identification of PINK1 kinase substrates are critical for understanding of the molecular mechanisms of PINK1-PD, for identifying potential therapeutic targets, and for drug screening and testing.