ROLE OF THE PINK1/PARKIN PATHWAY IN MITOCHONDRIAL DYNAMICS AND TISSUE INTEGRITY

描述（由申请人提供）：线粒体功能障碍是帕金森病的共同特征，但我们对其潜在分子机制的了解还很初级。最近，一种影响线粒体完整性的遗传途径已经从PINK1和帕金基因的研究中出现，其功能丧失突变是大多数早发性隐性帕金森病的原因。PINK1基因编码一种丝氨酸/苏氨酸激酶，定位于线粒体内膜空间和细胞质，而parkin基因编码一种泛素蛋白连接酶，广泛定位于整个细胞，包括细胞质和线粒体。对高度保守的PINK1和parkin果蝇同源基因的突变分析表明，PINK1在parkin的上游共同通路中起作用，促进一部分组织的线粒体完整性，包括中枢神经系统的间接飞行肌和多巴胺能神经元。我们最近的研究表明，PINK1/Parkin通路通过促进线粒体裂变来影响线粒体和组织的完整性。根据这一发现和其他发现，我们假设PINK1/Parkin通路通过磷酸化和/或泛素化线粒体形态发生机制的特定组分来促进线粒体裂变。我们进一步假设，parkin和PINK1突变体中伴随线粒体裂变减少的细胞死亡源于受损线粒体向自噬体的低效传递，扩大（融合）线粒体向突触前神经末梢的运输缺陷，和/或扩大的线粒体产生过多的活性氧。为了解决这些假设，我们提出了两个具体的目标：第一个目标将通过使用已建立的线粒体融合和裂变分析系统，以及通过测试PINK1/Parkin途径的候选底物，来测试PINK1/Parkin途径是激活线粒体裂变还是抑制线粒体融合。第二个目标涉及遗传、分子和细胞生物学实验，以区分PINK1和帕金突变体中受损的线粒体裂变影响组织完整性的特定模型。通过这些实验，我们希望推进我们的长期目标，阐明帕金森病线粒体功能障碍和选择性细胞死亡的机制。公共卫生相关性：帕金森氏病或pten诱导的激酶1 （PINK1）基因的功能丧失突变是常染色体隐性家族性帕金森氏病的最常见原因。我们的提议旨在了解Parkin和PINK1的生物学作用，以及它们的突变失活如何导致神经元丢失。从我们对PINK1和Parkin的研究中获得的见解可能与更常见的特发性帕金森病有关，这一见解可能会导致这种衰弱性疾病的预防性治疗的发展。

DESCRIPTION (provided by applicant): Mitochondrial dysfunction is a common feature of Parkinson's disease, but our knowledge of the underlying molecular mechanisms is rudimentary. Recently, a genetic pathway that influences mitochondrial integrity has emerged from studies of the PINK1 and parkin genes, loss-of-function mutations of which are responsible for the majority of early-onset recessive forms of parkinsonism. The PINK1 gene encodes a serine/threonine kinase that localizes to the mitochondrial inner membrane space and to the cytoplasm, whereas parkin encodes a ubiquitin-protein ligase that localizes broadly throughout the cell, including the cytoplasm and mitochondria. Mutational analyses of highly-conserved Drosophila orthologs of PINK1 and parkin indicate that PINK1 acts upstream of Parkin in a common pathway that promotes mitochondrial integrity in a subset of tissues, including indirect flight muscle and dopaminergic neurons in the central nervous system. Our recent work indicates that the PINK1/Parkin pathway influences mitochondrial and tissue integrity by promoting mitochondrial fission. We hypothesize from this and other findings that the PINK1/Parkin pathway promotes mitochondrial fission by phosphorylating and/or ubiquitinating particular components of the mitochondrial morphogenesis machinery. We further hypothesize that the cell death accompanying reduced mitochondrial fission in parkin and PINK1 mutants derives from inefficient delivery of damaged mitochondria to autophagosomes, defective trafficking of enlarged (fused) mitochondria to presynaptic nerve terminals, and/or excessive reactive oxygen species production by enlarged mitochondria. Two specific aims are proposed to address these hypotheses: the first aim will test whether the PINK1/Parkin pathway activates mitochondrial fission or inhibits mitochondrial fusion by using established mitochondrial fusion and fission assay systems, and by testing candidate substrates of the PINK1/Parkin pathway. The second aim involves genetic, molecular and cell biological experiments to distinguish between specific models by which impaired mitochondrial fission in PINK1 and parkin mutants influences tissue integrity. From these experiments, we hope to advance our long-term goal of elucidating the mechanisms underlying mitochondrial dysfunction and selective cell death in Parkinson's disease. PUBLIC HEALTH RELEVANCE: Loss-of-function mutations in the parkin or PTEN-induced kinase 1 (PINK1) genes are collectively the most frequent cause of autosomal recessive familial Parkinson's disease. Our proposal aims to understand the biological roles of Parkin and PINK1 and how their mutational inactivation results in neuronal loss. Insight from our studies of PINK1 and Parkin are likely to be relevant to more common idiopathic forms of Parkinson's disease and this insight could lead to the development of preventative treatments for this debilitating disorder.