PINK1 in the Regulation of Macroautophagy and Parkinsonian Neurodegeneration.

描述（由申请人提供）：神经退行性疾病折磨着数百万人，从儿童到老年人。由于没有已知的病因或有效的治疗方法，这类疾病超出了大脑补偿神经元可塑性的能力，导致身体虚弱。与神经退行性变有关的两种途径是线粒体功能障碍和巨噬。本研究计划的重点是这两种途径作为轴突和树突变性（神经突变性）的调节因子之间的相互作用。巨噬（以下简称自噬）是长寿命蛋白质和细胞器（包括线粒体）的大量分解代谢。虽然基础水平的自噬是健康所必需的，但失调已被认为是神经突变性的原因之一。线粒体是普遍存在的细胞器，在神经元的正常功能和可塑性中起着关键作用。巨噬调节失调和线粒体功能障碍/缺失与帕金森病（PD）的发病机制有关。最近的研究表明pten诱导的激酶1 （PINK1）具有神经保护活性。此外，PINK1与常染色体隐性遗传PD有关，可能是通过功能突变的丧失。我假设PINK1信号通过其对自噬/有丝自噬的影响来调节神经突变性。PINK1在调节神经突变性、自噬和线粒体丢失中的作用将通过体外生化和显微镜研究来测量。目的1将确定PINK1的表达水平是否调节自噬并防止引起神经突变性的神经元损伤。Aim 2将确定PINK1敲低或疾病相关突变是否会诱导自噬和神经突变性。我还将确定PINK1敲低或突变体是否需要自噬来治疗神经突变性。Aim 3将使用无偏见的蛋白质组学方法来鉴定PINK1的下游线粒体靶点。磷酸化蛋白谱将分别在PINK1缺陷小鼠或细胞和野生型小鼠或细胞之间进行比较。潜在的下游PINK1靶点将被鉴定为在PINK1缺乏样本中减少的野生型样品中的磷酸化蛋白。使用RNAi，我将确定假定的下游PINK1介质是否需要PINK1神经保护作用。通过确定调节pink1介导的神经保护和自噬“自我消化”的潜在途径，本研究结果将为帕金森病及相关疾病的神经突变性机制提供深入了解。除了为申请人提供博士前培训外，本研究计划旨在发现治疗神经退行性疾病的有希望的药物靶点。

DESCRIPTION (provided by applicant): Neurodegenerative diseases afflict millions of individuals ranging from children to the elderly. Without known causes or effective therapies, this class of diseases outstrips the brain's capacity for compensatory neuronal plasticity with debilitating results. Two pathways involved with neurodegeneration are mitochondrial dysfunction and macroautophagy. This research proposal focuses on the interplay between these two pathways as regulators of axonal and dendritic degeneration (neurite degeneration). Macroautophagy (hereafter, autophagy) is the bulk catabolism of long-lived proteins and organelles, including mitochondria. While basal levels of autophagy are required for health, dysregulation has been implicated as a cause for neurite degeneration. Mitochondria are ubiquitous organelles that play key roles in the proper function and plasticity of neurons. Dysregulated macroautophagy and mitochondrial dysfunction/loss have been implicated in the pathogenesis of Parkinson's disease (PD). Recent studies have illustrated the neuroprotective activity of the PTEN-induced kinase 1 (PINK1). Additionally, PINK1 has been linked to autosomal recessive PD, presumably through loss of function mutations. I hypothesize that PINK1 signaling regulates neurite degeneration through its effects on autophagy/mitophagy. The role of PINK1 in regulating neurite degeneration, autophagy, and mitochondrial loss will be measured in vitro by biochemical and microscopy studies. Aim 1 will determine whether levels of PINK1 expression regulate autophagy and protect against neuronal injuries that cause neurite degeneration. Aim 2 will determine whether PINK1 knockdown or disease-associated mutations induce autophagy and neurite degeneration. I will also determine whether PINK1 knockdown or mutants require autophagy for neurite degeneration. Aim 3 will use a non-biased proteomies approach to identify downstream mitochondrial targets of PINK1. Phosphorylated protein profiles will be compared between PINK1 deficient mice or cells and wild type mice or cells, respectively. Potential downstream PINK1 targets will be identified as phosphoproteins in wild-type samples that are reduced in PINK1 deficient samples. Using RNAi, I will determine if the putative downstream PINK1 mediators are required for the PINK1 neuroprotective effects. By identifying potential pathways that regulate PINK1-mediated neuroprotection and autophagic "selfdigestion", the results of this study will provide insight into the mechanisms of neurite degeneration in Parkinson's and related diseases. In addition to providing predoctoral training to the applicant, this research proposal is designed to uncover promising drug targets for the treatment of neurodegenerative diseases.