Gene Environment Interactions in Parkinson's Disease

描述(申请人提供)：我们的长期目标是研究环境神经毒物引起的神经变性的机制。这项建议是为了研究通过线粒体途径介导的帕金森病发病机制中的基因-环境相互作用。多年来，线粒体功能障碍一直被认为是帕金森病患者神经退行性变的主要机制，但在人类中直接证据不足，直到最近发现PTEN诱导的假定激酶1(PINK1)突变，该蛋白编码线粒体激酶。尽管PINK1突变与常染色体隐性遗传性帕金森病有关，但有趣的是，越来越多的帕金森病患者携带单一杂合突变。这些观察表明，PINK1杂合突变可能是与环境侮辱相互作用的易感因素。为了确定PINK1突变与环境毒物之间的相互作用，我们建立了稳定的细胞系，各种PINK1突变体可诱导过表达。根据我们的初步结果，我们假设PINK1突变通过一种新的机制增加细胞对环境毒物如百草枯(PQ)的敏感性：通过线粒体分裂/融合机制使线粒体碎裂。在第一个特定目标中，我们将通过线粒体分裂和融合途径，在PINK1突变(L347P)和空载体对照的n27细胞中，以及在siRNA介导的PINK1基因敲除的n27细胞中，表征PQ和MPP+(两种毒物代表不同的毒性机制)所引起的神经毒性。我们将评估细胞活力、功能结果(ATP产生、多巴胺释放和电子传输链活性)，以及线粒体碎裂(大小/形状)和线粒体分裂和融合蛋白的变化。在具体目标2中，我们将通过基因操作和小分子靶向线粒体分裂和融合途径，在突变的PINK1细胞中进行针对PQ和MPP+毒性的神经保护实验。我们将用相关的构建体转染细胞，并使用一种化学抑制剂来减弱线粒体的碎裂。这两种策略对MPP+和PQ毒性的神经保护作用将通过特定目的1中描述的细胞活性和功能分析来确定。这项研究可能通过解开一条仍未被认识的分子途径，为揭示帕金森病中细胞死亡原因中基因-环境相互作用的复杂性提供洞察力。此外，这一新的机制可能为开发帕金森氏病患者的神经保护疗法提供另一条途径。

DESCRIPTION (provided by applicant): Our long term goal is to study the mechanism of neurodegeneration induced by environmental neurotoxicants. This proposal is submitted to investigate gene-environment interactions in the pathogenesis of Parkinson's disease mediated through the mitochondrial pathway. Mitochondrial dysfunction has been proposed as a major mechanism of neurodegeneration in PD for years, but direct evidence in humans was inadequate until the recent discoveries of mutations in PTEN-induced putative kinase 1 (PINK1), which encodes a mitochondrial kinase. Although PINK1 mutations are associated with autosomal recessive PD, interestingly, an increasing number of PD patients carrying single heterozygous mutations have been reported. These observations suggest that a heterozygous PINK1 mutation may act as a susceptibility factor that interplays with environmental insults. To determine the interactions between PINK1 mutations and environmental toxicants, we have created stable cell lines with inducible over-expression of various PINK1 mutants. Based on our preliminary results, we hypothesize that PINK1 mutations increase cell susceptibility to environmental toxicants such as paraquat (PQ) through a novel mechanism: mitochondrial fragmentation via the mitochondrial fission / fusion machinery. In the first specific aim, we will characterize neurotoxicity induced by PQ and MPP+ (two toxicants representing different mechanisms of toxicity) through the mitochondrial fission and fusion pathway in N27 cells with a PINK1 mutation (L347P) and empty vector control, as well as in N27 cells with PINK1 knockdown mediated by siRNA. We will assess cell viability, functional outcomes (ATP production, dopamine release and electron transport chain activity), as well as mitochondrial fragmentation (size/shape) and alterations in mitochondrial fission and fusion proteins. In specific aim 2, we will perform neuroprotective experiments against PQ and MPP+ toxicity in mutant PINK1 cells by targeting the mitochondrial fission and fusion pathway, through genetic manipulations and a small molecule. We will transfect cells with relevant constructs and use a chemical inhibitor to attenuate mitochondrial fragmentation. The neuroprotective effects of these two strategies against MPP+ and PQ toxicity will be determined using cell viability and functional assays as described in specific aim 1. PUBLIC HEALTH RELEVANCE This research may provide insights into the complexity of gene-environment interactions in the cause of cell death as seen in Parkinson's disease by unraveling a still unrecognized molecular pathway. Furthermore, this novel mechanism may offer an additional avenue to develop neuroprotective therapy for patients with Parkinson's disease.