Role of Mitochondrial CYP2E1 in Chemical Exposure-Driven Neurodegeneration

线粒体 CYP2E1 在化学品暴露驱动的神经变性中的作用

基本信息

批准号：
9319548
负责人：
Jessica Helene Hartman
金额：
$ 5.67万
依托单位：
DUKE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-08-01 至 2019-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9319548
关键词：
Affect Age Biological Assay Bulla CYP2E1 gene Caenorhabditis elegans Cell Death Cells Cessation of life Chemical Exposure Clinical Development Disease Progression Early Diagnosis Endoplasmic Reticulum Environment Environmental Pollutants Environmental Risk Factor Exposure to Fellowship Fluorescence Microscopy Functional disorder Genetic Genetic Risk Genus Hippocampus Goals Health Hepatocyte Human In Situ Intervention Knowledge Lead Link Liver Longevity Measurement Measures Membrane Potentials Metabolic Metabolic Activation Metabolism Methanol Mitochondria Mitochondrial DNA Mitochondrial Proteins Modeling Mutation Nematoda Nerve Degeneration Neurodegenerative Disorders Neurons Organelles PC12 Cells Parkinson Disease Pathogenesis Persons Pesticides Pharmaceutical Preparations Play Population Preventive measure Research Respiration Risk Assessment Risk Factors Role Solvents Substantia nigra structure Symptoms Testing Toxic Environmental Substances Toxic effect Toxicant exposure Transgenic Organisms Trichloroethylene United States National Institutes of Health Whole Organism base cell injury dopaminergic neuron experimental study improved in vivo instrument mitochondrial dysfunction mitochondrial membrane nervous system disorder neuron loss neuroprotection parkin gene/protein prophylactic respiratory protein small molecule targeted treatment therapeutic target toxicant

项目摘要

Project Summary/Abstract Parkinson's Disease (PD) is one of the most common neurodegenerative disorders, affecting >1% of the population over age 60. PD pathophysiology includes preferential loss of dopaminergic neurons in the substantia nigra (SN), and has been linked to both genetic and environmental risk factors. However, genetics can only explain 5-10% of late-onset cases, so environment may play a larger role in most cases. PD has been demonstrated at the cellular level to involve mitochondrial dysfunction. Multiple environmental toxicants known to be toxic to mitochondria require metabolic activation to exert this toxicity through reactive metabolites that damage vulnerable mitochondrial proteins and DNA. Two well-known examples of metabolically activated compounds are trichloroethylene, a pervasive environmental pollutant, and methanol, a common solvent and fuel additive; both trichloroethylene and methanol are activated by cytochrome P450 2E1 (CYP2E1). CYP2E1 has traditionally been studied in the endoplasmic reticulum (ER) of liver hepatocytes; however, it is also expressed in SN dopaminergic neurons. Moreover, CYP2E1 is bimodally targeted to ER and mitochondria in those cells. Relatively little is known about the consequence of mitochondrial CYP2E1 localization on mitochondrial integrity and/or function and ultimately pathogenesis of neurodegenerative diseases. This NIH postdoctoral fellowship proposal will assess the role of mitochondrial CYP2E1 (mtCYP2E1) in mitochondrial dysfunction and neurodegeneration due to the activation of trichloroethylene and methanol in transgenic human CYP2E1-expressing PC-12 cells and C. elegans (in vivo). We hypothesize that activation of these toxicants by CYP2E1 within mitochondrial organelles will cause localized damage that triggers mitochondrial dysfunction and drives neurodegeneration. To test this hypothesis, mitochondrial dysfunction will be assessed in cells and in vivo through a battery of mitochondrial assays including whole-cell and whole-organism respiration assays using a Seahorse XF instrument, ATP measurements, and assessment of mitochondrial membrane potential. Neurodegeneration induced by methanol and trichloroethylene will be assessed in vivo by fluorescence microscopy of live C. elegans nematodes bearing GFP-tagged neurons. Overall, knowledge gained from this study will aid in development of more accurate risk assessments for neurodegeneration triggered by exposures to environmental toxicants and could provide new targets for intervention and/or neuroprotection. The following aims will be pursued: Specific Aim 1: Determine CYP2E1-dependent mitochondrial dysfunction induced by toxicants. Specific Aim 2: Assess the role of mitochondrial CYP2E1 in toxicant-induced neurodegeneration.

项目概要/摘要帕金森病 (PD) 是最常见的神经退行性疾病之一，影响超过 1% 的人 60 岁以上人群。PD 病理生理学包括多巴胺能神经元优先丧失黑质（SN），与遗传和环境风险因素有关。然而，遗传学只能解释 5-10% 的迟发病例，因此环境在大多数情况下可能发挥更大的作用。 PD 已在细胞水平上被证明涉及线粒体功能障碍。已知多种环境毒物对线粒体有毒需要代谢激活才能通过反应性代谢物发挥这种毒性损害脆弱的线粒体蛋白质和 DNA。代谢激活的两个著名例子化合物是三氯乙烯（一种普遍存在的环境污染物）和甲醇（一种常见溶剂）燃油添加剂；三氯乙烯和甲醇均被细胞色素 P450 2E1 (CYP2E1) 激活。 CYP2E1 传统上是在肝细胞的内质网（ER）中进行研究；然而，这也是在 SN 多巴胺能神经元中表达。此外，CYP2E1 双峰靶向 ER 和线粒体那些细胞。关于线粒体 CYP2E1 定位对线粒体的影响知之甚少。线粒体完整性和/或功能以及神经退行性疾病的最终发病机制。该 NIH 博士后奖学金提案将评估线粒体 CYP2E1 (mtCYP2E1) 在由于三氯乙烯和甲醇的活化导致线粒体功能障碍和神经变性转基因人表达 CYP2E1 的 PC-12 细胞和秀丽隐杆线虫（体内）。我们假设激活线粒体细胞器内的 CYP2E1 释放这些毒物会造成局部损伤，从而引发线粒体功能障碍并导致神经退行性变。为了验证这一假设，线粒体将通过一系列线粒体检测（包括全细胞检测）在细胞和体内评估功能障碍使用 Seahorse XF 仪器进行整个生物体呼吸测定、ATP 测量和线粒体膜电位的评估。甲醇诱导的神经变性三氯乙烯将通过活体线虫的荧光显微镜进行体内评估 GFP 标记的神经元。总体而言，从本研究中获得的知识将有助于开发更准确的风险对暴露于环境毒物引发的神经退行性疾病进行评估，可以提供新的干预和/或神经保护的目标。我们将追求以下目标：具体目标 1：确定毒物诱导的 CYP2E1 依赖性线粒体功能障碍。具体目标 2：评估线粒体 CYP2E1 在毒物诱导的神经变性中的作用。