Role of Mitochondrial CYP2E1 in Chemical Exposure-Driven Neurodegeneration

项目总结/摘要 帕金森病（PD）是最常见的神经退行性疾病之一，影响>1%的帕金森病患者。 60岁以上人口。PD的病理生理学包括帕金森氏病中多巴胺能神经元的优先损失。 黑质（SN），并与遗传和环境风险因素有关。然而，遗传学 仅能解释5-10%的迟发性病例，因此环境在大多数病例中可能起更大的作用。PD已经 在细胞水平上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活化以通过反应性代谢物发挥这种毒性， 破坏脆弱的线粒体蛋白质和DNA。两个众所周知的代谢活化的例子 化合物是三氯乙烯，一种普遍的环境污染物，和甲醇，一种常见的溶剂， 燃料添加剂;三氯乙烯和甲醇都被细胞色素P450 2 E1（CYP 2 E1）激活。CYP2E1 传统上在肝细胞的内质网（ER）中进行了研究;然而， 在SN多巴胺能神经元中表达。此外，CYP 2 E1是双峰靶向ER和线粒体， 这些细胞。关于线粒体CYP 2 E1定位对细胞凋亡的影响知之甚少。 线粒体完整性和/或功能以及最终神经变性疾病的发病机制。 这项NIH博士后奖学金提案将评估线粒体CYP 2 E1（mtCYP 2 E1）在以下方面的作用： 线粒体功能障碍和神经变性由于三氯乙烯和甲醇的激活， 转基因人CYP 2 E1表达PC-12细胞和C. elegans（in vivo）.我们假设激活 CYP 2 E1在线粒体细胞器内对这些毒物的毒性会引起局部损伤， 线粒体功能障碍和驱动神经变性。为了验证这一假设， 将通过一系列线粒体测定，包括全细胞测定， 和使用Seahorse XF仪器的全生物体呼吸测定，ATP测量，以及 线粒体膜电位的评估。甲醇诱导的神经变性和 三氯乙烯将通过活C的荧光显微镜在体内进行评估。线虫性线虫 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 评估暴露于环境毒物引发的神经变性，并可能提供新的 用于干预和/或神经保护的靶点。将努力实现以下目标： 具体目的1：确定毒物诱导的CYP 2 E1依赖性线粒体功能障碍。 具体目标2：评估线粒体CYP 2 E1在毒物诱导的神经变性中的作用。

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.