Role of Mitochondrial CYP2E1 in Chemical Exposure-Driven Neurodegeneration

项目摘要/摘要 帕金森氏病(PD)是最常见的神经退行性疾病之一，影响1%的 60岁以上的人口。帕金森病的病理生理学包括多巴胺能神经元的优先丢失。 黑质(SN)，并与遗传和环境风险因素有关。然而，遗传学 只能解释5%-10%的晚发性病例，因此环境可能在大多数病例中发挥更大的作用。警察局一直在 在细胞水平上被证明涉及线粒体功能障碍。已知的多种环境毒物 要对线粒体产生毒性，需要新陈代谢激活，通过下列反应代谢产物来施加这种毒性 破坏易受攻击的线粒体蛋白质和DNA。两个众所周知的代谢激活的例子 化合物是三氯乙烯，一种普遍存在的环境污染物，以及甲醇，一种常见的溶剂和 燃料添加剂：三氯乙烯和甲醇都被细胞色素P450 2E1(CYP2E1)激活。细胞色素P450-2 传统上是在肝细胞的内质网(ER)中进行研究；然而，它也是 在黑质多巴胺能神经元中有表达。此外，在内质网和线粒体中，CYP2E1是双峰靶向的。 那些细胞。线粒体CYP2E1基因定位的后果目前知之甚少。 线粒体的完整性和/或功能以及神经退行性疾病的最终发病机制。 这项美国国立卫生研究院博士后研究计划将评估线粒体细胞色素P450_2E_1(Mt_CYP_2E_1)在 三氯乙烯和甲醇激活引起的线粒体功能障碍和神经变性 转人CYP2E1的PC-12细胞和线虫(体内)。我们假设激活 线粒体细胞器中的CYP2E1会引起局部损伤，从而触发 线粒体功能障碍并导致神经退化。为了验证这一假设，线粒体 功能障碍将通过包括全细胞在内的一系列线粒体分析在细胞和体内进行评估 和使用SeaHorse XF仪器的整体呼吸分析，ATP测量，以及 线粒体膜电位的评估。甲醇和乙醇诱导的神经变性 三氯乙烯将通过活的线虫的荧光显微镜进行体内评估 绿色荧光蛋白标记的神经元。总体而言，从这项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变性的评估，并可能提供新的 干预和/或神经保护的目标。我们将会达致以下目标： 特异性目的1：检测毒物引起的细胞色素P450 2依赖的线粒体功能障碍。 具体目的2：评估线粒体细胞色素P450 2在毒物诱导的神经退行性变中的作用。

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.