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.