Parkinson's disease is a debilitating movement disorder characterized by death of dopaminergic midbrain neurons. Mutations in PTEN-induced putative kinase 1 (PINK1) have been recently identified in familial parkinsonism, and some populations of early onset sporadic Parkinson's disease. In order to assess potential causative effects of mutations, it is necessary to first understand the regulation and function of wild type PINK1. One key unresolved issue is whether PINK1 is a functional kinase. This proposal is designed to investigate the feasibility of a novel research direction focused upon the regulation of PINK1 phosphorylation, localization, and function under conditions of stress elicited by parkinsonian neurotoxins. Our previous studies suggest that mitochondrially targeted kinase pathways play a central role in toxin-induced dopaminergic cell death. Thus, we hypothesize that PINK1 is a functional kinase that interacts with a set of cell death- regulatory kinases that localize to mitochondria. To address this hypothesis, we will determine if PINK1 shows kinase activity that is regulated by and participates in known mitochondrially targeted signaling pathways, using both cell free and culture systems. We will determine whether subcellular localization of PINK1 is regulated in neurotoxin models of dopaminergic cell death, and compare the effects of PINK1 and a kinase dead PINK1 mutant on mitochondrial cell death in dopaminergic cell lines and primary midbrain cultures. Completion of these studies will lead to a better understanding of the role of PINK1 during dopaminergic neuronal cell injury, and how it participates in signaling networks that influence whether these cells live or die. Following this exploratory/developmental phase, more extensive studies will be aimed at identifying downstream targets of PINK1 activation and the effects of disease-associated mutations on PINK1 regulation, as these may lead to novel therapeutic approaches. In addition, a group of immunochemical and molecular reagents will have been created that can facilitate the work of other investigators studying mechanisms of Parkinson's and related diseases. [unreadable] [unreadable]