Parkinson's disease (PD) is a progressive neurodegenerative disorder characterized by both motor and non- motor disturbances. While the etiology of the disease is unknown, genetic mutations responsible for familial forms of PD likely provide critical molecular insights into the underlying mechanisms of idiopathic disease. Loss-of-function mutations in the ubiquitin E3 ligase parkin are the most common cause of autosomal recessive PD. Parkin is now widely recognized as a pro-survival protein that possesses broad ranging effects on mitochondrial biology, and beyond. A more complete comprehension of these functions will will lead to a firmer understanding of the neurodegenerative process in PD, as well as the identification of new pathways that may be targeted for disease modification. The long-term goal of our work is to uncover the molecular machinery responsible for parkin's protective effects in neurons. We propose that both the cytosolic and mitochondrial-localized pools of parkin play important roles in the maintenance of healthy neurons. This application will explore the role of cytosolic parkin in its neuro-protective function in human neurons. Using engineered isogenic and patient based WT and parkin null pluripotent stem cells differentiated to human neuronal fates, we will uncover the conditions that activate the cytoplasmic pool of parkin. We will identify the upstream proteins that control parkin activation and its protection against cell death in human neurons. Then, we will explore the role of BH3 domains in the recognition of an expanding class of putative substrates of cytoplasmic parkin E3 ligase activity. The pathways found to regulate neuronal parkin function may shed light on innovative targets for therapeutic intervention to up-regulate parkin activity and induce neuro-protection in neurologic disease.