Parkinson's disease (PD) is a disabling movement disorders caused by reduced dopamine levels in the striatum resulting from the death of dopaminergic neurons in the substantia nigra. Inactivating mutations in the parkin gene cause autosomal recessive-juvenile Parkinson disease with dystonia (PARK2), but can also cause a later onset tremor-predominant disease indistinguishable from sporadic PD. Using the yeast two-hybrid system, we have identified new parkin binding proteins: members of the synaptotagmin (syt) family, and a novel protein with homologies to the beta-adrenergic receptor. We will test the following hypotheses: Parkin regulates the level of synaptotagmin 1 (syt1) and protects against cell death induced by syt1 overexpression, whereas mutated parkin proteins do not. As syt1 is a key protein involved in vesicle docking, fusion, and recycling, changes in cellular parkin levels by overexpression or knockdown will alter synaptic vesicle release and uptake. Parkin deficiency results in increased levels of intracellular free dopamine, thus increasing oxidative stress in dopaminergic neurons. To further elucidate the role of parkin in neuronal dysfunction and cell death, three specific aims are proposed: 1) We will investigate the role of syt1 in causing cell death, and the protective role of parkin using MTT and trypan blue exclusion assays. We will use Tet-Off PC12 cells expressing regulated levels of syt1 or parkin and mouse primary dopaminergic neurons. We will co-express parkin with syt1 to determine if parkin protects these cells from syt1-induced cytotoxicity. Lentiviral vectors will be used to achieve consistent expression levels. 2) We will investigate the role of parkin on presynaptic vesicle release and uptake by measuring the release and reuptake of styryl fluorescent FM4- 64 dyes in response to overexpression and knockdown of parkin and syt1. We will use established parkin siRNAs to block the synthesis of endogenous parkin, and then determine its effect on synaptic vesicle release and reuptake. 3) We will investigate the role of parkin-synaptotagmin interaction and parkin deficiency on vesicular and cytoplasmic levels of dopamine in PC12 cells and dopaminergic neurons. The ultimate goal of this proposal is to elucidate how parkin deficiency results in dopaminergic dysfunction and cell death in Parkinson's disease.