) The goal of this project is to improve our understanding of the molecular mechanisms underlying the exo-endocytotic cycle of synaptic vesicles. More specifically, the project will address the role of phosphoinositide metabolism in the vesicle cycle. In the 1950's, Hokin and Hokin discovered for the first time that stimulation of secretion correlates with an increase in phosphoinositide metabolism. Since then numerous studies have established the key role of phosphoinositide turnover in receptor-mediated signaling pathways. However, there is also evidence that phosphoinositides are directly implicated in vesicular traffic, including synaptic vesicle exo-endocytosis, although their precise functions are unclear and deserve further investigation. Recently an inositol 5-phosphatase, synaptojanin, related to the OCRL protein (responsible for mental retardation in Lowe syndrome) was identified as a major nerve terminal protein that is associated with recycling synaptic vesicle membranes and that appears to act in concert with the GTPase dynamin and the SH3-containing protein amphiphysin in the endocytic process. Synaptojanin-like proteins are also expressed in non-neuronal cells and in yeast. The objects of this project are to test the hypothesis that phosphoinositide cleavage mediated by synaptojanin represents a crucial step in synaptic vesicle endocytosis and to identify this step. The applicants propose to characterize the biochemical properties of synaptojanin, to identify and characterize interacting proteins, to study effects of enhanced and decreased synaptojanin activity in a variety of assays, including cell free assays, cell transfection, antisense experiments, gene knockout in mice and yeast genetics. Yeast genetics will also be used to identify protein networks upstream and downstream to synaptojanin function. In addition, they plan to test the hypothesis that the phosphoinositide composition of synaptic vesicle membranes changes as they progress through the exo-endocytotic cycle and that synaptojanin is implicated in these changes. Since mechanisms of vesicular traffic are highly conserved, these studies will not only shed new light on mechanisms of synaptic transmission, but also, more generally, on vesicular transport along the secretory and endocytic pathways of all cells. Implications for the understanding and, possibly, the therapy of a variety of human conditions is anticipated, since abnormal traffic of synaptic vesicles is potentially implicated in several neurological and psychiatric diseases, and more generally, endocytosis from the plasmalemma plays an essential function in all cells and is implicated in internalization of a variety of pathogens.
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