The goal of this project is to improve our understanding of the molecular mechanisms underlying synaptic function. More specifically, the project will address the role of the phosphorylation-dephosphorylation of inositol phospholipids (phosphoinositides) in the regulation of membrane dynamics in the presynapse. Phosphoinositides, in addition to the well established roles as precursors of intracellular second messengers, mediate direct interactions between membranes and submembranous scaffolds, thus regulating a variety of cellular functions, including actin dynamics and membrane traffic. This application plans to further test the hypothesis that the vectorial progression of membranes from one compartment to the next correlates with the phosphorylation-dephosphorylation of specific phosphoinositides, and that a cycle of PI(4,5)P2 synthesis and hydrolysis is nested within the synaptic vesicle cycle exo-endocytic cycle. We will determine the contribution of specific kinases [PI 4-kinase and PI(4)P 5-kinases] and phosphatases (synaptojanin and type II phosphatase) to the synthesis and phosphorylation of P(4)P and PI(4,5)P2 needed for vesicle recycling, their sites of action and regulation, including the regulation by small GTPases. In addition, we begin to explore role of 3'-phosphorylated Pis and and type 14 phosphatase in endocytic reactions controlled by Rab5. These studies will involve mouse genetics, studies in cultured neurons, synaptosomes and cell-free systems. Given the fundamental nature of this work we expect implication for a variety of field of biology and medicine. The project will contribute to expand our knowledge on the role of membrane lipids as key regulatory molecules in brain, an area of research which is rapidly expanding. The importance of this field is underscored by several disorders of the nervous system in mice and humans that have already been shown to result from abnormal phosphoinositide metabolism.