Role of SNARE Interactions in Central Synapse Function

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DESCRIPTION (provided by applicant): Synaptobrevin2/VAMP2 (syb2) is a synaptic vesicle associated protein critical for normal synaptic transmission in central synapses. Its interaction with plasma membrane associated proteins syntaxin1 and SNAP-25 is thought to be an important step in synaptic vesicle fusion. These proteins are called SNAREs (acronym for soluble NSF attachment protein receptors). Despite extensive progress on characterization of molecular interactions between SNAREs, their precise role in synaptic transmission remains elusive. Knockout mice lacking syb2 exhibit a lethal phenotype and die at birth. In preliminary experiments, analysis of hippocampal cultures extracted from these mice revealed a persistent form of regulated exocytosis and synaptic vesicle recycling. This result supports a role for SNAREs in regulating the rate and Ca2+-dependence of neurotransmission rather than an executive function in driving fusion. The main objective of this project is to test the hypotheses that emerge from this initial analysis of syb2 deficient mice and elucidate the role of syb2 and its interactions with other SNAREs in synaptic vesicle exocytosis and recycling in mammalian central synapses. 1. To determine the molecular basis for synaptic vesicle fusion and recycling observed in syb2 deficient synapses, the involvement of other SNAREs or vesicle membrane associated proteins in the remaining neurotransmission will be tested. 2. The role of SNARE interactions in vesicle retrieval and recycling will be investigated using styryl dye imaging. 3. To define structural requirements for syb2 function, wild type and mutant syb2 constructs will be introduced to hippocampal cultures obtained from knockout mice to study in vitro rescue. Information attained from this study will also provide new insight to the disease process in psychiatric and neurological disorder such as mental retardation, schizophrenia and epilepsy leading to design of new therapeutic strategies.