The ability of neurons and endocrine cells to synthesize, package and secrete neurotransmitter or peptide hormones in response to the appropriate stimulus is critical to the proper function of both the nervous system and the endocrine system. Recent evidence from a number of laboratories suggests that synaptic vesicles and the presynaptic secretory apparatus may be the primary substrates for many forms of synaptic modulation, including facilitation, long term potentiation, and other molecular mechanisms potentially involved in memory. The long term goals of my research are to understand the molecular mechanisms underlying the biogenesis of synaptic vesicles, calcium-dependent exocytosis, membrane recycling, and vesicle transport, and to determine if these functions can be modulated by neuronal environment. This proposal is designed to specifically address the role of four synaptic vesicle-specific membrane proteins in the biogenesis and secretion of synaptic vesicles. These role that the proteins p38, SV2, p65, and synaptobrevin play in regulated or constitutive secretion will be assayed using two different techniques. First, antibodies to each of the proteins will be used to interfere with their function and to inactivate each protein using chromophore-assisted laser inactivation (CALI). Antibodies will be delivered to the cytoplasm of PC 12 cells and synaptosomes after permeabilization of the plasma membranes by electroporation. The calcium-dependent secretion of 3H-norepinephrine from cells and synaptosomes will be assayed after inactivation of each of these integral membrane proteins. The constitutive secretion of a heparan sulfate proteoglycan will also be measured in PC 1 2 cells. A second series of experiments will measure the effects of antisense RNAs encoding the synaptic vesicle proteins in PC 12 cells on regulated and constitutive secretion and endocytosis of the remaining vesicle proteins. To test the hypothesis that synaptic vesicle proteins play a role in the biogenesis of synaptic vesicles, each protein will be expressed by transfection of cDNA in nonneuronal cells. The ability of that protein to direct the subcellular localization of other synaptic vesicle proteins and two cytoplasmic proteins, synapsin I and rab3, will be assayed by immunocytochemistry, immunofluorescence, and subcellular fractionation.
