The research supported by our current grant has investigated the role of protein phosphorylation in the regulation of neurotransmitter release. Specifically, the data obtained from a variety of experimental approaches have provided support for a model by which synapsin I, a synaptic vesicle- associated phosphoprotein, regulates neurotransmitter release by affecting the movement of synaptic vesicles from a """"""""reserve"""""""" pool to a """"""""readily-releasable"""""""" pool. We have further demonstrated that the phosphorylation state of synapsin I regulates this biological function of the protein. We have established that synapsin I consists of two polypeptides, synapsin Ia and synapsin lb, which we have cloned and sequenced. The sequencing data indicate that synapsins Ia and lb are highly homologous to another pair of synaptic vesicle-associated phosphoproteins, synapsins IIa and lIb, which we have also cloned and sequenced. The current grant also supported the study of another phosphoprotein, MARCKS (myristoylated, alanine-rich, C-kinase substrate), previously called 87 k protein. This renewal application proposes to study the mechanisms by which the family of synapsin proteins (Ia, lb, IIa, and IIb) regulates neurotransmitter release. To date, most of our experiments have studied the effects of synapsins Ia and lb when present together. We now propose to study the potential functional differences among the four isoforms of synapsin. Using a variety of model neuronal systems, we will introduce each of the four synapsin isoforms alone and in various combinations. We will introduce the synapsins in both phosphorylated and dephosphorylated states, in order to determine the functional effects of phosphorylation on specific sites of the synapsin molecules. We will utilize mutated forms of synapsin to determine functional effects following deletions of phosphorylation sites and other functional domains. The principal measures of function will be neurotransmitter release, the development of facilitation, and the maintenance of long-term potentiation. We will study the molecular mechanisms underlying the function of the synapsins by using systems composed of purified components which have been reconstituted in vitro to examine the interactions between synapsin molecules, synaptic vesicles, and F-actin. Finally, we will study the differential distribution of the synapsins and the regulation of synapsin gene expression because our data indicate (a) that the four synapsin isoforms are differentially expressed in subpopulations of neurons, (b) that the expression of the synapsins can be regulated by transcriptional control via extracellular messengers (e.g., hormones), and (c) that the expression of the synapsins induces formation of presynaptic terminals.

National Institute of Health (NIH)
National Institute of Mental Health (NIMH)
Method to Extend Research in Time (MERIT) Award (R37)
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Neurosciences Research Review Committee (BPN)
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Rockefeller University
Other Basic Sciences
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New York
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Tomiyama, Katsunori; Makihara, Yasuyuki; Yamamoto, Hiroshi et al. (2006) Disruption of orofacial movement topographies in congenic mutants with dopamine D5 but not D4 receptor or DARPP-32 transduction 'knockout'. Eur Neuropsychopharmacol 16:437-45
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Hilfiker, Sabine; Benfenati, Fabio; Doussau, Frederic et al. (2005) Structural domains involved in the regulation of transmitter release by synapsins. J Neurosci 25:2658-69
Nally, Rachel E; Kinsella, Anthony; Tighe, Orna et al. (2004) Ethologically based resolution of D2-like dopamine receptor agonist-versus antagonist-induced behavioral topography in dopamine- and adenosine 3',5'-monophosphate-regulated phosphoprotein of 32 kDa ""knockout"" mutants congenic on the C57BL/6 genetic back J Pharmacol Exp Ther 310:1281-7
Giovedi, Silvia; Darchen, Francois; Valtorta, Flavia et al. (2004) Synapsin is a novel Rab3 effector protein on small synaptic vesicles. II. Functional effects of the Rab3A-synapsin I interaction. J Biol Chem 279:43769-79
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Giovedi, Silvia; Vaccaro, Paola; Valtorta, Flavia et al. (2004) Synapsin is a novel Rab3 effector protein on small synaptic vesicles. I. Identification and characterization of the synapsin I-Rab3 interactions in vitro and in intact nerve terminals. J Biol Chem 279:43760-8
Gitler, Daniel; Takagishi, Yoshiko; Feng, Jian et al. (2004) Different presynaptic roles of synapsins at excitatory and inhibitory synapses. J Neurosci 24:11368-80
Bloom, Ona; Evergren, Emma; Tomilin, Nikolay et al. (2003) Colocalization of synapsin and actin during synaptic vesicle recycling. J Cell Biol 161:737-47
Nally, Rachel E; McNamara, Fergal N; Clifford, Jeremiah J et al. (2003) Topographical Assessment of Ethological and Dopamine Receptor Agonist-Induced Behavioral Phenotype in Mutants with Congenic DARPP-32 'Knockout'. Neuropsychopharmacology 28:2055-63

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