Synapsins are a family of neuron-specific phosphoproteins that play a role in presynaptic regulation of neurotransmitter release and short-term plasticity. Recent studies in a number of experimental systems have provided compelling evidence that synapsins modulate axonal elongation, promote synaptogenesis and stabilize synaptic contacts. A greater understanding of the molecular mechanisms which account for these trophic effects of synapsins could lead to the identification of novel therapeutic targets for the treatment of Alzheimer's disease. Neurotrophins play essential roles in neuronal differentiation and survival, and more recently have been shown to regulate synaptic efficacy and activity- dependent plasticity. Thus, synapsins and neurotrophins share a common involvement in a number of neuronal processes. A major feature of neurotrophin-mediated signal transduction is the activation of mitogen- activated protein (MAP) kinase. We have recently demonstrated that synapsin I is a physiological substrate for MAP kinase, and have shown that this phosphorylation regulates the interactions between synapsin I and actin. As synapsin-actin interactions are believed to contribute to the effects of synapsins on synapse formation and maintenance, we now propose to further explore the possibility that MAP kinase-dependent phosphorylation of synapsins mediates some of the actions of the neurotrophins. Biochemical studies will be carried out with mammalian and Xenopus synapsin isoforms to identify the sites of phosphorylation and to characterize the enzymology and the physiological regulation of phosphorylation and dephosphorylation of synapsins I and II (Specific Aim 1). Phosphorylation dependent changes in structural and functional properties of the synapsins will be characterized (Specific Aim 2). Using neuronal culture models for synapse formation, the responsiveness to neurotrophins will be studied under conditions in which synapsin expression has been up or down-regulated, and in which phosphorylation site-mutated isoforms of synapsins are reintroduced (Specific Aim 3). Phosphorylation state-specific antibodies will be utilized to discern the temporal change, regional distribution, and subcellular localization of MAP kinase-dependent phosphorylation of synapsins I and II during normal development in vivo, and in cell culture systems during synapse formation, and will also be used to examine the distribution of MAP kinase- phosphorylated synapsins in post-mortem samples of Alzheimer's disease patients (Specific Aim 4).
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