Regulation of dense core vesicle trafficking and capture by synaptotagmin 4

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The transport and release of neurotrophins and neuropeptides by dense core vesicles (DCVs) is essential for a wide range of brain functions, including metabolism, pain perception and cognition. Unlike synaptic vesicles (SVs), which are locally refilled and recycled at synaptic terminals in neurons, DCVs are filled with cargo at the Golgi and trafficked from the cell soma to distal sites in neuronal processes. But the mechanism by which delivery to target sites occurs is unknown. In addition, many neurotrophins and neuropeptides are important for synaptic plasticity and are released during neuronal activity to modulate synapse and circuit function. Thus, the demand for DCVs at specific sites in conditions of high neuronal activity is necessarily greater. But the mechanisms of activity-dependent capture of DCVs, potentially at synaptic sites, are also unknown. We previously identified Syt4 as an integral vesicle protein present on brain-derived neurotrophic factor (BDNF)-harboring DCVs in neurons (Dean et al., 2009). In preliminary experiments, we uncovered an unexpected function of Syt4, in regulating the long-range trafficking and capture of DCVs at synapses. We found that the S135 site of Syt4 is a downstream target of activated JNK in hippocampal neurons, which may steer the activity-dependent capture of DCVs at synapses. Phosphorylation of the S135 site of Syt4 by active JNK at active synapses, appears to destabilize the interaction of Syt4 with Kif1A, leading to a switch from microtubule-dependent trafficking to capture of DCVs at synaptic sites by actin. This mechanism would potentially allow extremely fast recruitment of DCVs to active synapses, bypassing delays associated with signaling between synapses and the soma. The overall goal of this project is to determine how dense core vesicles are targeted to specific sites in neurons. Specifically, we aim to 1) Determine the role of Syt4 in the capture of dense core vesicles at synapses via JNK-dependent phosphorylation, 2) Determine if dense core vesicles in neurons are specifically recruited to active synapses, and 3) Determine the fate of dense core vesicles in neurons following fusion - can DCVs fuse multiple times at different sites? If successful, the proposed experiments would be the first to describe a "dense core vesicle cycle" in neurons, by which essential neurotrophins and neuropeptides are delivered to synaptic sites. This would significantly advance our understanding of how synapses and circuits are modulated by the action of neurotrophins and neuropeptides in the brain.