Regulation of dense core vesicle trafficking and capture by synaptotagmin 4

神经营养物质和神经肽通过致密核心囊泡（DCVs）的运输和释放对包括代谢、疼痛感知和认知在内的广泛脑功能至关重要。与突触囊泡（SVs）不同，突触囊泡（SVs）在神经元突触末端被局部填充和再循环，dcv在高尔基体充满货物，并从细胞体细胞运输到神经元突起的远端部位。但是，将其递送到目标部位的机制尚不清楚。此外，许多神经营养物质和神经肽对突触可塑性很重要，它们在神经元活动过程中释放，调节突触和回路功能。因此，在高神经元活动条件下，特定部位对dcv的需求必然更大。但是活动依赖性dcv捕获的机制，可能在突触位点，也是未知的。我们之前发现Syt4是神经元中脑源性神经营养因子(BDNF)-窝藏dcv的整体囊泡蛋白（Dean et al, 2009）。在初步实验中，我们发现sy4在调节突触上dcv的远程运输和捕获方面具有意想不到的功能。我们发现Syt4的S135位点是海马神经元中活化JNK的下游靶点，这可能引导突触中DCVs的活性依赖性捕获。Syt4的S135位点被活跃突触上的活性JNK磷酸化，似乎破坏了Syt4与Kif1A的相互作用，导致从微管依赖性转运到通过肌动蛋白捕获突触位点上的DCVs的转变。这种机制可能允许dcv极快地招募到活跃的突触，绕过突触和体细胞之间信号传导的延迟。这个项目的总体目标是确定密集的核心囊泡如何靶向神经元的特定部位。具体来说，我们的目标是：1)通过jnk依赖性磷酸化确定Syt4在突触致密核囊泡捕获中的作用；2)确定神经元致密核囊泡是否被特异性地招募到活跃的突触中；3)确定融合后神经元致密核囊泡的命运——DCVs能否在不同的位点融合多次？如果实验成功，这将是第一个描述神经元中“密集核心囊泡循环”的实验，通过这种循环，必需的神经营养物质和神经肽被传递到突触位点。这将极大地促进我们对突触和回路是如何被大脑中的神经营养因子和神经肽的作用所调节的理解。

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.