Regulation of dense core vesicle trafficking and capture by synaptotagmin 4

致密核心囊泡(DCV)转运和释放神经营养因子和神经肽对包括新陈代谢、痛觉和认知在内的一系列大脑功能是必不可少的。与突触小泡(SVS)不同的是，突触小泡在神经元的突触终末被局部再灌流和回收，DCV在高尔基体装满货物，从胞体运输到神经元突起的远端。但送货到目标地点的机制尚不清楚。此外，许多神经营养因子和神经肽对突触的可塑性很重要，并在神经元活动过程中释放出来，调节突触和回路功能。因此，在神经元活动较高的情况下，特定部位对DCV的需求必然更大。但依赖活动捕获DCV的机制，可能是在突触位置，也是未知的。我们之前发现Syt4是一种存在于神经元中携带DCV的脑源性神经营养因子(BDNF)上的完整囊泡蛋白(Dean等人，2009年)。在初步实验中，我们发现了Syt4在调节突触处DCV的远程运输和捕获方面的一个意想不到的功能。我们发现，Syt4的S135位点是海马神经元中激活的JNK的下游靶点，这可能指导了突触处DCV的活性依赖捕获。在活跃的突触上，活性JNK对Syt4的S135位点进行磷酸化，似乎破坏了Syt4与Kif1A的相互作用，导致从微管依赖的运输转变为通过肌动蛋白在突触部位捕获DCV。这种机制可能允许DCV极快地招募到活跃的突触，绕过与突触和胞体之间的信号传递相关的延迟。该项目的总体目标是确定致密的核心小泡如何定位于神经元中的特定位置。具体地说，我们的目标是1)确定Syt4在通过JNK依赖的磷酸化在突触捕获致密核心小泡中的作用，2)确定神经元中的致密核心小泡是否被特异性地招募到活跃的突触中，以及3)确定融合后神经元中致密核心小泡的命运-DCV能否在不同的位置多次融合？如果成功，拟议中的实验将是第一次描述神经元中的“密集核心囊泡周期”，通过这个周期，基本的神经营养因子和神经肽被运送到突触位置。这将极大地提高我们对突触和电路是如何由大脑中的神经营养素和神经肽的作用进行调节的理解。

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.