Molecular Mechanism and Regulation of Asynchronous Release

项目概要/摘要 神经递质的释放受到药物和某些精神障碍如抑郁症的影响，但具体情况是 不清楚它由SNARE复合物、复合蛋白（Cpx）、突触结合蛋白（Syt）和其他蛋白质介导。异步 释放是神经递质释放的一种方式。它受到越来越多的关注，因为它可以产生或补偿 神经活动异常，在突触前可塑性中起关键作用。虽然大多数神经元的交流依赖于 在由Ca 2+传感器Syt 1、2和9（统称为“快速Syt”）触发的同步释放后。异步 在一个动作电位或一系列动作电位之后，释放具有更长的可变延迟，并且它由Ca 2+介导。 传感器Syt 7。Fast Syts和Syt 7在其Ca 2+结合C2 A中共享相似的结构域结构和高度同源性 C2B领域最近，我已经确定了Syt 1 C2B结构域和C2B结构域之间的“主要”SNARE-Syt 1接口。 SNARE复杂，揭示了“主”接口是特定于同步发布的。相比之下，Syt 7不形成 类似的“主”接口。这些结果提出了以下问题：1）Syt 7如何与SNARE合作， Cpx触发“延迟”异步释放; 2）快速Syts和Syt 7之间的区别是什么，导致了 不同的神经递质释放模式; 3）Syts和Ca ~（2+）如何调节神经递质的异步释放。总目标 阐明了非同步释放的分子机制和调控。拟议 这项研究将研究Syt 7如何介导培养的皮层神经元和Syt 1/7脑切片的异步释放 基于序列分析和新解决的晶体设计的重新引入突变的双敲除小鼠 SNARE-Cpx-Syt 1复合物结构。最后，我将揭示Syt 1/7、Cpx、SNARE复合物和 细胞膜上，观察Syt 1、Syt 7和Ca ~（2+）对细胞膜非同步释放的调节作用。结合我的工作， 同步释放，这样的结果有望提供一个更好的理解神经递质释放的作用， 神经活动和精神障碍，可能会导致新的治疗方法，用于预防和治疗各种精神疾病， 紊乱此外，该结果还有望纵向推进对突触前可塑性的理解 它被认为与记忆、学习和行为有关，并从根本上推动了 神经科学我的长期职业目标是在突触生理学的前沿领导一个世界级的实验室。我打算 利用结构生物学、单分子光谱学和成像技术，以及 神经科学作为研究工具为了补充我在博士期间获得的结构生物学知识，我将 在阿克塞尔·布伦杰博士的实验室担任博士后研究员，完成该奖项的指导阶段 和托马斯·苏德霍夫我设计了一个雄心勃勃的研究职业发展计划，以实现我的近期目标， 指导期间：1）扩展我的先进科学和技术知识，2）准备我的过渡到 独立在Brunger和Südhof博士的指导下，我将遵循一个结构化的培训计划，以提高 我的专业能力，建立和运行自己的实验室。斯坦福大学医学院将为我提供理想的 环境充分受益于这个奖项，并成为一个成功的独立科学家。

Project Summary/Abstract Neurotransmitter release is influenced by drugs, and certain mental disorders such as depression, but the details are unclear. It is mediated by the SNARE complex, complexin (Cpx), synaptotagmin (Syt), and other proteins. Asynchronous release is a mode of neurotransmitter release. It has received increasing attention as it may generate or compensate abnormal neural activities, and play critical role in pre-synaptic plasticity. While most neuronal communication relies upon synchronous release triggered by the Ca2+ sensors Syt1, 2 and 9 (collectively called “fast Syts”). Asynchronous release has a longer, variable delay after an action potential or series of action potentials, and it is mediated by the Ca2+ sensor Syt7. Fast Syts and Syt7 share a similar domain structure and a high degree of homology in their Ca2+-binding C2A and C2B domains. Recently I have identified the "primary" SNARE-Syt1 interface between Syt1 C2B domain and the SNARE complex, revealing the "primary" interface is specific for synchronous release. In contrast, Syt7 does not form a similar "primary" interface. These results raise the following questions: 1) how does Syt7's cooperate with SNAREs and Cpx to trigger "delayed" asynchronous release; 2) what is the difference between fast Syts and Syt7 that gives rise to the different modes of neurotransmitter release; 3) how is asynchronous release regulated by Syts and Ca2+. The overall goal of this proposal is the elucidation of the molecular mechanism and regulation of asynchronous release. The proposed study will examine how Syt7 mediates asynchronous release in cultured cortical neurons and brain slices from Syt1/7 double knockout mice with re-introduction of mutates designed based on sequence analysis and a newly solved crystal structure of SNARE-Cpx-Syt1 complex. Finally, I will reveal the interaction among Syt1/7, Cpx, SNARE complex and membrane, and investigate the regulation of asynchronous release by Syt1, Syt7 and Ca2+. Combined with my work on synchronous release, such results are expected to provide a better understanding of the roles of neurotransmitter release in neural activity and mental disorders, may lead to new therapeutics for the prevention and treatment of a variety of mental disorders. In addition, the results are also expected to vertically advance the understanding of pre-synaptic plasticity which is believed to be related to memory, learning, and behavior, as well as fundamentally advance the field of neuroscience. My long-term career goal is to lead a world-class laboratory in the forefront of synaptic physiology. I intend to use techniques in structural biology, single molecule spectroscopy and imaging, as well as key techniques in neuroscience as investigation tools. To complement my knowledge of structural biology acquired during my PhD, I will carry out the mentored phase of this Award as a postdoctoral Research Fellow in the laboratories of Drs. Axel Brunger and Thomas Südhof. I have designed an ambitious research career development plan to achieve my immediate goals for the mentored period: 1) expand my advanced scientific and technical knowledge, and 2) prepare my transition to independence. Under the mentorship of Drs. Brunger and Südhof, I will follow a structured training program to enhance my professional abilities to establish and run my own laboratory. Stanford Medical School will provide me with ideal environment to fully benefit from this Award and become a successful independent scientist.