Molecular Mechanism and Regulation of Asynchronous Release

项目摘要/摘要 神经递质的释放会受到药物和某些精神障碍(如抑郁症)的影响，但细节是 不清楚。它由SNARE复合体、复合蛋白(CPX)、突触素(SYT)等蛋白质介导。异步 释放是神经递质释放的一种方式。它受到了越来越多的关注，因为它可能产生或补偿 异常的神经活动，并在突触前可塑性中起关键作用。而大多数神经元通讯依赖于 在由钙传感器Syt1、2和9(统称为“快速系统”)触发的同步释放时。异步 在一个或一系列动作电位之后，释放有一个更长的可变延迟，它是由钙调节的 传感器系统7。FAST Syts和Syt7在其与钙结合的C2a上具有相似的结构域结构和高度的同源性 和C2B域。最近，我发现了Syt1 C2B域和Syt1之间的“主”SNARE-Syt1接口 陷阱复杂，揭示了“主要”界面是特定于同步释放。相比之下，Syt7不会形成 类似的“主”界面。这些结果提出了以下问题：1)Syt7的S是如何配合SNARS和 Cpx触发“延迟的”异步释放；2)FAST系统和Syt7之间的区别是什么 神经递质释放的不同方式；3)Syts和Ca~(2+)是如何调节非同步释放的。总目标 这一建议的重点是阐明非同步释放的分子机制和调控。建议数 这项研究将研究Syt7如何在培养的皮层神经元和脑片中介导Syt1/7的异步释放 双基因敲除小鼠，重新引入基于序列分析的突变产物和一种新解决的晶体 SNARE-CPX-Syt1复合体的结构。最后，我将揭示Syt1/7、CPX、SNARE Complex和 并研究了Syt1、Syt7和钙离子对细胞非同步释放的调节。结合我在 同步释放，这样的结果有望提供更好的理解神经递质释放的作用。 神经活动和精神障碍，可能导致新的治疗方法来预防和治疗各种精神疾病 精神错乱。此外，这些结果也有望在垂直方向上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，并从根本上促进了 神经科学。我的长期职业目标是领导一个处于突触生理学前沿的世界级实验室。我打算 利用结构生物学、单分子光谱学和成像技术，以及 神经科学作为调查工具。为了补充我在博士期间学到的结构生物学知识，我将 作为阿克塞尔·布伦格博士实验室的博士后研究员，执行该奖项的指导阶段 托马斯·S·吕德霍夫。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 辅导期：1)扩展我的先进科技知识，2)为我的过渡做准备 独立。在布伦格博士和S博士的指导下，我将遵循一个有组织的培训计划，以提高 我的专业能力来建立和管理我自己的实验室。斯坦福医学院将为我提供理想的 环境，以充分受益于该奖项，并成为一名成功的独立科学家。

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.