Role of SNARE Interactions in Central Synapse Function

描述(由申请人提供)： Synaptobrevin2/VAMP2(Syb2)是一种突触小泡相关蛋白，对中枢突触的正常突触传递起关键作用。它与质膜相关蛋白Synaxin1和SNAP-25的相互作用被认为是突触囊泡融合的重要步骤。这些蛋白质被称为SNARES(可溶性NSF附着蛋白受体的首字母缩写)。尽管在表征SNARs之间的分子相互作用方面取得了广泛的进展，但它们在突触传递中的确切作用仍然不清楚。缺乏syb2的基因敲除小鼠表现出致命的表型，并在出生时死亡。在初步实验中，对从这些小鼠提取的海马区培养物的分析显示，这些小鼠持续存在受调节的胞吐作用和突触小泡回收。这一结果支持SNARES在调节神经传递的速率和钙依赖方面的作用，而不是在推动融合方面的执行功能。本项目的主要目的是验证对syb2缺陷小鼠的初步分析所产生的假说，并阐明syb2及其与其他SNARs的相互作用在哺乳动物中枢突触的突触囊泡吐出和再循环中的作用。1.为了确定在Syb2缺陷突触中观察到的突触囊泡融合和再循环的分子基础，将测试其他SNARs或囊泡膜相关蛋白是否参与剩余的神经传递。2.利用苯乙酰染料成像技术研究SNARE相互作用在囊泡回收和再循环中的作用。3.为了明确syb2功能的结构要求，将野生型和突变型syb2结构引入从基因敲除小鼠获得的海马区培养物中进行体外挽救研究。从这项研究中获得的信息还将为精神和神经疾病(如智力低下、精神分裂症和癫痫)的疾病过程提供新的见解，从而设计新的治疗策略。

DESCRIPTION (provided by applicant): Synaptobrevin2/VAMP2 (syb2) is a synaptic vesicle associated protein critical for normal synaptic transmission in central synapses. Its interaction with plasma membrane associated proteins syntaxin1 and SNAP-25 is thought to be an important step in synaptic vesicle fusion. These proteins are called SNAREs (acronym for soluble NSF attachment protein receptors). Despite extensive progress on characterization of molecular interactions between SNAREs, their precise role in synaptic transmission remains elusive. Knockout mice lacking syb2 exhibit a lethal phenotype and die at birth. In preliminary experiments, analysis of hippocampal cultures extracted from these mice revealed a persistent form of regulated exocytosis and synaptic vesicle recycling. This result supports a role for SNAREs in regulating the rate and Ca2+-dependence of neurotransmission rather than an executive function in driving fusion. The main objective of this project is to test the hypotheses that emerge from this initial analysis of syb2 deficient mice and elucidate the role of syb2 and its interactions with other SNAREs in synaptic vesicle exocytosis and recycling in mammalian central synapses. 1. To determine the molecular basis for synaptic vesicle fusion and recycling observed in syb2 deficient synapses, the involvement of other SNAREs or vesicle membrane associated proteins in the remaining neurotransmission will be tested. 2. The role of SNARE interactions in vesicle retrieval and recycling will be investigated using styryl dye imaging. 3. To define structural requirements for syb2 function, wild type and mutant syb2 constructs will be introduced to hippocampal cultures obtained from knockout mice to study in vitro rescue. Information attained from this study will also provide new insight to the disease process in psychiatric and neurological disorder such as mental retardation, schizophrenia and epilepsy leading to design of new therapeutic strategies.