Role of SNARE Interactions in Central Synapse Function

描述（由申请人提供）： 突触小泡蛋白2/VAMP 2（synaptobrevin 2/VAMP 2，syb 2）是一种突触囊泡相关蛋白，在中枢突触中对正常突触传递起关键作用。它与质膜相关蛋白syntaxin 1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SNARE（可溶性NSF附着蛋白受体的首字母缩写）。尽管在SNARE之间的分子相互作用的表征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难以捉摸。缺乏syb 2的基因敲除小鼠表现出致命的表型，出生时即死亡。在初步实验中，对从这些小鼠中提取的海马培养物的分析揭示了一种持久形式的受调节的胞吐作用和突触囊泡再循环。这一结果支持SNARE在调节神经传递的速率和Ca 2+依赖性中的作用，而不是在驱动融合中的执行功能。本项目的主要目标是测试的假设，出现从这个初步分析syb 2缺陷小鼠和阐明的作用syb 2和其他SNARE的相互作用在突触囊泡胞吐和回收在哺乳动物中枢突触。1.为了确定在syb 2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SNARE或囊泡膜相关蛋白在剩余神经传递中的参与。2. SNARE相互作用在囊泡回收和再循环中的作用将使用苯乙烯基染料成像进行研究。3.为了确定syb 2功能的结构要求，将野生型和突变syb 2构建体引入从敲除小鼠获得的海马培养物中以研究体外拯救。从这项研究中获得的信息也将提供新的见解，精神和神经系统疾病，如精神发育迟滞，精神分裂症和癫痫的疾病过程，导致新的治疗策略的设计。

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.