Role of SNARE Interactions in Central Synapse Function

描述(申请人提供)：Synaptobrevin-2/VAMP-2(Syb2)是一种丰富的突触囊泡蛋白，对大脑中正常的突触传递是必不可少的。Syb2‘S与质膜蛋白Synaxin 1和SNAP-25的相互作用是突触囊泡融合和神经递质释放的关键。这些蛋白统称为SNARES(可溶性N-乙基马来酰亚胺敏感因子附着蛋白受体的首字母缩写)，属于真核生物中介导囊泡运输和分泌途径融合的蛋白质家族。尽管在表征SNARs之间的分子相互作用及其在融合中的作用方面取得了广泛的进展，但它们在融合后突触小泡运输事件中的确切作用仍然不清楚。在神经传递过程中，囊泡融合和恢复的快速耦合使我们假设，驱动快速钙依赖融合的SNAR蛋白可能也负责确保突触小泡的快速恢复。事实上，我们在前一期的研究已经揭示了syb2在快速突触囊泡内吞作用中的重要作用。此外，我们的初步结果表明，SNAP-25可能不共享syb2的这一功能。这一观察表明，syb2在确保胞吐和内吞之间的忠实耦合方面发挥了特殊的作用。在下一个获奖期，我们的目标是利用荧光成像、电生理学和电子显微镜的强大组合，研究syb2和相关的v-SNARE在胞外耦合、内吞作用后突触小泡运输以及融合孔调节中的作用。为此，我们提出了三个目标。在第一个目标中，我们将通过监测荧光标记的v-SNARES突触小泡蛋白的运输来确定V-SNARES在胞吐和内吞结合中的作用。在第二个目标中，我们将通过检测摄取和释放荧光探针、监测神经递质释放和电子显微镜来确定v-SNARE在突触小泡内吞后运输中的作用。最后，我们将通过光学和电生理方法来确定v-SNARE对单一神经传递和谷氨酸释放动力学的影响。总之，这些实验将阐明中央突触中融合机制和内吞机制之间的重叠程度，以及SNARS在指导突触小泡提取、囊泡重复使用以及神经递质释放过程中的作用。从这些研究中获得的信息将为突触底物提供新的见解，这些底物可能会受到许多神经精神和神经疾病的影响，包括智力低下、自闭症和精神分裂症。与公共健康相关：为该项目提出的实验提出了一个系统和全面的努力，以解决关键的SNAR分子在调节突触小泡融合、回收和再循环中的作用。目前，对SNARs在囊泡融合后突触小泡运输中的作用缺乏深入的分析。在这个项目中，我们的目标是建立哺乳动物中央突触中突触小泡运输的圈套依赖调节的基本原理。从这些研究中获得的信息将为分子突触底物提供新的见解，这些分子突触底物可能受到许多神经精神和神经疾病的影响，包括智力低下、自闭症和精神分裂症。

DESCRIPTION (provided by applicant): Synaptobrevin-2/VAMP-2 (syb2) is an abundant synaptic vesicle protein essential for normal synaptic transmission in the brain. Syb2's interaction with the plasma membrane proteins, syntaxin 1 and SNAP-25, is critical for synaptic vesicle fusion and neurotransmitter release. These proteins are collectively called SNAREs (acronym for soluble N-ethylmaleimide-sensitive factor attachment protein receptors) and belong to a family of proteins that mediate vesicle trafficking and fusion in the secretory pathway in eukaryotes. Despite extensive progress in the characterization of molecular interactions among SNAREs and their role in fusion, their precise role in synaptic vesicle trafficking events after fusion remains elusive. Rapid coupling of vesicle fusion and retrieval during neurotransmission have led us to hypothesize that SNARE proteins that drive rapid Ca2+ dependent fusion may also be responsible for ensuring rapid synaptic vesicle retrieval. Indeed, our studies in the previous grant period have revealed an essential role for syb2 in rapid synaptic vesicle endocytosis. Moreover, our initial results suggest that this function of syb2 may not be shared by SNAP-25. This observation suggests a specific role for syb2 in ensuring faithful coupling between exocytosis and endocytosis. In the next award period, we aim to investigate the role of syb2 and related v-SNAREs in exo-endocytic coupling, synaptic vesicle trafficking after endocytosis as well as fusion pore regulation using a powerful combination of fluorescence imaging, electrophysiology and electron microscopy. For this purpose, we propose three aims. In the first aim, we will define the role of v-SNAREs in coupling exocytosis and endocytosis via monitoring trafficking of fluorescently-tagged v-SNAREs synaptic vesicle proteins. In the second aim, we will determine the function of v-SNAREs in postendocytic trafficking of synaptic vesicles by detecting uptake and release fluorescent probes, monitoring neurotransmitter release and electron microscopy. Lastly, we will determine the impact of v-SNAREs on unitary neurotransmission and glutamate release kinetics using optical and electrophysiological measures. Collectively, these experiments will elucidate the degree of overlap between the fusion machinery and endocytic machinery in central synapses and the role of SNAREs in directing synaptic vesicle trajectories during retrieval, vesicle reuse as well as neurotransmitter release. Information attained from these studies will provide new insight to the synaptic substrates that may be affected by a number of in neuropsychiatric and neurological disorders including mental retardation, autism and schizophrenia. PUBLIC HEALTH RELEVANCE: The experiments proposed for this project present a systematic and comprehensive effort to address the role of key SNARE molecules in the regulation of synaptic vesicle fusion, retrieval and recycling. Currently, a thorough analysis of the role of SNAREs in synaptic vesicle trafficking beyond vesicle fusion is lacking. In this project, we aim to establish the basic principles of SNARE-dependent regulation of synaptic vesicle trafficking in mammalian central synapses. Information attained from these studies will provide new insight to the molecular synaptic substrates that may be affected by a number of in neuropsychiatric and neurological disorders including mental retardation, autism and schizophrenia.