Role of SNARE Interactions in Central Synapse Function

描述（由申请人提供）：Synaptobrevin-2/VAMP-2 （syb2）是一种丰富的突触囊泡蛋白，是大脑正常突触传递所必需的。Syb2与质膜蛋白syntaxin 1和SNAP-25的相互作用对突触囊泡融合和神经递质释放至关重要。这些蛋白被统称为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），属于真核生物分泌途径中介导囊泡运输和融合的蛋白家族。尽管在表征SNAREs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囊泡运输事件中的确切作用仍然难以捉摸。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Ca2+依赖融合的SNARE蛋白也可能负责确保快速突触囊泡恢复。事实上，我们之前的研究已经揭示了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，syb2的这种功能可能不被SNAP-25共享。这一观察结果表明，syb2在确保胞吐作用和胞吞作用之间的忠实耦合方面具有特殊作用。在下一个奖励期，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来研究syb2和相关的v-SNAREs在胞外-内吞耦合、胞吞后突触囊泡运输以及融合孔调节中的作用。为此，我们提出三个目标。在第一个目标中，我们将通过监测荧光标记的v-SNAREs突触囊泡蛋白的运输来确定v-SNAREs在胞外作用和内吞作用耦合中的作用。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-SNAREs在突触囊泡吞后运输中的功能。最后，我们将通过光学和电生理测量来确定v-SNAREs对单一神经传递和谷氨酸释放动力学的影响。总的来说，这些实验将阐明中枢突触融合机制和内吞机制之间的重叠程度，以及SNAREs在检索、囊泡再利用和神经递质释放过程中指导突触囊泡轨迹的作用。从这些研究中获得的信息将为可能受到包括智力迟钝、自闭症和精神分裂症在内的许多神经精神和神经疾病影响的突触底物提供新的见解。

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.