Isolation of Recycling Synaptic Vesicles for Proteomic Analyses

描述（由申请人提供）：为响应RFA-MH-12-140，“开发探索突触组的工具”，我们建议开发一种程序来捕获和分离回收突触囊泡，作为分析囊泡蛋白组成如何随突触活性、药物治疗和病理条件的变化而变化的重要第一步。突触囊泡进行多轮融合的能力对于维持快速、有效的神经传递至关重要。突触囊泡形成并通过内吞作用再循环。在许多突触中，总囊泡群体的一小部分介导了大部分神经传递，而大部分仍在储备池中未被利用。因此，使用传统囊泡纯化方案的蛋白质组学分析不能跟踪回收池的蛋白质组成的变化。为了解决这个问题，我们将开发一个可扩展的，易于应用的纯化方案，回收突触囊泡，依赖于短暂捕获回收囊泡作为一种手段，将它们从总囊泡池分离。这种方法将打开一个新的领域的调查复杂的内吞机制，调节囊泡回收。它将提供一种手段来测试药物，遗传操作和病理条件对这一非常重要的过程的影响。

DESCRIPTION (provided by applicant): In response to RFA-MH-12-140, "Development of Tools to Explore the Synaptome", we propose to develop a procedure to trap and isolate recycling synaptic vesicles as an essential first step in analyzing how vesicle protein composition varies with changes in synaptic activity, drug treatment, and under pathological conditions. The ability of synaptic vesicles to undergo multiple rounds of fusion is crucial to maintaining fast, efficient neurotransmission. Synaptic vesicles are formed and recycle via endocytosis. At many synapses, a small proportion of the total vesicle population mediates the majority of neurotransmission while the larger portion remains untapped in a reserve pool. Therefore, proteomic analyses using traditional vesicle purification schemes cannot track changes in the protein composition of the recycling pool. To address this we will develop a scalable, readily applied purification protocol for recycling synaptic vesicles that relies on transiently trapping recycling vesicles as a means of separating them from the total vesicle pool. This approach will open a new field of investigation into the complex endocytotic machinery that regulates vesicle recycling. It will provide a means to test the effects of drugs, genetic manipulations, and pathological conditions on this very important process.