Isolation of Recycling Synaptic Vesicles for Proteomic Analyses

描述(申请人提供)：针对RFA-MH-12-140，“探索突触的工具的开发”，我们建议开发一种捕获和分离循环突触小泡的程序，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必要的第一步。突触小泡经历多轮融合的能力对于保持快速、有效的神经传递至关重要。突触小泡通过内吞作用形成和循环。在许多突触中，一小部分囊泡总人口负责大多数神经传递，而较大比例的囊泡在储备池中仍未被利用。因此，使用传统囊泡纯化方案的蛋白质组学分析无法跟踪回收池中蛋白质组成的变化。为了解决这个问题，我们将开发一种可扩展的、易于应用的回收突触小泡的纯化方案，该方案依赖于瞬时捕获回收小泡作为将它们从总的囊泡池中分离出来的一种手段。这一方法将开启一个新的研究领域，研究调控囊泡循环的复杂内吞机制。它将提供一种手段来测试药物、基因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.