Functional coupling between synaptic vesicle exocytosis and recycling in mammalian synapses

哺乳动物突触中突触小泡胞吐作用与回收之间的功能耦合

• 批准号: 445976607
• 负责人: Professor Volker Haucke, Ph.D.
• 金额: --
• 依托单位: Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP)
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2020
• 资助国家: 德国
• 起止时间: 2019-12-31 至 2022-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/445976607?language=en
• 关键词: Functional; coupling; between; synaptic; vesicle

Fast chemical synaptic transmission is at the basis of neuronal communication. It is mediated by the fusion of synaptic vesicles (SVs) containing neurotransmitter molecules with the presynaptic plasma membrane at the so-called exocytic active zone (AZ) facing post- synaptic sites, where post-synaptic neurotransmitter receptors are located. Exocytic SV fusion is tightly coupled in space and time to the endocytic retrieval of SV proteins and lipids and the reformation of release-ready SVs to enable sustained neurotransmission and thereby brain function. The basis for the coupling of SV exo- and endocytosis is poorly understood. The proposed collaborative project aims to define the structural and molecular basis of exo-endocytic coupling at synapses. We will develop new fluorescent probes and protocols to determine the location of SV exocytosis and endocytosis events and define a putative endocytic zone (EZ) relative to the AZ. We will use rat and transgenic mouse primary hippocampal neurons, as well as in human neurons differentiated from induced pluripotent stem cells (hiPSCs) that are easily amenable for genome editing strategies. Furthermore, we will use advanced live and super- resolution imaging including live gated stimulated emission depletion (gSTED) microscopy to define the molecular markers of the EZ and test how SV proteins regulate its formation. Finally, we will uncouple endocytosis from exocytosis by timed changes in membrane tension or by downregulating the expression of SV or associated proteins that may link the EZ to the AZ, and determine their consequences on synaptic physiology.

快速化学突触传递是神经元通讯的基础。它由含有神经递质分子的突触囊泡（SV）与突触前质膜在面向突触后位点的所谓胞吐活性区（AZ）处的融合介导，突触后神经递质受体位于所述突触后位点。外泌SV融合在空间和时间上与SV蛋白和脂质的内吞回收以及释放就绪SV的重组紧密耦合，以实现持续的神经传递，从而实现脑功能。SV胞外和胞吞作用耦合的基础知之甚少。这项合作计划的目的是确定突触外吞-内吞耦合的结构和分子基础。我们将开发新的荧光探针和协议，以确定SV胞吐和胞吞事件的位置，并定义一个推定的内吞区（EZ）相对于AZ。我们将使用大鼠和转基因小鼠原代海马神经元，以及从诱导多能干细胞（hiPSC）分化的人类神经元，这些细胞很容易适用于基因组编辑策略。此外，我们将使用先进的实时和超分辨率成像，包括实时门控受激发射耗尽（gSTED）显微镜，以定义EZ的分子标记物，并测试SV蛋白如何调节其形成。最后，我们将解偶联胞吞作用从胞吐作用的膜张力的定时变化，或通过下调SV或相关蛋白的表达，可能连接的EZ到AZ，并确定其后果突触生理。