Functional coupling between synaptic vesicle exocytosis and recycling in mammalian synapses

快速化学突触传递是神经元通信的基础。它是由含有神经递质分子的突触囊泡（SVs）与突触前质膜在面向突触后位置的所谓胞外活性区（AZ）融合介导的，突触后神经递质受体位于突触后位置。胞外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的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外胞和内吞作用耦合的基础尚不清楚。提出的合作项目旨在确定突触外-内吞耦合的结构和分子基础。我们将开发新的荧光探针和方案，以确定SV胞吞和内吞事件的位置，并确定相对于AZ的假定内吞区（EZ）。我们将使用大鼠和转基因小鼠的初级海马神经元，以及从诱导多能干细胞（hiPSCs）分化的人类神经元，这些神经元很容易适用于基因组编辑策略。此外，我们将使用先进的活成像和超分辨率成像，包括活门控受激发射损耗（gSTED）显微镜来定义EZ的分子标记，并测试SV蛋白如何调节其形成。最后，我们将通过膜张力的定时变化或下调SV或可能连接EZ和AZ的相关蛋白的表达来解除内吞作用和胞吐作用，并确定它们对突触生理学的影响。

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.