Presynaptic short- and long-term enhancement of neurotransmitter release: Molecular mechanisms and behavioral relevance

神经元之间的通信依赖于跨突触接触的快速化学传递，其中载有神经递质的突触囊泡（SV）的突触前胞吐激活突触后反应。胞吐作用由Ca 2+控制，发生在动态病灶，称为活性区（AZ）。随后的内吞作用通过支持囊泡再循环来确保突触前体内平衡和持续的活动。显然，胞吞和胞吐的同步化是有效的神经传递的保证。然而，令人惊讶的是，人们对这些反应的分子机制知之甚少。我的研究计划旨在弥合这一差距。AZ最适合胞吐作用;即使在刺激之前，囊泡也会靶向释放位点，并成熟到接近Ca 2+通道的可释放状态，Ca 2+通道会响应动作电位而打开，触发胞吐作用。细胞基质蛋白决定AZ-结构，并且这通常被认为协调胞吐激活剂（即Ca 2+通道）和靶标（SV）的偶联。然而，这些反应背后的分子拓扑结构和功能原理在很大程度上仍然不清楚。SV膜的内吞作用发生在紧邻AZ处。最近的数据表明，内吞作用不仅需要在长期运行，以补充SV池，但也需要释放网站的快速清除。此外，似乎内吞作用的模式取决于胞吐作用，但这些过程如何精确地连接仍然是难以捉摸的。最后，它是未知的重要监管机构的外吞和内吞在空间和时间上组织。我假设，细胞基质蛋白的AZ作为反应枢纽，需要协调有效的外吞，内吞和网站回收。我的计划将解决外吞耦合的机制相结合的理论（数学建模）和实验方法（即慢性和急性遗传学，电生理学，活细胞成像，电子显微镜，生物化学）使用果蝇神经肌肉接头以及小鼠海马神经元作为模型系统。协同实验工作和理论建模将使我能够测试AZ细胞基质是否以及如何整合胞吞作用。具体来说，我的目标是解开（一）AZ架构如何优化胞吐通过识别钙离子通道释放站点拓扑结构和（ii）定义神经元兴奋性的分子机制，通过研究当地的蛋白质组成和胞吐在单个AZ。此外（iii），我将表征的机制，内吞机制适应不同的模拟范例。最后（iv），我将直接测试是否细胞基质AZ-蛋白的反应枢纽，通过解剖他们的外吞和内吞功能。本提案中提出的工作将阐明突触神经传递的具体机制和一般原则，从而阐明大脑功能。

Communication between neurons relies on fast chemical transmission across synaptic contacts where presynaptic exocytosis of neurotransmitter-laden synaptic vesicles (SVs) activates postsynaptic responses. Exocytosis is controlled by Ca2+ and occurs at dynamic foci, called active zones (AZs). Subsequent endocytosis ensures presynaptic homeostasis and continued activity by supporting vesicle re-cycling. Evidently, synchronization of exo- and endocytosis is warranted for effective neurotransmission. However, surprisingly little is known about molecular mechanisms linking these reactions. My research program is designed to bridge this gap.AZs are optimized for exocytosis; even before stimulation, vesicles are targeted to release sites and mature to a readily releasable state close to Ca2+-channels, which open in response to action potentials to trigger exocytosis. Cytomatrix proteins determine the AZ-architecture and this is generally assumed to orchestrate coupling of exocytosis activators (i.e. Ca2+ channels) and targets (SVs). However, the molecular topologies and functional principles underlying these reactions remain largely unclear. Endocytosis of SV membranes occurs in close proximity to AZs. Recent data suggest that endocytosis not only is required on the long run to replenish SV pools but is also needed for fast clearance of release sites. Furthermore, it appears that the mode of endocytosis depends on exocytosis, but how precisely these processes are connected remains elusive. Finally, it is unknown how essential regulators of exo- and endocytosis are organized in space and time.I hypothesize that cytomatrix proteins of the AZ serve as reaction hubs that are needed to orchestrate effective exocytosis, endocytosis and site recycling. My program will address the mechanism of exo-endocytic coupling by combining theoretical (mathematical modelling) and experimental approaches (i.e. chronic and acute genetics, electrophysiology, live cell imaging, electron microscopy, biochemistry) using Drosophila melanogaster neuromuscular junctions as well as mouse hippocampal neurons as model systems. Synergizing experimental work and theoretical modeling will allow me to test whether and how the AZ cytomatrix integrates exo- with endocytosis. Specifically, I aim to unravel (i) how AZ-architecture optimizes exocytosis by identifying the Ca2+-channel release-site topology and to (ii) define the molecular mechanisms of neuronal excitability by studying local protein composition and exocytosis at single AZs. Moreover (iii), I will characterize the mechanisms by which the endocytosis machinery adapts to diverse simulation paradigms. Finally (iv), I will directly test whether cytomatrix AZ-proteins are reaction hubs by dissecting their exo- and endocytosis functions. The work presented in this proposal will shed light on the specific mechanisms and general principles that underlie synaptic neurotransmission and, thus, brain function.