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

神经元之间的交流依赖于突触接触的快速化学传输，在突触前，富含神经递质的突触小泡(SVS)的突触前胞吐激活突触后反应。胞吐作用是由钙离子控制的，发生在称为活动区(AZs)的动态灶上。随后的内吞作用通过支持小泡的再循环来确保突触前的动态平衡和持续的活动。显然，外吞和内吞的同步对于有效的神经传递是有保证的。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针对胞吐作用进行了优化；甚至在刺激之前，囊泡就以释放部位为目标，并成熟到靠近钙离子通道的易于释放的状态，钙通道根据动作电位打开以触发胞吐作用。细胞基质蛋白决定AZ-结构，这通常被认为是协调胞吐激活剂(即钙通道)和靶(SVS)的偶联。然而，这些反应背后的分子拓扑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靠近AZs的地方。最近的数据表明，从长远来看，不仅需要内吞作用来补充SV池，而且还需要内吞作用来快速清除释放部位。此外，内吞作用的模式似乎依赖于胞吐作用，但这些过程如何准确地联系在一起仍然难以捉摸。最后，尚不清楚外吞和内吞的基本调节因子在空间和时间上是如何组织的。我假设AZ的Cytomatrix蛋白作为反应中心，是协调有效的胞吞、内吞和部位回收所必需的。我的计划将以果蝇神经肌肉接头和小鼠海马神经元为模型系统，通过理论(数学建模)和实验(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)相结合的方法来研究细胞外-内耦合的机制。协同实验工作和理论建模将使我能够测试AZ Cytomatrix是否以及如何将外吞和内吞结合在一起。具体地说，我的目标是揭示(I)AZ-架构如何通过确定钙通道释放位点的拓扑结构来优化胞吐作用，以及(Ii)通过研究局部蛋白质组成和单个AZ的胞吐作用来确定神经元兴奋性的分子机制。此外(Iii)，我将描述内吞作用机制适应不同模拟范例的机制。最后(Iv)，我将通过解剖其外吞和内吞功能来直接测试Cytomatrix AZ-蛋白是否是反应中心。这项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理，从而揭示大脑功能。

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.