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

• Applicant: Dr. Alexander Walter
• Subject Area: Molecular Biology and Physiology of Neurons and Glial Cells
• Term: from 2015 to 2022
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 261020751

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.

• DFG Programme: Independent Junior Research Groups