Regulation of spontaneous and evoked release through essential proteins of the synaptic vesicle fusion

• Applicant: Professor Dr. Christian Rosenmund
• Subject Area: Molecular Biology and Physiology of Neurons and Glial Cells
• Term: since 2024
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 543999006

Rapid and efficient transduction of action potentials into synaptic vesicle (SV) fusion and neurotransmitter release is essential for adequate information processing in the brain. Additionally, it is important that spontaneous fusion of SV is minimal to maximize high signal to noise transmission at synapses. In this proposal, we aim to dissect the mechanisms governing neurotransmitter release by performing a systematic structure-function analysis of proteins essential for SV fusion. We will rely on state-of-the-art models of how the core SV fusion machinery, the SNARE proteins, and selected accessory proteins interact to ensure rapid and efficient action potential-evoked neurotransmitter release while limiting spontaneous fusion. We aim to elucidate the role that individual SNARE proteins play in regulated exocytosis in addition to enabling membrane fusion per se. In addition, we will incorporate pivotal crystal structure information into our functional analysis of how the SNARE complex interacts with its accessory proteins to modulate neurotransmitter release. In our first objective, we will determine critical structural features at the surface of the SNARE complex using chimers of related SNAREs as well as by altering individual residues. We will use mutagenesis combined with molecular replacement approaches and test synaptic properties using electrophysiology and electron microscopy. We will analyze how these alteration contribute to the regulation of SV priming, spontaneous SV fusion and the efficacy of Ca2+ triggered release. The main experimental model relies on cultured primary neurons derived from transgenic mice. Secondly, we will characterize the functional consequences of mutations in putative interaction sites at the primary interface between synaptotagmin 1 and the SNARE complex in regulating neurotransmitter release, and probe whether the closely related synaptotagmin 7 paralog modulates synaptic function through a similar molecular interaction. Using our established methods and mouse models, along with collaborations with key biochemists and biophysicists in the field, the proposed experiments stand to make a significant contribution to our understanding of SNARE complex-accessory protein interactions, and add to our understanding of the fundamental mechanism of regulated secretion in neuronal communication.

• DFG Programme: Research Grants