Analysis of Synaptic Protein Dynamics - Project Summary Synapses are fundamental to nervous system function and information processing in the normal and pathological brain. As highly dynamic biological structures, they display a broad range of activity and plasticity. A detailed picture of the molecular interactions occurring within a synapse is required to understand how synaptic protein dynamics ultimately shape activity in the nervous system in health and disease. In this proposal, we investigate Munc13, Munc18, and the neuronal SNAREs, highly conserved proteins that are crucial for proper synaptic function. Mice lacking any one of these core proteins die at birth, and there are numerous neurodegenerative and psychiatric disorders associated with defects in the function of these proteins. For instance, mutations in the human Munc13 ortholog (Unc13A) are associated with ALS, myasthenia, microcephaly, and severe autism. Mutations in the Munc18 ortholog (STXBP1) are associated with epileptic encephalopathies. We propose to study the molecular mechanisms underlying Munc13 and Munc18 function at the synapse using a unique and powerful combination of in vivo and in vitro approaches including physiology, quantitative imaging, behavioral assays, genetics, and protein/lipid biochemistry. Much of the detailed mechanistic work on the core proteins of synaptic transmission has been performed in vitro. To move forward on these molecular models, we need an in vivo testing platform that allows precise manipulations of the core machinery in their native environment with endogenous expression levels. To this end, we have built a collection of C. elegans mutants in the core machinery of synaptic transmission including the SNARE proteins, UNC-13, UNC-18, and CPX-1 (worm orthologs of Munc13, Munc18, and complexin, respectively) using CRISPR Cas9 gene editing to avoid over-expression issues that have plagued structure/function studies in the past. By combining various mutations using double and triple mutants, we are investigating detailed mechanistic questions in vivo using endogenous proteins. This step is essential in furthering our understanding of the molecular mechanisms underlying synaptic transmission. In addition, advances in protein structure prediction have enriched our hypotheses for how the core machinery operates as a functional unit, so we are also exploring novel interactions between these proteins with the goal of expanding the current models for the molecular underpinnings of neurotransmitter release. The experiments proposed here will provide new insights into the mechanisms that control neurotransmitter release, its modulation, and use-dependent plasticity in the brain.
