DECIPHERING THE PATHOGENIC MECHANISMS OF SYNAPTOPATHIES – COMPARISON OF SYT1 & VAMP2-ASSOCIATED NEURODEVELOPMENTAL DISORDERS

Synaptotagmin-1 (SYT1) and synaptobrevin-2 (VAMP2) are synaptic vesicle membrane proteins with essential but differing roles in vesicle exocytosis. SYT1 acts as a calcium-dependent trigger of evoked exocytosis, while VAMP2 is a key component of the core SNARE complex machinery that drives vesicle fusion. De novo heterozygous variants of SYT1 and VAMP2 cause neurodevelopmental disorders, with both overlapping and distinct features. Here, we explored the underlying mechanisms that may drive these divergent phenotypes using a variety of live cell fluorescence imaging assays in primary cultured neurons to assess comparative impacts on synaptic vesicle cycling dynamics and on presynaptic neurotransmitter release, at single synapse resolution. pHluorin assays revealed that both SYT1 and VAMP2 variants induce dominant negative impairment of evoked exocytosis kinetics. Further mechanistic insight revealed that SYT1 variants also reduced the size of the readily releasable pool (RRP) of synaptic vesicles, while VAMP2 variants did not. This indicates that SYT1 and VAMP2 pathogenic variants impact vesicle exocytosis through divergent mechanisms, particularly with respect to the docking, priming or fusogenicity of synaptic vesicles. Differences were also observed with presynaptic neurotransmitter release via iGluSnFR3 at the level of individual synapses. SYT1 variants exclusively induced a dominant negative reduction in the probability of single action potential-evoked glutamate release, demonstrating that impacts on the availability of docked or fusion-competent synaptic vesicles translate to impacts on the efficiency of neurotransmitter release at the level of single synapses. This provides a plausible mechanism for the exocytic impairment observed with SYT1 (but not VAMP2) variants.