Regulation of spontaneous neurotransmission and homeostatic synaptic plasticity by synaptotagmin-1 disease variants at the SNARE primary interface.

Abstract

De novo mutations in synaptotagmin-1 (syt1) cause a rare neurodevelopmental disorder, manifesting in global developmental delay, ophthalmic abnormalities, infantile hypotonia, facial dysmorphisms, absent speech, EEG abnormalities, and hyperkinetic movements, ranging from moderate to severe. Here, we evaluate eleven patient-relevant mutations spanning the Ca2+ binding domains of syt1--C2A and -C2B impact neurotransmission. We found that the mutation causing the most severe impact on neurotransmission, p.N341S, triggers aberrant spontaneous neurotransmission and occludes homeostatic synaptic plasticity signaling pathways. Our results suggest that potential phosphorylation of this newly introduced Ser residue underlies the functional change. A serine missense mutation creates a novel phosphorylation site as a broad spectrum protein kinase inhibitor rescues spontaneous neurotransmission. We identify key residues, localized to the primary interface between syt1 and SNAP-25, responsible for this shift in syt1 function in synaptic vesicle release. Substituting neutral amino acids at residue 341 alters the interaction of the Ser mutation, with double mutations in the surrounding amino acids in the primary interface rescuing synaptic function. These results provide a framework for how a syt1 point mutation introduces a substrate for phosphorylation and disrupts intermolecular interactions at the primary interface with SNAP-25 altering spontaneous neurotransmission and homeostatic plasticity. AUTHOR SUMMARYMutations in synaptotagmin-1 (SYT1), a protein essential for communication between neurons, cause a rare neurodevelopmental disorder marked by developmental delay, low muscle tone, abnormal movements, vision problems, and disrupted brain electrical activity. Disease severity varies, and how specific syt1 mutations alter brain signaling remains unclear. In this study, we examined 11 disease-associated syt1 mutations that affect regions of the protein responsible for sensing calcium, a key trigger for neurotransmitter release. Using a range of electrophysiological approaches, we measured how these mutations influence different modes of synaptic communication within neuronal networks. We found that one mutation, N341S, produced the most severe disruption. Neurons carrying this mutation released neurotransmitters abnormally at rest and were unable to engage normal homeostatic plasticity mechanisms that stabilize brain activity. These effects suggest a fundamental breakdown in how synapses regulate signaling strength. We investigated the molecular basis of this dysfunction and identified a likely explanation: the N341S mutation introduces a new serine residue that can be phosphorylated, a common regulatory modification in cells. Our data indicate that this newly created phosphorylation site alters syt1 function, as blocking phosphorylation pathways could modify the mutants effects. Importantly, we also show that N341 residue lies within a critical interaction interface between syt1 and another synaptic protein, SNAP-25. Adjusting nearby amino acids to neutralize this interaction restores wild-type levels of synaptic signaling. Together, these findings reveal how a single disease-associated mutation can rewire synaptic regulation by introducing a phosphorylation site, offering new insight into the underpinning of syt1-related neurodevelopmental disorders and potential therapeutic targets.