Abstracts

Rationale: Mutations in Munc18-1 are associated with Ohtahara, West, and Dravet syndromes, but the mechanistic relationship between mutations and these diseases is unknown. These devastating and often fatal infantile epileptic encephalopathies are characterized by cognitive, sensory and/or motor function deterioration due to unremitting epileptic activity. While proposed to be due to haploinsufficiency, it remains unclear how mutations in one gene result in diseases with different onsets and clinical presentations, how missense mutations cause a clinical phenotype indistinguishable from whole-gene deletions, and why some missense mutations are more harmful than others. We aim to determine how mutations in Munc18-1 cause neuronal defects. Munc18-1 controls neurotransmitter release by regulating the SNARE-protein syntaxin-1. We hypothesize that mutations in Munc18-1 cause defects in its folding, stability and localization, which subsequently impair syntaxin-1 and synapse function. The rationale for these studies is that clarifying how mutations in Munc18-1 trigger these seizure disorders will enable the development of novel treatments beyond the current limited symptom-based therapy.Methods: To examine the impact of mutations on Munc18-1, we measured folding of Munc18-1 mutants using recombinant purified proteins and circular dichroism spectroscopy. Stability and turnover of Munc18-1 mutants were analyzed in transfected N2a cells via cycloheximide chase experiments. Solubility profiling, limited proteolysis, and immunocytochemistry were used to assess aggregation of Munc18-1 mutants. To determine the effect of Munc18-1 mutants on syntaxin-1, we analyzed their interaction using GST pulldown and co-immunoprecipitation experiments. Syntaxin-1 stability, turnover, aggregation and subcellular targeting were assessed by same paradigms used for Munc18-1. To analyze the effect of Munc18-1 mutants on synapse function, we generated transgenic C. elegans strains overexpressing wild-type unc18 or the G544D mutant on an unc18 null background, and quantified the ability to rescue the unc18 null phenotype by locomotion and aldicarb assays.Results: We found that Munc18-1 mutants are unstable and are turned over faster than wild-type Munc18-1. Mutants also revealed decreased solubility and aggregated. The reduction in Munc18-1 levels triggered a reduction in syntaxin-1 levels and stability, suggesting a loss-of-function disease mechanism of both, Munc18-1 and syntaxin-1. In the worm, wild-type unc18 but not the G544D mutant restored the synaptic defects in unc18 null worms.Conclusions: Our research is significant, because it not only highlights the importance of Munc18-1 in regulating neurotransmitter release, but also clarifies how Munc18-1 mutants impair this function. Further, our research shifts focus from a symptom-centered perspective to an approach that focusses on convergent disease mechanisms pivoting on Munc18-1, thereby providing a basis for the development of novel therapies for three different diseases. This research is supported by the Epilepsy Foundation/AES (J.B.), and the IES Brain Research Foundation (J.M.).