DEVELOPMENTAL AND EPILEPTIC ENCEPHALOPATHY-ASSOCIATED NSF VARIANTS CAUSE A NOVEL SNAREOPATHY

N-ethylamaleimide sensitive factor (NSF) is an essential presynaptic protein that enables sustained neurotransmitter release by disassembling post-fusion cis-SNARE (Soluble N-ethylmaleimide Sensitive Factor Attachment Protein Receptor) and off-target SNARE complexes, thereby effectively maintaining a pool of fusogenic SNAREs. A recent report linked de novo missense variants in NSF with a severe developmental and epileptic encephalopathy (DEE), however, the presynaptic mechanism of pathogenicity remains unknown. In this study, we describe and examine the clinical and molecular impact of the 3 previously reported DEE-associated variants and 8 novel heterozygous de novo mutations in NSF as well as the functional impact of these variants on SNARE recycling. Though there is some heterogeneity in the clinical phenotypes, there is relative consistency in the severity and features associated with this DEE including severe to profound developmental delay and infant-onset, drug-resistant epilepsy. Protein modeling predicted pathogenicity for all missense variants. Expression of DEE-associated NSF variants in mouse hippocampal neurons revealed accurate trafficking of the mutant protein to the presynapse, no change in basal membrane localization of the v-SNARE synaptobrevin-II, but all variants demonstrated differential impairments to synaptobrevin-II exocytosis and retrieval. These clinical features and dominant negative in vitro neuronal impacts lead us to classify the NSF-associated DEE as a novel SNAREopathy.