SNAP25 variant I67N: synaptic phenotypes, drug response and proteome changes in human neurons.

SNAREopathies constitute a group of severe genetic neurodevelopmental disorders caused by de novo variants that disturb the synaptic release machinery. These neurodevelopmental disorders comprise highly diverse clinical phenotypes, usually including developmental delay, epilepsy, intellectual disability and sometimes autism spectrum disorder. Despite major progress in genetic testing, current treatments are limited to symptom-directed therapies. There is an urgent need to establish human experimental systems that improve translatability and help develop personalized mechanism-based treatment strategies. Here, we generated CRISPR/Cas9-engineered human-induced pluripotent stem-cell (hiPSC) lines carrying the pathogenic variant I67N in the SNAREopathy gene SNAP25. Using fast forward NGN2 (NEUROG2)-dependent programming into forebrain glutamatergic neurons, we examined synaptic phenotypes using electrophysiology and immunostaining, as well as protein changes by means of proteomics. The variant did not affect passive or active electrical properties, but caused changes in synaptic transmission, including reduced evoked and spontaneous release, decreased synaptic vesicle release probability and consequential changes in short-term plasticity towards facilitation. These phenotypes were distinct from those of human neurons differentiated from hiPSCs originating from a patient carrying the V48F variant, which displayed an increase in spontaneous release. The I67N variant phenotype could be ameliorated by the clinically approved K+-channel blocker 4-aminopyridine. Proteomic analysis of hiPSC-derived neurons revealed a significant enrichment in downregulated synaptic proteins (e.g. CALB1, SCN2A, LRRC4B, PPFIA2). Our study demonstrates the use of hiPSC-based models of neurodevelopmental disorders to identify disease mechanisms and to suggest personalized treatment options directly targeting specific synaptic defects.