Abstracts

Rationale: STXBP1 (Syntaxin-binding protein 1), also known as MUNC-18-1, is a protein involved in synaptic vesicles docking and fusion within the presynaptic membrane, enabling synaptic transmission. Mutations in STXBP1 gene have been associated with neurodevelopmental disorders in children, with >85% of patients developing seizures and severe to profound intellectual disability ^1-3.

No approved drug therapy addressing the underlying disease mechanism is available at this point. In this study we developed and characterized a human induced pluripotent stem cells (hiPSCs) derived model of STXBP1 haploinsufficiency.


Methods: Human iPSCs were CRISPR/Cas9-edited to obtain STXBP1 full KO (-/-) clones and two heterozygous (HET) relevant missense (R406 +/-) and nonsense mutations (R367X +/-). NGN2 transcription factor was introduced to AAVS1 locus by flipase – ligase system to differentiate into enriched glutamatergic neuronal population. STXBP1 protein expression was assessed by Western Blot (WB), as well as Syntaxin-1 (STX1A) protein, which is also a component of soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor (SNARE) complex and a downstream target of STXBP1 protein. The functional impact of STXBP1 was measured by recording extracellular spontaneous neuronal activity during maturation period, using a microelectrode array (MEA)–based assay. Glutamatergic neurons were then transduced between days In vitro (DIV) 12 and 16 with Lentiviral vectors (LVVs) containing wild-type (WT) human STXBP1.


Results: We confirmed that STXBP1 protein is absent in STXBP1 KO (-/-) neurons, while the glutamatergic neurons with the heterozygous mutations exhibited about 30 to 60% reduction. Moreover, when compared with isogenic control, KO (-/-) exhibited 90% reduction in STX1A expression, while the mutants showed 20 to 40% decrease. Recording spontaneous activity using MEA and after applying a multiparameter analysis, we observed that STXBP1 KO (-/-) glutamatergic neurons do not show spontaneous network burst activity, while STXBP1 mutant neurons showed impaired synchronization compared to the isogenic control. Overexpression of wild-type human STXBP1 partially restored STX1A levels, demonstrating the rescue of STXBP1 chaperone function. Additionally, spontaneous network activity was also restored after STXBP1 overexpression, suggesting that exogenous STXBP1 expression can lead to functional rescue of human STXBP1 KO (-/-) and mutant neurons.


Conclusions: We confirmed that STXBP1 gene-edited hiPSCs-derived glutamatergic neurons are a suitable model recapitulating the pathophysiology of STXBP1 In vitro. Moreover, the data demonstrates the potential of the hiPSCs model to be used to hypothesize a likely genotype/phenotype correlation and to explore novel approaches for disease modifying therapies.




Funding: GREAT GRANT (Wallonie recherche SPW)