1 research outputs found
Functional analysis of epilepsy-associated variants in STXBP1/Munc18-1 using humanised Caenorhabditis elegans
Objective:
Genetic variants in STXBP1 , which encodes the conserved exocytosis protein Munc18â1, are associated with a variety of infantile epilepsy syndromes. We aimed to develop an in vivo Caenorhabditis elegans model that could be used to test the pathogenicity of such variants in a costâeffective manner.
Methods:
The CRISPR/Cas9 method was used to introduce a null mutation into the uncâ18 gene (the C. elegans orthologue of STXBP1 ), thereby creating a paralyzed worm strain. We subsequently rescued this strain with transgenes encoding the human STXBP1/Munc18â1 protein (wildâtype and eight different epilepsyâassociated missense variants). The resulting humanized worm strains were then analyzed via behavioral, electrophysiological, and biochemical approaches.
Results:
Transgenic expression of wildâtype human STXBP1 protein fully rescued locomotion in both solid and liquid media to the same level as the standard wildâtype worm strain, Bristol N2. Six variant strains (E59K, V84D, C180Y, R292H, L341P, R551C) exhibited impaired locomotion, whereas two (P335L, R406H) were no different from worms expressing wildâtype STXBP1. Electrophysiological recordings revealed that all eight variant strains displayed less frequent and more irregular pharyngeal pumping in comparison to wildâtype STXBP1âexpressing strains. Four strains (V84D, C180Y, R292H, P335L) exhibited pentylenetetrazolâinduced convulsions in an acute assay of seizureâlike activity, in contrast to worms expressing wildâtype STXBP1. No differences were seen between wildâtype and variant STXBP1 strains in terms of mRNA abundance. However, STXBP1 protein levels were reduced to 20%â30% of wildâtype in all variants, suggesting that the mutations result in STXBP1 protein instability.
Significance:
The approach described here is a costâeffective in vivo method for establishing the pathogenicity of genetic variants in STXBP1 and potentially other conserved neuronal proteins. Furthermore, the humanized strains we created could potentially be used in the future for highâthroughput drug screens to identify novel therapeutics