Department of Cellular and Molecular Physiology, Institute of Translational Medicine, University of Liverpool, Liverpool, UK.
Max Planck Institute for Biology of Ageing, Cologne, Germany.
Epilepsia. 2020 Apr;61(4):810-821. doi: 10.1111/epi.16464. Epub 2020 Feb 29.
Genetic variants in STXBP1, which encodes the conserved exocytosis protein Munc18-1, 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 cost-effective manner.
The CRISPR/Cas9 method was used to introduce a null mutation into the unc-18 gene (the C. elegans orthologue of STXBP1), thereby creating a paralyzed worm strain. We subsequently rescued this strain with transgenes encoding the human STXBP1/Munc18-1 protein (wild-type and eight different epilepsy-associated missense variants). The resulting humanized worm strains were then analyzed via behavioral, electrophysiological, and biochemical approaches.
Transgenic expression of wild-type human STXBP1 protein fully rescued locomotion in both solid and liquid media to the same level as the standard wild-type 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 wild-type STXBP1. Electrophysiological recordings revealed that all eight variant strains displayed less frequent and more irregular pharyngeal pumping in comparison to wild-type STXBP1-expressing strains. Four strains (V84D, C180Y, R292H, P335L) exhibited pentylenetetrazol-induced convulsions in an acute assay of seizure-like activity, in contrast to worms expressing wild-type STXBP1. No differences were seen between wild-type and variant STXBP1 strains in terms of mRNA abundance. However, STXBP1 protein levels were reduced to 20%-30% of wild-type in all variants, suggesting that the mutations result in STXBP1 protein instability.
The approach described here is a cost-effective 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 high-throughput drug screens to identify novel therapeutics.
编码保守的胞吐蛋白 Munc18-1 的 STXBP1 中的遗传变异与多种婴儿癫痫综合征有关。我们旨在开发一种体内秀丽隐杆线虫模型，以便以具有成本效益的方式测试此类变异的致病性。
使用 CRISPR/Cas9 方法将无功能突变引入 unc-18 基因（秀丽隐杆线虫的 STXBP1 同源物），从而创建瘫痪的线虫株。随后，我们用编码人 STXBP1/Munc18-1 蛋白（野生型和 8 种不同的与癫痫相关的错义变异体）的转基因拯救了这种菌株。然后通过行为、电生理和生化方法分析产生的人源化线虫株。
野生型人 STXBP1 蛋白的转基因表达在固体和液体培养基中完全恢复了运动能力，与标准野生型线虫株 Bristol N2 相同。6 种变异株（E59K、V84D、C180Y、R292H、L341P、R551C）表现出运动障碍，而 2 种（P335L、R406H）与表达野生型 STXBP1 的线虫没有区别。电生理记录显示，与表达野生型 STXBP1 的菌株相比，所有 8 种变异株的咽部抽吸频率更低且更不规则。在急性癫痫样活动检测中，4 种菌株（V84D、C180Y、R292H、P335L）表现出戊四氮诱导的惊厥，而表达野生型 STXBP1 的线虫则没有。野生型和变异型 STXBP1 菌株在 mRNA 丰度方面没有差异。然而，所有变异体的 STXBP1 蛋白水平均降低至野生型的 20%-30%，表明突变导致 STXBP1 蛋白不稳定。
这里描述的方法是一种具有成本效益的体内方法，可用于确定 STXBP1 及其他保守神经元蛋白中遗传变异的致病性。此外，我们创建的人源化菌株将来可能用于高通量药物筛选，以确定新的治疗方法。
