Functional analysis of epilepsy-associated variants in STXBP1/Munc18-1 using humanized Caenorhabditis elegans.
Functional analysis of epilepsy-associated variants in STXBP1/Munc18-1 using humanized Caenorhabditis elegans.
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作者：
Zhu B;Mak JCH;Morris AP;Marson AG;Barclay JW;Sills GJ;Morgan A
STXBP1 编码保守的胞吐蛋白 Munc18-1，其遗传变异与多种婴儿癫痫综合征相关。我们的目标是开发一种体内秀丽隐杆线虫模型，该模型可用于以经济高效的方式测试此类变异的致病性。使用 CRISPR/Cas9 方法在 unc-18 基因（STXBP1 的线虫直系同源物）中引入无效突变，从而创建了瘫痪的蠕虫菌株。随后，我们用编码人类 STXBP1/Munc18-1 蛋白（野生型和八种不同的癫痫相关错义变体）的转基因拯救了该菌株。然后通过行为、电生理学和生化方法对所得的人源化蠕虫菌株进行分析。野生型人类 STXBP1 蛋白的转基因表达完全挽救了固体和液体培养基中的运动，达到与标准野生型蠕虫菌株 Bristol N2 相同的水平。六种变异菌株（E59K、V84D、C180Y、R292H、L341P、R551C）表现出运动受损，而两种（P335L、R406H）与表达野生型STXBP1的线虫没有什么不同。电生理记录显示，与表达 STXBP1 的野生型菌株相比，所有 8 个变异菌株的咽部抽吸频率更低且更不规则。与表达野生型 STXBP1 的线虫相比，四种菌株（V84D、C180Y、R292H、P335L）在癫痫样活动的急性测定中表现出戊四氮诱导的惊厥。野生型和变异型 STXBP1 菌株之间的 mRNA 丰度没有差异。然而，在所有变体中，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经济有效的体内方法，用于确定 STXBP1 和潜在的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有可能用于高通量药物筛选，以识别新的治疗方法。
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.
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