Abstract：
Mutation of the GABRA1 gene is associated with neurodevelopmental defects and epilepsy. GABRA1 encodes for the α1 subunit of the γ-aminobutyric acid type A receptor (GABAAR), which regulates the fast inhibitory impulses of the nervous system. Multiple model systems have been developed to understand the function of GABRA1, but these models have produced complex and, at times, incongruent data. Thus, additional model systems are required to validate and substantiate previous results. We sought to provide initial phenotypic analysis of a novel germline mutant allele. Our analysis provides a solid foundation for the future use of this allele to characterize gabra1 functionally and pharmacologically using zebrafish. We investigated the behavioral swim patterns associated with a nonsense mutation of the zebrafish gabra1 (sa43718 allele) gene. The sa43718 allele causes a decrease in gabra1 mRNA expression, which is associated with light induced hypermotility, one phenotype previously associated with seizure like behavior in zebrafish. Mutation of gabra1 was accompanied by decreased mRNA expression of gabra2, gabra3, and gabra5, indicating a reduction in the expression of additional α sub-units of the GABAAR. Although multiple sub-units were decreased, larvae continued to respond to pentylenetetrazole (PTZ), indicating that a residual GABAAR exists in the sa43718 allele. Proteomics analysis demonstrated that mutation of gabra1 is associated with abnormal expression of proteins that regulate synaptic vesicle fusion, vesicle transport, synapse development, and mitochondrial protein complexes. These data support previous studies performed in a zebrafish nonsense allele created by CRISPR/Cas9 and validate that loss of function mutations in the gabra1 gene result in seizure-like phenotypes with abnormal development of the GABA synapse. Our results add to the existing body of knowledge as to the function of GABRA1 during development and validate that zebrafish can be used to provide complete functional characterization of the gene.
摘要：
GABRA1基因的突变与神经发育缺陷和癫痫有关。GABRA1编码γ-氨基丁酸A型受体（GABAAR）的α1亚基，调节神经系统的快速抑制冲动。已经开发了多个模型系统来理解GABRA1的功能，但是这些模型产生了复杂的，有时,不一致的数据。因此，需要额外的模型系统来验证和证实以前的结果。我们试图提供新的种系突变等位基因的初始表型分析。我们的分析为将来使用该等位基因在功能和药理学上使用斑马鱼表征gabra1提供了坚实的基础。我们调查了与斑马鱼gabra1（sa43718等位基因）基因的无义突变相关的行为游泳模式。sa43718等位基因导致gabra1mRNA表达减少，这与光诱导的运动过度有关，一种表型以前与斑马鱼的癫痫样行为有关。gabra1的突变伴随着gabra2，gabra3和gabra5的mRNA表达降低，表明GABAAR的其他α亚基表达降低。尽管多个亚单位减少了，幼虫继续对戊四唑（PTZ）作出反应，表明在sa43718等位基因中存在残留的GABAAR。蛋白质组学分析表明，gabra1的突变与调节突触小泡融合的蛋白质的异常表达有关，囊泡运输，突触发育，和线粒体蛋白质复合物。这些数据支持以前在由CRISPR/Cas9创建的斑马鱼无义等位基因中进行的研究，并验证了gabra1基因中功能突变的丧失导致癫痫发作样表型与GABA突触的异常发育。我们的结果增加了关于GABRA1在发育过程中的功能的现有知识体系，并验证了斑马鱼可用于提供基因的完整功能表征。
