Shen Dingding, Chen Juan, Liu Dong, Shen Mi, Wang Xin, Wu Youjia, Ke Shuan, Macdonald Robert L, Zhang Qi
Department of Neurology & Collaborative Innovation Center for Brain Science, Ruijin Hospital Affiliated to Shanghai Jiaotong University School of Medicine, Shanghai, China.
Key Laboratory of Neuroregeneration of Jiangsu and Ministry of Education, Co-innovation Center of Neuroregeneration, Nantong University, Nantong, China.
Ann Transl Med. 2020 Dec;8(23):1560. doi: 10.21037/atm-20-3745.
Mutations in the γ-aminobutyric acid type A (GABA) receptor γ2 subunit gene, , have been associated frequently with epilepsy syndromes with varying severities. Recently, a mutation, c.T1027C, p.F343L, was identified in a patient with an early onset epileptic encephalopathy (EOEE). , we demonstrated that GABA receptors containing the mutant γ2(F343L) subunit have impaired trafficking to the cell surface. Here, we aim to validate an zebrafish model of EOEE associated with the mutation T1027C.
We generated a novel transgenic zebrafish (AB strain) that overexpressed mutant human γ2(F343L) subunits and provided an initial characterization of the transgenic Tg( ) zebrafish.
Real-time quantitative PCR and hybridization identified a significant up-regulation of in the mutant transgenic zebrafish, which has a well-established role in epileptogenesis. In the larval stage 5 days postfertilization (dpf), freely swimming Tg( ) zebrafish displayed spontaneous seizure-like behaviors consisting of whole-body shaking and hyperactivity during automated locomotion video tracking, and seizures can be induced by light stimulation. Using RNA sequencing, we investigated transcriptomic changes due to the presence of mutant γ2L(F343L) subunits and have found 524 genes that are differentially expressed, including up-regulation of 33 genes associated with protein processing. More specifically, protein network analysis indicated histone deacetylases (HDACs) as potential therapeutic targets, and suberanilohydroxamic acid (SAHA), a broad HDACs inhibitor, alleviated seizure-like phenotypes in mutant zebrafish larvae.
Overall, our Tg( ) overexpression zebrafish model provides the first example of a human epilepsy-associated mutation resulting in spontaneous seizures in zebrafish. Moreover, HDAC inhibition may be worth investigating as a therapeutic strategy for genetic epilepsies caused by missense mutations in and possibly in other central nervous system genes that impair surface trafficking.
γ-氨基丁酸A型（GABA）受体γ2亚基基因的突变经常与不同严重程度的癫痫综合征相关。最近，在一名早发性癫痫性脑病（EOEE）患者中鉴定出一种突变，即c.T1027C，p.F343L。我们证明，含有突变型γ2（F343L）亚基的GABA受体向细胞表面的转运受损。在此，我们旨在验证与突变T1027C相关的EOEE斑马鱼模型。
我们构建了一种新型转基因斑马鱼（AB品系），其过表达突变型人γ2（F343L）亚基，并对转基因Tg（ ）斑马鱼进行了初步表征。
实时定量PCR和杂交鉴定出突变型转基因斑马鱼中 显著上调，其在癫痫发生中具有既定作用。在受精后5天（dpf）的幼体阶段，自由游动的Tg（ ）斑马鱼在自动运动视频跟踪期间表现出自发性癫痫样行为，包括全身颤抖和多动，并且光刺激可诱发癫痫发作。使用RNA测序，我们研究了由于存在突变型γ2L（F343L）亚基而导致的转录组变化，发现有524个基因差异表达，包括33个与蛋白质加工相关的基因上调。更具体地说，蛋白质网络分析表明组蛋白脱乙酰酶（HDACs）是潜在的治疗靶点，而一种广泛的HDACs抑制剂辛二酰苯胺异羟肟酸（SAHA）减轻了突变斑马鱼幼体的癫痫样表型。
总体而言，我们的Tg（ ）过表达斑马鱼模型提供了人类癫痫相关 突变导致斑马鱼自发性癫痫发作的首个实例。此外，HDAC抑制作为由 错义突变以及可能由其他损害表面转运的中枢神经系统基因引起的遗传性癫痫的治疗策略可能值得研究。
