Department of Genetic and Behavioral Neuroscience, Gunma University Graduate School of Medicine, Maebashi, Japan.
Department of Physiology, Tohoku University Graduate School of Medicine, Sendai, Japan.
FASEB J. 2021 Feb;35(2):e21224. doi: 10.1096/fj.202001935R. Epub 2020 Nov 25.
GABA is synthesized by glutamate decarboxylase (GAD), which has two isoforms, namely, GAD65 and GAD67, encoded by the Gad2 and Gad1 genes, respectively. GAD65-deficient (Gad2 ) mice exhibit a reduction in brain GABA content after 1 month of age and show spontaneous seizures in adulthood. Approximately 25% of Gad2 mice died by 6 months of age. Our Western blot analysis demonstrated that the protein expression ratio of GAD65 to GAD67 in the brain was greater in rats than in mice during postnatal development, suggesting that the contribution of each GAD isoform to GABA functions differs between these two species. To evaluate whether GAD65 deficiency causes different phenotypes between rats and mice, we generated Gad2 rats using TALEN genome editing technology. Western blot and immunohistochemical analyses with new antibodies demonstrated that the GAD65 protein was undetectable in the Gad2 rat brain. Gad2 pups exhibited spontaneous seizures and paroxysmal discharge in EEG at postnatal weeks 3-4. More than 80% of the Gad2 rats died at postnatal days (PNDs) 17-23. GABA content in Gad2 brains was significantly lower than those in Gad2 and Gad2 brains at PND17-19. These results suggest that the low levels of brain GABA content in Gad2 rats may lead to epilepsy followed by premature death, and that Gad2 rats are more severely affected than Gad2 mice. Considering that the GAD65/GAD67 ratio in human brains is more similar to that in rat brains than in mouse brains, Gad2 rats would be useful for further investigating the roles of GAD65 in vivo.
GABA 是由谷氨酸脱羧酶 (GAD) 合成的，GAD 有两种同工型，即 GAD65 和 GAD67，分别由 Gad2 和 Gad1 基因编码。GAD65 缺陷型 (Gad2 ) 小鼠在 1 个月龄后大脑 GABA 含量减少，并在成年后出现自发性癫痫发作。大约 25%的 Gad2 小鼠在 6 月龄前死亡。我们的 Western blot 分析表明，在出生后发育过程中，大鼠大脑中 GAD65 与 GAD67 的蛋白表达比率高于小鼠，这表明两种物种中每种 GAD 同工型对 GABA 功能的贡献不同。为了评估 GAD65 缺乏是否导致大鼠和小鼠之间出现不同表型，我们使用 TALEN 基因组编辑技术生成了 Gad2 大鼠。使用新抗体的 Western blot 和免疫组织化学分析表明，Gad2 大鼠大脑中的 GAD65 蛋白无法检测到。Gad2 幼鼠在出生后第 3-4 周出现自发性癫痫发作和 EEG 阵发性放电。超过 80%的 Gad2 大鼠在出生后第 17-23 天死亡。Gad2 大脑中的 GABA 含量明显低于 Gad2 和 Gad2 大脑中的 GABA 含量。这些结果表明，Gad2 大鼠大脑中 GABA 含量低可能导致癫痫发作，随后死亡，而且 Gad2 大鼠比 Gad2 小鼠受到的影响更严重。考虑到人类大脑中的 GAD65/GAD67 比率与大鼠大脑中的比率更相似，而与小鼠大脑中的比率更不相似，因此 Gad2 大鼠将有助于进一步研究 GAD65 在体内的作用。
