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.

中文翻译：

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缺乏 GAD65 同种型的大鼠表现出癫痫和过早致死

GABA 由谷氨酸脱羧酶 (GAD) 合成，GAD 有两种亚型，即 GAD65 和 GAD67，分别由 Gad2 和 Gad1 基因编码。GAD65 缺陷型 (Gad2-/- ) 小鼠在 1 个月大后表现出脑 GABA 含量降低，并在成年后表现出自发性癫痫发作。大约 25% 的 Gad2-/- 小鼠在 6 个月大时死亡。我们的蛋白质印迹分析表明，在出生后发育期间，大鼠大脑中 GAD65 与 GAD67 的蛋白质表达比高于小鼠，这表明每种 GAD 亚型对 GABA 功能的贡献在这两个物种之间不同。为了评估 GAD65 缺乏是否会导致大鼠和小鼠之间的不同表型，我们使用 TALEN 基因组编辑技术生成了 Gad2-/- 大鼠。使用新抗体进行的蛋白质印迹和免疫组织化学分析表明，在 Gad2-/- 大鼠脑中检测不到 GAD65 蛋白。Gad2-/- 幼崽在出生后第 3-4 周的脑电图中表现出自发性癫痫发作和阵发性放电。超过 80% 的 Gad2-/- 大鼠在出生后天数 (PND) 17-23 时死亡。在 PND17-19 时，Gad2-/- 脑中的 GABA 含量显着低于 Gad2+/- 和 Gad2+/+ 脑中的 GABA 含量。这些结果表明 Gad2-/- 大鼠脑中低水平的 GABA 含量可能导致癫痫，继而导致过早死亡，并且 Gad2-/- 大鼠比 Gad2-/- 小鼠受到的影响更严重。考虑到人脑中的 GAD65/GAD67 比率比小鼠大脑中的更类似于大鼠脑中的比率，Gad2-/- 大鼠将有助于进一步研究 GAD65 在体内的作用。