OBJECTIVE Mutations of the cyclin-dependent kinase-like 5 (CDKL5) gene cause severe neurodevelopmental disorders characterized by intractable epilepsy, intellectual disability, and autism. Multiple mouse models generated for mechanistic studies have exhibited phenotypes similar to some human pathological features, but none of the models has developed one of the major symptoms affecting CDKL5 deficiency disorder (CDD) patients: intractable recurrent seizures. As disrupted neuronal excitation/inhibition balance is closely associated with the activity of glutamatergic and γ-aminobutyric acidergic (GABAergic) neurons, our aim was to study the effect of the loss of CDKL5 in different types of neurons on epilepsy. METHODS Using the Cre-LoxP system, we generated conditional knockout (cKO) mouse lines allowing CDKL5 deficiency in glutamatergic or GABAergic neurons. We employed noninvasive video recording and in vivo electrophysiological approaches to study seizure activity in these Cdkl5 cKO mice. Furthermore, we conducted Timm staining to confirm a morphological alteration, mossy fiber sprouting, which occurs with limbic epilepsy in both human and mouse brains. Finally, we performed whole-cell patch clamp in dentate granule cells to investigate cell-intrinsic properties and synaptic excitatory activity. RESULTS We demonstrate that Emx1- or CamK2α-derived Cdkl5 cKO mice manifest high-frequency spontaneous seizure activities recapitulating the epilepsy of CDD patients, which ultimately led to sudden death in mice. However, Cdkl5 deficiency in GABAergic neurons does not generate such seizures. The seizures were accompanied by typical epileptic features including higher amplitude spikes for epileptiform discharges and abnormal hippocampal mossy fiber sprouting. We also found an increase in spontaneous and miniature excitatory postsynaptic current frequencies but no change in amplitudes in the dentate granule cells of Emx1-cKO mice, indicating enhanced excitatory synaptic activity. SIGNIFICANCE Our study demonstrates that Cdkl5 cKO mice, serving as an animal model to study recurrent spontaneous seizures, have potential value for the pathological study of CDD-related seizures and for therapeutic innovation.

中文翻译：

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前脑谷氨酸能神经元中的 CDKL5 缺陷导致复发性自发性癫痫发作

目的 细胞周期蛋白依赖性激酶样 5 (CDKL5) 基因的突变会导致严重的神经发育障碍，其特征是顽固性癫痫、智力障碍和自闭症。为机制研究生成的多个小鼠模型表现出与某些人类病理特征相似的表型，但没有一个模型出现影响 CDKL5 缺陷症 (CDD) 患者的主要症状之一：顽固性复发性癫痫发作。由于神经元兴奋/抑制平衡被破坏与谷氨酸能和 γ-氨基丁酸能 (GABAergic) 神经元的活性密切相关，我们的目的是研究不同类型神经元中 CDKL5 缺失对癫痫的影响。方法 使用 Cre-LoxP 系统，我们生成了条件性敲除 (cKO) 小鼠品系，允许谷氨酸能或 GABA 能神经元中的 CDKL5 缺陷。我们采用无创视频记录和体内电生理学方法来研究这些 Cdkl5 cKO 小鼠的癫痫发作活动。此外，我们进行了 Timm 染色以确认形态学改变，苔藓纤维发芽，这在人类和小鼠大脑的边缘癫痫中都会发生。最后，我们在齿状颗粒细胞中进行了全细胞膜片钳以研究细胞内在特性和突触兴奋活动。结果我们证明，Emx1 或 CamK2α 衍生的 Cdkl5 cKO 小鼠表现出高频自发性癫痫发作活动，重演了 CDD 患者的癫痫，最终导致小鼠猝死。然而，GABA 能神经元中的 Cdkl5 缺陷不会产生此类癫痫发作。癫痫发作伴随着典型的癫痫特征，包括癫痫样放电的高振幅尖峰和异常的海马苔藓纤维发芽。我们还发现自发和微型兴奋性突触后电流频率增加，但 Emx1-cKO 小鼠齿状颗粒细胞的振幅没有变化，表明兴奋性突触活动增强。意义我们的研究表明，Cdkl5 cKO 小鼠作为研究复发性自发性癫痫发作的动物模型，对 CDD 相关癫痫发作的病理学研究和治疗创新具有潜在价值。我们还发现自发和微型兴奋性突触后电流频率增加，但 Emx1-cKO 小鼠齿状颗粒细胞的振幅没有变化，表明兴奋性突触活动增强。意义我们的研究表明，Cdkl5 cKO 小鼠作为研究复发性自发性癫痫发作的动物模型，对 CDD 相关癫痫发作的病理学研究和治疗创新具有潜在价值。我们还发现自发和微型兴奋性突触后电流频率增加，但 Emx1-cKO 小鼠齿状颗粒细胞的振幅没有变化，表明兴奋性突触活动增强。意义我们的研究表明，Cdkl5 cKO 小鼠作为研究复发性自发性癫痫发作的动物模型，对 CDD 相关癫痫发作的病理学研究和治疗创新具有潜在价值。