Haploinsufficiency of the CACNA1A gene, encoding the pore-forming α1 subunit of P/Q-type voltage-gated calcium channels, is associated with a clinically variable phenotype ranging from cerebellar ataxia, to neurodevelopmental syndromes with epilepsy and intellectual disability. To understand the pathological mechanisms of CACNA1A loss-of-function variants, we characterized a human neuronal model for CACNA1A haploinsufficiency, by differentiating isogenic induced pluripotent stem cell lines into glutamatergic neurons, and investigated the effect of CACNA1A haploinsufficiency on mature neuronal networks through a combination of electrophysiology, gene expression analysis, and in silico modeling. We observed an altered network synchronization in CACNA1A+/- networks alongside synaptic deficits, notably marked by an augmented contribution of GluA2-lacking AMPA receptors. Intriguingly, these synaptic perturbations coexisted with increased non-synaptically driven activity, as characterized by inhibition of NMDA and AMPA receptors on micro-electrode arrays. Single-cell electrophysiology and gene expression analysis corroborated this increased intrinsic excitability through reduced potassium channel function and expression. Moreover, we observed partial mitigation of the CACNA1A+/- network phenotype by 4-aminopyridine, a therapeutic intervention for episodic ataxia type 2. Positive modulation of KCa2 channels could reverse the CACNA1A+/- network electrophysiological phenotype. In summary, our study pioneers the characterization of a human induced pluripotent stem cell-derived neuronal model for CACNA1A haploinsufficiency, and has unveiled novel mechanistic insights. Beyond showcasing synaptic deficits, this neuronal model exhibited increased intrinsic excitability mediated by diminished potassium channel function, underscoring its potential as a therapeutic discovery platform with predictive validity.

中文翻译：

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CACNA1A单倍体不足导致突触功能降低和内在兴奋性增加


编码 P/Q 型电压门控钙通道的成孔 α1 亚基的 CACNA1A 基因单倍体不足与临床可变表型有关，范围从小脑性共济失调到伴有癫痫和智力障碍的神经发育综合征。为了了解CACNA1A功能丧失变异的病理机制，我们通过将同基因诱导的多能干细胞系分化为谷氨酸能神经元来表征CACNA1A单倍体不足的人类神经元模型，并通过电生理学、基因表达分析和计算机建模相结合，研究了CACNA1A单倍体不足对成熟神经元网络的影响。我们观察到 CACNA1A+/- 网络中的网络同步与突触缺陷一起改变，特别是以缺乏 GluA2 的 AMPA 受体的贡献增加为标志。有趣的是，这些突触扰动与增加的非突触驱动活动共存，其特征是抑制微电极阵列上的 NMDA 和 AMPA 受体。单细胞电生理学和基因表达分析证实了这种通过降低钾通道功能和表达而增加的内在兴奋性。此外，我们观察到 4-氨基吡啶（一种针对发作性共济失调 2 型的治疗干预）对 CACNA1A+/- 网络表型的部分缓解。KCa2 通道的正调制可以逆转 CACNA1A+/- 网络电生理表型。总之，我们的研究开创了人类诱导多能干细胞衍生的神经元模型对CACNA1A单倍体不足的表征，并揭示了新的机制见解。 除了显示突触缺陷外，这种神经元模型还表现出由钾通道功能减弱介导的内在兴奋性增加，强调了其作为具有预测有效性的治疗发现平台的潜力。