The axon initial segment (AIS) is a highly regulated subcellular domain required for neuronal firing. Changes in the AIS protein composition and distribution are a form of structural plasticity, which powerfully regulates neuronal activity and may underlie several neuropsychiatric and neurodegenerative disorders. Despite its physiological and pathophysiological relevance, the signaling pathways mediating AIS protein distribution are still poorly studied. Here, we used confocal imaging and whole-cell patch clamp electrophysiology in primary hippocampal neurons to study how AIS protein composition and neuronal firing varied in response to selected kinase inhibitors targeting the AKT/GSK3 pathway, which has previously been shown to phosphorylate AIS proteins. Image-based features representing the cellular pattern distribution of the voltage-gated Na+ (Nav) channel, ankyrin G, βIV spectrin, and the cell-adhesion molecule neurofascin were analyzed, revealing IV spectrin as the most sensitive AIS protein to AKT/GSK3 pathway inhibition. Within this pathway, inhibition of AKT by triciribine has the greatest effect on IV spectrin localization to the AIS and its subcellular distribution within neurons, a phenotype that Support Vector Machine classification was able to accurately distinguish from control. Treatment with triciribine also resulted in increased excitability in primary hippocampal neurons. Thus, perturbations to signaling mechanisms within the AKT pathway contribute to changes in IV spectrin distribution and neuronal firing that may be associated with neuropsychiatric and neurodegenerative disorders.

中文翻译：

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抑制 AKT 信号会改变 AIS 处的 IV 血影蛋白分布并增加神经元兴奋性

轴突初始段 (AIS) 是神经元放电所需的高度调节的亚细胞域。AIS 蛋白质组成和分布的变化是结构可塑性的一种形式，它有力地调节神经元活动，可能是几种神经精神和神经退行性疾病的基础。尽管具有生理和病理生理学相关性，但介导 AIS 蛋白分布的信号通路仍然缺乏研究。在这里，我们在原代海马神经元中使用共聚焦成像和全细胞膜片钳电生理学来研究 AIS 蛋白组成和神经元放电如何响应针对 AKT/GSK3 通路的选定激酶抑制剂而变化，该通路以前已被证明可以磷酸化 AIS 蛋白。分析了代表电压门控 Na+ (Nav) 通道、锚蛋白 G、βIV 血影蛋白和细胞粘附分子神经成束蛋白的细胞模式分布的基于图像的特征，揭示了 IV 血影蛋白是对 AKT/GSK3 最敏感的 AIS 蛋白通路抑制。在该途径中，曲西瑞滨对 AKT 的抑制对 IV 血影蛋白定位到 AIS 及其在神经元内的亚细胞分布具有最大影响，支持向量机分类能够准确地区分这种表型与对照。用曲西瑞滨治疗还导致原代海马神经元的兴奋性增加。因此，