Cellular Pathophysiology of Neuronal Na/K-ATPase Dysfunction

总结 ATP 1A 3基因的杂合性功能丧失突变，该基因编码神经元的催化（α3）亚基。 Na/K-ATP酶与一系列神经发育综合征有关，包括典型的 儿童交替性偏瘫（AHC），目前尚无有效的治疗方法。这些条件 与短暂性虚弱和肌张力障碍的急性发作相关， 神经发育和脑萎缩被认为继发于慢性神经元损失。ATP 1A 3虽然罕见， 突变引起常见疾病如癫痫和偏头痛所共有的神经功能障碍。而 关于这些疾病的遗传基础，ATP 1A 3的细胞后果，已经有了很多了解。 人类神经元的功能障碍和基本的病理生理学机制知之甚少。我们 已经使用从患者特异性诱导的神经元分化的神经元模拟了ATP 1A 3突变的细胞效应。 多能干细胞（iPSC）。我们建议利用这个模型来确定细胞的病理生理 与受损的Na/K泵活性相关的机制以及由此导致的离子稳态改变， 短期（偏瘫、肌张力障碍）和长期（发育迟缓、慢性神经元丢失）表现 ATP 1A 3功能障碍在目标1中，我们将检验神经元泵电流的直接测量 可以区分单倍功能不全和显性负性机制，并确定是否受损泵 可以用病毒ATP 1A 3转基因挽救活性。在目标2中，我们将检验一个假设， 跨膜K+浓度梯度引起去极化的神经元静息膜电位， 结果低于正常驱动力介导外向K+漏电流，影响神经元 兴奋性我们将通过确定增强K+渗漏通道活性是否会导致细胞内钾离子浓度升高来检验这一假设。 或遗传上在ATP 1A 3突变神经元中将补偿钝化的细胞内到细胞外的K+驱动 使静息电位正常化，防止去极化阻滞。单独的实验将调查 突变和非突变神经元之间对去极化阻滞敏感性和从去极化阻滞的恢复，以及相关性 这些发现与细胞内Na+动力学。在目标3中，我们将研究潜在的细胞病理生理学 负责ATP 1A 3的长期表现的机制。我们将检验ATP 1A 3 突变的神经元表现出延迟的GABA开关，这可以通过抑制或敲低 Na/K/2Cl协同转运蛋白（NKCC 1）。最后，我们将检验Na/K-ATP酶活性受损导致 神经元易受细胞内Na+超载的影响，这可触发胞质Ca 2+超载和细胞毒性。 总的来说，这项工作将揭示短期和长期神经元发病机制的重要方面， 与ATP 1A 3功能障碍，并促进机械驱动的方法，以寻找新的治疗策略。

SUMMARY Heterozygous loss-of-function mutations in ATP1A3, the gene encoding the catalytic (α3) subunit of the neuronal Na/K-ATPase, are associated with a spectrum of neurodevelopmental syndromes including the prototypical disorder Alternating Hemiplegia of Childhood (AHC), which has no effective therapy. These conditions are associated with acute attacks of transient weakness and dystonia, and poor long term outcome with delayed neurodevelopment and brain atrophy believed secondary to chronic neuron loss. Although rare, ATP1A3 mutations evoke neurological dysfunction shared by common disorders such as epilepsy and migraine. While much has been learned about the genetic basis of these disorders, the cellular consequences of ATP1A3 dysfunction in human neurons and fundamental pathophysiological mechanisms are poorly understood. We have modeled the cellular effects of ATP1A3 mutations using neurons differentiated from patient-specific induced pluripotent stem cells (iPSCs). We propose to exploit this model to determine cellular pathophysiological mechanisms associated with impaired Na/K pump activity and the resulting altered ion homeostasis that explain both short term (hemiplegia, dystonia) and long term (developmental delay, chronic neuron loss) manifestation of ATP1A3 dysfunction. In Aim 1, we will test the hypothesis that direct measurement of neuronal pump current can distinguish between haploinsufficiency and dominant-negative mechanisms, and determine if impaired pump activity can be rescued with a viral ATP1A3 transgene. In Aim 2, we will test the hypothesis that a blunted transmembrane K+ concentration gradient causes a depolarized neuronal resting membrane potential as a consequence of lower than normal driving force mediating outward K+ leak current, which impacts neuronal excitability. We will test this hypothesis by determining if potentiating K+ leak channel activity pharmacologically or genetically in ATP1A3 mutant neurons will compensate for the blunted intracellular to extracellular K+ driving force, normalize the resting potential and prevent depolarization block. Separate experiments will investigate susceptibility to and recovery from depolarization block between mutant and non-mutant neurons, and correlate these findings with intracellular Na+ dynamics. In Aim 3, we will investigate potential cellular pathophysiological mechanisms responsible for the long-term manifestations of ATP1A3. We will test the hypothesis that ATP1A3 mutant neurons exhibit a delayed GABA switch and this can be corrected by inhibition or knockdown of the Na/K/2Cl cotransporter (NKCC1). Finally, we will test hypothesis that impaired Na/K-ATPase activity renders neurons susceptible to intracellular Na+ overload, which can trigger cytosolic Ca2+ overload and cytotoxicity. Collectively, this work will reveal important aspects of short- and long-term neuronal pathogenesis associated with ATP1A3 dysfunction, and promote a mechanistically driven approach to finding new therapeutic strategies.