Cellular Pathophysiology of Neuronal Na/K-ATPase Dysfunction

摘要 编码神经元催化亚单位(α3)亚单位的基因ATP1A3的杂合性功能丧失突变 Na/K-ATPase与一系列神经发育综合征相关，包括典型的 儿童障碍交替性偏瘫(AHC)，目前尚无有效治疗方法。这些条件是 与一过性虚弱和肌张力障碍的急性发作有关，长期预后差且延迟 神经发育和脑萎缩被认为是慢性神经元丧失的继发性疾病。虽然罕见，但ATP1A3 突变会引发癫痫和偏头痛等常见疾病所共有的神经功能障碍。而当 关于这些疾病的遗传基础，ATP1A3的细胞后果，人们已经了解了很多 人类神经元功能障碍和基本的病理生理机制还知之甚少。我们 使用从患者特异性诱导的神经元分化的神经元来模拟ATP1A3突变的细胞效应 多能干细胞(IPSCs)。我们建议利用这一模型来确定细胞的病理生理。 与钠/钾泵活性受损和由此引起的离子稳态改变相关的机制解释 短期(偏瘫、肌张力障碍)和长期(发育迟缓、慢性神经元丧失)表现 ATP1A3功能障碍。在目标1中，我们将检验这样的假设：神经元泵电流的直接测量 可以区分单倍体功能不全和显性负性机制，并确定泵功能是否受损 病毒ATP1A3转基因可以挽救病毒的活性。在目标2中，我们将测试一个钝化的假设 跨膜K浓度梯度导致去极化的神经元静息膜电位 影响神经元的外向钾泄漏电流的驱动力低于正常的后果 兴奋性。我们将通过确定是否从药理上增强钾泄漏通道的活性来检验这一假说 或者在遗传上，ATP1A3突变神经元将补偿钝化的细胞内到细胞外K驱动 用力，使静息电位正常化，防止去极化阻断。单独的实验将调查 突变和非突变神经元之间的去极化阻断的敏感性和恢复，以及相关性 这些发现与细胞内钠的动态变化有关。在目标3中，我们将研究潜在的细胞病理生理学 ATP1A3长期表现的机制。我们将检验ATP1A3的假设 突变神经元表现出延迟的GABA开关，这可以通过抑制或敲除 Na/K/2Cl共转运体(NKCC1)。最后，我们将检验Na/K-ATPase活性受损的假设 神经元容易受到细胞内钠超载的影响，从而引发细胞内钙超载和细胞毒性。 总的来说，这项工作将揭示相关的短期和长期神经元发病机制的重要方面。 ATP1A3功能障碍，并促进一种机械驱动的方法来寻找新的治疗策略。

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.