Cellular Pathophysiology of Neuronal Na/K-ATPase Dysfunction

神经元 Na/K-ATP 酶功能障碍的细胞病理生理学

• 批准号: 10539624
• 负责人: Alfred L. George
• 金额: $ 40万
• 依托单位: NORTHWESTERN UNIVERSITY AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-06-15 至 2027-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10539624
• 关键词: ATP1A3 gene; Action Potentials; Acute; Address; Cell Survival; Cell model; Childhood; Chronic; Conflict (Psychology); Developmental Delay Disorders; Disease; Dominant-Negative Mutation; Dystonia; Epilepsy; Equilibrium; Exhibits; Functional disorder; Genes; Genetic; Goals; Hemiplegia; Homeostasis; Human; Impairment; Induced pluripotent stem cell derived neurons; Ions; Knock-out; Measurement; Mediating; Membrane; Membrane Potentials; Migraine; Modeling; Molecular; Monitor; Mutation; Na(+)-K(+)-Exchanging ATPase; Neurodevelopmental Disorder; Neurologic Dysfunctions; Neurologic Symptoms; Neuronal Dysfunction; Neurons; Outcome; Pathogenesis; Patients; Pharmacology; Physiological; Predisposition; Proteins; Pump; Recovery; Resources; Rest; Secondary to; Splice-Site Mutation; Symptoms; Syndrome; Testing; Time; Transgenes; Viral; Work; alternating hemiplegia; cerebral atrophy; cytotoxicity; driving force; effective therapy; excitatory neuron; experimental study; extracellular; gamma-Aminobutyric Acid; gene therapy; induced pluripotent stem cell; knock-down; loss of function; loss of function mutation; mutant; nervous system disorder; neurodevelopment; neuron loss; neuronal excitability; neurotoxicity; neurotransmission; novel therapeutic intervention; optogenetics; prevent; voltage

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.

概括 ATP1A3中杂合丧失功能突变，编码神经元的催化（α3）亚基的基因 Na/k-ATPase与一系列神经发育综合征有关 没有有效治疗的儿童偏瘫（AHC）交替偏瘫。这些条件是 与瞬态无力和肌张力障碍的急性发作有关，长期结局不良 神经发育和脑萎缩认为是慢性神经元丧失的继发性。尽管很少见，但ATP1A3 突变引起的神经功能障碍，由癫痫和偏头痛等常见疾病共享。尽管 关于这些疾病的遗传基础的知识已经很多，ATP1A3的细胞后果 人类神经元和基本病理生理机制的功能障碍知之甚少。我们 已经使用与患者特异性诱导的神经元建模了ATP1A3突变的细胞效应 多能干细胞（IPSC）。我们建议利用该模型来确定细胞病理生理学 与Na/k泵活性受损相关的机制以及由此解释的离子稳态改变的机制 短期（偏瘫，肌张力障碍）和长期（发育延迟，慢性神经元丧失）表现 ATP1A3功能障碍。在AIM 1中，我们将测试直接测量神经泵电流的假设 可以区分单倍不足和主导阴性机制，并确定泵是否受损 可以通过病毒ATP1A3变换来挽救活动。在AIM 2中，我们将检验一个钝的假设 跨膜K+浓度梯度引起去极化的神经元静息膜电位作为A 介导向外K+泄漏电流的低于正常驱动力的结果，这会影响神经元 兴奋性。我们将通过确定是否会增强K+泄漏通道活动来检验该假设 或在ATP1A3突变神经元中通常会补偿细胞内至细胞外K+驱动 强力，使静息电势归一化并防止沉积阻滞。单独的实验将调查 突变和非突变神经元之间沉积块的敏感性和恢复性，并相关 这些具有细胞内NA+动力学的发现。在AIM 3中，我们将研究潜在的细胞病理生理学 负责ATP1A3长期表现的机制。我们将测试ATP1A3的假设 突变神经元暴露了延迟的GABA开关，可以通过抑制或敲除纠正 NA/K/2CL共转运蛋白（NKCC1）。最后，我们将检验假设损害Na/K-ATPase活性渲染的假设 神经元易受细胞内Na+过载，这会触发胞质Ca2+过载和细胞毒性。 总的来说，这项工作将揭示与短期和长期神经元发病机理相关的重要方面 使用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。