Determining the effects of human KCC2 mutations on neuronal excitability

确定人类 KCC2 突变对神经元兴奋性的影响

• 批准号: 10018116
• 负责人: Paul Andrew Davies
• 金额: $ 20.63万
• 依托单位: TUFTS UNIVERSITY BOSTON
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-15 至 2021-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10018116
• 关键词: AMPA Receptors; Ablation; Action Potentials; Acute; Adult; Affect; Anatomy; Antiepileptic Agents; Architecture; Axon; Brain; Bumetanide; Cell Line; Cell surface; Cells; Chemosensitization; Clinical; Clinical Trials; Complement; Data; Dendritic Spines; Development; Developmental Delay Disorders; Diffusion; Disease; Environment; Epilepsy; Equilibrium; Event; Exhibits; Filopodia; Focal Seizure; Functional disorder; Future; Genes; Genetic; Glutamates; Hippocampus (Brain); Homeostasis; Human; Human Activities; Human Development; Hyperactive behavior; Impairment; Ion Channel; Ions; Lateral; Link; LoxP-flanked allele; Maintenance; Mediating; Membrane; Membrane Potentials; Mental disorders; Modification; Morphology; Mus; Mutation; N-terminal; Nervous system structure; Neurons; Output; Pathway interactions; Patients; Pattern; Periodicity; Permeability; Pharmacology; Phenotype; Phosphorylation; Phosphotransferases; Point Mutation; Population; Potassium; Protein Biochemistry; Proteins; Rodent; Role; Seizures; Signal Transduction; Site; Slice; Structure; Surface; Synaptic Transmission; System; Testing; Therapeutic; Translating; Up-Regulation; Variant; Vertebral column; Virus; cell immortalization; chloride-cotransporter potassium; de novo mutation; druggable target; experimental study; gamma-Aminobutyric Acid; glutamatergic signaling; human disease; improved; infancy; inhibitor/antagonist; insight; kinase inhibitor; mouse development; mutant; neuronal cell body; neuronal excitability; novel; novel therapeutics; overexpression; postnatal; postnatal development; prevent; protein expression; receptor; small molecule; synaptic inhibition; synaptogenesis; therapeutic evaluation; tool; trafficking; transmission process; treatment strategy; upstream kinase

Project Summary The level of activity in the nervous system is tightly controlled by the interplay between excitatory glutamatergic neurons and inhibitory GABA (γ–aminobutyric acid) neurons. When inhibition is compromised, the resultant hyperexcitability is linked to epilepsy, neurodevelopmental disease and psychiatric disorders. The efficacy of GABAergic inhibition is maintained by continuous extrusion of Cl– by the potassium cotransporter KCC2, which permits GABAA receptors to inwardly conduct those ions, resulting in neuron hyperpolarization. KCC2 also exhibits a structural role independent of its transporter function: it affects the maturation of dendritic spine morphology and glutamatergic signaling. KCC2 is upregulated during the first two postnatal weeks in mice, which is roughly equivalent to the final stages of preterm human development. In keeping with this, rare de novo mutations have recently been identified in the kcc2 gene that cause EIMFS, a form of epilepsy in infancy. In surviving patients, the epileptic phenotype persists into adulthood. We have evidence that the point mutations L288H, W318S, and ∆S748 impair the activity of KCC2. Mechanistically, each mutation decreased total protein expression; however, each differently affected the amount of the protein on the cell surface. While these and other experiments demonstrate that the mutations each confer a different pattern of changes to KCC2 that may account for their decreased function, they are limited in that they rely on overexpression of the gene in immortalized cell lines, which are fundamentally different from the neurons in which KCC2 normally functions. By developing a platform where novel mutations in the kcc2 gene can be rapidly characterized, we can guide the development of mouse lines which recapitulate the human disease state and screen for tailored therapeutics to each clinical population. We propose to generate neuron cultures where the normal KCC2 protein is removed and replaced by one of three above mutations found in patients with epilepsy. In addition to a characterization of these mutations in a neuronal context, our approach provides an opportunity to translate how altered KCC2 function as a result of these mutations may predispose to hyperexcitability and epilepsy. In the first aim we will determine how mutations affect the distribution and biochemistry of the protein, and if they disturb the neurons' anatomical maturation. We will also show that each mutation diminishes the capability of KCC2 to maintain hyperpolarizing inhibition. Importantly, KCC2 function and membrane trafficking is affected by phosphorylation at KCC2-T1007. This site is phosphorylated by SPAK kinase, which is phosphorylated and activated by WNK kinase. We have a recently developed WNK kinase inhibitor and our preliminary data indicate that it potentiates KCC2 activity by reducing KCC2-T1007 phosphorylation. In the second aim, we will show how mutations in KCC2 affect seizure activity and if WNK inhibitors will be beneficial. This platform can in the long term be a critical tool to gauge the appropriateness of current treatments and guide the development of future antiepileptic therapeutics for defined clinical populations.

项目摘要 神经系统的活动水平是由兴奋性神经递质之间的相互作用紧密控制的。 神经元和抑制性GABA（γ-氨基丁酸）神经元。当抑制作用受到损害时， 过度兴奋与癫痫、神经发育疾病和精神障碍有关。的疗效 GABA能抑制通过钾协同转运蛋白KCC 2持续排出Cl-来维持， 允许GABAA受体向内传导这些离子，导致神经元超极化。KCC 2也 表现出独立于其转运功能的结构作用：它影响树突棘的成熟 形态学和神经元信号传导。KCC 2在小鼠出生后的前两周内上调， 这大致相当于人类早产儿发育的最后阶段。与此同时，罕见的 最近在kcc 2基因中发现了新突变，该突变导致EIMFS，一种婴儿癫痫。 在存活的患者中，癫痫表型持续到成年。我们有证据表明 突变L288 H、W318 S和W318 S748损害KCC 2的活性。从机制上讲，每个突变都减少了 总蛋白质表达;然而，每种都不同地影响细胞表面上蛋白质的量。而 这些和其他实验表明，每种突变都赋予了不同的变化模式， KCC 2的过度表达可能导致其功能下降，但它们的局限性在于它们依赖于KCC 2的过度表达。 基因在永生化细胞系中，这是根本不同的神经元，其中KCC 2正常 功能协调发展的通过开发一个可以快速表征kcc 2基因中新突变的平台，我们 可以指导小鼠品系的开发，重现人类疾病状态， 每个临床人群的治疗方法。我们建议生成神经元培养物，其中正常的KCC 2 蛋白质被移除并被癫痫患者中发现的上述三种突变之一取代。除了 在神经元背景下对这些突变的表征，我们的方法提供了一个机会， 这些突变导致KCC 2功能改变可能导致过度兴奋和癫痫。在 第一个目标，我们将确定突变如何影响蛋白质的分布和生物化学，如果它们 干扰神经元的解剖成熟。我们还将证明，每一个突变都降低了 KCC 2维持超极化抑制。重要的是，KCC 2功能和膜运输受到影响， 通过KCC 2-T1007的磷酸化。该位点被SPAK激酶磷酸化，SPAK激酶被磷酸化， 由WNK激酶激活。我们有一个最近开发的WNK激酶抑制剂和我们的初步数据 表明其通过减少KCC 2-T1007磷酸化而增强KCC 2活性。第二个目标，我们将 显示KCC 2突变如何影响癫痫发作活动以及WNK抑制剂是否有益。该平台可以在 长期是衡量当前治疗方法是否适当并指导发展的关键工具 未来的抗癫痫治疗的定义临床人群。