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: 9894063
• 负责人: Tarek Ziad Deeb
• 金额: $ 24.75万
• 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/9894063
• 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; druggable target; experimental study; gamma-Aminobutyric Acid; glutamatergic signaling; human disease; immortalized cell; 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.

项目概要 神经系统的活动水平受到兴奋性谷氨酸能之间的相互作用的严格控制。 神经元和抑制​​性 GABA（γ-氨基丁酸）神经元。当抑制作用减弱时，所产生的 过度兴奋与癫痫、神经发育疾病和精神疾病有关。功效 GABA 能抑制是通过钾协同转运蛋白 KCC2 持续排出 Cl- 来维持的， 允许 GABAA 受体向内传导这些离子，导致神经元超极化。 KCC2也 表现出与其转运功能无关的结构作用：它影响树突棘的成熟 形态和谷氨酸信号传导。 KCC2 在小鼠出生后的前两周内表达上调， 这大致相当于早产人类发育的最后阶段。与此相一致，罕见的德 最近在 kcc2 基因中发现了新突变，该突变会导致 EIMFS（婴儿期癫痫的一种形式）。 在幸存的患者中，癫痫表型持续到成年。我们有证据表明这一点 突变 L288H、W318S 和 ΔS748 会损害 KCC2 的活性。从机制上讲，每次突变都会减少 总蛋白表达；然而，每种方法对细胞表面蛋白质含量的影响不同。尽管 这些和其他实验表明，每个突变都会赋予不同的变化模式 KCC2 可能是其功能下降的原因，但它们的局限性在于依赖于 KCC2 的过度表达 永生化细胞系中的基因与通常含有 KCC2 的神经元有根本不同 功能。通过开发一个可以快速表征 kcc2 基因新突变的平台，我们 可以指导重现人类疾病状态的小鼠品系的开发，并筛选定制的 针对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 KCC2 蛋白质被去除并被癫痫患者中发现的上述三种突变之一所取代。此外 在神经元环境中表征这些突变，我们的方法提供了翻译的机会 这些突变导致 KCC2 功能的改变如何可能导致过度兴奋和癫痫。在 我们的第一个目标是确定突变如何影响蛋白质的分布和生物化学，以及它们是否会影响蛋白质的分布和生物化学。 扰乱神经元的解剖成熟。我们还将证明，每个突变都会削弱 KCC2 维持超极化抑制。重要的是，KCC2 功能和膜运输受到影响 通过 KCC2-T1007 磷酸化。该位点被 SPAK 激酶磷酸化，SPAK 激酶被磷酸化并 由 WNK 激酶激活。我们最近开发了一种 WNK 激酶抑制剂以及我们的初步数据 表明它通过减少 KCC2-T1007 磷酸化来增强 KCC2 活性。在第二个目标中，我们将 显示 KCC2 突变如何影响癫痫发作活动以及 WNK 抑制剂是否有益。这个平台可以在 从长远来看，成为衡量当前治疗方法是否适当并指导发展的关键工具 针对特定临床人群的未来抗癫痫疗法。