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

基本信息

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
项目类别：
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

项目摘要

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性水平受兴奋性谷氨酸能之间的相互作用紧密控制神经元和抑制性GABA（γ-氨基丁酸）神经元。当抑制遭到损害时，结果过度兴奋与癫痫，神经发育疾病和精神疾病有关。效率通过连续扩展Cl-钾共转运蛋白KCC2来维持GABA能抑制作用，该抑制作用允许GABAA受体向内进行这些离子，从而导致神经元超极化。 KCC2也是如此表现出与其转运蛋白功能无关的结构作用：它影响树突状脊柱的成熟形态和谷氨酸能信号传导。 KCC2在出生后几周的小鼠中上调，这大致相当于早产的最后阶段。因此，稀有DE 最近在KCC2基因中鉴定出Novo突变，该基因导致EIMF是婴儿期癫痫的一种形式。在生存患者中，癫痫表型持续到成年。我们有证据表明重点突变L288H，W318和∆S748损害了KCC2的活性。从机械上讲，每个突变都减少总蛋白表达；但是，每个人都对细胞表面上蛋白质的量有所不同。尽管这些和其他实验表明，这些突变每个都赋予了不同的变化模式可能解释其功能降低的KCC2，它们受到限制，因为它们依靠过表达永生的细胞系中的基因，与KCC2通常不同的神经元有根本不同功能。通过开发一个可以快速表征KCC2基因中新型突变的平台，我们可以指导小鼠线的发展，这些小鼠线概括了人类疾病状态和筛选的筛选每个临床人群的治疗学。我们建议在正常KCC2的情况下产生神经元培养物蛋白质被去除并用癫痫患者的以上突变之一取代。此外在神经元背景下这些突变的特征，我们的方法提供了翻译的机会这些突变导致KCC2的作用如何改变可能会易于过度兴奋和癫痫。在第一个目的我们将确定突变如何影响蛋白质的分布和生物化学，以及它们是否是否干扰神经元的解剖学成熟。我们还将表明，每个突变都会降低 KCC2维持过度抑制。重要的是，KCC2功能和膜运输受到影响通过KCC2-T1007的磷酸化。该位点被SPAK激酶磷酸化，后者是磷酸化的，并且由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在第二个目标中，我们将展示KCC2中的突变如何影响癫痫发作的活性以及WNK抑制剂是否有益。这个平台可以长期是评估当前治疗的适当性并指导发展的关键工具针对定义的临床人群的未来抗癫痫疗法。