Deficits in KCC2 activity and the pathophysiology of Status Epilepticus

KCC2 活性缺陷和癫痫持续状态的病理生理学

基本信息

批准号：
8839921
负责人：
Stephen J Moss
金额：
$ 37.28万
依托单位：
TUFTS UNIVERSITY BOSTON
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-09-15 至 2019-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8839921
关键词：
Accounting Address Adult Alanine Aminobutyric Acids Animal Model Brain Brain Injuries Breeding C-terminal Cessation of life Data Development Disease Drug resistance Electroencephalography Emergency Situation Epilepsy Event Exhibits Functional disorder Genotype Health Homeostasis Intractable Epilepsy Lead Lysine Measures Mediating Medical Modification Morbidity - disease rate Mus Mutation Neurons Pathology Patients Phenotype Phospho-Specific Antibodies Phosphorylation Phosphotransferases Pilocarpine Play Potassium Chloride Protein Dephosphorylation Protein Isoforms Protein Kinase C Reagent Role Seizures Serine Soman Status Epilepticus Tertiary Protein Structure Testing Threonine Time Trauma clinically significant cost insight kainate mortality novel novel therapeutics prevent receptor research study selective expression symporter synaptic inhibition

项目摘要

DESCRIPTION (provided by applicant): The electroneutral K+/Cl- co-transporter 2 (KCC2) allows neurons to maintain low intracellular Cl- concentrations, an essential prerequisite for fast synaptic inhibition mediated by type A γ-aminobutyric acid (GABAAR). Consistent with this, deficits in KCC2 activity lead to seizures and are believed to be central to the pathology of Status Epilepticus (SE). SE is the most devastating form of epilepsy, and accounts for 42,000 deaths per year in the US, and hundreds of thousands more cases of severe brain damage. SE becomes less tractable with time, leading to the development of drug resistant seizures, resulting in increased mortality and morbidity. SE is associated with a cost of $4.8 billion per year in the US. Consistent with its essential role in regulating neuronal Cl- homeostasis, deficits in KCC2 activity are seen in patients with intractable epilepsy, and in animal models of SE. Therefore, understanding the mechanisms by which SE leads to inactivation of KCC2 is of clear clinical significance. KCC2 function is subject to both positive and negative modulation via phosphorylation of key regulatory residues within the C-terminal intracellular domain of this protein. Specifically, phosphorylation of serine 940 (S940) by protein kinase C enhances KCC2 activity, while phosphorylation of the adjacent threonine residues 906 and 1007 by with-no-lysine kinases (WNKs) decreases transporter activity (Lee et al., 2007; 2011; Riehart, 2009). Thus, one mechanism that may contribute to KCC2 inactivation during SE is modifications in the phosphorylation of these key regulatory residues. To address this issue, we have utilized phospho-specific antibodies against S940 and T906. In addition, we have created mice in which the phosphorylation of these key regulatory residues has been prevented via mutation to alanines. Finally, we have made use of mice deficient in WNK3, the principle WNK isoform expressed in the adult brain. Preliminary studies using these novel reagents have allowed us to formulate an overarching hypothesis that will be tested here; "Persistent elevations in neuronal activity during SE lead to dephosphorylation of S940, but enhanced phosphorylation of T906/1007, events that lead to rapid inhibition of KCC2, reductions in the efficacy of GABAergic inhibition that directly contribute to the pathophysiology of SE". Our studies will focus on the following specific aims. Specific Aim 1. To test the hypothesis that deficits in KCC2 phosphorylation contribute to the development and lethality of SE Specific Aim 2. To test the hypothesis that S940A mice exhibit enhanced T906 phosphorylation and a selective deficit in KCC2 activity during SE Specific Aim 3. To test the hypothesis that reducing WNK dependent phosphorylation of KCC2 prevents the development of SE. Collectively these experiments will provide key mechanistic insights into the pathophysiology of SE, and may aid the development of novel therapeutics to limit the impact of this devastating disorder.

描述（由申请人提供）：电中性 K+/Cl- 协同转运蛋白 2 (KCC2) 使神经元能够维持较低的细胞内 Cl- 浓度，这是快速反应的必要先决条件。与此一致的是，KCC2 活性缺陷会导致癫痫发作，并且被认为是癫痫持续状态 (SE) 病理学的核心，SE 是最具破坏性的癫痫形式。每年导致美国 42,000 人死亡，并且随着时间的推移，数十万严重脑损伤病例变得越来越难处理，导致耐药性的产生。癫痫发作，导致死亡率和发病率增加，在美国每年造成 48 亿美元的费用，这与其在调节神经元 Cl-稳态中的重要作用相一致，在难治性癫痫患者和儿童中也发现了 KCC2 活性的缺陷。因此，了解 SE 导致 KCC2 失活的机制具有明确的临床意义。KCC2 功能受到磷酸化的正向和负向调节。该蛋白 C 端胞内结构域内的关键调节残基具体而言，蛋白激酶 C 对丝氨酸 940 (S940) 的磷酸化增强了 KCC2 活性，而无赖氨酸激酶 (WNK) 对邻近的苏氨酸残基 906 和 1007 进行磷酸化。 ) 降低转运蛋白活性 (Lee et al., 2007; 2011; Riehart, 2009）因此，SE 期间可能导致 KCC2 失活的一种机制是这些关键调节残基的磷酸化，为了解决这个问题，我们利用了针对 S940 和 T906 的磷酸特异性抗体。其中，这些关键调节残基的磷酸化通过突变为丙氨酸而被阻止。最后，我们利用了 WNK3 缺陷的小鼠，WNK3 是在成人中表达的主要 WNK 亚型。使用这些新试剂的初步研究使我们能够制定一个总体假设，并将在此进行测试；“SE 期间神经元活动的持续升高导致 S940 去磷酸化，但增强了 T906/1007 的磷酸化，这些事件导致快速抑制。 KCC2 的 GABA 能抑制功效降低，直接导致 SE 的病理生理学。我们的研究将集中于以下具体目标。具体目标 1. 检验 KCC2 磷酸化缺陷导致 SE 特定目标的发生和致死的假设 2. 检验 S940A 小鼠在 SE 特定目标 3 期间表现出 T906 磷酸化增强和 KCC2 活性选择性缺陷的假设减少 KCC2 的 WNK 依赖性磷酸化可以防止 SE 的发展的假设，这些实验将为 SE 的病理生理学提供关键的机制见解，以及可能有助于开发新的疗法来限制这种破坏性疾病的影响。