Rescuing KCC2 Dysfunction in CDKL5 Deficiency Disorder to Restore GABA(A) Receptor-Mediated Hyperpolarization and Seizure Protection.

挽救 CDKL5 缺乏症中的 KCC2 功能障碍，以恢复 GABA(A) 受体介导的超极化和癫痫保护。

• 批准号: 10581661
• 负责人: Paul Andrew Davies
• 金额: $ 20.43万
• 依托单位: TUFTS UNIVERSITY BOSTON
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-03-01 至 2025-02-28
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10581661
• 关键词: Ablation; Address; Affect; Affinity Chromatography; Age Months; Animal Model; Appearance; Benzodiazepines; Brain; CDKL5 disorder; Child; Consensus; Coupled; Cyclin-Dependent Kinases; Development; Disease; Electroencephalography; Epilepsy; Event; Focal Seizure; Functional disorder; Genotype; Hippocampus; Impaired cognition; Intervention; Ion Channel Gating; Knockout Mice; Ligands; Liquid Chromatography; LoxP-flanked allele; Mass Spectrum Analysis; Mediating; Membrane; Mus; Myoclonic Epilepsies; Neurons; Patients; Permeability; Persons; Phosphorylation; Phosphorylation Site; Phosphotransferases; Predisposition; Process; Proline; Prosencephalon; Protein-Serine-Threonine Kinases; Proteins; Proteome; Proteomics; Role; Seizures; Site; Sleep disturbances; Testing; Up-Regulation; Work; early onset; epileptic encephalopathies; gamma-Aminobutyric Acid; gel electrophoresis; gene therapy; insight; kainate; loss of function; novel; novel therapeutics; patch clamp; positive allosteric modulator; postnatal; postnatal development; prevent; protein complex; pup; receptor; small molecule; synaptic inhibition; targeted treatment; trafficking

Abstract. Cyclin-dependent kinase-like 5 (CDKL5) deficiency disorder (CDD) is a neurodevelopmental epileptic encephalopathy that is characterized by early-onset epilepsy, sleep disturbances, and developmental challenges. Pharmaco-resistant epilepsy with generalized, multifocal, and myoclonic seizures are a major issue for CDD patients. Currently, no targeted treatment or gene therapy exists for CDKL5 disorder. CDKL5 is a serine/threonine protein kinase and although it is known to be essential for normal brain development and function it is unknown which proteins are targeted by CDKL5. KCC2 is the principal Cl--extrusion mechanism employed by developing and mature neurons in the CNS. Its activity is a prerequisite for the efficacy of fast synaptic inhibition mediated by γ-aminobutyric acid type A receptors (GABAAR), which are Cl- permeable ligand- gated ion channels. The postnatal development of canonical hyperpolarizing GABAAR currents reflects the progressive decrease of intraneuronal Cl- levels that is caused by the upregulation of KCC2 expression and subsequent activity. The developmental appearance of hyperpolarizing GABAAR currents is determined by the phosphorylation status of KCC2, a process that facilitates its membrane trafficking and activity. Deficits in KCC2 expression levels and activity have been detailed in patient and animal models of epilepsy. Furthermore, we have demonstrated that KCC2 loss of function is strongly correlated with cognitive impairment, and the development of pharmaco-resistant seizures that are insensitive to GABAAR positive allosteric modulators such as benzodiazepines. To address this issue, we have developed novel small molecule activators that potentiate KCC2 activity which, in preliminary studies, have been shown to effectively terminate pharmaco-resistant seizures. CDKL5 null mice have seizure-like EEG events at postnatal day 12 and had a significant decrease in the amount of phosphorylated KCC2. We will further examine this relationship between KCC2-S1022 phosphorylation and CDKL5. Our working hypothesis is:Loss of CDKL5 results in a reduced phosphorylation of S1022 KCC2, decreased KCC2 activity, and an increased susceptibility to pharmacoresistant seizures. Small molecule activators of KCC2 will increase KCC2 activity and prevent pharmacoresistance seizures. To test this hypothesis, we propose the following aims: Aim1. To determine the effects of ablating CDKL5 expression on KCC2 phosphorylation. Aim 2. To determine the effects of ablating CDKL5 expression on KCC2 activity. Specific Aim 3. Determine the consequences of ablating KCC2 S1022 phosphorylation on KCC2 function and expression. The result of this study will provide new insights into the targets of CDKL5 and the mechanisms that result in the debilitating pharmaco-resistant epilepsy occurring in CDD. Such insights may promote the development of new therapeutics to alleviate the burdens of pharmaco- resistant epilepsies that affect many epileptic patients.

摘要：细胞周期蛋白依赖性激酶样 5 (CDKL5) 缺乏症 (CDD) 是一种神经发育性癫痫症。 以早发性癫痫、睡眠障碍和发育障碍为特征的脑病 全身性、多灶性和肌阵挛性癫痫发作的耐药性癫痫是一个主要问题。 对于 CDD 患者，目前尚无针对 CDKL5 疾病的靶向治疗或基因治疗。 丝氨酸/苏氨酸蛋白激酶，尽管已知它对正常大脑发育至关重要， 目前尚不清楚哪些蛋白质是 CDKL5 的主要 Cl- 挤出机制。 中枢神经系统中正在发育和成熟的神经元所使用，其活性是快速发挥功效的先决条件。 由 γ-氨基丁酸 A 型受体 (GABAAR) 介导的突触抑制，GABAAR 是 Cl-可渗透配体- 典型超极化 GABAAR 电流的出生后发展反映了 由 KCC2 表达上调引起的神经元内 Cl- 水平进行性降低 超极化 GABAAR 电流的发育表现由 KCC2 的磷酸化状态，这是一个促进其膜运输和活性的过程。 此外，我们在癫痫患者和动物模型中详细说明了表达水平和活性。 已经证明 KCC2 功能丧失与认知障碍密切相关，并且 发生对 GABAAR 正变构调节剂不敏感的耐药性癫痫发作，例如 为了解决这个问题，我们开发了新型小分子激活剂。 初步研究表明 KCC2 活性可有效终止耐药性 CDKL5缺失小鼠在出生后第12天出现癫痫样脑电图事件，并且癫痫发作显着减少。 我们将进一步检查 KCC2-S1022 之间的这种关系。 我们的工作假设是：CDKL5 的缺失导致磷酸化减少。 S1022 KCC2 的缺失，KCC2 活性降低，耐药性癫痫发作的易感性增加。 KCC2 小分子激活剂将增加 KCC2 活性并防止耐药性 为了检验这一假设，我们提出以下目标： 目的 1. 确定消融的效果。 CDKL5 表达对 KCC2 磷酸化的影响 目标 2. 确定消除 CDKL5 的影响。 具体目标 3. 确定消除 KCC2 S1022 的后果。 磷酸化对KCC2功能和表达的影响这项研究的结果将为我们提供新的见解。 CDKL5 的靶点以及导致衰弱性耐药性癫痫发生的机制 CDD 的见解可能会促进新疗法的开发，以减轻药物负担。 影响许多癫痫患者的难治性癫痫。