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

抽象的。细胞周期蛋白依赖性激酶样5（CDKL 5）缺乏症（CDD）是一种神经发育性癫痫 一种以早发性癫痫、睡眠障碍和发育障碍为特征的脑病， 挑战具有全身性、多灶性和肌阵挛性发作的耐药性癫痫是一个主要问题 对于CDD患者。目前，没有针对CDKL 5病症的靶向治疗或基因治疗。CDKL 5是一种 丝氨酸/苏氨酸蛋白激酶，尽管已知其对于正常脑发育是必需的， 目前尚不清楚CDKL 5靶向哪些蛋白质。KCC 2是主要的Cl-挤出机制 在中枢神经系统中发育和成熟的神经元中使用。它的活性是速效的先决条件 γ-氨基丁酸A型受体（GABAAR）介导的突触抑制， 门控离子通道典型超极化GABAAR电流的出生后发育反映了 由KCC 2表达上调引起的神经元内Cl-水平的进行性降低， 后续活动。超极化GABAAR电流的发育外观由以下因素决定： KCC 2的磷酸化状态，这是一个促进其膜运输和活性的过程。KCC 2的赤字 表达水平和活性已经在癫痫患者和动物模型中详细描述。而且我们 已经证明KCC 2功能丧失与认知障碍密切相关， 对GABAAR阳性变构调节剂不敏感的药物抗性癫痫发作的发展， 苯二氮卓类药物。为了解决这个问题，我们已经开发了新的小分子活化剂， KCC 2活性，在初步研究中，已显示可有效终止药物耐药性 癫痫发作CDKL 5缺失小鼠在出生后第12天具有脑电样EEG事件，并且在出生后第12天的脑电活动中显著降低。 磷酸化KCC 2的量。我们将进一步研究KCC 2-S1022之间的这种关系 磷酸化和CDKL 5。我们的工作假设是：CDKL 5的缺失导致磷酸化减少 S1022 KCC 2，KCC 2活性降低，耐药性癫痫发作的易感性增加。 KCC 2的小分子活化剂将增加KCC 2活性并防止耐药性 癫痫发作为了验证这一假设，我们提出了以下目标：目标1。为了确定消融的效果， KCC 2磷酸化对CDKL 5表达的影响。目标2.为了确定消融CDKL 5 KCC 2活性的表达。具体目标3。确定消融KCC 2 S1022的后果 磷酸化对KCC 2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， CDKL 5的靶点以及导致在癫痫患者中发生的使人衰弱的药物抗性癫痫的机制。 CDD。这些见解可能会促进新疗法的开发，以减轻药物治疗的负担。 难治性癫痫，影响许多癫痫患者。

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.