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蛋白5(CDKL5)缺乏症是一种神经发育性癫痫 以早发性癫痫、睡眠障碍和发育为特征的脑病 挑战。具有全身性、多灶性和肌阵挛发作的耐药癫痫是一个主要问题。 适用于CDD患者。目前，还没有针对CDKL5疾病的靶向治疗或基因治疗。CDKL5是一种 丝氨酸/苏氨酸蛋白激酶，尽管已知它对正常的大脑发育和 功能尚不清楚CDKL5针对的是哪些蛋白质。KCC2是主要的氯离子挤出机理 被中枢神经系统发育和成熟的神经元所利用。它的活性是FAST疗效的先决条件 γ-氨基丁酸A型受体介导的突触抑制，是一种氯离子通透性配体- 门控离子通道。正则超极化伽巴电流的后天发展反映了 KCC2表达上调引起的神经元内氯离子水平进行性降低 后续活动。超极化伽巴电流的发展形态是由 KCC2的磷酸化状态，这是一个促进其膜运输和活性的过程。KCC2的赤字 在癫痫的患者和动物模型中，表达水平和活性都得到了详细的研究。此外，我们 已经证明KCC2功能丧失与认知障碍密切相关，而 对GABAAR阳性变构调节剂不敏感的耐药癫痫的研究进展 作为苯二氮卓类药物。为了解决这个问题，我们开发了新型的小分子激活剂，它可以增强 KCC2活性，在初步研究中已被证明能有效地终止抗药性 癫痫发作。CDKL5基因缺失的小鼠在出生后第12天出现癫痫样脑电事件，并在出生后12天显著减少 磷酸化KCC2的量。我们将进一步研究KCC2-S1022之间的这种关系 磷酸化和CDKL5。我们的工作假设是：CDKL5的缺失会导致磷酸化水平的降低 在S1022KCC2中，KCC2活性降低，对耐药癫痫的易感性增加。 KCC2小分子激活剂可提高KCC2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：要确定消融的效果 CDKL5在KCC2磷酸化中的表达。目的2.确定消融CDKL5的效果 KCC2活性的表达。具体目标3.确定KCC2 S1022烧蚀的后果 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对 CDKL5的靶点及其致衰性耐药癫痫的机制 CDD。这些见解可能会促进新疗法的发展，以减轻药物治疗的负担。 影响许多癫痫患者的难治性癫痫。

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.