Epilepsy and dendritic excitability

描述（由申请人提供）：超极化激活的环核苷酸门控（HCN）通道是电压门控离子通道，可调节参与癫痫发病机制的几个脑区（包括海马、新皮质和丘脑）的兴奋性。大量证据表明，Ih（HCN通道产生的电流）的下调与神经元过度兴奋和癫痫相关。在该项目的前期资助期间，发现动物模型中癫痫的发作与HCN通道表达的丧失和HCN通道门控的下调（即Ih电压依赖性激活的超极化）相关。HCN通道特性的后一种变化在癫痫发作的发生中可能特别重要，因为抗癫痫药物拉莫三嗪作为其抗癫痫作用的一部分产生HCN通道门控的上调。一种新的HCN通道门控调节剂，p38丝裂原活化激酶（p38 MAPK），其特征在于，抑制p38 MAPK导致HCN门控下调。在本提案中，磷酸化（即激酶或磷酸酶）信号和HCN通道之间的联系将被探索。总的假设是，在癫痫中发生的HCN通道门控的下调可能是由于激酶活性的丧失，如p38 MAPK的活性，或磷酸酶活性的增加。我们还将探讨是否直接调制p38 MAPK活性在体内发挥抗癫痫作用。具体而言，我们将回答以下问题：1）癫痫中HCN通道门控的改变是否与激酶活性的丧失有关？2)癫痫患者HCN通道门控改变是否与磷酸酶活性增加有关？3)通过激活p38 MAPK信号上调HCN门控在颞叶癫痫动物模型中是否有效？为了回答这些问题，我们将使用细胞电生理技术在脑切片制备，生化技术来测定激酶和磷酸酶活性，并在匹鲁卡品癫痫动物模型的长期视频脑电图（VEEG）。这些实验的结果应该增加我们的理解癫痫发作改变离子通道生物物理特性的分子机制，确定癫痫是否与磷酸化信号通路紊乱，并探索可能的新的抗癫痫治疗策略。 公共卫生相关性：目前的提案将在细胞和分子水平上研究癫痫的机制。癫痫是最常见的神经系统疾病之一，影响近1%的人口，并导致30%的癫痫患者严重残疾，这些患者的癫痫发作不受现有药物的控制。这项研究的结果可能会确定特定的生化途径，这些途径可以作为开发新型抗癫痫药物的目标，提高目前控制不良的患者有一天可能无癫痫的可能性。

DESCRIPTION (provided by applicant): Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are voltage-gated ion channels that modulate excitability in several brain regions involved in the pathogenesis of epilepsy, including hippocampus, neocortex, and thalamus. The preponderance of evidence shows that downregulation of Ih, the current generated by HCN channels, is associated with neuronal hyperexcitability and epilepsy. In the prior funding period of this project, the onset of epilepsy in an animal model was found to be associated with loss of HCN channel expression, and downregulation of HCN channel gating (i.e. hyperpolarization of Ih voltage-dependent activation). This latter change in HCN channel properties may be particularly important in the generation of seizures, as lamotrigine, an antiepileptic drug, produces upregulation of HCN channel gating as part of its antiepileptic action. A novel modulator of HCN channel gating, p38 mitogen- activated kinase (p38 MAPK), was characterized as well, with inhibition of p38 MAPK causing downregulation of HCN gating. In the present proposal, the links between phosphorylation (i.e. kinase or phosphatase) signaling and HCN channels will be explored. The overall hypothesis is that the downregulation of HCN channel gating that occurs in epilepsy may be due to loss of kinase activity, such as that of p38 MAPK, or to increased phosphatase activity. We will also explore whether direct modulation of p38 MAPK activity exerts an antiepileptic action in vivo. Specifically, we will answer the following questions: 1) Is altered HCN channel gating in epilepsy associated with loss of kinase activity? 2) Is altered HCN channel gating in epilepsy associated with increased phosphatase activity? 3) Is upregulation of HCN gating via activation of p38 MAPK signaling effective in an animal model of temporal lobe epilepsy (TLE)? To answer these questions, we will use cellular electrophysiology techniques in the brain slice preparation, biochemical techniques to assay kinase and phosphatase activity, and long-term video-electroencephalography (VEEG) in the pilocarpine animal model of epilepsy. The outcome of these experiments should increase our understanding of the molecular mechanisms by which seizures alter ion channel biophysical properties, determine whether epilepsy is associated with derangement of phosphorylation signaling pathways, and explore possible novel antiepileptic treatment strategies. PUBLIC HEALTH RELEVANCE: The current proposal will study the mechanisms of epilepsy at cellular and molecular levels. Epilepsy is one of the most common neurological diseases, affecting nearly 1% of the population, and causing significant disability in the 30% of epilepsy patients whose seizures are uncontrolled by existing medication. The outcome of this study may identify specific biochemical pathways that could be targeted for development of novel antiepileptic drugs, improving the likelihood that currently poorly-controlled patients may one day be seizure-free.