Epilepsy and dendritic excitability

描述(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，调节参与癫痫发病机制的几个大脑区域的兴奋性，包括海马体、新皮质和丘脑。大量证据表明，hcn通道产生的电流ih的下调与神经元过度兴奋和癫痫有关。在该项目的早期资助期间，动物模型中癫痫的发作被发现与HCN通道表达的丧失和HCN通道门控的下调(即ih电压依赖激活的超极化)有关。HCN通道特性的后一种变化在癫痫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丝裂原活化蛋白激酶(P38MAPK)也被鉴定，抑制p38MAPK导致HCN门控下调。在本提案中，将探索磷酸化(即，激酶或磷酸酶)信号与HCN通道之间的联系。总体的假设是，癫痫时HCN通道门控的下调可能是由于p38MAPK等激酶活性的丧失，或者是由于磷酸酶活性的增加。我们还将探讨直接调节p38MAPK活性是否在体内发挥抗癫痫作用。具体来说，我们将回答以下问题：1)在癫痫中，HCN通道门控是否与激酶活性丧失有关？2)在癫痫中，HCN通道门控是否与磷酸酶活性增加有关？3)通过激活p38MAPK信号上调HCN门控在颞叶癫痫(TLE)动物模型中是否有效？为了回答这些问题，我们将在脑片制备中使用细胞电生理学技术，在匹罗卡品癫痫动物模型中使用生化技术测定激酶和磷酸酶活性，并进行长期视频脑电图(VEEG)。这些实验的结果将增加我们对癫痫改变离子通道生物物理性质的分子机制的理解，确定癫痫是否与磷酸化信号通路的紊乱有关，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，影响着近1%的人口，并在30%的癫痫患者中导致严重残疾，这些患者的癫痫发作无法通过现有的药物控制。这项研究的结果可能会确定特定的生化途径，可以作为开发新型抗癫痫药物的靶点，从而提高目前控制不佳的患者有朝一日可能没有癫痫发作的可能性。

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.