Epilepsy and dendritic excitability

 描述(申请人提供)：超极化激活环核苷酸门控(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。越来越多的证据表明，在获得性癫痫模型中，HCN通道产生的电流Ih的下调导致神经元过度兴奋，并且主要的皮质和海马亚型HCN1通道的基因缺失加速了癫痫的发生。最近的证据表明，在一些患有严重癫痫和发育迟缓的儿童中，HCN1基因突变是导致早期癫痫脑病的原因。因此，在人类遗传性癫痫和获得性癫痫的动物模型中都会发生HN1通道病。在我们实验室最近的工作中，化学惊厥诱导的癫痫持续状态(SE)与HCN1通道表达的丢失有关，这种表达在SE后1小时内开始，并持续到慢性癫痫。在癫痫发作后的第一个小时内，HCN1通道从海马锥体树突的表面膜上迅速内化，直到几天后HCN1 mRNA转录下调的开始。我们现在首次表明，HCN1通道表面的表达是由蛋白激酶C(PKC)活性双向控制的，PKC对HCN1的磷酸化增加导致HCN1表面表达降低。使用质谱学技术的新的初步数据表明，在静止状态下，HCN1通道的磷酸化程度最低，但在S867和S868残基上发生了更高的磷酸化，在PKC激活的条件下，这两个残基在幼稚组织中也被磷酸化。这些数据表明，在癫痫发生过程中，HCN1通道功能障碍的最早表现是PKC介导的表面表达的丧失以及伴随而来的HCN1介导的电流(Ih)的丧失。我们在R01更新应用中的目标是进一步了解癫痫发展过程中HN1通道功能障碍的机制。我们建议进一步研究SE后HCN1膜转运的急性变化背后的磷酸化信号机制；确定这些早期事件是否导致或启动慢性HCN1通道病；并表征人类HCN1突变对HCN1通道生物物理特性的影响。为此，我们将使用最先进的技术，包括：质谱学；病毒转导体内突变的HCN1通道表达；CA1海马区锥体神经元树突的细胞电生理学；以及对800名原因不明的癫痫性脑病儿童的新型HCN1突变数据集的分析。

 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 the hippocampus, neocortex, and thalamus. Accumulated evidence shows that downregulation of Ih, the current generated by HCN channels, causes neuronal hyperexcitability, and that genetic deletion of HCN1 channels, the main cortical and hippocampal subtype, accelerates the rate of epileptogenesis in acquired epilepsy models. More recent evidence shows that mutations in HCN1 underlie early life epileptic encephalopathy in some children with severe epilepsy and developmental delay. Thus HCN1 channelopathy occurs in both human genetic epilepsy and animal models of acquired epilepsy. In recent work from our laboratory, epilepsy induced by chemoconvulsant-induced status epilepticus (SE) was associated with loss of HCN1 channel expression that began within 1 hour post-SE, and persisted into chronic epilepsy. HCN1 channels were acutely internalized from the surface membrane of hippocampal pyramidal dendrites within the first hour following SE, well before the onset several days later of transcriptional downregulation of HCN1 mRNA expression. We now show for the first time that HCN1 channel surface expression is governed in a bidirectional fashion by protein kinase C (PKC) activity, with increased phosphorylation of HCN1 by PKC leading to decreased HCN1 surface expression. New preliminary data using mass spectrometry techniques show that HCN1 channels are minimally phosphorylated at rest, but undergo increased phosphorylation at S867 and S868 residues, which are also phosphorylated in naive tissue under conditions of PKC activation. These data suggest that the earliest manifestation of HCN1 channels dysfunction during epileptogenesis is PKC-mediated loss of surface expression and concomitant loss of HCN1-mediated current (Ih). Our goal in this R01 renewal application is to further understand the mechanisms of HCN1 channel dysfunction during the development of epilepsy. We propose to further study the phosphorylation signaling mechanisms underlying acute changes in HCN1 membrane trafficking after SE; determine whether these early events contribute to or initiate chronic HCN1 channelopathy; and characterize the effects of human HCN1 mutations on HCN1 channel biophysical properties. To do so, we will use state-of-the-art techniques including: mass spectrometry; viral transduction of mutant HCN1 channel expression in vivo; cellular electrophysiology in the dendrites of CA1 hippocampal pyramidal neurons; and analysis of a novel dataset of HCN1 mutations in a cohort of 800 children affected with epileptic encephalopathy of unknown cause.