Role of TRIP8b in epilepsy

描述（由申请人提供）：药物和手术治疗难治性癫痫发作对许多癫痫患者的生活产生负面影响，并导致显著的发病率和死亡率，但治疗难治性癫痫发作的基本机制尚不清楚。在不同类型的遗传性和获得性癫痫中，控制大脑兴奋性的一个候选是超极化激活的环核苷酸门控（HCN）通道（h通道）家族蛋白。h通道介导超极化激活电流（Ih），这是控制神经元兴奋性的关键。四个亚单位，HCN1-4，结合形成h通道，并在整个大脑中表达差异。小鼠的Hcn2基因突变导致癫痫发作，类似于人类的癫痫发作。HCN1和HCN2也与颞叶癫痫（TLE）有关，颞叶癫痫是医学上难治性癫痫发作的最常见原因。在大鼠TLE模型中，海马树突中的Ih下调，这种变化导致兴奋性增加，并可能导致这些癫痫动物的癫痫发作倾向增加。TLE的Ih降低可以解释为h通道的错误定位，远离远端树突，进入体细胞内的亚细胞区室。这种h通道运输缺陷与HCN1与脑内h通道辅助亚基-含Rab8b相互作用蛋白（TRIP8b）的相互作用减少有关。TRIP8b存在于多种可选择的剪接变体中，对h通道运输和功能具有“上调”或“下调”作用，但主要的脑TRIP8b亚型上调了h电流密度和HCN1表面表达。由于有证据表明TRIP8b在控制h通道功能中起着关键作用，我们通过对编码TRIP8b的基因进行操作，生成了三种不同的小鼠品系，1)完全敲除TRIP8b， 2)条件敲除TRIP8b， 3)选择性删除外显子，将表达限制在“上调”的TRIP8b亚型。初步研究表明，完全敲除TRIP8b可导致小鼠癫痫缺失。我们假设TRIP8b小鼠的癫痫是由于远离质膜的h通道定位错误导致的丘脑和皮质h通道病变。我们将通过完成以下具体目标来解决这一假设：1)确定丘脑和皮层中TRIP8b的区域特异性消除是否导致失神癫痫，2)证明h通道表面表达水平控制失神癫痫的易感性。本项目将利用遗传、生化、免疫组织化学和电生理等工具，对携带TRIP8b基因编码基因突变的小鼠进行癫痫发病机制的表征，以确定未知的癫痫病因，为未来开发新的癫痫治疗方法奠定基础。

DESCRIPTION (provided by applicant): Seizures refractory to medical and surgical management negatively affect the lives of many patients with epilepsy and lead to significant morbidity and mortality, but basic mechanisms underlying therapy-resistant seizures remain elusive. One candidate for controlling brain excitability in different types of genetic and acquired epilepsy is the hyperpolarization-activated cyclic nucleotide-gated (HCN) channel (h channel) family of proteins. h channels mediate hyperpolarization-activated current, (Ih), which is critical for control of neuronal excitability. Four subunits, HCN1-4, combine to form h channels and are expressed differentially throughout the brain. Mutation of the Hcn2 gene in mice results in seizures resembling those in human absence epilepsy. HCN1 and HCN2 have also been implicated in temporal lobe epilepsy (TLE), the most common cause of medically refractory seizures. In a rat model of TLE, Ih is downregulated in hippocampal dendrites, a change that leads to increased excitability and may contribute to increased seizure propensity in these epileptic animals. Reduced Ih in TLE can be explained by mislocalization of h channels away from distal dendrites and into subcellular compartments within the soma. This h channel trafficking defect is associated with reduced interaction between HCN1 and tetratricopeptide repeat (TPR)-containing Rab8b interacting protein (TRIP8b), the h channel auxiliary subunit in brain. TRIP8b exists in multiple alternative splice variants with "upregulating" or "downregulating" effects on h channel trafficking and function, but the predominant brain TRIP8b isoforms upregulate Ih current density and HCN1 surface expression. Because of evidence that TRIP8b plays a critical role in controlling h channel function, we generated three distinct lines of mice with manipulations in the gene encoding TRIP8b, 1) Total knockout of TRIP8b, 2) conditional knockout of TRIP8b, and 3) selective deletion of exons, limiting expression to "upregulating" TRIP8b isoform. Preliminary studies reveal that total knockout of TRIP8b leads to absence epilepsy in mice. We hypothesize that epilepsy in TRIP8b mice results from thalamic and cortical h channelopathy due to mislocalization of h channels away from the plasma membrane. We will address this hypothesis by completing the following specific aims: 1) to determine if region-specific elimination of TRIP8b in thalamus and cortex leads to absence epilepsy, and 2) to demonstrate that h channel surface expression levels control susceptibility to absence seizures. This project will utilize genetic, biochemical, immunohistochemical and electrophysiological tools to characterize the mechanisms of epilepsy in mice with mutations of the gene encoding TRIP8b gene, with the overarching goal of characterizing a previously unknown cause of epilepsy to build the foundation for future development of novel epilepsy treatments. PUBLIC HEALTH RELEVANCE: Despite numerous existing medical and surgical treatments, seizures resistant to medical intervention remain a significant cause of disability and death in patients with epilepsy. Our project will characterize a new animal model with a mutation in a gene that leads to epilepsy, and will use cutting-edge techniques to understand why and how the gene mutation leads to seizures. From a public health perspective, the most important benefit of this project is the characterization of a previously unknown cause of epilepsy that will be the foundation for development of novel treatments to improve the lives of patients with epilepsy.