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仍然难以捉摸。在不同类型的遗传性和获得性癫痫中控制脑兴奋性的一个候选者是超极化激活的环核苷酸门控（HCN）通道（h通道）蛋白质家族。h通道介导超极化激活电流（Ih），其对于控制神经元兴奋性至关重要。四个亚基HCN 1 -4联合收割机形成h通道，并在整个脑中差异表达。小鼠Hcn 2基因突变导致类似于人类失神癫痫的癫痫发作。HCN 1和HCN 2也与颞叶癫痫（TLE）有关，TLE是医学难治性癫痫发作的最常见原因。在TLE大鼠模型中，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中的Ih减少可以通过h通道远离远端树突并进入索马内的亚细胞区室的错误定位来解释。这种h通道运输缺陷与HCN 1和含有tetratricopeptide repeat（TPR）的Rab 8b相互作用蛋白（TRIP 8b）（脑中h通道辅助亚基）之间的相互作用减少有关。TRIP 8b存在于多种可变剪接变体中，对h通道运输和功能具有“上调”或“下调”作用，但主要的脑TRIP 8b同种型上调Ih电流密度和HCN 1表面表达。由于TRIP 8b在控制h通道功能中起关键作用的证据，我们产生了三种不同的小鼠品系，其在编码TRIP 8b的基因中进行操作，1）TRIP 8b的完全敲除，2）TRIP 8b的条件敲除，和3）外显子的选择性缺失，将表达限制为“上调”TRIP 8b同种型。初步研究表明，TRIP 8b的完全敲除导致小鼠的失神癫痫。我们假设TRIP 8b小鼠癫痫是由于h通道远离质膜的错误定位导致丘脑和皮质h通道病。我们将通过完成以下具体目标来解决这一假设：1）确定丘脑和皮质中TRIP 8b的区域特异性消除是否导致失神癫痫，以及2）证明h通道表面表达水平控制对失神癫痫发作的易感性。该项目将利用遗传学、生物化学、免疫组织化学和电生理学工具来表征TRIP 8b基因突变小鼠的癫痫机制，其总体目标是表征以前未知的癫痫病因，为未来开发新的癫痫治疗方法奠定基础。 公共卫生相关性：尽管有许多现有的医疗和手术治疗，癫痫发作抵抗医疗干预仍然是一个重要的原因，残疾和死亡的癫痫患者。我们的项目将描述一种新的动物模型，该模型具有导致癫痫的基因突变，并将使用尖端技术来了解基因突变为什么以及如何导致癫痫发作。从公共卫生的角度来看，该项目最重要的好处是描述了一种以前未知的癫痫病因，这将成为开发新疗法以改善癫痫患者生活的基础。

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.