The Circadian Molecular Clock is a Biomarker for Epilepsy in Focal Cortical Dysplasia

项目摘要 母体R 01是基于我们对医学难治性小儿癫痫的研究，我们发现 转录因子，昼夜运动输出周期Kaput（时钟）的mRNA水平，与非 癫痫大脑在兴奋性神经元中靶向缺失Clock基因的小鼠具有自发性癫痫发作， 这让我们假设生物钟的丢失会导致电路功能障碍和癫痫。在父R 01中， 和新的实验，我们研究小儿癫痫。在这篇增刊中，我们调查了一种罕见的， 一种新诊断的遗传性婴儿癫痫和SLC 13 A5突变引起的发育迟缓， 钠偶联柠檬酸转运蛋白，使用我们在实验室中已有的模型， 我们在母基金中使用的技术，即视频EEG和全细胞膜片钳电生理学 海马神经元中。slc 13 a5突变导致细胞内柠檬酸盐水平降低，表明代谢 缺损由于我们已经将Clock基因的研究扩展到代谢组学，因此建议的研究是 与代谢缺陷导致回路功能障碍的范围和总体假设一致， 癫痫将Slc 13 a5作为一种不太复杂的单基因疾病进行分析，将有助于我们了解 代谢特征和癫痫之间的联系我们制作了小鼠模型，其中包含两个最重要的 在儿科患者中常见的Slc 13 a5错义突变。初步特征显示， 钠结合结构域错义突变的意外功能获得效应，即更严重的癫痫发作， 符合癫痫持续状态的定义，与Slc 13 a5基因消融形成鲜明对比，Slc 13 a5基因消融不符合癫痫持续状态的定义。 导致癫痫发作，表明基因功能改变我们假设异常的皮层和海马 活动是由兴奋性和兴奋性神经递质水平和电生理特性的改变引起的， 抑制性突触我们将在两个目标中检验我们的假设。在目标1中，我们将研究癫痫的变化 与Slc 13 a5消融相比，与Slc 13 a5突变相关。使用视频脑电图 (EEG)，我们计划测量这些小鼠的癫痫发作阈值，发作间期癫痫样异常，癫痫 严重程度和基线脑电图模式。在目标2中，我们将确定神经递质的变化与 Slc 13 a5突变，并通过膜片钳电生理学确定柠檬酸盐或TCA循环如何改变 中间体导致谷氨酸和GABA水平耗尽。我们还将研究动作电位 产生阈值，放电模式和膜特性，以确定兴奋-抑制性 平衡对杂合和纯合小鼠的无效和错义突变体的分析将有助于确定 突变等位基因获得功能或干扰正常基因活性。这些研究构成了 为我们的URM研究生的论文项目，其多学科的培训计划的职业发展， 神经科学研究者在这个建议中概述。了解遗传和代谢机制 可能导致癫痫及其相关认知和行为症状的新治疗方法。

PROJECT SUMMARY The parent R01 is based on our study of medically refractory pediatric epilepsy, where we identified decreased mRNA levels of the transcription factor, Circadian Locomotor Output Cycles Kaput (Clock), compared with non- epileptic brain. Mice with targeted deletion of the Clock gene in excitatory neurons have spontaneous seizures, leading us to hypothesize that loss of Clock leads to circuit dysfunction and epilepsy. In both the parent R01 and the proposed new experiments, we study pediatric epilepsy. In this supplement, we investigate a rare, newly diagnosed form of genetic infantile epilepsy and developmental delay caused by mutations in SLC13A5, a sodium-coupled citrate transporter, using models that we already have in the laboratory, and using the same techniques that we are using in the parent grant i.e. video EEG and whole cell patch-clamp electrophysiology in hippocampal neurons. Slc13a5 mutations result in decreased intracellular citrate levels, indicating metabolic defect. Since we have extended our parent studies of Clock gene into metabolomics, the proposed studies are in keeping with the scope and overall hypothesis that metabolic defects underlie circuit dysfunction in epilepsy. Analysis of Slc13a5 as a less complex, single gene disorder will help us understand important links between metabolic signatures and epilepsy. We generated mouse models containing two of the most commonly found Slc13a5 missense mutations in pediatric patients. Preliminary characterization revealed an unexpected gain-of-function effect of a sodium-binding domain missense mutation i.e. more severe seizures, meeting the definition of status epilepticus, in striking contrast to Slc13a5 gene ablation, which does not produce seizures, indicating altered gene function. We hypothesize that aberrant cortical and hippocampal activity arises from altered neurotransmitter levels and electrophysiological properties at excitatory and inhibitory synapses. We will test our hypothesis in two Aims. In Aim 1, we will investigate changes in epilepsy associated with Slc13a5 mutations in comparison with Slc13a5 ablation. Using video electroencephalogram (EEG), we plan to measure seizure thresholds in these mice, interictal epileptiform abnormalities, epilepsy severity, and baseline EEG patterns. In Aim 2, we will determine neurotransmitter changes associated with Slc13a5 mutations, and identify, by patch-clamp electrophysiology, how altered citrate or TCA cycle intermediates lead to depletion of glutamate and GABA levels. We will also investigate action potential generation threshold, firing patterns, and membrane properties to determine changes in excitatory-inhibitory balance. Analysis of hetero- and homozygous mouse null and missense mutants would help determine how the mutant allele gains function or interferes with normal gene activity. These studies constitute a major part of a thesis project for our URM graduate student, whose multi-disciplinary training plans for career growth as a neuroscience investigator are outlined in this proposal. Understanding the genetic and metabolic mechanisms may lead to new treatments for epilepsy and its associated cognitive and behavioral symptoms.