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

项目总结 父母R01是基于我们对儿童难治性癫痫的研究，我们发现减少了 转录因子、昼夜节律运动输出循环Kaput(Clock)的mRNA水平，与非 癫痫样的大脑。兴奋性神经元中时钟基因定向缺失的小鼠有自发性癫痫发作， 这让我们假设，时钟丢失会导致电路功能障碍和癫痫。在两个父R01中 在提出的新实验中，我们研究了儿童癫痫。在本增刊中，我们调查了一种罕见的、 新诊断的遗传性婴儿癫痫和SLC13A5基因突变所致的发育迟缓， 一种钠偶联柠檬酸转运体，使用我们已经在实验室中拥有的模型，并使用相同的 我们在家长资助中使用的技术，即视频脑电和全细胞膜片钳电生理学 在海马神经元中。SLC13a5突变导致细胞内柠檬酸水平下降，表明代谢 叛逃。由于我们已将母公司对时钟基因的研究扩展到代谢组学，建议的研究包括 为了与代谢缺陷导致脑循环功能障碍的范围和总体假设保持一致 癫痫。分析SLC13a5作为一种不那么复杂的单基因疾病将有助于我们理解重要的联系 代谢特征和癫痫之间的关系。我们生成了包含两个最重要的 在儿科患者中常见的SLc13a5错义突变。初步表征显示， 钠结合结构域错义突变的意外功能增强效应，即更严重的癫痫发作， 符合癫痫持续状态的定义，与SLc13a5基因消融形成鲜明对比，后者不 导致癫痫发作，表明基因功能发生了变化。我们假设异常的大脑皮层和海马区 兴奋性和非兴奋性的神经递质水平和电生理特性的改变引起活动。 抑制性突触。我们将在两个目标上检验我们的假设。在目标1中，我们将研究癫痫的变化。 与SLc13a5消融术相比，与SLc13a5突变相关。使用视频脑电图仪 (EEG)，我们计划测量这些小鼠的癫痫阈值、发作间期癫痫样异常、癫痫 严重程度和基线脑电模式。在目标2中，我们将确定相关的神经递质变化 SLC13a5突变，并通过膜片钳电生理学确定柠檬酸或TCA循环如何改变 中间体会导致谷氨酸和GABA水平的耗尽。我们还将调查行动潜力 产生阈值、放电模式和膜特性以确定兴奋性抑制的变化 平衡。对杂合和纯合子小鼠零和错义突变的分析将有助于确定 突变的等位基因获得功能或干扰正常的基因活动。这些研究构成了 我们的URM研究生的毕业论文项目，他的多学科培训计划作为一名 这份提案概述了神经科学研究人员的情况。了解遗传和代谢机制 可能导致治疗癫痫及其相关的认知和行为症状的新疗法。

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.