Epilepsy Mechanisms and the Path to Intervention

项目摘要/摘要 编码一种质膜柠檬酸转运蛋白的SLC13A5基因突变导致一种新的 遗传性癫痫的诊断形式称为早期婴儿癫痫性脑病，其特征是 新生儿多灶性惊厥。这些婴儿随后会出现认知和行为缺陷。人类 遗传学已经发现了常见的错义突变和缺失突变，但尚不清楚是如何发生的 不同的基因突变会影响疾病的表现和癫痫的严重程度。我们正在通过以下方式解决这个问题 一组携带SLC13A5突变小鼠模型的特征：i)内源性小鼠SLC13A5的消融 基因(基因敲除)，II)患者最常见的突变，G222R点突变(相当于人类 突变G219R)，以及iii)第二常见的患者突变，T230M(相当于人类 突变T227M)。我们的初步数据表明，纯合子Slc13a5基因敲除小鼠 癫痫样脑电(EEG)异常，而G222R有癫痫发作，并明显 更严重的癫痫样活动。初步的组织病理学检查显示， 基因敲除和G222R，G222R还表现为少突胶质细胞丢失。这些结果是 与我们关于特定错义突变可能获得显性功能增益效应的解释一致 这会加剧脆弱性和神经元的过度兴奋性。淘汰赛表现出的兴奋性和 突触后抑制活动提示神经递质水平降低。我们将进一步描述这些特性 用突变小鼠模型进行以下实验：实验1.1，发作间期的特征 脑电检测突变等位基因的放电和癫痫发作，同时进行脑组织病理学分析。在……里面 实验1.2，我将应用电生理技术来研究细胞内的缺陷 SLC13a5基因敲除和错义突变中的柠檬酸转运通过改变谷氨酸影响神经元功能 和GABA浓度。将进行神经递质的代谢组学筛查以证实 功能数据。在实验1.3中，我将研究这些SLC13A5突变蛋白对细胞的影响 病理学。结合起来，这些实验将研究分子和细胞机制 SLC13A5疾病的癫痫表型，这反过来将为治疗方法提供信息。 我将接受电生理学和分子生物学方面的培训，以完成这些任务。 实验。我们的研究培训计划和持续的专业发展将为我提供技能 让我进入科学生涯的下一个阶段。在博士后阶段，我计划积累经验 神经系统疾病实验模型中的基因治疗策略。我会继续调查手机 和大脑疾病的分子机制，并将这些发现应用于优化基因治疗 接近了。此外，我将继续培养成为独立人士所需的专业技能 学术研究机构的首席研究员。

PROJECT SUMMARY/ABSTRACT Mutations in the SLC13A5 gene, which encodes a plasma membrane citrate transporter, result in a newly diagnosed form of genetic epilepsy termed early infantile epileptic encephalopathy, which is characterized by multi-focal seizures in neonates. These infants subsequently develop cognitive and behavioral deficits. Human genetics has identified both commonly occurring missense and deletion mutations, but it is not known how distinct genetic mutations affect disease presentation and seizure severity. We are addressing this question by characterizing an array of SLC13A5 mutant mouse models carrying: i) ablation of its endogenous murine Slc13a5 gene (knockout), ii) the most common patient mutation, the G222R point mutation (equivalent to the human mutation G219R), and iii) the second most common patient mutation, the T230M (equivalent to the human mutation T227M). Our preliminary data demonstrates that homozygous Slc13a5 knockout mouse demonstrates abnormal epileptiform electroencephalogram (EEG) profiles, while the G222R has seizures and significantly more severe epileptiform activity. Preliminary histopathology reveals differential interneuron reduction between the knockout and G222R, with the G222R additionally showing oligodendroglial loss. These results are consistent with our interpretation that specific missense mutations may acquire dominant gain-of-function effects which exacerbate vulnerability and neuronal hyperexcitability. The knockout has shown reduced excitatory and inhibitory postsynaptic activity suggestive of reduced neurotransmitter levels. We will further characterize these mutant mouse models with the following experiments: experiment 1.1, the characterization of interictal discharges and seizures in the mutant alleles using EEG, in parallel with analysis of brain histopathology. In experiment 1.2, I will apply electrophysiological techniques to investigate whether a deficiency in intracellular citrate transport in Slc13a5 knockout and missense mutations affects neuronal function by altering glutamate and GABA concentrations. A metabolomics screen for neurotransmitters will be performed to corroborate functional data. In experiment 1.3, I will investigate the effect of these SLC13A5 mutant proteins on cellular pathologies. Combined, these experiments will investigate the molecular and cellular mechanisms that contribute to the seizure phenotype in the SLC13A5 disease, which in turn will inform therapeutic approaches. I will acquire training in electrophysiology and molecular biology to follow through with these experiments. Our research training plan and ongoing professional development will provide me with skills to transition me to the next stages of my scientific career. In the postdoctoral stage, I plan to gain experience with gene therapy strategies in experimental models of nervous system disease. I will continue to investigate cellular and molecular mechanisms underlying brain disease, and to apply these findings to optimize gene therapy approaches. Furthermore, I will continue to develop the professional skills required to become an independent primary investigator at an academic research institution.