Epilepsy Mechanisms and the Path to Intervention

癫痫机制和干预途径

• 批准号: 10704607
• 负责人: Kelvin A DeLeon
• 金额: $ 4.87万
• 依托单位: BROWN UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-15 至 2024-09-14
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10704607
• 关键词: Ablation; Address; Adult; Affect; Alleles; Animal Model; Behavioral; Biochemical; Biological Assay; Birth; Brain; Brain Diseases; Cell membrane; Cells; Cellular Stress; Citrates; Citric Acid Cycle; Cognitive; Coupled; Cytoplasm; DNA Sequence Alteration; Data; Deletion Mutation; Development; Disease; Disease model; Early Infantile Epileptic Encephalopathy; Electroencephalography; Electrophysiology (science); Epilepsy; Event; Experimental Models; Fellowship; Focal Seizure; Frequencies; Genes; Genetic; Genotype; Glutamates; Hippocampus; Histologic; Histopathology; Human; Human Genetics; Impairment; Infant; Institution; Intellectual functioning disability; Interneurons; Intervention; Knock-out; Knockout Mice; Laboratories; Measures; Metabolic; Metabolic Marker; Missense Mutation; Mitochondria; Modeling; Molecular; Molecular Biology; Mus; Mutant Strains Mice; Mutation; Neurons; Neurotransmitters; Newly Diagnosed; Nonsense Mutation; Oligodendroglia; Pathogenicity; Pathologic; Patients; Pattern; Phenotype; Point Mutation; Postdoctoral Fellow; Production; Property; Proteins; Reactive Oxygen Species; Recombinant Proteins; Recombinants; Research; Research Personnel; Research Training; Seizures; Severities; Slice; Sodium; Synapses; Techniques; Therapeutic; Training; Work; career; cellular pathology; citrate carrier; clinical phenotype; design; endoplasmic reticulum stress; epileptiform; experience; experimental study; gain of function; gamma-Aminobutyric Acid; gene replacement therapy; gene therapy; hippocampal pyramidal neuron; histological studies; in vitro Model; in vivo; interdisciplinary approach; knock-down; loss of function; metabolomics; mitochondrial dysfunction; mouse model; mutant; mutant mouse model; natural flow; neonate; nervous system disorder; novel; post-doctoral training; postsynaptic; pre-clinical; precision medicine; protein misfolding; response; skills

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.

项目总结/摘要 SLC 13 A5基因的突变，编码质膜柠檬酸转运蛋白，导致新的 诊断形式的遗传性癫痫称为早期婴儿癫痫性脑病，其特征在于， 新生儿多灶性癫痫发作。这些婴儿随后出现认知和行为缺陷。人类 遗传学已经确定了常见的错义和缺失突变，但不知道如何发生 不同的基因突变影响疾病表现和癫痫发作的严重程度。我们正在通过以下方式解决这个问题： 表征一系列SLC 13 A5突变小鼠模型，所述小鼠模型携带：i）其内源性鼠Slc 13 a5 基因（敲除），ii）最常见的患者突变，G222 R点突变（相当于人类 突变G219 R），和iii）第二最常见的患者突变，T230 M（相当于人突变G219 R）。 突变T227 M）。我们的初步数据表明，纯合子Slc 13 a5敲除小鼠表现出 异常的癫痫样脑电图（EEG）曲线，而G222 R具有癫痫发作， 更严重的癫痫样活动初步组织病理学显示， 敲除和G222 R，其中G222 R另外显示少突胶质细胞损失。这些结果 这与我们的解释一致，即特定的错义突变可能获得显性的功能获得性效应 这会加剧脆弱性和神经元过度兴奋。敲除显示兴奋性降低， 暗示神经递质水平降低的抑制性突触后活动。我们将进一步描述这些 突变小鼠模型，进行以下实验：实验1.1，发作间期的表征 放电和癫痫发作的突变等位基因使用脑电图，在平行的脑组织病理学分析。在 实验1.2，我将应用电生理技术来研究是否细胞内缺乏 Slc 13 a5敲除和错义突变中柠檬酸盐转运通过改变谷氨酸影响神经元功能 GABA浓度。将对神经递质进行代谢组学筛查， 功能数据在实验1.3中，我将研究这些SLC 13 A5突变蛋白对细胞凋亡的影响。 病理学结合起来，这些实验将研究分子和细胞机制，有助于 SLC 13 A5疾病中的癫痫发作表型，这反过来将为治疗方法提供信息。 我将获得电生理学和分子生物学的培训， 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，我计划获得经验， 神经系统疾病实验模型的基因治疗策略。我会继续调查 以及脑部疾病的分子机制，并将这些发现应用于优化基因治疗 接近。此外，我将继续发展成为一个独立的专业技能， 学术研究机构的首席研究员。