Epilepsy Mechanisms and the Path to Intervention

癫痫机制和干预途径

基本信息

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
项目类别：
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

项目摘要

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.

项目概要/摘要编码质膜柠檬酸转运蛋白的 SLC13A5 基因的突变导致了一种新的诊断出的遗传性癫痫称为早期婴儿癫痫性脑病，其特征是新生儿多灶性癫痫发作。这些婴儿随后会出现认知和行为缺陷。人类遗传学已识别出常见的错义突变和缺失突变，但尚不清楚如何进行不同的基因突变会影响疾病的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 SLC13A5 突变小鼠模型，这些模型携带： i) 消除其内源性小鼠 Slc13a5 基因（敲除），ii) 最常见的患者突变，G222R 点突变（相当于人类突变 G219R)，以及 iii) 第二个最常见的患者突变，T230M（相当于人类突变 T227M）。我们的初步数据表明，纯合子 Slc13a5 敲除小鼠表现出异常癫痫样脑电图 (EEG) 特征，而 G222R 有癫痫发作且明显更严重的癫痫样活动。初步组织病理学揭示了之间的差异性中间神经元减少敲除和 G222R，G222R 还显示少突胶质细胞损失。这些结果是与我们的解释一致，即特定的错义突变可能获得显性的功能获得效应这加剧了脆弱性和神经元过度兴奋。淘汰赛显示出兴奋性和抑制性突触后活动表明神经递质水平降低。我们将进一步描述这些突变小鼠模型进行如下实验：实验1.1，发作间期的表征使用脑电图分析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。在实验1.2，我将应用电生理技术来研究细胞内是否缺乏 Slc13a5 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，这些实验将研究有助于的分子和细胞机制 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，以完成这些工作实验。我们的研究培训计划和持续的专业发展将为我提供以下技能：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。我将继续研究蜂窝和脑疾病的分子机制，并应用这些发现来优化基因治疗接近。此外，我将继续发展成为独立人士所需的专业技能学术研究机构的主要研究员。