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Mechanisms of seizure resistance in a mouse genetic model with altered metabolism

代谢改变的小鼠遗传模型的癫痫抵抗机制

基本信息

批准号：
10307554
负责人：
GARY I YELLEN
金额：
$ 38.46万
依托单位：
HARVARD MEDICAL SCHOOL
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-03-01 至 2023-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10307554
关键词：
ATP sensitive potassium channel complex Acute Address Affect Alleles Animals Anticonvulsants Apoptosis Regulation Gene Apoptotic Attention Bad protein Brain Brain region Carbohydrates Caregivers Cells Cellular Metabolic Process Cytoplasmic Granules Diet Diet therapy Disease Effectiveness Enterobacteria phage P1 Cre recombinase Epilepsy Event Exhibits Gene Mutation Genes Genetic Genetic Models Glucose Hippocampus (Brain)Hydrocarbons Individual Intractable Epilepsy Ion Channel Kainic Acid Ketone Bodies Knock-out Learning Mediating Metabolic Metabolism Modeling Modification Mus Mutation Neurons Pentylenetetrazole Permeability Persons Pharmacology Point Mutation Resistance Reverse engineering Seizures Series Signal Transduction Site Slice Status Epilepticus Testing base brain cell brain metabolism cell type cellular targeting conditional knockout dentate gyrus effective therapy entorhinal cortex experience genetic manipulation in vivo ketogenic diet knockout animal mimetics mouse genetics mouse model prevent public health relevance response restoration stapled peptide

项目摘要

PROJECT SUMMARY/ABSTRACT Drug-resistant epilepsy is seriously debilitating and very common, affecting about one-third of the 1-2% of people who experience epilepsy during their lifetime. One of the most effective treatments for drug-resistant epilepsy is dietary therapy, in the form of a very-low-carbohydrate, ketogenic diet. Despite its effectiveness, this diet is not very widely used because of the stringency of the diet and the high commitment required of clinicians and other caregivers. It would be very valuable to understand the mechanism by which altered metabolism produces resistance to epileptic seizures, to “reverse-engineer” it, and to discover alternative pharmacologic ways of tapping into this potent and apparently unique anti-seizure mechanism. We have identified a mouse model that recapitulates the seizure resistance seen in ketogenic diet, but that involves a mutation in a single gene, Bad. The seizure resistance in this genetic model is due to alteration in brain cell metabolism, with less glucose utilization and better utilization of alternative fuels such as ketone bodies, similar to the metabolic changes on a ketogenic diet. We have also discovered a downstream mechanism that is altered both by Bad alteration and by ketogenic diet: a metabolically-sensitive class of ion channels, the ATP-sensitive potassium channels (KATP channels), become more activated in response to metabolic changes. These channels are critical for seizure resistance of the Bad-altered mice, and we have also found that they are responsible for anti-seizure effects of BAD knockout in a brain slice model of seizure. We have also recently learned that KATP channel activation depends on the expression of the BAD protein in individual neurons, which means that the effects of BAD can be genetically targeted to individual cell types or to specific brain regions. This ability to target the genetic manipulation of the BAD protein – which cannot be done for a global manipulation like diet – creates the opportunity to learn the cellular sites of action where BAD modification is required to produce seizure resistance. We now have a conditional knockout allele of the Bad gene (Bad flox/flox) that can be used in combination with various “driver lines” that express Cre recombinase in specific cells. We will determine whether BAD knockout is effective in slice seizure models or against seizures in mice, when the knockout is restricted to certain targets, for instance, to neurons in specific brain regions like the dentate gyrus that are hypothesized to function as “seizure gates”. We will also test a pharmacological approach to producing the anti-seizure effects of BAD, by asking whether a specific class of BAD-mimetic compounds is capable of reversing or mimicking the effect of BAD knockout on seizure-like events in slices. These studies will advance our mechanistic understanding of metabolic seizure resistance and more generally of endogenous “seizure gates”, and will explore new pharmacologic approaches to drug-resistant epilepsy.

项目总结/摘要耐药性癫痫是严重的衰弱和非常常见的，影响约三分之一的1-2%的癫痫病患者在一生中治疗耐药的最有效的方法之一癫痫是饮食疗法，以非常低碳水化合物的生酮饮食的形式。尽管它很有效，这种饮食并没有被广泛使用，因为饮食的严格性和高承诺要求。临床医生和其他护理人员。了解改变的机制是非常有价值的，新陈代谢产生对癫痫发作的抵抗力，对其进行“反向工程”，并发现替代方案药理学方法来利用这种有效的和明显独特的抗癫痫机制。我们已经确定了一种小鼠模型，该模型重现了生酮饮食中观察到的癫痫发作抵抗，但都涉及一个单一基因的突变，坏基因这种遗传模型中的癫痫抵抗是由于脑细胞代谢，葡萄糖利用率更低，替代燃料（如酮）利用率更高身体，类似于生酮饮食的代谢变化。我们还发现了一个下游通过不良改变和生酮饮食改变的机制：一类代谢敏感的离子通道，ATP敏感性钾通道（KATP通道），变得更加活跃，以响应代谢变化这些通道对于Bad改变小鼠的癫痫抵抗至关重要，还发现它们负责BAD敲除在癫痫发作的脑切片模型中的抗癫痫发作作用。我们最近还了解到，KATP通道的激活依赖于BAD蛋白的表达。单个神经元，这意味着BAD的影响可以在遗传上靶向单个细胞类型，到特定的大脑区域这种针对BAD蛋白的遗传操作的能力-这在全球范围内是无法做到的。操纵，如饮食-创造了机会，以了解细胞的行动地点，其中不良修改是产生抗癫痫性所必需的。我们现在有一个条件性敲除Bad基因的等位基因 (Bad flox/flox），其可以与表达Cre重组酶的各种“驱动系”组合使用，所述“驱动系”特异性地表达Cre重组酶。细胞我们将确定BAD基因敲除是否在切片癫痫发作模型或小鼠癫痫发作中有效，当敲除仅限于某些靶点时，例如，特定脑区的神经元，如齿状回被假设为“癫痫发作门”。我们还将测试一种药物一种产生BAD抗癫痫作用的方法，通过询问特定类别的BAD模拟物是否化合物能够逆转或模拟BAD基因敲除对切片中的类凋亡事件的作用。这些研究将推进我们对代谢性癫痫抵抗的机制理解，内源性“癫痫发作门”，并将探索新的药物耐药性癫痫的方法。