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：
10057397
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 2022-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10057397
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 Engineering 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 Peptides Permeability Pharmacology Point Mutation Resistance 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

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.

项目概要/摘要耐药性癫痫会严重使人衰弱，而且非常常见，影响 1-2% 患者中的约三分之一。一生中患有癫痫的人最有效的耐药治疗方法之一。癫痫是一种饮食疗法，采用极低碳水化合物的生酮饮食，尽管它很有效。由于饮食的严格性和对饮食的高度承诺，这种饮食并没有被广泛使用。了解侄子和其他照顾者的机制非常有价值。新陈代谢产生对癫痫发作的抵抗力，对其进行“逆向工程”，并发现替代方案利用这种有效且明显独特的抗癫痫机制的药理学方法。我们已经确定了一种小鼠模型，可以概括生酮饮食中观察到的癫痫发作抵抗力，但是涉及单个基因的突变，坏，这种遗传模型中的癫痫抵抗是由于基因的改变。脑细胞新陈代谢，减少葡萄糖利用率，更好地利用酮等替代燃料身体，类似于生酮饮食的代谢变化，我们还发现了下游。因不良改变和生酮饮食而改变的机制：代谢敏感的一类离子通道，即 ATP 敏感钾通道（KATP 通道），会因响应而变得更加激活这些通道对于 Bad 突变小鼠的癫痫抵抗能力至关重要，我们已经发现。还发现，在癫痫发作的脑切片模型中，它们与 BAD 敲除的抗癫痫作用有关。我们最近还了解到，KATP 通道的激活取决于 BAD 蛋白的表达单个神经元，这意味着 BAD 的影响可以针对单个细胞类型或到特定的大脑区域。这种针对 BAD 蛋白进行基因操纵的能力——这在全球范围内是不可能实现的像饮食一样的操纵——创造了了解不良修饰的细胞作用位点的机会我们现在有一个 Bad 基因的条件性敲除等位基因。（Bad flox/flox）可与以特定方式表达 Cre 重组酶的各种“驱动线”组合使用我们将确定 BAD 敲除对于切片癫痫模型或小鼠癫痫发作是否有效，当敲除仅限于某些目标时，例如特定大脑区域的神经元，例如齿状回被袭击以发挥“癫痫门”的作用。我们还将测试药理学。通过询问是否有特定类别的 BAD 模拟物来产生 BAD 的抗癫痫作用化合物能够逆转或模拟 BAD 敲除对切片中癫痫样事件的影响。这些研究将增进我们对代谢性癫痫抵抗的机制理解，并且更广泛地理解内源性“癫痫门”的研究，并将探索治疗耐药性癫痫的新药理学方法。