Epilepsy in Focal Cortical Malformations

局灶性皮质畸形中的癫痫

• 批准号: 10371098
• 负责人: Angelique Bordey
• 金额: $ 50.73万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-04-01 至 2024-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10371098
• 关键词: Address; Adult; Affect; Age; Aging; Binding; Biochemical; Biology; Birth; CCI-779; Cells; Cortical Dysplasia; Cortical Malformation; Cyclic AMP; Cyclic Nucleotides; Data; Development; Disease; Dose; Electroporation; Epilepsy; Excision; FRAP1 gene; Generations; Genes; Genetic Diseases; Heart; Human; Hyperactivity; Individual; Lead; Life; Light; Medial; Messenger RNA; Modeling; Mus; Mutation; Neurobiology; Neurons; Operative Surgical Procedures; Pathway interactions; Patients; Peripheral; Phosphorylation; Physiological; Post-Translational Protein Processing; Prefrontal Cortex; Protein Isoforms; Proteins; Refractory; SDZ RAD; Science; Seizures; Serious Adverse Event; Sirolimus; TSC1 gene; Testing; Time; Tissues; Translations; Treatment Protocols; Tuberous Sclerosis; base; beta-adrenergic receptor; conventional therapy; genetic variant; hippocampal pyramidal neuron; improved; in utero; in vivo; mouse model; mutant; new therapeutic target; novel; patient population; personalized medicine; population based; prevent; receptor; side effect; therapeutic target

Abstract Tuberous sclerosis complex (TSC) and focal cortical dysplasia type II (FCDII) are caused by mutations in mTOR pathway genes leading to mTOR hyperactivity, focal malformations of cortical development (fMCD), and seizures in 80-90% of the patients. The current definitive treatments for epilepsy are surgical resection or treatment with everolimus, which inhibits mTOR activity (only approved for TSC). Because both options have severe limitations, there is a major need to better understand the mechanisms leading to seizures to improve life-long epilepsy treatment in TSC and FCDII. To investigate such mechanisms, we recently developed a murine model of fMCD-associated epilepsy that recapitulates the human TSC and FCDII disorders. fMCD are defined by the presence of misplaced, dysmorphic cortical neurons expressing hyperactive mTOR – for simplicity we will refer to these as “mutant” neurons. In our model and in human TSC tissue, we made a surprising finding that mutant neurons express HCN4 channels, which are not normally functionally expressed in cortical neurons. These data led us to ask several important questions based on the known biology of HCN4 channels: (1) As HCN4 channels are responsible for the pacemaking activity of the heart, can HCN4 channel expression lead to repetitive firing of mutant neurons resulting in seizures? (2) HCN4 is the most cAMP-sensitive of the four HCN isoforms. Do coincident increases in cAMP (e.g., β-adrenergic receptors) and hyperpolarization or depolarizations drive HCN4 channel opening and neuronal firing? (3) HCN4 channel mRNA is expressed in cortical neurons. Is the abnormal HCN4 expression in mutant neurons due to increased translation via mTOR? (4) Seizures can start at any age in patients that have been seizure-free for decades, but we do not know why. Can this be explained by worsening of mTOR hyperactivity with age leading to a progressive increase in HCN4 expression until there is enough HCN4 channels to depolarize cells and reach firing threshold upon activation? (5) There is no selective blocker of the HCN4 channel and blocking other HCN channels would have serious central and peripheral side-effects. Identifying the mechanisms responsible for functional HCN4 expression may therefore provide alternative therapeutic targets. Do binding partners and/or post-translational modifications contribute to HCN4 abnormal expression in mutant neurons? We will address these questions in three aims testing our central hypothesis that abnormal mTOR- and translation- dependent expression of HCN4 channels leads to repetitive neuronal firing and seizures in TSC and FCDII. Aim 1: Test the hypothesis that abnormal HCN4 channel expression in murine TSC/FCDII mutant neurons contribute to neuron excitability and seizure activity. Aim2: Test the hypothesis that abnormal HCN4 expression is mTOR- and translation-dependent and increases with age and seizures. Aim 3: Test the hypothesis that HCN4 binding partners and posttranslational modifications are necessary for its functional expression and function. The proposed studies will be performed through a collaborative effort between the Bordey and Calderwood labs that together combine unique and extensive expertise in in vivo neurobiology, and biochemical and protein science.

抽象的 结节硬化症复合物（TSC）和局灶性皮质发育不良II型（FCDII）是由突变引起的 在导致MTOR多动症的MTOR途径基因中，皮质发育的局灶性畸形 （FMCD）和80-90％的患者癫痫发作。当前对癫痫的权威治疗方法是 依维莫司（Everolimus）的手术切除或治疗，该切除层抑制了MTOR活性（仅批准用于TSC）。 因为这两种选择都有严重的局限性，所以主要需要更好地了解 导致提高TSC和FCDII终身癫痫治疗的癫痫发作的机制。到 研究这种机制，我们最近开发了与FMCD相关癫痫的鼠模型 这概括了人类TSC和FCDII疾病。 FMCD由存在 放错位置的，营养不良的皮质神经元表达多动MTOR - 为简单起见，我们将参考 这些是“突变”神经元。在我们的模型和人类TSC组织中，我们惊讶地发现 突变神经元表达HCN4通道，通常在皮质中表达该通道 神经元。这些数据导致我们根据HCN4的已知生物学提出了几个重要问题 通道：（1）由于HCN4通道负责心脏的起搏活动，可以HCN4 渠道表达导致突变神经元的重复发射导致癫痫发作？ （2）HCN4是 四个HCN同工型的营地敏感。 cAMP的同时增加（例如β-肾上腺素 受体）和超极化或去极化驱动HCN4通道开口和神经元放电？ （3）HCN4通道mRNA在皮质神经元中表达。是突变体中HCN4异常的表达 神经元由于MTOR的翻译增加而引起的？ （4）癫痫发作可以从任何年龄开始的患者开始 数十年来一直没有癫痫发作，但我们不知道为什么。可以通过想知道 MTOR多动症随着年龄的增长，导致HCN4表达逐渐增加直到有 足够的HCN4通道可以扩大细胞并在激活时达到阈值？ （5）没有 HCN4通道的选择性阻滞剂并阻止其他HCN通道将具有严重的中央 和外围副作用。确定负责功能性HCN4表达的机制 因此，可以提供替代性治疗靶标。进行约束伙伴和/或翻译后 修饰有助于突变神经元中的HCN4异常表达？我们将解决这些 在三个目的中的问题测试了我们的中心假设，即MTOR和翻译异常 - HCN4通道的依赖表达导致TSC中的重复性神经元发射和癫痫发作 FCDII。 AIM 1：测试鼠TSC/FCDII中异常HCN4通道表达的假设 突变神经元有助于神经元兴奋性和癫痫活性。 AIM2：检验以下假设 异常的HCN4表达是mTOR和翻译依赖性的，并且随着年龄的增长而增加 癫痫发作。目标3：检验HCN4结合伴侣和翻译后修改的假设 对于其功能表达和功能是必需的。拟议的研究将进行 通过Bordey和Calderwood Labs之间的合作努力，将其结合在一起 体内神经生物学方面的广泛专业知识以及生化和蛋白质科学。