Epilepsy in focal cortical malformations

局灶性皮质畸形中的癫痫

• 批准号: 9902570
• 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/9902570
• 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; Hyperactive behavior; 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 protein complex; 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 结节性硬化症 (TSC) 和 II 型局灶性皮质发育不良 (FCDII) 是由突变引起的 mTOR 通路基因导致 mTOR 过度活跃、皮质发育局灶性畸形 (fMCD)，80-90% 的患者会出现癫痫发作。目前治疗癫痫的明确方法是 手术切除或依维莫司治疗，抑制 mTOR 活性（仅批准用于 TSC）。 由于这两种选择都有严重的局限性，因此迫切需要更好地了解 导致癫痫发作的机制，以改善 TSC 和 FCDII 的终身癫痫治疗。到 为了研究此类机制，我们最近开发了 fMCD 相关癫痫的小鼠模型 概括了人类 TSC 和 FCDII 疾病。 fMCD 的定义是存在 表达过度活跃的 mTOR 的错位、变形的皮质神经元 – 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，我们有了一个令人惊讶的发现： 突变神经元表达 HCN4 通道，这些通道通常在皮质中不进行功能性表达 神经元。这些数据让我们根据 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，HCN4 可以 通道表达导致突变神经元重复放电，导致癫痫发作？ (2) HCN4 是 四种 HCN 亚型中对 cAMP 最敏感的。 cAMP 是否同时增加（例如，β-肾上腺素能 受体）和超极化或去极化驱动 HCN4 通道开放和神经元放电？ (3)HCN4通道mRNA在皮质神经元中表达。突变体中HCN4表达是否异常 神经元是由于 mTOR 翻译增加所致？ (4) 癫痫发作可以发生在任何年龄的患者中， 几十年来一直没有癫痫发作，但我们不知道为什么。这可以用恶化来解释吗 mTOR 随着年龄的增长而过度活跃，导致 HCN4 表达逐渐增加，直到出现 是否有足够的 HCN4 通道使细胞去极化并在激活时达到放电阈值？ (5) 没有 选择性阻断 HCN4 通道并阻断其他 HCN 通道会产生严重的中枢性副作用。 和外周副作用。确定 HCN4 功能表达的机制 因此可能提供替代的治疗靶点。结合伴侣和/或翻译后 修饰导致突变神经元中 HCN4 异常表达？我们将解决这些 三个目标的问题测试我们的中心假设，即异常的 mTOR- 和翻译- HCN4 通道的依赖性表达导致 TSC 和 TSC 中重复的神经元放电和癫痫发作 FCDII。目标 1：检验小鼠 TSC/FCDII 中 HCN4 通道表达异常的假设 突变神经元有助于神经元兴奋性和癫痫发作活动。目标 2：检验假设 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饰的假设 是其功能表达和功能所必需的。将进行拟议的研究 通过 Bordey 和 Calderwood 实验室之间的合作，将独特的技术结合在一起 以及体内神经生物学、生化和蛋白质科学方面的广泛专业知识。