Epilepsy in Focal Cortical Malformations

摘要 结节性硬化症(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多动的mTOR通路基因中，皮质发育的局灶性畸形 (FMCD)和80-90%的患者癫痫发作。目前对癫痫的权威治疗方法是 手术切除或用抑制mTOR活性的伊维莫司治疗(仅批准用于TSC)。 由于这两种选择都有严重的限制，因此有必要更好地了解 在TSC和FCDII中改善终身癫痫治疗的导致癫痫发作的机制。至 为了研究这种机制，我们最近建立了一种fmcd相关性癫痫的小鼠模型。 这概括了人类的TSC和FCDII疾病。FMCD由以下条件定义： 错位、变形的皮质神经元表达高度活跃的mTOR-为简单起见，我们将参考 这些被称为“突变”神经元。在我们的模型和人类TSC组织中，我们有一个令人惊讶的发现 突变的神经元表达HCN4通道，而这些通道在大脑皮层中没有正常的功能表达 神经元。这些数据让我们根据已知的HCN4生物学提出了几个重要的问题 通道：(1)由于HCN4通道负责心脏的起搏活动，所以可以 通道表达导致突变神经元的重复放电导致癫痫发作？(2)HCN4是 在四种HCN亚型中，cAMP最敏感。CAMP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)皮质神经元表达HCN4通道基因。HCN4在突变体中的异常表达 通过mTOR增加翻译所致的神经元？(4)符合以下条件的患者可在任何年龄开始癫痫发作 几十年来一直没有癫痫发作，但我们不知道为什么。这可以用经济形势恶化来解释吗 随着年龄的增长，mTOR多动导致HCN4表达进行性增加，直到 是否有足够的HCN4通道使细胞去极化并在激活时达到激活阈值？(5)没有 选择性阻断HCN4通道和阻断其他HCN通道将有严重的中枢 和外周副作用。确定功能性HCN4表达的机制 因此可以提供替代的治疗靶点。有约束力的合作伙伴和/或翻译后 基因修饰导致突变神经元中HCN4的异常表达？我们将解决这些问题 三个目的的问题验证了我们的中心假设，即异常的mTOR-和翻译- HCN4通道的依赖表达导致TSC和TSC反复神经元放电和癫痫发作 FCDII。目的1：验证小鼠TSC/FCDII中HCN4通道异常表达的假说 突变神经元有助于神经元的兴奋性和癫痫发作活动。目标2：测试假设 HCN4的异常表达依赖于mTOR和翻译，并随着年龄和年龄的增加而增加 癫痫发作。目的3：验证HCN4结合伙伴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 通过波尔迪和考德伍德实验室之间的合作，将独特的 以及在体内神经生物学、生化和蛋白质科学方面的广泛专业知识。

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.