mTORC1-dependent translational control in developmental epilepsy

发育性癫痫中 mTORC1 依赖性翻译控制

• 批准号: 10090615
• 负责人: LENA NGUYEN
• 金额: $ 7.05万
• 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: 2019-02-01 至 2022-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10090615
• 关键词: Affinity Chromatography; Animal Model; Antiepileptic Agents; Biochemical; Brain region; Cell physiology; Cells; Child; Complement; Complex; Cortical Dysplasia; Cortical Malformation; Defect; Development; Disease; Electroencephalography; Electrophysiology (science); Epilepsy; FRAP1 gene; Generations; Genetic; Genetic Translation; Goals; Intractable Epilepsy; Knowledge; Link; Messenger RNA; Microarray Analysis; Molecular; Mus; Mutation; Neurodevelopmental Disorder; Neurons; Operative Surgical Procedures; Pathogenesis; Pathogenicity; Pathway interactions; Pharmacology; Protein Biosynthesis; Proteins; Research; Resistance; Ribosomes; Role; Seizures; Side; Signal Pathway; Signal Transduction; Sirolimus; Testing; Therapeutic; Translating; Translations; Tuberous sclerosis protein complex; Up-Regulation; Validation; Western Blotting; analog; brain abnormalities; effective therapy; hemimegalencephaly; human model; improved; in vivo; inhibitor/antagonist; insight; knock-down; mRNA capping; mouse model; neuronal excitability; novel; novel strategies; novel therapeutics; overexpression; patch clamp; prevent; relating to nervous system; side effect

PROJECT SUMMARY Malformation of cortical development (MCD) is the leading cause of intractable epilepsy in children. Seizures due to MCD are severe and frequently resistant to antiepileptic drugs, and the only definitive treatment option is often surgery to remove the abnormal brain regions responsible for seizure generation. These therapeutic limitations highlight the need for novel approaches to treat MCD-related epilepsy. Recent studies in humans and animal models have identified pathogenic mutations leading to aberrant hyperactivation of the mechanistic target of rapamycin complex 1 (mTORC1) signaling pathway in several MCD subtypes that are highly linked to epilepsy, including tuberous sclerosis complex, focal cortical dysplasia, and hemimegalencephaly, demonstrating an important role for mTORC1 dysregulation in the pathogenesis of epilepsy. However, the mechanisms by which mTORC1 dysregulation contributes to epilepsy are not well understood. Since current mTORC1 inhibitors such as rapamycin and its analogs can have serious side effects, do not fully block all of mTORC1’s functions, and have limited efficacy, a more specific understanding of the downstream molecular players responsible for epilepsy development is crucial in order to develop more effective therapies. mTORC1 regulates many cellular processes, with the best studied function being mRNA cap-dependent translational control. Activation of mTORC1 promotes mRNA translation via inactivation of the translational suppressor 4EBP. Thus, one working hypothesis is that upregulated mTORC1-dependent translation leads to exaggerated protein synthesis that contributes to the neuronal defects underlying epilepsy. Interestingly, normalizing translation via 4EBP overexpression during cortical development prevented mTORC1-induced cytoarchitectural abnormalities associated with MCD. However, the effects on neuronal excitability and seizures have not been investigated. The overall goal of this research is to delineate the contribution of increased mTORC1-dependent translation to hyperexcitability and seizures (Aim 1) and to identify the specific molecules that are abnormally expressed as a result of this aberrant increase in translation (Aim 2). A canonical activator of mTORC1, Rheb, will be constitutively expressed in neurons to mimic the persistent activation of mTORC1 in disease states in mouse models. Complementing genetic and pharmacological approaches will then be used to target mTORC1-induced translation and the resulting effects on neuronal excitability and seizures will be assessed. In parallel, mRNAs that are actively translated in neurons under different conditions of mTORC1 activity will be isolated and identified by microarray analysis and biochemical validation. The proposed research will advance our understanding of how altered intracellular signaling pathways and translational control mechanisms modulate neuronal excitability and seizures and provide mechanistic insights into the pathogenesis of epilepsy associated with MCD. Knowledge gained from this study will help uncover novel targets for the treatment of intractable epilepsy and potentially other neurodevelopmental disorders caused by mTORC1 dysregulation.

项目总结 皮质发育畸形(MCD)是儿童难治性癫痫的主要原因。癫痫发作 由于MCD严重且经常对抗癫痫药物产生抗药性，唯一确定的治疗选择是 通常通过手术移除导致癫痫发作的异常脑区。这些治疗方法 局限性凸显了治疗MCD相关癫痫的新方法的必要性。最近对人类和 动物模型已发现致病突变导致机械靶标异常过度激活 雷帕霉素复合体1(MTORC1)信号通路在几种与癫痫高度相关的MCD亚型中， 包括结节性硬化症、局灶性皮质发育不良和半大脑畸形，表现为 MTORC1异常在癫痫发病机制中的重要作用。然而，通过这些机制 MTORC1调节失调导致癫痫的机制还不是很清楚。由于目前的mTORC1抑制剂 由于雷帕霉素及其类似物可能有严重的副作用，请不要完全阻断mTORC1‘S的所有功能，以及 疗效有限，对下游分子玩家有更具体的了解 为了开发更有效的治疗方法，癫痫的发展至关重要。MTORC1调节许多细胞 过程，研究最好的功能是mRNA帽依赖的翻译控制。激活 MTORC1通过失活翻译抑制因子4EBP促进mRNA的翻译。因此，一个人在工作 假说是上调的mTORC1依赖的翻译会导致蛋白质合成的夸大 导致癫痫潜伏的神经元缺陷。有趣的是，通过4EBP标准化翻译 皮质发育过程中过表达可阻止mTORC1诱导的细胞结构异常 与MCD相关。然而，对神经元兴奋性和癫痫发作的影响还没有被研究。 这项研究的总体目标是描绘增加依赖mTORC1的翻译对 高兴奋性和癫痫发作(目标1)，并识别异常表达的特定分子 这是翻译异常增加的结果(目标2)。MTORC1的典型激活剂Rheb将是 在神经元中组成性表达以模拟小鼠疾病状态下mTORC1的持续激活 模特们。然后将使用互补的遗传和药理学方法来针对mTORC1诱导的 将评估翻译及其对神经元兴奋性和癫痫发作的影响。同时，mRNAs 将分离和鉴定在不同条件下在神经元中活跃翻译的mTORC1 通过基因芯片分析和生化验证。这项拟议的研究将增进我们对 改变的细胞内信号通路和翻译控制机制如何调节神经元的兴奋性 并为与MCD相关的癫痫的发病机制提供了机械性的见解。 从这项研究中获得的知识将有助于发现治疗难治性癫痫和 可能由mTORC1失调引起的其他神经发育障碍。