Functional analysis of the microRNA-induced silencing complex in epilepsy

microRNA诱导的沉默复合物在癫痫中的功能分析

• 批准号: 10059274
• 负责人: Christina Gross
• 金额: $ 34.78万
• 依托单位: CINCINNATI CHILDRENS HOSP MED CTR
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-12-01 至 2024-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10059274
• 关键词: Address; Affect; Biological Assay; Brain; Cells; Chronic; Complement; Complex; Core Facility; Data; Development; Disease; Drug resistance; Epilepsy; Epileptogenesis; Event; Frequencies; Future; Gene Expression; Genes; Genetic; Goals; Immunoprecipitation; Impairment; Knowledge; Lead; Life; Mediating; Messenger RNA; MicroRNAs; Mission; Modeling; Molecular; Molecular Target; Mus; Nature; Pathway Analysis; Pathway interactions; Pharmaceutical Preparations; Predisposition; Process; Protein Analysis; Proteins; Public Health; RNA; RNA-Induced Silencing Complex; Recurrence; Regulation; Regulator Genes; Research; Research Personnel; Ribosomes; Rodent; Rodent Model; Role; Seizures; Status Epilepticus; Testing; Therapeutic; Translating; Translations; United States National Institutes of Health; Work; acquired epilepsy; base; cell type; excitotoxicity; improved; innovation; knock-down; mouse model; neuron loss; neuronal excitability; neuronal survival; novel; prevent; protein expression; recruit; therapeutic candidate; therapeutic miRNA; therapeutic target; transcriptome sequencing; translatome; treatment strategy

SUMMARY: Acquired epilepsy is a chronic condition that requires life-long medication and is often drug- resistant. The development of more efficient drugs to prevent or reduce epilepsy is currently hindered by a gap in knowledge of how an epilepsy-precipitating event turns a healthy brain into a brain that produces spontaneous recurrent seizures. This process, known as epileptogenesis, is thought to be highly complex. Halting or preventing epileptogenesis thus most likely requires the concerted manipulation of many different molecular networks and pathways. It is therefore plausible that treatment strategies targeting regulatory mechanisms that control multiple of these cellular pathways at once will be most successful. This research will address this challenge by analyzing how a potent regulator of the expression of hundreds of genes, the microRNA-induced silencing complex (RISC), contributes to epileptogenic processes after status epilepticus (SE). Previous work supports a role of microRNAs and the RISC in epilepsy development by showing that select microRNAs and mRNAs are recruited to the RISC after seizure, and that inhibition of single microRNAs reduces seizure susceptibility and epileptogenesis in rodent epilepsy models. Yet, the molecular mechanisms and pathways underlying the seizure-mitigating effects of microRNA manipulation are largely unknown. The overall hypothesis of this research is that SE induces changes in RISC and microRNA function that enhance epileptogenic and decrease neuroprotective pathways. Inhibiting these changes may prevent or impair the development of epilepsy. This hypothesis will be tested with two aims. Aim 1 will follow an unbiased approach using cell type- specific immunoprecipitation of the RISC, RNA sequencing and genetic knockdown strategies to reveal the nature and functional relevance of molecular pathways that are differentially regulated by the RISC after SE in mice. Aim 2 will follow a candidate-based approach using ribosomal tagging and functional assays together with antisense-mediated microRNA inhibition to reveal the cell-specific translatome of a pro-convulsive microRNA and how it contributes to epileptogenesis after SE. Based on complementing expertise of neuroscientists and computational biologists, the approach to perform screens of RISC and microRNA target regulation after SE paired with pathway and functional analyses is expected to generate unique information about the complex processes regulating epileptogenesis. The innovative strategy using RISC association as a surrogate for microRNA function, and association of mRNAs with the RISC or actively translating ribosomes as a surrogate for their silencing or translation, respectively, is expected to provide an improved functional assessment of the silencing activity of microRNAs and the effect on target mRNAs compared to previous expression analyses. This study will fill a crucial gap in the understanding of RISC function, protein expression and pathway dysregulation in epileptogenesis, which will be vital to advance microRNA-induced silencing as therapeutic target. Seen from a broader perspective, this strategy could serve as a blueprint for RISC analysis in other diseases.

摘要：获得性癫痫是一种慢性疾病，需要终生药物治疗，并且通常是药物引起的。 抵抗的。目前，开发更有效的药物来预防或减少癫痫的工作因差距而受到阻碍 了解癫痫诱发事件如何将健康的大脑转变为产生自发性癫痫的大脑 反复发作。这个过程被称为癫痫发生，被认为是非常复杂的。停止或 因此，预防癫痫发生很可能需要许多不同分子的协同操作 网络和途径。因此，针对调节机制的治疗策略是合理的。 同时控制多个细胞途径将是最成功的。本研究将解决这个问题 通过分析数百种基因表达的有效调节因子（microRNA 诱导的）如何应对挑战 沉默复合体（RISC）有助于癫痫持续状态（SE）后的癫痫发生过程。以前的工作 通过证明选择的 microRNA 和 RISC 在癫痫发展中的作用，支持了 microRNA 和 RISC 癫痫发作后 mRNA 被招募到 RISC，抑制单个 microRNA 可减少癫痫发作 啮齿动物癫痫模型的易感性和癫痫发生。然而，分子机制和途径 microRNA 操作的癫痫发作缓解作用背后的机制尚不清楚。总体假设 这项研究的重点是 SE 会诱导 RISC 和 microRNA 功能的变化，从而增强癫痫发作和 减少神经保护途径。抑制这些变化可能会阻止或损害 癫痫。该假设将通过两个目标进行检验。目标 1 将遵循使用细胞类型的公正方法- RISC 的特异性免疫沉淀、RNA 测序和基因敲低策略来揭示 SE后受RISC差异调节的分子途径的性质和功能相关性 老鼠。目标 2 将遵循基于候选的方法，使用核糖体标记和功能测定以及 反义介导的 microRNA 抑制揭示促惊厥 microRNA 的细胞特异性翻译组 以及它如何促进 SE 后的癫痫发生。基于神经科学家的专业知识补充和 计算生物学家，SE 后进行 RISC 和 microRNA 靶标调控筛选的方法 与途径和功能分析相结合，预计将生成有关该复合物的独特信息 调节癫痫发生的过程。以RISC协会为替代的创新策略 microRNA 功能，以及 mRNA 与 RISC 的关联或作为替代物的主动翻译核糖体 分别针对它们的沉默或翻译，预计将提供改进的功能评估 与之前的表达分析相比，microRNA 的沉默活性以及对靶 mRNA 的影响。这 研究将填补对 RISC 功能、蛋白质表达和通路失调理解的关键空白 癫痫发生过程中，这对于推进 microRNA 诱导的沉默作为治疗靶点至关重要。看到自 从更广泛的角度来看，该策略可以作为其他疾病的 RISC 分析的蓝图。