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: 10225865
• 负责人: Christina Gross
• 金额: $ 12.49万
• 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/10225865
• 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，需要终身药物治疗，通常是药物治疗。 抵抗开发更有效的药物来预防或减少癫痫目前受到一个差距的阻碍 癫痫诱发事件是如何将健康的大脑转变为自发性 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协调操纵许多不同的分子， 网络和路径。因此，针对调节机制的治疗策略似乎是合理的， 同时控制多个细胞通路将是最成功的。这项研究将解决这一问题 通过分析数百个基因表达的有效调节剂，microRNA诱导的 沉默复合体（RISC）有助于癫痫持续状态（SE）后的致癫痫过程。以前的工作 支持microRNA和RISC在癫痫发展中的作用，通过显示选择microRNA和 癫痫发作后，mRNA被募集到RISC中，抑制单个microRNA可以减少癫痫发作 在啮齿动物癫痫模型中的易感性和癫痫发生。然而，分子机制和途径 microRNA操作的潜在的减轻感染的作用在很大程度上是未知的。总体假设 SE诱导RISC和microRNA功能的变化，这些变化增强了癫痫的发生， 减少神经保护途径。抑制这些变化可能会阻止或损害 癫痫这个假设将以两个目标进行检验。Aim 1将采用无偏的方法，使用细胞类型- RISC的特异性免疫沉淀，RNA测序和基因敲除策略，以揭示 SE后RISC差异调节的分子途径的性质和功能相关性， 小鼠目标2将遵循基于候选人的方法，使用核糖体标记和功能测定， 反义介导的microRNA抑制以揭示促惊厥microRNA的细胞特异性翻译组 以及它如何促进SE后癫痫的发生。基于补充神经科学家的专业知识， 计算生物学家，在SE后进行RISC和microRNA靶向调节筛选的方法 与途径和功能分析相结合，预计将产生关于复合物的独特信息 控制癫痫发生的过程使用RISC协会作为替代的创新策略， microRNA功能，以及mRNA与RISC或作为替代物的积极翻译核糖体的关联 对于它们的沉默或翻译，分别，预计将提供一个改进的功能评估， 与先前的表达分析相比，微RNA的沉默活性和对靶mRNA的影响。这 这项研究将填补理解RISC功能、蛋白质表达和通路失调的关键空白 这对于促进microRNA诱导沉默作为治疗靶点至关重要。所示 从更广泛的角度来看，这一策略可以作为其他疾病RISC分析的蓝图。