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: 10302281
• 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/10302281
• 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; gene network; 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：获得的癫痫是一种慢性病，需要终身药物，通常是药物 - 抵抗的。目前，开发更有效的药物以预防或减少癫痫 在了解癫痫病如何将健康的大脑变成自发的大脑中 复发性癫痫发作。这种称为癫痫发生的过程被认为是高度复杂的。停止或 因此，防止癫痫发生很可能需要对许多不同分子进行协同操作 网络和路径。因此，针对监管机制的治疗策略是合理的 这些细胞途径的多个控制将最成功。这项研究将解决这个问题 通过分析如何表达数百个基因表达的有效调节因子，MicroRNA诱导的 沉默复合物（RISC）在癫痫持续状态后有助于癫痫发作（SE）。以前的工作 通过表明选择microRNA和 癫痫发作后将mRNA招募到RISC，并且抑制单microRNA会减少癫痫发作 啮齿动物癫痫模型中的敏感性和癫痫发生。然而，分子机制和途径 MicroRNA操纵的癫痫发作作用的基本是未知的。总体假设 这项研究是SE诱导RISC和MicroRNA功能的变化，从而增强了癫痫发生和 减少神经保护途径。抑制这些变化可能会阻止或损害发展 癫痫。该假设将以两个目标进行检验。 AIM 1将遵循使用细胞类型的公正方法 - 对RISC的特定免疫沉淀，RNA测序和遗传敲低策略，以揭示 分子途径的性质和功能相关性。 老鼠。 AIM 2将使用核糖体标记和功能测定法遵循基于候选的方法 反义介导的microRNA抑制作用，以揭示促磁力microRNA的细胞特异性翻译组 以及在SE之后如何促进癫痫发生。基于神经科学家和 计算生物学家，在SE之后执行RISC和MicroRNA目标调节筛选的方法 预计与途径和功能分析将生成有关复合物的独特信息 调节癫痫发生的过程。使用RISC协会作为代理的创新策略 microRNA功能，以及mRNA与RISC的关联或主动将核糖体作为替代物转化 预计他们的沉默或翻译分别将提供改进的功能评估 与先前的表达分析相比，microRNA的沉默活性和对靶mRNA的影响。这 研究将填补理解RISC功能，蛋白质表达和途径失调的关键空白 在癫痫发生中，这对于推动微洋诱导的沉默作为治疗靶点至关重要。从 更广泛的观点，该策略可以作为其他疾病中RISC分析的蓝图。