Functional analysis of the microRNA-induced silencing complex in epilepsy

摘要：获得性癫痫是一种需要终生服药的慢性疾病，通常是药物- 抵抗力强。目前，预防或减少癫痫的更有效药物的开发受到一个缺口的阻碍 关于癫痫突发事件如何将健康的大脑转变为自发的大脑的知识 反复发作。这一过程被称为癫痫发生，被认为是非常复杂的。暂停或 因此，预防癫痫的发生很可能需要许多不同分子的协同操作 网络和路径。因此，有可能以调控机制为目标的治疗策略 同时控制这些细胞通路中的多个将是最成功的。这项研究将解决这一问题 通过分析数百个基因的表达的有效调节者，microRNA如何诱导 沉默复合体(RISC)参与癫痫持续状态(SE)后的致痫过程。以前的工作 支持microRNAs和RISC在癫痫发展中的作用，通过显示选择的microRNAs和 癫痫发作后，mRNAs被招募到RISC，抑制单个microRNAs可以减少癫痫发作 啮齿动物癫痫模型的易感性和致痫机制。然而，分子机制和途径 MicroRNA操作减轻癫痫发作的潜在作用在很大程度上是未知的。总体假设 本研究的主要内容是SE诱导RISC和microRNA功能的变化，从而增强癫痫的发生和发展 减少神经保护途径。抑制这些变化可能会阻止或损害 癫痫。这一假设将通过两个目标进行检验。目标1将遵循公正的方法，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揭示 SE后受RISC差异调控的分子通路的性质和功能相关性 老鼠。目标2将遵循基于候选人的方法，使用核糖体标记和功能分析，以及 反义介导的microRNA抑制揭示前惊厥microRNA的细胞特异性翻译组 以及它在SE后癫痫发生中的作用。基于补充神经科学家和 计算生物学家，在SE后进行RISC和microRNA靶标调控的筛选方法 结合途径和功能分析有望产生关于复合体的独特信息 调控癫痫发生的过程。以RISC协会为代理的创新战略 MicroRNA的功能，以及mRNAs与RISC的关联或主动翻译核糖体作为替代 对于它们的消音或翻译，预计将提供改进的功能评估 与以前的表达分析相比，microRNAs的沉默活性以及对靶mRNAs的影响。这 研究将填补了解RISC功能、蛋白质表达和途径失调的关键空白 在癫痫发生中的作用，这将是推进microRNA诱导的沉默作为治疗靶点的关键。从以下位置查看 从更广泛的角度来看，这一战略可以作为其他疾病的RISC分析的蓝图。

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.