Using Human iDNs to Study Translational Control of Neuronal Function and Survival

项目摘要 神经元表现出高度极化的形态，并与脑内其他细胞进行错综复杂的突触连接 尸体。这种连接的强度会对神经元的活动做出反应，并可以根据个体的不同而进行调节。 突触水平。这种独特的极化形态、错综复杂的连通性和功能可塑性 有必要在神经元中精确控制基因表达。翻译控制已成为一个关键问题 赋予神经元基因表达时空精确度的调控机制。此外，由 影响细胞的能量消耗-考虑到蛋白质合成是一种非常耗能的 过程，并通过调节错误折叠或聚集的蛋白质的水平，翻译控制密切相关 与能量代谢和蛋白平衡有关，这两个过程对神经元维持至关重要。它是这样的 预计翻译控制在正常的神经生物学过程中将具有特殊的重要性 如突触可塑性、学习和记忆，以及神经疾病的发病机制。 然而，与转录调控等其他基因表达调控机制相比，我们的 对翻译控制在健康和疾病中的机制和功能的理解滞后 在后面。在拟议的项目中，我们的目标是定义LRRK2(富含亮氨酸的重复序列)的作用机制 激酶2)是家族性和散发性帕金森病中最常突变的基因，在调节 人类疾病相关多巴胺能神经元的信使核糖核酸翻译。基于强有力的初步研究， 我们假设LRRK2参与了人多巴胺能神经元mRNAs的翻译控制。 神经元通过不同的底物和/或效应器来调节起始和 伸长步骤。为了验证这一假设，我们将使用人类诱导的多巴胺能神经元(IDN)。 从患者成纤维细胞和强大的CRISPR/Cas9基因组编辑技术重新编程以 确定LRRK2控制翻译起始和延伸的机制和功能(目标1)， 并描述LRRK2调节的mRNAs和蛋白质的分子特征(目标2)。 该项目的实施将通过创新的技术和研究战略来促进 重新编程衍生的人类神经元的翻译控制。这个项目的成功完成将 为LRRK2的生物学和病理生物学提供新的见解，并验证一个新的平台 使用患者来源的神经元和CRISPR/Cas9对人类神经系统疾病的机制研究。这个 因此，从这个项目中产生的信息有望成为基础神经科学的基础 研究并具有很高的临床相关性。

Project Summary Neurons exhibit highly polarized morphology and make intricate synaptic connections with other cells in the body. The strength of such connections responds to neuronal activity and can be modulated at individual synapse level. Such unique features of polarized morphology, intricate connectivity, and functional plasticity necessitate precisely controlled gene expression in neurons. Translational control has emerged as a critical regulatory mechanism that confers spatiotemporal precision to neuronal gene expression. In addition, by influencing energy expenditure in cells - considering that protein synthesis is a very energy-consuming process, and by modulating levels of misfolded or aggregated proteins, translational control is intimately linked to energy metabolism and proteostasis, two processes essential for neuronal maintenance. It is thus expected that translational control will assume particular importance in normal neurobiological processes such as synaptic plasticity, learning, and memory, and in the pathogenesis of neurological disorders. However, compared to other regulatory mechanisms of gene expression such as transcriptional control, our understanding of the mechanism and function of translational control in health and disease is lagging behind. In the proposed project, we aim to define the mechanism of action of LRRK2 (leucine-rich repeat kinase 2), a gene most frequently mutated in familial and sporadic Parkinson's disease, in the regulation of mRNA translation in disease-relevant human dopaminergic neurons. Based on strong preliminary studies, we hypothesize that LRRK2 participates in the translational control of mRNAs in human dopaminergic neurons by acting through distinct substrates and/or effectors to regulate translation at the initiation and elongation steps. To test this hypothesis, we will use human induced dopaminergic neurons (iDNs) reprogrammed from patient fibroblasts and the powerful CRISPR/Cas9 genome editing technique to determine the mechanisms and function of translation initiation and elongation control by LRRK2 (Aim 1), and to profile the molecular signatures of LRRK2-regulated mRNAs and proteins (Aim 2). Execution of this project will be facilitated by innovative technologies and strategies for studying translational control in reprogramming-derived human neurons. Successful completion of this project will provide new insights into the biology and pathobiology of LRRK2 and validate a new platform for mechanistic studies of human neurological diseases using patient-derived neurons and CRISPR/Cas9. The information to be generated from this project is therefore expected to be fundamental to basic neuroscience research and of high clinical relevance.