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，并与脑内其他细胞形成复杂的突触连接。 身体这种连接的强度响应于神经元活动，并且可以在个体中调节。 突触水平。这种独特的特征极化形态，错综复杂的连接，和功能可塑性 需要精确控制神经元中的基因表达。翻译控制已经成为一个关键的 调节机制，赋予神经元基因表达的时空精度。另外通过 影响细胞的能量消耗-考虑到蛋白质合成是一个非常消耗能量的过程， 过程，并通过调节水平的错误折叠或聚集的蛋白质，翻译控制是密切相关的 与能量代谢和蛋白质稳态有关，这两个过程对神经元的维持至关重要。因此 预期翻译控制在正常的神经生物学过程中具有特别重要的意义 如突触可塑性、学习和记忆，以及神经系统疾病的发病机制。 然而，与基因表达的其他调控机制如转录控制相比，我们的 对翻译控制在健康和疾病中的机制和功能的理解是滞后的 后面在拟议的项目中，我们的目标是确定LRRK 2（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 疾病相关的人类多巴胺能神经元中的mRNA翻译。基于强有力的初步研究， 我们假设LRRK 2参与了人多巴胺能神经元中mRNA的翻译控制。 神经元通过不同的底物和/或效应物起作用，在起始时调节翻译， 延伸步骤。为了验证这一假设，我们将使用人类诱导多巴胺能神经元（iDN） 从患者成纤维细胞重编程和强大的CRISPR/Cas9基因组编辑技术， 确定LRRK 2（Aim 1）控制翻译起始和延伸的机制和功能， 并分析LRRK 2调节的mRNA和蛋白质的分子特征（Aim 2）。 该项目的执行将通过创新技术和研究战略来促进 在重编程衍生的人类神经元中的翻译控制。该项目的成功完成将 为LRRK 2的生物学和病理学提供新的见解，并验证一个新的平台， 使用患者来源的神经元和CRISPR/Cas9的人类神经系统疾病的机制研究。的 因此，从该项目中产生的信息预计将成为基础神经科学的基础 研究和临床相关性高。

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.