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

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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.