Roles for TDP-43 and FUS in ALS Using Motor Neurons from Embryonic Stem Cells

描述（由申请人提供）：肌萎缩性侧索硬化症（ALS）是一种成人发病的神经退行性疾病，其上部和下部运动神经元过早丧失导致致命的瘫痪，典型病程为1至5年。两种DNA/RNA结合蛋白的突变，TAR DNA结合蛋白（TDP-43）和融合肉瘤（FUS）最近被确定为遗传性ALS的主要原因，并可能导致理解ALS发病机制的范式转变。然而，仍有必要检查这些蛋白质在与ALS进展相关的细胞类型中的功能，包括运动神经元和神经胶质细胞。虽然在SOD1突变引起的遗传性ALS中，周围细胞的作用已经得到证实，但在TDP-43或FUS突变后疾病进展中的非细胞自主机制的影响是完全未知的。本研究旨在利用来自现有转基因小鼠的胚胎干细胞来检测纯化运动神经元中TDP-43或FUS的减少或突变的后果。这些基因改变的细胞内在后果将通过诱导胚胎干细胞分化为缺乏或表达突变TDP-43或FUS的运动神经元来确定。而不是采取候选人的方法去追求一些基因已经被确认为目标TDP-43或付在增生细胞系,孤立运动神经元文化将被用来确定完整的rna所改变TDP-43损失或在目标1付,然后在目标2问rna是否改变的突变TDP-43或付,如果是这样的话,如果他们观察到的相同功能丧失。通过从纯化的起始材料中采取这种全面和系统的方法，可能会发现受影响的特定靶点和信号通路，这些发现可能会阐明疾病的潜在机制，并为未来的治疗发展提供基础。这些目标的成功将导致对ALS疾病机制的更深入了解，并将为未来的胶质细胞类型研究提供理论依据，以确定操作支持的非神经元细胞是否会为TDP-43或FUS突变的ALS患者提供治疗益处。

DESCRIPTION (provided by applicant): Amyotrophic lateral sclerosis (ALS) is an adult-onset neurodegenerative disorder in which premature loss of upper and lower motor neurons leads to fatal paralysis with a typical disease course of one to five years. Mutations in two DNA/RNA binding proteins, TAR DNA-binding protein (TDP-43) and Fused in Sarcoma (FUS) have recently been identified as primary causes of inherited ALS and have led to what is likely to be a paradigm shift in efforts to understand the pathogenesis of ALS. Yet there remains a need to examine the functions of these proteins in cell types relevant for ALS progression, both motor neurons and glial cells. While a contribution from surrounding cells is well established in inherited ALS caused by SOD1 mutations, the impact of non-cell autonomous mechanisms in disease progression following mutation in TDP-43 or FUS is completely unknown. This proposal seeks to use embryonic stem cells derived from existing transgenic mice to examine the consequences of either reduction or mutation of TDP-43 or FUS in purified motor neurons. Cell intrinsic consequences of alterations in these genes will be determined by inducing differentiation of embryonic stem cells into motor neurons which either lack or express mutant TDP-43 or FUS. Rather than take a candidate approach to pursue the few genes that have already been identified as targets of TDP-43 or FUS in proliferating cell lines, isolated motor neuron cultures will be used to identify the complete set of RNAs that are altered by loss of TDP-43 or FUS in Aim 1, and then in Aim 2 to ask whether any RNAs are altered upon mutation of TDP-43 or FUS and if so, if they are the same as those observed upon loss of function. By taking such a comprehensive and systematic approach from purified starting material it is likely that the specific targets and signaling pathways affected can be uncovered - discoveries that might elucidate underlying mechanisms for disease and provide a basis for future therapeutic developments. Success in these goals will lead to greater understanding of ALS disease mechanism and will provide rationale for future studies in glial cell types to determine whether manipulation of supporting non-neuronal cells would provide therapeutic benefit in ALS patients with mutations in TDP-43 or FUS. PUBLIC HEALTH RELEVANCE: Amyotrophic lateral sclerosis (ALS) is an adult-onset neurodegenerative disorder that affects neurons of the brain and spinal cord controlling muscle movement and leads to fatal paralysis within the course of one to five years. Mutations in two proteins have recently been identified as a primary cause of ALS, yet the mechanism by which they contribute to disease is unknown. This project seeks to understand the functions of normal or mutant forms of these proteins in purified motor neurons to uncover the specific pathways affected, and may have wider implications for other nervous system diseases with similar pathology.