FUS/TLS GAIN AND LOSS OF FUNCTION IN ALS: ANIMAL AND CELLULAR MODELS OF DISEASE

描述（由申请人提供）：肌萎缩性侧索硬化症（ALS）是一种进行性神经退行性疾病，运动神经元（MNs）的优先丧失导致瘫痪和死亡。尽管肌萎缩侧索硬化症在很大程度上是一种散发性疾病，但由于临床和病理证据表明有共同的致病机制，研究一直集中在这种疾病的遗传形式上。最近在罕见的ALS家族中报道了FUS基因（或TLS）的突变，并且在散发性ALS中也发现了FUS病理，这表明FUS可能在家族性和散发性疾病之间提供了机制联系。FUS和TDP-43（另一种参与散发性和家族性ALS发病机制的RNA/ dna结合蛋白）之间的结构和功能相似性也导致了这两种因子调控的分子通路对我们理解常见疾病机制的重要作用。我们对FUS突变如何引起运动神经元变性知之甚少。FUS突变的显性遗传提示一种新的功能增益，对运动神经元具有选择性毒性。或者，突变的FUS可能作为显性阴性，抑制野生型蛋白的正常活性，可能是通过将其隔离在异常的FUS阳性细胞质聚集体中，这是散发性和家族性ALS的标志。如果ALS是由于FUS缺乏导致的，那么更多地了解FUS在中枢神经系统中的正常功能，特别是在疾病中受影响的运动回路中的功能是至关重要的。在这个项目中，功能策略的丧失和获得被用来探索FUS在动物和细胞模型中正常运动神经元发育中的作用，并将该功能与突变的FUS介导的ALS联系起来。体外研究将利用我们大量产生FUS突变胚胎干细胞衍生的运动神经元的能力。在Aim 1中，FUS基因敲除小鼠将被用来支持ALS中运动神经元退化是FUS缺乏的结果的假设。我们将测试FUS损失对运动神经元分化和存活的影响，以及对正常运动活动所需的脊髓运动回路功能发育的影响。通过高通量RNA测序（RNA Seq），我们将探讨FUS在正常情况下对神经系统基因表达的调控作用。在Aim 2中，我们将使用突变FUS的过表达研究来表征对体内运动神经元存活和功能的影响，并确定突变如何改变ALS中FUS的功能特性。RNA序列分析将用于确定参与疾病发病机制的分子途径。在Aim 3中，我们将使用来自小鼠胚胎干细胞的运动神经元来研究fus介导的运动神经元变性的细胞和分子机制。该项目将解决关于FUS在ALS中的作用的基本问题，并在小鼠和培养的运动神经元中产生FUS介导的疾病的新模型，这将是ALS研究界未来研究疾病机制和药物发现的关键工具。

DESCRIPTION (provided by applicant): Amyotrophic Lateral Sclerosis (ALS) is a progressive neurodegenerative disorder in which preferential loss of motor neurons (MNs) results in paralysis and death. Although ALS is largely a sporadic disease, research has focused on heritable forms of the disorder because clinical and pathological evidence suggests common pathogenic mechanisms. Mutations in the gene FUS (or TLS) were recently reported in rare ALS families, and FUS pathology has since been found in sporadic ALS, suggesting that FUS may provide a mechanistic link between familial and sporadic disease. Structural and functional similarities between FUS and TDP-43 - another RNA/DNA-binding protein involved in the pathogenesis of sporadic and familial ALS - have also led to speculation that the molecular pathways regulated by both of these factors are vital to our understanding of common disease mechanisms. We know very little about how mutations in FUS cause motor neuron degeneration. Dominant inheritance of FUS mutations suggests a novel gain of function that is selectively toxic to motor neurons. Alternatively, mutant FUS may act as a dominant negative, inhibiting the normal activity of wild type protein, perhaps by sequestering it in abnormal FUS-positive, cytoplasmic aggregates that are a hallmark of sporadic and familial ALS. If ALS results as a consequence of FUS deficiency, then it is critical to understand more about the normal functions of FUS in the central nervous system, and specifically in the motor circuits affected in the disease. In this project, loss and gain of function strategies are used to explore the role of FUS in normal motor neuron development in animal and cellular models, and to relate that function to mutant FUS-mediated ALS. In vitro studies will take advantage of our ability to generate FUS mutant embryonic stem cell-derived motor neurons in large numbers. In Aim 1, FUS knockout mice will be used to support the hypothesis that motor neuron degeneration in ALS is a consequence of FUS deficiency. We will test the effect of FUS loss on motor neuron differentiation and survival and on the functional development of spinal motor circuits required for normal motor activity. By high-throughput RNA sequencing (RNA Seq), we will explore the normal role of FUS in the regulation of gene expression in the nervous system. In Aim 2, we will use overexpression studies of mutant FUS to characterize the effect on motor neuron survival and function in vivo, and to determine how mutations alter the functional properties of FUS in ALS. RNA Seq analysis will be used to identify molecular pathways involved in the pathogenesis of disease. In Aim 3, we will use motor neurons derived from mouse embryonic stem cells to study cellular and molecular mechanism of FUS-mediated motor neuron degeneration. This project will address fundamental questions about the role of FUS in ALS, and generate novel models of FUS-mediated disease in mice and cultured motor neurons that will be critical tools for future studies of disease mechanism and drug discovery in the ALS research community.