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

ALS 中 FUS/TLS 功能的获得和丧失：疾病的动物和细胞模型

• 批准号: 8856371
• 负责人: Neil Alan Shneider
• 金额: $ 35万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-09-01 至 2016-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8856371
• 关键词: Address; Affect; Alleles; Alternative Splicing; Amyotrophic Lateral Sclerosis; Animal Disease Models; Animal Model; Behavioral; Birth; Brain; Candidate Disease Gene; Cell model; Cells; Cessation of life; Clinical; Communities; DNA-Binding Proteins; Data Set; Development; Disease; Dominant-Negative Mutation; Familial Amyotrophic Lateral Sclerosis; Family; Future; Gene Expression; Gene Expression Regulation; Genes; Genetic; Genetic Transcription; High-Throughput Nucleotide Sequencing; High-Throughput RNA Sequencing; Human; In Vitro; Knock-in Mouse; Knock-out; Knockout Mice; Link; Mediating; Modeling; Molecular; Molecular Analysis; Morphology; Motor; Motor Activity; Motor Neuron Disease; Motor Neurons; Mus; Mutant Strains Mice; Mutation; Neonatal; Nervous system structure; Neuraxis; Neurodegenerative Disorders; Neurons; Paralysed; Pathogenesis; Pathology; Pathway interactions; Pattern; Perinatal; Phenotype; Physiological; Property; Proteins; RNA; RNA Processing; Reporting; Research; Role; Series; Spinal; Spinal Cord; Synapses; Testing; cell type; differential expression; disease mechanisms study; drug discovery; embryonic stem cell; gain of function; genetic analysis; high throughput analysis; in vivo; in vivo Model; loss of function; mRNA Expression; motor neuron degeneration; motor neuron development; mouse model; mutant; neuron loss; neurotoxicity; novel; overexpression; protein TDP-43; stem cell technology; therapeutic target; tool; transcriptome sequencing

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.

描述（由申请人提供）：肌萎缩性侧性硬化症（ALS）是一种进行性神经退行性疾病，其中运动神经元（MNS）的优先丧失导致瘫痪和死亡。尽管ALS在很大程度上是一种零星疾病，但研究集中在可遗传的疾病形式上，因为临床和病理证据表明了常见的致病机制。最近在罕见的ALS家族中报道了基因FUS（或TLS）中的突变，此后在零星的ALS中发现了FUS病理学，这表明FUS可能会提供家族性和零星疾病之间的机械联系。 FUS和TDP -43之间的结构和功能相似性 - 参与零星和家族性ALS发病机理的另一种RNA/DNA结合蛋白 - 也导致人们猜测，这些因素受调节的分子途径对于我们对常见疾病机制的理解至关重要。我们对FUS中的突变如何引起运动神经元变性。 FUS突变的主要遗传表明，具有选择性毒性的运动神经元有毒的新功能增益。另外，突变型FU可以作为主要阴性，抑制野生型蛋白的正常活性，也许是通过在异常的FUS阳性，细胞质聚集体中隔离，这是零星和家族性ALS的标志。如果由于FUS缺乏而导致ALS，那么更多地了解中枢神经系统中FUS的正常功能，特别是在疾病影响的运动电路中，至关重要。在该项目中，功能策略的丧失和增益用于探索FUS在动物和细胞模型中正常运动神经元发展中的作用，并将该功能与突变体FUS介导的ALS相关联。体外研究将利用我们大量产生FUS突变体干细胞衍生运动神经元的能力。在AIM 1中，FUS敲除小鼠将用于支持以下假设：ALS中的运动神经元变性是FUS缺乏的结果。我们将测试FUS丧失对运动神经元分化和存活的影响以及正常运动活动所需的脊柱运动电路的功能发展。通过高通量RNA测序（RNA SEQ），我们将探讨FUS在神经系统中基因表达调节中的正常作用。在AIM 2中，我们将使用突变FUS的过表达研究来表征对运动神经元存活和体内功能的影响，并确定突变如何改变ALS中FUS的功能特性。 RNA SEQ分析将用于鉴定疾病发病机理涉及的分子途径。在AIM 3中，我们将使用源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。该项目将解决有关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新颖的FUS介导疾病模型，这将是对ALS研究社区中疾病机制和药物发现的未来研究的关键工具。