FUS Gain-of-Function Mechanisms in Animal and Cellular Models of ALS

ALS 动物和细胞模型中的 FUS 功能获得机制

• 批准号: 9513163
• 负责人: Neil Alan Shneider
• 金额: $ 55.68万
• 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 2019-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9513163
• 关键词: Adolescent; Alleles; Amyotrophic Lateral Sclerosis; Animal Model; Astrocytes; Automobile Driving; Axon; Binding; C-terminal; Cell Differentiation process; Cell model; Cells; Cessation of life; Clinical; Cytoplasmic Granules; Data; Data Analyses; Data Set; Defect; Development; Disease; Disease Pathway; Disease Progression; Disease model; Dose; Familial Amyotrophic Lateral Sclerosis; Fluorescent in Situ Hybridization; Funding; Gene Activation; Gene Expression; Generations; Genes; Genetic; Genetic Transcription; Genetic study; Heritability; Heterogeneity; Heterogeneous-Nuclear Ribonucleoprotein U; Immunofluorescence Immunologic; In Vitro; Knock-in; Knock-in Mouse; Label; Lasers; Measures; Mediating; Messenger RNA; Metabolic; Methodology; Microfluidics; Modeling; Molecular; Molecular Genetics; Motor Neuron Disease; Motor Neurons; Mus; Mutant Strains Mice; Mutation; Natural Increases; Nerve Degeneration; Nervous system structure; Neurodegenerative Disorders; Paralysed; Pathogenicity; Pathologic; Pathology; Patients; Pattern; Phase Transition; Phenotype; Play; Population; Protein Biosynthesis; Proteins; RNA; RNA-Binding Proteins; Research; Ribonucleoproteins; Role; Series; Spinal; System; Testing; Time; Toxic effect; Transgenic Organisms; Translational Repression; Translations; Work; differential expression; early onset; embryonic stem cell; experimental study; gain of function; hnRNP A1; in vivo; in vivo Model; induced pluripotent stem cell; insight; loss of function; motor neuron degeneration; mouse model; mutant; neurotoxic; new therapeutic target; novel; novel therapeutics; prion-like; protein TDP-43; selective expression; single cell sequencing; success; therapeutic target; transcriptome; transcriptome sequencing; treatment strategy; ubiquilin

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 cause some of the most aggressive early-onset forms of ALS. In a recent study, our lab demonstrated in a mouse model of disease that mutant FUS causes motor neuron degeneration not by a loss-of-function, by a toxic gain-of-function that does not involve an excess of FUS activity. FUS is one of a number of RNA binding proteins – including TDP-43 and hnRNP A1 – that have been causally related to ALS. Our recent work – together with related studies from several labs – has led to a disease model in which the low complexity (LC) “prion-like” domain of FUS and related proteins drives its phase transition to an irreversible, neurotoxic assembly. Our data demonstrates that ALS-related mutations in FUS increase the natural tendency of the protein to form these toxic assemblies, which trap and sequester other ribonucleoprotein granule components, including proteins involved in translational control. In this project we will explore the mechanisms of FUS toxicity in a series of transgenic and knock-in mutant mice with which we have modelled key aspect of the FUS-ALS phenotype. In addition, in vitro studies using motor neurons and astrocytes derived from these mouse models will be used to investigate cell autonomous and non-autonomous mechanisms of disease. In Aim 1, we will use a conditional knock-in mouse model to express mutant FUS in MNs or astrocytes, or more broadly in the nervous system to explore the effects of regulated mutant FUS expression on MN survival and function, and on gene expression changes that may underlie MN degeneration in FUS-ALS. We will also analyze mice with ALS-causing mutations in the LC domain of FUS to test the role of this critical domain in the disease. In Aim 2, we will use microfluidics to isolate MN axons and test the idea that FUS-dependent defects in axonal protein synthesis contribute to MN degeneration, and we will pursue our finding that hnRNP U selectively interacts with ALS-mutant FUS by exploring in vivo the functional role of this RNA binding protein in disease progression. Finally in Aim 3, we will apply a combination of single-cell RNA sequencing and topological data analysis to a mixed distribution of in vitro differentiated MNs derived from our FUS knock-in mutant mice. This sophisticated integration of in vivo and in vitro experimental systems, combined with our integrative computational and analytical approach will allow us to elucidate pathways of disease in vulnerable subpopulations of MNs and to identify potential therapeutic targets for the treatment of FUS-ALS and related forms of motor neuron disease.

肌萎缩性外侧硬化症（ALS）是一种进行性神经退行性疾病，在其中优先 运动神经元（MN）的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 由于临床和病理证据表明常见 致病机制。基因FUS中的突变引起了一些最激进的早期发作形式 ALS。在最近的一项研究中，我们的实验室在疾病的小鼠模型中证明了突变型FUS引起运动神经元 变性不是通过功能丧失，而不是涉及过量的FUS活性的功能收益。 FUS是许多RNA结合蛋白之一，包括TDP-43和HNRNP A1，它们一直是因果关系 与ALS有关。我们最近的工作以及来自多个实验室的相关研究导致了疾病模型 其中，FUS和相关蛋白的低复杂性（LC）“类似prion的”结构域将其相变到一个 不可逆的神经毒性组装。我们的数据表明，FUS中与ALS相关的突变增加了 蛋白质形成这些有毒组件的自然趋势，这些组件会捕获和隔离其他核糖核蛋白 颗粒成分，包括参与翻译控制的蛋白质。在这个项目中，我们将探索 我们已经建模的一系列转基因和敲门突变小鼠中FUS毒性的机制 FUS-ALS表型的关键方面。此外，使用运动神经元和星形胶质细胞的体外研究 这些小鼠模型将用于研究细胞自主和非自主机制 疾病。 在AIM 1中，我们将使用有条件的敲入小鼠模型来表达MNS或星形胶质细胞中的突变FUS， 或在神经系统中更广泛地探索受调节突变FUS表达对MN存活的影响 和功能，以及基因表达的变化，可能是FUS-ALS中MN变性的基础。我们也会 在FUS的LC结构域中用ALS引起的突变分析小鼠，以测试该关键域中的作用 疾病。在AIM 2中，我们将使用微流体分离Mn轴突并测试FUS依赖性缺陷的想法 轴突蛋白合成有助于MN变性，我们将选择HNRNP U选择地 通过在体内探索该RNA结合蛋白在疾病中的功能作用，与ALS突变的FUS相互作用 进展。最后，在AIM 3中，我们将使用单细胞RNA测序和拓扑数据的组合 分析从我们的FUS敲门突变小鼠衍生的体外分化MN的混合分布。这 体内和体外实验系统的软化整合，并结合我们的综合 计算和分析方法将使我们能够阐明易受伤害的疾病途径 MN的亚群，并确定用于治疗FUS-ALS及其相关的潜在治疗靶标 运动神经元疾病的形式。