Role of FUS in ALS

FUS 在 ALS 中的作用

• 批准号: 8234739
• 负责人: Haining Zhu
• 金额: $ 32.48万
• 依托单位: UNIVERSITY OF KENTUCKY
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-08-15 至 2015-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8234739
• 关键词: Amyotrophic Lateral Sclerosis; Attenuated; C-terminal; Cell Nucleus; Cells; Cessation of life; Collaborations; Complex; Cytoplasm; DNA Repair; Data; Dendrites; Denervation; Disease; Drosophila genus; Elements; Etiology; Familial Amyotrophic Lateral Sclerosis; Future; Gemin3; Genes; Knowledge; Lead; Locomotion; Mediating; Messenger RNA; Modeling; Molecular; Motor; Motor Neurons; Muscle Weakness; Mutation; Names; Neurodegenerative Disorders; Neuroglia; Neuromuscular Junction; Neurons; Nuclear; Nuclear Structure; Outcome; Pathology; Pathway interactions; Phenotype; Physiological; Play; Process; Proteins; Proteomics; Publishing; RNA; RNA Binding; RNA Processing; RNA Splicing; RNA-Binding Proteins; Reagent; Regulation; Reporting; Research; Role; Small Nuclear Ribonucleoproteins; Spliceosome Assembly Pathway; Spliceosomes; Symptoms; Testing; Toxic effect; Transcriptional Regulation; Transgenic Organisms; Translations; design; fly; in vivo; insight; interest; liposarcoma; mRNA Precursor; motor neuron degeneration; mutant; nerve supply; neuromuscular; novel; nucleocytoplasmic transport; protein TDP-43; research study; sarcoma; tool; wasting

DESCRIPTION (provided by applicant): Amyotrophic lateral sclerosis (ALS, also known as Lou Gehrig's disease) is a progressive and fatal neurodegenerative disease. A general symptom of ALS is muscle weakness and wasting triggered by denervation at neuromuscular junctions. The majority of ALS cases are sporadic, and approximately 10% are familial. Several ALS genes have been identified as their mutation can lead to familial ALS, including two genes encoding RNA processing proteins TDP-43 and fused in sarcoma/translocated in liposarcoma (FUS/TLS). FUS is a ubiquitously expressed multi-domain RNA-binding protein. In neurons and glial cells, FUS is almost exclusively localized to the nucleus but is also reported to transport mRNA for local translation in dendrites in neurons. In addition, FUS plays a role in a variety of processes including nucleocytoplasmic shuttling of mRNA, transcriptional regulation and mRNA splicing. However, little is known regarding how FUS mutations cause motor neuron degeneration and ALS, which is the focus of this study. We recently published that the C-terminus of FUS, where the ALS-causing mutations are clustered, functions as an effective nuclear localization sequence (NLS). Our newly generated data suggest that a FUS- interacting protein Gemin3 plays a critical role in the perturbations caused by FUS mutations. Gemin3 can be sequestered by ALS mutant FUS, which causes reduced Gemin3-positive nuclear structures (Gems), decreased assembly of snRNPs, and attenuated spliceosome activity. The Drosophila model we established showed motor function deficiency when FUS was over-expressed in motor neurons. Interestingly, Gemin3 was also reported to be required for larval motor function in Drosophila. Moreover, we generated FUS/Gemin3 double transgenic flies and showed that expression of Gemin3 rescued the phenotypes of FUS transgenic flies. We thus hypothesize that the ALS-related FUS mutants or WT FUS with deregulated over-expression can accumulate in cytoplasm and sequester Gemin3, which results in decreased assembly of snRNPs in cytoplasm and compromised spliceosome function in the nucleus. To test the central hypothesis, three specific aims have been designed to determine the role of FUS in ALS. Aim 1 is to understand the regulation of FUS subcellular localization by the localization sequence elements within FUS as well as by its RNA binding ability. In Aim 2, we will first determine the molecular mechanism how FUS and Gemin 3 interact. We will further characterize how FUS mutations disturb Gemin 3- mediated snRNP assembly and spliceosome activity. Aim 3 will test the molecular mechanisms defined in Aims 1 and 2 using the Drosophila model. We will first determine whether motor neuron death and neuromuscular denervation are prominent in the transgenic flies with motor neuron-specific FUS expression. FUS-mediated Gemin3 sequestering and subsequent spliceosome changes will be especially tested in flies since Gemin3 over-expression rescued the motor function deficit phenotype caused by FUS. Furthermore, the significance of FUS subcellular localization and RNA binding in producing toxicity in motor neurons will be investigated. Lastly, we will carry out RNA-Seq experiment to determine the FUS-mediated splicing alterations. This project will utilize the combination of cellular and Drosophila models to investigate the FUS- mediated ALS etiology. The findings are expected to provide critical insights into the mechanisms by which FUS mutations perturb the RNA processing pathways and ultimately lead to the disease. PUBLIC HEALTH RELEVANCE: Several amyotrophic lateral sclerosis (ALS) genes have been identified as their mutation can lead to familial ALS, including two genes encoding RNA processing proteins TDP-43 and fused in sarcoma (FUS). The major challenge in the field is that little is known how FUS mutations cause motor neuron degeneration in ALS. We propose to test the hypothesis that the ALS-related FUS mutants or WT FUS with deregulated over- expression can accumulate in cytoplasm and sequester Gemin3, which results in decreased assembly of snRNPs in cytoplasm and compromised spliceosome function in the nucleus. To test the central hypothesis, three specific aims have been designed to determine the role of FUS in ALS. We have produced novel preliminary data, developed unique tools, generated nearly all critical reagents, and established collaborations with leading experts. We propose to use the combination of cellular and Drosophila models to test the hypothesis. The findings are expected to provide critical insights into the mechanisms by which FUS mutations perturb the RNA processing pathways and ultimately lead to the disease. The knowledge generated in the proposed research will also provide much-needed future direction for developing ALS treatment.

描述（由申请人提供）：肌萎缩侧索硬化症（ALS，也称为Lou Gehrig病）是一种进行性和致命的神经退行性疾病。ALS的一般症状是由神经肌肉接头处的去神经支配触发的肌肉无力和消耗。大多数ALS病例是散发性的，大约10%是家族性的。几个ALS基因已被鉴定为它们的突变可导致家族性ALS，包括编码RNA加工蛋白TDP-43和在肉瘤中融合/在脂肪肉瘤中易位（FUS/TLS）的两个基因。FUS是一种广泛表达的多结构域RNA结合蛋白。在神经元和神经胶质细胞中，FUS几乎仅定位于细胞核，但也报道转运mRNA用于神经元树突中的局部翻译。此外，FUS在多种过程中发挥作用，包括mRNA的核质穿梭、转录调节和mRNA剪接。然而，关于FUS突变如何导致运动神经元变性和ALS，这是本研究的重点，目前知之甚少。 我们最近发表了FUS的C-末端，其中ALS引起的突变聚集，作为一个有效的核定位序列（NLS）的功能。我们新产生的数据表明，FUS相互作用蛋白Gemin 3在FUS突变引起的扰动中起着关键作用。Gemin 3可以被ALS突变体FUS隔离，其导致Gemin 3阳性核结构（Gems）减少、snRNP组装减少和剪接体活性减弱。当FUS在运动神经元中过度表达时，我们建立的果蝇模型显示运动功能缺陷。有趣的是，Gemin 3也被报道是果蝇幼虫运动功能所必需的。此外，我们产生了FUS/Gemin 3双转基因果蝇，并表明Gemin 3的表达拯救了FUS转基因果蝇的表型。因此，我们假设ALS相关的FUS突变体或WT FUS与失调的过度表达可以积累在细胞质中，并隔离Gemin 3，这导致减少组装的snRNP在细胞质中和受损的剪接体功能的核。为了检验中心假设，三个具体的目标已被设计来确定FUS在ALS中的作用。 目的1是了解FUS内定位序列元件及其RNA结合能力对FUS亚细胞定位的调控。在目标2中，我们将首先确定FUS和Gemin 3相互作用的分子机制。我们将进一步表征FUS突变如何干扰Gemin 3介导的snRNP组装和剪接体活性。目标3将使用果蝇模型测试目标1和2中定义的分子机制。我们将首先确定运动神经元死亡和神经肌肉去神经支配是否在运动神经元特异性FUS表达的转基因果蝇中突出。FUS介导的Gemin 3隔离和随后的剪接体变化将特别在果蝇中进行测试，因为Gemin 3过表达挽救了由FUS引起的运动功能缺陷表型。此外，FUS亚细胞定位和RNA结合在运动神经元中产生毒性的意义将被研究。最后，我们将进行RNA-Seq实验以确定FUS介导的剪接改变。 本计画将利用细胞模型与果蝇模型相结合的方式来探讨FUS介导的ALS病因.这些发现有望为FUS突变扰乱RNA加工途径并最终导致疾病的机制提供重要见解。 公共卫生相关性：几个肌萎缩侧索硬化症（ALS）基因已被确定，因为它们的突变可导致家族性ALS，包括编码RNA加工蛋白TDP-43的两个基因，并在肉瘤（FUS）中融合。该领域的主要挑战是，很少有人知道FUS突变如何导致ALS运动神经元变性。我们提出检验以下假设：ALS相关FUS突变体或具有失调的过表达的WT FUS可以在细胞质中积累并螯合Gemin 3，这导致snRNP在细胞质中的组装减少和细胞核中剪接体功能受损。为了检验中心假设，三个具体的目标已被设计来确定FUS在ALS中的作用。我们已经产生了新的初步数据，开发了独特的工具，生成了几乎所有的关键试剂，并与领先的专家建立了合作关系。我们建议使用细胞和果蝇模型的组合来检验这一假设。这些发现有望为FUS突变扰乱RNA加工途径并最终导致疾病的机制提供重要见解。在拟议的研究中产生的知识也将为开发ALS治疗提供急需的未来方向。