Role of FUS in ALS

FUS 在 ALS 中的作用

• 批准号: 8449217
• 负责人: Haining Zhu
• 金额: $ 31.35万
• 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/8449217
• 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 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; transcriptome sequencing; 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.

描述（由申请人提供）：肌萎缩性侧索硬化症（ALS，也称为Lou Gehrig's病）是一种进行性和致命的神经退行性疾病。肌萎缩侧索硬化症的一般症状是由神经肌肉连接处的失神经支配引起的肌肉无力和消瘦。大多数ALS病例是散发的，大约10%是家族性的。一些ALS基因的突变可导致家族性ALS，包括两个编码RNA加工蛋白TDP-43的基因，它们在肉瘤中融合/在脂肪肉瘤中易位（FUS/TLS）。FUS是一种普遍表达的多结构域rna结合蛋白。在神经元和神经胶质细胞中，FUS几乎完全定位于细胞核，但也有报道称其在神经元树突中转运mRNA进行局部翻译。此外，FUS还参与核细胞质中mRNA的穿梭、转录调控和mRNA剪接等多种过程。然而，关于FUS突变如何导致运动神经元变性和ALS，我们知之甚少，这是本研究的重点。我们最近发表了FUS的c端，即引起als的突变聚集的地方，作为一个有效的核定位序列（NLS）。我们新生成的数据表明，FUS相互作用蛋白Gemin3在FUS突变引起的扰动中起关键作用。Gemin3可以被ALS突变体FUS隔离，导致Gemin3阳性核结构（Gems）减少，snRNPs组装减少，剪接体活性减弱。我们所建立的果蝇模型在运动神经元中过表达FUS时出现运动功能缺陷。有趣的是，据报道，Gemin3也是果蝇幼虫运动功能所必需的。此外，我们培育了FUS/Gemin3双转基因果蝇，结果表明，Gemin3的表达挽救了FUS转基因果蝇的表型。因此，我们假设als相关的FUS突变体或过度表达失调的WT FUS可以在细胞质中积累并隔离Gemin3，从而导致细胞质中snRNPs的组装减少，细胞核中的剪接体功能受损。为了验证中心假设，我们设计了三个具体目标来确定FUS在ALS中的作用。目的1是了解FUS内部的定位序列元件及其RNA结合能力对FUS亚细胞定位的调控。在Aim 2中，我们将首先确定FUS和Gemin 3相互作用的分子机制。我们将进一步描述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和目标2中定义的分子机制。我们将首先确定运动神经元特异性表达FUS的转基因果蝇中运动神经元死亡和神经肌肉失神经支配是否突出。FUS介导的Gemin3分离和随后的剪接体变化将在果蝇中进行特别测试，因为Gemin3过表达挽救了FUS引起的运动功能缺陷表型。此外，我们还将研究FUS亚细胞定位和RNA结合在运动神经元中产生毒性的意义。最后，我们将进行RNA-Seq实验来确定fus介导的剪接改变。本项目将利用细胞和果蝇模型的结合来研究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关键见解。