Role of FUS in ALS

FUS 在 ALS 中的作用

基本信息

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
项目类别：
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

项目摘要

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）：肌萎缩侧索硬化症（ALS，也称为卢伽雷氏病）是一种进行性且致命的神经退行性疾病。 ALS 的一般症状是神经肌肉接头去神经支配引发的肌肉无力和消瘦。大多数 ALS 病例是散发性的，大约 10% 是家族性的。一些 ALS 基因已被确定，因为它们的突变可导致家族性 ALS，其中包括编码 RNA 加工蛋白 TDP-43 并在肉瘤中融合/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，但据报道也可转运 mRNA，在神经元树突中进行局部翻译。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 却知之甚少，而这正是本研究的重点。我们最近发表了 FUS 的 C 末端（引起 ALS 的突变聚集于此）的功能，可作为有效的核定位序列 (NLS)。我们新生成的数据表明，FUS 相互作用蛋白 Gemin3 在 FUS 突变引起的扰动中发挥着关键作用。 Gemin3 可以被 ALS 突变体 FUS 隔离，从而导致 Gemin3 阳性核结构 (Gems) 减少、snRNP 组装减少以及剪接体活性减弱。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出运动功能缺陷。有趣的是，据报道，果蝇幼虫运动功能也需要 Gemin3。此外，我们生成了 FUS/Gemin3 双转基因果蝇，并表明 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 snRNP 组装减少并损害细胞核中的剪接体功能。为了检验中心假设，设计了三个具体目标来确定 FUS 在 ALS 中的作用。目标 1 是了解 FUS 内的定位序列元件及其 RNA 结合能力对 FUS 亚细胞定位的调节。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 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在具有运动神经元特异性 FUS 表达的转基因果蝇中是否显着。 FUS 介导的 Gemin3 隔离和随后的剪接体变化将在果蝇中进行特别测试，因为 Gemin3 过度表达可以挽救 FUS 引起的运动功能缺陷表型。此外，还将研究 FUS 亚细胞定位和 RNA 结合在运动神经元产生毒性中的重要性。最后，我们将进行RNA-Seq实验来确定FUS介导的剪接改变。该项目将利用细胞和果蝇模型的组合来研究 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重要见解。