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）：肌萎缩性侧面硬化症（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的一般症状是肌肉无力，在神经肌肉连接处的神经膜引起的浪费。大多数ALS病例是零星的，大约10％是家族性的。已经确定了几种ALS基因，因为它们的突变会导致家族性ALS，包括两个编码RNA加工蛋白TDP-43并在肉瘤中融合/在脂肪肉瘤（FUS/TLS）中易位的基因。 FUS是一种普遍表达的多域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，但也据报道将mRNA转运为神经元中树突中的局部翻译。此外，FUS在包括mRNA的核质穿梭，转录调控和mRNA剪接的各种过程中起作用。但是，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，知之甚少。我们最近发表了说，FUS的C末端是由ALS引起的突变聚集的，作为有效的核定位序列（NLS）。我们新生成的数据表明，FUS相互作用的蛋白质Gemin3在FUS突变引起的扰动中起关键作用。 Gemin3可以通过ALS突变型FUS隔离，ALS突变FUS会导致Gemin3阳性核结构（GEM），SNRNPs的组装减少和减弱剪接体活性。当FUS在运动神经元中过表达时，我们建立的果蝇模型显示出运动功能缺乏。有趣的是，据报道，果蝇中的幼虫运动功能是必需的。此外，我们产生了FUS/GEMIN3双转基因蝇，并表明Gemin3的表达营救了FUS转基因蝇的表型。因此，我们假设与ALS相关的FUS突变体或WT FUS具有失调的过表达可以积聚在细胞质中和隔离GEMIN3中，从而导致SNRNPs在细胞质中的组装减少，并损害了核中SNRNP的组装。为了检验中心假设，已经设计了三个特定目标来确定FUS在ALS中的作用。目的1是通过FUS内的定位序列及其RNA结合能力来了解FUS亚细胞定位的调节。在AIM 2中，我们将首先确定FUS和GEMIN 3相互作用的分子机制。我们将进一步表征FUS突变如何干扰双子3-介导的SNRNP组装和剪接体活性。 AIM 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果蝇在运动神经元特异性FUS表达中是否突出运动神经元死亡和神经肌肉神经神经。 FUS介导的GEMIN3隔离和随后的剪接体变化将在苍蝇中进行特别测试，因为Gemin3的过表达挽救了由FUS引起的运动功能不足表型。此外，将研究FUS亚细胞定位和RNA在产生运动神经元毒性中的重要性。最后，我们将进行RNA-Seq实验，以确定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融合介导的ALS病因。这些发现有望提供有关FUS突变扰动RNA加工途径并最终导致该疾病的机制的关键见解。