Role of FUS in ALS

描述(申请人提供)：肌萎缩侧索硬化症(ALS，也称为卢格里克病)是一种进行性和致命性的神经退行性疾病。肌萎缩侧索硬化症的一个普遍症状是肌肉无力和消瘦，这是由神经肌肉接头处的失神经引起的。大多数肌萎缩侧索硬化症病例是散发的，大约10%是家族性的。一些ALS基因的突变可导致家族性ALS，包括编码RNA加工蛋白TDP-43和融合于肉瘤/转位于脂肪肉瘤(FUS/TLS)的两个基因。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几乎只定位于细胞核，但也有报道在神经元的树突中运输mRNA进行局部翻译。此外，FUS还参与了多种过程，包括核质穿梭、转录调控和mRNA剪接。然而，关于FUS突变如何导致运动神经元退化和ALS，这是本研究的重点，人们知之甚少。我们最近发表了FUS的C末端，在那里导致ALS的突变聚集在一起，作为一个有效的核定位序列(NLS)。我们新产生的数据表明，FUS相互作用蛋白Gmin3在FUS突变引起的扰动中起着关键作用。ALS突变体fus可以隔离Gin3，导致Gin3阳性核结构(Gem)减少，SnRNPs组装减少，剪接体活性减弱。我们建立的果蝇模型，当FUS在运动神经元中过度表达时，显示出运动功能缺陷。有趣的是，据报道，果蝇幼虫的运动功能也需要GMin3。此外，我们还获得了FUS/Gin3双转基因果蝇，结果表明Gin3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突变体或过度表达的WT FUS可以积聚在细胞质中，隔离Gin3，从而导致SnRNPs在细胞质中的组装减少，并损害核内的剪接体功能。为了验证这一中心假说，我们设计了三个特定的目标来确定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亚细胞定位的调节。在目标2中，我们将首先确定FUS和Gumin3相互作用的分子机制。我们将进一步研究FUS突变是如何干扰Gmin3介导的SnRNP组装和剪接体活性的。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FUS表达的转基因果蝇中运动神经元死亡和神经肌肉失神经是否显著。FUS介导的Gin3隔离和随后的剪接体变化将特别在果蝇中进行测试，因为Gin3的过度表达挽救了FUS导致的运动功能缺陷表型。此外，还将研究FUS亚细胞定位和RNA结合在产生运动神经元毒性中的意义。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组合来研究FUS介导的ALS病因学。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的见解。 公共卫生相关性：几个肌萎缩侧索硬化症(ALS)基因已被确认为其突变可导致家族性ALS，包括编码RNA加工蛋白TDP-43和融合肉瘤(FUS)的两个基因。该领域的主要挑战是，人们对FUS突变如何导致ALS运动神经元退化知之甚少。我们建议检验这样一种假设，即过度表达的ALS相关FUS突变体或WT FU可以在细胞质中积聚并隔离Gin3，从而导致SnRNPs在细胞质中的组装减少，并损害核中的剪接体功能。为了验证这一中心假说，我们设计了三个特定的目标来确定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，产生了几乎所有的关键试剂，并与领先的专家建立了合作。我们建议使用细胞模型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关键的见解。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。

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.