Cellular and Molecular Mechanisms of FUS-related Amyotrophic Lateral Sclerosis

FUS相关肌萎缩侧索硬化症的细胞和分子机制

• 批准号: 9230443
• 负责人: Udai B Pandey
• 金额: $ 32.92万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-03-01 至 2019-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9230443
• 关键词: ATP Synthesis Pathway; Alternative Splicing; Amyotrophic Lateral Sclerosis; Behavioral Assay; Binding; Binding Proteins; Biochemical; Biological Models; Biology; Cell Culture Techniques; Cell Line; Cell Nucleus; Cells; Clinical; Cytoplasm; DNA; Data; Disease; Drosophila eye; Drosophila genus; Ectopic Expression; Enzymes; Exons; Eye; Functional disorder; Genes; Genetic; Genetic Screening; Goals; Hereditary Disease; Homologous Gene; Human; Hypersensitivity; Impairment; Lead; Link; Mammalian Cell; Mammals; Mediating; Messenger RNA; Metabolism; Mission; Mitochondria; Modeling; Molecular; Morphology; Motor Neurons; Muscle; Mutate; Mutation; Myotonic Dystrophy; Nematoda; Nerve Degeneration; Neurodegenerative Disorders; Neurons; Nuclear Export; Paralysed; Paraquat; Pathogenesis; Pathogenicity; Pathologic; Pathway interactions; Patients; Phenotype; Proteins; Publishing; Pupa; RNA; RNA Binding; RNA Splicing; RNA-Binding Proteins; Research; Research Personnel; Role; Rotenone; Series; Signal Transduction; Testing; Therapeutic Intervention; Toxic effect; Toxin; Transgenic Organisms; Translating; Ubiquitin; United States National Institutes of Health; Validation; Wing; disease-causing mutation; effective therapy; experimental study; fly; human disease; in vivo; insight; mRNA Precursor; motor neuron degeneration; muscle degeneration; mutant; neurodegenerative phenotype; novel; novel therapeutics; nucleocytoplasmic transport; polyglutamine; protein TDP-43; public health relevance; trafficking

DESCRIPTION (provided by applicant): Abstract Amyotrophic lateral sclerosis (ALS) is a late onset neurodegenerative disorder characterized by the loss of upper and lower motor neurons. FUS and TDP-43 are DNA/RNA binding proteins found to be mutated in both sporadic and familial forms of ALS. To investigate the pathogenesis of ALS caused by FUS/TLS mutations, we established a series of transgenic Drosophila lines that ectopically express human wild type and mutant FUS/TLS. Targeted expression of mutant, but not wild type FUS, in Drosophila eyes causes the formation of ubiquitinated aggregates and an external eye degenerative phenotype. Interestingly, ectopic expression of mutant FUS in motor neurons resulted in larval paralysis, pupal lethality, whereas wild type FUS expression had minimal effect. Mutant FUS localized to both the cytoplasm and nucleus, whereas wild type FUS localized only to the nucleus, suggesting that the cytoplasmic localization of FUS/TLS is required for toxicity. Furthermore, we found that deletion of the nuclear export signal strongly suppressed toxicity, suggesting that cytoplasmic localization is necessary for neurodegeneration. We found that expression of mutant FUS in the fly muscles leads to held-up wing phenotype, muscle degeneration and mitochondrial degeneration. We also found that mutating RNA binding residues of FUS strongly suppress mutant FUS toxicity in vivo. While doing an unbiased genetic screening, we discovered muscleblind as a novel modifier of mutant FUS toxicity. In our proposed studies, we will determine the role of mitochondrial functions in mediating FUS- related ALS and which functions of mitochondria are disrupted by the ALS causing mutations. We will systematically determine the role of RNA binding ability of FUS in causing ALS pathogenesis. We will also investigate if muscleblind regulates alternative splicing and sub- cellular distribution of FUS and contributes to the onset and progression of FUS-related ALS. Our proposed studies are expected to provide novel insights into the molecular mechanism of FUS-related ALS that would help in developing a therapeutic interventions for ALS for which currently no cure available.

肌萎缩性侧索硬化症（ALS）是一种迟发性神经退行性疾病，其特征是上下运动神经元的丧失。FUS和TDP-43是发现在散发性和家族性ALS中突变的DNA/RNA结合蛋白。为了研究FUS/TLS突变引起ALS的发病机制，我们建立了一系列异位表达人野生型和突变型FUS/TLS的转基因果蝇品系。果蝇眼睛中突变型而非野生型FUS的靶向表达导致形成泛素化聚集体和外部眼睛变性表型。有趣的是，在运动神经元中异位表达突变FUS导致幼虫麻痹，蛹死亡，而野生型FUS表达的影响很小。突变型FUS定位于细胞质和细胞核，而野生型FUS仅定位于细胞核，表明FUS/TLS的细胞质定位是毒性所必需的。此外，我们发现，核输出信号的删除强烈抑制毒性，这表明细胞质定位是必要的神经变性。我们发现，突变FUS在果蝇肌肉中的表达导致了抱翅表型、肌肉变性和线粒体变性。我们还发现，突变的RNA结合残基的FUS强烈抑制突变FUS在体内的毒性。在进行无偏遗传筛选时，我们发现肌盲是突变FUS毒性的一种新修饰剂。在我们提出的研究中，我们将确定线粒体功能在介导FUS相关ALS中的作用，以及线粒体的哪些功能被ALS引起的突变破坏。我们将系统地确定FUS的RNA结合能力在导致ALS发病机制中的作用。我们还将研究肌盲是否调节FUS的选择性剪接和亚细胞分布， 有助于FUS相关ALS的发作和进展。我们提出的研究有望为FUS相关ALS的分子机制提供新的见解，这将有助于开发目前无法治愈的ALS治疗干预措施。