Cellular and Molecular Mechanisms of FUS-related Amyotrophic Lateral Sclerosis

描述（由申请人提供）：肌萎缩性侧索硬化症（ALS）是一种以上下运动神经元丧失为特征的晚发性神经退行性疾病。FUS和TDP-43是发现在散发性和家族性ALS中发生突变的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类野生型和突变型FUS的转基因果蝇系。突变型而非野生型FUS在果蝇眼睛中的靶向表达导致泛素化聚集体的形成和外眼退行性表型。有趣的是，运动神经元中突变型FUS的异位表达导致幼虫瘫痪和蛹致死，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，这在患有FUS突变的ALS患者中可见，这表明FUS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缺失强烈抑制毒性，这表明细胞质定位是神经变性所必需的。我们发现突变的FUS在果蝇肌肉中的表达导致撑翅表型、肌肉变性和线粒体变性。我们还发现突变的FUS RNA结合残基在体内强烈抑制突变的FUS毒性。根据这些观察结果，我们进行了无偏倚的遗传筛选，发现肌肉盲是突变型FUS毒性的新修饰因子。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ALS发病机制中的作用。我们还将在我们的苍蝇模型中研究肌肉盲介导的突变FUS毒性抑制的分子机制，然后在哺乳动物细胞培养系统和携带FUS突变的人类患者细胞中进行验证。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的治疗干预措施。

DESCRIPTION (provided by applicant): 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. 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 as seen in human ALS patients with FUS mutations, suggesting that the cytoplasmic localization of FUS 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. Following up these observations, we performed an unbiased genetic screening and 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 molecular mechanisms of muscleblind mediated suppression of mutant FUS toxicity in our fly model followed by validation in mammalian cell culture system and human patient cells carrying FUS mutations. 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.