Defining the role of FUS phosphorylation in neurodegeneration

定义 FUS 磷酸化在神经变性中的作用

• 批准号: 9533703
• 负责人: THOMAS L KUKAR
• 金额: $ 32.22万
• 依托单位: EMORY UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-08-01 至 2020-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9533703
• 关键词: Affect; Age of Onset; Amyotrophic Lateral Sclerosis; Arginine; Astrocytes; Binding; Biochemical; Brain; Brain Sarcoma; C-terminal; Cell Nucleus; Cells; Chemicals; Cytoplasm; Cytoplasmic Granules; Cytoplasmic Inclusion; DNA; DNA Damage; DNA Double Strand Break; DNA Markers; DNA Repair; DNA Repair Pathway; DNA-dependent protein kinase; Data; Defect; Disease; Event; Frontotemporal Dementia; Frontotemporal Lobar Degenerations; Genes; Genetic Transcription; Goals; Human; Huntington Disease; Impairment; In Vitro; Inherited; Karyopherins; Label; Lead; Link; Maps; Mass Spectrum Analysis; Mediating; Messenger RNA; Metabolism; Methods; Methylation; Mus; Mutation; Nerve Degeneration; Neurodegenerative Disorders; Neurons; Nuclear Import; Nuclear Localization Signal; Nuclear Protein; Pathogenesis; Pathogenicity; Pathologic; Pathology; Phosphorylation; Phosphorylation Site; Phosphotransferases; Protein Family; Protein Kinase; Proteins; RNA; RNA Binding; RNA Splicing; RNA-Binding Proteins; Regulation; Research; Role; Severities; Stress; TAF15 gene; Testing; Tissues; Toxic effect; Toxin; base; chemical genetics; drug development; ds-DNA; gain of function; genetic approach; human disease; innovation; insight; mimetics; mouse model; mutant; neurotoxic; neurotoxicity; new therapeutic target; novel; novel strategies; polyglutamine; prevent; protein TDP-43; protein aggregate; protein aggregation; public health relevance; receptor; recruit; sarcoma; tau Proteins

 DESCRIPTION (provided by applicant): Fused in Sarcoma (FUS) is a ubiquitous multifunctional RNA-binding protein (RBP) located in the nucleus. The abnormal and pathogenic aggregation of FUS in the cytoplasm of neurons defines subtypes of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD), termed FTLD-FUS or ALS-FUS. ALS- FUS cases are caused by mutations in the FUS gene. In these cases, the accumulation of FUS is thought to be driven by long-term increases in cytoplasmic FUS caused by mutations that decrease nuclear import through disruption of a conserved nuclear localization signal (NLS). However, it is unknown why FUS accumulates in FTLD-FUS cases. Moreover, it is unclear what causes cytoplasmic FUS in ALS to aggregate and become insoluble. We have discovered a novel mechanism that may explain both phenomena. We find that FUS can be phosphorylated and this event causes the cytoplasmic redistribution of FUS in multiple cells including human astrocytes and neurons. In particular, we find the DNA-damage, caused by chemical toxins, is a potent inducer of FUS phosphorylation. Furthermore, DNA-damage also causes cytoplasmic accumulation of EWS, TAF15, and TRN, which mimics a unique aspect of FTLD-FUS pathology. Preliminary evidence suggests that phosphorylation of FUS leads to increased amounts of FUS in the cytoplasm by disrupting a novel nuclear localization signal in the N-terminus. Consistent with this mechanism, we find that a FUS phospho-mimetic accumulates in the cytoplasm and forms aggregates. These aggregates co-label with markers of stress granules (SGs), RNA/protein granules that have been linked to the formation of inclusions in ALS-FUS and other forms of neurodegeneration. We theorize that cytoplasmic phosphorylated FUS can cause disease through a toxic gain of function by inducing granules that sequester RNA and RNA-binding proteins, impeding normal function. Importantly, we find that phosphorylated FUS occurs in the biochemically insoluble fraction of brains of human and mice with FUS inclusions. Finally, we find a large increase in -H2AX, a marker of DNA damage, in FTLD-FUS brains, supporting the idea that DNA damage and phosphorylation of FUS is a key component of disease pathogenesis. In this proposal we focus on the hypothesis that double-strand DNA damage induces phosphorylation of FUS by the DNA-dependent protein kinase (DNA-PK) causing FUS accumulation in the cytoplasm by impairing nuclear import. We will test this hypothesis by 1) Defining the kinase and types of DNA damage responsible for FUS phosphorylation, 2) Determining how phosphorylation of FUS causes cytoplasmic translocation and affects function, and 3) Determining the role of FUS phosphorylation in neurodegeneration. This research will provide insight into how FUS accumulation causes neurodegeneration and inform drug development strategies for ALS and FTLD. Our data suggest that methods to prevent FUS from forming pathogenic RNA/stress granules, potentially through modulation of the DNA- repair pathway or DNA-PK, may yield treatments for these devastating neurodegenerative diseases.

 描述（由适用提供）：肉瘤（FUS）中的融合是位于核中的无处不在的多功能RNA结合蛋白（RBP）。神经元细胞质中FUS的异常和致病性聚集定义肌萎缩性侧面硬化症（ALS）和额叶Lobar变性（FTLD）的亚型，称为FTLD-FUS或ALS-FUS。 ALS-FUS病例是由FUS基因突变引起的。这些情况下，FUS的积累被认为是由于突变引起的长期增加的胞质FUS驱动的，这些突变通过破坏保守的核定位信号（NLS）而减少核进口的突变。但是，尚不清楚为什么FUS在FTLD-FUS情况下积聚。此外，目前尚不清楚是什么原因导致ALS中的细胞质Fus聚集并变得不溶性。我们发现了一种新的机制，发现FUS可以磷酸化，并且该事件导致多个细胞中FUS的细胞质重新分布，包括人星形细胞和神经元。特别是，我们发现由化学毒素引起的DNA破坏是FUS磷酸化的潜在诱导剂。此外，DNA破坏还会引起EWS，TAF15和TRN的细胞质积累，这模仿了FTLD-FUS病理学的独特方面。初步证据表明，通过破坏N末端中新型的核定位信号，FUS的磷酸化导致细胞质中的FUS增加。与这种机制一致，我们发现FUS磷酸模拟物在细胞质中积聚并形成聚集体。这些聚集体与应力颗粒（SG），RNA/蛋白颗粒的标记共同标记，这些颗粒与ALS和其他形式的神经变性生成中的夹杂物形成有关。我们理论上，细胞质磷酸化的FUS可以通过诱导的颗粒造成的功能增益来引起疾病，从而隔离RNA和RNA结合蛋白，从而阻碍正常功能。重要的是，我们发现磷酸化的FU发生在人和小鼠带有FUS夹杂物的大脑的生化不溶性中。最后，我们发现在FTLD-FUS大脑中，DNA损伤的标志物的-H2AX大幅增加，这支持了DNA损伤和FUS的磷酸化是疾病发病机理的关键组成部分。在此提案中，我们集中于以下假设：双链DNA损伤通过DNA依赖性蛋白激酶（DNA-PK）诱导FUS的磷酸化，从而通过损害核进口而导致FUS积累。我们将通过1）定义负责磷酸化的DNA损伤的激酶和类型来检验该假设。2）确定FUS的磷酸化如何导致细胞质易位并影响功能，以及3）确定FUS磷酸化在NeuroDegeneration中的作用。这项研究将提供有关FUS积累如何引起神经变性并为ALS和FTLD提供药物开发策略的见解。我们的数据表明，防止FU的方法可能通过调节DNA修复途径或DNA-PK形成致病性RNA/应激颗粒，可能会产生这些毁灭性神经退行性疾病的处理。