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（由申请方提供）：融合肉瘤（FUS）是一种位于细胞核中的普遍存在的多功能RNA结合蛋白（RBP）。FUS在神经元细胞质中的异常和致病性聚集定义了肌萎缩侧索硬化（ALS）和额颞叶变性（FTLD）的亚型，称为FTLD-FUS或ALS-FUS。肌萎缩侧索硬化-肌萎缩侧索硬化病例是由肌萎缩侧索硬化基因突变引起的。在这些情况下，FUS的积累被认为是由细胞质FUS的长期增加驱动的，细胞质FUS的长期增加是由突变引起的，突变通过破坏保守的核定位信号（NLS）来减少核输入。然而，尚不清楚FUS在FTLD-FUS病例中蓄积的原因。此外，目前还不清楚是什么原因导致ALS细胞质FUS聚集并变得不溶。我们发现了一种新的机制，可以解释这两种现象。我们发现FUS可以被磷酸化，并且这一事件导致FUS在包括人星形胶质细胞和神经元在内的多种细胞中的胞质重新分布。特别是，我们发现由化学毒素引起的DNA损伤是FUS磷酸化的有力诱导剂。此外，DNA损伤还导致EWS、TAF 15和TRN的细胞质积累，这模拟了FTLD-FUS病理学的独特方面。初步证据表明，FUS的磷酸化通过破坏N-末端的新的核定位信号而导致细胞质中FUS的量增加。与这种机制一致，我们发现FUS磷酸模拟物在细胞质中积累并形成聚集体。这些聚集体与应激颗粒（SG）、RNA/蛋白质颗粒的标记物共标记，所述应激颗粒、RNA/蛋白质颗粒与ALS-FUS中的包涵体的形成和其他形式的神经变性有关。我们的理论是，胞质磷酸化FUS可以通过诱导螯合RNA和RNA结合蛋白的颗粒，阻碍正常功能，通过毒性获得功能引起疾病。重要的是，我们发现磷酸化的FUS发生在具有FUS内含物的人和小鼠的脑的生化不溶性部分中。最后，我们发现FTLD-FUS脑中DNA损伤的标志物β-H2 AX大量增加，支持DNA损伤和FUS的磷酸化是疾病发病机制的关键组成部分的观点。在这个建议中，我们专注于双链DNA损伤诱导FUS的磷酸化的DNA依赖性蛋白激酶（DNA-PK），导致FUS积累在细胞质中，通过损害核输入的假设。我们将通过以下方式检验这一假设：1）确定负责FUS磷酸化的激酶和DNA损伤类型，2）确定FUS磷酸化如何引起细胞质易位并影响功能，3）确定FUS磷酸化在神经变性中的作用。这项研究将深入了解FUS积累如何导致神经变性，并为ALS和FTLD的药物开发策略提供信息。我们的数据表明，可能通过调节DNA修复途径或DNA-PK来防止FUS形成致病性RNA/应激颗粒的方法可以产生对这些破坏性神经退行性疾病的治疗。