Mechanistic dissection of novel regulators of TDP-43 aggregation identified in a genome-wide CRISPR-Cas9 knockout screen

全基因组 CRISPR-Cas9 敲除筛选中发现的 TDP-43 聚集新型调节因子的机制剖析

• 批准号: 10404620
• 负责人: Katelyn Sweeney
• 金额: $ 3.42万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-06-01 至 2023-05-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10404620
• 关键词: ALS patients; Address; Affect; Alzheimer' s Disease; Amyotrophic Lateral Sclerosis; Astrocytes; Binding; Biology; CRISPR/Cas technology; Cell Line; Cells; Cellular Morphology; Client; Coculture Techniques; Collaborations; Coupled; Cyclic AMP-Dependent Protein Kinases; Data; Development; Diffuse; Disease; Disease Progression; Dissection; Double-Stranded RNA; Enzyme-Linked Immunosorbent Assay; Fluorescence-Activated Cell Sorting; Generations; Genes; Genetic Predisposition to Disease; Global Change; Goals; Homeostasis; Immune; Immune response; Immune signaling; Importins; In Vitro; Incidence; Interferon-alpha; Knock-out; Link; Mass Spectrum Analysis; Measures; Mediating; Messenger RNA; Methyltransferase; Microglia; Mind; Modeling; Modification; Molecular Chaperones; Motor Neurons; Mutation; Neurodegenerative Disorders; Nuclear; Nuclear Export; Pathogenesis; Pathologic; Pathology; Pathway interactions; Phase; Physiologic pulse; Prevalence; Proteins; Quality Control; RNA; RNA Binding; RNA methylation; RNA-Binding Proteins; Regulation; Reporter; Ribosomal RNA; Role; Signal Pathway; Signal Transduction; Small RNA; Stains; Structure; Testing; Therapeutic; Time; Toxic effect; Translations; Western Blotting; Work; Yeast Model System; Yeasts; base; crosslinking and immunoprecipitation sequencing; disease-causing mutation; follow-up; frontotemporal degeneration; genome-wide; in vitro testing; interest; novel; prevent; protein TDP-43; protein kinase R; shape analysis; therapeutically effective

Project Summary The mislocalization and aggregation of RNA-binding proteins (RBP) is a pathological hallmark of Amyotrophic Lateral Sclerosis (ALS). The RBP TAR DNA-binding protein 43 (TDP-43) is of particular interest, as pathological aggregation of TDP-43 is observed in nearly 97% of ALS patients, despite the fact that disease-causing mutations in TDP-43 explain fewer than 5% of disease incidence. This observation suggests that misregulation or mutation of other genes converge on TDP-43 pathology, however it remains poorly understood how the aggregates arise or whether they can be reversed or prevented. To this end, we have developed Pulse-Shape Analysis-based aggregation reporters for the TDP-43 that, when coupled to fluorescence activated cell sorting, quantifies aggregation at the single cell level. This reporter was leveraged against a genome-wide CRISPR- Cas9 knockout screen to identify regulators of TDP-43 aggregation. Reassuringly, this screen revealed known interactors and pathways involved in TDP-43 regulation and pathology. Additionally, this work revealed several novel proteins not previously implicated in TDP-43 biology or aggregation. Initial follow up on one top hit, SRRD, revealed that this protein reduces TDP-43 aggregation in a mammalian aggregation model, and reduces TDP- 43-associated toxicity in a yeast model, indicating that these genes indeed modify TDP-43 aggregation. The goal of this proposal is to mechanistically dissect how these novel regulators modify TDP-43 aggregation. Using an induced motor neuron model of TDP-43 aggregation, yeast toxicity models, and in vitro studies, I will assess the interactors, signaling pathways, and mode of TDP-43 modification for the following four genes: METTL5, EIF2AK2, XPO4, and SRRD. This work will not only reveal the function of uncharacterized proteins, but dissect their heretofore unknown mechanisms in regulating TDP-43 aggregation. Through better understanding of how TDP-43 aggregation is regulated, the field can begin to answer questions regarding the role of TDP-43 aggregation in disease pathogenesis and progression. By understanding the regulatory network of underlying TDP-43 aggregation, we can begin to therapeutically modulate the pathways that contribute to disease.

项目摘要 RNA结合蛋白(RBP)的错误定位和聚集是肌营养不良的一个病理标志 侧索硬化(ALS)。RBP TAR DNA结合蛋白43(TDP-43)是一种特殊的病理性蛋白 在近97%的ALS患者中观察到TDP-43的聚集，尽管事实上 TDP-43的突变解释了不到5%的疾病发病率。这一观察表明，监管不善 或其他基因突变汇聚在TDP-43的病理上，然而仍然鲜为人知 聚集出现了，或者它们是否可以逆转或防止。为此，我们开发了脉冲形 TDP-43的基于分析的聚集报告，当与荧光激活的细胞分选相结合时， 在单个单元级别量化聚合。这位记者被用来对抗全基因组的CRISPR- Cas9基因敲除筛选以识别TDP-43聚集的调节因子。令人放心的是，这个屏幕显示了已知的 参与TDP-43调控和病理的相互作用因子和通路。此外，这项工作揭示了几个 以前未涉及TDP-43生物或聚集的新蛋白质。对一首热门歌曲SRRD的初步跟进， 在哺乳动物聚集模型中，该蛋白降低TDP-43的聚集，并降低TDP-43的聚集。 酵母模型中的43相关毒性，表明这些基因确实改变了TDP-43的聚集。 该提案的目标是机械地剖析这些新型调节剂是如何修改TDP-43的 聚合。使用TDP-43聚集的诱导运动神经元模型、酵母菌毒性模型和体外 研究中，我将评估以下四个方面的相互作用、信号通路和TDP-43修饰模式 基因：METTL5、EIF2AK2、XPO4和SRRD。这部作品不仅将揭示未刻画的功能 蛋白质，但剖析了它们迄今未知的调节TDP-43聚集的机制。通过更好的 了解TDP-43聚集是如何调节的，该领域可以开始回答关于 TDP-43聚集在疾病发生发展中的作用。 通过了解潜在的TDP-43聚合的调控网络，我们可以开始 从治疗上调节导致疾病的途径。