Spatiotemporal analysis of TDP-43 toxicity and endolysosomal turnover mechanisms

TDP-43毒性和内溶酶体周转机制的时空分析

• 批准号: 10626673
• 负责人: Sami Barmada
• 金额: $ 39.3万
• 依托单位: UNIVERSITY OF ARIZONA
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-07-01 至 2024-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10626673
• 关键词: ALS patients; Acute; Affect; Alzheimer' s Disease; Amyotrophic Lateral Sclerosis; Biological Assay; Cells; Cessation of life; Characteristics; Chemicals; Chronic; Complex; DNA; Data; Defect; Degradation Pathway; Development; Diagnosis; Disease; Dot Immunoblotting; Epitopes; Event; Exhibits; Frontotemporal Dementia; Gene Expression; Genes; Genetic; Genetic Screening; Genome engineering; Goals; Grant; Homeostasis; Human; In Situ; Inclusion Body Myositis; Label; Lead; Life Expectancy; Link; Maps; Measurement; Mediating; Membrane; Messenger RNA; Modeling; Molecular Chaperones; Monitor; Motor Neurons; Mutation; Nature; Nerve Degeneration; Neurodegenerative Disorders; Neurons; Nuclear; Nuclear Export; Nuclear RNA; Optics; Paralysed; Pathogenesis; Pathologic; Pathology; Pathway interactions; Patients; Phenotype; Physiologic pulse; Physiological; Prosencephalon; Proteins; Quality of life; RNA; RNA Splicing; RNA metabolism; RNA-Binding Proteins; Reporter; Role; Solubility; Sorting - Cell Movement; Supporting Cell; System; TDP-43 aggregation; Testing; Therapeutic; Time; Toxic effect; Translations; Ubiquitination; Yeasts; base; drug candidate; effective therapy; frontotemporal lobar dementia-amyotrophic lateral sclerosis; fused in sarcoma; gain of function; innovation; insight; limbic-predominant age-related TDP-43 encephalopathy; loss of function; motor neuron degeneration; neuron loss; neuronal survival; new therapeutic target; novel; novel strategies; novel therapeutic intervention; nucleocytoplasmic transport; optogenetics; protein TDP-43; protein metabolism; proteostasis; response; ribosome profiling; small molecule; spatiotemporal; stress granule; targeted treatment; therapy development; tool; transcriptome sequencing; ubiquitin-protein ligase; yeast genetics

Amyotrophic Lateral Sclerosis (ALS) is a devastating disease in which progressive degeneration of motor neurons leads to paralysis, usually resulting in death 2-5 years after diagnosis. No therapies exist that significantly increase quality of life or life expectancy. Mutations in >30 genes are linked to ALS onset, albeit >90% of all ALS cases are sporadic. However, a unifying cellular hallmark in >95% of all ALS cases is the cytoplasmic mis-localization, accumulation, and aggregation of the nuclear RNA binding protein TDP-43 (TAR DNA/RNA binding protein 43) in motor neurons and support cells. Similar TDP-43 pathology is observed in other neurodegenerative diseases, including in forebrain neurons of ~50% of Frontotemporal dementia patients (FTD). TDP-43 pathology confers a toxicity to neurons, but the nature of this toxicity remains fiercely debated. Loss of nuclear function (LOF) and gain of cytoplasmic function (GOF) mechanisms have been proposed, though separating such mechanisms and identifying the earliest impacts of TDP-43 pathology has remained elusive. Regardless, a therapeutic strategy that has shown promise in some ALS models is promoting the degradation of cytoplasmic TDP-43. Recently, cytoplasmic TDP-43 was shown to be degraded via a novel endolysosomal pathway, which, when induced, suppresses TDP-43 toxicity. However, understanding of this degradation pathway remains limited. Key gaps in understanding include determining, in an ALS-relevant neuronal model, the earliest and most disease-relevant impacts of TDP-43 pathology and defining how endolysosomal TDP-43 degradation occurs. The aims of this grant are: 1.) Establish a novel endogenous TDP-43 reporter system in neurons that allows precise control of TDP-43 abundance and cellular localization via small molecule and optogenetic means. Using this system, the impacts of altered TDP-43 levels and localization on TDP-43 itself, ALS phenotypes and gene expression, focusing on RNA abundance and translation, will be examined. 2.) Test an endolysosomal degradation model involving TDP-43 ubiquitination and endosomal membrane invagination in neurons using an optical pulse labelling approach. TDP-43 degradation mechanisms will also be defined in patient-derived ALS models using similar means. Finally, a novel high throughput yeast dot-blot assay will be used to identify genetic and chemical regulators of TDP-43 and Fused in Sarcoma (FUS) abundance, which is also implicated in ALS and FTD pathology. This grant is innovative in that a novel approach to exert spatiotemporal control of TDP-43 expression, which promises separation of TDP43 LOF and GOF toxicity effects, and a means to identify regulators of TDP-43 and FUS abundance via dot blot, are proposed. Finally, mechanistically defining endolysosomal-based means of cytoplasmic TDP-43 degradation promises new basic insight into proteostasis for TDP-43 and other substrates. In summary, TDP-43 and FUS are logical entry points for the study of ALS and FTD. This grant will enhance disease understanding and may lead to identification of new therapeutic targets with broad patient applicability.

肌萎缩性外侧硬化症（ALS）是一种毁灭性疾病，运动​​的进行性变性 神经元导致瘫痪，通常在诊断后2 - 5年导致死亡。没有疗法存在 显着提高生活质量或预期寿命。 > 30个基因中的突变与ALS发作有关，尽管 在所有ALS病例中，> 90％是零星的。但是，在所有ALS案件中，> 95％的统一的蜂窝标志是 核RNA结合蛋白TDP-43的细胞质错误定位，积累和聚集（TAR 运动神经元和支持细胞中的DNA/RNA结合蛋白43）。在其他中观察到类似的TDP-43病理 神经退行性疾病，包括约50％额颞痴呆患者（FTD）的前脑神经元中。 TDP-43病理学赋予了对神经元的毒性，但这种毒性的性质仍然激烈争论。损失 已经提出了核功能（LOF）和细胞质功能（GOF）机制的增益 分离这种机制并确定TDP-43病理的最早影响仍然难以捉摸。 无论如何，在某些ALS模型中表现出希望的治疗策略正在促进降解 细胞质TDP-43。最近，细胞质TDP-43被证明是通过新型内溶液体降解的 途径，当诱导时，它会抑制TDP-43的毒性。但是，了解这种退化 路径仍然有限。理解的关键差距包括在与ALS相关的神经元模型中确定， TDP-43病理学最早，最重要的影响，并定义了内溶性TDP-43 降解发生。该赠款的目的是：1。）在 神经元可以通过小分子和 光遗传学手段。使用此系统，TDP-43级别和TDP-43本身的定位变化的影响， 将检查ALS表型和基因表达，重点是RNA丰度和翻译。 2.）测试 涉及TDP-43泛素化和内体膜的内溶性降解模型 在神经元中使用光脉冲标记方法。 TDP-43降解机制也将在 使用类似方法的患者来源的ALS模型。最后，一种新颖的高吞吐量酵母点斑点分析将是 用于鉴定TDP-43的遗传和化学调节剂并在肉瘤（FUS）丰度中融合 也与ALS和FTD病理有关。这笔赠款具有创新性，因为这种新颖的方法 TDP-43表达的时空控制有望分离TDP43 LOF和GOF毒性 提出了效果，以及通过DOT印迹鉴定TDP-43和FUS丰度调节剂的方法。最后， 机械上定义基于内溶血的细胞质TDP-43降解的方法有望新基本 深入了解TDP-43和其他底物的蛋白质量。总而言之，TDP-43和FUS是逻辑入口点 用于研究ALS和FTD。该赠款将增强疾病的理解，并可能导致对 具有广泛患者适用性的新治疗靶标。