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.

肌萎缩侧索硬化症 (ALS) 是一种破坏性疾病，患者运动神经进行性退化 神经元导致瘫痪，通常导致诊断后 2-5 年内死亡。不存在任何治疗方法 显着提高生活质量或预期寿命。尽管超过 30 个基因的突变与 ALS 发病有关 >90% 的 ALS 病例是散发的。然而，超过 95% 的 ALS 病例的一个统一的细胞特征是 核 RNA 结合蛋白 TDP-43 (TAR 运动神经元和支持细胞中的 DNA/RNA 结合蛋白 43)。在其他疾病中也观察到类似的 TDP-43 病理学 神经退行性疾病，包括约 50% 额颞叶痴呆患者 (FTD) 的前脑神经元。 TDP-43 病理学赋予神经元毒性，但这种毒性的性质仍然存在激烈争论。损失 尽管已经提出了核功能（LOF）和细胞质功能获得（GOF）机制 分离这些机制并确定 TDP-43 病理学的最早影响仍然难以捉摸。 无论如何，在某些 ALS 模型中显示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 被证明可通过一种新型内溶酶体降解 途径，当被诱导时，抑制 TDP-43 毒性。然而，对这种退化的理解 途径仍然有限。理解方面的主要差距包括在 ALS 相关神经元模型中确定， TDP-43 病理学的最早和最相关的影响以及定义内溶酶体 TDP-43 发生降解。这笔赠款的目的是： 1.) 建立一种新型内源性 TDP-43 报告系统 神经元可以通过小分子精确控制 TDP-43 丰度和细胞定位 光遗传学手段。使用该系统，改变 TDP-43 水平和定位对 TDP-43 本身的影响， 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 泛素化和内体膜内陷的内溶酶体降解模型 使用光脉冲标记方法在神经元中进行标记。 TDP-43 降解机制也将在 使用类似方法衍生的患者 ALS 模型。最后，一种新型的高通量酵母斑点印迹测定将是 用于鉴定 TDP-43 和肉瘤融合 (FUS) 丰度的遗传和化学调节因子， 也与 ALS 和 FTD 病理有关。这笔赠款的创新之处在于，它采用了一种新颖的方法来发挥作用 TDP-43 表达的时空控制，有望分离 TDP43 LOF 和 GOF 毒性 提出了一种通过斑点印迹识别 TDP-43 和 FUS 丰度调节因子的方法。最后， 机械地定义基于内溶酶体的细胞质 TDP-43 降解方法有望提供新的基础 深入了解 TDP-43 和其他底物的蛋白质稳态。综上所述，TDP-43和FUS是逻辑切入点 用于 ALS 和 FTD 的研究。这笔赠款将增进对疾病的了解，并可能导致识别 具有广泛患者适用性的新治疗靶点。