Mechanistic analysis of TDP-43-mediated RNA localization in neurons and its misregulation in ALS

TDP-43介导的神经元RNA定位及其在ALS中的错误调节的机制分析

• 批准号: 10659532
• 负责人: Holger A. Russ
• 金额: $ 59.33万
• 依托单位: UNIVERSITY OF COLORADO DENVER
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-19 至 2028-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10659532
• 关键词: ALS pathology; ALS patients; Affect; Amyotrophic Lateral Sclerosis; Axon; Biological Assay; Brain; Cells; Data; Defect; Dendrites; Disease; Drosophila genus; Engineering; Ensure; Eukaryotic Cell; FMRP; Face; Fragile X Syndrome; Genes; Human; Image; Imaging Techniques; Knockout Mice; Label; Language; Learning; Length; Location; Mediating; Messenger RNA; Microscopy; Modality; Molecular; Molecular Target; Monitor; Morphology; Motor Neurons; Mus; Mutation; Neurons; Orthologous Gene; Patients; Phenotype; Physiological; Process; Proteins; Qualifying; RNA; RNA Sequences; RNA Transport; RNA-Binding Proteins; Regulation; Reporter; Signal Transduction; Site; Sorting; Spinal Muscular Atrophy; Techniques; Testing; Transcript; Translating; Visualization; disease phenotype; experimental study; fly; frontotemporal lobar dementia amyotrophic lateral sclerosis; insight; live cell microscopy; meter; molecular phenotype; mutant; nervous system disorder; novel; protein TDP-43; protein function; research and development; single molecule; stem cell biology; stem cell differentiation; therapeutic development; transcriptome; transcriptomics

PROJECT SUMMARY The proper functioning of essentially all cells is due in part to spatially defined regions that are populated by specific proteins to perform specific activities. Neurons, due to their elongated size and morphology, must deal with large distances when navigating protein localization with regard to axons and dendrites. This problem is solved by transporting RNA molecules to specific locations within neurons. When these RNA molecules are translated, they immediately produce correctly localized proteins. The importance of this key process is evidenced by multiple neurological diseases that are associated with its misregulation. The RNA-binding protein TDP-43 is known to be involved in RNA localization, and mutations in TDP-43 are associated with Amyotrophic Lateral Sclerosis (ALS). Yet the RNAs that depend on TDP-43 for localization, how TDP-43 recognizes these RNAs and promotes their transport, and the phenotypic consequences of their mislocalization are all currently unknown. This means that an entire regulatory modality contributing to ALS pathology may be undiscovered. Here, we propose to use our combined expertises in subcellular transcriptomics, stem cell differentiation, and live cell microscopy to engage these critical problems and provide novel insights into ALS disease mechanisms that likely will result in novel treatment modalities. To do so we have developed a novel technique to monitor neuronal RNA mislocalization transcriptome-wide, allowing us to identify transcripts that need functional TDP-43 for proper transport. By combining this approach with our expertise in stem cell differentiation, we will use this technique to probe TDP-43-mediated RNA localization in functional human motor neurons. We will identify mislocalized RNAs in engineered TDP-43-null motor neurons, motor neurons expressing ALS-associated TDP-43 mutants, and motor neurons derived from ALS patients. We will use this information to derive the RNA sequence requirements that define transcripts that depend on TDP-43 for transport and test their necessity and sufficiency with reporter transcripts. We have also developed a technique for imaging the transport of single RNA and protein molecules in live Drosophila brains. We will use this approach to visualize TDP-43-mediated RNA transport in live brains and learn about its dynamics and regulation. Drosophila TDP-43 mutants display phenotypes similar to those seen in ALS patients. However, as of now data attributing cellular or organismal phenotypes to the mislocalization of specific transcripts for ALS or any other neurological disease is missing. This may be due in part to the fact that RNA localization is often overlooked and the transcripts that are mislocalized in disease states are not known. To engage this problem, we will mislocalize specific transcripts in Drosophila brains that depend on TDP-43 for transport by removing sequences that TDP-43 recognizes within the transcript. We will then ask if these flies display the locomotor and physiological defects that characterize both TDP-43 mutant flies and ALS patients. Overall, we intend to define new connections between misregulated molecular mechanisms and ALS patient phenotypes, thus opening up new avenues of research and therapeutic development.

项目概要 基本上所有细胞的正常功能部分归因于空间定义的区域 由特定蛋白质填充以执行特定活动。神经元由于其细长的尺寸和 形态学，在导航关于轴突和蛋白质的定位时必须处理长距离 树突。这个问题可以通过将 RNA 分子运输到神经元内的特定位置来解决。什么时候 这些RNA分子被翻译后，它们立即产生正确定位的蛋白质。的重要性 与其失调相关的多种神经系统疾病证明了这一关键过程。这 RNA 结合蛋白 TDP-43 已知参与 RNA 定位，TDP-43 的突变 与肌萎缩侧索硬化症（ALS）有关。然而，依赖 TDP-43 进行定位的 RNA， TDP-43 如何识别这些 RNA 并促进它们的运输，以及它们的表型后果 错误定位目前都是未知的。这意味着有助于 ALS 的整个监管模式 病理学可能未被发现。 在这里，我们建议利用我们在亚细胞转录组学、干细胞 分化和活细胞显微镜来解决这些关键问题并为 ALS 提供新的见解 疾病机制可能会导致新的治疗方式。为此，我们开发了一部小说 监测神经元 RNA 转录组范围内错误定位的技术，使我们能够识别转录本 需要功能性 TDP-43 才能正确运输。通过将这种方法与我们在干细胞方面的专业知识相结合 分化，我们将使用这种技术来探测功能性人类中 TDP-43 介导的 RNA 定位 运动神经元。我们将在工程 TDP-43 缺失的运动神经元、运动神经元中识别错误定位的 RNA 表达 ALS 相关的 TDP-43 突变体和来自 ALS 患者的运动神经元。我们将使用这个 导出 RNA 序列要求的信息，这些要求定义了依赖于 TDP-43 的转录本 运输并用记者笔录测试其必要性和充分性。 我们还开发了一种对单个 RNA 和蛋白质分子的运输进行成像的技术 在活的果蝇大脑中。我们将使用这种方法来可视化活体大脑中 TDP-43 介导的 RNA 运输 并了解其动态和监管。果蝇 TDP-43 突变体表现出与那些相似的表型 见于 ALS 患者。然而，到目前为止，将细胞或有机体表型归因于 ALS 或任何其他神经系统疾病的特定转录本的错误定位缺失。这可能是由于 部分原因是 RNA 定位经常被忽视，并且转录本在疾病中定位错误 状态未知。为了解决这个问题，我们将果蝇大脑中的特定转录本定位错误 通过去除转录物中 TDP-43 识别的序列，依赖 TDP-43 进行运输。我们将 然后询问这些果蝇是否表现出 TDP-43 突变体特有的运动和生理缺陷 苍蝇和 ALS 患者。 总体而言，我们打算定义失调分子机制与 ALS 之间的新联系 患者表型，从而开辟新的研究和治疗开发途径。