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.

项目摘要 基本上所有细胞的正常功能部分是由于空间上限定的区域， 由特定的蛋白质组成以执行特定的活动。神经元，由于其细长的尺寸和 形态学，必须处理大的距离时，导航蛋白定位关于轴突和 树突这个问题通过将RNA分子运送到神经元内的特定位置来解决。当 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。的 已知RNA结合蛋白TDP-43参与RNA定位，并且TDP-43中的突变是RNA结合蛋白TDP-43的突变。 与肌萎缩侧索硬化症（ALS）相关。然而，依赖TDP-43定位的RNA， TDP-43如何识别这些RNA并促进它们的转运，以及它们的表型后果。 错误定位目前都是未知的。这意味着导致ALS的整个监管模式 病理可能未被发现。 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，我们开发了一种新的 技术，以监测神经元RNA的错误定位转录组范围内，使我们能够确定转录本， 需要功能正常的TDP-43来进行适当的运输。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TDP-43介导的RNA定位在功能性人类 运动神经元我们将在工程化的TDP-43缺失的运动神经元，运动神经元， 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 信息，以获得定义转录本的RNA序列要求，转录本依赖于TDP-43， 运输和测试其必要性和充分性与记者成绩单。 我们还开发了一种技术，用于成像单个RNA和蛋白质分子的运输 在果蝇的大脑中我们将使用这种方法来可视化TDP-43介导的RNA在活脑中的转运 并了解它的动态和调节。果蝇TDP-43突变体表现出类似于 在ALS患者中观察到。然而，到目前为止，将细胞或生物体表型归因于 ALS或任何其他神经系统疾病的特异性转录物的错误定位缺失。这可能是由于在 部分原因是RNA定位经常被忽视， 国家不知道。为了解决这个问题，我们将在果蝇大脑中错误定位特定的转录本， 依赖TDP-43通过去除转录物中TDP-43识别的序列进行转运。我们将 然后询问这些果蝇是否表现出TDP-43突变体的运动和生理缺陷， 苍蝇和ALS患者。 总的来说，我们打算确定失调的分子机制和ALS之间的新联系， 患者表型，从而开辟了研究和治疗发展的新途径。