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

项目总结 基本上所有细胞的正常功能部分归因于空间定义的区域，这些区域是 由特定的蛋白质填充以执行特定的活动。神经元，由于它们拉长的大小和 形态，在导航蛋白质定位时必须处理大距离的轴突和 树枝状结构。这个问题可以通过将RNA分子输送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，会立即产生正确定位的蛋白质。重要的是 这一关键过程的证据是与其调控不当相关的多种神经系统疾病。这个 RNA结合蛋白TDP-43已知与RNA定位有关，TDP-43的突变是 肌萎缩侧索硬化症(ALS)。然而，依赖TDP-43进行本地化的RNA， TDP-43如何识别这些RNA并促进它们的运输，以及它们的表型结果 本地化错误目前都是未知的。这意味着导致肌萎缩侧索硬化症的整个监管模式 病理可能还未被发现。 在这里，我们建议使用我们在亚细胞转录学、干细胞方面的综合专业知识 分化和活细胞显微镜来研究这些关键问题并提供对ALS的新见解 可能导致新的治疗模式的疾病机制。为此，我们创作了一部小说 在全基因组范围内监测神经元RNA错误定位的技术，使我们能够识别 需要功能正常的TDP-43才能正常运输。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，我们将使用这项技术来探索TDP-43介导的RNA在功能性人类中的定位 运动神经元。我们将在工程TDP-43缺失的运动神经元、运动神经元中识别定位错误的RNA 表达肌萎缩侧索硬化症相关的TDP-43突变体和肌萎缩侧索硬化症患者的运动神经元。我们将使用这个 用于推导定义依赖于TDP-43的转录本的RNA序列要求的信息 运输和测试他们的必要性和充分性与记者的成绩单。 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 在活的果蝇脑中。我们将使用这种方法来可视化TDP-43介导的RNA在活脑中的运输 并了解其动态和监管。果蝇TDP-43突变体的表型与 见于肌萎缩侧索硬化症患者。然而，到目前为止，将细胞或生物表型归因于 肌萎缩侧索硬化症或任何其他神经系统疾病的特定转录本的错误定位缺失。这可能是在 部分原因是RNA定位经常被忽视，而在疾病中错误定位的转录本 州政府目前还不得而知。为了解决这个问题，我们将在果蝇大脑中错位定位特定的转录本 通过去除转录本中TDP-43识别的序列来依赖TDP-43进行运输。我们会 然后问这些果蝇是否表现出TDP-43突变体的运动和生理缺陷 苍蝇和肌萎缩侧索硬化患者。 总体而言，我们打算定义调控不当的分子机制和ALS之间的新联系 患者表型，从而开辟了研究和治疗开发的新途径。

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.