Molecular Origins of Neurodegeneration through Force Detangling of Toxic RNA

通过强制解开有毒 RNA 导致神经退行性变的分子起源

• 批准号: 10667873
• 负责人: Christian Kaiser
• 金额: $ 24.17万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-03-15 至 2025-02-28
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10667873
• 关键词: Address; Affect; Amyotrophic Lateral Sclerosis; Base Pairing; Binding Proteins; Biological; Biological Assay; Brain; CAG repeat; Cations; Cell Aggregation; Cell Death; Cell Separation; Cells; Cellular biology; Chemicals; Complex; Deterioration; Development; Disease; Future; Gel; Genes; Goals; Growth; Human; Huntington Disease; Huntington gene; In Vitro; Inherited; Kinetics; Knowledge; Lateral; Link; Liquid substance; Measures; Messenger RNA; Methods; Microfluidics; Molecular; Mutation; Nerve Degeneration; Neurodegenerative Disorders; Neurofibrillary Tangles; Neuroglia; Neurons; Nucleotides; Pathogenesis; Pathogenicity; Pathologic; Physical condensation; Process; Proteins; RNA; RNA Probes; RNA-Binding Proteins; RNA-Protein Interaction; Reaction; Research; Risk; Spectrum Analysis; Spinocerebellar Ataxias; Stress; Stretching; Structure; Testing; Time; Toxic effect; Trinucleotide Repeats; Triplet Multiple Birth; Variant; Work; X-Ray Crystallography; base; confocal imaging; flexibility; fluidity; fluorescence imaging; fluorophore; genome wide association study; imaging capabilities; laser tweezer; mechanical properties; method development; molecular imaging; molecular pathology; mouse model; mutant; neurotoxicity; new therapeutic target; novel strategies; optic trap; optic tweezer; optical traps; physical property; polyglutamine; protein complex; single molecule; small molecule; small molecule inhibitor; tool

Nucleotide repeat expansion mutations cause progressive and lethal neurodegenerative diseases such as Huntington’s Disease (HD) and amyotrophic lateral schlerosis (ALS). The molecular reasons for the pathological effects of these mutations remain elusive. Most research has focused on the toxic effects of the mutant proteins. The expanded repeat RNA, however, has more recently been shown to be toxic to neurons and to contribute to disease. In Huntington’s Disease, RNA containing 40 or more CAG repeats is known to associate with itself, condensing into gel-like assemblies or aggregates. Nevertheless, the stability and dynamics of the RNA interactions inside these condensates and aggregates are little known. It is also not known what features of the RNA, if any, correlate with disease propensity. This lack of progress is in part owing to the difficulty of studying interactions that are repetitive, heterogeneous, and changeable. This project will develop a new single molecule tool to study the structures and mechanical properties of abnormal CAG sequences in huntingtin mRNA. Sensitive optical traps will be used to manipulate and unfold single RNA molecules. Integrated real-time confocal fluorescence imaging of fluorophore-tagged RNA with microfluidics control of the reaction components will track the build-up or dissolution of the RNA and protein complexes. The first aim will determine the structure and stability of normal and expanded RNA and compare them with the localization, aggregation, and toxicity of huntingtin RNA and protein in cells. The second aim is to develop a real-time single molecule confocal assay to study the mechanism of aggregation. The third aim is to develop a method for pulling on natural huntingtin RNA complexes or aggregates isolated from cells, to understand how these aberrant structures contribute to pathogenesis. Although this single force-spectroscopy method for studying RNA aggregation is untried, the results have the potential to provide new information on the molecular pathology of nucleotide repeat diseases. In the future, our single molecule tools can be applied to many different RNA repeats and used to test small molecule inhibitors. The long- term goal of this research is to identify a physical signature of huntingtin mRNA interactions that predict neurotoxicity and that can be targeted by new therapies.

核苷酸重复扩展突变导致进行性和致命的神经退行性变 亨廷顿病 (HD) 和肌萎缩侧索硬化症 (ALS) 等疾病。这 这些突变的病理作用的分子原因仍然难以捉摸。大多数研究 重点关注突变蛋白的毒性作用。然而，扩增的重复RNA， 最近被证明对神经元有毒并导致疾病。在 亨廷顿病，含有 40 个或更多 CAG 重复序列的 RNA 已知与 本身凝结成凝胶状组件或聚集体。尽管如此，稳定性和 这些凝聚物和聚集体内部 RNA 相互作用的动力学鲜为人知。它 也不知道 RNA 的哪些特征（如果有的话）与疾病倾向相关。这个缺乏 进展的部分原因在于研究重复性相互作用的困难， 异质的、多变的。该项目将开发一种新的单分子工具来研究 亨廷顿蛋白 mRNA 中异常 CAG 序列的结构和机械特性。 敏感的光陷阱将用于操纵和展开单个 RNA 分子。融合的 通过微流体控制对荧光团标记的 RNA 进行实时共焦荧光成像 反应组分将跟踪 RNA 和蛋白质复合物的形成或溶解。 第一个目标是确定正常和扩增 RNA 的结构和稳定性，以及 将它们与亨廷顿蛋白 RNA 和蛋白质的定位、聚集和毒性进行比较 细胞。第二个目标是开发一种实时单分子共聚焦测定法来研究 聚合机制。第三个目标是开发一种拉动天然亨廷顿蛋白的方法 从细胞中分离出 RNA 复合物或聚集体，以了解这些异常结构是如何发生的 有助于发病。虽然这种用于研究 RNA 的单一力谱方法 聚合未经尝试，结果有可能提供有关 核苷酸重复疾病的分子病理学。未来，我们的单分子工具可以 可应用于许多不同的 RNA 重复序列并用于测试小分子抑制剂。长- 这项研究的长期目标是确定亨廷顿蛋白 mRNA 相互作用的物理特征， 预测神经毒性，并且可以作为新疗法的目标。