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.

核苷酸重复扩增突变导致进行性和致命的神经退行性变 疾病如亨廷顿氏病（HD）和肌萎缩性侧索硬化症（ALS）。的 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 都集中在突变蛋白质的毒性作用上。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 亨廷顿氏病，已知含有40个或更多CAG重复的RNA与 本身，冷凝成凝胶状组件或聚集体。然而，稳定性和 这些凝聚物和聚集体内部的RNA相互作用的动力学知之甚少。它 也不知道RNA的哪些特征（如果有的话）与疾病倾向相关。这种缺乏 部分原因是研究重复的相互作用的困难， 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mRNA中异常CAG序列的结构和力学性质。 灵敏的光学陷阱将用于操纵和展开单个RNA分子。集成 用微流体控制荧光团标记的RNA的实时共聚焦荧光成像 反应组分将跟踪RNA和蛋白质复合物的积累或溶解。 第一个目标是确定正常和扩增RNA的结构和稳定性， 将它们与亨廷顿蛋白RNA和蛋白质在细胞中的定位、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分析来研究 聚集机制。第三个目标是开发一种方法， 从细胞中分离的RNA复合物或聚集体，以了解这些异常结构 有助于发病机制。虽然这种研究RNA的单力光谱方法 聚合是未经尝试的，结果有可能提供新的信息， 核苷酸重复疾病的分子病理学。在未来，我们的单分子工具可以 可以应用于许多不同的RNA重复序列，并用于测试小分子抑制剂。很长的- 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，并可以通过新的治疗靶向。