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

基本信息

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
项目类别：
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

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

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项目摘要

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.

核苷酸重复膨胀突变引起进行性和致命神经退行性诸如亨廷顿氏病（HD）和肌萎缩性侧面造成病（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。大多数研究已关注突变蛋白的毒性作用。但是，扩展的重复RNA 最近，最近证明对神经元有毒并为疾病做出贡献。在亨廷顿氏病，已知包含40或更多CAG重复序列的RNA与本身，凝结成凝胶状的组件或聚集体。然而，稳定性和这些冷凝物和聚集体内RNA相互作用的动力学鲜为人知。它也不知道RNA的特征（如果有的话）与疾病改善相关。这个缺乏进步的部分原因是研究重复的相互作用的困难异质，可变。该项目将开发一种新的单分子工具来研究 Huntingtin mRNA中异常CAG序列的结构和机械性能。灵敏的光学陷阱将用于操纵和展开单个RNA分子。融合的用微流体控制的荧光团标记的RNA的实时共聚焦荧光成像反应成分将跟踪RNA和蛋白质复合物的堆积或溶解。第一个目的将确定正常和扩展的RNA的结构和稳定性以及将它们与Huntingtin RNA和蛋白质的定位，聚集和毒性进行比较细胞。第二个目的是开发实时的单分子共聚焦测定，以研究聚集机理。第三个目的是开发一种拉动自然亨廷顿的方法从细胞分离出的RNA复合物或聚集体，以了解这些异常结构如何尽管这种用于研究RNA的单一力谱镜法，但对发病机理的贡献聚合未经尝试，结果有可能提供有关核肽重复疾病的分子病理。将来，我们的单分子工具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。长期这项研究的一项目标是确定亨廷顿mRNA相互作用的物理签名预测神经毒性，可以通过新疗法来靶向。