ESTABLISHING AN ASSAY FOR DETECTING SINGLE TAU FIBRILS

建立单个 TAU 原纤维的检测方法

• 批准号: 9014868
• 负责人: Martin Margittai
• 金额: $ 21.52万
• 依托单位: UNIVERSITY OF DENVER (COLORADO SEMINARY)
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-04-01 至 2018-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9014868
• 关键词: Affect; Alzheimer' s Disease; Amino Acids; Base Sequence; Biological; Biological Assay; Biological Markers; Blood; Brain; Cells; Cerebrospinal Fluid; Complex; Data; Deposition; Diagnosis; Disease; Early Diagnosis; Early Intervention; Electron Microscopy; Environment; Enzyme-Linked Immunosorbent Assay; Enzymes; Extracellular Fluid; Fluorescence Spectroscopy; Foundations; Future; Goals; Growth; Human; Individual; Investigation; Knowledge; Lead; Link; Methodology; Methods; Mission; Molecular; Monitor; Monoclonal Antibodies; Mutation; Neurodegenerative Disorders; Neurofibrillary Tangles; Neurons; Outcome; Pathology; Pathway interactions; Peptide Hydrolases; Preventive; Preventive Intervention; Prions; Protein Engineering; Proteolysis; Protocols documentation; Public Health; Recruitment Activity; Research; Resistance; Sedimentation process; Source; Staging; Structure; Testing; Therapeutic; Therapeutic Intervention; Work; base; brain tissue; diagnostic assay; extracellular; improved; innovation; monomer; neuron loss; novel; novel strategies; pre-clinical; public health relevance; research study; tau Proteins; tau aggregation; tool

 DESCRIPTION (provided by applicant): Currently there are no biomarkers for the earliest preclinical stage of Alzheimer's disease. This constitutes an important problem because future therapeutic interventions will rely on early diagnosis. Tau fibrils are spreading throughout the brain via transfer from one neuron to another. Unless new methods are developed that detect these fibrils, the early stages in the pathology of preclinical Alzheimer's disease will remain largely illusive. The long-term goal is to understand the pathways of Tau propagation and how they can be manipulated for preventive and therapeutic purposes. The objective of this proposal is to use newly developed amplification methodology in conjunction with sequence-based protein design and enzyme-linked immunodetection to detect single Tau fibrils. The central hypothesis is that single Tau fibrils can be detected after repetitive cycles of shearing and growth. This hypothesis has been formulated based on data produced in the applicant's lab that reveal shear-induced amplification of synthetic and brain-derived Tau fibrils. The rationale for this project is that once single fibril sensitivities are obtained, biofluidics can be tested for A diagnosis, and mechanistic studies into the release of Tau fibrils can be initiated. Supported by strong preliminary data the central hypothesis will be tested by pursuit of the following three specific aims. 1) Suppress self-nucleation of Tau monomers through sequence-guided protein design. Select residues in the hydrophobic core of Tau will be individually substituted to suppress self-nucleation, but not growth. Aggregation will be monitored by fluorescence spectroscopy, sedimentation, and electron microscopy. 2) Develop proteolysis-enhanced immunodetection protocols for distinguishing fibrils from monomers. Proteolysis will be used to completely degrade unfolded Tau monomers. The protease resistant core will be captured by monoclonal antibodies and detected by enzyme-linked immunosorbent assays. 3) Amplify Tau fibrils from Alzheimer's disease brain tissue. AD brain tissue will serve as a source of authentic Tau fibrils. Amplification of single Tau fibrils will be achieved by repetitive cycles of shearing nd growth. Immunodetection will be used to enhance sensitivity. The research is innovative, because it uses a new approach to detect single Tau fibrils, namely cyclic amplification combined with sequence-guided protein design and immunodetection. The proposed research is significant because the results will provide a robust protocol for amplifying and detecting single Tau fibrils. Such capability has the potential to lead to new assays for early diagnosis of Alzheimer's disease.

 描述（由申请人提供）：目前没有阿尔茨海默病最早临床前阶段的生物标志物。这构成了一个重要的问题，因为未来的治疗干预将依赖于早期诊断。Tau原纤维通过从一个神经元转移到另一个神经元而遍布整个大脑。除非开发出检测这些纤维的新方法，否则临床前阿尔茨海默病的早期病理学阶段将在很大程度上仍然是虚幻的。长期目标是了解Tau传播的途径以及如何操纵它们以达到预防和治疗目的。本提案的目的是使用新开发的扩增方法结合基于序列的蛋白质设计和酶联免疫检测来检测单个Tau原纤维。中心假设是在剪切和生长的重复循环后可以检测到单个Tau原纤维。该假设是基于申请人实验室中产生的数据制定的，该数据揭示了合成的和脑来源的Tau原纤维的剪切诱导的扩增。该项目的基本原理是，一旦获得单原纤维敏感性，就可以测试生物流体用于A诊断，并且可以启动对Tau原纤维释放的机制研究。在强有力的初步数据的支持下，将通过追求以下三个具体目标来检验中心假设。1)通过序列指导的蛋白质设计抑制Tau单体的自成核。Tau疏水核心中的选择残基将被单独取代以抑制自成核，但不抑制生长。将通过荧光光谱法、沉降法和电子显微镜法监测聚集。2)开发蛋白水解增强的免疫检测方案，用于区分原纤维和单体。蛋白水解将用于完全降解未折叠的Tau单体。蛋白酶抗性核心将被单克隆抗体捕获，并通过酶联免疫吸附试验进行检测。3)放大阿尔茨海默病脑组织中的Tau纤维。AD脑组织将充当真实Tau原纤维的来源。单个Tau原纤维的扩增将通过剪切和生长的重复循环来实现。免疫检测将用于提高灵敏度。这项研究具有创新性，因为它使用了一种新的方法来检测单个Tau原纤维，即循环扩增结合序列指导的蛋白质设计和免疫检测。所提出的研究是重要的，因为结果将提供用于扩增和检测单个Tau原纤维的稳健方案。这种能力有可能导致阿尔茨海默病早期诊断的新检测方法。