ESTABLISHING AN ASSAY FOR DETECTING SINGLE TAU FIBRILS

 描述(申请人提供)：目前还没有阿尔茨海默病最早的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纤维通过从一个神经元转移到另一个神经元，在整个大脑中传播。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阶段在很大程度上仍将是虚幻的。长期目标是了解牛磺酸的传播途径，以及如何将其用于预防和治疗目的。这项建议的目的是将新开发的扩增方法与基于序列的蛋白质设计和酶联免疫检测相结合来检测单个Tau纤维。中心假设是，在重复的剪切和生长周期之后，可以检测到单一的Tau纤维。这一假设是基于申请人实验室产生的数据提出的，这些数据揭示了合成和脑源性Tau纤维的剪切诱导放大。这个项目的基本原理是，一旦获得了单一纤维的敏感性，就可以对生物流体进行A诊断测试，并可以启动对Tau纤维释放的机理研究。在强劲的初步数据的支持下，中心假说将通过追求以下三个具体目标来检验。1)通过序列引导的蛋白质设计抑制Tau单体的自成核。Tau疏水核心中的部分残基将被单独取代以抑制自成核，但不会生长。聚集性将通过荧光光谱、沉淀和电子显微镜进行监测。2)开发蛋白质降解增强的免疫检测方法，以区分纤维和单体。蛋白水解法将用于完全降解未折叠的Tau单体。抗蛋白酶核心将被单抗捕获，并通过酶联免疫吸附试验进行检测。3)从阿尔茨海默病患者脑组织中扩增Tau纤维。AD脑组织将作为正宗Tau纤维的来源。单个Tau原纤维的放大将通过重复的剪切和生长循环来实现。将使用免疫检测来提高灵敏度。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纤维，即循环扩增结合序列引导的蛋白质设计和免疫检测。这项拟议的研究具有重要意义，因为该结果将为扩增和检测单个Tau纤维提供一个健壮的方案。这种能力有可能为阿尔茨海默病的早期诊断带来新的分析方法。

 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.