LINKING TAU FILAMENT STRUCTURE TO PHENOTYPIC DIVERSITY IN HUMAN TAUOPATHIES

描述（由申请人提供）：在理解tau蛋白的错误折叠与人类tau病中观察到的巨大表型多样性之间的关系方面，存在根本性的差距。这一差距的持续存在构成了一个重要的问题，因为在它被填补之前，对决定疾病进展的过程的分子理解在很大程度上仍然是虚幻的。长期目标是了解tau蛋白错误折叠的途径和神经元间扩散的过程。本提案的目的是使用新开发的播种方法结合双电子-电子共振（DEER）光谱（一种测量两个顺磁报告分子之间距离的方法）来确定tau细丝中多肽链的构象。中心假设是构象不同的tau蛋白丝与不同的疾病表型相关。这一假设是基于申请人实验室产生的初步数据制定的，该数据揭示了不同的tau亚型和由相同亚型组成的细丝的结构多态性之间存在强大的播种屏障。该项目的基本原理是，一旦了解了丝结构的差异，就可以测试不同构象对细胞转移机制的影响，并可以研究这些过程的抑制作用。在强有力的初步数据的支持下，中心假设将通过追求以下三个具体目标来检验。1)确定tau细丝的播种特性。一种新的基于丙烯丹的荧光分析将用于研究合成和脑源性tau蛋白细丝的播种特性，并表征新发现的tau亚型之间的播种屏障。2)识别tau蛋白细丝之间的构象差异。DEER光谱和种子细丝生长将用于确定tau细丝在分子水平上的构象差异。3)确定磷酸化tau蛋白和tau蛋白病突变体的丝特性。磷酸化和疾病相关突变对纤维构象的影响将被研究。tau同工异构体之间种子屏障的变化将有助于识别结构变化。初步数据已经确定了一种突变，这种突变会导致种子特性的改变。该突变体的细丝将使用顺磁报告群之间的距离测量来详细分析。这项研究具有创新性，因为它采用了一种新的、灵敏的方法来解决细丝之间的结构差异，即结合双电子-电子共振光谱的构象模板。这项研究具有重要意义，因为其结果将提供第一个纤维多样性及其与人类牛头病关系的一般模型。这些知识有可能导致新的治疗策略，包括生产新的单克隆抗体和小分子抑制剂，选择性地干扰特定的传播途径，减缓或逆转tau介导的疾病的进展。

DESCRIPTION (provided by applicant): There is a fundamental gap in understanding of how the misfolding of tau relates to the enormous phenotypic diversity observed in human tauopathies. Persistence of this gap constitutes an important problem because, until it is filled, a molecular understanding of the processes that determine disease progression will remain largely illusive. The long-term goal is to understand the pathways of tau misfolding and the processes of interneuronal spreading. The objective of this proposal is to use newly developed seeding methodology in conjunction with double electron-electron resonance (DEER) spectroscopy (a method that measures the distances between two paramagnetic reporter molecules) to determine the conformations of polypeptide chains in tau filaments. The central hypothesis is that conformationally distinct tau filaments are associated with different disease phenotypes. This hypothesis has been formulated based on preliminary data produced in the applicant's lab that reveal a robust seeding barrier between distinct isoforms of tau and structural polymorphism of filaments composed of identical isoforms. The rationale for this project is that once differences in filament structure are known, the effects of distinct conformers on cell transfer mechanisms can be tested, and the inhibition of these processes can be investigated. Supported by strong preliminary data the central hypothesis will be tested by pursuit of the following three specific aims. 1) Determine the seeding properties of tau filaments. A novel acrylodan-based fluorescence assay will be used to investigate the seeding properties of synthetically and brain-derived tau filaments and to characterize a newly discovered seeding barrier between tau isoforms. 2) Identify conformational differences between tau filaments. DEER spectroscopy and seeded filament growth will be used to determine conformational differences between tau filaments at the molecular level. 3) Determine filament properties of phosphorylated tau and tau disease mutants. The effects of phosphorylations and disease-related mutations on filament conformation will be investigated. Changes in seeding barriers between tau isoforms will serve to identify structural changes. Preliminary data have identified a mutation that causes a switch in seeding properties. Filaments of this mutant will be analyzed in detail using distance measurements between paramagnetic reporter groups. The research is innovative, because it uses a new, sensitive approach to resolve structural differences between filaments, namely conformational templating combined with double electron-electron resonance spectroscopy. The proposed research is significant, because the results will provide a first general model of filament diversity and its relationship to human tauopathies. Such knowledge has the potential to lead to new therapeutic strategies, including the production of novel monoclonal antibodies and small molecule inhibitors that selectively interfere with specific propagation routes, slowing or reversing the progression of tau-mediated diseases.