Structural characterization of tau aggregation variability and maturity in isolated cell types of the brain

摘要 最近的结构和生化研究表明，微管相关蛋白tau(MAPT/tau)采用 不同的毒丝构象或菌株，专用于不同的变态反应疾病。装配 这些有毒的tau构象被认为是通过prion类型的机制发生的，在这种机制中tau形成交叉β。 片状淀粉样蛋白聚集该模板并催化可溶性tau的转化。低温电子显微镜 (CRYO-EM)已被证明是从 ADRD脑源性组织，为确定与疾病相关的药物靶点建立了一个关键平台 设计。此外，最近的低温EM研究已经发现了ADRD tau的独特的、疾病特异性的构象 并提出了新解决的结构可能在更精确的药物设计中有用的希望。 然而，很难接近淀粉样蛋白平坦、重复的表面，也很难发现淀粉样蛋白- 结合诊断化合物仅限于随机筛选。此外，还推导了现有的结构 来自晚期的大量组织制剂，主要是零星样本。因此，我们假设 Tau在治疗上的相关状态还不完全清楚，包括在不同脑细胞中出现的状态。 疾病类型的或处于疾病初期的。重要的是，tau纤维的结构还没有从 病理上相关的ADRD动物模型，阻碍了针对tau病的治疗的发展 构象。我们推测，这是由于现有的、结构上易于处理的tau纤维丰度较低。 老鼠模型。这些具有挑战性的问题促使我们使用组合成像方法， 结构敏感的tau结合小分子染料和低温EM。我们计划给体外实验带来革命性的变化 通过我们分离和分离神经胶质细胞和神经胶质细胞的方法的进步来表征tau脑沉积 人脑组织神经细胞类型及其致病大鼠模型的研究 结构。这些目标是建立在我们强有力的前期工作基础上的，在这些工作中，我们确定了第一个高点-- 与疾病相关的tau特异结合的医学相关小分子结合部位的分辨结构 并开发了结构敏感的染料成像方法，以揭示不同物种中tau的不同状态 细胞类型和疾病。此外，我们开发了定制的抗体功能化EM网格，用于纯化 来自小体积散装组织的生化相关的ADRD tau细丝将：1)使冷冻-EM研究成为可能 在具有稀少tau沉积的宝贵早期大脑区域，2)基于已知的PTM提纯tau细丝 Tau成熟度的标记，以及3)减少了对容易发生结构不忠的扩增方法的需要。这 创新的提议建立在索斯沃斯和康德洛之间建立的合作之上，他们的实验室将 利用冷冻-EM结构、细胞生物学和组织学方法的综合优势来预测， 区分和确定疾病、细胞类型和年龄特定的tau丝和配体结合的结构 以足够的分辨率进行配基建模和结构导向设计。

Abstract Recent structural and biochemical work reveals the microtubule-associated protein tau (MAPT/tau) adopts different toxic filament conformations or strains that are specific to different tauopathy diseases. Assembly of these toxic tau conformations is thought to occur through a prion-type mechanism in which tau forms cross-β sheet amyloid aggregates that template and catalyze the conversion of soluble tau. Cryo-electron microscopy (cryo-EM) has proven indispensable for determining high-resolution structures of these conformations from ADRD brain-derived tissue, establishing a critical platform for identifying the disease-relevant targets for drug design. Further, recent cryo-EM efforts have revealed distinct, disease-specific conformations of ADRD tau filaments, and raised the hope that the newly solved structures could be useful in more precise drug design. However, it has been difficult to approach the flat, repetitive surfaces of amyloids and the discovery of amyloid- binding diagnostic compounds has been limited to random screening. Moreover, existing structures are derived from bulk tissue preparations from late stage, primarily sporadic samples. Thus, we hypothesize that therapeutically relevant states of tau are not fully understood, including states that arise in different brain cell types or during initial stages of disease. Importantly, tau filament structures have not been determined from a pathologically relevant ADRD animal model, hindering development of therapies that target tau disease conformations. We hypothesize this is due to low abundance of structurally tractable tau fibrils from existing mouse models. These challenging questions prompted us to use combined imaging approaches involving novel, structurally sensitive tau-binding small molecule dyes and cryo-EM. We plan to revolutionize ex vivo characterization of tau brain deposits through the advancement of our methods to separate and isolate glial and neuronal cell types from human brain tissue and characterization of our novel rat model for pathogenic tau structures. These goals are based on our strong preliminary work in which we have determined the first high- resolution structure of a medically-relevant small molecule bound site-specifically to disease-relevant tau filaments, and developed structurally-sensitive dye imaging methods that reveal distinct states of tau in different cell types and diseases. Furthermore, we developed custom antibody-functionalized EM grids for purification of biochemically-relevant ADRD tau filaments from small volumes of bulk tissue that will: 1) enable cryo-EM studies on precious early stage brain regions with sparse tau deposits, 2) purify tau filaments based on known PTM markers of tau maturity, and 3) reduce the need for amplification methods prone to structural infidelity. This innovative proposal is built on the established collaboration between Southworth and Condello, whose labs will leverage combined strengths in cryo-EM structural, cell biological and histological approaches for predicting, prioritizing and determining structures of disease-, cell type-, and age-specific tau filaments and ligand-bound co-complexes at a sufficient resolution for ligand modeling and structure-guided design.