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

摘要 最近的结构和生物化学工作揭示了微管相关蛋白tau（MAPT/tau）采用 不同的毒性丝状体构象或对不同的tau蛋白病疾病特异的菌株。大会 这些有毒的tau构象被认为是通过朊病毒型机制发生的，其中tau形成交叉β 折叠淀粉样蛋白聚集模板并催化可溶性tau转化。低温电子显微镜 （冷冻EM）已被证明是必不可少的，以确定这些构象的高分辨率结构， ADRD脑源性组织，为确定药物治疗的疾病相关靶点建立关键平台 设计此外，最近的冷冻电镜研究揭示了ADRD tau蛋白独特的疾病特异性构象， 细丝，并提出了希望，新解决的结构可以在更精确的药物设计有用。 然而，很难接近淀粉样蛋白的平坦、重复的表面， 结合诊断化合物已限于随机筛选。此外，现有结构是由 来自晚期的大量组织制备物，主要是零星的样品。因此，我们假设， tau的治疗相关状态尚未完全了解，包括在不同脑细胞中出现的状态， 在疾病的初期或初期阶段。重要的是，tau细丝结构还没有从一个细胞中确定。 病理学相关的ADRD动物模型，阻碍了靶向tau疾病的疗法的开发 构象我们假设这是由于现有的结构上易处理的tau纤维丰度低。 小鼠模型。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结合小分子染料和冷冻EM。我们计划彻底改变体外培养 通过我们的方法的进步来分离和分离神经胶质细胞和神经胶质细胞， 来自人脑组织的神经元细胞类型和我们用于致病性tau蛋白的新型大鼠模型的表征 结构.这些目标是基于我们强大的初步工作，我们已经确定了第一个高- 与疾病相关tau位点特异性结合的医学相关小分子的解析结构 纤维，并开发了结构敏感的染料成像方法，揭示了不同的tau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 来自小体积的大块组织的生物化学相关的ADRD tau细丝，其将：1）使得能够进行冷冻EM研究 在具有稀疏tau沉积物的宝贵的早期脑区域上，2）基于已知的PTM纯化tau细丝， tau成熟度的标记，和3）减少对易于结构失真的扩增方法的需要。这 创新的建议是建立在Southworth和Condello之间建立的合作，其实验室将 利用冷冻EM结构、细胞生物学和组织学方法的综合优势进行预测， 优先考虑和确定疾病，细胞类型和年龄特异性tau细丝和配体结合的结构 以足够的分辨率进行配体建模和结构指导设计。

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.