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

大脑分离细胞类型中 tau 聚集变异性和成熟度的结构表征

• 批准号: 10721681
• 负责人: CARLO L CONDELLO
• 金额: $ 241.65万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-08-01 至 2026-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10721681
• 关键词: Adopted; Advanced Development; Age; Alzheimer disease screening; Alzheimer like pathology; Alzheimer' s Disease; Alzheimer' s disease brain; Alzheimer' s disease related dementia; Amyloid; Animal Model; Antibodies; Binding; Binding Sites; Biochemical; Biological; Biological Assay; Brain; Brain Diseases; Brain region; Cell Separation; Cells; Cessation of life; Collaborations; Complex; Cryoelectron Microscopy; Custom; Data; Deposition; Development; Diagnosis; Diagnostic; Disease; Drug Design; Drug Targeting; Dyes; Filament; Fluorescence-Activated Cell Sorting; Functional disorder; Goals; Heterogeneity; Histologic; Human; Image; Image Analysis; Label; Lateral; Ligand Binding; Ligands; MAPT gene; Medical; Methods; Modeling; Molecular Conformation; Neuroglia; Neurons; Outcome; Oxides; Pathologic; Patients; Pick Disease of the Brain; Positron-Emission Tomography; Preparation; Prions; Progressive Supranuclear Palsy; Proteomics; Rat Transgene; Rattus; Research Proposals; Resolution; Sampling; Sorting; Specificity; Structure; Surface; Tauopathies; Techniques; Technology; Therapeutic; Tissues; Tracer; Vertebral column; Work; beta pleated sheet; brain cell; brain tissue; cell type; corticobasal degeneration; design; fluorescence imaging; graphene; human tissue; imaging approach; imaging modality; innovation; mouse model; novel; novel diagnostics; optical imaging; screening; small molecule; structural imaging; success; targeted treatment; tau Proteins; tau aggregation; tau conformation; tau mutation; therapeutic development; therapy development; tissue preparation

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.

抽象的 最近的结构和生化工作揭示了微管相关蛋白 tau (MAPT/tau) 采用 不同的毒性丝构象或菌株是不同tau蛋白病疾病所特有的。 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 片状淀粉样蛋白聚集体模板并催化可溶性 tau 蛋白的转化。 （冷冻电镜）已被证明对于确定这些构象的高分辨率结构是必不可少的 ADRD 脑源组织，为识别疾病相关药物靶点建立关键平台 此外，最近的冷冻电镜研究揭示了 ADRD tau 独特的疾病特异性构象。 丝，并提出了新解决的结构可用于更精确的药物设计的希望。 然而，接近淀粉样蛋白的平坦、重复表面以及淀粉样蛋白的发现一直很困难。 此外，结合诊断化合物仅限于随机筛选。 来自后期的大量组织制剂，主要是零星样品。 tau 蛋白的治疗相关状态尚未完全了解，包括不同脑细胞中出现的状态 重要的是，tau 丝的结构尚未确定。 病理相关的 ADRD 动物模型，阻碍了针对 tau 疾病的疗法的开发 我们探索这是由于现有结构上易处理的 tau 原纤维丰度较低。 这些具有挑战性的问题促使我们使用涉及新颖、 结构敏感的 tau 结合小分子染料和冷冻电镜我们计划彻底改变体外。 通过改进我们分离和分离神经胶质和神经胶质的方法来表征 tau 脑沉积物 人脑组织的神经元细胞类型以及致病性 tau 蛋白新型大鼠模型的表征 这些目标是基于我们强有力的前期工作，我们在这些工作中确定了第一个高目标。 与疾病相关 tau 蛋白位点特异性结合的医学相关小分子的解析结构 丝，并开发了结构敏感的染料成像方法，揭示了不同状态下 tau 的不同状态 此外，我们开发了定制的抗体功能化 EM 网格，用于纯化细胞类型和疾病。 来自小体积组织的生化相关 ADRD tau 丝，将：1) 实现冷冻电镜研究 在具有稀疏 tau 沉积物的珍贵早期大脑区域，2）根据已知的 PTM 纯化 tau 丝 tau 成熟度标记，3) 减少对容易出现结构不忠实的扩增方法的需求。 创新提案建立在 Southworth 和 Condello 之间已建立的合作基础上，他们的实验室将 利用冷冻电镜结构、细胞生物学和组织学方法的综合优势来预测、 优先考虑并确定疾病、细胞类型和年龄特异性 tau 丝和配体结合的结构 具有足够分辨率的共复合物，可用于配体建模和结构指导设计。