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

基本信息

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
项目类别：
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&apos s Disease Alzheimer&apos s disease brain Alzheimer&apos 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

项目摘要

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的蛋白质TAU（MAPT/TAU）采用不同的毒性细丝构象或特定于不同tauopathy疾病的菌株。组装这些有毒的tau构象被认为是通过prion型机制发生的，其中tau形成了交叉β 薄淀粉样蛋白聚集体，该模板并催化固体tau的转化。冷冻电子显微镜（Cryo-Em）已证明是必不可少的，对于确定这些构象的高分辨率结构是必不可少的 ADRD大脑衍生的组织，建立了一个关键平台，以识别与疾病相关的药物靶标设计。此外，最近的低温EM努力揭示了Adrd Tau的独特，特定疾病的构象细丝，并提出了希望新解决的结构在更精确的药物设计中有用的。但是，很难接近淀粉样蛋白的平坦，重复的表面以及淀粉样蛋白的发现结合诊断化合物仅限于随机筛选。此外，现有结构是派生的来自后期的散装组织制剂，主要零星样品。那我们假设 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类型或疾病初始阶段。重要的是，尚未确定tau细丝结构病理学相关的ADRD动物模型，阻碍靶向TAU疾病的疗法的发展构象。我们假设这是由于现有的结构可处理的tau原纤维的抽象较低鼠标模型。这些挑战性问题促使我们使用涉及小说的组合成像方法，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内通过我们的方法扩展tau脑沉积的表征，以分离和分离神经胶质和分离来自人类脑组织的神经元细胞类型以及我们的新型大鼠模型的致病性TAU表征结构。这些目标是基于我们的强大初步工作，在该工作中，我们确定了第一个高级与疾病与疾病相关的tau的分辨率与医学相关的小分子结构细丝和开发的结构敏感的染料成像方法，这些方法揭示了不同的Tau的不同状态细胞类型和疾病。此外，我们开发了自定义抗体功能化的EM网格，以纯化来自少量散装组织的生化与生化相关的Adrd Tau丝：1）启用冷冻EM研究在稀疏tau沉积物的珍贵早期大脑区域上，2）基于已知的PTM净化tau细丝 Tau成熟度的标记和3）减少了容易发生结构不忠的扩增方法的需求。这创新的提案建立在索斯沃思和康德洛之间既定的合作中，他们的实验室将杠杆作用在冷冻EM结构，细胞生物学和组织学方法中的联合优势，用于预测，优先和确定疾病，细胞类型和特定年龄特异性tau丝和配体结构的结构以足够的分辨率进行配体建模和结构引导设计的共复合。