Super-Resolution Imaging of Alzheimer's Disease Hyperphosphorylated Tau Aggregates

阿尔茨海默病过度磷酸化 Tau 聚集体的超分辨率成像

• 批准号: 10603421
• 负责人: Adriana Naomi Santiago-Ruiz
• 金额: $ 4.77万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-01 至 2025-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10603421
• 关键词: Address; Alzheimer' s Disease; Alzheimer' s disease brain; Alzheimer' s disease diagnosis; Antibodies; Autopsy; Brain region; Clinical; Color; Cytosol; Data; Dementia; Disease; Disease Progression; Evaluation; Frequencies; Goals; Heterogeneity; Human; Human Pathology; Huntington gene; Immunofluorescence Immunologic; Knowledge; Label; Link; MAPT gene; Machine Learning; Methodology; Microtubules; Modeling; Modification; Molecular; Molecular Profiling; Morphology; Nerve Degeneration; Neurofibrillary Tangles; Neurons; Patients; Pattern; Phosphorylation; Pick Disease of the Brain; Play; Post-Translational Protein Processing; Proteins; Research; Role; Severities; Shapes; Site; Symptoms; Tauopathies; Testing; Tissue Sample; Tissues; alpha synuclein; brain tissue; classification algorithm; combinatorial; corticobasal degeneration; human tissue; hyperphosphorylated tau; innovation; light microscopy; monomer; nervous system disorder; neuropathology; superresolution imaging; superresolution microscopy; tau Proteins; tau aggregation; tau function; therapy development; tool

Project Summary Alzheimer’s disease (AD) is a neurological disorder characterized by the accumulation of microtubule- associated protein tau into many abnormal intraneuronal aggregates detected at distinct brain regions. Their emergence strongly correlates with the progressive severity of AD diagnosed patients’ clinical symptoms and neuropathological features. These observations suggest tau aggregation plays a crucial and toxic role in AD. Tau is a monomeric highly soluble protein that maintains the assembly and stability of microtubules. Tau’s function is regulated through post-translational modifications (PTMs) of a specific number of residues, primarily phosphorylation. Previous studies indicate that the tau proteins that make up polymorphous insoluble tau aggregates (e.g., neurofibrillary tangles) have abnormal hyperphosphorylation. Based on these observations, it is suggested that ubiquitous hyperphosphorylation of tau promotes its aggregation in disease. However, the precise link between the pattern and degree of tau hyperphosphorylation with aggregation is unclear. Furthermore, how a heavily molecularly modified protein can form a wide range of morphologically diverse aggregates within one disease is not well-understood. These questions have not been investigated thoroughly due to the diffraction limit of conventional light microscopy (~250nm), in which aggregates of a size well below this limit are not resolvable. By labeling human postmortem AD brain tissues with multiple hyperphosphorylation- specific phosphor-tau antibodies and super-resolution imaging, I have been able to identify tau oligomers ( 20- 30 nm), linear fibrils (30-250 nm), branched fibrils (50-350 nm), and NFTs (>1μm). Based on this preliminary data, I hypothesize that the tau oligomers/small fibrils present in AD have unique PTM profiles that arise from the combination of tau proteins hyperphosphorylated at different residues. I also hypothesize that morphologically distinct insoluble tau aggregates in AD have unique PTM profiles and that these profiles match the PTM profiles of tau oligomers/small fibrils. To test these hypotheses, the aims established in this proposal will determine the relative frequency of tau proteins carrying a particular array of hyperphosphorylated sites within tau oligomers, fibrils, and NFTs. I will achieve this by combinatorial immunostaining with established phosphor-tau antibodies, multicolor super-resolution imaging, and advanced quantitative analysis using machine learning to rigorously evaluate the hyperphosphorylation profiles of all tau aggregates present in human AD tissues. These studies will demonstrate that it is possible to determine the PTM profiles of tau oligomers and morphologically distinct insoluble tau aggregates in human AD tissue. Furthermore, these research strategies will open the door to compare tau oligomer PTM heterogeneity across several tauopathies, including Cortical basal degeneration and Pick’s disease. Furthermore, the research strategies developed herein can be applied to study the PTM profiles of other aggregation-prone proteins, including α-synuclein, huntingtin, and others.

项目概要 阿尔茨海默病（AD）是一种神经系统疾病，其特征是微管积聚 相关的 tau 蛋白进入许多在不同大脑区域检测到的异常神经元内聚集体。 AD 诊断患者临床症状的严重程度与出现密切相关 这些观察结果表明 tau 蛋白聚集在 AD 中发挥着至关重要的毒性作用。 Tau 是一种单体高度可溶性蛋白质，可维持微管的组装和稳定性。 功能是通过特定数量的残基的翻译后修饰（PTM）来调节的，主要是 先前的研究表明，构成多态不溶性 tau 的 tau 蛋白。 根据这些观察，聚集体（例如神经原纤维缠结）具有异常的过度磷酸化。 有人认为，普遍存在的 tau 蛋白过度磷酸化会促进其在疾病中的聚集。 tau 过度磷酸化与聚集的模式和程度之间的精确联系尚不清楚。 此外，经过大量分子修饰的蛋白质如何形成广泛的形态多样性 一种疾病内的聚集体尚未得到充分研究。 由于传统光学显微镜（~250nm）的衍射极限，其中尺寸远低于 通过对人类死后 AD 脑组织进行多重过度磷酸化标记，这一限制是无法解决的。 通过特异性磷酸化 tau 抗体和超分辨率成像，我已经能够识别 tau 寡聚体（ 20- 30 nm）、线性原纤维（30-250 nm）、分支原纤维（50-350 nm）和 NFT（>1μm）。 根据数据，我认为 AD 中存在的 tau 寡聚体/小原纤维具有独特的 PTM 特征，这些特征源自 tau 蛋白在不同残基处过度磷酸化的组合。 AD 中形态不同的不溶性 tau 聚集体具有独特的 PTM 配置文件，并且这些配置文件匹配 tau 寡聚物/小原纤维的 PTM 概况 为了测试这些假设，本提案确立了目标。 将确定携带特定超磷酸化位点阵列的 tau 蛋白的相对频率 在 tau 寡聚体、原纤维和 NFT 中，我将通过与已建立的组合免疫染色来实现这一目标。 荧光 tau 抗体、多色超分辨率成像以及使用机器进行的高级定量分析 学习严格评估人类 AD 中所有 tau 蛋白聚集体的过度磷酸化特征 这些研究将证明确定 tau 寡聚体的 PTM 谱是可能的。 此外，这些研究策略还揭示了人类 AD 组织中形态不同的不溶性 tau 聚集体。 将为比较多种 tau 病（包括皮质病）的 tau 寡聚体 PTM 异质性打开大门 此外，本文开发的研究策略可以应用 研究其他易聚集蛋白的 PTM 图谱，包括 α-突触核蛋白、亨廷顿蛋白等。