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

项目摘要 阿尔茨海默病(AD)是一种以微管堆积为特征的神经系统疾病。 在不同的大脑区域检测到许多异常的神经细胞内聚集的相关蛋白tau。他们的 出现与AD诊断患者的临床症状和进行性严重程度密切相关 神经病理特征。这些观察表明，tau聚集在AD中起着关键和有毒的作用。 Tau是一种单体的高度溶解的蛋白质，它维持微管的组装和稳定。陶氏病 功能主要是通过特定数量的残基的翻译后修饰(PTM)来调节的 磷酸化。先前的研究表明，构成多态不溶性tau的tau蛋白 聚集体(例如，神经原纤维缠结)具有异常的过度磷酸化。基于这些观察，它 提示在疾病中，普遍存在的tau蛋白过度磷酸化促进了它的聚集。然而， Tau蛋白过度磷酸化的模式和程度与聚集之间的确切联系尚不清楚。 此外，一种高度分子修饰的蛋白质如何形成广泛的形态多样性 一种疾病中的聚集体还没有被很好地理解。这些问题还没有得到彻底的调查 由于常规光学显微镜(~250 nm)的衍射限制，其中尺寸远低于 这一限制是无法解决的。通过标记人类死后阿尔茨海默病脑组织的多重过度磷酸化- 通过特异性的磷酸化tau抗体和超分辨率成像，我已经能够识别tau寡聚体(20- 30 nm)、线状纤维(30-250 nm)、分支纤维(50-350 nm)和NFT(&gt；1μm)。根据这一初步情况 数据，我假设AD中存在的tau低聚物/小纤维具有独特的PTM图谱，其起源于 Tau蛋白在不同残基上过度磷酸化的结合。我还假设 AD中形态上不同的不溶性tau聚集体具有独特的Ptm谱，并且这些谱匹配 Tau齐聚物/小纤维的PTM图谱。为了检验这些假设，本提案中确立的目标 将确定携带一组特定的过度磷酸化位点的tau蛋白的相对频率 在tau齐聚物、纤维和NFT中。我将通过组合免疫染色来实现这一点 磷酸-tau抗体、多色超分辨率成像和先进的机器定量分析 学会严格评估人类AD中存在的所有tau聚合体的过度磷酸化特征 纸巾。这些研究将证明，有可能确定tau齐聚物和 人类阿尔茨海默病组织中形态不同的不溶性tau聚集体。此外，这些研究策略 将开启比较几种tau病的tau齐聚物PTM异质性的大门，包括Cortical 基底变性和皮克氏病。此外，本文开发的研究策略也可以应用 研究其他易于聚集的蛋白质，包括α-突触核蛋白、亨廷顿蛋白等的pTm图谱。

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.