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

项目摘要 阿尔茨海默病（AD）是一种神经系统疾病，其特征在于微管- 在不同的脑区域检测到许多异常的神经元内聚集体。他们的 出现与AD诊断患者临床症状的进展严重程度密切相关， 神经病理学特征这些观察结果表明tau聚集在AD中起着至关重要的毒性作用。 Tau是一种单体的高度可溶性蛋白质，其维持微管的组装和稳定性。Tau的 功能通过特定数量的残基的翻译后修饰（PTM）来调节，主要是 磷酸化先前的研究表明，构成多形不溶性tau蛋白的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。根据这些观察， 提示普遍存在的tau过度磷酸化促进其在疾病中的聚集。但 tau过度磷酸化的模式和程度与聚集之间的精确联系尚不清楚。 此外，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不太清楚。这些问题没有调查清楚 由于常规光学显微镜的衍射极限（~ 250 nm），其中尺寸远低于 这个极限是无法解决的。通过标记人类死后AD脑组织的多重过度磷酸化- 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- 在一些实施方案中，所述纳米纤维包括纳米纤维（30- 30 nm）、直链原纤维（30-250 nm）、支链原纤维（50-350 nm）和NFT（>1μm）。根据初步的 根据这些数据，我假设AD中存在的tau寡聚体/小纤维具有独特的PTM谱， tau蛋白在不同残基处过度磷酸化的组合。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寡聚体/小原纤维的PTM谱。为了验证这些假设，本提案中确立的目标 将确定携带特定高度磷酸化位点阵列的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 磷酸化tau抗体，超分辨率成像，以及使用机器的高级定量分析 学习严格评估人类AD中存在的所有tau聚集体的过度磷酸化特征 组织中这些研究将证明有可能确定tau寡聚体的PTM谱， 在人AD组织中存在形态学上不同的不溶性tau聚集体。此外，这些研究策略 将打开大门，比较tau寡聚体PTM异质性在几个tau病变，包括皮质 基底变性和皮克病。此外，本文提出的研究策略可以应用于 研究其他聚集倾向蛋白的PTM谱，包括α-突触核蛋白、亨廷顿蛋白等。

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.