BACKGROUND Alzheimer's disease is associated with the accumulation of intracellular Tau tangles within neurons and extracellular amyloid-β plaques in the brain parenchyma, which altogether results in synaptic loss and neurodegeneration. Extracellular concentrations of oligomers and aggregated proteins initiate microglial activation and convert their state of synaptic surveillance into a destructive inflammatory state. Although Tau oligomers have fleeting nature, they were shown to mediate neurotoxicity and microglial pro-inflammation. Due to the instability of oligomers, in vitro experiments become challenging, and hence, the stability of the full-length Tau oligomers is a major concern. METHODS In this study, we have prepared and stabilized hTau40WT oligomers, which were purified by size-exclusion chromatography. The formation of the oligomers was confirmed by western blot, thioflavin-S, 8-anilinonaphthaalene-1-sulfonic acid fluorescence, and circular dichroism spectroscopy, which determine the intermolecular cross-β sheet structure and hydrophobicity. The efficiency of N9 microglial cells to phagocytose hTau40WT oligomer and subsequent microglial activation was studied by immunofluorescence microscopy with apotome. The one-way ANOVA was performed for the statistical analysis of fluorometric assay and microscopic analysis. RESULTS Full-length Tau oligomers were detected in heterogeneous globular structures ranging from 5 to 50 nm as observed by high-resolution transmission electron microscopy, which was further characterized by oligomer-specific A11 antibody. Immunocytochemistry studies for oligomer treatment were evidenced with A11+ Iba1high microglia, suggesting that the phagocytosis of extracellular Tau oligomers leads to microglial activation. Also, the microglia were observed with remodeled filopodia-like actin structures upon the exposure of oligomers and aggregated Tau. CONCLUSION The peri-membrane polymerization of actin filament and co-localization of Iba1 relate to the microglial movements for phagocytosis. Here, these findings suggest that microglia modified actin cytoskeleton for phagocytosis and rapid clearance of Tau oligomers in Alzheimer's disease condition.

中文翻译：

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通过激活小胶质细胞的肌动蛋白重塑全长Tau寡核苷酸的吞噬作用。

背景技术阿尔茨海默氏病与神经实质内的细胞内Tau缠结和脑实质内的细胞外淀粉样蛋白-β斑块的积聚有关，它们总计导致突触丧失和神经变性。细胞外浓度的低聚物和聚集蛋白会启动小胶质细胞活化，并将其突触监视状态转变为破坏性的炎症状态。尽管Tau低聚物具有短暂的性质，但已证明它们可介导神经毒性和小胶质细胞促炎症。由于低聚物的不稳定性，体外实验变得具有挑战性，因此，全长Tau低聚物的稳定性是主要问题。方法在本研究中，我们制备并稳定了hTau40WT低聚物，并通过尺寸排阻色谱法进行了纯化。寡聚体的形成通过蛋白质印迹，硫黄素-S，8-苯胺基萘-苯磺酸-1-磺酸荧光和圆二色光谱法确定，其确定了分子间交叉-β片层结构和疏水性。N9小胶质细胞吞噬hTau40WT寡聚体和随后的小胶质细胞激活的效率通过用免疫球蛋白显微镜与potome研究。进行单向方差分析用于荧光分析和显微镜分析的统计分析。结果通过高分辨率透射电子显微镜观察，在5至50 nm的异质球状结构中检测到全长Tau寡聚体，并进一步以寡聚体特异性A11抗体为特征。A11 + Iba1高小胶质细胞证明了用于寡聚体治疗的免疫细胞化学研究，提示胞外Tau低聚物的吞噬作用会导致小胶质细胞活化。同样，在低聚物和聚集的Tau暴露后，观察到小胶质细胞具有重塑的丝状伪足样肌动蛋白结构。结论肌动蛋白丝的跨膜聚合和Iba1的共定位与吞噬作用的小胶质细胞运动有关。在这里，这些发现表明，小胶质细胞修饰的肌动蛋白细胞骨架可用于吞噬作用和在阿尔茨海默氏病条件下快速清除Tau低聚物。结论肌动蛋白丝的跨膜聚合和Iba1的共定位与吞噬作用的小胶质细胞运动有关。在这里，这些发现表明，小胶质细胞修饰的肌动蛋白细胞骨架可用于吞噬作用和在阿尔茨海默氏病条件下快速清除Tau低聚物。结论肌动蛋白丝的跨膜聚合和Iba1的共定位与吞噬作用的小胶质细胞运动有关。在这里，这些发现表明，小胶质细胞修饰的肌动蛋白细胞骨架可用于吞噬作用和在阿尔茨海默氏病条件下快速清除Tau低聚物。