Recent studies suggest that microglia contribute to tau pathology progression in Alzheimer’s disease. Amyloid plaque accumulation transforms microglia, the primary innate immune cells in the brain, into neurodegenerative microglia (MGnD), which exhibit enhanced phagocytosis of plaques, apoptotic neurons and dystrophic neurites containing aggregated and phosphorylated tau (p-tau). It remains unclear how microglia promote disease progression while actively phagocytosing pathological proteins, therefore ameliorating pathology. Adeno-associated virus expressing P301L tau mutant (AAV-P301L-tau) was stereotaxically injected into the medial entorhinal cortex (MEC) in C57BL/6 (WT) and humanized APP mutant knock-in homozygote (AppNL-G-F) mice at 5 months of age. Mice were fed either chow containing a colony stimulating factor-1 receptor inhibitor (PLX5622) or control chow from 4 to 6 months of age to test the effect of microglia depletion. Animals were tested at 6 months of age for immunofluorescence, biochemistry, and FACS of microglia. In order to monitor microglial extracellular vesicle secretion in vivo, a novel lentiviral EV reporter system was engineered to express mEmerald-CD9 (mE-CD9) specifically in microglia, which was injected into the same region of MEC. Expressing P301L tau mutant in the MEC induced tau propagation to the granule cell layer of the hippocampal dentate gyrus, which was significantly exacerbated in AppNL-G-F mice compared to WT control mice. Administration of PLX5622 depleted nearly all microglia in mouse brains and dramatically reduced propagation of p-tau in WT and to a greater extent in AppNL-G-F mice, although it increased plaque burden and plaque-associated p-tau+ dystrophic neurites. Plaque-associated MGnD microglia strongly expressed an EV marker, tumor susceptibility gene 101, indicative of heightened synthesis of EVs. Intracortical injection of mE-CD9 lentivirus successfully induced microglia-specific expression of mE-CD9+ EV particles, which were significantly enhanced in Mac2+ MGnD microglia compared to Mac2− homeostatic microglia. Finally, consecutive intracortical injection of mE-CD9 lentivirus and AAV-P301L-tau into AppNL-G-F mice revealed encapsulation of p-tau in microglia-specific mE-CD9+ EVs as determined by super-resolution microscopy and immuno-electron microscopy. Our findings suggest that MGnD microglia hyper-secrete p-tau+ EVs while compacting Aβ plaques and clearing NP tau, which we propose as a novel mechanistic link between amyloid plaque deposition and exacerbation of tau propagation in AppNL-G-F mice.

中文翻译：

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斑块相关小胶质细胞过度分泌细胞外囊泡并加速人源化 APP 小鼠模型中的 tau 传播

最近的研究表明，小胶质细胞有助于阿尔茨海默病的 tau 病理学进展。淀粉样蛋白斑块的积累将小胶质细胞（大脑中的主要先天免疫细胞）转化为神经退行性小胶质细胞 (MGnD)，其表现出增强的斑块、凋亡神经元和含有聚集和磷酸化 tau (p-tau) 的营养不良神经突的吞噬作用。目前尚不清楚小胶质细胞如何在积极吞噬病理蛋白的同时促进疾病进展，从而改善病理。表达 P301L tau 突变体 (AAV-P301L-tau) 的腺相关病毒在 5 个月时被立体定向​​注射到 C57BL/6 (WT) 和人源化 APP 突变敲入纯合子 (AppNL-GF) 小鼠的内侧内嗅皮层 (MEC)年龄。给小鼠喂食含有集落刺激因子-1 受体抑制剂 (PLX5622) 的食物或 4 至 6 个月大的对照食物，以测试小胶质细胞耗竭的影响。动物在 6 个月大时接受了小胶质细胞的免疫荧光、生物化学和 FACS 测试。为了监测体内小胶质细胞外囊泡的分泌，设计了一种新型慢病毒 EV 报告系统，以在小胶质细胞中特异性表达 meEmerald-CD9 (mE-CD9)，并将其注射到 MEC 的同一区域。在 MEC 中表达 P301L tau 突变体可诱导 tau 增殖到海马齿状回的颗粒细胞层，与 WT 对照小鼠相比，AppNL-GF 小鼠的这种情况显着加剧。PLX5622 的施用几乎耗尽了小鼠大脑中的所有小胶质细胞，并显着减少了 WT 中 p-tau 的传播，并且在 AppNL-GF 小鼠中的作用更大，尽管它增加了斑块负荷和斑块相关的 p-tau + 营养不良性神经突。斑块相关的 MGnD 小胶质细胞强烈表达一种 EV 标记物，即肿瘤易感基因 101，表明 EV 的合成增加。皮质内注射 mE-CD9 慢病毒成功诱导了 mE-CD9+ EV 颗粒的小胶质细胞特异性表达，与 Mac2− 稳态小胶质细胞相比，Mac2+ MGnD 小胶质细胞中的 mE-CD9+ EV 颗粒显着增强。最后，连续皮层内注射 mE-CD9 慢病毒和 AAV-P301L-tau 到 AppNL-GF 小鼠中，通过超分辨率显微镜和免疫电子显微镜确定小胶质细胞特异性 mE-CD9+ EV 中 p-tau 的封装。