Despite its identification as a key checkpoint regulator of microglial activation in Alzheimer’s disease, the overarching role of CX3CR1 signaling in modulating mechanisms of Aβ driven neurodegeneration, including accumulation of hyperphosphorylated tau is not well understood. Accumulation of soluble and insoluble Aβ species, microglial activation, synaptic dysregulation, and neurodegeneration is investigated in 4- and 6-month old 5xFAD;Cx3cr1+/+ and 5xFAD;Cx3cr1−/− mice using immunohistochemistry, western blotting, transcriptomic and quantitative real time PCR analyses of purified microglia. Flow cytometry based, in-vivo Aβ uptake assays are used for characterization of the effects of CX3CR1-signaling on microglial phagocytosis and lysosomal acidification as indicators of clearance of methoxy-X-04+ fibrillar Aβ. Lastly, we use Y-maze testing to analyze the effects of Cx3cr1 deficiency on working memory. Disease progression in 5xFAD;Cx3cr1−/− mice is characterized by increased deposition of filamentous plaques that display defective microglial plaque engagement. Microglial Aβ phagocytosis and lysosomal acidification in 5xFAD;Cx3cr1−/− mice is impaired in-vivo. Interestingly, Cx3cr1 deficiency results in heighted accumulation of neurotoxic, oligomeric Aβ, along with severe neuritic dystrophy, preferential loss of post-synaptic densities, exacerbated tau pathology, neuronal loss and cognitive impairment. Transcriptomic analyses using cortical RNA, coupled with qRT-PCR using purified microglia from 6 month-old mice indicate dysregulated TGFβ-signaling and heightened ROS metabolism in 5xFAD;Cx3cr1−/− mice. Lastly, microglia in 6 month-old 5xFAD;Cx3cr1−/− mice express a ‘degenerative’ phenotype characterized by increased levels of Ccl2, Ccl5, Il-1β, Pten and Cybb along with reduced Tnf, Il-6 and Tgfβ1 mRNA. Cx3cr1 deficiency impairs microglial uptake and degradation of fibrillar Aβ, thereby triggering increased accumulation of neurotoxic Aβ species. Furthermore, loss of Cx3cr1 results in microglial dysfunction typified by dampened TGFβ-signaling, increased oxidative stress responses and dysregulated pro-inflammatory activation. Our results indicate that Aβ-driven microglial dysfunction in Cx3cr1−/− mice aggravates tau hyperphosphorylation, neurodegeneration, synaptic dysregulation and impairs working memory.

中文翻译：

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CX3CR1 缺乏会加重阿尔茨海默病中淀粉样蛋白驱动的神经元病理和认知能力下降


尽管 CX3CR1 信号被确定为阿尔茨海默病中小胶质细胞激活的关键检查点调节因子，但其在调节 Aβ 驱动的神经变性机制（包括过度磷酸化 tau 蛋白积累）中的总体作用尚不清楚。使用免疫组织化学、蛋白质印迹、转录组和实时定量研究 4 个月和 6 个月大的 5xFAD;Cx3cr1+/+ 和 5xFAD;Cx3cr1−/− 小鼠中可溶性和不溶性 Aβ 物质的积累、小胶质细胞激活、突触失调和神经变性纯化的小胶质细胞的 PCR 分析。基于流式细胞术的体内 Aβ 摄取测定用于表征 CX3CR1 信号传导对小胶质细胞吞噬作用和溶酶体酸化的影响，作为甲氧基-X-04+ 纤维状 Aβ 清除的指标。最后，我们使用 Y 迷宫测试来分析 Cx3cr1 缺陷对工作记忆的影响。 5xFAD;Cx3cr1−/− 小鼠疾病进展的特点是丝状斑块沉积增加，显示小胶质细胞斑块接合有缺陷。 5xFAD;Cx3cr1−/− 小鼠中小胶质细胞 Aβ 吞噬作用和溶酶体酸化在体内受损。有趣的是，Cx3cr1 缺陷会导致神经毒性寡聚 Aβ 的高度积累，以及严重的神经炎性营养不良、突触后密度优先丧失、tau 病理加剧、神经元丧失和认知障碍。使用皮质 RNA 进行的转录组分析，结合使用来自 6 个月大小鼠的纯化小胶质细胞进行的 qRT-PCR，表明 5xFAD;Cx3cr1−/− 小鼠中 TGFβ 信号传导失调，并且 ROS 代谢增强。 最后，6 个月大的 5xFAD;Cx3cr1−/− 小鼠中的小胶质细胞表达“退行性”表型，其特征是 Ccl2、Ccl5、Il-1β、Pten 和 Cybb 水平升高，同时 Tnf、Il-6 和 Tgfβ1 mRNA 水平降低。 Cx3cr1 缺陷会损害小胶质细胞对纤维状 Aβ 的摄取和降解，从而引发神经毒性 Aβ 物质的积累增加。此外，Cx3cr1 的缺失会导致小胶质细胞功能障碍，其典型表现是 TGFβ 信号传导减弱、氧化应激反应增加和促炎激活失调。我们的结果表明，Cx3cr1−/− 小鼠中 Aβ 驱动的小胶质细胞功能障碍会加剧 tau 蛋白过度磷酸化、神经变性、突触失调并损害工作记忆。