Microglia are highly dynamic cells that play a critical role in tissue homeostasis through the surveillance of brain parenchyma and response to cues associated with damage. Aging and APOE4 genotype are the strongest risk factors for Alzheimer’s disease (AD), but how they affect microglial dynamics remains unclear. Using ex vivo confocal microscopy, we analyzed microglial dynamic behaviors in the entorhinal cortex (EC) and hippocampus CA1 of 6-, 12-, and 21-month-old mice APOE3 or APOE4 knock-in mice expressing GFP under the CX3CR1 promoter. To study microglia surveillance, we imaged microglia baseline motility for 20 min and measured the extension and retraction of processes. We found that APOE4 microglia exhibited significantly less brain surveillance (27%) compared to APOE3 microglia in 6-month-old mice; aging exacerbated this deficit. To measure microglia response to damage, we imaged process motility in response to ATP, an injury-associated signal, for 30 min. We found APOE4 microglia extended their processes significantly slower (0.9 µm/min, p < 0.005) than APOE3 microglia (1.1 μm/min) in 6-month-old animals. APOE-associated alterations in microglia motility were observed in 12- and 21-month-old animals, and this effect was exacerbated with aging in APOE4 microglia. We measured protein and mRNA levels of P2RY12, a core microglial receptor required for process movement in response to damage. We found that APOE4 microglia express significantly less P2RY12 receptors compared to APOE3 microglia despite no changes in P2RY12 transcripts. To examine if the effect of APOE4 on the microglial response to ATP also applied to amyloid β (Aβ), we infused locally Hi-Lyte Fluor 555-labeled Aβ in acute brain slices of 6-month-old mice and imaged microglia movement for 2 h. APOE4 microglia showed a significantly slower (p < 0.0001) process movement toward the Aβ, and less Aβ coverage at early time points after Aβ injection. To test whether P2RY12 is involved in process movement in response to Aβ, we treated acute brain slices with a P2RY12 antagonist before Aβ injection; microglial processes no longer migrated towards Aβ. These results provide mechanistic insights into the impact of APOE4 genotype and aging in dynamic microglial behaviors prior to gross Aβ pathology and could help explain how APOE4 brains are more susceptible to AD pathogenesis.

中文翻译：

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APOE4 基因型和衰老通过 P2RY12 损害脑切片中损伤诱导的小胶质细胞行为，包括针对 Aβ 的行为

小胶质细胞是高度动态的细胞，通过监视脑实质和对损伤相关线索的反应，在组织稳态中发挥关键作用。衰老和 APOE4 基因型是阿尔茨海默病 (AD) 的最强危险因素，但它们如何影响小胶质细胞动态仍不清楚。使用离体共聚焦显微镜，我们分析了在 CX3CR1 启动子下表达 GFP 的 6、12 和 21 月龄小鼠 APOE3 或 APOE4 敲入小鼠的内嗅皮层 (EC) 和海马 CA1 中的小胶质细胞动态行为。为了研究小胶质细胞的监测，我们对小胶质细胞基线运动进行了 20 分钟的成像，并测量了过程的延伸和缩回。我们发现，与 6 个月大的小鼠中的 APOE3 小胶质细胞相比，APOE4 小胶质细胞表现出明显较少的大脑监视能力 (27%)；老龄化加剧了这种赤字。为了测量小胶质细胞对损伤的反应，我们对 ATP（一种损伤相关信号）反应的过程运动进行了 30 分钟的成像。我们发现，在 6 个月大的动物中，APOE4 小胶质细胞的延伸速度明显慢于 APOE3 小胶质细胞 (1.1 μm/min) (0.9 µm/min，p < 0.005)。在 12 个月和 21 个月大的动物中观察到与 APOE 相关的小胶质细胞运动性改变，并且这种影响随着 APOE4 小胶质细胞的衰老而加剧。我们测量了 P2RY12 的蛋白质和 mRNA 水平，P2RY12 是响应损伤过程运动所需的核心小胶质细胞受体。我们发现，尽管 P2RY12 转录本没有变化，但与 APOE3 小胶质细胞相比，APOE4 小胶质细胞表达的 P2RY12 受体显着减少。为了检查 APOE4 对小胶质细胞对 ATP 反应的影响是否也适用于淀粉样蛋白 β (Aβ)，我们将 Hi-Lyte Fluor 555 标记的 Aβ 局部注入 6 个月大小鼠的急性脑切片中，并对小胶质细胞运动进行了 2 小时的成像。 H。APOE4 小胶质细胞显示出向 Aβ 移动的速度明显较慢 (p < 0.0001)，并且在 Aβ 注射后的早期时间点 Aβ 覆盖范围较小。为了测试 P2RY12 是否参与响应 Aβ 的过程运动，我们在注射 Aβ 之前用 P2RY12 拮抗剂处理急性脑切片；小胶质细胞过程不再向 Aβ 迁移。这些结果提供了关于 APOE4 基因型和衰老对 Aβ 病理发生之前动态小胶质细胞行为的影响的机制见解，并有助于解释 APOE4 大脑如何更容易受到 AD 发病机制的影响。