Department of Pathology and Laboratory Medicine, School of Medicine, University of California, Irvine, CA, USA.
Department of Developmental and Cell Biology, University of California, Irvine, CA, USA.
Mol Neurodegener. 2017 Sep 18;12(1):66. doi: 10.1186/s13024-017-0210-z.
Pharmacologic inhibition of C5aR1, a receptor for the complement activation proinflammatory fragment, C5a, suppressed pathology and cognitive deficits in Alzheimer's disease (AD) mouse models. To validate that the effect of the antagonist was specifically via C5aR1 inhibition, mice lacking C5aR1 were generated and compared in behavior and pathology. In addition, since C5aR1 is primarily expressed on cells of the myeloid lineage, and only to a lesser extent on endothelial cells and neurons in brain, gene expression in microglia isolated from adult brain at multiple ages was compared across all genotypes.
C5aR1 knock out mice were crossed to the Arctic AD mouse model, and characterized for pathology and for behavior performance in a hippocampal dependent memory task. CX3CR1 and CCR2 reporter mice were bred to C5aR1 sufficient and knockout wild type and Arctic mice to enable sorting of microglia (GFP-positive, RFP-negative) isolated from adult brain at 2, 5, 7 and 10 months of age followed by RNA-seq analysis.
A lack of C5aR1 prevented behavior deficits at 10 months, although amyloid plaque load was not altered. Immunohistochemical analysis showed no CCR2 monocytes/macrophages near the plaques in the Arctic brain with or without C5aR1. Microglia were sorted from infiltrating monocytes (GFP and RFP-positive) for transcriptome analysis. RNA-seq analysis identified inflammation related genes as differentially expressed, with increased expression in the Arctic mice relative to wild type and decreased expression in the Arctic/C5aR1KO relative to Arctic. In addition, phagosomal-lysosomal gene expression was increased in the Arctic mice relative to wild type but further increased in the Arctic/C5aR1KO mice. A decrease in neuronal complexity was seen in hippocampus of 10 month old Arctic mice at the time that correlates with the behavior deficit, both of which were rescued in the Arctic/C5aR1KO.
These data are consistent with microglial polarization in the absence of C5aR1 signaling reflecting decreased induction of inflammatory genes and enhancement of degradation/clearance pathways, which is accompanied by preservation of CA1 neuronal complexity and hippocampal dependent cognitive function. These results provide links between microglial responses and loss of cognitive performance and, combined with the previous pharmacological approach to inhibit C5aR1 signaling, support the potential of this receptor as a novel therapeutic target for AD in humans.
C5aR1 是补体激活炎症片段 C5a 的受体，对其进行药理学抑制可抑制阿尔茨海默病（AD）小鼠模型的病理和认知缺陷。为了验证拮抗剂的作用是通过 C5aR1 抑制特异性实现的，生成了缺乏 C5aR1 的小鼠并对其进行了行为和病理比较。此外，由于 C5aR1 主要表达于髓系细胞，而在脑内皮细胞和神经元中仅少量表达，因此比较了来自不同基因型成年大脑的小胶质细胞中的基因表达。
将 C5aR1 敲除小鼠与北极 AD 小鼠模型杂交，并对其进行病理学和海马依赖性记忆任务中的行为表现进行特征分析。将 CX3CR1 和 CCR2 报告基因小鼠与 C5aR1 充足和野生型以及北极野生型和 AD 小鼠杂交，以便对来自 2、5、7 和 10 个月龄成年大脑的小胶质细胞（GFP 阳性，RFP 阴性）进行分选，然后进行 RNA-seq 分析。
缺乏 C5aR1 可防止 10 个月时的行为缺陷，尽管淀粉样斑块负荷没有改变。免疫组织化学分析显示，在北极大脑中，无论是否存在 C5aR1，CCR2 单核细胞/巨噬细胞都不在斑块附近。从小胶质细胞中分离出浸润的单核细胞（GFP 和 RFP 阳性）进行转录组分析。RNA-seq 分析确定了差异表达的炎症相关基因，与野生型相比，北极小鼠中的表达增加，与北极小鼠相比，北极/C5aR1KO 中的表达减少。此外，与野生型相比，北极小鼠的吞噬溶酶体基因表达增加，但在北极/C5aR1KO 小鼠中进一步增加。在与行为缺陷相关的时间点，10 个月大的北极小鼠的海马神经元复杂性降低，而在北极/C5aR1KO 中得到挽救。
这些数据与 C5aR1 信号缺失时小胶质细胞极化一致，反映了炎症基因诱导减少和降解/清除途径增强，同时伴有 CA1 神经元复杂性和海马依赖性认知功能的保留。这些结果将小胶质细胞反应与认知功能丧失联系起来，结合之前抑制 C5aR1 信号的药理学方法，支持该受体作为人类 AD 的新型治疗靶点的潜力。
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