APOE4 genotype confers transcriptomic and functional alterations to primary mouse microglia

Relative to E3 microglia, E4 microglia exhibit altered morphology, increased numbers of endosomes and lysosomes, increased cytokine/chemokine production, and increased lipid accumulation at baseline. These changes were accompanied by increased activation of kinases that mediate global repression of translation. In a subsequent phagocytosis assay, E4 microglia exhibited increased accumulation of a variety of substrates when compared to E3 microglia. While transcriptomic profiling of myelin-challenged microglia revealed a largely overlapping response profile across genotypes, differential regulation of several pathways including interferon-related signaling, translation-related processes was identified in E4 versus E3 microglia both at baseline and following myelin challenge. Here we identify and describe several important functional alterations in E4 relative to E3 microglia. Together, our results suggest E4 genotype confers elevated levels of baseline stress to microglia relative to E3 genotype, even prior to treatment with exogenous stimuli. This work provides insight into the molecular mechanisms by which E4 genotype may increase risk for AD. The major genetic risk factor for AD is Apolipoprotein E (APOE) genotype, with the ε4/ε4 genotype increasing risk for AD by ten fold compared to the most common ε3/ε3 genotype. However, the impact of APOE genotype on microglial function has not been thoroughly investigated. To address this, we cultured primary microglia from mice in which both alleles of the mouse Apoe gene have been humanized to encode either human APOE ε3 (E3) or APOE ε4 (E4). Using a combination of immunocytochemistry, immunoblotting, and flow cytometry we profiled functional differences in the endolysosomal system, lipid accumulation, phagocytosis, and cytokine secretion across E3 and E4 microglia both at baseline and in response to an acute (~3 hour) myelin challenge. RNA sequencing was used to assess differences in transcriptional profiles across genotypes and treatment conditions.

与E3小胶质细胞（microglia）相比，E4小胶质细胞在基础状态下即表现出形态学改变、内体与溶酶体数量增多、细胞因子/趋化因子生成增加以及脂质蓄积加重。这些变化同时伴随介导翻译全局抑制的激酶激活水平升高。在后续的吞噬功能实验中，与E3小胶质细胞相比，E4小胶质细胞对多种底物的蓄积量更高。尽管对髓鞘刺激后的小胶质细胞进行转录组分析显示，不同基因型的应答谱整体高度重合，但无论是在基础状态还是髓鞘刺激后，E4与E3小胶质细胞在干扰素相关信号通路、翻译相关进程等多条通路的调控上均存在差异。本研究鉴定并阐述了E4相较于E3小胶质细胞的多项重要功能改变。综合来看，我们的研究结果表明，即便未施加外源性刺激，E4基因型也会使小胶质细胞相较于E3基因型处于更高水平的基础应激状态。本研究为阐明E4基因型增加阿尔茨海默病（AD, Alzheimer's Disease）发病风险的分子机制提供了新的见解。阿尔茨海默病最主要的遗传风险因素为载脂蛋白E（APOE, Apolipoprotein E）基因型，其中ε4/ε4基因型相较于最常见的ε3/ε3基因型，可使AD发病风险升高10倍。然而，APOE基因型对小胶质细胞功能的影响尚未得到全面深入的研究。为填补这一研究空白，我们通过基因人源化改造小鼠的Apoe基因，使其两个等位基因分别编码人类APOE ε3（记为E3）或APOE ε4（记为E4），并以此培养原代小胶质细胞。我们联合运用免疫细胞化学、免疫印迹及流式细胞术，对基础状态下以及经急性（约3小时）髓鞘刺激后的E3与E4小胶质细胞，在内体溶酶体系统功能、脂质蓄积、吞噬功能及细胞因子分泌等方面的差异进行了系统分析。我们通过RNA测序技术，评估了不同基因型及处理条件下小胶质细胞的转录组差异。