Alzheimer’s-associated PU.1 expression levels regulate microglial inflammatory response

More than forty loci contribute to genetic risk for Alzheimer’s disease (AD). These risk alleles are enriched in myeloid cell enhancers suggesting that microglia, the brain-resident macrophages, contribute to AD risk. We have previously identified SPI1/PU.1, a master regulator of myeloid cell development in the brain and periphery, as a genetic risk factor for AD. Higher expression of SPI1 is associated with increased risk for AD, while lower expression is protective. To investigate the molecular and cellular phenotypes associated with higher and lower expression of PU.1 in microglia, we used stable overexpression and knock-down of PU.1 in BV2, an immortalized mouse microglial cell line. Transcriptome analysis suggests that reduced PU.1 expression suppresses expression of homeostatic genes similar to the disease-associated microglia response to amyloid plaques in mouse models of AD. Moreover, PU.1 knock-down resulted in activation of protein translation, antioxidant action and cholesterol/lipid metabolism pathways with a concomitant decrease of pro-inflammatory gene expression. PU.1 overexpression upregulated and knock-down downregulated phagocytic uptake in BV2 cells independent of the nature of the engulfed material. However, cells with reduced PU.1 expression retained their ability to internalize myelin similar to control albeit with a delay, which aligns with their anti-inflammatory profile. Here we identified several microglial responses that are modulated by PU.1 expression levels and propose that risk association of PU.1 to AD is driven by increased pro-inflammatory response due to increased viability of cells under cytotoxic conditions. In contrast, low expression of PU.1 leads to increased cell death under cytotoxic conditions accompanied by reduced pro-inflammatory signaling that decreased A1 reactive astrocytes signature supporting the protective effect of SPI1 genotype in AD. These findings inform future in vivo validation studies and design of small molecule screens for therapeutic discovery in AD. We performed bulk RNA-sequencing of FACS-sorted BV2 cells with transient shRNA-mediated knock-down and cDNA overexpression of mouse Spi1.

超过40个基因座与阿尔茨海默病（Alzheimer’s disease, AD）的遗传易感性相关。这些风险等位基因在髓系细胞增强子（myeloid cell enhancers）中富集，提示脑驻留巨噬细胞——小胶质细胞（microglia）参与了AD的发病风险。我们此前已鉴定出SPI1/PU.1——脑与外周髓系细胞发育的主调控因子——作为AD的遗传风险因子。SPI1的高表达与AD风险升高相关，而其低表达则具有疾病保护作用。为探究小胶质细胞中PU.1表达水平异常相关的分子与细胞表型，我们在永生化小鼠小胶质细胞系BV2中实现了PU.1的稳定过表达与敲低。转录组分析（transcriptome analysis）显示，PU.1表达降低会抑制稳态基因的表达，该特征与AD小鼠模型中疾病相关小胶质细胞（disease-associated microglia）对淀粉样斑块（amyloid plaques）的应答模式相似。此外，PU.1敲低会激活蛋白质翻译（protein translation）、抗氧化作用（antioxidant action）及胆固醇/脂质代谢通路（cholesterol/lipid metabolism pathways），同时伴随促炎基因表达（pro-inflammatory gene expression）的下调。PU.1过表达会上调BV2细胞的吞噬摄取（phagocytic uptake）能力，而PU.1敲低则下调该能力，且该效应与被吞噬物质（engulfed material）的类型无关。不过，PU.1表达降低的细胞仍保留了与对照组相似的髓鞘（myelin）吞噬能力，仅吞噬过程略有延迟，这与其抗炎表型相符。本研究鉴定出若干受PU.1表达水平调控的小胶质细胞应答反应，并提出PU.1与AD的风险关联，是由细胞在细胞毒性条件下的存活能力增强所引发的促炎反应加剧所驱动。与之相反，PU.1低表达会在细胞毒性条件下增加细胞死亡，同时伴随促炎信号通路的减弱，且A1型反应性星形胶质细胞（A1 reactive astrocytes）的特征标志物表达降低，这进一步印证了SPI1基因型对AD的保护作用。上述发现为未来的体内验证研究（in vivo validation studies）以及针对AD治疗靶点发现的小分子筛选方案设计提供了重要参考。我们对经荧光激活细胞分选（FACS, fluorescence-activated cell sorting）的BV2细胞进行了批量RNA测序（bulk RNA-sequencing），这些细胞分别接受了瞬时短发夹RNA（shRNA, short hairpin RNA）介导的Spi1敲低及小鼠Spi1的互补DNA（cDNA, complementary DNA）过表达处理。