Transcriptomic and functional analysis of Ab1-42 oligomer-stimulated human monocyte-derived microglia-like cells

Dysregulation of microglial function contributes to Alzheimer’s disease (AD) pathogenesis. Several genetic and transcriptome studies have revealed microglia specific genetic risk factors, and changes in microglia expression profiles in AD pathogenesis, viz. the human-Alzheimer’s microglia/myeloid (HAM) profile in AD patients and the disease-associated microglia profile (DAM) in AD mouse models. The transcriptional changes involve genes in immune and inflammatory pathways, and in pathways associated with Aβ clearance. Aβ oligomers have been suggested to be the initial trigger of microglia activation in AD. To study the direct response to Aβ oligomers exposure, we assessed changes in gene expression in an in vitro model for microglia, the human monocyte-derived microglial-like (MDMi) cells. We confirmed the initiation of an inflammatory profile following LPS stimulation, based on increased expression of IL1B, IL6, and TNFα. In contrast, the Ab1-42 oligomers did not induce an inflammatory profile or a classical HAM or DAM profile. Interestingly, we observed a specific increase in the expression of metallothioneins in the Aβ1-42 oligomer treated MDMi cells. Metallothioneins are involved in metal ion regulation, protection against reactive oxygen species, and have anti-inflammatory properties. In conclusion, our data suggests that Aβ1-42 oligomers may trigger a protective response both in vitro and in vivo. Bulk RNAseq analysis on MDMi cells stimulated with AB1-42 oligomers, LPS, PBS and vehicle. Libraries were de-multiplexed and raw reads were aligned to the hg19 human RefSeq transcriptome with Burrows-Wheeler Aligner (BWA) (Li and Durbin, 2010). Duplicate reads and reads that mapped equally well to multiple locations were discarded. The quality control, normalization, and identification of differentially expressed genes were done with DESeq2, an R (version 3.6.3) based package (Love MI et al 2014).

小胶质细胞（microglia）功能失调参与阿尔茨海默病（Alzheimer’s disease, AD）的发病机制。多项遗传学与转录组学研究已揭示了小胶质细胞特异性遗传风险因子，以及AD发病进程中小胶质细胞表达谱的改变，即AD患者体内的人阿尔茨海默病小胶质细胞/髓系细胞（human-Alzheimer’s microglia/myeloid, HAM）特征，以及AD小鼠模型中的疾病相关小胶质细胞（disease-associated microglia, DAM）特征。此类转录组变化涉及免疫、炎症通路以及与Aβ清除相关通路中的基因。淀粉样β蛋白寡聚体（Aβ oligomers）被认为是AD中小胶质细胞活化的初始触发因素。为探究细胞对Aβ寡聚体暴露的直接应答，本研究以人单核细胞源性类小胶质细胞（human monocyte-derived microglial-like, MDMi）这一小胶质细胞体外模型为对象，评估其基因表达的变化。研究人员通过检测IL1B、IL6及肿瘤坏死因子α（Tumor Necrosis Factor α, TNFα）的表达上调，证实脂多糖（Lipopolysaccharide, LPS）刺激可诱导炎症特征谱的激活。与之相反，Aβ1-42寡聚体并未诱导炎症特征谱，亦未诱导典型的HAM或DAM特征谱。值得注意的是，本研究观察到经Aβ1-42寡聚体处理的MDMi细胞中，金属硫蛋白（metallothioneins）的表达出现特异性上调。金属硫蛋白参与金属离子调控、活性氧（reactive oxygen species, ROS）清除，并具备抗炎特性。综上，本研究数据表明Aβ1-42寡聚体可能在体外与体内环境中触发保护性应答。本研究对经Aβ1-42寡聚体、LPS、磷酸盐缓冲液（PBS）及溶剂对照处理的MDMi细胞进行了批量RNA测序（bulk RNAseq）分析。测序文库经解多重处理后，使用Burrows-Wheeler比对工具（Burrows-Wheeler Aligner, BWA）将原始读段比对至hg19版本的人类RefSeq转录组（Li和Durbin, 2010）。本研究剔除了重复读段以及可同等比对至多基因组位点的读段。基因表达的质量控制、标准化以及差异表达基因的筛选，均通过基于R语言（version 3.6.3）的DESeq2软件包完成（Love MI等, 2014）。