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 Aß clearance. Aß oligomers have been suggested to be the initial trigger of microglia activation in AD. To study the direct response to Aß 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 TNFa. 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 Aß1-42 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 Aß1-42 oligomers may trigger a protective response both in vitro and in vivo. Overall design: 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。多项遗传与转录组研究已揭示小胶质细胞特异性遗传风险因子，以及AD发病过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（human-Alzheimer's microglia/myeloid, HAM）特征与AD小鼠模型中的疾病相关小胶质细胞（disease-associated microglia, DAM）特征。转录组变化涉及免疫、炎症通路及与Aβ清除相关的通路。Aβ寡聚体被认为是AD中小胶质细胞活化的初始触发因素。
为探究Aβ寡聚体暴露后的直接应答，我们在小胶质细胞体外模型——人单核细胞源性小胶质细胞样（human monocyte-derived microglial-like, MDMi）细胞中评估了基因表达的变化。我们证实脂多糖（lipopolysaccharide, LPS）刺激可启动炎症特征，表现为IL1B、IL6及TNFα的表达上调。与之相反，Aβ1-42寡聚体并未诱导炎症特征，也未触发典型的HAM或DAM特征。值得注意的是，我们观察到经Aβ1-42寡聚体处理的MDMi细胞中，金属硫蛋白的表达出现特异性升高。金属硫蛋白参与金属离子调节、活性氧防护，并具有抗炎活性。
综上，本研究数据表明Aβ1-42寡聚体可能在体外及体内触发保护性应答。
整体实验设计：对经Aβ1-42寡聚体、LPS、磷酸盐缓冲液（phosphate-buffered saline, PBS）及溶剂处理的MDMi细胞进行批量RNA测序（Bulk RNAseq）分析。对文库进行解多路复用，并使用Burrows-Wheeler比对工具（Burrows-Wheeler Aligner, BWA）（Li与Durbin, 2010）将原始测序读段比对至hg19人类RefSeq转录组。剔除重复读段及比对到多个基因组位置的读段。质量控制、标准化及差异表达基因的鉴定通过基于R（版本3.6.3）的DESeq2工具完成（Love MI等, 2014）。