TREM2 Alzheimer's variant R47H causes similar transcriptional dysregulation to knockout, yet only subtle functional phenotypes in human iPSC-derived macrophages

TREM2 is a microglial cell surface receptor, with risk mutations linked to Alzheimer's disease (AD), including R47H. TREM2 signalling via SYK aids phagocytosis, chemotaxis, survival, and changes to microglial activation state. In AD mouse models, knockout (KO) of TREM2 impairs microglial clustering around amyloid, and prevents microglial activation. The R47H mutation is proposed to reduce TREM2 ligand binding. We investigated cell phenotypes of the R47H mutant and TREM2 KO in a model of human microglia, and compared their transcriptional signatures, to determine the mechanism by which R47H TREM2 disrupts function. We generated human microglia-like iPSC-macrophages (pMac) from isogenic induced pluripotent stem cell (iPSC) lines, with homozygous R47H mutation or TREM2 knockout (KO). We firstly validated the effect of the R47H mutant on TREM2 surface and subcellular localization in pMac. To assess microglial phenotypic function we measured phagocytosis of dead neurons, cell morphology, directed migration, survival, and LPS-induced inflammation. We performed bulk RNA-seq, comparing significant differentially-expressed genes (DEGs: p<0.05) between the R47H and KO versus WT, and bioinformatically predicted potential upstream regulators of TREM2-mediated gene expression. Altered gene expression in the R47H TREM2 pMac overlapped by 90% with the TREM2 KO, and was characterized by dysregulation of genes involved with immunity, proliferation, activation, chemotaxis and adhesion. Downregulated mediators of ECM adhesion included the vitronectin receptor aVß3, and consequently R47H TREM2 pMac adhered weakly to vitronectin compared with WT pMac. To counteract these transcriptional defects, we investigated TGFß1, as a candidate upstream regulator. TGFß1 failed to rescue vitronectin adhesion of pMac, although improved aVß3 expression. The R47H mutation is not sufficient to cause gross phenotypic defects of human pMac under standard culture conditions. However, overlapping transcriptional defects with TREM2 KO supports the hypothesised partial loss-of-function effects of the R47H mutation. Furthermore, transcriptomics can guide us to more subtle phenotypic defects in the R47H cells, such as reduced cell adhesion, and can be used to predict targets for therapeutic intervention. Overall design: We generated human microglia-like iPSC-macrophages (pMac) from isogenic induced pluripotent stem cell (iPSC) lines, with homozygous TREM2 R47H mutation (R47H), TREM2 knockout (KO) or a wildtype condtion (WT). Bulk RNA sequencing with full length transcripts and polyA library prepration was perfromed on a total of 9 samples with 3 replicates harvested at 7,8, and 9 differentiation weeks for each of the conditions.

TREM2是一种小胶质细胞表面受体，其风险突变与阿尔茨海默病（Alzheimer's disease, AD）相关，其中包括R47H突变。TREM2通过SYK（脾酪氨酸激酶，Spleen tyrosine kinase, SYK）介导的信号转导，可促进吞噬作用、趋化作用与细胞存活，并调控小胶质细胞的激活状态。在AD小鼠模型中，敲除（knockout, KO）TREM2会损害小胶质细胞在淀粉样蛋白斑块周围的聚集，并阻止小胶质细胞激活。研究推测R47H突变会降低TREM2的配体结合能力。本研究在人类小胶质细胞模型中探究了R47H突变体与TREM2敲除（KO）细胞的表型，并比对二者的转录组特征，以阐明R47H突变型TREM2破坏细胞功能的分子机制。

我们从同基因诱导多能干细胞（induced pluripotent stem cell, iPSC）系中构建了人类小胶质细胞样iPSC-巨噬细胞（pMac），这些细胞分别携带纯合R47H突变或TREM2敲除（KO）基因型。我们首先验证了R47H突变对pMac中TREM2的表面及亚细胞定位的影响。为评估小胶质细胞的表型功能，我们检测了其对死亡神经元的吞噬能力、细胞形态、定向迁移能力、细胞存活情况以及脂多糖（Lipopolysaccharide, LPS）诱导的炎症反应。我们开展了批量RNA测序（bulk RNA-seq），比对R47H突变组、KO组与野生型（wildtype, WT）组间具有显著差异的表达基因（differentially-expressed genes, DEGs：p<0.05），并通过生物信息学方法预测了TREM2介导的基因表达的潜在上游调控因子。R47H突变型TREM2 pMac中的基因表达改变与TREM2 KO组的重叠度达90%，其特征为免疫、增殖、激活、趋化及黏附相关基因的表达失调。细胞外基质（extracellular matrix, ECM）黏附的下调介导因子包括玻连蛋白受体αVβ3，因此与WT pMac相比，R47H突变型TREM2 pMac对玻连蛋白的黏附能力较弱。为逆转这些转录组缺陷，我们将转化生长因子β1（Transforming growth factor beta 1, TGFβ1）作为潜在上游调控因子进行了探究。尽管TGFβ1可提升αVβ3的表达，但未能挽救pMac对玻连蛋白的黏附能力。在标准培养条件下，R47H突变不足以引发人类pMac的显著表型缺陷。但与TREM2 KO组重叠的转录组缺陷，支持了R47H突变存在部分功能丧失效应的假说。此外，转录组学分析可帮助我们发现R47H突变细胞中更细微的表型缺陷，比如细胞黏附能力降低，还可用于预测治疗干预的靶点。

整体实验设计：我们从同基因诱导多能干细胞（iPSC）系中构建了人类小胶质细胞样iPSC-巨噬细胞（pMac），分别为纯合TREM2 R47H突变组（R47H）、TREM2敲除组（KO）以及野生型组（WT）。我们对所有组别分别在分化第7、8、9周收集的3份生物学重复样本共计9个样品，开展了带有全长转录本和polyA文库制备的批量RNA测序。