James Martin Stem Cell Facility, Sir William Dunn School of Pathology, University of Oxford, Oxford, OX1 3RE, UK.
Nuffield Department of Medicine Research Building, Alzheimer's Research UK Oxford Drug Discovery Institute, University of Oxford, Oxford, OX3 7FZ, UK.
Alzheimers Res Ther. 2020 Nov 16;12(1):151. doi: 10.1186/s13195-020-00709-z.
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.
R47H modified surface expression and shedding of TREM2, but did not impair TREM2-mediated signalling, or gross phenotypes that were dysregulated in the TREM2 KO (phagocytosis, motility, survival). However, altered gene expression in the R47H TREM2 pMac overlapped by 90% with the TREM2 KO and was characterised by dysregulation of genes involved with immunity, proliferation, activation, chemotaxis, and adhesion. Downregulated mediators of ECM adhesion included the vitronectin receptor αVβ3, and consequently, R47H TREM2 pMac adhered weakly to vitronectin compared with WT pMac. To counteract these transcriptional defects, we investigated TGFβ1, as a candidate upstream regulator. TGFβ1 failed to rescue vitronectin adhesion of pMac, although it improved αVβ3 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.
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（AD）有关，包括 R47H。TREM2 通过 SYK 进行信号转导有助于吞噬、趋化、存活以及改变小胶质细胞的激活状态。在 AD 小鼠模型中，TREM2 的敲除（KO）会损害淀粉样蛋白周围小胶质细胞的聚集并阻止小胶质细胞的激活。R47H 突变被认为会降低 TREM2 配体的结合。我们研究了 R47H 突变体和 TREM2 KO 在人类小胶质细胞模型中的细胞表型，并比较了它们的转录特征，以确定 R47H TREM2 破坏功能的机制。
我们从小胶质细胞诱导多能干细胞（iPSC）系中生成具有同源纯合 R47H 突变或 TREM2 KO 的人类小胶质细胞样 iPSC-巨噬细胞（pMac）。我们首先验证了 R47H 突变对 pMac 中 TREM2 表面和亚细胞定位的影响。为了评估小胶质细胞表型功能，我们测量了死神经元的吞噬作用、细胞形态、定向迁移、存活和 LPS 诱导的炎症。我们进行了批量 RNA-seq 分析，比较了 R47H 和 KO 与 WT 之间显著差异表达基因（DEG；p < 0.05），并从生物信息学上预测了 TREM2 介导的基因表达的潜在上游调节剂。
R47H 改变了 TREM2 的表面表达和脱落，但并未损害 TREM2 介导的信号转导，或 TREM2 KO 失调的表型（吞噬作用、运动、存活）。然而，R47H TREM2 pMac 的改变基因表达与 TREM2 KO 重叠了 90%，其特征是参与免疫、增殖、激活、趋化和黏附的基因失调。细胞外基质黏附的下调调节剂包括 vitronectin 受体 αVβ3，因此，与 WT pMac 相比，R47H TREM2 pMac 对 vitronectin 的黏附能力较弱。为了对抗这些转录缺陷，我们研究了 TGFβ1，作为候选的上游调节剂。尽管 TGFβ1 改善了 αVβ3 的表达，但它未能挽救 pMac 对 vitronectin 的黏附。
在标准培养条件下，R47H 突变不足以引起人类 pMac 的明显表型缺陷。然而，与 TREM2 KO 重叠的转录缺陷支持 R47H 突变部分丧失功能的假说。此外，转录组学可以帮助我们发现 R47H 细胞中更微妙的表型缺陷，例如细胞黏附能力降低，并可用于预测治疗干预的靶点。
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