TREM2 Alzheimer's variant R47H causes similar transcriptional dysregulation to knockout, yet only subtle functional phenotypes in human iPSC-derived macrophages.
TREM2 Alzheimer's variant R47H causes similar transcriptional dysregulation to knockout, yet only subtle functional phenotypes in human iPSC-derived macrophages.
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DOI：
10.1186/s13195-020-00709-z
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发表时间：
2020-11-16
期刊：
影响因子：
--
通讯作者：
Cowley SA
中科院分区：
文献类型：
--
作者：
Hall-Roberts H;Agarwal D;Obst J;Smith TB;Monzón-Sandoval J;Di Daniel E;Webber C;James WS;Mead E;Davis JB;Cowley SA
TREM2是一种小胶质细胞表面受体，具有与阿尔茨海默病(AD)相关的风险突变，包括R47H。TREM2信号通过SYK帮助吞噬、趋化、存活和改变到小胶质细胞的激活状态。在AD小鼠模型中，TREM2基因敲除(KO)损害了淀粉样蛋白周围的小胶质细胞聚集，并阻止了小胶质细胞的激活。R47H突变被认为是减少TREM2配体结合的原因。我们在人小胶质细胞模型中研究了R47H突变体和TREM2 KO的细胞表型，并比较了它们的转录特征，以确定R47H TREM2干扰功能的机制。我们从R47H纯合子突变或TREM2基因敲除(KO)的同源诱导多能干细胞(IPSC)系中产生了人小胶质细胞样IPSC-巨噬细胞(PMAC)。我们首先验证了R47H突变体对TREM2表面和PMAC亚细胞定位的影响。为了评估小胶质细胞的表型功能，我们测量了死亡神经元的吞噬功能、细胞形态、定向迁移、存活和内毒素诱导的炎症。我们进行了批量RNA-SEQ，比较了R47H和KO与WT之间显著差异表达的基因(degs；p <；TREM20.05)，并通过生物信息预测了 介导的基因表达的潜在上游调控因子。R47H改善了TREM2的表面表达和脱落，但不影响TREM2介导的信号传递，也不影响TREM2 KO中失调的大体表型(吞噬、运动、存活)。然而，R47H TREM2 PMAC中基因表达的变化与TREM2 KO重叠了90%，其特征是与免疫、增殖、激活、趋化和黏附相关的基因调控失调。玻璃连结蛋白受体αVβ3是细胞外基质黏附下调的介质，因此，与WT PMAC相比，R47H TREM2 PMAC与玻璃连结蛋白的黏附能力较弱。为了抵消这些转录缺陷，我们研究了转化生长因子β1作为候选上游调控因子。转化生长因子β1虽能促进αVβ3的表达，但不能挽救PMAC的玻璃连结素粘附力。R47H突变不足以在标准培养条件下引起人PMAC的严重表型缺陷。然而，与TREM2 KO重叠的转录缺陷支持R47H突变的部分功能丧失效应。此外，转录学可以引导我们找到R47H细胞中更细微的表型缺陷，如细胞粘附性降低，并可用于预测治疗干预的靶点。
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.
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DOI：
10.1056/nejmoa1211851
发表时间：
2013-01-10
期刊：
The New England journal of medicine
影响因子：
--
作者：
Guerreiro R;Wojtas A;Bras J;Carrasquillo M;Rogaeva E;Majounie E;Cruchaga C;Sassi C;Kauwe JS;Younkin S;Hazrati L;Collinge J;Pocock J;Lashley T;Williams J;Lambert JC;Amouyel P;Goate A;Rademakers R;Morgan K;Powell J;St George-Hyslop P;Singleton A;Hardy J;Alzheimer Genetic Analysis Group
通讯作者：
Alzheimer Genetic Analysis Group
影响因子：
8.8
作者：
Garcia-Reitboeck P;Phillips A;Piers TM;Villegas-Llerena C;Butler M;Mallach A;Rodrigues C;Arber CE;Heslegrave A;Zetterberg H;Neumann H;Neame S;Houlden H;Hardy J;Pocock JM
通讯作者：
Pocock JM
影响因子：
14.8
作者：
Dunkley, Peter R.;Jarvie, Paula E.;Robinson, Phillip J.
通讯作者：
Robinson, Phillip J.
影响因子：
14.9
作者：
Chatr-Aryamontri A;Oughtred R;Boucher L;Rust J;Chang C;Kolas NK;O'Donnell L;Oster S;Theesfeld C;Sellam A;Stark C;Breitkreutz BJ;Dolinski K;Tyers M
通讯作者：
Tyers M
在GBA-N370S帕金森氏菌IPSC衍生的多巴胺神经元中，ER应力和自噬扰动导致细胞外α-突触核蛋白升高。
影响因子：
5.9
作者：
Fernandes HJ;Hartfield EM;Christian HC;Emmanoulidou E;Zheng Y;Booth H;Bogetofte H;Lang C;Ryan BJ;Sardi SP;Badger J;Vowles J;Evetts S;Tofaris GK;Vekrellis K;Talbot K;Hu MT;James W;Cowley SA;Wade-Martins R
通讯作者：
Wade-Martins R