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A reporter cell system for the triggering receptor expressed on myeloid cells 2 reveals differential effects of disease‐associated variants on receptor signaling and activation by antibodies against the stalk region
Glia ( IF 5.4 ) Pub Date : 2020-12-14 , DOI: 10.1002/glia.23953 Melanie Ibach 1 , Mona Mathews 2 , Bettina Linnartz-Gerlach 3 , Sandra Theil 1 , Sathish Kumar 1 , Regina Feederle 4, 5 , Oliver Brüstle 2, 6 , Harald Neumann 3 , Jochen Walter 1
Glia ( IF 5.4 ) Pub Date : 2020-12-14 , DOI: 10.1002/glia.23953 Melanie Ibach 1 , Mona Mathews 2 , Bettina Linnartz-Gerlach 3 , Sandra Theil 1 , Sathish Kumar 1 , Regina Feederle 4, 5 , Oliver Brüstle 2, 6 , Harald Neumann 3 , Jochen Walter 1
Affiliation
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The triggering receptor expressed on myeloid cells 2 (TREM2) is an immune receptor expressed on myeloid‐derived cell types. The extracellular immunoglobulin‐like domain of TREM2 binds anionic ligands including Apolipoprotein E and Amyloid‐β. The transmembrane domain interacts with its adaptor protein DAP12/TYROBP that is responsible for propagation of downstream signaling upon ligand interaction. Several sequence variants of TREM2 have been linked to different neurodegenerative diseases including Alzheimer's disease. Here, we generated HEK 293 Flp‐In cell lines stably expressing human TREM2 and DAP12 using a bicistronic construct with a T2A linker sequence allowing initial expression of both proteins in stoichiometric amounts. Cell biological and biochemical analyses revealed transport of TREM2 to the cell surface, and canonical sequential proteolytic processing and shedding of TREM2 (sTREM2). The functionality of this cell system was demonstrated by detection of phosphorylated spleen tyrosine kinase (SYK) upon stimulation of TREM2 with the anionic membrane lipid phosphatidylserine or anti‐TREM2 antibodies. Using this cell model, we demonstrated impaired signaling of disease associated TREM2 variants. We also identified a monoclonal antibody against the stalk region of TREM2 with agonistic activity. Activation of TREM2‐DAP12 signaling with the monoclonal antibody and the partial loss of function of disease associated variants were recapitulated in induced pluripotent stem cell derived microglia. Thus, this reporter cell model represents a suitable experimental system to investigate signaling of TREM2 variants, and for the identification of ligands and compounds that modulate TREM2‐DAP12 signaling.
中文翻译：
用于在骨髓细胞上表达的触发受体的报告细胞系统 2 揭示了疾病相关变体对受体信号传导和针对茎部区域的抗体激活的不同影响
骨髓细胞 2 (TREM2) 上表达的触发受体是一种在骨髓衍生细胞类型上表达的免疫受体。TREM2 的细胞外免疫球蛋白样结构域结合阴离子配体，包括载脂蛋白 E 和淀粉样蛋白-β。跨膜结构域与其衔接蛋白 DAP12/TYROBP 相互作用，后者负责在配体相互作用时传播下游信号。TREM2 的几个序列变体与不同的神经退行性疾病有关，包括阿尔茨海默病。在这里，我们使用具有 T2A 接头序列的双顺反子构建体生成了稳定表达人 TREM2 和 DAP12 的 HEK 293 Flp-In 细胞系，允许两种蛋白质以化学计量的初始表达。细胞生物学和生化分析显示 TREM2 转运到细胞表面，以及 TREM2 (sTREM2) 的典型顺序蛋白水解加工和脱落。该细胞系统的功能通过在用阴离子膜脂质磷脂酰丝氨酸或抗 TREM2 抗体刺激 TREM2 后检测磷酸化脾酪氨酸激酶 (SYK) 来证明。使用这种细胞模型，我们证明了与疾病相关的 TREM2 变体的信号传导受损。我们还鉴定了一种具有激动活性的针对 TREM2 茎区的单克隆抗体。用单克隆抗体激活 TREM2-DAP12 信号传导和疾病相关变体的部分功能丧失在诱导的多能干细胞衍生的小胶质细胞中得到了概括。因此，这个报告细胞模型代表了一个合适的实验系统来研究 TREM2 变体的信号传导，
更新日期：2020-12-14
中文翻译：
用于在骨髓细胞上表达的触发受体的报告细胞系统 2 揭示了疾病相关变体对受体信号传导和针对茎部区域的抗体激活的不同影响
骨髓细胞 2 (TREM2) 上表达的触发受体是一种在骨髓衍生细胞类型上表达的免疫受体。TREM2 的细胞外免疫球蛋白样结构域结合阴离子配体，包括载脂蛋白 E 和淀粉样蛋白-β。跨膜结构域与其衔接蛋白 DAP12/TYROBP 相互作用，后者负责在配体相互作用时传播下游信号。TREM2 的几个序列变体与不同的神经退行性疾病有关，包括阿尔茨海默病。在这里，我们使用具有 T2A 接头序列的双顺反子构建体生成了稳定表达人 TREM2 和 DAP12 的 HEK 293 Flp-In 细胞系，允许两种蛋白质以化学计量的初始表达。细胞生物学和生化分析显示 TREM2 转运到细胞表面，以及 TREM2 (sTREM2) 的典型顺序蛋白水解加工和脱落。该细胞系统的功能通过在用阴离子膜脂质磷脂酰丝氨酸或抗 TREM2 抗体刺激 TREM2 后检测磷酸化脾酪氨酸激酶 (SYK) 来证明。使用这种细胞模型，我们证明了与疾病相关的 TREM2 变体的信号传导受损。我们还鉴定了一种具有激动活性的针对 TREM2 茎区的单克隆抗体。用单克隆抗体激活 TREM2-DAP12 信号传导和疾病相关变体的部分功能丧失在诱导的多能干细胞衍生的小胶质细胞中得到了概括。因此，这个报告细胞模型代表了一个合适的实验系统来研究 TREM2 变体的信号传导，
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