Skip to main content
Fig. 1 | Alzheimer's Research & Therapy

Fig. 1

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

Fig. 1

Reduced cell surface localization of R47H TREM2 does not impair antibody-mediated activation of TREM2 in pMac. a Schematic of microglia phenotypes investigated in this study. b, c Reduced cell surface expression of R47H TREM2. Cell surface proteins on pMac were biotinylated and pulled down, the level of TREM2 protein enrichment was measured by Western blotting vs whole cell lysate, probed on separate blots. c Means ± SEM, for N = 5 harvests measured on separate Western blots. 2-tailed paired t-test: ** p = 0.0011 for R47H versus WT. d Increased sTREM2 production from R47H TREM2 pMac. sTREM2 were measured from unstimulated pMac supernatants by ELISA. Means ± SEM, for N = 3 harvests measured on same ELISA plate, data for each harvest was normalised to the average cell count. 1-way ANOVA, with Dunnett’s post hoc test: p = 0.0005 R47H versus WT (***), p = 0.0066 KO versus WT (**). e, f Co-localization of TREM2 with subcellular compartment markers in fixed and permeabilized pMac, images are a confocal slice at 4 μm, taken with Opera Phenix microscope. Inset panels are × 3 magnification of a selected cell. Calnexin used as a marker for ER, TGN46 for TGN, and LAMP1 for lysosomes. f Co-localization expressed as a ratio of TREM2 intensity to compartment marker intensity, in regions automatically segmented by high marker staining. Means ± SEM, for N = 3 harvests, each in triplicate wells. 2-way ANOVA, with Bonferroni’s post hoc test, no significant differences. g, h TREM2-activating antibody stimulation (used at concentration of 2.4 μg/1 × 106 cells and 3.84 μg/mL, for 10 min) of both WT and R47H TREM2 pMac caused SYK phosphorylation, measured by Western blotting. No response seen in TREM2 antibody-stimulated TREM2 KO cells, or cells treated with a goat IgG isotype control. h Means ± SEM, for N = 3 harvests measured on separate Western blots. 2-way ANOVA, with Sidak’s post hoc test, pairwise comparisons to WT for each treatment: p < 0.001 for KO stimulated with TREM2 Ab versus WT (****). i Calcium response in response to TREM2 antibody is similar in WT and R47H TREM2 pMac, measured by peak Fluo4-AM fluorescence, normalised to minimum fluorescence and cell number. Means ± SEM, for N = 3 harvests. 2-way ANOVA, with Dunnett’s multiple comparison test, pairwise comparisons to WT for each treatment: p = 0.0049 for KO stimulated with TREM2 Ab versus WT (**)

Back to article page