Journal: Alzheimer's Research & Therapy
Article Title: TREM2 Alzheimer’s variant R47H causes similar transcriptional dysregulation to knockout, yet only subtle functional phenotypes in human iPSC-derived macrophages
Figure Lengend Snippet: Extracellular matrix-adhesion modifiers and adhesion to vitronectin are dysregulated in R47H TREM2 and TREM2 KO pMac. TGFβ treatment does not rescue adhesion deficits. a Validation of selected RNA-seq hits by qRT-PCR of unstimulated pMac. Means ± SEM, for N = 7 harvests, including the 3 samples originally used for RNA-seq (open symbols) plus 4 samples harvested independently from a separate differentiation (filled symbols). Repeated-measures 1-way ANOVA, with Dunnett’s post hoc test, pairwise comparisons to the WT. b Effect of TGFβ-stimulation (50 ng/mL, 24 h) on mRNA levels of selected RNA-seq hits, measured by qRT-PCR. Means ± SEM, for N = 3 samples harvested independently to Fig. 5 a. Two-way ANOVA, with Sidak’s post hoc test. c TREM2 KO pMac secrete reduced levels of TGFβ1 compared with WT, and TGFβ1 secretion is partly SYK-dependent. Total (inactive and active) TGFβ1 measured from supernatants by ELISA, cells treated ± OXSI-2 (2 μM, 24 h) to inhibit SYK. Means ± SEM, for N = 3 harvests. Two-way ANOVA, with Sidak’s post hoc test. d Fibronectin protein expression is reduced in both R47H TREM2 and TREM2 KO versus WT. Fibronectin measured by Western blotting of pMac ± TGFβ1 stimulation (50 ng/mL, 24 h). Means ± SEM, quantified for N = 3 harvests on separate blots. Two-way ANOVA, with Sidak’s post hoc test. TGFβ1 vs unstimulated was not significant. e αVβ3 complex formation is reduced in both R47H TREM2 and TREM2 KO versus WT. Intact surface integrins αVβ3 and αVβ5 measured by flow cytometry in pMac ± TGFβ1 stimulation (50 ng/mL, 24 h). Data is expressed as the difference in median fluorescence intensity of the specific antibody versus isotype control, normalised (by subtraction) to the average for the harvest. Means ± SEM, for N = 3 harvests. Two-way ANOVA, with Sidak’s post hoc test. f Adhesion to vitronectin is reduced for both R47H TREM2 and TREM2 KO versus WT, and treatment with TGFβ (50 ng/mL, 24 h prior to assay) increases αVβ3/5-dependent adhesion. Adhesion measured after 3 h by crystal violet colorimetric assay, and normalised to BSA-blocked wells (by division, and the result subtracted from 1). αVβ3/5 inhibitor (10 μM cilengitide) was added at the start of the assay to determine αVβ3/5-specific adhesion to vitronectin (striped bars). Means ± SEM, for N = 3 harvests. Two-way ANOVA, with Dunnett’s post hoc test. Black annotations compare stimulations to unstimulated control. Grey annotations compare R47H or KO versus WT for each condition. WT = grey circles, R47H = orange squares, TREM2 KO = burgundy triangles. * p
Article Snippet: TREM2 functions at the cell surface; therefore, proteins were purified by cell surface biotinylation and TREM2 detected by Western blotting, with comparison to the input cell homogenate (Fig. b).
Techniques: RNA Sequencing Assay, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Expressing, Western Blot, Flow Cytometry, Fluorescence, Colorimetric Assay