Epistatic analysis of hMGL mutations and characterization of a SORL1–TREM2–APOE regulatory axis in Aβ phagocytosis in microglia. (A) Epistasis model: TREM2 KO down-regulates APOE and other induced AD-associated DEG signatures (yellow box). SORL1 KO may up-regulate TREM2; TREM2R47H may induce APOE and other APOE-responsive or AD-related microglial DEGs (gray box). (B) SORLA, TREM2, and APOE levels in the hMGL lines indicated were detected by immunoblot; relative SORLA, TREM2, and APOE levels were normalized to actin and quantified by densitometry in the lower graphs. All values were normalized to WT hMGLs (set to 1.0). (C) WT or SORL1 KO hMGLs were transfected with 25 nM control (con) or TREM2-targeting siRNAs, and lysates were immunoblotted for the components indicated. Graphs depict SORLA, TREM2, and APOE levels individually normalized to actin in WT (black bars) and SORL1 hMGLs (purple); values were compared to WT in control or TREM2 siRNA groups (set to 1.0). (D) WT, TREM2 KO or R47H hMGLs were transfected with 25 nM control or SORL1-targeting siRNAs, and lysates were subsequently immunoblotted for the components indicated. Graphs depict SORLA, TREM2, and APOE levels individually normalized to actin in WT (black bars), TREM2 KO (yellow bars), and TREM2R47H hMGLs (green bars); values were compared with WT in control or SORL1 siRNA groups (set to 1.0). (E–G) Characterizing effects of modulating APOE on hMGL-dependent Aβ binding and uptake. (E) WT or mutant hMGLs were treated with 5 µg/ml recombinant human APOEε3 or APOEε4 or left untreated (control) in the presence of 555-Aβ1-42 oligomers. Phagocytosis of 555-Aβ1-42 oligomers was measured in real time, and phagocytic index (PI) was determined by measuring average fluorescence intensity in individual hMGL lines in comparison to the 15-min time point and normalized to WT at the 15 min time point (set to 1.0). Values represent mean ± SEM from n = 3 independent experiments. Mean phagocytotic index values for each hMGL line was quantified at the 3 h time point, as shown in the bar graph. (F) Mutant hMGL lines transfected with 25 nM control or APOE siRNAs were assayed for Aβ phagocytosis as in E. (G) Mutant hMGL lines with endogenous APOEε3/ε4 allele combinations, or edited APOEε3/ε3 alleles were assayed for Aβ phagocytosis as in E. (H) WT, TREM2 KO, or R47H hMGL lines were transfected with 25 nM control or SORL1 siRNA oligos, and assayed for Aβ phagocytosis as in E. In B–H, analyses were validated using two independently derived hMGL clones; individual plots in B–D represent results from independent experiments (B, n = 4; C and D, n = 3), and plots in E–H represent phagocytosis results from averages of three replicate values from each independent experiment. All graphs depict mean ± SEM. Statistical significance in B and D were calculated using one-way ANOVA with Tukey’s multiple comparison; statistical significance in E–H was determined by two-way ANOVA with Dunnett’s multiple comparison (*, P < 0.05; **, P < 0.01; ***, P < 0.001); and statistical significance in C was determined by Student’s t test (*, P < 0.05; **, P < 0.01; ***, P < 0.001). Gray asterisks in E, F, and H depict statistical differences between WT control, as determined by two-way ANOVA with Bonferroni–Dunn correction (*, P < 0.05; **, P < 0.01; ***, P < 0.001).