Fig. 7: Microglia in APPPS1 and APPPS1.Il12b^−/− mice share gene signatures associated with enhanced microgliosis but exhibit distinct phagocytotic phenotypes.

a, Distinct homeostatic (yellow) and disease-associated (blue) microglia clusters were found in the combined snRNA-seq dataset with the disease-associated clusters present only in APPPS1 and APPPS1.Il12b^−/− mice. b, Scatterplot comparing the gene expression in the disease-associated clusters versus the homeostatic clusters. c, GO analysis of differentially upregulated genes per indicated genotypes. The dot size illustrates gene ratio, and the color denotes P value. Violin plot showing log₂FC of certain specific genes to the corresponding GO term. Fisher’s exact test and the GO algorithm ‘elim’. d,e, Volcano plots showing differentially expressed genes in microglia of APPPS1 mice compared to APPPS1.Il12b^−/− mice (downregulated: blue; upregulated: red) known to be involved in phagocytosis of microglia (d) or to be myelin related or amyloid related (e). Adjusted P value by Benjamini–Hochberg. A cluster of selected AD risk genes involved in phagocytosis in ex vivo human microglia and human brain lysates^68,69 served as reference for assessing phagocytosis-related microglial transcriptome changes, which, upon conversion into their mouse orthologs, resulted in 27 genes comprising Bin1, Ptk2b, Trem2, Zyx, Apbb3, Clu, Rin3, Cd33, Ms4a4a, Cr1l, Grn, Apoe, Picalm, Cd2ap, Plcg2, Sorl1, Fermt2, Ap4e1, Zkscan1, Abca7, Siglech, Trp53inp1, Abi3, Rabep1, Cass4, Ap4m1 and Sppl2a. Myelin-related or amyloid-related transcriptome changes in microglia (right) were defined by referencing the gene list described by Depp et al.³² (Supplementary Table 1, tab 6) depicting differentially expressed genes of DAM derived from 6-month-old mice with amyloid pathology and/or mutant myelin, followed filtering by logFC > 0.25 and FDR < 0.01. Genes that were altered significantly are shown as filled circles (FDR < 0.05); open circles indicate differences that did not reach statistical significance. Il12b served as internal control. f–h, Phagocytic activity of microglia in adult acute brain slices of WT and APPPS1 mice with and without IL-12 signaling. Organotypic brain slices prepared from 90-day-old WT (Il12b^+/+), Il12b^−/−, APPPS1 and APPPS1.Il12b^−/− mice were incubated with fluorescent microbeads to analyze phagocytic microglia. Representative views from 15-μm confocal z-stacks showing uptake of fluorescent microbeads (in green) by microglia (labeled with Iba1, red) in brain slices of mice with the indicated genotypes (f). Percentage of phagocytic microglia with engulfed microbeads (P = 0.0104) (g) and phagocytic index (P = 0.0314) (h). For the calculation of the phagocytic index, phagocytic cells were grouped according to the number of ingested microbeads, with 1–3 microbeads = grade 1; 4–6 microbeads = grade 2; 7–10 microbeads = grade 3; and more than 10 microbeads = grade 4. Each grade (1–4) was multiplied with the respective percentage of phagocytic microglia to calculate the phagocytic index. Scale bar, 50 μm. n = 4 mice per group (mean ± s.e.m., one-way ANOVA with Dunnett’s post hoc test with WT as control group). i, Representative immunohistochemical image of Clec7a, Iba1 and 4G8 staining in APPPS1.Il12b^−/− mouse brain cortical tissue. Scale bar, 100 µm. j, Clec7a staining intensity within plaque‐associated Iba1⁺ microglia in WT and APPPS1.Il12b^−/− mice (n = 6). Mean ± s.e.m., statistical analysis: two‐tailed unpaired t‐test with Bonferroni correction for each single bin, P = NS. NS, not significant.