Fig 2: Dynamics and phenotypic specification of DAM in APP/PS1 mice(A) Time course of the microglial (IBA1+) density in APP/PS1 mice and age-matched controls, analyzed by IHC.(B) Representation of the cumulative number of microglial division cycles from early colonization of the brain primordium to aging (20 months of age) in WT and APP/PS1 mice, based on retrospective analysis of published data (Askew et al., 2017; Olmos-Alonso et al., 2016; Alliot et al., 1999; Nikodemova et al., 2015).(C–E) Time course of the density and plaque association (%) of DAM, identified as CLEC7A+ (C), CD11C+ (D), or MHCII+ (E) cells, in APP/PS1 mice and WT littermate controls, analyzed by immunohistochemistry (IHC) (black). Aß plaques labeled with Congo Red (C)–(E).(F) Confocal imaging of the co-localization of MHCII, CD11C, and CLECL7A in plaque-associated microglia (IBA1+) in APP/PS1 mice. Nuclei stained with DAPI, shown in grayscale.(G–I) Flow cytometry analysis of the expression of markers of DAM (CLEC7A, CD11C) in microglia (CD11B+CSF1R+) in APP/PS1 and WT controls. Immunonegative versus immunopositive gates for CD11C represented as cell count in a histogram plot. Co-expression pattern of CLEC7A and CD11C in microglia (H), analyzed as in (G). Quantification of the frequency of CLEC7A+ or CD11C+ microglia shown in (I). IHC data collected from average of parietal, auditory, and entorhinal cortex. Flow performed in samples from cerebral cortex.Scale bars in (C)–(F), 20 µm, shown in (C) and (F). Data shown in (A), (C)–(E), and (I) represented as means SEMs. N = 4–5 (A–F), N = 6–8 (G–I). Statistical differences: *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 versus age-matched controls. Data were analyzed with a 2-way ANOVA and post hoc Tukey tests.

Fig 3: Microglial senescence in APP/PS1 mice(A) Time course of the density of ßgal+ (blue) microglia (IBA1+; brown) in APP/PS1 mice and age-matched controls, analyzed by IHC. Representative ßgal+IBA1+ cells identified with an arrowhead. Time course of the plaque association (%) of ßgal+IBA1+ microglia, in APP/PS1 mice.(B) Correlation of the density of senescent microglia (ßgal+IBA1+) with the total density of microglia (IBA1+) or the density of DAM (CLEC7A+). R2 of linear regression analysis shown in plots.(C) Expression of ßgal in CLEC7A+ cells in APP/PS1 mice and age-matched controls, analyzed by IHC and shown as percentage of total CLEC7A+ cells. Representative ßgal+CLEC7A+ cells identified with an arrowhead.(D–F) Flow-FISH analysis of the telomere length (Cy-5 probe) in microglia (CD11B+CD45low), identifying DAM by CD11C+ expression in APP/PS1 mice and WT littermates. Immunonegative versus immunopositive gates for the telomere probe in WT and APP/PS1 microglia, as well as negative controls, represented as cell count in a histogram plot. Relative telomere length (percentage corrected to internal T1301 signal and relative to WT) in total microglia (E) and in homeostatic (Hom; CD11C-) versus DAM (CD11C+) in APP/PS1 mice (F).(G–I) Analysis of the relative telomere length in specific subpopulations of microglia (CD11B+CD45low), identified by CD11C expression as negative (CD11Cneg), low (CD11Clow), intermediate (CD11Cint), and high (CD11Chigh).(G) Representative gates and their cell count for the telomere probe shown in a histogram plot.(H) Quantification of the relative telomere length (percentage relative to individual average CD11C MFI) in 4 CD11C gates in APP/PS1 mice.(I) Correlation of CD11C intensity versus telomere probe intensity in a representative APP/PS1 mouse.R2 of the correlation analyses shown in (H and I). IHC data collected from average of parietal, auditory, and entorhinal cortex.Scale bars in (A and C), 20 µm. Flow cytometry performed in samples from cerebral cortex. Data represented as means ± SEMs. N = 4–5 (A–C), N = 8–14 (D–I). Statistical differences: **p < 0.01 versus age-matched control (A); *p < 0.05, **p < 0.01, and ***p < 0.001 correlation analysis, linear regression (B, H, and I); **p < 0.01 versus homeostatic microglia (Hom; F).Data were analyzed with a 2-way ANOVA and post hoc Tukey tests (A) and unpaired t test (E and F).

Fig 4: DAM display a senescent transcriptional signature(A) Heatmap representation of the log2 fold expression of genes from the DAM signature (Keren-Shaul et al., 2017) in WT CD11C- microglia (blue), APP/PS1 CD11C- microglia (green), and APP/PS1 CD11C+ microglia (red), using the pheatmap package.(B) Correlation analysis of the top 100 genes with highest and lowest fold change from Keren-Shaul et al. (2017) alongside the log2 fold change comparison of CD11C+ versus CD11C- microglia from APP/PS1 mice, using the ggplot2 package.(C) Correlation analysis of the fold change of genes from the core senescence signature (Hernandez-Segura et al., 2017), with low read genes filtered out, alongside the log2 fold change comparison of APP/PS1 CD11C+ microglia versus WT CD11C- microglia, using pheatmap and corrplot packages.(D) Correlation analysis of the genes from the senescence-associated signature of melanocytes, keratinocytes, astrocytes, fibroblasts, and core senescence signature (boxed in green) (Hernandez-Segura et al., 2017), with low read genes filtered out, with microglia from APP/PS1 and WT mice, using the corrplot package.(E) Gene set enrichment analysis (Mootha et al., 2003; Subramanian et al., 2005) of signatures upregulated or downregulated in senescence cells (Hernandez-Segura et al., 2017; Fridman and Tainsky, 2008; Casella et al., 2019; Kamminga et al., 2006), as well as a custom signature of genes highly associated with senescent cells (see Results section). Normalized enrichment score (NES) shown for the comparison of DAM (CD11C+) versus homeostatic microglia (CD11C-) from APP/PS1 mice. NES reaching a p < 0.05 and FDR < 0.25 highlighted by a squared NES.(F–J) Analysis of the single-cell dataset from Van Hove et al. (2019).(F) Uniform manifold approximation and projection (UMAP) plot of the microglial clusters identified from the original dataset after subsetting based on enriched expression of Sall1, Gpr34, Tmem119, Hexb, P2ry12, and Cx3cr1 from the whole brains of 16-month-old APP/PS1 and WT mice.(G) Feature plot of the DAM signature (Cst7, Csf1, Lpl, Apoe, Spp1, Cd74, Itgax), identifying cluster 2 as DAM. Further annotation of the clusters (see Figure S5) identified cluster 1 as homeostatic microglia in C57BL/6 mice, cluster 0 as homeostatic microglia in APP/PS1 mice, cluster 3 as white matter microglia (highly expressing Usp18; Goldmann et al., 2015), and cluster 2 as DAM.(H) Feature plot of the custom senescence signature (Cdkn2a, Cdkn1a, Cdkn2d, Casp8, Il1b, Glb1, Serpine1) identifying the DAM cluster 2 as enriched in senescent genes.(I) Bar plots of the percentage of cells per genotype (left) or per cluster (right), with a low, medium, or high enrichment in the custom senescence signature (from all cells with senescence scores > 0).(J) Dot plot representing the expression of the individual genes of the custom senescence signature in the 4 clusters identified in (F).N = 3–4 (A–E).