Fig. 6: Tbk1 loss in brain microglia induces an early shift to a pro-inflammatory, aged-like signature.

a Setup to compare microglia from young and aged (female) mice with full or partial microglial specific Tbk1 deletion, to control littermates. Created in BioRender. Lenoel, I. (2025) https://BioRender.com/ct8qnd6. b PCA reveals clustering of all samples by age. c Significant DEGs in Tbk1-KO microglia at 4 and 22 months vs control microglia and overlapping DEGs between the two ages (DESeq2, log₂FC ≥ 0.5, adjusted p values (FDR) < 0.05, see “Methods”) (for Tbk1-HET, see Supplementary Fig. 9j). d Venn diagram of DEGs comparing genotypes and ageing (with partial and total sums indicated). e GSEA showing the top 5 Hallmark pathways upregulated by Tbk1 deletion at 4 and 22 months compared to control microglia, and during ageing in control microglia (adjusted p values < 0.05, see “Methods”). f WGCNA revealed only one KO relevant module (see also Supplementary Fig. 9g, h). Data represented as single data points (corresponding to a single mouse sample, see Fig. 6a) with mean. Adjusted p values were determined by a two-tailed Wilcoxon test. g, h Upper panels: Red/blue squares indicate significant UP/DOWNregulations (red/blue DEGs). White squares indicate not significant (ns) changes. Lower panels: Gene heatmaps with unbiased clustering (centered VST expression values, see “Methods”). g Heatmap of the top 20 DEGs (ranked by adjusted p values; marked with a white cross in the upper panel) upregulated in microglia by Tbk1 deletion at either 4 or 22 months compared to control microglia, and their regulation during ageing in control microglia. Of note, as 10 DEGs were overlapping, this resulted in a total of 30 DEGs shown. h Heatmap of the top 30 DEGs (ranked as in (g)) upregulated during ageing in control microglia and compared to their regulation in microglia with Tbk1 deletion at 4 or 22 months (vs control microglia). Purple arrowheads highlight 3 disease-associated microglia (DAM) markers, Axl, Cst7, and Itgax. i, j GSEA to assess how full microglial Tbk1 deletion compares to partial. GSEA plots comparing KO to WT (i), and HET to WT (j), at 4 and 22 months, with the top 100 genes significantly upregulated by ageing in control microglia (genes ranked based on adjusted p values).