Fig. 4: Non-PAM show transcriptional plasticity toward peripheral stimuli and gut dysbiosis at early stages of amyloid pathology.

a, UMAP plot visualizing CD11c⁻ non-PAM (blue) and CD11c⁺ PAM (red) from untreated, LPS-treated and ABX-treated Cx3cr1^CreERT2R26^Confetti5×FAD⁺ animals at an early stage of disease. Each dot represents one cell (N = 1,095). b, UMAP plot of cell clusters (C0–C4) identified by unsupervised clustering of all analyzed cells from untreated, LPS-treated and ABX-treated Cx3cr1^CreERT2R26^Confetti5×FAD⁺ animals at an early stage of disease. Each color represents one cell cluster; each cluster is outlined by dotted lines. Each dot represents one cell (N = 1,095). c, Left: UMAP plot visualizing CD11c⁻ and CD11c⁺ cells from untreated, LPS-treated and ABX-treated Cx3cr1^CreERT2R26^Confetti5×FAD⁺ animals at an early stage of disease relative to the identified cell clusters (C0–C4, dotted outlines) (see color legend, right). Right: stacked bar plot depicting the relative composition of microglial clusters with respect to the cells’ CD11c signal and their treatment group (see color legend). d, Heatmap presenting normalized expression of the 20 most differentially expressed genes per cluster. Expression levels are encoded by color as shown in the legend. e, Volcano plots with pseudobulk gene expression comparing differential gene expression between PAM and non-PAM (top), between PAM cluster 0 and non-PAM cluster 2 (middle) and between PAM cluster 1 and non-PAM cluster 2 (bottom). The –log₁₀-transformed adjusted P value (P adjusted, y axis) is plotted against the log₂-transformed FC in expression between the indicated cell groups or clusters (x axis). Genes under log₂FC and –log₁₀ P value cutoff are shown in gray (NS). Genes above log₂FC but under P value cutoff are shown in green, and genes above log₂FC and –log₁₀ P value cutoff are shown in red. f, GSEA of the pseudobulk differential gene expression in non-PAM versus non-PAM after LPS treatment. HALLMARK_INFLAMMATORY_RESPONSE: normalized enrichment score (NES) = 2, P = 0.0, false discover rate (FDR) q-value = 0.0. HALLMARK_INTERFERON_GAMMA_RESPONSE: NES = 1.62, P = 0.0, FDR q = 0.002. HALLMARK_TNFA_SIGNALING_VIA_NFKB: NES = 2.66, P = 0.0, FDR q = 0.0. Enrichment scores and gene hits are plotted. g, GSEA of the pseudobulk differential gene expression in non-PAM compared to non-PAM after ABX treatment. GOBP_REGULATION_OF_INNATE_IMMUNE_RESPONSE: NES = −1.51, P = 0.002, FDR q = 0.13. GOBP_REGULATION_OF_MACROPHAGE_ACTIVATION: NES = −1.34, P = 0.1, FDR q = 0.15. Enrichment scores and gene hits are plotted. h–j, Violin plots depicting the indicated Gene Ontology modules in PAM and non-PAM across treatment groups. The module expression was quantified using the UCell algorithm. The indicated P values represent the results of pairwise two-tailed Mann–Whitney U-tests. Specific P values of statistical tests are indicated. NS, not significant.