Extended Data Fig. 9: snRNA-seq analysis of Cnp−/− 5xFAD and respective single mutant controls.
From: Myelin dysfunction drives amyloid-β deposition in models of Alzheimer’s disease
Continuation of Fig. 5. (a) Left panel shows Western blot analysis of Trem2 levels in WT vs 5xFAD mice demonstrating clear induction of full length Trem2 (30–60 kDa, glycosylated forms) and enhanced Trem2 cleavage (~12 kDa) in 5xFAD mice. Lanes present single animals/biological replicates/n (n = 3 per group). The right panel shows Western blot analysis of Trem2 levels in Cnp−/− 5xFAD and 5xFAD mice. No differences in Trem2 cleavage could be detected. Total protein fastgreen staining is shown as loading control. Lanes present single animals/biological replicates/n (n = 3 per group). Source data are given in Supplementary Fig. 3. (b) Cell type distribution and annotations from 6-month-old mouse brain hemisphere snRNA-seq data visualised in the UMAP space. In total 4 genotypes (WT, Cnp−/−, 5xFAD, and Cnp−/−5xFAD) were included and 61,949 cells passed quality controls for downstream analysis. Right panel shows microglial cells highlighted in yellow in UMAP space. (c) Heatmap visualisation of scaled marker gene expressions at single-cell level. For each cell population, a random n = 1000 cells were selected to be displayed. For NFOL and pericyte, all cells are shown (total cell numbers from both populations were under 1000). (d) Violin plots showing gene expressions across oligodendroglia and microglia subpopulations (microglia detailed subpopulations annotated in Fig. 5b). (e) Ms4a7 fluorescent single molecule in situ hybridisation (RNAscope) in combination with Iba1 immunostaining on 5xFAD and Cnp−/−5xFAD brain tissue. The same observations were made in different independent samples/mice per group (n = 3 per group). (f) 2D zoom in on UMAP representation of amyloid and myelin DAM colour coded to cluster annotation (left panel) or genotype (right panel). (g) 3D representation of DAM clusters colour-coded according to genotype. (h) Volcano plot representation of DEGs between DAM subclusters and between genotypes within Amyloid- and Myelin-DAM clusters. (i) Heatmap representation of genes commonly regulated in-between DAM clusters and between genotypes in specific DAM clusters. (j) Immunofluorescence analysis of myelin phagocytosis (PLP+) by microglia (Iba1+) in the cortex of Cnp−/−5xFAD mice. Quantification on the right shows the number of myelin-positive(PLP+) microglia (Iba1+). Bars represent means, dots represent single mice/biological replicates/n (n = 3 for 5xFAD, n = 4 for Cnp−/−5xFAD). Statistical analysis: Two-sided, unpaired Student’s t-test (p-value is given in the graph).
