Extended Data Fig. 10: Lack of microglia activation and APP metabolism changes in forebrain shiverer 5xFAD mice and MS4A cluster gene expression in human AD microglia.
From: Myelin dysfunction drives amyloid-β deposition in models of Alzheimer’s disease
(a) Experimental setup to study microglia states in FB-shiv (forebrain shiverer) in comparison to wildtype controls and Cnp−/− brain by snRNAseq at 3 months of age. (b) Identification of microglia/macrophage subpopulations and genotype distribution visualised in the UMAP space. (c) Marker gene expression for the four identified clusters visualised in violin plots. (d) Number of microglia per microglia subtype and genotype. Different bars represent single animals/biological replicates (n = 2 per group). (e) Assessment of microglia coverage and morphology by Iba1 stainings in 3-month-old FB shiverer 5xFAD mice. Lower panel shows closeups of cells delineated by dashed lines in the upper panel. As a comparison and positive control for activated microglia a micrograph of Cnp−/−5xFAD is shown on the right. Bargraphs show quantification of Iba1+ area and microglia number in the cortex (CTX) and corpus callosum (CC). Bars represent means, dots represent animals/biological repolicates/n (n = 4 for control and n = 3 for FB shiverer mice). Statistical analysis: two-sided Student’s t-test (p-value is given in the graph). (f) Assessment of APP+ axonal swellings in the fimbria (FIM) of 3-month-old FB shiverer 5xFAD by 4G8 (APP/Aβ) immunolabeling. In both control 5xFAD mice and FB shiverer 5xFAD APP+ swellings were rarely or not found. The contoured arrow in the left micrograph highlights an amyloid plaque. As a comparison and positive control for APP+ swellings a micrograph of Cnp−/−5xFAD is shown on the right. Swellings are highlighted by arrowheads. Bars represent means, dots represent animals/biological repolicates/n (n = 4 for control and n = 3 for FB shiverer mice). Statistical analysis: two-sided Student’s t-test (p-value is given in the graph). (g) Western blot analysis of APP processing in FB shiverer 5xFAD mice. Bands for full-length APP (fAPP) and c-terminal fragments (CTFs) are shown. n = 3 biological replicates/mice/lanes per group. Source data are given in Supplementary Fig. 1. Molecular weight marker (in kDa) is given on the left. (h-k) Reanalysis of microglia subcluster of Mathys et al.46. (h) Genotype and clustering distribution of the microglia/leukocyte subset in the Mathys et al.46 snRNA-seq dataset visualised in the UMAP space. Hom = homeostatic microglia, DAM = Disease-associated microglia, Ribo high = high in ribosomal transcripts. (i) Histogram representation of cell numbers in each subcluster according to sample condition (control versus AD). Bars represent cell numbers. (j) Expression analysis of classical DAM marker genes (TREM2, SPP1, APOE) and MS4A6A in the main microglial subcluster (leukocytes not shown) visualised in the UMAP space (left) and by dot plot representation (right). (k) Co-expression analysis of MS4A6A and SPP1 at the single-cell level visualised in the UMAP space. Colour indicates gene expression and co-expression level according to the colour scale shown on the right. (l-o) Reanalysis of microglia subcluster of and Zhou et al.35. (l) Genotype and clustering distribution of the microglia/leukocyte subset in the Zhou et al.35 snRNA-seq dataset visualised in the UMAP space. Hom = homeostatic microglia, DAM = Disease-associated microglia, Histone high = high in histone transcript, MyTE = Myelin transcript enriched. (m) Histogram representation of cell numbers in each subcluster according to sample condition (control versus AD). (n) Expression analysis of classical DAM marker genes (TREM2, APOE), ABCA1, and MS4A6A in the main microglial subcluster (leukocytes, monocytes and MyTE not shown) visualised in the UMAP space (left) and by dot plot representation (right). (o) Co-expression analysis of MS4A7 and TREM2 at the single-cell level visualised in the UMAP space. Colour indicates gene expression and co-expression level according to the colour scale shown on the right. The results published here are in whole or in part based on data obtained from the AD Knowledge Portal (https://adknowledgeportal.org).
