S100A8-enriched microglia populate the brain of tau-seeded and accelerated aging mice

Abstract

Long considered to fluctuate between pro- and anti-inflammatory states, it has now become evident that microglia occupy a variegated phenotypic landscape with relevance to aging and neurodegeneration. However, whether specific microglial subsets converge in or contribute to both processes that eventually affect brain function is less clear. To investigate this, we analyzed microglial heterogeneity in a tauopathy mouse model (K18-seeded P301L) and an accelerated aging model (Senescence-Accelerated Mouse-Prone 8, SAMP8) using cellular indexing of transcriptomes and epitopes by sequencing. We found that widespread tau pathology in K18-seeded P301L mice caused a significant change in the number and morphology of microglia, but only a mild overrepresentation of disease-associated microglia. At the cell population-level, we observed a marked upregulation of the calprotectin-encoding genes S100a8 and S100a9. In 9-month-old SAMP8 mice, we identified a unique microglial subpopulation that showed partial similarity with the disease-associated microglia phenotype and was additionally characterized by a high expression of the same calprotectin gene set. Immunostaining for S100A8 revealed that this population was enriched in the hippocampus, correlating with the cognitive impairment observed in this model. However, incomplete colocalization between their residence and markers of neuronal loss suggests regional specificity. Importantly, S100A8-positive microglia were also retrieved in brain biopsies of human AD and tauopathy patients as well as in a biopsy of an aged individual without reported pathology. Thus, the emergence of S100A8-positive microglia portrays a conspicuous commonality between accelerated aging and tauopathy progression, which may have relevance for ensuing brain dysfunction.

Keywords: Alzheimer's disease; CITE‐seq; SAMP8; microglia; tau.

FIGURE 1

Microglia depletion reduces regional phospho‐tau load in the contralateral hemisphere of P301L+K18 mice. (a) Representative contrast‐matched images of phosphotau (AT8) staining of sagittal slices of the contralateral hemisphere of PLX3397 treated P301L+K18 and non‐treated FVB+PBS, P301L+PBS, and P301L+K18 male mice. Insets represent zoomed‐in views on the isocortex. Scale bar 400 μm. (b) Quantification of phospho‐tau (AT8) load in different regions of the contralateral hemisphere of P301L+K18 after 104 days (14 days pre‐ + 3 months p.i.) of feeding with modified AIN76A supplemented with vehicle (green dot) or PLX3397 (orange square). Positive pixels were quantified and normalized over the total number of pixels (% of positive pixels) in different regions (represented in black). (n = 7 mice/condition) (c) Representative images and quantification of microglia morphology in the hippocampal region and the isocortex (somatomotor area) of both P301L+PBS and P301L+K18 mice. The images feature IBA1 and DAPI staining, accompanied by zoomed‐in sections to emphasize microglial morphology. White arrows indicate ameboid morphology, which is not depicted in the zoomed image. Scale bar 100 μm. The Feret diameter is the longest distance between any two points along the boundary of a structure (n = 4–5 mice/condition). Values are mean ± SEM. Outliers were detected using ROUT test (Q = 1%) and excluded from analysis. Statistical differences (*p < 0.05, **p < 0.01, and ***p < 0.001) were determined by non‐parametric one‐tailed Mann–Whitney U test or parametric one‐tailed t‐test depending on normality checks. Striat. dors. region = striatum dorsal region; P = P301L; F = FVB.

FIGURE 2

Characterization of microglial subpopulations in injected FVB and P301L mice. (a) Schematic diagram showing the CITE‐Seq timeline. (b) UMAP plot of CD11b⁺ selected cells depicting the different cell types across all different groups (59,129 cells). (c) UMAP plot of microglia depicting the different microglial subpopulations of all different groups (51,163 cells). (d) Dot plot representing the top 10 differentially expressed genes of each microglial subpopulation. (e) Feature plot depicting the relative expression of two representative genes of each microglial cluster. (f) Dot plot displaying the representative protein (epitope) markers of each microglial subpopulation determined by CITE‐seq (n = 4 mice/condition). D = Day; M = Months; W = Weeks.

FIGURE 3

The DAM subpopulation is increased in P301L+K18 mice at 3 months p.i.. (a) UMAP plots depicting the different microglial subpopulation in FVB+PBS, P301L+PBS, and P301L+K18 mice. (b) Quantification of the percentage of cells in each microglial subpopulation over the different models at 1‐month and 3‐months p.i.. (c) CD63 protein level projected onto the UMAP plot shows its enrichment in DAM. (d) Representative images at 3‐month p.i. of CD63⁺IBA1⁺ cells. Images represent a merge of a whole‐hemisphere slice (left) and a zoom in the delineated region were CD63⁺IBA1⁺ cells were localized. (e) Quantification of the percentage of CD63⁺IBA1⁺ cells over the total number of IBA1⁺ cells in the delimited region (d dotted line) at 1‐ and 3‐month p.i. in the different mice models (2–3 slices/mouse). Scale bar 100 μm. Values are mean ± SEM. Statistical difference (*p < 0.05) was determined by one‐way ANOVA with Dunn's multiple comparisons test for each time point. (n = 4–7 mice/condition) P = P301L; F = FVB.

FIGURE 4

In P301L+K18 mice, microglia exhibit high expression levels of S100a8, S100a9, Apoe, and Lyz2. (a) Volcano plot depicting the differentially expressed genes between P301L+PBS and P301L+K18 mice at 1‐month and 3‐month p.i.. (b,c) Feature plot depicting the relative level of expression of Lyz2, Apoe, S100a8, and S100a9 genes in the different group at 1‐ (b) and 3‐ (c) Month p.i.. P = P301L; F = FVB, M = Month.

FIGURE 5

Accelerated Aging‐Associated Microglia (A3M), characterized by the high expression of S100a8 and S100a9, are specifically present in SAMP8 mice at 9 months. (a) Schematic diagram showing the CITE‐Seq timeline. (b) Volcano plot illustrating the genes differentially expressed in microglia between SAMP8 and SAMR1 mice. (c) UMAP plot depicting the different microglial subpopulations among all different groups (47,200 cells). (d) Dot plot representing the top 10 differentially expressed genes of each microglial subpopulation. (e) Feature plot depicting the relative expression of two representative genes of each microglial cluster. (f) Dot plot displaying the representative protein markers of each microglial subpopulation determined by CITE‐Seq. (g) Quantification of the percentage of cells in each microglial subpopulation in the different mice and at different time points. (n = 4 mice/condition) (h) Pseudotime trajectory demonstrating the shift in microglial state. The black arrowheads indicate two separate tracks that end in the aging‐associated microglia population. Values are mean ± SEM. Statistical differences (*p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001) were determined by two‐way ANOVA with Tukey's multiple comparisons test. M = Months.

FIGURE 6

S100A8⁺IBA1⁺ A3M cells are present in mice and human samples showing aging, AD or tauopathy. (a) Average enrichment score of the A3M signature in FVB and P301L‐injected mice at 3‐month p.i., derived from CITE‐seq data. The A3M signature is defined by the top 30 genes differentiating A3M from DAM in SAMP8 mice. Enrichment in individual cells was determined using the AddModuleScore function, which evaluates the collective expression of this specific gene set. (b) Representative images of P2RY12 staining of the S100A8⁺IBA1⁺ positive cells. (c) Representative image of IBA1, S100A8, and DAPI staining in P301L+PBS and P301L+K18 mice. Montages on the right show a zoom of the delineated region presented on the left image. The first square (1) represents a zoom in the cortex where S100A8⁺IBA1⁺ cells (white arrowheads) were localized in P301L+K18. The second square (2) represents a zoom in the hippocampus where an intense S100A8 signal was observed in the P301L+K18 compared to P301L+PBS. Scale bar 200 μm. (d) Quantification of the percentage of S100A8⁺IBA1⁺ cells over the total number of IBA1⁺ cells in the isocortex at 3‐month p.i. in P301L+PBS and P301L+K18 (1 slice/mouse). (e) Representative images showing S100A9 staining of S100A8⁺IBA1⁺ cells in the brain (left part) and S100A8, S100A9, DAPI immunostaining in the spleen (right part), using the same antibodies, serving as a positive control. Scale bar 50 μm. (f) Representative, contrast‐enhanced images of two S100A8⁺IBA1⁺ cells (upper section) and quantification (lower section) of S100A8⁺IBA1⁺ positive cells over the total number of IBA1⁺ cells in the hippocampus of SAMP8 and SAMR1 mice at the age of 9 months (1 slice/mouse, n = 5–6 mice/condition). Red arrows show intracellular staining. The top cell displays a punctate, vesicular like staining, while the bottom cell shows a denser cytoplasmic staining. Scale bar = 20 μm. (g–i) Representative image of S100A8⁺IBA1⁺ double positive staining (red arrows) of an aged non‐neuropathologic patient (69 years old) (g), AD (h), and tauopathy (i) diagnosed patients. Scale bar 50 μm. Values are mean ± SEM. Statistical differences (*p < 0.05, **p < 0.01, and ***p < 0.001) were determined by non‐parametric one‐tailed Mann–Whitney U test. P = P301L.