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.


TUNEL staining
For the detection of apoptosis in situ, tissue sections were subjected to the Click-iT™ Plus TUNEL Assay for In Situ Apoptosis Detection (C10619, Invitrogen), utilizing the Alexa Fluor™ 647 dye according to the manufacturer's protocol.Fresh PFA-fixed brains were dissected into coronal 30 µm sections.These sections were then re-fixed with paraformaldehyde, permeabilized with proteinase K, and then incubated with TdT enzyme for the labeling of free 3'-OH termini in DNA fragments, a hallmark of apoptosis.Following the labeling step, the sections were exposed to the Click-iT™ Plus reaction cocktail containing the Alexa Fluor™ 647 azide, which specifically reacts with the TdT-mediated dUTP incorporated in the nicked DNA ends via a copper-catalyzed click reaction.After thorough washing to remove unbound components and staining with DAPI (5 µg/ml), the stained tissue sections were mounted with a coverslip using Citifluor TM Mounting solution AF-1.Confocal images were acquired of the slices (as tile scan) with a Nikon Ti2 W1 spinning disk confocal using a 20x/NA 0.75, stitched, flattened and quantified with QuPath as described in previously in the material & method section of the article.

Senescent staining
For the in situ detection of senescent cells, brain tissue sections underwent staining using the CellEvent™ Senescence Green Detection Kit (C10850, Invitrogen), designed to identify senescent cells based on β-galactosidase activity, following the manufacturer's protocol.
Briefly, freshly PFA-fixed brains were dissected into coronal 30 µm sections and subjected to incubation with the CellEvent™ Senescence Green Probe (1X) for 2 hours at 37°C in a humidified chamber.Following washing, epitope retrieval, immunostaining, and mounting, the slices were captured using a Nikon Ti2 W1 spinning disk confocal with a 20x/NA 0.75 objective.Subsequently, images were stitched, flattened, and analyzed in ImageJ, as detailed in the material and methods section of the article.

Immunochemistry staining on slices
After perfusion and PFA 4% fixation, brains were embedded in paraffin and sliced into sagittal 5 µm sections using microtome (Leica).Following antigen retrieval with 70% formic acid for 10 minutes at RT and endogenous peroxidase and biotin/avidin blocking, the tissue was incubated with AG8 antibody (1:2000, Biolegend, 800704) overnight at RT.For detection, the HRP conjugated streptavidin from Invitrogen and DAB from Dako (K3468) were used and counterstained with hematoxylin.For the Alcian blue-Periodic Acid Schiff (PAS), we used the kit from Abcam (ab245876), according to the manufacturer recommendation.Images were acquired on Axiophot (Zeiss, 20x/NA 0.60, pixel size: 0.16 x 0.16 µm 2 ).

Behavioural tests
Behavioural deficits of SAMR1 and SAMP8 mice at the age of 9 months were assessed using Morris Water Maze (MWM), Y-Maze, elevated plus maze and open-field test at the Laboratory of Neurochemistry & Behaviour (Wilrijk, Belgium).

Morris Water Maze
The MWM assesses hippocampus-dependent visuo-spatial learning and memory (Hendrickx et al., 2022).The setup consisted of a circular pool (diameter: 150 cm, height: 30 cm) filled with opacified water using non-toxic white paint and was kept at 25 °C.Invariable visual cues were placed around the pool.The MWM consisted of an acquisition phase and a probe trial.The acquisition phase was performed over a period of 4 days and consisted of 2 daily trial blocks (at 10:30 AM and at 03:00 PM) of 4 trials with a 15 min inter-trial interval.
During the acquisition phase, a round acrylic glass platform (diameter 15 cm) was placed 1 cm below the water surface in a fixed position in the centre of one of the pool's quadrants.
Mice were placed in the water facing the wall and were recorded while trying to find the hidden platform for a maximum duration of 120 s.If the mouse was not able to reach the platform within 120 s, it was guided to the platform, where it had to stay for 15 s before being returned to their home cage.The starting positions varied in a semi-random order.The probe trial followed 4 days after the final acquisition trial.For this trial, the platform was removed, mice were placed in the MWM at a fixed position, and swimming trajectories were recorded for a period of 100 s.During both acquisition and probe trials, the animals' trajectories were recorded using a computerized video-tracking system (Ethovision), with path length, escape latency, and swimming speed recorded.

Y-Maze
Y-Maze Spontaneous Alternation is a behavioural test to measure the willingness of rodents to explore new environments.Rodents typically prefer to investigate a new arm of the maze rather than returning to one that was previously visited.It is used to investigate exploratory behaviour and cognitive function related to spatial working memory.Testing occurred in a Yshaped maze with three white, opaque plastic arms (length: 33 cm, width: 5 cm) at a 120° angle from each other.After introduction to the centre of the maze, the animal is allowed to freely explore the three arms.Over the course of multiple arm entries, the subject should show a tendency to enter a less recently visited arm.On day 1, the mouse was allowed to freely explore the maze for 8 min, while on day 2, one arm was closed off and the animal was allowed to explore for 10 min (trial run).After 4h, the animal was subjected to a probe trial where it was allowed again to explore the complete maze (5 min).The number of arm entries and the number of triads were recorded (Ethovision, Noldus, Wageningen, The Netherlands) to calculate the percentage of alternation.An entry occured when all four limbs were within the arm.

Elevated plus-maze
The elevated plus-maze apparatus was used to evaluate anxiety (Vloeberghs, Van Dam, Franck, Staufenbiel, & De Deyn, 2007).It consisted of four cross-shaped arms, of which two were brightly lit and open, and the other two dark and enclosed (30 cm × 5 cm × 15 cm; length × width × height of the enclosed arms).The maze itself was elevated 60 cm from the floor and mice were always placed in the central area (5 cm × 5 cm), facing the left enclosed arm.All mouse movements were tracked by camera for 5 min (Ethovision).The number of entries, latencies to the first arm entry, the duration of the time spent in open and closed arms, the total distance moved, and velocity were the parameters measured.

Open-field
Open-field behaviour was measured for 10 min during the dark phase of the animal's activity cycle in a brightly lit 50×50 cm 2 arena (Van Dam et al., 2003).Mice always started from the same corner of the arena and were allowed 1 min of acclimation to the setup before recording.A computerized video tracking system (Ethovision) was used to record trajectories and calculate path length and number of entries in the centre circle or the 7 × 7 cm 2 corners of the open field.Table S1.Overview of mouse models, injections and treatments schemes used in this study.References to the mouse models are provided in the material and method section.M = months.

Figure S1 .
Figure S1.Actinomycin D and Brefeldin A treatment reduce microglia activation during neural dissociation.The fold change of early immediate genes (a) and proinflammatory cytokine genes (b) expression was measured using RT-PCR after brain dissociation and Percoll gradient in the absence (CTRL) or presence of Brefeldin A (1x) and Actinomycin D (5 µM) (+BrefA +ActD).Values are mean ± SEM.Statistical differences (*P < 0.05) were determined by nonparametric one-tailed Mann-Whitney U test.(n = 4-5 mice/group)

Figure S5 .
Figure S5.Characterization of isolated CD11b + cells from FVB and P301L mice.(a) UMAP plot of CD11b + cells depicting the different cell types of all treated groups (59129 cells) of FVB and P301L injected mice.(b) Dot plot displaying the representative protein markers of the different cluster.(c) Dot plot depicting the top 10 differentially expressed genes of each cluster.(d) Feature plot representing specific markers of each cell type.

Figure S6 .
Figure S6.Characterization of CD63 + IBA1 + cells.(a) Feature plot depicting the relative expression of Cd74 in FVB and P301L mice.(b) Representative image of co-staining of cells positive for CD63 and IBA1 with CD74 in P301L+K18 mice.(c) Representative images of CD63, IBA1, and AT8 staining.The first two images show individual (top image) or clustered (middle image) CD63 + IBA1 + cells in P301L+K18 mice, with no AT8 staining observed in or around the cells.The third image illustrates AT8 -positive staining in the same slice but without positive CD63 + microglia.Scale bar = 50 µm.

Figure S10 .
Figure S10.SAMP8 mice exhibit reduced anxiety and spatial memory and learning impairment at the age of 9 months.At 9 months, the behavioural results of SAMP8 mice (orange square, n = 10 mice) were compared to those of SAMR1 mice (blue circle, n = 12 mice).Schematic diagram (left) and results (middle and right) of the Morris Water Maze (MWM) trials (a), the MWM probe (b), the Y-maze trials (c), the Y-maze probe (d), the elevated plus maze test (e), the open-field test (f).Values are mean ± SEM.Statistical differences (*P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001) were determined by non-parametric one-tailed Mann-Whitney U test or parametric one-tailed t-test depending on normality checks or two-way ANOVA with Sidak's multiple comparisons test for comparisons involving more than two groups.Outliers were detected using ROUT test (Q = 1 %).S = Start, F = Familiar, N = Novel.

Figure S11 .
Figure S11.Characterization of isolated CD11b + cells from SAMR1 and SAMP8 mice.(a) UMAP plot of CD11b + cells depicting the different cell types of all treated groups (51182 cells) of SAMP8 and SAMR1 mice.(b) Dot plot displaying the representative protein markers of the different cluster.(c) Dot plot depicting the top 10 differentially expressed genes of each cluster.(d) Feature Plot representing specific markers of each cell type.

Figure S12 .
Figure S12.Differential expression between SAMR1 and SAMP8 at different time

Figure S13 .
Figure S13.No change in DAM fraction between SAMR1 and SAMP8 strains.

Table S3 . Summary of sequencing parameters and cell counts after quality filtration.
/c = gene per cell, M = months, ncells = number of cells; Seq = sequencing; W = weeks g