All animal procedures were reviewed and approved by the University Committee on Animal Resources of the University of Rochester Medical Center and performed according to the Institutional Animal Care and Use Committee and guidelines from the National Institute of Health (NIH). Animals were housed in a 12-hour light/12-hour dark cycle with food ad libitum. 14-month-old male and female Tg(APPswe, PSEN1dE9)85Dbo mice (also known as APP/PS1) were obtained from an established colony (JAX stock no. 005864) maintained at the University of Rochester vivarium. Tg(APPSwe, tauP301L)1Lfa Psen1tm1Mpm mice (also known as 3xTg) were initially obtained from Frank M. LaFerla and Salvatore Oddo by Howard Federoff and maintained at the University of Rochester as a homozygous line. The 3xTg mice express mutated human APP Swedish, MAPT P301L under the control of the Thy1.2 promoter and PS1 M146V under the Psen1 promoter. Age-matched non-transgenic (NTg) mice, bred continuously in a parallel colony to 3xTgs with a similar genetic background, were used as wild-type controls to 3xTg mice in RNA sequencing experiments. With age, the 3xTg mice develop Aβ plaque deposits and intraneuronal hyperphosphorylated Tau aggregates. Our studies used two cohorts of 21-22-month-old male 3xTg mice.
Microglia depletion and repopulation
Mice received a chow diet (AIN-76A-D1001i, Research Diets) containing 1200 mg/kg PLX5622 (Chemgood) ad libitum for 2 weeks to deplete microglia. Control chow with the same base formula without PLX5622 was given to the control group. After the 2-week treatment, mice were returned to the standard chow diet: AIN-76A (Research Diets) for the 3xTg Cohort 1 or 5053-Rodent Diet 20 (currently in use at the University of Rochester vivarium) for other experiments to allow microglial repopulation for 1 month. All of these formulations were irradiated by the vendor.
Open field (OF):
14 days before behavioral testing, mice were switched to a reverse light/dark cycle room. For 2 days before behavioral testing, mice were transported from the colony room to the behavior room, handled for ~5 minutes, and returned to the colony room on that day. On the day of testing, individual mice were placed in the center of a 31x31 cm box. After 20s, the animal’s behavior was video recorded for 5 minutes. Mouse entries and time spent in the center zone and outside the zone were quantified using AnyMaze software (Stoelting Co).
Novel Object Recognition (NOR):
For the novel object recognition testing, mice were allowed to freely explore a 31 x 31 cm box containing two identical objects for 10 minutes. Doorknobs (5-6 cm in height and ~3 cm in width) were used. The testing chamber was sanitized between each trial with 70% ethanol. An hour after the habituation phase, mice were returned to the same box with one of the previously exposed, familiar objects and a novel object (i.e., a different doorknob). Placement of the novel object was randomized for each test. Mice were allowed to explore the box again for 5 minutes. Mice were videotaped during habituation and testing trials. For scoring, the time mice moved toward the object with the head facing the object and the neck extended was counted as exploratory behavior. Mice that spent less than 8 seconds exploring both objects were excluded from the analysis. Novel object discrimination index (DI) was defined by the following formula:
The Lashley III maze was used to test spatial memory in a stress-free environment (34). The maze consisted of a start box, three interconnected alleys, and a pseudo-home cage. The alley was divided into zones. Mice were allowed to explore the maze for 10 mins per day. The maze was sanitized with 70% ethanol between each trial and each animal. For 8 days, the number of zone entries was recorded under the assumption that mice that correctly remember the route from starting box to the pseudo-home cage will not enter zones outside of the direct path. Anymaze was used to record the videos.
Contextual Fear conditioning (CFC):
After the behavioral tests above were conducted, mice underwent cued and contextual fear conditioning as previously described (35). Briefly, on the conditioning day, mice were allowed to explore the context compromised of an enclosed Plexi-glass chamber and a metal floor grid (model H10-11M, Coulbourn Instruments) inside an isolation chamber (Model H10-24T, Coulbourn Instruments). After 3 min, 15s of white noise was presented, followed by a 2s, 0.75mA foot shock. The noise-shock pairing was repeated twice for a total of 3 shocks with 30s intervals. The next day, mice were exposed to the same chamber for 5 minutes, and freezing behavior was quantified with AnyMaze. Four hours later, the mice were placed in a novel context (a plastic cylinder with bedding and red light) within the same Plexi-glass chamber. After 3 minutes, the conditioned tone stimulus was played, and freezing behavior was quantified.
Flow cytometry / FACS
Mice were injected 24 hours before sacrifice with Methoxy-X04 (MeX04, i.p., 4mg/kg, Tocris Biosciences), a brain-permeable Aβ fluorescent marker (36, 37). On the day of sacrifice, animals were deeply anesthetized with a mixture of xylazine (i.p., 10 mg/kg) and ketamine (i.p., 100 mg/kg) and perfused intracardially with 0.15M phosphate buffer (PB) containing 0.5% sodium nitrite and 2 IU heparin/ml. After perfusion, hemispheres were separated: one was either immediately submerged in fixative solution (4% paraformaldehyde (PFA), pH 7.2 in PB, 4°C) to be used for immunofluorescence experiments or flash-frozen in cold isopentane for ELISA (both as described below), and the other was processed for flow cytometry as follows. The hippocampus from each half brain was dissected and homogenized in 3 ml FACS buffer (1X Phosphate Buffered Saline (PBS) + 0.5% BSA). Homogenates were filtered through a 70 µm cell strainer into a 15 ml tube containing 3 ml FACS buffer. The strainer was washed with an additional 3 ml of FACS buffer, and the cell suspensions were centrifuged at 400xg for 5 min at 4°C. The supernatants were discarded, and the remaining pellets were resuspended in 40% percoll (Cytiva) prepared with PBS, then centrifuged at 400g for 30 min with no braking. After removing the supernatants, the pellets were resuspended in 90 µl FACS buffer with 1:100 Fc block (2.4G2, 1:100, BioLegend) and transferred to a 96 well-plate. After a 15 min incubation with Fc block at 4°C, the following antibodies were added in a 10 µl master mix: CD11b-FITC (M1/70, Biolegend), CD45-APC/Cy7 (30F11, Biolegend), 7AAD (Invitrogen), P2Ry12-APC (S16007D, Biolegend) & TMEM119-PE (106-6, Abcam). The latter two cell surface molecules are considered homeostatic microglial markers (4). The plate was then incubated for 30 mins at 4°C in the dark. The samples were washed once with FACS buffer and transferred to 5 ml tubes containing 7AAD such that its final dilution was 1:80. Appropriate fluorescent-minus-one (FMO) and single-stained bead controls (Ultracomp eBeads, Invitrogen) were prepared in tandem with samples. After excluding debris, doublets, and dead cells, CD45lo/CD11b+ was used to gate for microglia on a FACSAria II (BD). MeX04+ and MeX04- microglia were sorted. Samples from APP/PS1 mice were analyzed the same way, but with a LSR II flow cytometer (BD) without sorting. All events were recorded, and data were analyzed with FCS Express 7 (DeNovo Software).
Half-brains were fixed overnight in 4% PFA at 4°C, dehydrated in 30% sucrose overnight, frozen in cold isopentane, and stored at -80°C until sectioning on a -25°C freezing stage microtome into 30 µm thick coronal slices stored in cryoprotectant solution. For immunofluorescence, sections were washed extensively in PBS and blocked with 10% normal donkey or goat serum for 1 h at RT.
For amyloid pathology analysis, sections were immunolabeled for amyloid-beta (Aβ), microglia (Iba1 or P2RY12), a common microglial activation marker CD68, and a widely-used marker for neuritic damage LAMP1 (38). The following primary antibodies were used: biotin anti-Aβ (clone 6E10, 1:3000, BioLegend), rabbit anti-Iba1 (1:2000, Wako), rabbit anti-P2RY12 (1:2000, Anaspec), rat anti-CD68 (1:500, Bio-Rad) and rat anti-LAMP1 (1:2000, Abcam). Sections were incubated in primary antibodies for 48 h at 4°C. The sections were washed and incubated in fluorescently-labeled secondary antibodies/reagents (Alexa Fluor 488, Alexa Fluor 594 streptavidin conjugate and Alexa Fluor 647, Invitrogen; all at 1:1000) for 3 h at RT, then mounted and coverslipped (Prolong Gold, ThermoFisher Scientific).
For Tau pathology analysis, sections were incubated in biotinylated mouse anti-HT7 (1:1000, Invitrogen) and either rabbit anti-pT205 (1:1000, Invitrogen) or rabbit anti-pS396 (1:1000, RayBiotech) for 2 h at RT then overnight at 4°C. They were washed, incubated in fluorescently-labeled secondary antibodies (Alexa Fluor 488, Invitrogen) and Alexa fluor 594 streptavidin conjugate (Invitrogen; all at 1:1000) for 3 h at RT, and then mounted and coverslipped.
Image acquisition and analysis
For each animal, 3-4 coronal tissue sections that included the subiculum (S) and CA1 field of the hippocampus (CA1) were imaged with a Nikon A1R HD confocal microscope using a 10x (Plan Apo Lambda, NA: 0.40), 20x (Plan Apo VC, NA: 0.75) or 40x water-submersion (Apo LWD, NA: 1.15) objective lens as indicated in figure legends. Imaging parameters were kept constant across all sections for each set of immunofluorescent labels. All image analysis was performed using ImageJ FIJI (NIH) with semi-automated custom macros. Experimenters were blinded to treatment.
Analysis of amyloid pathology and associated neuritic damage
For plaque area fraction and number analysis, regions of interest (ROIs) outlining the above-mentioned structures (S and CA1 for APP/PS1 and S for 3xTg) were drawn on maximum z-projections of the acquired 6E10 images. Images were subsequently thresholded and binarized using automated ImageJ’s Otsu thresholding algorithm, which was used for all other thresholding steps in this manuscript (except for MeX04 analysis, in which MaxEntropy was used). The plaque area fraction was calculated as the ratio between the number of pixels above the threshold over all pixels in the ROIs. The number of plaques per ROI was computed using automated ImageJ’s analyze particles function with a cut-off size of 50 µm2.
For quantification of plaque-associated neuritic damage, 6E10 z-stacks were thresholded and binarized for analysis taking into account individual z planes. Subsequently, plaques detected by the analyze particles algorithm were dilated 25 µm to encompass the surrounding tissue for quantification of plaque-associated LAMP1. The overlap between LAMP1 and microglia was measured by multiplying the binarized LAMP1 and Iba1 (or P2RY12) image stacks. The resultant image was subtracted from the binarized LAMP1 image to obtain a non-microglial LAMP1 image, which was multiplied with the image containing dilated plaques to compute the overlap between LAMP1 and areas surrounding plaques. The number of colocalized signal pixels was calculated and divided by the number of thresholded 6E10 pixels (non-dilated) to obtain the ratio of LAMP1/plaque as quantification of plaque-associated dystrophic neurites.
Analysis of microglia
To measure the total volume occupied by microglia, Iba1 or P2RY12 z-stacks were thresholded and binarized using the same algorithm as described above. The percentage thresholded pixels was recorded as % microglia coverage. The 6E10 thresholded z-stacks mentioned above were dilated 5 µm, then plaque outlines were overlayed on Iba1 or P2RY12 z-stacks. The percentage of microglial area associated with plaque was calculated as the number of colocalized signal pixels divided by all microglial pixels.
To assess microglial activation, CD68 and P2RY12 markers were quantified. CD68 analysis was performed on APP/PS1 and the first cohort of 3xTg animals, while P2RY12 analysis was done on the second 3xTg cohort. CD68 z-stacks were thresholded and binarized within the same region. The overlap between CD68 and microglia was measured by multiplying the binarized CD68 and Iba1 z-stacks. The number of colocalized signal pixels was divided by the total microglia pixels to get the fraction of CD68-expressing microglia. For quantification of P2RY12 intensity as a proxy for microglia activation state, P2RY12 slices were summed in the z-direction and duplicated. One P2RY12 image was thresholded, and the P2RY12-negative region was chosen as the background ROI. On the other image, background intensity was measured on the P2RY12 z-sum projection within the pre-defined background ROI. The pixel value for background intensity was then subtracted from the entire image. Subsequently, P2RY12 intensity was measured.
Analysis of Tau pathology
For 3xTg animals that exhibit tauopathy, images containing CA1, the region with the highest accumulation of pathological Tau, were analyzed. The area fraction of total Tau (HT7) and two phospho-Tau epitopes (pT205 and pS396) were computed in a similar manner as described above for plaque analysis. The ratio between pT205 or pS396 and HT7 was calculated and reported.
Single-cell RNA sequencing
Generation of microglia single-cell suspension for sequencing
3xTg control-chow treated, 3xTg PLX-repopulated, and non-transgenic control-chow treated (NTg) mice were perfused and processed as described above in Flow Cytometry. All of the equipment was maintained at 4°C, and the processing steps were done on ice. The only modifications were that the Fc block was incubated for 10 min, and primary antibodies were incubated for 20 min. The primary antibodies used were CD11b (M1/70) and CD45 (30F11) from BioLegend. DAPI (BD) was used as a viability stain. DAPI-CD45int/+ events were sorted on a BD FACSAria II using an 85-micron nozzle. Each sample took approximately 3-7 minutes to sort. Throughout the protocol, samples were kept on ice, and the FACSAria II was operated in a 4°C environment. In our preliminary experiments, we identified over 85% viability with this method (data not shown). The samples were immediately processed for single-cell capture as described below.
Cellular suspensions containing 50,000-90,000 CD45int/+ events were loaded on a Chromium Single-Cell Instrument (10x Genomics, Pleasanton, CA, USA) to generate single-cell Gel Bead-in-Emulsions (GEMs). Single-cell RNA-Seq libraries were prepared using Chromium Next GEM Single Cell 3′ GEM, Library & Gel Bead Kit v3.1 (10x Genomics). The beads were dissolved, and cells were lysed per the manufacturer’s recommendations. GEM reverse transcription (GEM-RT) was performed to produce a barcoded, full-length cDNA from poly-adenylated mRNA. After incubation, GEMs were broken, and the pooled post-GEM-RT reaction mixtures were recovered, and cDNA was purified with silane magnetic beads (DynaBeads MyOne Silane Beads, PN37002D, ThermoFisher Scientific). The entire purified post GEM-RT product was amplified by PCR. This amplification reaction generated sufficient material to construct a 3’ cDNA library. Enzymatic fragmentation and size selection was used to optimize the cDNA amplicon size, and indexed sequencing libraries were constructed by End Repair, A-tailing, Adaptor Ligation, and PCR. Final libraries contain the P5 and P7 priming sites used in Illumina bridge amplification. Sequence data were generated using Illumina’s NovaSeq 6000.
scRNAseq Data Analysis
CellRanger v3.1.0 pipeline was used to demultiplex, make fastq files and generate gene counts of expression data referenced to mm10-3.0.0. It was determined that pooled samples had 150,000-200,000 mean reads per cell. Over 95% of reads were mapped to the genome, and over 93% of reads were above the quality control score of Q30. The gene expression matrix was analyzed by Seurat v3.1.5 package. Genes detected in less than 3 cells, and ribosomal genes were excluded from the analysis. Cells expressing less than 200 unique genes/features, more than mean+3*standard deviation number of transcripts, or more than 5% mitochondrial genes were excluded from the analysis. Overall, this approach yielded 5943 cells for 3xTg control-chow group and 9885 cells for the 3xTg PLX-repopulated group. These two groups of cells were further compared for findings reported in the main figures. In Supplemental data, we also show a direct comparison of these two groups to the 4979 cells identified from the non-transgenic (NTg) group.
Following the above filtering criteria, the data was normalized, 2000 most variable features were selected, and their expression was scaled with the built-in functions of the Seurat package. The top 20 Principal Components (PCs) were used for subsequent clustering (resolution = 0.25) and UMAP dimension reduction. To directly compare 3xTg microglia with NTg microglia, we used the anchoring algorithm of the Seurat package since these mouse lineages are bred to homozygosity, and the mice are not littermates. No anchoring algorithm was used for comparisons between control and PLX-repopulated 3xTg microglia. The scMCA package was used for the initial annotation of the cells (39). The clusters encompassing perivascular macrophages and microglia (PVMMicro) were manually annotated according to the list of differentially expressed features that were determined by the FindMarkers() function with default Wilcoxon rank-sum test and |logFC| > 0.25. Significantly (padj < 0.05), up-and down-regulated features were used as input to ClusterProfiler v3.16.0 for overrepresentation analysis to identify significantly enriched gene sets. FindMarkers() function was also used to identify differentially expressed features between the two chow treatments.
In situ hybridization
PFA-fixed brain slices were used for in situ hybridization. RNAScope multiplex V2 Assay (ACD Bio) was used to detect Cxcl13 (ACD Bio, 406311) transcripts per manufacturer’s instructions with slight modifications. Specifically, tissue mounted on SuperFrost Plus slides (Fisher Scientific) was subjected to 5 min of antigen retrieval at ~100°C and was digested for 30 min with Protease Plus (ACD Bio). Opal 520 dye (Akoya Biosciences) was used at 1:800 for the detection of transcripts. Negative and positive control probes and spleen tissue (data not shown) were stained in tandem with experimental samples.
ELISA and Western Blot
Frozen hippocampi were homogenized in Tissue Protein Extraction Reagent (ThermoFisher Scientific) at a concentration of 50 mg/ml with 1X Halt Protease and Phosphatase Inhibitor Single-Use Cocktail (ThermoFisher Scientific), vortexed and sonicated. The homogenates were centrifuged for 100,000xg for 1 hour. The supernatant was collected as the soluble fraction; whereas the pellet was incubated in guanidinium-HCl pH 6.0 for 4 h and centrifuged at 100,000xg for 1 hour. This new supernatant was collected as the insoluble fraction. For Aβ40 ELISAs (ThermoFisher Scientific), the soluble fraction was diluted at 1:20, and the insoluble fraction was diluted at 1:3000. For Aβ42 ELISAs (ThermoFisher Scientific), the soluble fraction was diluted 1:2, and the insoluble was diluted 1:30. The soluble fraction diluted at 1:3 was used as input to the CXCL13 ELISA kit (R&D Systems). All dilutions were established empirically.
All statistical analyses were performed in Graphpad Prism v7.04. Comparisons between PLX and control-treated groups in male-only experiments were made using Student’s t-test. Comparisons of MeX04 and MFI of several homeostatic markers were made using two-way ANOVA with Bonferroni correction. All data points that represent individual animal averages are presented as mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001.