Two mouse models were used in this study: heterozygous 5xFAD transgenic mice (on a C57/BL6-SJL background), which overexpress familial AD mutant forms of human APP (the Swedish mutation, K670N/M671L; the Florida mutation, I716V; and the London mutation, V717I) and PS1 (M146L/L286V) transgenes under the transcriptional control of the neuron-specific mouse Thy-1 promoter (19) (5xFAD line Tg6799; The Jackson Laboratory), and Trem2-/-5xFAD mice. Trem2-/-5xFAD and Trem2+/+5xFAD were obtained from the laboratory of Marco Colonna (Washington University, St. Louis), where they were generated as previously described (69). For bone-marrow transplantation assays, donor cells were isolated from C57BL/6 CD45.2 Ub-GFP mice in which GFP is ubiquitously expressed (45). All mice were bred and maintained at the animal breeding center of the Weizmann Institute of Science. For the period of cognitive assessments, mice were kept on a reversed light-dark cycle. All experiments described complied with the regulations formulated by the Institutional Animal Care and Use Committee (IACUC) of the Weizmann Institute of Science. In all experiments, mice were anesthetized and transcardially perfused with phosphate buffered saline (PBS) before tissue dissection.
Preparation of BM chimeras:
Chimeras were prepared by subjecting recipient mice to lethal irradiation (950 rad), directing the beam to the lower part of the body, and avoiding the head (22). The following day, bone marrow (BM) cells were isolated from the tibiae and femur leg bones of Ub-GFP mice and filtered through a 70 µm cell strainer. Each recipient mouse was then reconstituted with 5×106 BM intravenously injected cells of gender-matched donors. The recipient mice were analyzed 5-8 weeks after BM transplantation to determine the extent of chimerism.
For PD-L1 blockade, PD-L1-blocking antibody directed to mouse PD-L1 (anti-PD-L1 antibody, clone 10F.9G2 BIOX-CELL) or an isotype control (anti-keyhole limpet hemocyanin; clone LTF-2 BIOXCELL), were administered i.p. at an effective dose of 1.5 mg/mouse (40).
Single cell sorting:
After perfusion with PBS, supplemented with 1% L-glutamine, brains were excised without olfactory bulb and brain stem, and manually chopped to pieces (0.5-1 mm2 in size), prior to a software-controlled dissociation by gentle MACS™ in PBS. For density gradient separation, the pellet was resuspended with 40% Percoll, and centrifuged at 750 G for 20 min at 20 °C; the supernatant was then discarded. Cells were suspended in ice-cold sorting buffer (PBS supplemented with 2mM EDTA and 2% FCS) supplemented with anti-mouse CD16/32 (BD Bioscience) to block Fc receptors before labeling with fluorescent antibodies against cell-surface epitopes. Samples were stained using the following antibodies: BV421-conjugated CD45, PE-conjugated CD45, PE-conjugated CD11b. For sorting, samples were gated for CD45+ after exclusion of debris and doublets, and further gated according to the experimental design. Cell populations were sorted using either SORP-aria (BD Biosciences) or ARIA-III instruments (BD Biosciences), and analyzed using BD FACSDIVA (BD Biosciences) software. Isolated single cells were sorted into 384-well cell capture plates containing 2 µL of lysis solution and barcoded poly(T) reverse-transcription (RT) primers for single-cell RNA-seq (42). Four empty wells were kept in each 384-well plate as a no-cell control. Immediately after sorting, each plate was spun down to ensure cell immersion in the lysis solution, snap frozen on dry ice, and stored at –80°C until processing.
Massively Parallel Single-Cell RNA-seq library preparation (MARS-seq2.0):
Single-cell libraries were prepared according to the MARS-seq2.0 protocol (43). In brief, mRNA was isolated from cells sorted into capture plates, barcoded and converted into cDNA, and pooled using an automated pipeline. The pooled sample was then linearly amplified by T7 in-vitro transcription, and the resulting RNA was fragmented and converted into a sequencing-ready library by tagging the samples with pool of barcodes and Illumina sequences during ligation, RT, and PCR. Each pool of cells was tested for library quality and concentration, assessed as previously described (43).
Analysis of MARS-seq data:
Single cell RNA-seq libraries (pooled at equimolar concentrations) were sequenced on an Illumina NextSeq 500 at a median sequencing depth of ~20,000 reads per cell. Sequences were mapped to the mouse genome (mm10), demultiplexed and filtered as previously described (42, 43) with the following adaptations: mapping of reads was done using HISAT (v.0.1.6) and reads with multiple mapping positions were excluded. Reads were associated with genes if they were mapped to an exon, using the UCSC Genome Browser for reference. The level of spurious unique molecular identifiers (UMIs) in the data were estimated by using statistics on empty MARS-seq wells, and excluded rare cases with estimated noise > 5%. We used the R package ‘‘MetaCell’’ (44) to generate homogenous and robust groups of cells in each analysis. We removed specific mitochondrial genes, immunoglobulin genes, and genes linked with poorly supported transcriptional models (annotated with the prefix ‘‘Rp-’’). We then filtered cells with less than 400 UMIs. Feature genes were selected using the parameter Tvm = 0.8 and a minimum total UMI count > 50. In all the analysis of all experiments, microglia were identified according to the expression of Hexb gene.
Aβ1-40 and Aβ1-42 ELISA:
For these experiments, tissues from 7-10 month-old mice were used. Following intracardial PBS perfusion of the mice, the hippocampi were collected from one or both brain hemispheres, as indicated in the legends, and immediately frozen and stored at -80°C. The samples were homogenized in TBS solution [Tris, pH 7.4 (50 mM), NaCl (150 mM)], EDTA (2mM), and 1% Protease Inhibitor Cocktail (Sigma Aldrich) using the Micro Tube homogenizer with plastic pestles. The lysates were then centrifuged for 40 minutes at 350,000 g in 500 μl Polycarbonate centrifuge tubes (Beckman Coulter) at 4°C in an Optima MAX-XP Ultracentrifuge with a TLA 120.1 rotor (Beckman Coulter). The supernatant was collected and stored at -80°C as “TBS-soluble fraction” ready for further ELISA assay. The pellet was completely resuspended in Triton-X-100 solution [Tris, pH 7.4 (50 mM), NaCl (150 mM), 1% Triton X-100 (Sigma Aldrich), 1% Protease Inhibitor Cocktail (Sigma Aldrich)]. After 15 min incubation at 4°C, the samples were centrifuged for 40 minutes at 350,000 g at 4°C, and the supernatant was collected as “Triton-X-100-soluble fraction” and stored at -80°C. BCA assay (Pierce BCA Protein Assay Kit) was performed to determine the protein concentrations in both TBS-soluble (also referred as “soluble”) and Triton-X-100-soluble (or “insoluble”) fractions. In the experiments presented in Figure 2, LEGEND MAX β-Amyloid x-40 and x-42 ELISA Kits (BioLegend) were used to measure Aβ1-40 and Aβ1-42 peptide levels, respectively, following the manufacturer’s instructions, in the experiments presented in Figure 4, human Aβ42 Ultrasensitive ELISA Kit (Invitrogen) was used to measure Aβ1-42 levels, according to the manufacturer’s instructions.
For depletion of CCR2-expressing cells, anti-CCR2 monoclonal antibody, MC21was injected i.p. (400μg) every 4 days. No effect on behavior was observed in wild type animals.
After perfusion with PBS, brain tissues were excised and fixated. Paraffin-embedded sections were prepared, as previously described (31). The following primary antibodies were used: mouse anti-Aβ (1:300, Covance, #SIG-39320) and rabbit anti-synaptophysin (1:100, Abcam, #32127). Secondary antibodies included: Cy2-conjugated donkey anti-mouse and Cy3-conjugated donkey anti-rabbit antibodies (1:200; all from Jackson Immunoresearch) Microscopic analysis was performed using a fluorescence microscope (E800; Nikon) equipped with a digital camera (DXM 1200F; Nikon), and with a ×20 NA 0.50 objective lens (Plan Fluor; Nikon). Representative images were taken using confocal microscopy (Zeiss, LSM880), with 20x objective lens.
Aβ plaque quantitation:
From each brain, 6 µm sagittal slices were made, and four sections per mouse were immunostained. Histogram-based segmentation of positively stained pixels was performed using Image-Pro Plus software (Media Cybernetics, Bethesda, MD, USA). The segmentation algorithm was manually applied to each image, in the DG area, and the percentage of the area occupied by total Aβ immunostaining, selected for minimal size, was determined. Plaque numbers were quantified from the same 6 µm sagittal brain slices, and are presented as the average number of plaques per brain region, in the region of interest (ROI), identically marked on all slides from all animals examined. Prior to quantification, slices were coded to mask the identity of the experimental groups, and were quantified by an observer blinded to the identity of the groups.
Radial Arm Water Maze (RAWM):
RAWM was used to test hippocampal-dependent spatial learning, following the protocol of Alamed and colleagues (70) with some modifications. Briefly, six stainless-steel inserts were placed in a plastic pool, forming six open and connected arms. A hidden platform was placed at the end of a ‘goal arm’ (arm 6; Supplementary Figure 2a). Milk powder was used to make the water opaque, and the water was maintained at a temperature of 23±1°C. On day 1, training phase, mice were subjected to 15 trials. In each trial, a mouse was given 60 sec to find the platform. A mouse that failed in finding the platform, was placed on it by the experimenter. Inter-trial interval (ITI) was, on average, 20 min. Trials alternated between a visible and hidden platform. However, from trial 12 and throughout the second day, the platform was hidden. Spatial learning and memory were measured by an investigator who was blinded to the treatment of the mice, and who recorded the number of arm entry errors (error was defined as entrance to an incorrect arm, or a failure to enter any arm within 15 sec) as well as the escape latency of the mice on each trial. The 30 trials were grouped into 3-trial bins – 5 bins on day 1, and 5 bins on day 2. Data were analyzed by a team member who did not perform the experiment.
Novel Object Recognition (NOR) task
The NOR protocol was modified from Bevins and Besheer (71), and used a 41.5 x 41.5 cm gray apparatus. The experiment spanned 2 days and included three trials: a habituation trial – a 20 min session in the empty apparatus (day 1), familiarization trial – a 10 min session presenting two identical objects located 15 cm apart (day 2); and a test trial – following a 1 hour training-to-testing interval, each mouse was returned to the apparatus for a 6 min session in which one of the objects was replaced by a novel one (see supplementary, Figure 2b). Mouse behavior was recorded and analyzed by an investigator who was blinded to the treatment group. Novel object preference was defined as “discrimination ratio” = time (sec) spent with novel object / (time spent with familiar object + time spent with novel object). Locomotor activity was measured as distance moved (cm/day) in the empty arena during the day of the habituation. The time spent in the center of the arena was recorded to assess anxiety.
A paired student’s t-test (one-tail) was performed to assess the difference in percentage of DAM out of total microglia cells (Fig. 1c). Unpaired student’s t-tests (one tail) were used to analyze the data of immunostaining measurements (Fig. 2f, Supplementary Figure 2d) and ELISA with two groups (Fig. 4e). One-way ANOVAs were used to analyze the data of ELISA (Fig. 2d,e, Supplementary Figure 2e,f), and NOR (Fig. 2c, Fig. 4d and Supplementary Figure 4c), as well as of activity (Supplementary Fig. 4d) and anxiety (Supplementary Figure 4e) measurements collected at NOR test habituation phase. Two-way ANOVAs with repeated measures (one for latency and one for errors were used to analyze RAWM data (Fig. 2b, Fig. 4b,c and Supplementary Fig. 2c ). ANOVAs identifying a significant result were followed by a Fisher’s LSD test for multiple comparisons. Pearson correlation was used to determine the correlation between RAWM performance (time to platform) and the levels of soluble Aβ1-42 (Fig. 4f). Differential gene expression analysis (Fig. 3e, Supplementary Fig. 3) was performed upon down-sampling of the UMI matrix as part of the MetaCell package on molecules/1,000 UMIs by Mann-Whitney U test with false-discovery rate (FDR) correction.