RESOURCE AVAILABILITY
Lead Contact
Further information and requests for resources and reagents should be directed to and will be addressed by the Lead Contact, Rada Koldamova ([email protected]).
Materials Availability
This study did not generate new unique reagents.
Data and Code Availability
The RNA-seq expression data has been deposited in the GEO database under the accession number: To Be Determined.
METHOD DETAILS
Experimental models
All animal experiments were performed in accordance with the NIH Guide for Care and Use of Animals and approved by the University of Pittsburgh Institutional Animal Care and Use Committee. The following mouse lines were bred to generated experimental mice: Wild-type C57BL/6J (WT; JAX), human APOE3 and APOE4 targeted replacement mice (E3 & E4; Taconic), Trem2em2ADiuj/J mice (Trem2ko; JAX) and 129P2(Cg)-Cx3cr1tm1Litt/J (Cx3cr1GFP; JAX). Human APOE3 and APOE4 targeted replacement mice were bred to Abca1tm1Jdm/J mice (Abca1ko; JAX) to generate APOE3/Abca1het and APOE4/Abca1het (E3het & E4het). Likewise, human APOE3 and APOE4 targeted replacement mice were bred to Trem2em2ADiuj/J mice (Trem2ko; JAX) to generate APOE3/Trem2ko and APOE4/Trem2ko (E3/Trem2ko & E4/Trem2ko). All experimental mice were on the C57BL/6 genetic background, kept on a 12 h light-dark cycle with ad libitum access to food and water and randomly assigned to an experimental group. All reagents were purchased from Fisher Scientific unless documented otherwise.
AD brain samples
Human samples (Suppl. Table 1) were provided by the University of Pittsburgh Alzheimer’s Disease Research Center (ADRC) brain bank and the Sanders-Brown Center on Aging at the University of Kentucky. Braak staging was performed on Bielschowsky-stained sections and APOE allelic polymorphism determined by a PCR-based assay. Gray matter samples of APOEε3/3 and APOEε4/4 genotypes from the right inferior parietal lobule were dissected and used for Multi-Dimensional Mass Spectrometry Shotgun Lipidomics (MDMS-SL). Age and Postmortem intervals (PMI) matching was confirmed by t test.
Native APOE generation and characterization
Cultures of primary astrocytes were established from one-day-old E3, E4, E3het and E4het targeted replacement pups as previously shown42, 43. Briefly, the cortices and hippocampi were mechanically dissociated using a sterile pasteur pipette and cultivated in DMEM/F12 medium supplemented with 10% bovine growth serum, l-glutamine and antibiotics. Cells were cultured on poly-d-lysine (100 μg/mL) coated T75 Costar flasks (Corning). Confluent primary astrocytes were incubated with treatment medium (Neurobasal medium with antibiotics and glutamine as above minus serum) for 48 h. Conditioned medium were collected, immediately filtered through 0.22 μm filter and concentrated using Amicon® Ultra Centrifugal Filters (10kD cutoff). Native APOE lipoproteins were washed 3 times with cold PBS and their concentration measured by quantitative Western Blotting (NUPAGE) using commercial human APOE (Meridian Life Science) as a standard. Native APOE particles were resolved on Novex™ 4-20% Tris-Glycine gels using an Amersham™ HMW calibration kit as native ladder (GE Healthcare). The size of APOE lipoprotein particles were measured by dynamic light scattering with Zetasizer Nano Z (Malvern). APOE lipoprotein cholesterol content was assessed with the Cholesterol Quantitation Kit (Sigma) according to the manufacturer’s instructions.
Multi-Dimensional Mass Spectrometry Shotgun Lipidomics (MDMS-SL)
The MDMS-SL assay44, 45, 46 was performed to determine differences in the lipid composition of the native E3 and E4 particles isolated from primary astrocyte cultures and human AD brain samples described above. APOE particles isolated from E3het and E4het primary astrocytes, exhibiting diminished APOE lipidation states, were used as negative controls. Quantitative analysis was performed on a triple-quadrupole mass spectrometer (Thermo Fisher Scientific) equipped with an automated nanospray apparatus NanoMate and Xcalibur system. Internal standards for the quantification of individual molecular species of the major lipid classes were added to each sample at the start of the extraction procedure. Lipid extraction was performed by the methyl-tert-butyl ether (MTBE) method with resuspension in chloroform/methanol (1:1 v/v) solution with nitrogen flush. Identification and quantification of all reported lipid molecular species were performed using an in-house automated software program45. The resulting MDMS-SL data was analyzed and visualized in R (v3.6.0) and GraphPad Prism (v8.3.1). Phosphatidylcholine = PC; Phosphatidylinositol = PI; Phosphatidylethanolamine = PE; Phosphatidylserine = PS; Sphingomyelin = SM; Lysophosphatidylcholine = LPC; Lysophosphatidylethanolamine = LPE; Phosphatidylglycerol = PG; and Phosphatidic acid = PA.
Liquid Chromatography-Mass Spectrometry (LCMS) Lipidomics
Phospholipids were extracted and separated as before47. Briefly, MS analysis of phospholipids was performed on a Q-Exactive hybrid-quadrupole-orbitrap mass spectrometer (Thermo Fisher Scientific) as previously described47 using a normal phase column (Luna 3 µm Silica 100Å, 150 x 2.0 mm, (Phenomenex)) at a flow rate of 0.2 mL/min on a Dionex Ultimate 3000 HPLC system (maintained at 35°C). The analysis was performed using A and B gradient solvents containing 10 mM ammonium acetate and 0.5% triethylamine (A - propanol:hexane:water (285:215:5, v/v/v) and B - propanol:hexane:water (285:215:40, v/v/v)). The column was eluted for 0.5 min isocratically at 25% B, then from 0.5 to 6.5 min with a linear gradient from 25% to 40% solvent B, from 6.5–25 min using a linear gradient of 40-55% solvent B, from 25-38 min with a linear gradient of 55-70% solvent B, from 38-48 min using a linear gradient of 70%-100% solvent B, then isocratically from 48-55 min at 100% solvent B followed by a return to initial conditions from 55-70 min from 100% to 25% B. The column was then equilibrated at 25% B for an additional 5 min. Analysis was performed in negative ion mode at a resolution of 140,000 for the full MS scan in a data-dependent mode. The scan range for MS analysis was 400-1800 m/z with a maximum injection time of 128 ms using 1 microscan. An isolation window of 1.0 Da was set for the MS and MS2 scans. Capillary spray voltage was set at 3.5 kV and capillary temperature was 320oC. The S-lens Rf level was set to 60. The phospholipid content for each APOE isoform was normalized to the amount of APOE protein as determined by SDS NuPAGE. The analysis of the resulting LCMS data was performed and visualized in R (v3.6.0) and GraphPad Prism (v8.3.1). See lipid class abbreviations in prior section.
Aβ oligomer preparation
Aβ42 peptide (American Peptide Company), HiLyte™ Fluor 488-Aβ42 or HiLyte™ Fluor 555-Aβ42 (AnaSpec) were used for all Aβ oligomerizations and injections as before27. Under a fume hood, 0.1 mg Aβ peptide was dissolved in ice cold 1,1,1,3,3,3-Hexafluoro-2-Propanol (HFIP, Fluka) then vortex for a few seconds. The solution was dried with a gentle stream of nitrogen to obtain a peptide film at the bottom of the vial. Prior to use, the film was re-suspended in anhydrous dimethyl sulfoxide (DMSO) to form a 5 mM solution, sonicated in a water bath for 10 min and diluted in sterile phosphate-buffered saline (PBS) to a final concentration of 100 μM. HiLyte™ Fluor 488- or 555-Aβ or Aβ42 peptide and E3 or E4 native particles were combined, vortexed, held under oligomer forming conditions (room temperature) for 24 h at a final concentration of 50 μM Aβ and 5 μM APOE and stored at -20° C until use (abbreviated AβE3 or AβE4). The same molar concentration (10:1) of scrambled Aβ (AnaSpec) was dissolved in vehicle, combined with isolated E3 or E4 particles, held under oligomer forming conditions and stored at -20° C until use (abbreviated scrAβE3 or scrAβE4). scrAβE3 or scrAβE4 was utilized as negative controls throughout the subsequent experiments. Furthermore, Aβ42 peptide and scrambled Aβ was incubated with PBS vehicle as negative control for APOE particles.
Aβ oligomer characterization
Western Blot
Aβ oligomers were examined using western blotting, proteins were resolved on 4-12% Bis-Tris gels (Invitrogen) and transferred onto nitrocellulose membranes. These membranes were probed with 6E10 antibody (Biolegend) and anti-APOE antibody (Invitrogen) with immunoreactive signals visualized using enhanced chemiluminescence.
Electron Microscopy
For electron microscopy, 5 μL of each sample was placed on a freshly glow-discharged carbon-coated grid, adsorbed for 2 min and excess solution was blotted using filter paper27. The grid was washed with deionized water before staining with 5 μL of freshly filtered uranyl acetate solution (1%, w/v) for 15 s. Excess stain was blotted and the grid was allowed to air-dry. Grids were imaged on a Tecnai T12 microscope (FEI) operating at 120 kV and ×30,000 magnification and equipped with an UltraScan 1000 CCD camera (Gatan) with post column magnification of 1.4x.
Guide cannula implantation
To examine the cognitive effects in WT male mice (3 mo.), Aβ oligomers co-incubated with native APOE lipid particles were infused directly into the brain through implanted guide cannulas27. Following anesthesia with isoflurane, the head was shaven and sterilized with two separate povidone-iodine – alcohol washes. A 50% mixture of bupivacaine and lidocaine was applied to the surgical site and ophthalmic ointment applied to the eyes. The head was leveled in a stereotaxic frame and an incision made exposing the dorsal aspect of the skull. Two holes were drilled into the skull (coordinates: AP = -2.46 mm, L =±1.50 mm) and 26-gauge guide cannulas (Plastics One) were lowered to a depth of 1.0 mm. Cannulas were fixed to the skull with acrylic dental cement attached to two bone anchoring screws and the surgical opening sutured closed. After suturing, animals were administered buprenorphine and sterile saline, body temperature was maintained until sterna recumbence and animals were allowed to recover for 8 days prior to the start of behavioral testing.
Behavioral testing
All stages of behavioral testing were performed at the same time of the day (during the light phase), ensuring 24 h between each phase of testing. Fear conditioning was started 24 h after the completion of novel object recognition. Prior to behavioral testing, mice were placed into individual containers, taken to the behavioral testing room and handled for 3 min for three consecutive days to reduce anxiety. Thirty minutes before a training stage, animals received an infusion of Aβ oligomer co-incubated with either E3 or E4 (Fig. 2a-d). Scrambled Aβ co-incubated with either E3 or E4 was used as a negative control. Mice were randomly assigned to either the AβE3, AβE4, scrAβE3 or scrAβE4 group. The dummy cannulas were removed and infusion cannulas, attached to microsyringe pump by polyethylene tubing, were placed in the guide cannula. Aβ oligomer (final volume of 1 μL per hemisphere) was infused over 1 min, the cannulas were left in place for 1 min to allow for diffusion of the sample and finally dummy cannulas replaced. Each animal received 5 infusions of Aβ oligomer starting on day 2 of novel object recognition until the completion of behavioral testing (day 6). Between behavioral trials, the paradigms were cleaned with 70% ethanol to eliminate any olfactory cues. Performance was recorded and scored using ANY-maze software (Stoelting Co.) during all phases of testing.
Novel Object Recognition
Novel object recognition (NOR) was performed over three consecutive days as previously described24. On Day 1, habituation phase, each animal was allowed to freely explore an open arena (40 cm X 40 cm X 30 cm white plastic box) for two 5 min trials with a 5 min inter-trial interval. On Day 2, familiarization phase, each animal was returned to the arena containing two identical objects (tower of LEGO® bricks) located in opposite diagonal corners for two 5 min trials separated with a 5 min inter-trial interval. On Day 3, testing phase, the animal was returned to the arena with two objects in the same positions as previously, but one object was replaced with a novel object (metal bolt and nut of similar size). Mice were allowed to explore the two objects for 10 min. The exploration of both objects was defined as the mouse sniffing or interacting while facing an object within 3 cm. Mice were consistently placed into the middle of the arena facing the posterior wall to prevent any object preference. The percent exploration, an indicator of recognition memory, was determined by dividing the time exploring the novel object by the total time exploring both objects. Locomotor activity was assessed by measuring the total distance traveled in the open field during day 1 of testing.
Contextual and Cued Fear Conditioning
Contextual and Cued Fear Conditioning (CCFC) was performed over three consecutive days as previously described24. On Day 1, training phase, mice were placed in a conditioning chamber (Stoelting Co.) for 5.5 min. The first 2 mins were silent, allowing the mouse to acclimate to the chamber; this was followed by a 30 sec tone (2,800 Hz; Intensity 85 dB, conditioned stimulus (CS)) ending in a 2 sec foot shock (0.7 mA, unconditioned stimulus (US)) through the floor of the conditioning chamber. The process was repeated one more time and ended with 30 sec of re-acclimation. On Day 2, contextual phase, mice were placed in the same conditioning chamber for 5 min with no tone or shock administered, to measure contextual fear conditioning. On Day 3, the gray walls of the chamber were replaced with black and white striped walls to introduce a novel environment for assessing cued fear conditioning. Mice were placed in the conditioning chamber for 5 min. After the first 2 min of silence, the tone was administered for 3 min, to measure cued fear conditioning. Freezing time was defined as the absence of movement except for respiration and calculated as percent freezing of the total time in the chamber during each phase of testing.
Cortical infusion
WT male mice (3 mo.) received cortical infusions of un-labeled AβE3 or AβE4. In a separate cohort of Cx3cr1GFP, WT and Trem2ko male mice (3 mo.), HiLyte™ Fluor 555-labeled Aβ combined with native E3 and E4 particles was infused into the cortex. Mice were randomly assigned to either the scrambled Aβ, Aβ alone, AβE3 or AβE4 group. For the bilateral infusion, a 28-gauge infusion cannula (Plastics One) was connected to a 10 µL glass syringe (Hamilton) with vinyl tubing and placed in a micro syringe pump. Following anesthesia with isoflurane, the head was shaven and sterilized with two separate povidone-iodine-alcohol washes. A 50% mixture of bupivacaine and lidocaine was applied to the surgical site and ophthalmic ointment applied to the eyes. The head was leveled in a stereotaxic frame and an incision made, exposing the dorsal aspect of the skull. Four holes were drilled into the skull (coordinates: AP= -1 and -2.5 mm, L= +/-2.0 mm) and the infusion cannula was lowered into the cortex (DV= -1.0 mm). At each infusion site, 2 µL of Aβ preparation was infused at a rate of 0.5 µL/min and the cannula remained in place for 4 min following the infusion. Following the infusions, the surgical opening was sutured closed, animals were administered buprenorphine and sterile saline and placed on a heating pad until fully recovered.
Animal tissue processing
4 or 24 h following the cortical infusion, mice were perfused and tissues collected. Mice were anesthetized with Avertin (250 mg/kg of body weight, i.p.) and transcardially perfused with 25 mL of cold 0.1 M PBS, pH 7.4. For Cx3cr1GFP and Trem2ko mice used for FACS, flow cytometry and single cell RNAseq the infusion sites in both hemispheres were excised for cellular isolation as described below. For WT mice, one hemisphere was drop fixed in 4% phosphate-buffered paraformaldehyde at 4°C for 48 h before storage in 30% sucrose. The other hemisphere was used for cellular isolation as described below. Fixed hemibrains were mounted in O.C.T. and cut in the coronal plane at 30 µm sections using a frozen cryotome (Thermo Scientific) and stored in glycol-based cryoprotectant at -20°C until histological staining. Prior to staining, sections containing the infusion site were selected under a dissection microscope (Olympus).
Immunohistochemistry
To characterize changes in activated microglia in the vicinity of the infusion site, a series of 6 brain sections from each animal was immunostained with anti-IBA1 antibody (WAKO). Free-floating sections were washed, followed by antigen retrieval in sodium citrate buffer at 80°C for 60 min, blocked in Normal Donkey Serum (Jackson Lab) for 1 h and finally incubated in IBA1 antibody (1:1000) overnight at 4°C. Sections were washed and transferred into secondary donkey anti-rabbit Alexa 594 antibody (Invitrogen) for 1 h, before being washed, mounted on superfrost plus slides and coverslipped. Fluorescent confocal images of the infusion site were taken using a Nikon A1 confocal microscope at 60x magnification with 1.0 μm step size. Four sequential images were captured flanking either side of the infusion site. Analysis was performed as in Marsh et al.48 with modifications. The FilamentTracer module (Imaris, version 7.1.1, Bitplane) was utilized to trace processes of cortical IBA1 positive microglia and determine process length and number of branch points as indicators of microglial morphology complexity. Using the 3D mapped filament tracings, we oriented all microglia so their central axis was positioned facing the infusion site and generated a heatmap depicting the location of microglia from an experimental group utilizing the ImageJ - heatmap from image stack plugin (National Institutes of Health). The number of overlapping microglial processes increases the pixel saturation metric. The percent of all processes which occupy space in each of the 4 quadrants was used to determine the percent coverage of that quadrant.
A second series of 6 brain sections around the infusion site was used for F4/80 immunostaining. First, we quenched endogenous peroxidases with 0.3% hydrogen peroxide, tissues were blocked in 3% normal goat serum (Vector), then blocked for endogenous avidin and biotin. Sections were incubated in F4/80 antibody (Abcam) overnight at 4°C. Sections were washed and transferred into secondary biotinylated anti-rat antibody (Vector) for 90 min before being washed and subsequently developed using the Vector ABC kit and DAB substrate kit (Vector). Sections were mounted onto superfrost plus slides and coverslipped. Bright-field images were taken using a Nikon Eclipse 90i microscope at 20x magnification encompassing the infusion site. Image intensity threshold was established to detect the F4/80 staining compared to background using NIS Elements software (Nikon Instruments Inc.) and values were represented as the area of staining normalized to total image area or percentage of area covered.
Magnetic-activated cell sorting (MACS)
For WT mice infused with unlabeled Aβ, we isolated microglial and neuronal cellular populations utilizing MACS column-based protocols according to manufacturer instructions (Miltenyi Biotec). First, the tissue was dissociated utilizing the Neural Tissue Dissociation kit (Miltenyi Biotec), a gentle two-step enzymatic dissociation (papain and trypsin) that yields a high number of viable cells. Briefly, the cortical tissue was enzymatically and mechanically lysed at 37°C for 35 min to obtain a cell suspension. Then the suspension was passed through a 40 µm cell strainer to remove debris and ensure a single-cell suspension. After a 10 min centrifugation at 300 g, the pellet was gently resuspended with ice-cold PBS + 0.5% BSA buffer and incubate for 15 min at 4°C with Myelin Removal Beads II (Miltenyi Biotec). After washing with PBS + 0.5% BSA and 10 min centrifugation at 300 g, the pellet was resuspended with PBS + 0.5% BSA. The single-cell suspension was applied to a column placed on a magnetic stand (LS column and magnets from Miltenyi Biotec) to deplete the myelin fragments by magnetic separation and washed with PBS + 0.5% BSA. Finally, the cells were centrifuged for 5 min at 300 g and the pellet was resuspended with 1 mL of PBS + 0.5% BSA. After obtaining a myelin-free single-cell suspension, the microglia were labeled with anti-CD11b immunomagnetic beads and isolated using magnetic columns (Miltenyi Biotec). To isolate the neurons, the cell suspension depleted of microglia cells was incubated with a cocktail of antibodies from the Neuron Isolation Kit (Miltenyi Biotec). Non-neuronal cells like astrocytes, oligodendrocytes, endothelial cells or fibroblasts were magnetically labeled and depleted through the magnetic columns; therefore, neurons were isolated by negative selection. To confirm the purity of isolated microglia, we performed RT-qPCR with P2ry12 and Tmem119, microglia-specific markers. To further ensure microglia and neuron purity when using MACS, following sequencing and alignment, all genes with low expression (average raw read count < 30.9) were removed from further analysis.
Fluorescence-activated cell sorting (FACS) and flow cytometry
For the Cx3cr1GFP mice infused with HiLyte™ Fluor 555-labeled Aβ, microglia were isolated utilizing FACS. After removing the cerebellum, subcortical area and olfactory bulbs, cortical tissue within 1 mm of either side of the infusion site was processed into a single-cell suspension using the Neural Tissue Dissociation kit followed by the Myelin Removal Beads II (Miltenyi Biotec) as described above. For WT and TREM2ko mice, after the myelin removal steps the pellet were resuspended with 100 µl of PBS and the microglia cells were labeled with 5 µl of anti-mouse/human CD11b antibody (clone M1/70) conjugated with Alexa FluorÒ 647 (BioLegend) for 20 min on ice. All cells were then washed and prepared with 1 mL of PBS + 0.5% BSA for FACS sorting.
A bio-contained BD FACSAria™ III sorter (BD Biosciences) was used to sort microglia cells for the two experimental populations. First, live cells were separated from the debris according to their forward scatter and side scatter properties and a second gate was used on individual cells only. The GFP fluorescence was detected with a 525 nm filter (488 nm laser) for the microglia isolated from the Cx3cr1GFP mice, the CD11b/APC fluorescence was collected with a 668 nm filter (647 nm laser) for the WT and TREM2ko mice and the 555-labeled Aβ fluorescence was detected with a 613 nm filter (555 nm laser). 2,000 microglia with high GFP signal but low 555-labeled Aβ signal, were considered unexposed to Ab and termed single-positive cells (single+). 2,000 microglia cells with a high signal for both labels (GFP and 555-labeled Ab) were considered exposed to Ab and termed dual-positive cells (dual+). The single+ and dual+ microglial populations were sorted into 1.5 mL tubes containing 350 µL of RLT lysis buffer from the RNeasy Micro kit (Qiagen) and 3.5 µL of 2-Mercaptoethanol. After the FACS sorting, the bio-contained BD FACSAria™ III sorter was used with the same gates to count and estimate the percentages of the total microglia population and single+ and dual+ microglia population in a total of 1,000,000 live cells.
RNA isolation and RNA-sequencing
RNA was isolated from the MACS-isolated microglia and neurons and FACS single+ and dual+ microglia following the manufacturer instructions for the RNeasy Mini kit and RNeasy Micro kit (Qiagen) respectively. After RNA isolation, concentration was measured with a Qubit 3.0 Fluorometer (Thermofisher) before quality assessment with the 2100 Bioanalyzer instrument (Agilent Technologies). Sequencing libraries for MACS-isolated cells were generated using mRNA Library Prep Reagent set (Illumina) as previously described49. The total RNA from sorted cells was fragmented and converted into cDNA using the SMART-Seq® v4 Ultra® Low Input RNA Kit for Sequencing (Takara Bio). Briefly, a minimum of 10 pg of purified total RNA was used to perform a first-strand cDNA synthesis in a PCR clean workstation. After amplification by LD PCR and purification using the Agencourt AMPure XP beads (Beckman Coulter), 1 μL of the amplified cDNA was used for validation using the Agilent 2100 Bioanalyzer on a High Sensitivity DNA chip. 1 ng of the cDNA was used with the Nextera DNA Library Preparation Kit (Illumina) to prepare the libraries. After tagmentation, the cDNA was linked with two different index adapters from the Nextera XT Index Kit (Illumina) during the amplification by PCR. Each sample received a unique combination of two specific index/barcodes during the library generation. Finally, the libraries were purified using Agencourt AMPure XP beads that provide a size selection to remove short library fragments. Again, 1 μL of the libraries was used for validation using the Agilent 2100 Bioanalyzer on a High Sensitivity DNA chip. After preparation of the libraries, sequencing was performed by the Next Generation Sequencing Center (University of Pennsylvania, https://ngsc.med.upenn.edu/) on HiSeq 2500 machine.
mRNA-seq data processing
Following initial processing and quality control, the sequencing data was aligned to the mouse genome mm10 using Subread/featureCounts (v1.5.3; https://sourceforge.net/projects/subread/files/subread-1.5.3/) with an average read depth of 21,554,242 successfully aligned reads. Statistical analysis was carried out using Rsubread (v1.34.2; https://bioconductor.org/packages/release/bioc/html/Rsubread.html), DEseq2 (1.24.0; https://bioconductor.org/packages/release/bioc/html/DESeq2.html), and edgeR (v3.26.5; https://bioconductor.org/packages/release/bioc/html/edgeR.html), all in the R environment (v3.6.0; https://www.r-project.org/). Functional annotation clustering was performed using the Database for Annotation, Visualization and Integrated Discovery (DAVID v6.8, https://david.ncifcrf.gov). All GO terms were considered significant if p<0.05 following corrections for multiple comparisons using the Benjamini-Hochberg method to control the FDR. t-Distributed Stochastic Neighbor Embedding (t-SNE) plots were generated to visualize patterns of similarity between groups. To generate the t-SNE, normalized data from all genes in the analysis were submitted and the perplexity set as high as possible given the total number of samples in the analysis with the theta set to 0.5.
Single-cell isolation and library creation
Separate biological replicates from AβE3 or AβE4 infused WT and Trem2ko mice were used for single cell RNA-seq (scRNA-seq) and infused as described above. 24 h after the injections mice were perfused with PBS. After the brain extraction, cortical tissue around the injection site were excised using adult mouse brain matrix at 1 mm before and after the injection site. The brain tissue was then dissociated according to manufacturer instructions (Miltenyi Biotec) as described above. CD11b-purified cells were resuspended in 0.04% BSA/PBS solution and passed through a 40-μm nylon and a 20-μm nylon cell strainer to obtain single cell suspension. The cell viability and number were assessed using an Countess II FL Automated Cell Counter (ThermoFisher) with Live/Dead viability/Cytotoxicity kit (Invitrogen). The single cell suspension with >90% viability was immediately loaded onto the Chromium controller (10x Genomics) in order to capture ~5,000 cells per sample. Libraries were generated with Chromium single cell 3’ chip kit v3 for scRNA-seq (10x Genomics) according to the manufacturer’s instructions and then the quality assessed using Agilent Bioanalyzer 2100. Sequencing was performed using NovaSeq (Illumina) by MedGenome Inc. with a custom sequencing setting (28bp for Read1 and 91bp for Read2) to obtain an average sequencing depth of 45K reads/cell.
scRNA-seq data processing
After libraries were sequenced and quality control was performed, samples were aligned to the mm10 mouse reference genome using the Cell Ranger 3.0.1 pipeline (https://support.10xgenomics.com/single-cell-gene-expression/software/pipelines/latest/what-is-cell-ranger). Each sample was then aggregated using the cellranger aggr function to produce a raw UMI count matrix containing the number of reads for genes in each cell per sample. The expression matrix was then loaded into R for further analysis and visualization using Seurat v3.0.250. Genes expressed in fewer than 200 cells were excluded from further analysis. The number of genes expressed in each cell (nGene), the number of reads in each cell (nCount) and the percentage of reads mapped to mitochondrial genes (percent.mito) were obtained for each cell. Cells were then filtered to reduce the potential of including doublet and low-quality cells using the following criteria: 200 < nGene < 8500; 500 < nCount < 90000; and percent.mito <25%. Feature counts were normalized using LogNormalize method with a scale factor of 10000; and the effects of nGene, nCount and percent.mito were regressed out using the ScaleData method. A shared nearest neighbor (SNN) graph was constructed using FindNeighbors function with default parameters. Using the Louvain algorithm implemented in FindClusters function with a clustering resolution of 0.2 and the first 50 PCs, we identified 16 clusters. To determine the cell types, present in each cluster, we examined the expression levels of cell type specific markers across each cluster and identified clusters containing unique populations of microglia, neurons, astrocytes, vascular cells, ependymal cells and myeloid cells. We used FindAllMarkers function to identify genes that act as markers for each cluster, using the Wilcoxon rank-sum test. A gene was considered the marker of a cluster if it had a Bonferroni-adjusted p-value < 0.01 and an average log fold change > 0.1. The data were then filtered to contain clusters containing only microglia and cells with a normalized expression < 1 for Aldoc, F13a1, Mylk, Meg3, Gfap, Thy1, Slc17a7, Olfm1, Aqp4, Dlx1, Olig2, Foxj1, Nkg7, Vtn, Flt1, Acta2 and S100a9 were removed, resulting in the retention of 36,244 microglial cells. To control for the potential of batch effect we regressed out the percentage of UMI counts in each cell belonging to several sex-linked genes: Ddx3y, Eif2s3y, Xist, Kdm5d and Uty. Using a clustering resolution of 0.1 and the first 50 PCs, we identified 5 separate clusters. To perform differential expression analysis, we used Seurat’s FindMarkers function and performed Wilcoxon rank-sum tests. A gene was considered differentially expressed if it had a Bonferroni-corrected p-value < 0.05 and a natural log fold change (logFC) > 0.1. Seurat data objects were reformatted for pseudotime analysis using the SingleCellExperiment package and trajectory analysis was performed using Slingshot51 establishing cluster 1a as the starting cluster and clusters 2, 3 and 4 as terminal clusters.
Fluorescence in situ hybridization (FISH)
In a separate infused cohort, mice were perfused, tissue fixed and sectioned as documented above. RNAscope experiments were performed using the Multiplex Fluorescent Reagent kit v2 (Advanced Cell Diagnostics) following the manufacturer’s recommendations with minor adjustments. Six freshly sectioned tissues per animal were mounted onto superfrost plus slides within a 0.75” x 0.75” square and baked at 60°C for 60 min. Slides were dehydrated using a series of ethanol dilution steps, then submerged in target retrieval reagent at 100°C for 10 min. Protease digestion was performed at 40°C for 30 min using Protease III and probe hybridization was carried out at 40°C for 2 h. We used probe sets available from ACD for Adgre1 and Tmem119. Following the amplification steps, the sections were counterstained with DAPI and coverslipped. Imaging was carried out using a Nikon Eclipse 90i microscope at 20X magnification with tiled imaging of the entire infusion site and analyzed using NIS Elements software (Nikon Instruments Inc.). Four 200 µm x 200 µm ROIs were drawn and a threshold established for each probe to determine the area of puncta coverage. The 4 ROI were averaged together to create one data point per infusion site representing the average area of puncta coverage within 200 µm of the edge of the infusion site.
Two-photon imaging and statistical analysis
We used Cx3cr1GFP male mice (3 mo.) to determine the microglial response of AβE3 or AβE4 utilizing two-photon imaging52 . Briefly, the animals were anesthetized I.P. with a cocktail of ketamine/xylazine (75/10 mg/kg) and placed in a stereotaxic frame. Body temperature was maintained at 37.6°C. The skin above the skull was removed and a well of 2 cm diameter was built over both hemispheres with a light-curable cement (Composite Flowable) to hold a saline immersion for the microscope objective. The craniotomy was conducted with a high-speed dental drill over the parietal cortex bilaterally. The craniotomy site was regularly flushed with saline to wash out bone fragments and prevent thermal damage. The exposed cortex was injected with Hi-Lyte™ Fluor 555-labeled Aβ (Anaspec) combined with native E3 or E4 particles pre-incubated for 24 h. The injection was performed at an approximate depth of 300 µm below the surface of the cortex with a borosilicate glass capillary tube (WPI) pulled with a two-stage vertical pipette puller (Narishige) to a tip diameter of approximately 10 µm and coupled with flexible tubing to a picospritzer microinjection dispense system (Parker). Mice were randomly assigned to either the AβE3 or AβE4 group. After the injection, the mice were moved under the two-photon microscope within 5 minutes and anesthesia was maintained with 40 mg/ml/h ketamine.
In vivo imaging was conducted with an Ultima IV two-photon laser scanning microscope (Bruker) with an InSight DS Laser system (Spectra-Physics) tuned at a wavelength of 920 nm. Two channels were collected red (595/50) and green (525/50 nm) to visualize the 555-labeled Aβ and microglia, respectively. Images were collected with 16x Nikon objective lens on Prairie View software using a time series of 11 slices with 1024 x 1024 matrix size, dwell time 4.4 microseconds and 3 µm step size. The effective in-plane resolution was 0.4 μm per pixel. The Z-stack images were collected in Galvo acquisition mode such that each Z-stack was collected every minute for a total duration of 80 min.
For each animal, Z-stack images were analyzed to quantify the microglial approach and microglial injection site coverage over time. Initially, the data was loaded in ImageJ (National Institutes of Health) and a rigid-body co-registration with the StackReg plugin was used to account for global temporal drifts of the field of view. The Z-stacks were transformed to sum-based XY projections by linear interpolation using the 3D Project function, allowing for time series analysis of specific regions of interest. We generated the panels illustrating cell displacement from those datasets by assigning red to the beginning of the time series (t=0) and green to subsequent time points (10 min or 80 min). Then, the two color-coded images were merged in ImageJ to reveal spatial details, with red showing the original location, green for the new cellular location after a particular segment of time has elapsed (0-10 min or 0-80 min) and yellow at the regions where the two channels overlap. The 555-labeled Aβ infusion site was shown in white to display the increasing coverage as cell processes approach. The time series were also saved as movies presented in the supplementary material.
The datasets were then converted to mat structures and all subsequent analyses were performed in MATLAB (Math Works), using standard image processing and statistical functions. Briefly, the red channel showing the infusion site was thresholded using the Otsu’s method with the multithresh Matlab function. The resulting images were binarized and small features outside of the infusion site were removed using the bwareaopen function. The mask was then dilated to smooth the borders and account for the lower contrast of the diffusion gradient around the borders. The percent coverage by the microglial processes over time was then computed as the increase of the green channel intensity above 1.5 times the standard deviation baseline of the green channel within the binary mask.
The mean distance of the cell processes to the infusion site over time was computed by eroding the binary mask described above and computing the Euclidean distance transformation of the full binary image using the bwdist function. Then, each pixel in the green channel over 1.5 standard deviations of the intensity was mapped with that transformation and assigned a number that is the distance between that pixel and the edge of the infusion site forming progressive concentric level sets with increasing proximity to the infusion site. The change of this Euclidean distance over time was converted from pixels to microns using the effective in-plane resolution of the imaging data and plotted as a time series.
Primary microglia cell culture
Primary microglial culture and Aβ uptake assay were performed using WT, E3, E4, E3/Trem2ko and E4/Trem2ko pups (1-3 days old) as described before42, 43. Briefly, the cortices and hippocampi were mechanically dissociated using a sterile Pasteur pipette. The dissociated cells were plated in 75 cm2 flasks with DMEM/F12 medium (Thermo Fisher) containing 10% FBS at DIV0. 24 h after plating (DIV1), the media was replaced to remove debris. Microglia were collected by tapping at DIV14 and plated on 0.01% poly-L-lysine (Sigma) coated 12mm circular coverslips in 24-well plates with the density of 60,000 cells/well. 24 h after plating, cells in the treatment groups were treated with 1µM Hi-LyteTM Fluor 488-labeled Aβ (Anaspec) at 37 °C for 1 h. Following the treatment, cells were washed three times with PBS, fixed in 4% PFA and permeabilized with 0.2% Triton X-100 at room temperature. Microglia were then labeled with Anti-IBA1 antibody (1:500) at 4 °C for 18 h and followed by 2 h incubation with horse anti-rabbit 594 secondary antibody and stained with DAPI to visualize nuclei.
Fluorescent images of in vitro Ab-treated microglia were taken on a Nikon Eclipse 90i microscope (20X magnification) and analyzed in NIS elements (Nikon Instruments Inc.). Exposure levels for each channel were consistent across all genotypes and samples. Images were thresholded to identify microglia, nuclei and Ab. To assess the Aβ uptake in microglia, the percentage of microglia containing Ab ((IBA1+ colocalized with 488-labeled Aβ+) / (IBA1+ and 488 Aβ-)) was calculated and averaged across all images in each genotype. To assess the morphology of the microglia, the circularity of thresholded IBA1+ microglia was calculated and averaged across all images in each genotype on a scale of 0 to 1, with 1 being a perfect circle.
QUANTIFICATION AND STATISTICAL ANALYSIS
Sample sizes (n) indicated in the figure legend 1 indicate number of separate measurements. Sample sizes (n) indicated in the figure legend 2-7 correspond to the number of biological replicates analyzed. All researchers were blinded to experimental groups during the analysis. All results are reported as means ± SEM.
Unless otherwise indicated, all statistical analyses were performed in GraphPad Prism (v 8.2.0), or in R (v 3.6.0) and significance was determined as p<0.05. Number of experiments and statistical information are stated in the corresponding figure legends. In figures, asterisks denote statistical significance marked by * p<0.05; ** p<0.01; *** p<0.001.