Trem2 knockout (KO) (Stock no. 027197) and Wild-type (WT) C57BL/6 J mouse strains were purchased from The Jackson Laboratory (JAX). Both KO and WT animals were maintained as homozygous lines. Postnatal day 2–3 WT and Trem2 KO mice were used for dissection and generation of primary microglial cultures. Since hormonal effects are negligible at this stage, mixed primary microglial cultures were taken from both males and females. Animal procedures did not include additional drug or treatment regimen other than that described. Mouse lines were housed with littermates with free access to food and water under a 12 hour light/day cycle. All animal procedures, including husbandry were performed under the guidelines of the Institutional Animal Care and Use Committee at Sanford Burnham Prebys Medical Discovery Institute.
Stereotaxic AAV injection
Control and recombinant AAV-P301L tau (AAV9) vectors have been described previously; AAV-GFP and AAV-P301L tau plasmid vectors were generously provided by Dr. Tsuneya Ikezu . 1 × 109 AAV particles were stereotaxically/bilaterally injected into the MEC (layer II/III) using the following coordinates: anteroposterior, − 4.75 mm; lateral, + 2.9; dorsoventral, − 4.6. Mice were then subjected to behavioral analysis, or histological staining 14 (behavior only) or 35 (histology and behavior) days following stereotaxic injection.
Immunohistological staining and analysis
Mice stereotaxically injected with AAV particles were sacrificed for immunohistological analysis at 35 days post-injection. Mice were anesthetized with 4% isoflurane and intracardially perfused with PBS. Brain tissues were harvested and fixed in 4% paraformaldehyde at 4C for 24 h. Tissues were washed in PBS and cryoprotected in PBS containing 30% sucrose. Tissues were embedded in OCT containing 30% sucrose (at 1:1 v/v) and free-floating coronal brain cryostat sections (25 mm) were collected. Data were collected and analyzed in a double-blind fashion.
For detection of Iba1, ptau, and tau, brain slices were stained using antibodies as follows: Goat anti-Iba1 (1:400, ab5076, Abcam), AT8 (1:500, MN1020 ThermoFisher), AT180 (1:500, MN1040 ThermoFisher), T13 (1:500, #835201 Biolegend), PSD95 (1:200, #3450 s Cell Signaling), Cd68 (1:250, MCA341R BioRad), P2y12 (1:200, #69766 Cell Signaling Technology), Cd9 (1:100, 20,597-I-AP Proteintech), Tmem119 (1:150, #90840 Cell Signaling), Clec7a (1:100, Invivogen), GFP (1:250, NB600–308 Novus). Alexa Fluor 488, 568 or 647 secondary antibodies (1:400, ThermoFisher) were used, DAPI counterstains were applied to the sections, and image z stacks were acquired from multiple sections (up to 9) from each animal using a Zeiss LSM 710 laser-scanning confocal microscope.
Staining intensity of human tau in cultured neurons was quantified using Imaris image analysis software (Bitplane, Oxford Instruments), and phospho-tau epitopes quantified in histological sections were scored manually by identifying AT8 or AT180-positive cells in imaged MEC or DG regions. Quantification of PSD95 puncta in the stratum moleculare within the hippocampus was also performed using Imaris by defining a region of interest (ROI) defined immediately above of the granular cell layer (GCL). The number of PSD95 puncta in each ROI was measured using Imaris, and calculated using a set intensity threshold, expressed as number of PSD95 puncta/mm2. Percentage of P2y12, Cd68, Tmem119, Clec7a, CD9 positive microglia and P2y12/CD68, Tmem119/Clec7a, CD9/Iba1 double positive microglia was quantified using Imaris.
Barnes maze test was performed as described previously . Briefly, WT or Trem2 KO mice stereotaxically injected with AAV-tau or AAV control (AAV9-synapsin GFP) were habituated on day 1, where mice were placed in the center of the maze underneath a clear 3500-ml glass beaker for 30 s. Mice were slowly guided to the target hole leading to the escape cage by moving the glass beaker over the target hole within a span of 10–15 s. Mice were then given 3 min to enter the target hole, and gently forced to enter in the event no entry was apparent. Training was initiated the next day. Mice were placed inside an opaque cardboard cylinder, 10″ tall and 7″ in diameter, in the center of the Barnes maze for 15 s. The cylinder was then removed, and mice were allowed to explore the maze for 2 mins. Upon entering the escape cage, the mouse was allowed to remain in the escape cage for 1 min; otherwise, the mouse was gently guided to the escape hole using a glass beaker and allowed to freely enter the escape cage. In case the mouse did not enter the escape cage within 3 min, it was gently nudged with the beaker to enter. Five trials were performed during training, with 3 trials on day 1, and 2 trials on day 2. Forty-eight hours after the last training session, a probe test to analyze the behavioral characteristics of the mice seeking the target area was monitored using a digital camera controlled by the ANY-maze video tracking system (Stoelting Co.). Subsequent analyses of the probe test parameters were processed using ANY-maze software, where statistical analyses and significance values were calculated using GraphPad Prism (Dotmatics, Boston, MA).
Contextual fear conditioning behavior tests were performed using the Freeze Detector System (San Diego Instruments, CA). Twenty-four hours before training initiated, mice were placed in the conditioning chamber and allowed to freely explore the chamber for 5 mins. On the first day of training, mice were placed in the conditioning chamber and allowed to freely explore for 120 s, where a 0.4 mA electrical foot-shock was subsequently applied to the mice for 2 s. After 60 s, another 0.4 mA electric shock was given to the mouse for 2 s. Following shocks, mice were left in the chamber for an additional 120 s. Twenty-four hours after training, each mouse was monitored in the same chamber for 5 min. Freezing time was automatically recorded and analyzed by the Freeze Detector System.
Following behavior analysis, ex vivo hippocampal slices were prepared from WT and Trem2 KO mice stereotaxically injected with either AAV-control or AAV-tau using methods described previously . Briefly, mice were decapitated under deep terminal anesthesia, and brains were surgically removed in ice-cold, sucrose-based artificial cerebrospinal fluid (aCSF) (190 mM sucrose, 25 mM D-glucose; 25 mM NaHCO3, 3 mM KCl, 1.25 mM NaH2PO4, 5 mM MgSO4, 10 mM NaCl, and 0.5 mM CaCl2) saturated with carbogen (95% O2/5% CO2) at pH 7.4. A vibrating-blade microtome (Leica VT1000S) was used to cut 400-μm-thick coronal slices containing both cortex and hippocampus. Slices were transferred to a holding chamber containing a warmed (32 °C) aCSF formulation for recording (125 mM NaCl, 25 mM NaHCO3, 3.0 mM KCl, 1.25 mM NaH2PO4, 2.0 mM CaCl2, 1.0 mM MgSO4, and 10 mM D-glucose) saturated with carbogen (95% O2/5% CO2) at pH 7.4. Slices were left to recover at room temperature in oxygenated aCSF for at least 30 min before recording. Population spike amplitude was measured to determine synaptic transmission within the excitatory perforant pathway in acute hippocampal slices. Concentric bipolar stimulating electrodes were positioned in the middle molecular layer of the DG while recording electrodes were positioned in the dentate granule cell body layer. Stimuli (0.1 ms in duration) were applied at 0.05 Hz in increments of 20 μA from 0 to 200 μA, at each time-point, five recordings of evoked responses were averaged.
Primary microglial and neuronal culture
Primary microglial cultures were prepared as described previously [3, 68]. Briefly, brains were removed from WT or Trem2 KO mice at postnatal day 2–3. After removal of the meninges, brains were treated with a Papain Dissociation System (Worthington Biochemical Corporation) according to manufacturer’s specifications. Mixed glial cells were plated in flasks coated with poly-D-lysine and grown in DMEM containing 10% FBS (VWR Life Science Seradigm). Twenty-five nanograms per milliliter GM-CSF (R&D Systems) was added into the cultures after 5 days and removed before harvesting. Microglial cells were harvested twice by shaking (200 rpm, 60 min) 10–14 days after plating and subjected to various treatments within 24 h of harvest.
Neurons were dissected as described previously . Primary neurons were dissected from embryonic days 17–18 (E17-E18) embryos from pregnant female C57BL/6 mice, and hippocampal and cortical neurons were isolated by microdissection from the cerebral cortex and hippocampus using a stereomicroscope. Tissue was dispersed by trypsin and DNase 1 digestion for 30 minutes at 37 °C, followed by trituration in DMEM+ penicillin /streptomycin + HEPES. Neurons were maintained separately on poly-D-lysine coated coverslips or seeded onto poly-D-lysine coated layers in microfluidic chamber systems. Neurons were cultured in Neurobasal Medium Plus supplemented with B27 Plus, glutamine, and penicillin/streptomycin, where half of the media was replaced every 2–3 days.
3-chamber interneuronal tau dispersion assay
A three-layer microfluidic chamber (TCND1000, Xona Microfluidics) was designed to reconstitute intraneuronal tau dispersion through cells within an intermediary layer, with microgrooves adjoining the three chambers. Two side-reservoirs connected the poly-D-lysine coated culture chambers to facilitate neuronal adhesion. Primary cortical neurons from wildtype C57BL/6 J E18 embryos were dissected as previously described  (see “Primary microglial and neuronal culture”) and 2 × 105 cells were plated in chamber layers 1 and 3 in 50% neurobasal with B27 medium and 50% DMEM/F12 with FBS medium, and allowed to adhere overnight. The following day, medium was changed to complete neurobasal with B27 and maintained at 37 °C in 5% CO2. Neurons in layer 1 were transduced with 1 × 1010 VP/ml AAV-tau particles at DIV2. At DIV7, primary WT or Trem2 KO microglia were detached from mixed glial cultures and 1 × 104 cells were seeded into chamber layer 2 in 50% Neurobasal with B27 containing/50% DMEM with 10% FBS, 25 ng/ml GM-CSF (a control without microglia in layer 2 was also included); and neurons/microglia were cultured for another 7 days. At DIV14, chambers were fixed and stained to visualize tubulin (#5568, Cell Signaling Technology), Iba1 (ab5076, Abcam) and human tau (T13, Biolegend) by fluorescence microscopy.
Intensity of human tau (T13) staining and neuronal density in layers 1 and 3 were calculated from intensity measurements using Imaris (Bitplane) in imaged fields, and neuronal number were quantified by DAPI measurements in layers 1 and 3 (normalized per cm2). Iba1-positive microglia were also quantified in chamber layer 2, and overlap in human tau with Iba-1 positive microglia were quantified, and normalized to Iba1/tau-positive microglia in WT layer 2 (set to 1.0).
Microglia tau uptake
Binding/uptake of tau oligomers in cultured WT and Trem2 KO microglia was measured using purified recombinant 2N4R human tau 1–441 (500 μg/ml, #AS-55556-50, AnaSpec) oligomerized in 30 μM heparin (#07980, StemCell Technologies Inc.) for 24 h. Tau oligomers were subsequently conjugated to Alex555 (“tau-555”) using an Alexa Fluor 555 Microscale Protein Labeling Kit (#A30007, ThermoFisher) according to the manufacturer’s instructions. Binding/uptake of tau-Alexa Fluor 555 was assayed in microglia cultures seeded at 50,000 cells in 24-well plates, and tau-555 binding/uptake was measured in real time at a final concentration of 10 μg/ml where a fixed area in each well was serially imaged every 15 min using a Nikon N-SIM microscope. Serial confocal images were acquired for 10 h, and tau-555 intensity/area was quantified. Fluorescence intensity was normalized to microglia cell number using automated IMARIS imaging software (Bitplane); fluorescence thresholds for tau-555 were set to a value of 10, and individual cells were identified by differential interference contrast (DIC) imaging. Fluorescence measurements normalized to cell number from 9 total confocal images from 3 independent microglia batches/experiments (three independent wells per batch) at time points ranging from 15 to 180 min (for all experiments). The phagocytic index (PI) for varying time points was then calculated using the following formula: PI = It / I15, where It represents averaged fluorescence intensity at various time points and I15 represents fluorescence intensity at 15 min following addition of tau-555. The resulting value represents fold change of tau-555over the 15 min time point for each microglia culture. Ratios were then normalized to the PI value in WT microglia under control conditions at 15 min (where WT microglia under control conditions/15 min are set to 1.0) for each experiment.
Flow cytometry and TUNEL analysis
Flow cytometry for tau oligomer uptake
Tau oligomers were conjugated to an Alexa-488 fluor label using an Alexa Fluor™ 488 Protein Labeling Kit (A10235, ThermoFisher); WT and Trem2 KO microglia were incubated with tau-Alexa488 (tau488) for 6 hours. Microglia were washed in 1xPBS, harvested and fixed with 4% PFA. Fluorescent signals were detected and quantified by flow cytometry (Novocyte, ACEA Bioscience). WT microglia (“cell only”) without tau uptake was used as an unlabeled control.
Effects of LPS and ATP treatment on cell death in WT and Trem2 KO microglia were quantified by TUNEL labeling and flow cytometry analysis. 1 × 106 WT and Trem2 KO microglia left untreated or treated with 1 μg/ml LPS (#L3024, Sigma-Aldrich) for 3 h, and 5 mM ATP, or WT microglia exposed to 10 μM camptothecin for 3 h were processed for TUNEL staining using the Apo-Direct TUNEL Assay Kit (APT110, Millipore Sigma) according to the manufacturer’s recommendations, and subjected to flow cytometry and FlowJo analysis. TUNEL staining in WT and Trem2 KO microglia under varying treatments was performed using the Click-IT Plus TUNEL assay system (C10617, ThermoFisher), and stained cells were imaged by confocal microscopy.
Induction and purification of microglia exosomes
Exosomes were purified from primary microglia cultures as described previously [2, 54]. Briefly, 1 × 107 WT and Trem2 KO microglia cultured under untreated conditions or incubated with 2.5 μg/ml tau oligomers for 24 h; cells were then washed in 1xPBS and incubated in DMEM with 10% exosome-depleted FBS (A2720803, ThermoFisher) and treated with 1 μg/ml LPS (#L3024, Sigma-Aldrich) for 3 h, and 5 mM ATP for 15 mins. Conditioned media (5 ml) was collected and centrifuged at 2000×g, and supernatant subsequently centrifuged at 10,000×g for 30 min at 4 °C to remove cell debris. Supernatants were then diluted to 10 ml in 1x PBS and centrifuged at 100,000×g for 90 mins. To precipitate exosomes, pellets were then resuspended in 10 ml 1xPBS and re-centrifuged at 100,000×g to remove contaminants and non-exosomal debris. Resulting pellets were then resuspended in 50 μl 1xPBS for electron microscopy (EM), ELISA, or RIPA buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% NP-40, 0.1% SDS, 0.5% DOC) for immunoblot analysis.
Formvar-carbon-coated copper grids (100 mesh, Electron Microscopy Sciences, Hatfield, PA) were placed on 20 μl drops of each sample solution displayed on a Parafilm sheet. After allowing material to adhere to the grids for 10 minutes, grids were washed 3 times by rinsing through 200 μl drops of milli-Q water before being left for 1 min on 2% (wt/vol) uranyl acetate (Ladd Research Industries, Williston, VT). Excess solution was removed with Whatman 3MM blotting paper, and grids were left to dry for a few minutes before viewing. Grids were examined using a JEOL JEM-1400Plus transmission electron microscope operating at 80 kV. Images were recorded using a Gatan OneView 4 K digital camera.
Immunoblot and ELISA analysis
Exosome preparations or lysates from primary microglia were generated/resuspended in RIPA buffer in the presence of protease and phosphatase inhibitors (#78430, ThermoFisher). Proteins were separated by SDS-PAGE using 4–20% gradient gels, transferred onto nitrocellulose, and blocked in 5% non-fat milk in 1xPBS. Blots were then probed with primary antibodies overnight in 5% BSA/1xPBS, washed in 1xPBS with 0.1% tween-20, and probed with HRP-conjugated secondary antibodies. Blots were then incubated with ECL and immunoblot signals were acquired using a Chemidoc imaging system (BioRad). ELISA measurements for exosomal human tau were performed using ELISA kits for tau (KHB0041, ThermoFisher) according to specifications supplied by the manufacturer. Primary antibodies used for immunoblot analysis include Alix (1:1000, 2171 Cell Signaling), T13 (htau) and actin (1:5000, A5441 Sigma).
Sample preparation for proteomics analysis and LC-MS/MS
Primary Trem2 KO or WT microglia were left untreated, or treated with tau oligomers (2.5 μg/ml for 24 h), or 1 μg/ml LPS for 3 h, and 5 mM ATP for 15 mins (run 2), and cell pellets were lysed in UAB buffer (8 M urea, 50 mM ammonium bicarbonate (ABC) and Benzonase 24 U/100 ml). Protein concentration was determined using BCA assays (ThermoFisher) according to the manufacturer’s instructions. Proteins were then reduced by the addition of 5 mM tris(2-carboxyethyl) phosphine (TCEP) at 30 °C for 60 min, followed by alkylation of cysteines with 15 mM iodoacetamide (IAA) for 30 minutes in the dark at room temperature. Urea concentration was reduced to 1 M by adding 50 mM ammonium bicarbonate. Samples were digested overnight with Lys-C/trypsin (Promega) at room temperature with constant agitation at a 1:25 enzyme:protein ratio. Following digestion, samples were acidified using 0.1% FA and desalted using AssayMap C18 cartridges mounted on an Agilent AssayMap BRAVO liquid handling system. Cartridges were sequentially conditioned with 100% acetonitrile (ACN) and 0.1% FA; samples were then loaded, washed with 0.1% FA, and eluted with 60% ACN, 0.1% FA. Peptide concentration was determined using a NanoDrop spectrophotometer (Thermo Fisher).
Samples were subjected to mass spectrometry analysis using an EASY nanoLC system (ThermoFisher). Buffer A consisted of H2O/0.1% FA; Buffer B consisted of 80% ACN/0.1% FA. Samples were separated over a 90-min gradient of increasing Buffer B on analytical C18 Aurora column (75 μm × 250 mm, 1.6 μm particles; IonOpticks) at a flow rate of 300 nL/min. The mass spectrometer was operated in positive data-dependent acquisition mode, and the Thermo FAIMS Pro device was set to standard resolution with the temperature of FAIMS inner and outer electrodes set to 100 °C. A three-experiment method was set up where each experiment utilized a different FAIMS Pro compensation voltage: – 50, − 70, and − 80 Volts, and each of the three experiments had a 1 second cycle time. A high resolution MS1 scan in the Orbitrap (m/z range 350 to 1500, 60 k resolution at m/z 200, AGC 4e5 with maximum injection time of 50 ms, RF lens 30%) was collected in top speed mode with 1-second cycles for the survey and the MS/MS scans. For MS2 spectra, ions with charge state between + 2 and + 7 were isolated with the quadrupole mass filter using a 0.7 m/z isolation window, fragmented with higher-energy collisional dissociation (HCD) with normalized collision energy of 30% and the resulting fragments were detected in the ion trap as rapid scan mode with AGC of 5e4 and maximum injection time of 35 ms. The dynamic exclusion was set to 20 sec with a 10 ppm mass tolerance around the precursor.
Proteomic data analysis
Raw files were searched with SpectroMine software (Biognosys, version 2.7.210226.47784) using the BGS default settings. The search criteria were set as follows: full tryptic specificity was, 2 missed cleavages were allowed, carbamidomethylation (C) was set as fixed modification and oxidation (M) as a variable modification. The false identification rate was set to 1%. Spectra were searched against the curated Uniprot mus musculus database including common contaminants from the GPM cRAP sequences. Data was further processed using the MSstats package (version 4.2) in R . We avoided use of imputation of missing values prior to statistical test using MSstats; instead we calculated a pseudo Log2 fold-change (L2FC), adj.pvalue and pvalue of proteins completely missing in one condition after failing to perform the statistical test. The imputed (pseudo) L2FC was calculated as the sum of intensities of the protein (i.e., sum of feature intensities of a given protein within a given sample) across all replicates of the same group that it was detected, divided by 3.3. On the other hand, the imputed pvalue and adj.pvalue was calculated by dividing 0.05 or 0.1, respectively, by the number of replicates that a given protein was confidently identified multiplied by the number of features quantified. Therefore, the imputed L2FC gives an estimate of the protein abundance in condition that it is detected, while the imputed pvalue or adj.pvalue reports the confidence of the imputation in the sense of consistency of protein detection in the group that it is detected.
Proteins with an adjusted p value< 0.05 (adjp< 0.05) were selected as significantly differentially expressed proteins (DEPs). GO analysis was performed by entering DEPs into the GO DAVID input interface, and KEGG, Biological Process (BP), Cellular Component (CC) and Molecular Function (MF) categories were retrieved for each query [29, 30]. Principal Component Analysis (PCA) was carried out in R version 4.1.2 with PCATools package (version 2.6.0) using log2 protein intensity for all proteins summarized by dataProcess function from MSstats (version 4.2). Contaminant proteins (non-murine, “Bos taurus” proteins) or proteins with gene names that failed to map from gene ID’s were manually removed from the datasets prior to analysis. To calculate z-score values within each replicate, row (protein-wise) z-scores were computed in R version 4.0.2 using the scale function by subtracting mean intensity of each protein from the corresponding intensities of the biological replicates, and dividing the resulting values by the standard deviation of the intensities.
Tau internalization and quantification
WT and Trem2 KO microglia were seeded at a density of 0.2 million cells on coverslips on 24-well plates, and treated with 2.5 μg/ml tau oligomers for 0.5, 2, 4 and 24 h. Cells were then fixed and stained with antibodies to visualize colocalization of recombinant human tau (T13) with Rab5, Rab7, LAMP1, CD63 and Tsg101. Images were acquired by confocal microscopy and overlapping signals from tau and intracellular markers were quantified; manual identification, demarcation and quantification of regions of overlap were performed using Imaris, and normalized to the total area of tau staining in fluorescence images.
In vitro exosomal tau seeding assay
Tau seeding was performed using a FRET biosensor HEK293T cell line stably expressing tau-RD P301S-CFP and P301S-YFP (tau-RD cells) as described previously . To assay tau seeding capacity of exosomes purified from cultured microglia, exosomes were purified from WT or Trem2 KO microglia pre-incubated with tau oligomers and induced with LPS/ATP and resuspended in 50 μl PBS. Tau-RD cells were then exposed to 20 μl purified exosomes and transduced with Lipofectamine 2000 (#11668019, ThermoFisher) for 48 hours, and FRET activity from tau aggregation was visualized by excitation at 405 nm and consequent fluorescence at 525/50 nm was imaged using a Zeiss LSM 710 microscope. DIC images were concurrently acquired in FRET images and used to normalize FRET activity according to cell number. FRET area was calculated from positive cell signals in tau-RD cells through defined FRET-positive regions in Imaris, and FRET-positive area (μm2) was normalized over cell number; cells without FRET signals were included in the analyses.
Stereotaxic exosome injection and analysis in vivo
Exosomes were prepared from 3 × 107 tau-loaded and untreated (control), LPS/ATP-treated WT and Trem2 KO microglia as described under “Induction and purification of microglia exosomes”. Exosome pellets were resuspended in 20 μl sterile 1xPBS and 2 μl of the exosome preparations purified from WT and Trem2 KO microglia were stereotaxically injected into the DG region (coordinates: AP, − 2.0 mm; ML, ± 1.3 mm; DV, 2.1 mm) in 6 month C57BL/6 WT mice. Three weeks post-injection, mice were perfused with 4% PFA and stained with AT8 (MN1020, Thermo Fisher), β3-Tubulin (5568, Cell Signaling Technology) and DAPI to examine tau pathology. AT8-positive cells were scored in 2.5 × 105 μm2 imaging areas in independently-injected animals.
All statistical analyses were performed using R scripts (proteomics) or Graphpad Prism as indicated.