Reagents
Lipopolysaccharide (LPS), interferon-γ (IFN-γ), JWH-133 (JWH), and SR-144528 (SR) were purchased from Sigma-Aldrich (St Louis, MO, USA). N-Methyl-D-aspartate (NMDA), MK-801 (MK), and forskolin (FK) were purchased from Tocris (Bristol, UK). Tau and p-Tau proteins were kindly provided by Prof. J. Avila (CBM, UAM-CSIC, Madrid, Spain). Detailed descriptions of the elaboration and processing of proteins can be found elsewhere [42, 43]
HEK-293T cells and primary cultures
Human embryonic kidney HEK-293T (lot 612968) cells were acquired from the American Type Culture Collection (ATCC). They were amplified and frozen in liquid nitrogen in several aliquots. Cells from each aliquot were used until passage 12.
HEK-293T cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco) supplemented with 2 mM L-glutamine, 100 μg/ml sodium pyruvate, 100 U/ml penicillin/streptomycin, MEM non-essential amino acids solution (1/100), and 5% (v/v) heat-inactivated fetal bovine serum (FBS) (all supplements were from Invitrogen, Paisley, Scotland, UK). Cells were maintained at 37 °C in a humid atmosphere of 5% CO2.
To prepare mice hippocampal primary microglial cells, the brain was removed from C57BL/6 mice of 2–4 days of age. Microglial cells were isolated as described in [44]. Briefly, the brain was dissected, carefully stripped of its meninges and the hippocampus was digested with 0.25% trypsin for 20 min at 37 °C. Trypsinization was stopped by washing the tissue. Cells were brought to a cell suspension by passage through 0.9 mm and 0.5 mm nails followed by passage through a 100 μm pore mesh. Glial cells were resuspended in medium and seeded at a density of 1 × 106 cells/ml in 6-well plates for cyclic adenylic acid (cAMP) assays, in 12-well plates with coverslips for in situ proximity ligation assays (PLA) and in 96-well plates for mitogen-activated protein kinase (MAPK) activation experiments. Cultures were grown in DMEM medium supplemented with 2 mM L-glutamine, 100 U/ml penicillin/streptomycin, MEM non-wssential amino acids preparation (1/100) and 5% (v/v) heat-inactivated fetal bovine serum (FBS) (Invitrogen, Paisley, Scotland, UK) and maintained at 37°C in humidified 5% CO2 atmosphere and, unless otherwise stated, medium was replaced once a week.
For culturing primary neurons, the hippocampus from mouse embryos (E19) was removed and the neurons were isolated as described by [45] [45] and plated at a density of circa 120,000 cells/cm2. Cells were grown in a neurobasal medium supplemented with 2 mM L-glutamine, 100 U/mL penicillin/streptomycin, and 2% (v/v) B27 supplement (Gibco) in a 6-, 12- or 96-well plate for 12 days. Cultures were maintained at 37oC in a humidified 5% CO2 atmosphere and the medium was replaced every 4–5 days.
Immunodetection of specific markers (Neu N for neurons and CD-11b for microglia) showed that neuronal preparations contained >98% neurons and microglia preparations contained, at least, 98% microglial cells [28].
Preparation of brain cortex slices
Mouse brains were extracted in a cold chamber at a temperature of 4°C. Brain slices (BS) with a thickness of 500 μm were made with the aid of a mouse coronal matrix (Agnthos, ref. 69-2165) and subsequently, the cortical region was isolated. Brain slices were maintained in Krebs’s buffer (124 mM NaCl, 4 mM KCl, 1,25 mM KH2PO4, 1,5 mM MgSO4, 10 mM glucose, 26 mM NaHCO3, 1,5 mM CaCl2 and carbogen). Brain slices were incubated for 2 h at 32 °C in a humidified 5% CO2 atmosphere, Krebs’s buffer was replaced once in the first 30 min. After that, BSC were treated or not for 15 min with the selective antagonist for CB2R (SR-144528 (1 μM)) followed by 15 min treatment with the selective agonists (NMDA (15 μM) and/or JWH-133 (100 nM)). After treatment, the slices were immediately frozen in dry ice to stop the metabolic activity.
On the one hand, ERK1/2 phosphorylation was determined by Western blot. Samples were sonicated on ice with 10-s pulse, 20-s rest, and 10-s pulse, using lysis buffer for tissue disaggregation and cell lysis. Final lysates total protein was adjusted to 2 μg/μL with SDS and lysis buffer using BCA quantification. Equivalent amounts of protein (40 μg) were subjected to electrophoresis (10% SDS-polyacrylamide gel) and transferred onto PVDF membranes (Immobilon-FL PVDF membrane, MERK, St. Louis, MO, USA) for 30 min using Trans-Blot Turbo system (Bio-Rad). Then, the membranes were blocked for 2 h at room temperature (constant shaking) with Odyssey Blocking Buffer (LI-COR Biosciences, Lincoln, NE, USA) and labeled with a mixture of primary mouse anti-phospho-ERK 1/2 antibody (1:2500, MERK, Ref. M8159), primary rabbit anti-ERK 1/2 antibody (1:40,000, MERK, Ref. M5670), which recognizes both phosphorylated and non-phosphorylated ERK1/2 overnight at 4 °C with shaking. Then, the membranes were washed three times with PBS containing 0.05% tween for 10 min and subsequently were incubated by the addition of a mixture of IRDye 800 anti-mouse antibody (1:10,000, MERK, Ref. 92632210) and IRDye 680 anti-rabbit antibody (1:10,000, MERK, Ref. 926-68071) for 2 h at room temperature, light-protected. Membranes were washed 3 times with PBS-tween 0.05% for 10 minutes and once with PBS and left to dry. Bands were analyzed using Odyssey infrared scanner (LI-COR Biosciences). Band densities were quantified using Fiji software, and the level of phosphorylated ERK1/2 was normalized using the total ERK 1/2 protein band intensities. Results obtained are represented as the percent over basal (non-stimulated cells).
APPSw/Ind transgenic mice
APPSw/Ind transgenic mice (line J9; C57BL/6 background) expressing the human APP695 harboring the FAD-linked Swedish (K670N/M671L) and Indiana (V717F) mutations under the platelet-derived growth factor subunit B (PDGFβ) promoter were obtained by crossing heterozygous APPSw/Ind to non-transgenic (control) mice [46]. Control and APPSw;Ind embryos (E16.5) and adult mice (6 months) were genotyped individually, and hippocampus/cortex dissected and prepared for microglia and neuron primary cultures as described elsewhere [22, 28]. All experimental procedures were conducted according to the approved protocols from the Animal and Human Ethical Committee of the Universitat Autònoma de Barcelona (CEEAH 2895) and Generalitat de Catalunya (10571) following the experimental European Union guidelines and regulations (2010/63/EU)
Fusion proteins
Human cDNAs for the GluN1 NMDA receptor subunit, for the CB2 receptor, and for the ghrelin GHS1a receptor, all cloned into pcDNA3.1 were amplified without their stop codons using sense and antisense primers harboring either BamHI and HindIII restriction sites to amplify GluN1, BamHI, and KpnI restriction sites to amplify CB2 receptor or EcoRI and KpnI restriction sites to amplify GHS1a receptor. Amplified fragments were then subcloned to be in frame with an enhanced yellow fluorescent protein (pEYFP-N1; Clontech, Heidelberg, Germany) or a RLuc (pRLuc-N1; PerkinElmer, Wellesley, MA) on the C-terminal end of the receptor to produce GluN1-RLuc, CB2R-YFP, and GHSR1a-YFP fusion proteins.
Cell transfection
HEK-293T cells were transiently transfected with the corresponding cDNA by the PEI (PolyEthylenImine, Sigma-Aldrich) method. Briefly, the corresponding cDNA diluted in 150 mM NaCl was mixed with PEI (5.5 mM in nitrogen residues) also prepared in 150 mM NaCl for 10 min. The cDNA-PEI complexes were transferred to HEK-293T cells and were incubated for 4 h in a serum-starved medium. Then, the medium was replaced by a fresh supplemented culture medium, and cells were maintained at 37 °C in a humid atmosphere of 5% CO2. 48 h after transfection, cells were washed, detached, and resuspended in the assay buffer.
Immunocytochemistry
HEK-293T cells were seeded on glass coverslips in 12-well plates. Twenty-four hours after, cells were transfected with CB2-YFP cDNA (1 μg), GluN1-RLuc cDNA (1 μg), and GluN2B cDNA (0.75 μg). Forty-eight hours after, cells were fixed in 4% paraformaldehyde for 15 min and washed twice with PBS containing 20 mM glycine before permeabilization with PBS-glycine containing 0.2% Triton X-100 (5 min incubation). Cells were blocked during 1 h with PBS containing 1% bovine serum albumin. HEK-293T cells were labeled with a mouse anti-RLuc antibody (1/100; Millipore, Darmstadt, Germany) and subsequently treated with Cy3-conjugated anti-mouse (1/200; Jackson ImmunoResearch (red)) antibody (1 h each). The CB2R-YFP expression was detected by the YFP’s own fluorescence. Nuclei were stained with Hoechst (1/100 from stock 1 mg/mL; Sigma-Aldrich). Samples were washed several times and mounted with 30% Mowiol (Calbiochem).
Images were obtained in a Zeiss LSM 880 confocal microscope (ZEISS, Germany) with the 40X and 63X oil objectives.
Bioluminescence resonance energy transfer (BRET) assay
For BRET assay, HEK-293T cells were transiently cotransfected with a constant amount of cDNA encoding for GluN1-RLuc (0.25 μg) and GluN2B (0.15 μg) and with increasing amounts of cDNA corresponding to CB2R-YFP (0.25 to 1.25 μg). As a negative control, HEK-293T cells were transiently cotransfected with a constant amount of cDNA encoding for GluN1-RLuc (0.25 μg) and GluN2B (0.15 μg) and with increasing amounts of cDNA corresponding to GHSR1a-YFP (0.25 to 1.5 μg). To control the cell number, sample protein concentration was determined using a Bradford assay kit (Bio-Rad, Munich, Germany) using bovine serum albumin (BSA) dilutions as standards. To quantify fluorescent proteins, cells (20 μg of total protein) were distributed in 96-well microplates (black plates with a transparent bottom) and fluorescence was read in a Fluostar Optima Fluorimeter (BMG Labtech, Offenburg, Germany) equipped with a high-energy xenon flash lamp, using a 10-nm bandwidth excitation filter at 485 nm. For BRET measurements, the equivalent of 20 μg of total protein cell suspension was distributed in 96-well white microplates with a white bottom (Corning 3600, Corning, NY). BRET was determined one minute after adding coelenterazine H (Molecular Probes, Eugene, OR), using a Mithras LB 940 plate reader (Berthold Technologies, DLReady, Germany), which allows the integration of the signals detected in the short-wavelength filter at 485 nm and the long-wavelength filter at 530 nm. To quantify GluN1-RLuc expression, luminescence readings were obtained 10 min after the addition of 5 μM coelenterazine H. MilliBRET units (mBU) are defined as:
$$\mathrm{mBU}=\left[\ \frac{\uplambda_{530}\left(\mathrm{long}-\mathrm{wavelength}\ \mathrm{emission}\right)}{\uplambda_{485}\left(\mathrm{short}-\mathrm{wavelength}\ \mathrm{emission}\right)}-{\mathrm{C}}_{\mathrm{f}}\ \right]\times 1000$$
where Cf corresponds to [(long-wavelength emission)/(short-wavelength emission)] for the RLuc construct expressed alone in the same experiment.
cAMP level determination
The analysis of cAMP levels was performed in HEK-293T cells cotransfected with the cDNA for two subunits of the NMDA receptor, GluN1 (1 μg) and GluN2B (0.75 μg) or/and/or the cDNA for the CB2R (1 μg). Similar assays were also performed in primary microglia and primary neurons prepared from wild-type mice or the transgenic APPSw/Ind AD mice model. In the case of microglia cells were first activated using 1 μM LPS and 200 U/mL IFN-γ (48 h). Two hours before the experiment, the medium was substituted by serum-starved DMEM medium. Cells growing in a medium containing 50 μM zardaverine were distributed in 384-well microplates (2000 HEK-293T cells or 4000 hippocampal neurons or microglial cells per well) followed by the stimulation with the NMDA and/or CB2R agonists (NMDA (15 μM) and/or JWH-133 (100 nM)) for 15 min before adding 0.5 μM forskolin or vehicle for an additional 15 min period. When indicated cells were pre-treated (15 min) with the NMDA or CB2R antagonists, respectively, MK-801 (1 μM) or SR-144528 (1 μM). Homogeneous time-resolved fluorescence energy transfer (HTRF) measures were performed using the Lance Ultra cAMP kit (PerkinElmer). Fluorescence at 665 nm was analyzed on a PHERAstar Flagship microplate reader equipped with an HTRF optical module (BMG Labtech). A standard curve for cAMP was obtained in each experiment.
MAP kinase pathway activation is measured by ERK1/2 phosphorylation
Hippocampal neurons, microglial cells, or HEK-293T cells cotransfected with the cDNA for the protomers of the NMDA receptor, GluN1 (1 μg) and GluN2B (0.75 μg), and/or with the cDNA for CB2R (1 μg) were plated in transparent Deltalab 96-well microplates. Primary microglial cells were activated by incubating cells with 1 μM LPS and 200 U/mL IFN-γ during 48 h. Two hours before the experiment, the medium was substituted by serum-starved DMEM medium. Cells were treated or not for 10 min with the selective antagonists (MK-801 (1 μM) or SR-144528 (1 μM)) followed by 7 min treatment with the selective agonists (NMDA (15 μM) and/or JWH-133 (100 nM)). Cells were then washed twice with cold PBS before the addition of lysis buffer (15 min treatment). Ten microliters of each supernatant was placed in white ProxiPlate 384-well microplates and ERK1/2 phosphorylation was determined using an AlphaScreen®SureFire® kit (Perkin Elmer) following the instructions of the supplier and using an EnSpire® Multimode Plate Reader (PerkinElmer).
Detection of cytoplasmic calcium levels
HEK-293T cells were cotransfected with the cDNA for the protomers of the NMDA receptor channel GluN1 (1 μg) and GluN2B (0.75 μg), with thee cDNA for CB2R (1 μg) and with the cDNA for the GCaMP6 calcium sensor (1 μg) [47] by the use of PEI method (Section “Cell Transfection”). 48 hours after transfection, HEK-293T cells plated in 6-well black, clear bottom plates, were incubated with Mg2+-free Locke’s buffer (154 mM NaCl, 5.6 mM KCl, 3.6 mM NaHCO3, 2.3 mM CaCl2, 5.6 mM glucose, 5 mM HEPES, 10 μM glycine, pH 7.4). Online recordings were performed right after the addition of agonists. When indicated cells were pre-treated with receptor antagonists for 10 min. Fluorescence emission intensity due to complexes GCaMP6 was recorded at 515 nm upon excitation at 488 nm on the EnSpire® Multimode Plate Reader for 150 s every 5 s at 100 flashes per well.
Dynamic mass redistribution (DMR) label-free assays
Cell signaling was explored using an EnSpire® Multimode Plate Reader (PerkinElmer) by a label-free technology. Cellular cytoskeleton redistribution induced upon receptor activation was detected by illuminating the underside of the plate with polychromatic light and measured as changes in wavelength of the reflected monochromatic light. The magnitude of this wavelength shift (in picometers) is directly proportional to the amount of DMR. To determine the label-free DMR signal, 10,000 HEK-293T cells cotransfected with cDNAs for the protomers of the NMDA receptor channel, GluN1 (1 μg) and GluN2B (0.75 μg) and/or with the cDNA for the CB2R (1 μg). Similar assays were performed using 10,000 primary neurons from wild type or transgenic APPSw/Ind mice. Transparent 384-well fibronectin-coated microplates were used until obtaining 70-80% confluent monolayers (kept in the incubator for 24 h). Previous to the assay, cells were washed twice with assay buffer (HBSS with 20 mM HEPES, pH 7.15, 0.1% DMSO) and incubated in the reader with assay buffer for 2 h at 24 °C. Hereafter, the sensor plate was scanned and a baseline optical signature was recorded for 10 min before adding 10 μL of selective agonists (NMDA (15 μM) and/or JWH-133 (100 nM)) also dissolved in assay buffer. When indicated cells were pre-treated with antagonists (MK-801 (1 μM) or SR-144528 (1 μM); 10 μL in volume). Real-time DMR responses were monitored for a minimum of 3600 s.
Proximity Ligation Assay (PLA)
Detection in natural sources of clusters formed by the NMDA and CB2 receptors was addressed in slices of in primary hippocampal microglia and hippocampal neurons of wild type mice or the transgenic APPSw/Ind mice model. When assays were performed in slices they were embedded in O.C.T. compound (OCT; Tissue Tek Products, Ames Division, Miles Laboratories, Inc., Elkhart, IN, USA) to allow cryostat sectioning (Leica CM3050S; 40 μm-thick sections). When using cells, they were grown on glass coverslips, were fixed in 4% paraformaldehyde for 15 min, washed twice with PBS containing 20 mM glycine to quench the aldehyde groups, permeabilized with the same buffer containing 0.05% Triton X-100 between 5 and 15 min and washed with PBS. After 1 h incubation at 37 °C with the blocking solution in a pre-heated humidity chamber, samples were incubated overnight at 4 °C with a mixture of a rabbit monoclonal anti-GluN1 antibody (1/100, ab52177, Abcam, Cambridge, UK) and a mouse monoclonal anti-CB2R antibody (1/100, sc-293188, Santa Cruz Biotechnology, TX, USA). Nuclei were stained with Hoechst (1/100 from 1 mg/mL stock; Sigma-Aldrich). The antibodies were validated following the method in the technical brochure of the vendor with fairly similar results. Cells were further processed using the PLA probes detecting primary antibodies (Duolink In Situ PLA probe Anti-Mouse plus and Duolink In Situ PLA probe Anti-Rabbit minus) (1/5 v:v for 1 h at 37 °C). Ligation and amplification were done as indicated by the supplier (Sigma-Aldrich) and cells were mounted using the mounting medium Mowiol (30%) (Calbiochem). To detect red dots corresponding to CB2-NMDA-Hets, samples were observed in a Zeiss LSM 880 confocal microscope (ZEISS, Germany) equipped with an apochromatic 63X oil-immersion objective, and 405-nm and 561-nm laser lines. For each field of view, a stack of two channels (one per staining) and 3 Z-planes with a step size of 1 μm were acquired. Andy’s algorithm, a specific ImageJ macro for reproducible and high-throughput quantification of the total PLA foci dots and total nuclei, was used for data analysis [48].
Statistical analysis
The data in graphs are the mean ± SEM (at least n=5). GraphPad Prism 9 software (San Diego, CA, USA) was used for data fitting and statistical analysis. One-way ANOVA followed by post hoc Bonferroni’s test were used when comparing multiple values. Experiments performed in samples from transgenic mice and age-matched controls were analyzed independently, i.e., quantitative inter-group differences were not addressed. When a pair of values were compared, Student’s t test was used. Significant differences were considered when the p value was <0.05.