Cell line and primary microglial cultures
A murine microglial cell line, N9 (a kind gift from Professor Claudia Verderio, CNR Institute of Neuroscience, Cellular and Molecular Pharmacology, Milan, Italy), was grown in an RPMI medium supplemented with 30 mM glucose (Sigma, Sintra, Portugal), 100 U/mL penicillin and 100 μg/mL streptomycin (GIBCO, Invitrogen, Porto, Portugal).
Primary microglial cultures were prepared as previously described . Briefly, primary cultures of glial cells were obtained from a postnatal (P1-P5) C57BL6 mouse and maintained for 15 days in the DMEM-F12 medium with glutamax (Invitrogen) containing 10% fetal bovine serum (Invitrogen), 0.25% gentamycin (Invitrogen) and 0.25 ng/mL M-CSF (murine-colony stimulating factor, Peprotech, Rocky Hill, New Jersey, USA). Microglia were then separated from the mixed primary culture by shaking (200 rpm for 2 hours), and plated in the DMEM-F12 medium with glutamax containing 0.25% gentamycin (Invitrogen).
Cells were kept at 37°C under a humidified atmosphere with 95% O2 and 5% CO2. Viable cells (identified by counting trypan-blue-excluding cellular elements) were plated at a density of 5 × 105 cells per cm2 in 6 well trays for Western blotting and enzyme-linked immunosorbent assay (ELISA) or 1 × 105 cells per cm2 in 12 well trays for proliferation and immunocytochemistry studies.
Microglial cell pharmacological treatment
In order to evaluate the ability of an inflammatory trigger to control the cellular content of BDNF over time, N9 cells were challenged with 100 ng/mL LPS (from Escherichia coli, serotype 055:B5 from Sigma) for 3, 6 and 12 hours. This concentration of LPS was chosen since it was previously shown to induce changes of A2AR density in microglial cells , which we now aim to pharmacologically manipulate to modulate BDNF secretion/function. Thus, microglial cells were pre-incubated (20 minutes before LPS) with a supra-maximal and selective concentration (50 nM) of an A2AR antagonist, SCH58261 , which was present until the end of the experiment (3, 6 or 12 hours). Given that we were able to identify the tipping time point of the changes in BDNF levels and A2AR modulation effects, subsequent experiments were carried out at this time point (6 hours). In all these experiments, 20 to 30 minutes before adding LPS, N9 cells were incubated with different modulators used in supra-maximal and selective concentrations gauged from our previous experience in different preparations [25, 26]: adenosine deaminase (ADA, 1 U/mL, which removes endogenous adenosine; Sigma), H89 (1 μM, a protein kinase A (PKA) inhibitor; Tocris, Madrid, Spain), chelerythrine (6 μM, a protein kinase C (PKC) inhibitor; Calbiochem, Lisbon, Portugal), or anti-human BDNF polyclonal antibody (10 μg/mL; Promega, Lisbon, Portugal), a concentration chosen according to . ADA and the anti-human BDNF polyclonal antibody were directly diluted in the culture medium; H89 and chelerythrine were made up to a 25 mM stock solution in water to dilute in the culture medium.
In the experiments carried out in the absence of LPS, N9 cells were incubated with PKA upstream modulators used in concentrations gauged from our previous experience in different preparations (for example [25, 26]), namely: CGS21680 (30 nM; Sigma), an A2AR selective agonist; forskolin (1 μM; Ascent Scientific, Cambridge, UK), an activator of adenylate cyclase; 8-Br-AMP (5 μM; Tocris), a cyclic AMP (cAMP) analog, or with exogenously added BDNF (Sigma) used in a concentration (20 ng/mL) able to modulate A2AR-mediated neuronal functions . BDNF was directly diluted in the culture medium, whereas CGS21680 and forskolin were made up in dimethyl sulfoxide (100 mM) and 8-Br-AMP was made up in water (100 mM) to dilute in the culture medium. We always tested the impact of the vehicles of the drugs and modulators on each measure and found that all the used vehicles were devoid of effects (data not shown).
Cell lysates were obtained in a lysis solution containing 150 mM NaCl, 50 mM Tris–HCl, 1 mM ethylenediamine tetraacetic acid (EDTA), 1% NP-40 Igepal (Sigma, Sintra, Portugal), 0.1% sodium dodecyl sulfate (SDS), 0.5% sodium deoxycolate, 1 mM (phenylmethylsulfonyl fluoride (PMSF), 1 mM sodium ortovanadate, 1 mM NaF, 1 μg/mL CLAP (protease inhibitor cocktail; Sigma). After cell scraping for homogenization, the total amount of protein was quantified using the bicinchoninic acid (BCA; Thermo Scientific, Loures, Portugal) method. Samples were then loaded onto gels with 7.5% (to detect A2AR, 50 kDa and pro-BDNF, 37.5 kDa) or 15% (to detect mBDNF, 13 kDa) of acrylamide plus bisacrylamide (BioRad, Amadora, Portugal); proteins were separated by electrophoresis (100 to 120 V for 1 hour) using a bicine-buffered solution (20 mM Tris, 192 mM bicine and 0.1% SDS, pH 8.3) and then transferred (300 mV, 100 minutes, 4°C) to polyvinylidene difluoride (PVDF) membranes (0.45 μm pore diameter) (GE Healthcare, Little Chalfont, Buckinghamshire, UK). Blots were then blocked for 1 hour at room temperature (RT) with 5% low-fat milk in Tris-buffered saline (20 mM Tris, 140 mM NaCl, pH 7.6, TBS) with 0.1% Tween 20 (TBS-T) and incubated overnight at 4°C with primary antibodies diluted in TBS-T with 0.5% low-fat milk. The tested primary antibodies were mouse monoclonal anti-BDNF (1:1000; Sigma) and mouse anti-A2AR (1:1000; Millipore, Lisbon, Portugal). After rinsing three times with 0.5% low-fat milk in TBS-T, membranes were incubated for 1 hour at RT with alkaline phosphatase-conjugated secondary antibodies (1:2000; Amersham, Piscataway, New Jersey, USA). Protein immunoreactive bands were visualized in a Versa Doc Imaging System (Model 3000, BioRad Laboratories), after the incubation of the membrane with enhanced chemofluorescence reagent (ECF; GE Healthcare).
Re-probing of the same membranes with a different antibody was achieved by washing the ECF in 40% methanol for 30 minutes and stripping the previous antibodies in a solution of 0.2 M glycine with 0.1% SDS and 1% (v/v) Tween 20, pH 2.2, for 1 hour. After washing (3 times with TBS-T for 20 minutes), membranes were blocked and incubated with primary and respective secondary antibodies, namely for mouse anti-α-tubulin (1:20000; Sigma) to confirm that similar amounts of sample were loaded to the different lanes.
Cells were fixed with 4% paraformaldehyde (Sigma) and permeabilized for 20 minutes in 0.25% Triton X-100 (Sigma) in a phosphate-buffered saline (PBS) solution (137 mM NaCl, 2.7 mM KCl, 10 mM NaH2PO4, 1.8 mM KH2PO4, pH 7.4). Unspecific binding was prevented by incubating cells in PBS with 3% bovine serum albumin (BSA) and 5% normal horse serum. Cells were incubated overnight at 4°C in PBS with 3% BSA and 5% normal horse serum and including the primary antibody (mouse anti-A2A, 1:250; Millipore). Cells were then washed and incubated for 1 hour at RT with an Alexa Fluor 488 donkey anti-mouse (1:400; Molecular Probes, Lisbon, Portugal) secondary antibody. Membrane ruffling, which is characteristic of activated microglia, was probed using a marker for filamentous actin, phalloidin, by incubating cells for 20 minutes at RT with PBS containing actin-stain 670 fluorescent phalloidin (1:75; Cytoskeleton, Denver, USA). For nuclear labeling, N9 cells were stained with DAPI (0.1 mg/mL; Invitrogen). The coverslips were mounted in the Prolong Gold Antifade fluorescent medium (Invitrogen). In order to check for non-specific labeling of the secondary antibodies or cross-reactivity between secondary antibodies, staining was tested in the absence of each primary antibody and in the absence of both primary antibodies. Fluorescent images were acquired using a Zeiss Imager Z2 fluorescence microscope equipped with an AxioCam HRm and 63x Plan-ApoChromat oil objective (1.4 numerical aperture), with Axiovision SE64 4.8.2 software. Ten images were randomly taken from each coverslip (three per condition in each experiment).
BrdU incorporation assay
Microglial proliferation was evaluated by measuring the incorporation of 5-bromo-2'-deoxyuridine (BrdU; Sigma), a synthetic nucleoside that can be incorporated into newly synthesized DNA, replacing thymidine during cell replication. Cells were incubated with BrdU (10 μM) for the last 2 hours of pharmacological treatment, fixed in 4% paraformaldehyde, washed in TBS with 0.3% Triton X-100 and maintained in 1 M HCl at 37°C for 30 minutes. Non-specific binding was prevented by incubation for 1 hour in TBS with 3% BSA and 1% Triton X-100. Cells were incubated overnight at 4°C with a primary rat antibody anti-BrdU (1:100; Serotec, Oxford, UK) in a 0.1% Triton X-100 and 0.3% BSA solution, washed and incubated for 2 hours at RT with an Alexa Fluor 594 donkey anti-rat secondary antibody (1:200; Molecular Probes). For nuclear staining, cells were incubated for 5 minutes at RT with Hoechst 33342 (10 μg/mL; Molecular Probes) in 0.3% BSA, and mounted in Dakocytomation fluorescent medium (Dakocytomation Inc., California, USA). Fluorescent images were acquired using an Axioskop 2 Plus fluorescence microscope (Zeiss, PG-Hitec, Lisbon, Portugal). The number of proliferating cells (BrdU-positive) was counted and expressed as a percentage of the total cells stained with Hoechst 33342 .
BDNF enzyme-linked immunosorbent assay
The extracellular levels of BDNF were measured in the supernatant of N9 cells using the BDNF Emax Immunoassay System (Promega, Madison, USA). Each well of a 96-well polystyrene plate was incubated overnight at 4°C with 80 μL of anti-BDNF monoclonal antibody (1:1000) in coating carbonate buffer (50 mM, pH 9.7). Non-adsorbed antibody was discarded and rinsed off by washing once in a TBS-T buffer (20 mM Tris–HCl, pH 7.6, 150 mM NaCl and 0.05% (v/v) Tween 20). Unspecific binding was prevented by blocking with 80 μL Promega 1x Block and Sample Buffer (BB 1x) for 1 hour at RT. Plates were then washed (as mentioned before) and 80 μL of each sample or standard (7.8 pg/mL to 500 pg/mL) were loaded in triplicate to the plates (2 hours with 300 rpm shaking at RT). After washing (5 times in TBS-T wash buffer), 80 μL of anti-human BDNF polyclonal antibody (1:500 in BB 1x) was added to each well and the plates were incubated at RT (2 hours with 300 rpm shaking). After washing (5 times in TBS-T wash buffer), the plates were incubated for 1 hour with shaking (300 rpm) with 80 μL anti-IgY horseradish peroxidase conjugate (1:200 in BB 1x). After the last wash with the TBS-T buffer, 80 μl of TMB One solution was used as developer and the reaction was stopped by adding 80 μL of HCl 1 M. Absorbance was measured at 450 nm. BDNF levels are reported as pg/mL normalized per total amount of protein.
Values are presented as mean ± standard error of the mean (SEM) of n experiments. Either a Student’s t test for independent means or a one-way analysis of variance (ANOVA) followed by a Newman–Keuls post hoc test, was used to define statistical differences between absolute values, which were considered significant at P < 0.05 unless otherwise specified. Note that although the impact of several drugs and modulators are presented as percentage values for the sake of clarity, the statistical comparisons were always carried out using the absolute values.