Escherichia coli 0111:B4 lipopolysaccharide (LPS), zymosan from Saccharomyces cerevisiae, Z-Leu-Leu-Leu-al (MG-132), and D-desthiobiotin were purchased from Sigma-Aldrich (St. Louis, MO, USA). CpG oligodeoxynucleotides were purchased from Invivogen (Toulouse, France). N-Acetyl-Leu-Leu-Norleu-al (ALLN) was purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Puromycin dihydrochloride was purchased from Gold Biotechnology (St. Louis, MO, USA).
BV-2 microglia were cultured in RPMI1640 with 5% fetal calf serum (FCS) supplemented with 2 mM L-glutamine and 195 nM β-mercaptoethanol at 37°C in a humidified atmosphere of 5% CO2 as previously described . BV-2 cells were preincubated for 24 h with 10 μg/ml recombinant AMWAP or PBS as vehicle control, unless stated otherwise. BV-2 cells were stimulated with 50 ng/ml LPS, 50 μg/ml zymosan, or 4 μg/ml CpG oligodeoxynucleotides. BV-2 cells were preincubated with the proteasome inhibitor ALLN (100 μg/ml) for 30 min before LPS stimulation to allow for accumulation of phosphorylated proteins. Generation and culture conditions of embryonic stem cell-derived microglia (ESdM) and mouse brain microglia have been described previously [34,35]. ESdM and primary microglia were preincubated for 24 h with 10 μg/ml recombinant AMWAP or PBS as vehicle control and stimulated with 500 ng/ml and 50 ng/ml LPS, respectively. 661W photoreceptor-like cells were a kind gift from Prof. Muayyad Al-Ubaidi (Department of Cell Biology, University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA), and the culture conditions have been described elsewhere . HEK293 EBNA cells were cultured in DMEM low glucose with 1 mM sodium pyruvate and 10% FCS. All media were supplemented with 1% penicillin/streptomycin.
Recombinant protein expression
Recombinant expression and purification of C-terminally His-tagged AMWAP in E. coli was carried out as described previously . For eukaryotic expression of C-terminally Strep(II)-tagged AMWAP, the AMWAP ORF was amplified from BV-2 microglial cDNA with primers forward 5′-cccgctagccacctatgtagtgtcttgccc-3′ and reverse 5′-cccctcgagaaagacaggagttttgcaga-3′ and subcloned into the pCEP-Pu plasmid at restriction sites NheI and XhoI. This plasmid includes the BM-40 signaling peptide which leads to secretion and is then cleaved off from the recombinant protein. The clone was validated by DNA sequencing. HEK293 EBNA cells were transfected with the expression plasmid using the Turbofect™ reagent (Thermo Scientific, Waltham, MA, USA). After 24 h, 3 μg/ml puromycin was added to select for plasmid-positive cells. After expansion of the cells, FCS-free supernatants containing secreted AMWAP were collected every 48 h, centrifuged for 7 min at 3,500 rpm at 4°C and 1 mM PMSF was added. For affinity chromatography, a Strep-Tactin® Sepharose (Iba Life Sciences, Goettingen, Germany) column was prepared and the supernatant loaded onto and passed through the column at a velocity of 0.25 ml/min at 4°C using a peristaltic pump P-1 (GE Healthcare, Little Chalfont, UK). Thereafter, the column was washed with PBS (pH 7.8) and recombinant AMWAP eluted with PBS containing 2.5 mM D-desthiobiotin. Protein-containing fractions were identified using a NanoDrop 2000 (Thermo Scientific), and protein concentration was determined by Bradford assay (Roti®-Quant, Roth, Karlsruhe, Germany) and BCA assay (Roti®-Quant universal, Roth). Recombinant protein was then stored at −20°C until further use.
Generation of an AMWAP antiserum and Western blot analysis
Rabbit antibodies were raised against an N-terminally GST-tagged recombinant AMWAP peptide. The AMWAP ORF was cloned into a pGEX-4-T1 vector system to express an N-terminally GST-tagged AMWAP protein in prokaryotic cells. The AMWAP ORF was PCR amplified from a plasmid that was described previously . GST-tagged AMWAP protein was expressed and purified as described above. Immunization of rabbits, isolation of serum, and affinity purification was performed by Davids Biotechnologie GmbH (Neutraubling, Germany). For extraction of total cellular protein, BV-2 cells were lysed in cold RIPA buffer (20 mM Na-phosphate buffer, 150 mM NaCl, 5 mM EDTA, 1% Triton X-100, and protease inhibitors) and insoluble debris was removed by centrifugation for 15 min at 13,200 rpm. Nuclear and cytosolic protein extraction was carried out using the NE-PER kit (Thermo Scientific) according to the manufacturer’s instructions. Serum-free microglial supernatants were concentrated 50-fold using Amicon® Ultra-4 centrifugal filter units with a molecular weight cut-off at 3 kDa (Merck-Millipore, Billerica, MA, USA). Protein concentrations were determined by Bradford assay (Roti®-Quant, Roth). A 10 to 20 μg of protein was separated by SDS-PAGE on 10% to 15% gels with PageRuler pre-stained protein ladder (Thermo Scientific). Proteins were then transferred to 0.45 μm nitrocellulose membranes (Bio-Rad, Munich, Germany). After blocking in TBS-T containing 5% nonfat dry milk or bovine serum albumin, membranes were incubated with primary antibodies against AMWAP, NFκB p65 (sc-372, Santa Cruz Biotechnology), phosphorylated NFκB p65 (sc-33020, Santa Cruz Biotechnology), IRAK-1 (sc-7883, Santa Cruz Biotechnology), IκBα (sc-371, Santa Cruz Biotechnology), phosphorylated IκBα (sc-101713, Santa Cruz Biotechnology), ubiquitin (ab7780, Abcam, Cambridge, UK), actin (sc-1616, Santa Cruz Biotechnology), or GAPDH (sc-48166, Santa Cruz Biotechnology). Blots were then incubated with secondary goat anti-rabbit IgG-HRP or rabbit anti-goat IgG-HRP antibodies (sc-2004, sc-2768, Santa Cruz Biotechnology). Enhanced chemiluminescence signals were visualized and imaged with the MultiImage II system (Alpha Innotech, Santa Clara, CA, USA). Total protein visualization was done by Ponceau S staining.
RNA isolation and reverse transcription
Total RNA was extracted from microglia cells according to the manufacturer’s instructions using the NucleoSpin® RNA Mini Kit (Macherey-Nagel, Dueren, Germany). RNA was quantified spectrophotometrically using a NanoDrop 2000 (Thermo Scientific) and then stored at −80°C. First-strand cDNA synthesis was performed with the RevertAid™ H Minus First strand cDNA Synthesis Kit (Thermo Scientific).
Quantitative real-time RT-PCR
Amplifications of 50 ng cDNA were performed with an ABI7900HT machine (Applied Biosystems, Carlsbad, CA, USA) in 10 μl reaction mixtures containing 1× TaqMan Universal PCR Master Mix (Applied Biosystems), 200 nM of primers, and 0.125 μl of dual-labeled UPL probe (Roche Applied Science, Basel, Switzerland). The reaction parameters were as follows: 2 min 50°C hold, 30 min 60°C hold, and 5 min 95°C hold, followed by 40 cycles of 20 s 94°C melt and 1 min 60°C anneal/extension. Primer sequences and UPL probe numbers were as follows: IL6, forward primer 5′-gatggatgctaccaaactggat-3′, reverse primer 5′-ccaggtagctatggtactccaga-3′, probe #6; iNOS, forward primer 5′-ctttgccacggacgagac-3′, reverse primer 5′-tcattgtactctgagggctga-3′, probe #13; CCL2, forward primer 5′-catccacgtgttggctca-3′, reverse primer 5′-gatcatcttgctggtgaatgagt-3′, probe #62; CASP11, forward primer 5′-gatcgggcaaccttgaca-3′, reverse primer 5′-tgagattcagttgcttgttgc-3′, probe #72; TNFα, forward primer 5′-ctgtagcccacgtcgtagc-3′, reverse primer 5′-ttgagatccatgccgttg-3′, probe #78; ATP5B, forward primer 5′-ggcacaatgcaggaaagg-3′, reverse primer 5′-tcagcaggcacatagatagcc-3′, probe #77. ATP5B expression was used as the most stable reference gene. Measurements were performed in triplicates, and results were analyzed with the ABI sequence detector software version 2.4 using the ΔΔCt method for relative quantification.
Fluorescence labeling of recombinant AMWAP
Recombinant AMWAP was fluorescently labeled using the Fluorescein-EX Protein Labeling Kit (Life Technologies, Carlsbad, CA, USA) according to the manufacturer’s instructions. This dye has a seven-atom spacer between the succinimidyl ester group and the fluorophore to minimize unwanted interaction between the fluorophore and the labeled protein. After labeling, the protein solution was passed five times through an Amicon® Ultra-4 centrifugal filter unit with a molecular weight cut-off at 3 kDa (Merck-Millipore) at 4°C and extensively washed with PBS to get rid of excess fluorescent dye.
BV-2 cells were seeded on glass cover slips and incubated with 10 μg/ml fluorescently labeled AMWAP or vehicle for different time spans prior to stimulation with 50 ng/ml LPS for 1 h. Cells were then fixed with 4% formaldehyde, washed with PBS, and incubated in blocking buffer containing 10% goat serum and 0.3% Triton X-100. Subsequently, cells were incubated with antibodies against Iba1 or the p65 subunit of NFκB in a solution containing 2.5% goat serum and 0.1% Triton X-100 for 1 h at room temperature. After 30-min incubation with goat anti-rabbit Alexa-594 (A-11012, Life Technologies), slides were washed with PBS and stained with 4′,6-diamidino-2-phenylindole (DAPI). Cover slips were mounted with fluorescent mounting medium (Dako Cytomation, Hamburg, Germany), and fluorescence photomicrographs were taken with an AxioImager.M2 plus ApoTome2 microscope (Carl Zeiss, Oberkochen, Germany). ImageJ software (National Institutes of Health, Bethesda, MD, USA) was used to determine the ratio of nuclear to cytosolic NFκB p65 after quantifying pixel intensities of both cellular compartments and subtracting background fluorescence.
In a total volume of 200 μl, cytosolic extracts (200 μg of protein), supplemented with 1× complete protease inhibitor mix (Roche Applied Science), were incubated with 30 μl Protein A-sepharose® 4B (Sigma-Aldrich) for 60 min at 4°C and subsequently centrifuged for 10 min at 2,000 g. The pre-cleared samples were then incubated at 4°C overnight with 2 μg of anti-IκBα antibody (sc-371, Santa Cruz Biotechnology). Thereafter, 30 μl fresh Protein A-sepharose® 4B was added and the mixture was incubated overnight at 4°C to allow for immunoprecipitation. The beads were then pelleted by centrifugation at 2,000 g and the pellet washed five times with PBS. Beads were then incubated in SDS-PAGE sample buffer at 95°C for 5 min and electrophoresed on a 10% SDS-PAGE. The gel was blotted and the membrane incubated with an anti-ubiquitin antibody at 4°C overnight followed by incubation with secondary goat anti-rabbit IgG-HRP at room temperature for 1 h. Enhanced chemiluminescence signals were then visualized and imaged with the MultiImage II system (Alpha Innotech).
20S proteasome activity assay
Trypsin-, chymotrypsin-, and caspase-like peptidase activities associated with the 20S proteasome were measured using the Proteasome-Glo™ Assay System (Promega, Madison, WI, USA). A 1 μg/ml of purified 20S proteasome (Enzo Life Sciences, Farmingdale, NY, USA) was incubated at room temperature for 30 min with vehicle, 20 μg/ml AMWAP or 1 μM MG-132 peptidase inhibitor positive control in a 96-well plate containing the substrate for one specific peptidase. Luminescence was then measured with an Infinite F200 Pro plate reader (Tecan, Crailsheim, Germany). A blank reaction without 20S proteasome was used to determine background luminescence associated with the vehicle and Proteasome-Glo™ reagent. The values of the blank reactions were subtracted from all experimental values. Relative luciferase units (RLUs) correspond to the levels of peptidase activity.
Nitric oxide concentrations were determined by measurement of nitrite released into BV-2 culture supernatants using the Griess reagent system (Promega). Fifty microliter cell culture supernatants from control, 50 ng/ml LPS-, 10 μg/ml AMWAP-, or 50 ng/ml LPS + 10 μg/ml AMWAP-treated BV-2 microglia were incubated with 100 μl Griess reagent in each well of a translucent 96-well plate. After incubation for 30 min at room temperature, absorbance was measured at 540 nm on an Infinite F200 Pro plate reader (Tecan). Nitrite concentrations were calculated on the basis of a sodium nitrite reference curve.
661W photoreceptor apoptosis assay
To investigate microglial neurotoxicity, 661W photoreceptor cells were incubated for 48 h with culture supernatants from control, 50 ng/ml LPS-, 10 μg/ml AMWAP-, or 50 ng/ml LPS + 10 μg/ml AMWAP-treated BV-2 microglia. Apoptotic cell death was determined using the Caspase-Glo® 3/7 Assay (Promega). Cells were lysed and incubated with a luminogenic caspase-3/7 substrate, which contains the tetrapeptide sequence DEVD. After incubation at room temperature for 1 h, the generated luminescence was measured on an Infinite F200 Pro plate reader (Tecan). A blank reaction without cells was used to determine background luminescence associated with the cell culture system and Caspase-Glo® 3/7 reagent. The values of the blank reactions were subtracted from all experimental values. Negative control reactions were performed to determine the basal caspase activity of 661W cells. Relative luciferase units (RLUs) reflect the level of apoptotic cell death.
BV-2 cells were seeded on cover slips in six-well plates, and attachment was allowed overnight. Cells were then preincubated with 10 μg/ml AMWAP or vehicle for 24 h before incubation with 50 ng/ml LPS for 24 h. Thereafter, cells were fixed with 4% formaldehyde, permeabilized with 0.1% Triton X-100, and F-actin was fluorescently labeled using 0.1 μg/ml Phalloidin-TRITC (Sigma-Aldrich). Nuclei were stained using DAPI, and the cover slips were mounted with fluorescent mounting medium (Dako Cytomation). Photomicrographs were taken with an AxioImager.M2 plus ApoTome2 microscope (Carl Zeiss).
For carboxyfluorescein diacetate succinimidyl ester (CFSE) proliferation assay, BV-2 microglia were labeled with 5 μM CFSE (e-Bioscience, San Diego, CA, USA) and cultured in a six-well plate. After 24 h of AMWAP or vehicle pretreatment and subsequent 24 h of LPS treatment, cells were stained with a fixable viability dye (e-Bioscience) to exclude dead cells from the analysis. The fluorescence intensity of CFSE-labeled BV-2 cells was analyzed by flow cytometry on a FACS Canto II (BD Biosciences, San Jose, CA, USA). Analysis of cell division rate was performed using FlowJo software (Treestar Inc., Ashland, OR, USA).
661W photoreceptor cells were starved by serum deprivation and harvested and fluorescently labeled using CellTracker CM-DiI (Invitrogen, Carlsbad, CA, USA). For phagocytosis assay, BV-2 cells were seeded on cover slips in six-well plates and attachment was allowed overnight. After pretreatment with 10 μg/ml AMWAP for 24 h and subsequent stimulation with 50 ng/ml LPS for 24 h, 500 μl-stained apoptotic 661W cell suspension was added for 6 h. After extensive washing with PBS to get rid of remaining extracellular 661W debris, cells were fixed and nuclei were stained with DAPI. Fluorescence micrographs with constant exposure times were taken with an AxioImager.M2 plus ApoTome2 microscope (Carl Zeiss). ImageJ software (National Institutes of Health) was used to determine the ratio of phagocytosed apoptotic photoreceptor fragments (background-corrected red signal) relative to the total microglia cell number (background-corrected DAPI signal).
Quantitative real-time RT-PCR data were analyzed with the ΔΔCt method using an unpaired Student’s t-test. Assays for NFκB localization, 20S proteasome inhibition, nitrite secretion, caspase 3/7, and phagocytic activity were analyzed with a Mann–Whitney U-test. P < 0.05 was considered statistically significant.