Animals
TRAIL-R-/- animals were kindly provided by Prof. H. Walczak, Dept of Cancer Biology, CRUK-UCL Cancer Ctr., UCL Cancer Inst., London, United Kingdom. TRAIL-R-/- animals and wild-type (WT) littermates in the B6 background were maintained on a 12 h light/dark cycle in temperature and humidity-controlled rooms, and food and water were available ad libitum. All experiments were approved by the Italian Ministry of Health (authorization n.86/2015 PR) and conducted following the European Community directive guidelines for the use of animals in laboratory (2010/63/EU) and the Italian law (D.Lgs. 26/2014).
Preparation of Aβ1-42 Oligomers
Aβ1–42 oligomers were generated as the previously described method [34]. Briefly, the Aβ1–42 lyophilized peptide (Sigma-Aldrich, St. Louis, MO, USA) was initially dissolved in 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP; Sigma-Aldrich) to a final concentration of 1 mM and incubated at room temperature for 2 h. The peptide solution was aliquoted and dried in the fume hood. Traces of HFIP was removed under vacuum in a SpeedVac centrifuge (800 g, RT), and the thin clear peptide film was stored over desiccant at −80°C. For aggregation, the aliquoted peptide film was dissolved in dimethyl sulfoxide (DMSO) to 5 mM. The peptide in DMSO was diluted directly into sterile phosphate buffered saline (PBS, 1×) at 100 µM and incubated at 4°C for 12 h to make the oligomeric form of Aβ1–42. Following incubation, Aβ1-42 samples were immediately used for the cell treatment or aliquoted and stored at –20°C until their use.
Experimental Groups and Drug Administration
Twenty TRAIL-R-/- and 20 wild-type C57BL/6J male mice were enrolled at nine months of age and four study groups were used: (i) Wild-type plus vehicle; (ii) Wild-type plus Aβ1-42; (iii) TRAIL-R-/- plus vehicle; and (iv) TRAIL-R-/- plus Aβ1-42.
As previously described [35], mice were positioned on a stereotaxic frame and a Hamilton syringe with a 29 G needle was implanted into the dentate gyrus of the hippocampus using the following stereotaxic coordinates from the bregma: AP, -2.00 mm; ML, ±1.3 mm; DV, -2.2 mm. Animals were treated with oligomeric Aβ1-42 or vehicle and sacrificed after 2 weeks.
Primary Cultures of Mouse Hippocampal Neurons
Sixteen embryonic day mice were obtained from surgically sacrificed pregnant mouse and the hippocampus was sepa-rated under surgical stereomicroscope. Separated tissues were isolated and dissociated by manual dispersion with a fire-polished Pasteur pipette. Cells were plated at a density of 1.5 × 105 cells/cm2. The cells were plated in Neurobasal medium (Invitrogen Corporation, Waltham, MA, USA) supplemented with 2% B27 (Invitrogen Corporation), 0.5 mM L-glutamine, and 50 U/ml penicillin/streptomycin (Invitrogen Corporation). Three days after plating, 50% of the medium was changed with fresh medium and subsequently 50% of the medium was changed twice a week, until 11 days in vitro. To inhibit glial cell outgrowth, cytosine arabinoside (1 µM) was added at the moment of media change.
Cell Viability Assay
Cell viability was determined by 3-[4,5 dimethylthiazol-2-yl]-2,5- diphenyltetrazolium bromide assay. At the end of each treatment, cell viability was measured by the reduction of 3-[4,5 dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide solution (0.5 mg/mL). The solution was removed after 3 h of incubation at 37°C and dimethylsulfoxide was added to ob-tain cell lysis and solubilization of blue formazan crystals resulting from MTT reduction by viable cells’ mitochondrial activity. Optical density of the blue formazan was measured at 570 nm with a VarioskanTM Flash Multimode Reader (Thermo Fisher Scientific, Waltham, MA, USA).
Free-Floating Fluorescence Immunohistochemistry
Mice were deeply anesthetized by i.p. injection of Zoletil 100 (40 mg/kg) (Virbac S.r.l., Milan, Italy) and perfused trancardially with 4% paraformaldehyde (PFA) solution in 0.1 M phosphate buffer (PBS; pH 7.4). The brains were removed, post-fixed overnight in the same 4% PFA and then transferred into a 30% sucrose in PBS as cryo-protective solution at 4°C for 2–3 days. Serial 25 µm frozen hippocampal sections of the brains were cut and subjected to immunohistochemical assay. Briefly, free-floating sections were washed three times in PBS and then blocked at room temperature for 1 h in 5% normal goat serum (NGS) in PBS. They were then incubated overnight at 4°C with the following antibodies: a mouse monoclonal anti-Iba1 (1:200, Abcam, Cambridge, United Kingdom) as microglial marker and a mouse anti-GFAP antibody (Cell Signaling Technology, Inc., Danvers, MA, United States). For fluorescence visualization, after washing in PBS three times for 5 minutes each, sections were incubated in the dark for 1h at room temperature with the corresponding fluorescent-labelled secondary antibodies: Alexa Fluor 546 donkey anti-mouse IgG (1:500, Thermo Fisher Scientific) and Alexa Fluor 488 goat anti-mouse IgG (1:500, Thermo Fisher Scientific). Finally, sections were washed in PBS three times for 5 minutes each and mounted on gelatinated slides. Digital images were captured with a Zeiss Observer.Z1 microscope equipped with the Apotome.2 acquisition system (Zeiss, Oberkochen, Germany).
Protein Extraction
Freshly isolated hippocampal tissues were lysed in a buffer containing 150mM NaCl, 50mM Tris–HCl (pH 7.5), 5mM EDTA, 1mM Na3VO4, 30mM sodium pyrophosphate, 50mM NaF, 1mM acid phenyl-methyl-sulphonyl-fluoride, 5 µg/ml aprotinin, 2 µg/ml leupeptin, 1 µg/ml pepstatin, 10% glycerol, and 0.2% Triton X-100. The homogenates were then centrifuged at 14000 rpm for 10 min at 4°C and supernatants were collected. Protein concentration of the supernatant was determined by the Bradford method [36].
Western Blot Analysis
Equal amounts of protein (50 µg) were separated by 8%-12% SDS-PAGE gels and transferred onto Hybond ECL nitrocellulose membranes (Amersham Life Science, Buckinghamshire, UK). The membranes were blocked with 5% non-fat dry milk in PBST for 1 hour at RT and were then probed overnight at 4°C on orbital shaker with the following appropriate primary antibodies: rabbit anti-DR5 (1:500; Abcam, Cambridge, United Kingdom), mouse anti-p53 (1:1000; Cell Signaling Technology, Inc., Danvers, MA, United States), rabbit anti-DcR1 (1:1000; Abcam), mouse anti-p-JNK (1:500; Santa Cruz Biotechnology Inc.), mouse anti-JNK (1:500; Santa Cruz Biotechnology), rabbit anti-p-AKT (1:1000; Cell Signaling Technology, Inc.), rabbit anti-AKT (1:1000; Cell Signaling Technology, Inc.), mouse anti-p-GSK3β (1:500; Becton Dickinson, Franklin Lakes, NJ, USA), mouse anti-GSK3β (1:500; Santa Cruz Biotechnology Inc.), mouse anti-p-TAU (1:250; Santa Cruz Biotechnology Inc.), rabbit anti-TAU (1:500; Santa Cruz Biotechnology Inc.), mouse anti-Iba-1 (1:500; Abcam), mouse anti-GFAP (1:500; Cell Signaling Technology), rabbit anti-NOS2 (1:500; Santa Cruz Biotechnology Inc.), mouse anti-COX2 (1:500; Santa Cruz Biotechnology Inc.), rabbit anti-IL-1β (1:250; Santa Cruz Biotechnology Inc.) and rabbit anti-TNF-α (Novus Biologicals, Centennial, CO, USA). Beta-tubulin (Santa Cruz Biotechnology Inc.) primary antibody was used as an internal control to validate the right amount of protein loaded in the gels. Then the membranes were washed with PBS-T and probed with the appropriate horseradish peroxidase-conjugated secondary antibodies (GE Healthcare, Milan, Italy) for 1 hour at room temperature in 5% non-fat dry milk. After washing with PBS-T, protein bands were visualized by enhanced chemiluminescence (Thermo Fisher Scientific) and scanned with the iBright FL1500 Imaging System (Thermo Fisher Scientific). Densitometric analysis of band intensity was performed with the aid of ImageJ software (https://imagej.nih.gov/ij/).
Caspase Colorimetric Assay
Caspase activities were measured with the Caspase Colorimetric Substrate Set II Plus kit (BioVision Inc., Milpitas, CA, USA) as previously described [37]. In brief, protein extracts (100 µg) were added to a reaction buffer containing a p-nitroanilide-labelled specific caspase substrate and incubated for 2h at 37°C. Relative caspase activity was measured as optical density at 405 nm in a microplate reader (Bio-Rad Laboratories, Inc., Italy). Fold-increase in caspase activity was determined by comparing results with the level of the uninduced control.
Nitrite Assay
Nitric oxide production in the primary embryonic hippocampal neurons was assessed by the Griess method, as previously described [13]. Briefly, after 24 h treatments, 100µL aliquots of culture supernatants were incubated with 100 µL of Griess reagent (1% sulphanilamide, 0,1%N-(1-naphtil)ethyl-enediamine dihydrochloride and 5% of phosphoric acid) at room temperature for 20 min. Optical density at 540 nm was determined using a microplate reader (Bio-Rad Laboratories). The nitrite concentration was determined from a sodium nitrite standard curve.
Statistical Analysis
Data were analyzed by the one-way analysis of variance (ANOVA) test, followed by the Bonferroni post-hoc test. Data were represented as means ± standard error mean (SEM). Significance was set at a p<0.05. Graph design and statistical analyses were carried out with the dedicated software GraphPad Prism (La Jolla, CA, USA).