Materials
Rotenone, 6-hydroxydopamine (6-OHDA), poly-D-lysine (PDL), 2′7′-dichlorodihydrofluorescein diacetate (H2DCFDA), 4′,6-diamidino-2-phenylindole (DAPI), catalase (CAT), apocynin, and protease inhibitor cocktail were purchased from Sigma (St Louis, MO, USA). Mito-TEMPO was acquired from ALEXIS Biochemicals Corporation (San Diego, CA, USA). Compound C and 1-methyl-4-phenylpyridin-1-ium (MPP+) were provided by Calbiochem (San Diego, CA, USA). Dulbecco's Modified Eagle's Medium (DMEM), 0.05% Trypsin–EDTA, NEUROBASAL™ Media, and B27 Supplement were from Invitrogen (Grand Island, NY, USA). Horse serum and fetal bovine serum (FBS) were supplied by Hyclone (Logan, UT, USA). Enhanced chemiluminescence solution was from Sciben Biotech Company (Nanjing, China). The following antibodies were used: p-Akt (Ser473), p-Akt (Thr308), p-S6K1 (Thr389), p-4E-BP1 (Thr70), 4E-BP1, cleaved caspase-3 (Cell Signaling Technology, Danvers, MA, USA); β-tubulin, p-mTOR (Ser2448), mTOR, p-AMPKα (Thr172), AMPKα, p40phox, p47phox, HA (Sigma); Akt, S6K1, p22phox (Santa Cruz Biotechnology, Dallas, TX, USA); NOX2, p67phox (Epitomics, Burlingame, CA, USA), Rac1 (Cytoskeleton, Denver, CO, USA); Goat anti-rabbit IgG-horseradish peroxidase (HRP), goat anti-mouse IgG-HRP, and rabbit anti-goat IgG-HRP (Pierce, Rockford, IL, USA). Other chemicals were purchased from local commercial sources and were of analytical grade, unless stated elsewhere.
Cell culture
Rat pheochromocytoma (PC12) cell line (RRID: CVCL_0481) was obtained from American Type Culture Collection (ATCC) (Manassas, VA, USA). Because of the replicative nature and cost effectiveness, the cell line is widely used as neuronal cell models, so it was employed in this study. Cells, seeded in a 6-well plate (5×105 cells/well) or 96-well plate (1×104 cells/well) pre-coated with 0.2 μg/ml PDL, were cultured in antibiotic-free DMEM supplemented with 10% horse serum and 5% FBS in a humidified incubator of 5% CO2 at 37°C. To verify the data obtained from PC12 cells, primary neurons were also used in this study. For this, primary murine neurons were isolated from fetal mouse cerebral cortexes of 16–18 days of gestation in female ICR mice (being pregnant) as described [22], and seeded in a 6-well plate (5×105 cells/well) or 96-well plate (1×104 cells/well) coated with 10 μg/mL PDL for experiments after 6 days of culture. All procedures used in this study were approved by the Institutional Animal Care and Use Committee, and were in compliance with the guidelines set forth by the Guide for the Care and Use of Laboratory Animals.
Recombinant adenoviral constructs and infection of cells
The recombinant adenoviral vectors encoding HA-tagged myristoylated, constitutively active Akt (Ad-myr-Akt), HA-tagged dominant negative AMPKα (Ad-dn-AMPKα), and the control adenovirus expressing -galactosidase (Ad-LacZ) were described previously [25,26,22]. The viruses were amplified, titrated and used as described [27,22]. For experiments, PC12 cells were cultured in the growth medium, and infected with the individual adenovirus for 24 h at 5 of multiplicity of infection (MOI = 5). Subsequently, cells were used for experiments. Cells infected with Ad-LacZ alone served as a control. Expression of HA-tagged myr-Akt or dn-AMPKα was determined by Western blotting with antibodies to HA.
Lentiviral shRNA cloning, production, and infection
Lentiviral shRNAs to NOX2 and GFP (for control) were generated as described [25,28]. For use, a monolayer of PC12 cells, when grown to about 70% confluence, were infected with the corresponding lentivirus-containing medium in the presence of 8 mg/ml polybrene for 12 h twice at an interval of 6 h. Uninfected cells were eliminated by exposure to 2 mg/mL puromycin for 48 h before use. After 5 days of culture, cells were used for experiments.
Assays for cell caspase-3/7 activity
PC12 cells and primary neurons were seeded in a PDL-coated 96-well plate (1×104 cells/well). The next day, cells were treated with/without 6-OHDA (30, 60 and/or 120 μM), MPP+ (0.5, 1 and/or 1.5 mM) or rotenone (0.5, 1 and/or 2 μM) for 6, 12 and/or 24 h with five replicates of each treatment. Subsequently, caspase-3/7 activity was detected using Caspase-Glo®3/7 Assay Kit (Promega, Madison, WI, USA), according to the manufacturer’s protocol.
DAPI staining
PC12 cells and primary neurons, or PC12 cells infected with lentiviral shRNA to NOX2 or GFP, or PC12 cells infected with Ad-myr-Akt, Ad-dn-AMPKα or Ad-LacZ, respectively, were seeded at a density of 5 × 105 cells/well in a 6-well plate containing a PDL-coated glass coverslip per well. The next day, cells were treated with/without 6-OHDA (30, 60 and/or 120 μM), MPP+ (0.5, 1 and/or 1.5 mM) or rotenone (0.5, 1 and/or 2 μM) for 6, 12 and/or 24 h, or treated with/without 6-OHDA (120 μM), MPP+ (1 mM) or rotenone (1 μM) for 24 h following pre-incubation with/without a H2O2-scavenging enzyme CAT (350 U/ml), a mitochondria-targeted antioxidant Mito-TEMPO (10 μM), or a NOX2 inhibitor apocynin (50 μM) for 1 h, or an AMPK inhibitor compound C (20 μM) for 2 h with 5 replicates of each treatment. Afterwards, the cells with fragmented and condensed nuclei were determined using DAPI staining as described [29]. Finally, slides were mounted in glycerol/phosphate buffered saline (PBS) (1:1, v/v) containing 2.5% 1,4-diazabiclo-(2,2,2) octane. Photographs were taken under a fluorescence microscope (Leica DMi8, Wetzlar, Germany) equipped with a digital camera.
Intracellular H2O2 imaging
Intracellular H2O2 level was imaged by using a nonfluorescent probe, H2DCFDA. This peroxide-selective dye can penetrate into the intracellular matrix of cells, where it is cleaved by intracellular esterases and oxidized by H2O2 to form fluorescent DCF [30]. In brief, the indicated cells, after treatment as described above, were loaded with H2DCFDA (20 M) for 1 h. Subsequently, all stained specimens were rinsed three times with PBS, followed by imaging under a fluorescence microscope, and quantitatively measuring integral optical density (IOD) of the fluorescence intensity by Image-Pro Plus 6.0 software (Media Cybernetics Inc., Newburyport, MA, USA).
Western blot analysis
The indicated cells, after treatments, were briefly washed with cold PBS, and then on ice, lysed in the radioimmunoprecipitation assay buffer. Afterwards, Western blotting was performed as described previously [22].
Statistical analysis
All data were expressed as means ± standard error of the mean (means ± SEM). Student’s t-test for non-paired replicates was used to identify statistically significant differences between treatment means. Group variability and interaction were compared using either one-way or two-way ANOVA followed by Bonferroni’s post-tests to compare replicate means. A level of P < 0.05 was considered to be significant.