Cell cultures, transfections and treatments
HEK-293 and SH-SY5Y cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM, Sigma) supplemented with 10% of heat-inactivated fetal bovine serum (FBS, HyClone), 2 mM L-glutamine (Gibco-Invitrogen), 50 IU/ml penicillin (Omega Scientific) and 50 μg/ml streptomycin (Omega Scientific). Cells were cultured in 100 mm culture plates at 37°C in a water-saturated atmosphere of 95% air and 5% CO2. The SH-SY5Y cell line stably overexpressing parkin (Parkin-SY5Y cells) was prepared by selecting the transfectants with geneticin (Gibco-Invitrogen). Parkin-SY5Y cells were maintained in DMEM supplemented with 10% FBS, 2 mM l-glutamine, 50 IU/ml penicillin, 50 μg/ml streptomycin and 100 μg/ml geneticin. Transfections were performed in 6-well plates using Lipofectamine LTX and Plus Reagent (Invitrogen) according to the manufacturer’s instructions.
Cultures of neuroblastoma cell lines were grown in 6-well plates. When indicated, cells were exposed for 6–24 hours with 200–600 μM of the short-half life nitric oxide donor S-nitrosocysteine (SNOC), or control solution from which NO had been previously dissipated (designated ‘old’ SNOC). Additional exposures included 200 μM of the long-half life NO donor S-nitrosoglutathione (GSNO) or glutathione (GSH) as its negative control. The neuroblastoma cell line and mesencephalic primary cultures containing substantia nigra cells were exposed to pesticides previously linked to PD in epidemiological studies, including 100 μM of the herbicide paraquat (PQ; Fluka) and 10 μM of the fungicide maneb (MB; Fluka). These concentrations were chosen as approximately two-fold the maximal allowed exposure level from EPA data (US EPA codes CASRN 1910-42-5 and CASRN 12427-38-2, respectively). In some cases, 1 mM of the broad spectrum NOS inhibitor NG-nitro-l-Arginine (NNA; Alexis) was added. Cells were exposed for 6 hours at 37°C in a water-saturated atmosphere of 95% air and 5% CO2. Recombinant proteins were exposed to 200 μM SNOC or old SNOC for 10 min at room temperature.
Primary mesencephalic cultures
Mixed mesencephalic primary cultures, including substantia nigra, were prepared from embryonic day-13 (E13) fetuses of Sprague–Dawley rats. The mesencephalon was dissected in ice-cold EBSS (Gibco-Invitrogen) and subsequently digested in 0.25% trypsin (Gibco-Invitrogen) for 35 minutes at 37°C in a 7% CO2 humidified incubator. Tissues were washed with pre-incubation medium containing DMEM Ham’s F-12 (1:1) (Omega Scientific), 10% fetal bovine serum (FBS, HyClone), 50 IU/ml penicillin (Omega Scientific) and 50 μg/ml streptomycin (Omega Scientific) and dissociated by trituration. Cells were plated on 24-well plates coated with poly-l-lysine (Sigma) in 1:1 pre-incubation medium combined with incubation medium, the latter containing DMEM Ham’s F-12, 0.5 mM l-glutamine (Gibco-Invitrogen), 1% B27 (Gibco-Invitrogen), 50 IU/ml penicillin and 50 μg/ml streptomycin, and maintained at 37°C in a 5% CO2 humidified incubator; 24 hours after plating, the entire medium was replaced with incubation medium. Subsequently, half of the incubation medium was replaced once every four days. At 21 days in vitro (DIV), cells were fixed with 4% paraformaldehyde or treated according to the specific experimental design. The percentage of dopaminergic neurons in the mixed mesencephalic primary culture was 35 ± 4.5%, as measured by immunocytochemistry for the dopamine transporter.
Human brain tissue
Human brain tissues were provided by the Banner Sun Health Research Institute Brain and Body Donation Program. All sample groups correspond to the posterior temporal cortex, from the middle temporal gyrus at the coronal level of the end of the lateral fissure. Samples were frozen (−80°C) within less than 5 hours postmortem (Table 1) and homogenized with HENTS buffer, pH 7.2 (100 mM Hepes, 1 mM EDTA, 0.1 mM neocuproine, 1% Triton X-100, 0.1% sodium dodecyl sulfate) prior to biochemical analysis. The “control” group included samples from elderly patients with no history of dementia or other neurological diseases (Table 1). The “Parkinson’s disease” (PD) group included samples from 5 different brains obtained from patients whose clinical diagnosis in life was verified at postmortem examination (males and females, 69–85 years old; Table 1). The “incidental Lewy body disease” (ILBD) group included samples from 2 clinically normal individuals but whose autopsy revealed the presence of Lewy bodies in the central nervous system by means of α-synuclein immunohistochemistry (males, 91–97 years old; Table 1).
Mouse model of sporadic PD from pesticide exposure
Animal care was conducted in accordance with the United States Public Health Service Guide for the Care and Use of the Laboratory Animals, and all experiments were approved by the University of California San Diego Institutional Animal Care and Use Committee. Exposure to the combination of pesticides paraquat (PQ) and maneb (MB) has been linked to sporadic cases of human PD in epidemiological studies [27–29] and can produce selective loss of nigrostriatal dopaminergic cell bodies and reduction in dopamine levels in the rodent striatum; the combination of paraquat and maneb has therefore been used as an experimental model of PD induced by pesticides in rodents [30–33, 66]. Female mice weighing 20–30 g were injected intraperitoneally (i.p.) with freshly prepared paraquat (Fluka; 5 mg/kg) and maneb (Fluka; 15 mg/kg) twice a week for four weeks. Additional groups of animals also received the relatively specific neuronal NOS inhibitor 3-bromo-7-nitroindazole (3-Br-7-NI, Enzo Life Sciences; 30 mg/kg) or vehicle (60% DMSO in PBS) twice a week on alternate days for four weeks. Animals were sacrificed after treatment. Half of each brain was frozen at −80°C) and homogenized in HENTS buffer prior to biochemical analysis. The remaining halves were fixed in 4% paraformaldehyde for histological assessment of neuronal damage.
Postmortem processing and immunohistochemistry
To evaluate the neurodegenerative histological alterations after pesticide exposure, blind-coded 40-μm thick vibratome sections from mouse brains fixed in 4% paraformaldehyde were immunolabeled for different markers. To assess the integrity of the dendritic system, sections were immunolabeled with the mouse monoclonal antibody against the dendritic marker microtubule-associated protein 2 (MAP2; 1:40; Chemicon, Temecula, CA), as previously described . To evaluate neuronal damage, blind-coded sections were immunolabeled with mouse monoclonal antibody against the neuronal marker NeuN (1:500; Millipore, Billerica, MA). To assess astroglial response, tissue was incubated with the astroglial marker glial fibrillary acidic protein (GFAP; 1:500; Dako, Fort Collins, CO), as previously described [62, 67]. For the evaluation of dopamine synthesis, sections were incubated with mouse anti-TH antibody (1:1000; Pel-Freez). To assess proliferation capacity of adult stem cells that are critical for neurogenesis, tissue was pre-treated with formamide and HCl to denature DNA. Blind-coded sections were then immunolabeled with mouse monoclonal antibody against PCNA (1:500; Santa Cruz Biotechnology, Santa Cruz, CA).
After overnight incubation with the specific primary antibody, for the detection of MAP2, sections were incubated with FITC-conjugated horse anti-mouse IgG secondary antibody (1:75, Vector Laboratories, Burlingame, CA), transferred to SuperFrost slides (Fisher Scientific, Tustin, CA) and mounted under glass coverslips with anti-fading media (Vector). The immunolabeled blind-coded sections were imaged with the laser scanning confocal microscope (MRC1024, Bio-Rad, Hercules, CA) and analyzed with the Image 1.43 program (NIH), as previously described [33, 59]. For the detection of TH, NeuN, PCNA and GFAP, sections were incubated with biotinylated secondary antibody and avidin-conjugated peroxidase (both Vector). Staining was visualized by incubating in diaminobenzidine solution according to the manufacture’s directions (all from Vector Laboratories, Burlingame, CA) and analyzed with the Quantimet 570C densitometer .
All sections were processed under the same standardized conditions. For each mouse, a total of 3 sections were analyzed and for each section, 4 fields in the specific brain region were examined. Results for all images were averaged and expressed as the percentage area covered by immunoreactive dendrites in the case of MAP2, as number of positive neurons per square millimeter in the case of TH, NeuN and PCNA, and as corrected optical density in the case of GFAP.
Luciferase reporter gene activity assay
Cells were seeded in 6-well plates, allowed to adhere overnight, and then co-transfected in each well with 1.5 μg of p53-responsive luciferase reporter plasmid DNA (kindly donated by Dr. Stephen H. Safe, Department of Veterinary Physiology and Pharmacology at Texas A&M University) and 0.5 μg of Renilla luciferase cDNA; in some experiments1 μg of wild-type human parkin cDNA construct was also transfected. The total amount of plasmid DNA per transfection was kept constant (3 μg per well) by balancing the total transfected DNA amount with empty vector pcDNA3 (Invitrogen). After incubation for 48 hours, cells were exposed to various protocols, according to the specific experimental design. Approximately 72 hours after transfection, cells were lysed in 1x reporter lysis buffer (Promega), 20 μl of cell extract was placed in each well of a 96-well plate, and firefly and Renilla luciferase activity were measured in a luminometer with a dual-luciferase reporter assay system following the manufacturer’s instructions (Promega). Firefly luminescence was normalized to the Renilla luminescence signal.
Protein extraction and western blot analyses
Cultured cells were harvested and lysed in RIPA buffer (50 mM Tris–HCl pH 8.0, 150 mM NaCl, 1% Nonidet P-40, 0.5% deoxycholate, 0.1% sodium dodecyl sulphate) for 10 min at 4°C. Protein concentrations were determined using the Bio-Rad protein assay dye according to the manufacturer’s specifications. For immunoblotting, typically 5–30 μg of proteins were resolved on 4-12% polyacrylamide NuPAGE-MES gels (Invitrogen) and then wet-transferred to PVDF membranes (Millipore). Membranes were blocked in 2% skim milk in Tris-buffered saline (TBS) containing 1% Tween-20 (TBS-T). Immunoblotting was performed using mouse anti-parkin monoclonal antibody (1:1,000; MAB5512, Millipore), rabbit anti-p53 polyclonal antibody (1:1,000; 9282, Cell Signaling), mouse anti-p53 monoclonal antibody (1:1,000; SC-99, Santa Cruz), mouse monoclonal anti-β-actin antibody (1:5,000; MAB1501, Millipore), or rabbit polyclonal anti-β-actin antibody (1:5,000; 4967, Cell Signaling) for mouse samples. For cell line experiments, immunological complexes were detected with goat anti-mouse (1:3,000; Jackson) or goat anti-rabbit (1:3,000; BioRad) IgG secondary antibodies coupled to peroxidase, followed by electrochemiluminescence using the Super Signal substrate kit (Thermo Scientific) as directed by the manufacturer. For human and mouse brain lysates, goat anti-mouse at 680 LT (1:20,000; Li-Cor) or goat anti-rabbit at 800 CW (1:15,000; Li-Cor) IR-dye-conjugated secondary antibodies were used, followed by infrared analysis with Odyssey (Li-Cor). When p53 was analysed in the human neuroblastoma cell line, polyclonal antibody anti-p53 was used. In human and mouse brain samples, p53 was detected using the monoclonal antibody SC-99 (Santa Cruz).
Densitometric analysis of protein bands was performed using Adobe Photoshop. All values were normalized to their respective loading controls.
Biotin-switch assay for detection of S-nitrosylated proteins
Analysis of S-nitrosylated parkin (SNO-parkin) with the biotin-switch assay was performed as previously described [26, 68, 69]. Briefly, fresh brain tissue samples were homogenized in HEN buffer, pH 7.2 (100 mM Hepes, 1 mM EDTA, 0.1 mM neocuproine) with 1% Triton X-100 and 0.1% sodium dodecyl sulfate. A total of 1 mg of protein per sample was used for the assay. Free thiol groups were blocked by incubation with 20 mM methyl methanethiol-sulfonate (MMTS; Aldrich) for 30 min at 50°C. Cell extracts were then precipitated with acetone and resuspended in HEN buffer with 1% SDS. S-Nitrosothiol groups were then selectively reduced by 75 mM ascorbate to free thiols, which were subsequently biotinylated with 4 mM N-[6-(biotinamido)hexyl]-3′-(2′-pyridyldithio)-propionamide (biotin-HPDP; Soltec Ventures). The biotinylated proteins were pulled down with streptavidin-agarose beads and analyzed by immunoblotting. Total protein as a loading control was quantified by standard immunoblot analysis. Results were expressed as SNO-parkin relative to total parkin for each sample.
Immunocytochemistry and nuclear staining
Cell cultures were fixed in 4% paraformaldehyde in PBS for 10 min, and then permeabilized with 0.1% Triton X-100 and blocked with 5% goat serum for 30 min. Mouse anti-myc (46–0603, Invitrogen), mouse anti-parkin (MAB5512, Millipore) or rat anti-dopamine transporter (AB5990, Abcam) monoclonal antibodies were used at 1:500 dilution (4°C overnight incubation). Goat anti-mouse FITC- (115-095-166, Jackson ImmunoResearch) or goat anti-rat rhodamine red-conjugated (112-295-075, Jackson ImmunoResearch) secondary antibodies were used for fluorescence labeling at a dilution of 1:500 (RT, 1 hour). Nuclei were detected by co-staining with 12 μM Hoechst 33342 (Molecular Probes) for 30 min. Coverslips were mounted for immunofluorescence analysis using Fluoro Gel mounting medium (Electron Microscopy Sciences), and analysis of samples was performed on a deconvolution epifluorescence microscope (Axio, Carl Zeiss Microimaging GmbH).
Terminal-deoxynucleotidyl-transferase dUTP nick end labeling (TUNEL) assay
For neuroblastoma cultures, cells were collected and TUNEL assay was performed in suspension. For primary neuronal cultures, TUNEL assay was performed on coverslips. Briefly, cells were fixed with 4% paraformaldehyde in PBS for 10 min, and then permeabilized with 0.1% Triton X-100 and 0.1% sodium citrate for 2 min. Apoptotic cells were labeled using an in situ cell death detection kit (Roche) according to the manufacturer’s instructions. Coverslips were mounted using Fluoro Gel mounting medium, and apoptotic cells were observed and quantified on a deconvolution epifluorescence microscope.
RNAi-mediated knockdown of p53
psiRNA vector expressing short hairpin RNA for efficiently targeting and silencing human p53 gene (shRNA-p53) was purchased from InvivoGen (psirna42-hp53). Briefly, shRNA-p53 was produced with the psiRNA-h7SKGFPzeo plasmid from the human 7SK RNA pol III promoter, featuring a GFP-Zeo fusion gene for monitoring transfection efficiency. psiRNA plasmid targeting luciferase-GL3, whose sequence was not found in the mouse, human or rat genome databases [70, 71], was used as a non-targeting control (shRNA-ctrl; InvivoGen). shRNA vectors were amplified in competent E. coli GT116 cells according to the manufacturer’s instructions. Transfection of neuroblastoma cells with shRNAs was performed in 6-well plates using Lipofectamine LTX and Plus Reagent (Invitrogen). A total of 3 μg of plasmid DNA per well was used for each transfection.
Recombinant protein expression and purification
Recombinant GST-tagged parkin protein was expressed and purified as previously described .
Chromatin immunoprecipitation (ChIP) assay
ChIP assays were performed using the ChIP-IT EXPRESS assay kit protocol (Active Motif, USA). Briefly, 1 × 107 SH-SY5Y cells that had been transfected with pcDNA or parkin were exposed to 200 μM GSNO or GSH (as a control) for 4 hours. Cells were then cross-linked with 1% formaldehyde at room temperature for 10 min, washed with PBS and lysed, followed by centrifugation at 2400 × g. The nuclear pellet was resuspended in shearing buffer and sonicated with 8 pulses of 20 s each to shear DNA into 200–1000 bp fragments. Ten percent of the mixture containing protein/DNA complexes was used for “input DNA” analysis. An equal amount of the protein/DNA complex mixture was then incubated at 4°C overnight with magnetic beads and control IgG or an anti-parkin antibody (MAB5512, Chemicon International, USA). Immunoprecipitated DNA was then eluted from the magnetic beads and the cross-linking was reversed. Input and ChIP DNA were analyzed using quantitative real-time PCR. The human p53 promoter region was amplified using forward primer 5′-CTCCAAAATGATTTCCACCAA-3′ and reverse primer 5′-GGAAGCAAAGGAAATGGAGTT-3′ for determination of parkin binding. For quantitative ChIP, the PCR was performed using EXPRESS SYBR GreenERTM (Invitrogen, USA) on the Stratagene Mx3000P Q-PCR system. Levels of enrichment (n-fold) were calculated using the comparative cycle threshold method.
Electrophoretic mobility shift assay (EMSA)
A synthetic oligonucleotide covering the region of the human p53 promoter (Pp53 forward and reverse; see below)  labeled with biotin at the 5′ end was obtained from IDT Integrated DNA Technologies and subsequently annealed. For detection of binding of parkin to p53, 1 μM recombinant, wild-type parkin was incubated with 0.1 nM biotin-labeled DNA oligonucleotide. The specificity of the reaction was verified by incubation of Pp53 with increasing concentrations of recombinant parkin, typically from 100 nM to 1 μM. Binding reactions were performed at room temperature for 20 min in 1x binding buffer (Panomics). Then, protein-DNA complexes were resolved by electrophoresis on 6% polyacrylamide Novex DNA retardation gels (Invitrogen) at 100 V in 0.5x TBE buffer, pH 8.3 (90 mM Tris–HCl, 90 mM boric acid, 2 mM EDTA), and wet-transferred to biodyne B nylon membranes (Pall Life Sciences). Oligonucleotides were fixed to the membrane by UV crosslinking at 254 nm for 5 min. After blocking the membrane with 1x blocking buffer (Thermo Scientific), biotinylated oligonucleotides were detected by incubation with stabilized streptavidin-horseradish peroxidise conjugate (Thermo Scientific), followed by electrochemiluminescence analysis using a substrate kit (Thermo Scientific) as directed by the manufacturer.
Pp53 forward: 5′-GGCACCAGGTCGGCGAGAATCCTGACTCTGCACCCTCCTCCC CAACTCCATTTCCTTTGCTTCCTCCGGC-3′.
Pp53 reverse: 5′-GCCGGAGGAAGCAAAGGAAATGGAGTTGGGGAGGAGGGTGC AGAGTCAGGATTCTCGCCGACCTGGTGCC-3′.
Data were quantified and stored in Excel software format (Microsoft, Redmond, WA). Graphs depicting data from quantitative analyses were generated with PRISM software (GraphPad Software). All experimental points are expressed as mean ± SEM. Statistical differences among different groups were determined by Student’s t-test (for single comparisons) or ANOVA (for multiple comparisons) using PRISM software.