Chemicals and reagents
RPMI, neurobasal medium, B27 supplement, fetal bovine serum, l-glutamine, Sytox assay dye, IR-dye-tagged secondary antibodies, penicillin, streptomycin and other cell culture reagents were purchased from Invitrogen (Gaithersburg, MD, USA). Recombinant rat TNF, LPS (Escherichia coli 0111:B4) and cytosine arabinoside were purchased from Sigma-Aldrich (St Louis, MO, USA). Recombinant murine TNF and the tumor necrosis factor receptor 1 (TNFR1) neutralizing antibody were from R&D Systems (Minneapolis, MN, USA). Etanercept (Enbrel) was purchased from Amgen, Inc. (Thousand Oaks, CA, USA). Antibodies for rabbit PKCδ and caspase-8 were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Tyrosine hydroxylase (TH) antibody was purchased from Chemicon (Temecula, CA, USA) and microtubule-associated protein 2 (MAP-2) antibody from Cell Signaling Technologies (Beverly, MA, USA). 32P-ATP was purchased from Perkin Elmer (Boston, MA, USA) and the AMAXA Nucleofector kit from Lonza (Basel, Switzerland). Caspase assay substrates and inhibitors were purchased from MP Biomedicals (Solon, OH, USA). The DNA fragmentation assay kit was purchased from Roche Applied Science and the Bradford protein assay kit was purchased from Bio-Rad Laboratories (Hercules, CA, USA).
Culture and treatment paradigm for rat dopaminergic N27 cells
The development and culture conditions of the N27 clonal dopaminergic cell line have been described previously [21, 24, 25]. Similar culture conditions were used in this study. Briefly, cells were cultured in RPMI 1640 medium containing 10 % heat inactivated fetal bovine serum, 2 mM l-glutamine, penicillin (100 units/ml), and streptomycin (100 μg/ml). Cells were maintained in a humidified atmosphere of 5 % CO2 at 37°C. RPMI medium containing 2 % fetal bovine serum was used for the TNF treatment. Cells were washed twice in 2 % RPMI serum and then treated with the indicated doses of recombinant rat TNF.
Primary mouse mesencephalic neuron cultures
Primary neurons were cultured from ventral mesencephalon tissue of gestational 14-day (E14) mouse embryos, as described previously [21, 26] with some modifications. The ventral mesencephalon was dissected under a microscope and collected in ice-cold Dulbecco’s modified Eagle medium F-12 complete medium (DMEM-F12 supplemented with 10 % heat-inactivated fetal bovine serum (FBS), 50 U/mL penicillin, 50 μg/mL streptomycin, 2 mM l-glutamine, 100 μM non-essential amino acids, and 2 mM sodium pyruvate). The tissue was then dissociated using trypsin-ethylenediaminetetra-acetic acid (EDTA) (0.25 %) for 15 minutes at 37°C. Trypsinization was stopped by adding an equal volume of DMEM-F12 complete medium and dissociated tissue was washed in the same medium to remove residual trypsin. The DMEM-F12 medium was aspirated out and the tissue triturated in neurobasal medium containing B-27 antioxidant supplement, 500 μM l-glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin. After a single cell suspension was obtained, cells were passed through a 70 μm nylon mesh cell strainer to remove tissue debris and aggregates. Cells were counted using a Beckman Coulter ViCell XR automated cell counter and then plated at an equal density (0.8 × 106 cells per well) in 24 well plates containing coverslips precoated with poly-d-lysine (100 μg/ml). Cultures were maintained in neurobasal medium with B-27 antioxidant supplements and cytosine arabinoside (5 μM) was added to inhibit glial proliferation. Cultures were grown in a humidified CO2 incubator (5 % CO2, 37°C) and the medium was changed every 2 days. Approximately 4-day-old to 5-day-old cultures were used for treatment. The neuronal cultures were verified to be around 98 % free of glial cells at the time of treatment by using immunocytochemistry for MAP-2, glial fibrillary acidic protein (GFAP) and ionized calcium binding adaptor molecule 1 (iba1) as markers of neurons, astrocytes and microglia, respectively . For TNF treatment, cultures were switched to serum-free neurobasal medium without antioxidant supplements and treated for 48 h. Recombinant murine TNF (30 ng/ml) was added at the beginning of the treatment and re-added again 24 h later. At the end of the treatment, primary cultures were processed for TH immunocytochemistry and neurotransmitter uptake assays.
DNA fragmentation and Sytox assays
DNA fragmentation was quantified using the Cell Death Detection ELISA Plus assay kit (Roche Applied Science), as described in our previous publications [21, 22, 24]. This highly sensitive and reliable assay detects and quantifies early changes in apoptosis based on the amounts of histone-associated low molecular weight DNA released into the cytoplasm of cells. Briefly, N27 cells were plated in six-well plates at 0.8 × 106 cells/well and treated the next day with TNF for 16 h. Cells were collected using a cell scraper and centrifuged at 400 × g for 5 minutes. The cells were gently lysed using the lysis buffer provided with the kit. The lysate then was spun down again at 200 g for 10 minutes to pellet the nuclear fraction, and the supernatant was collected and used to measure DNA fragmentation according to the manufacturer's instructions for the ELISA protocol. The absorbance at 405 nm was measured against an 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS) solution as a blank (reference wavelength approximately 490 nm) using a Synergy-2 Multi-Mode Microplate Reader (BioTek Instruments, Inc). The absorbance values were normalized to the amount of protein present in the lysates and the data expressed as percent control. The Sytox cytotoxicity assay was performed using the Sytox green dye (Molecular Probes). The assay is based on the principle that live cells with intact plasma membranes can exclude the Sytox dye, which selectively enters cells with a compromised membrane and emits bright green fluorescence on binding to DNA . N27 cells were grown in 24 well plates and 1 μM of the Sytox dye was added at the time of treatment. Cells were treated for 16 h with TNF and pretreated with etanercept. The increase in green fluorescence, as a result of TNF cytotoxicity, was measured using a fluorescence microplate reader (Spectramax Gemini, Molecular Devices) at an excitation of 485 nm and 538 nm emission. Phase contrast and fluorescence images of matching fields were captured on the same cells to visualize the toxicity in N27 cells.
Caspase-3 enzymatic activity assays
Enzymatic assays for caspase-3 activity were performed as described previously [22, 24] using acetyl-DEVD-amino-4-trifluoromethylcoumarin (Ac-DEVD-AFC, 25 μm) as the fluorometric substrate for the reaction. N27 cells were treated with TNF (30 ng/ml) for 6 h or pretreated with etanercept (5 μg/ml) for 30 minutes. Following treatment, 100 μl of cell extract was incubated with 5 μl of the fluorescence substrate and then incubated at 37°C for 1 h while being protected from light. The fluorescence signal generated upon cleavage of the AFC peptide substrate by caspase-3 was measured at 510 nm with an excitation of 400 nm using a Synergy-2 Multi-Mode microplate reader. Protein concentrations were determined using the Bradford assay. Raw values were normalized using protein concentrations and expressed as percent control.
Confocal immunofluorescence microscopy for PKCδ translocation
N27 dopaminergic cells were plated on poly-d-lysine (100 μg/ml) coated coverslips in 24 well plates at 0.2 × 106 cells per well. The next day, the cells were treated with 30 ng/ml of TNF for 6 h in 2 % serum RPMI medium. Cells were fixed in 4 % paraformaldehyde for 20 minutes and washed five times in PBS. The cells were blocked and permeabilized with blocking buffer (5 % goat serum, 0.2 % Triton X, and 0.05 % Tween-20 in PBS) for 1 h and then incubated overnight at 4°C with a rabbit polyclonal PKCδ antibody (1:1,000) that recognizes a C-terminal epitope present in both the native and the proteolytically cleaved protein. The cells were then washed five times in PBS and then incubated with secondary antibody (1:2,000, Alexa 488 goat anti-rabbit) for 1 h at room temperature. Negative controls for non-specific staining that contained secondary antibody alone were used on parallel wells to ensure specificity of the fluorescent signals obtained. The cells were then washed five times in PBS and the nucleus was labeled using the TOPRO 3 dye. The cells were then washed three more times in PBS and coverslips were mounted using the ProLong Gold antifade reagent (Molecular Probes). Confocal images were acquired with a Nikon EZ-C1 confocal system using the 488 nm and 633 nm lasers to visualize PKCδ and TOPRO 3, respectively. Fluorescence spatial intensity plots along the XY plane for PKCδ localization (green channel) and the nucleus (blue channel) were generated using the Nikon EZC1 software to show nuclear translocation.
Fluorescent Western blotting
Lysates from N27 cells and brain tissue were prepared using a modified radio immunoprecipitationassay (RIPA) buffer and normalized for equal amounts of protein using the BCA protein assay (Pierce Biotechnology). Gel loading dye was added to the lysates and stored in aliquots at −80°C. Equal amounts of protein (30 to 60 μg) were loaded for each sample and separated on either 12 % or 15 % SDS-PAGE gels, depending on the molecular weight of the target protein. After separation, the proteins were transferred to nitrocellulose membranes and non-specific binding sites were blocked by incubating the membranes in fluorescent Western blocking buffer (Rockland Immunochemicals) for 1 h, and then they were probed with primary antibodies overnight at 4°C. Primary antibodies used were rabbit polyclonal PKCδ (1:500), mouse monoclonal TH (1:2,000), rabbit polyclonal caspase-8 (1:200). β-Actin (1:5,000) was used as the loading control. After incubation, membranes were washed three times with PBS containing 0.05 % Tween and IR-dye tagged secondary antibodies (1:5,000; Molecular Probes) were added. Membranes were visualized on the Odyssey infra-red imaging system.
PKCδ immunoprecipitation (IP)-kinase assays
The PKCδ enzymatic kinase activity assay was performed as described previously [21, 22, 29]. Briefly, N27 cells or substantia nigra tissues were washed in ice-cold PBS and then resuspended in a mild RIPA lysis buffer containing protease and phosphatase inhibitor cocktail (Pierce Biotechnology). The lysates were placed on ice for 20 minutes, sonicated gently and centrifuged at 13,000 rpm for 45 minutes. The supernatant was collected and protein concentration was determined using the Bradford assay . All samples were made up to a concentration of 2 μg/ml, and 500 μg of total protein in a 250 μl volume was immunoprecipitated overnight at 4°C using 5 μg of the PKCδ antibody. The next day, the protein A-agarose beads (Sigma-Aldrich) were added and the samples were incubated for 1 h at room temperature. The protein A-bound antibody complexes were then washed three times in 2 × kinase assay buffer (40 mM Tris, pH 7.4, 20 mM MgCl2, 20 μM ATP, and 2.5 mM CaCl2), and then resuspended in 40 μl of the same buffer. The kinase reaction was started by adding 40 μl of the reaction buffer containing 0.4 mg of histone H1, 50 μg/ml phosphatidylserine, 4 μM dioleoylglycerol, and 10 μCi of [γ-32P] ATP at 3,000 Ci/mM to the immunoprecipitated samples. The samples were incubated for 10 minutes at 30°C. The kinase reaction was stopped by adding 2 × SDS loading buffer and boiling the samples for 5 minutes. The proteins were separated on a 12 % SDS-PAGE gel and the phosphorylated histone H1 bands were scanned using a Fujifilm FLA 5000 imager. Image analysis and band quantification were performed using the Fujifilm Multigauge software package (Fujifilm USA, Stamford, CT).
Transfection with siRNA and cleavage-resistant PKCδD327A-CRM mutant
Design and synthesis of PKCδ siRNA are described in our previous publications [21, 31]. N27 cells were transfected with 40 to 50 nM of either PKCδ or scramble siRNA duplexes using the AMAXA Nucleofector kit, according to the manufacturer’s instructions. Transfected cells were counted using a Vi-Cell XR automated cell counter and seeded at equal density (0.5 × 106 cells/well) into six-well plates. The cells were treated 24 h after transfection to allow for optimal knockdown of gene expression. At the end of the treatment, cells were collected and used for the DNA fragmentation assay as described above. For studies with the PKCδ cleavage-resistant mutant (PKCδD327A-CRM), stable N27 cell lines overexpressing either the mutant PKCδD327A protein or the β-galactosidase (Lac Z) protein as the vector control were prepared as described in our publications . Both cell lines were cultured in the presence of blasticidin for three passages before treatment. Expression of the PKCδ CRM-mutant and the Lac Z vector control at the time of treatment was confirmed by staining for the V5-epitope. Cells were plated at equal density (0.7 × 106 cells per well) in six-well plates and allowed to grow overnight. The next day, cells were treated with 30 ng/ml of TNF for 16 h and processed for the DNA fragmentation assay.
Dopamine uptake assays
Uptake assays for tritiated dopamine on primary EVM cultures were performed according to previously published protocols [17, 33, 34]. After TNF treatment, the primary neuron cultures from PKCδ wild type (+/+) and knockout (−/−) mice were washed three times with 0.5 ml of warm Krebs-Ringer buffer (16 mM sodium phosphate, 120 mM NaCl, 4.7 mM KCl, 1.8 mM CaCl2, 1.2 mM MgSO4, 1.3 mM EDTA, and 5.6 mM glucose; pH 7.4). The cultures were then incubated with 5 μM 3 H]-dopamine (30 Ci/mmol) in Krebs-Ringer buffer at 37°C for 20 minutes. The cells were then washed three times with ice-cold Krebs-Ringer buffer and collected in 1 ml of 1 N NaOH. Scintillation fluid was added to a total volume of 5 ml and radioactivity counts were measured using a Tri-Carb Liquid Scintillation counter (Packard). In parallel wells, the nonspecific uptake of dopamine was determined by incubation with 10 μM mazindol. The nonspecific uptake values were subtracted to obtain the data for high affinity neurotransmitter uptake. Data from dopamine uptake studies were expressed as percent control.
TH-positive cell counts and immunofluorescence
After TNF treatment, primary neuron cultures from PKCδ wild type (+/+) and PKCδ knockout (−/−) mice were fixed in 4 % paraformaldehyde and permeabilized with PBS containing 2 % bovine serum albumin (BSA), 0.2 % Triton X-100 and 0.05 % Tween-20. Blocking buffer (PBS with 2 % BSA) was added for 1 h at room temperature and primary antibodies for TH (1:1,000) and MAP-2 (1:1,000) were diluted in blocking buffer and incubated overnight at 4°C. The next day, cells were washed in PBS four times and incubated with appropriate Alexa-dye conjugated secondary antibodies for 1 h at room temperature. After several washes, the samples were counterstained with Hoechst to label the nucleus, and coverslips were mounted using the Prolong Antifade (Molecular Probes) mounting medium. Images were acquired using a Nikon inverted fluorescence microscope (model TE-2000U) equipped with a SPOT digital camera system (Diagnostic Instruments, Sterling Heights, MI, USA). Image analysis was performed using the Metamorph software package (Universal Imaging Systems, PA, USA).
Stereotaxic infusion of LPS into the mouse substantia nigra
A single dose LPS injection model that was previously described  was used to induce delayed, progressive loss of dopaminergic neurons in the substantia nigra. C57/BL/6 mice (8 weeks old, n = 6 per group) were anesthetized with a mixture of ketamine-xylazine (100 mg/kg, 10 mg/kg) and carefully immobilized on a stereotaxic apparatus (Benchmark Angle One, Leica Microsystems). The skin above the skull was prepped with alcohol and an incision was made to expose the skull. A single burr hole was carefully drilled at the injection site for the right SN and ophthalmic gel was used to protect the eyes. The stereotaxic coordinates for the injection site were anteroposterior (AP) -3.3 mm, mediolateral (ML) -1.2 mm, dorsoventral (DV) -4.6 mm from bregma . A stainless steel cannula attached to a 5 μl Hamilton syringe was carefully inserted into the hole drilled at the injection site and a single dose of 5 μg LPS in a 1 μl volume was injected at the rate of 0.5 μl per minute. The needle was left in place for another 5 minutes to prevent retrograde flow of liquid along the needle track. Control mice were injected in an identical manner with equal volumes of saline. After surgery, the skin was sutured and carefully held in place using sterile, non-pyrogenic stainless steel clips (Autoclip Wound Closing System, Braintree Scientific, Inc). Mice were allowed to recover on a heating pad (Braintree Scientific, Inc) and were carefully monitored through recovery from anesthesia. All animal procedures were approved by the Iowa State University Institutional Animal Care and Use Committee (IACUC).
Data analysis was performed using the Prism 4.0 software package (GraphPad Software, San Diego, CA, USA). The data were first analyzed using one-way analysis of variance (ANOVA) and then Bonferroni’s post-test was performed to compare all treatment groups. Differences of P <0.05 were considered statistically significant. The Student’s t test was used when differences between two groups were being compared.