- Open Access
Proteolytic activation of proapoptotic kinase protein kinase Cδ by tumor necrosis factor α death receptor signaling in dopaminergic neurons during neuroinflammation
© Gordon et al.; licensee BioMed Central Ltd. 2012
- Received: 12 November 2011
- Accepted: 27 April 2012
- Published: 27 April 2012
The mechanisms of progressive dopaminergic neuronal loss in Parkinson’s disease (PD) remain poorly understood, largely due to the complex etiology and multifactorial nature of disease pathogenesis. Several lines of evidence from human studies and experimental models over the last decade have identified neuroinflammation as a potential pathophysiological mechanism contributing to disease progression. Tumor necrosis factor α (TNF) has recently emerged as the primary neuroinflammatory mediator that can elicit dopaminergic cell death in PD. However, the signaling pathways by which TNF mediates dopaminergic cell death have not been completely elucidated.
In this study we used a dopaminergic neuronal cell model and recombinant TNF to characterize intracellular signaling pathways activated during TNF-induced dopaminergic neurotoxicity. Etanercept and neutralizing antibodies to tumor necrosis factor receptor 1 (TNFR1) were used to block TNF signaling. We confirmed the results from our mechanistic studies in primary embryonic mesencephalic cultures and in vivo using the stereotaxic lipopolysaccharide (LPS) model of nigral dopaminergic degeneration.
TNF signaling in dopaminergic neuronal cells triggered the activation of protein kinase Cδ (PKCδ), an isoform of the novel PKC family, by caspase-3 and caspase-8 dependent proteolytic cleavage. Both TNFR1 neutralizing antibodies and the soluble TNF receptor Etanercept blocked TNF-induced PKCδ proteolytic activation. Proteolytic activation of PKCδ was accompanied by translocation of the kinase to the nucleus. Notably, inhibition of PKCδ signaling by small interfering (si)RNA or overexpression of a PKCδ cleavage-resistant mutant protected against TNF-induced dopaminergic neuronal cell death. Further, primary dopaminergic neurons obtained from PKCδ knockout (−/−) mice were resistant to TNF toxicity. The proteolytic activation of PKCδ in the mouse substantia nigra in the neuroinflammatory LPS model was also observed.
Collectively, these results identify proteolytic activation of PKCδ proapoptotic signaling as a key downstream effector of dopaminergic cell death induced by TNF. These findings also provide a rationale for therapeutically targeting PKCδ to mitigate progressive dopaminergic degeneration resulting from chronic neuroinflammatory processes.
- dopaminergic degeneration
- Parkinson’s disease
- proteolytic activation
- nuclear translocation
Parkinson’s disease (PD) is a debilitating neurodegenerative movement disorder affecting around 2 % of the population over the age of 60 [1, 2]. The key pathological hallmark of the disease is a loss of dopaminergic (DA) neurons in the substantia nigra of the midbrain, resulting in a depletion of striatal dopamine that is clinically manifest as a range of motor and non-motor deficits . Recent studies indicate that a sustained neuroinflammatory response is initiated in the substantia nigra pars compacta (SNpc) during early stages of dopaminergic degeneration, and remains evident in postmortem PD brains with increased microgliosis, dystrophic astrocytes and lymphocyte infiltration [4–6]. Compelling evidence over the last decade from animal models, in vitro studies and PD patients has demonstrated that the protracted neuroinflammation that occurs in the SNpc can exacerbate the degeneration of vulnerable dopaminergic neurons. However, the molecular mediators and mechanisms underlying the neuroinflammatory processes remain to be established.
Tumor necrosis factor α (TNF) has recently emerged as an important neuroinflammatory mediator linked to dopaminergic degeneration in PD. Increased levels of TNF are present in the SNpc and the CSF of postmortem PD patients, and genetic polymorphisms in the TNF gene locus have been linked to the development of PD [7–12]. Increased TNF is also well documented in rodent and nonhuman primate models of PD induced by neurotoxic insults and in transgenic models [13, 14]. Importantly, ablation of TNF signaling using knockout mice for TNFα or its receptors has been shown to attenuate 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced dopaminergic degeneration [15, 16]. Furthermore, genetic or pharmacological inhibition of soluble TNF signaling with dominant negative mutants is protective against 6-hydroxydopamine and lipopolysaccharide (LPS)-induced dopaminergic degeneration in vivo [17, 18]. Conversely, the chronic expression of low levels of TNF in the substantia nigra of rats induced by a viral vector causes progressive dopaminergic degeneration with delayed motor deficits . Together, these independent findings strongly implicate TNF in the progressive loss of dopaminergic neurons in PD. However, the signaling mechanisms by which TNF can mediate dopaminergic degeneration have not been characterized.
Protein kinase Cδ (PKCδ), a member of the novel PKC isoform family, is highly expressed in nigral dopaminergic neurons . Recent studies from our laboratory and others have shown that PKCδ is proteolytically activated by caspase-3 during dopaminergic cell death and that genetic or pharmacological targeting of PKCδ can protect against dopaminergic degeneration in PD models [21–24]. In this paradigm, increased oxidative stress in dopaminergic neurons results in proteolytic activation of PKCδ by caspase-3 downstream of the intrinsic mitochondrial apoptotic pathway. The proteolytic cleavage of PKCδ by caspase-3 persistently activates the kinase as an effector of dopaminergic cell death. However, PKCδ activation by extrinsic neuroinflammatory mediators such as TNF has not been studied and would be relevant to the progression of PD by neuroinflammatory mechanisms. Since TNF also activates caspase-3 by signaling through its death receptors, we determined if PKCδ is proteolytically activated during TNF-induced dopaminergic cell death and in the mouse substantia nigra (SN). Our findings herein identify PKCδ as a key downstream target of TNF death receptor signaling in dopaminergic neurons and demonstrate a novel link between neuroinflammatory mechanisms and progressive dopaminergic degeneration.
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.
TNF is neurotoxic to dopaminergic N27 cells and activates caspase-8 and caspase-3
TNF induces proteolytic activation of protein kinase Cδ in dopaminergic neuronal cells
Proteolytic activation of PKCδ by TNF signaling activates the kinase
PKCδ translocates to the nucleus during TNF-induced cell death
Overexpression of a cleavage-resistant mutant (PKCδD327A-CRM) or siRNA knockdown of PKCδ protects against TNF-induced dopaminergic cell death
Primary dopaminergic neurons obtained from PKCδ knockout (−/−) mice are protected from TNF neurotoxicity
PKCδ is activated by proteolytic cleavage in the mouse substantia nigra following stereotaxic infusion of LPS
The results from our mechanistic studies using N27 rat dopaminergic cells indeed demonstrate for the first time that soluble TNF induces a time-dependent and dose-dependent proteolytic cleavage of PKCδ by a caspase-8 and caspase-3 signaling pathway, indicative of canonical TNF death receptor signaling via caspase-8. Using the anti-TNF drug Etanercept and neutralizing antibodies to the TNFR1 (p55) death receptor, we show that proteolytic cleavage of PKCδ by soluble TNF is dependent on TNFR1 signaling and is accompanied by a substantial increase in PKCδ kinase activity (Figure 3), consistent with proteolytic activation. Previous reports from our laboratory and other independent research groups have shown that PKCδ proteolytic activation contributes to apoptosis induced by diverse neurotoxic stimuli that converge on the intrinsic mitochondrial apoptotic pathway primarily through intracellular oxidative stress mechanisms [21–24, 31, 32, 54–56]. Taken together, our present results suggest that proteolytic activation of PKCδ could be a common effector of cell death via both intrinsic and extrinsic cascades in dopaminergic neurons.
Rapid and dynamic translocation to distinct subcellular locations following activation is a characteristic feature of PKCδ in different cell types, and typically delineates the unique functional role of this kinase . Various substrates have been identified as targets of PKCδ phosphorylation in different subcellular locations that participate in a range of multiple cellular functions, from apoptosis to cell migration and activation of immune cells . In particular, studies have demonstrated that nuclear translocation of PKCδ is required for induction of apoptosis and is driven by a bipartite nuclear localization sequence at its C-terminus [48, 49, 59]. Our confocal immunofluorescence experiments reveal a distinct localization of PKCδ to the nucleus 6 h after TNF treatment, the time point at which maximum proteolytic cleavage of PKCδ was evident, consistent with its proapoptotic function (Figure 4). While intense PKCδ staining is evident throughout the nucleus in TNF-treated cells, there is also a distinct pattern of accumulation of PKCδ at specific sites around the perinuclear region and within the nucleus. Interestingly, in a previous study, TNF superfamily ligands, such as BAFF (B cell-activating factor belonging to the TNF family), have been shown to prevent nuclear accumulation of full length PKCδ and thus prolong cell survival in B cells . The substrates of PKCδ and the functional significance of nuclear localization of the kinase during neuroinflammation are currently under investigation in our laboratory. However, the kinetics of PKCδ proteolytic activation and its translocation to the nucleus observed in our present study, both indicate of a proapoptotic role for PKCδ downstream of TNF. Indeed, suppressing total PKCδ protein levels using siRNA or blocking proteolytic activation using a PKCδ cleavage-resistant mutant (PKCδD327A-CRM) affords robust protection against TNF neurotoxicity (Figure 5). The results from our mechanistic studies are further supported by our experiments in primary mesencephalic neuronal cultures treated with soluble TNF, which induces degeneration of dopaminergic neurons in these cultures [17, 26]. Although TNF was less toxic to primary dopaminergic neurons than the classical Parkinsonian neurotoxicant 1-methyl-4-phenylpyridinium (MPP+) , we observed a substantial reduction in TH neuron numbers and functional impairment as determined by dopamine uptake studies in cultures from PKCδ wild type (+/+) mice (Figure 6). Importantly, dopaminergic neurons in mesencephalic cultures obtained from PKCδ knockout (−/−) mice were protected against TNF toxicity, indicating that PKCδ signaling is essential for dopaminergic degeneration triggered by TNF.
Evidence from previous studies [17, 26] and our own results here, indicate that TNF can induce dopaminergic neurotoxicity by a direct mechanism, possibly via the TNFR1 (TNFRSF1) death receptor, which has been shown to be highly expressed on these cells. However, additional lines of evidence suggest that TNF can also contribute to dopaminergic degeneration indirectly by potentiating microglial reactive oxygen species (ROS) and other neurotoxic mediators, which can increase local oxidative stress levels in the SNpc [19, 52, 61]. The elevated TNF levels in the SN in both animal models and human PD patients and the concomitant microglial activation indicate that potentiation of microglial neurotoxic responses by TNF likely occurs in vivo, although it may not be the primary mechanism of dopaminergic neurotoxicity, as suggested by McCoy et al. using mixed neuron-glial cultures. A chronic increase in TNF levels in the nigrostriatal system may drive the progressive loss of compromised and vulnerable dopaminergic neurons in the pro-oxidant environment of the SNpc, both by TNF apoptotic signaling and by potentiation of microglial neurotoxic responses, including ROS and RNS production [17, 19, 26, 52, 62]. Nonetheless, our data demonstrating that PKCδ is proteolytically activated in the substantia nigra in the neuroinflammatory LPS model (Figure 7) of PD, implicate proapoptotic PKCδ signaling as a common downstream effector of dopaminergic cell death triggered by convergent neuroinflammatory mechanisms involving TNF and other neurotoxic mediators in the mouse brain.
Despite extensive research into the etiology of PD for several decades, a cogent basis for the vulnerability and extensive loss of SNpc dopaminergic neurons still remains elusive. The emerging consensus is that PD results from complex interactions between environmental, genetic and cellular processes that induce dopaminergic cell death over time [3, 63]. This highlights the multifactorial nature of the disease process and underscores the importance of identifying therapeutic targets that are relevant across these multiple mechanisms of disease pathogenesis. While oxidative stress, mitochondrial dysfunction and proteasomal impairment have been identified as causative factors that can trigger dopaminergic cell death; reactive microgliosis and dysregulated neuroinflammation can sustain this process and result in progressive neurodegeneration [6, 52, 53]. We previously showed that proteasomal impairment, mitochondrial dysfunction and oxidative stress induce proteolytic activation of PKCδ to drive dopaminergic cell death [24, 45, 64]. Our results herein suggest a crucial role for proapoptotic PKCδ signaling during dopaminergic cell death induced by extracellular TNF and neuroinflammatory mechanisms. Pharmacological targeting of PKCδ with kinase inhibitors such as rottlerin has shown efficacy in protecting against dopaminergic degeneration in vitro and in the MPTP mouse model of PD [21, 55]. Our results from this study provide a rationale for pharmacological targeting of PKCδ to mitigate neuroinflammatory events associated with progressive degeneration of dopaminergic neurons in PD. Taken together, our results herein suggest that proteolytic activation of PKCδ could be a common downstream target for multiple mediators of dopaminergic degeneration by both extrinsic and intrinsic cell death mechanisms, making it an attractive therapeutic target during the progressive phase of PD.
This study was supported by grants from the National Institutes of Health NS 65167, NS078247 and NS74443.
- Moore DJ, West AB, Dawson VL, Dawson TM: Molecular pathophysiology of Parkinson’s disease. Annu Rev Neurosci 2005, 28:57–87.View ArticlePubMedGoogle Scholar
- Schapira AH: Neurobiology and treatment of Parkinson’s disease. Trends Pharmacol Sci 2009, 30:41–47.View ArticlePubMedGoogle Scholar
- Obeso JA, Rodriguez-Oroz MC, Goetz CG, Marin C, Kordower JH, Rodriguez M, Hirsch EC, Farrer M, Schapira AH, Halliday G: Missing pieces in the Parkinson’s disease puzzle. Nat Med 2010, 16:653–661.View ArticlePubMedGoogle Scholar
- Hirsch EC, Hunot S: Neuroinflammation in Parkinson’s disease: a target for neuroprotection? Lancet Neurol 2009, 8:382–397.View ArticlePubMedGoogle Scholar
- Tansey MG, Goldberg MS: Neuroinflammation in Parkinson’s disease: its role in neuronal death and implications for therapeutic intervention. Neurobiol Dis 2010, 37:510–518.View ArticlePubMedGoogle Scholar
- Glass CK, Saijo K, Winner B, Marchetto MC, Gage FH: Mechanisms underlying inflammation in neurodegeneration. Cell 2010, 140:918–934.View ArticlePubMedPubMed CentralGoogle Scholar
- Bialecka M, Klodowska-Duda G, Kurzawski M, Slawek J, Gorzkowska A, Opala G, Bialecki P, Sagan L, Drozdzik M: Interleukin-10 (IL10) and tumor necrosis factor alpha (TNF) gene polymorphisms in Parkinson’s disease patients. Parkinsonism Relat Disord 2008, 14:636–640.View ArticlePubMedGoogle Scholar
- Nishimura M, Mizuta I, Mizuta E, Yamasaki S, Ohta M, Kaji R, Kuno S: Tumor necrosis factor gene polymorphisms in patients with sporadic Parkinson’s disease. Neurosci Lett 2001, 311:1–4.View ArticlePubMedGoogle Scholar
- Wu YR, Feng IH, Lyu RK, Chang KH, Lin YY, Chan H, Hu FJ, Lee-Chen GJ, Chen CM: Tumor necrosis factor-alpha promoter polymorphism is associated with the risk of Parkinson’s disease. Am J Med Genet B Neuropsychiatr Genet 2007, 144B:300–304.View ArticlePubMedGoogle Scholar
- Boka G, Anglade P, Wallach D, Javoy-Agid F, Agid Y, Hirsch EC: Immunocytochemical analysis of tumor necrosis factor and its receptors in Parkinson’s disease. Neurosci Lett 1994, 172:151–154.View ArticlePubMedGoogle Scholar
- Nagatsu T, Mogi M, Ichinose H, Togari A: Changes in cytokines and neurotrophins in Parkinson’s disease. J Neural Transm Suppl 2000, 60:277–290.PubMedGoogle Scholar
- Mogi M, Harada M, Riederer P, Narabayashi H, Fujita K, Nagatsu T: Tumor necrosis factor-alpha (TNF-alpha) increases both in the brain and in the cerebrospinal fluid from parkinsonian patients. Neurosci Lett 1994, 165:208–210.View ArticlePubMedGoogle Scholar
- Barcia C, de Pablos V, Bautista-Hernandez V, Sanchez-Bahillo A, Bernal I, Fernandez-Villalba E, Martin J, Banon R, Fernandez-Barreiro A, Herrero MT: Increased plasma levels of TNF-alpha but not of IL1-beta in MPTP-treated monkeys one year after the MPTP administration. Parkinsonism Relat Disord 2005, 11:435–439.View ArticlePubMedGoogle Scholar
- Mogi M, Togari A, Tanaka K, Ogawa N, Ichinose H, Nagatsu T: Increase in level of tumor necrosis factor (TNF)-alpha in 6-hydroxydopamine-lesioned striatum in rats without influence of systemic L-DOPA on the TNF-alpha induction. Neurosci Lett 1999, 268:101–104.View ArticlePubMedGoogle Scholar
- Sriram K, Matheson JM, Benkovic SA, Miller DB, Luster MI, O’Callaghan JP: Mice deficient in TNF receptors are protected against dopaminergic neurotoxicity: implications for Parkinson’s disease. FASEB J 2002, 16:1474–1476.PubMedGoogle Scholar
- Ferger B, Leng A, Mura A, Hengerer B, Feldon J: Genetic ablation of tumor necrosis factor-alpha (TNF-alpha) and pharmacological inhibition of TNF-synthesis attenuates MPTP toxicity in mouse striatum. J Neurochem 2004, 89:822–833.View ArticlePubMedGoogle Scholar
- McCoy MK, Martinez TN, Ruhn KA, Szymkowski DE, Smith CG, Botterman BR, Tansey KE, Tansey MG: Blocking soluble tumor necrosis factor signaling with dominant-negative tumor necrosis factor inhibitor attenuates loss of dopaminergic neurons in models of Parkinson’s disease. J Neurosci 2006, 26:9365–9375.View ArticlePubMedPubMed CentralGoogle Scholar
- McCoy MK, Ruhn KA, Martinez TN, McAlpine FE, Blesch A, Tansey MG: Intranigral lentiviral delivery of dominant-negative TNF attenuates neurodegeneration and behavioral deficits in hemiparkinsonian rats. Mol Ther 2008, 16:1572–1579.View ArticlePubMedPubMed CentralGoogle Scholar
- De Lella Ezcurra AL, Chertoff M, Ferrari C, Graciarena M, Pitossi F: Chronic expression of low levels of tumor necrosis factor-alpha in the substantia nigra elicits progressive neurodegeneration, delayed motor symptoms and microglia/macrophage activation. Neurobiol Dis 2010, 37:630–640.View ArticlePubMedGoogle Scholar
- Zhang D, Kanthasamy A, Yang Y, Anantharam V, Kanthasamy A: Protein kinase C delta negatively regulates tyrosine hydroxylase activity and dopamine synthesis by enhancing protein phosphatase-2A activity in dopaminergic neurons. J Neurosci 2007, 27:5349–5362.View ArticlePubMedPubMed CentralGoogle Scholar
- Zhang D, Anantharam V, Kanthasamy A, Kanthasamy AG: Neuroprotective effect of protein kinase C delta inhibitor rottlerin in cell culture and animal models of Parkinson’s disease. J Pharmacol Exp Ther 2007, 322:913–922.View ArticlePubMedGoogle Scholar
- Anantharam V, Kitazawa M, Wagner J, Kaul S, Kanthasamy AG: Caspase-3-dependent proteolytic cleavage of protein kinase Cdelta is essential for oxidative stress-mediated dopaminergic cell death after exposure to methylcyclopentadienyl manganese tricarbonyl. J Neurosci 2002, 22:1738–1751.PubMedGoogle Scholar
- Hanrott K, Murray TK, Orfali Z, Ward M, Finlay C, O’Neill MJ, Wonnacott S: Differential activation of PKC delta in the substantia nigra of rats following striatal or nigral 6-hydroxydopamine lesions. Eur J Neurosci 2008, 27:1086–1096.View ArticlePubMedGoogle Scholar
- Kaul S, Kanthasamy A, Kitazawa M, Anantharam V, Kanthasamy AG: Caspase-3 dependent proteolytic activation of protein kinase C delta mediates and regulates 1-methyl-4-phenylpyridinium (MPP+)-induced apoptotic cell death in dopaminergic cells: relevance to oxidative stress in dopaminergic degeneration. Eur J Neurosci 2003, 18:1387–1401.View ArticlePubMedGoogle Scholar
- Clarkson ED, Rosa FG, Edwards-Prasad J, Weiland DA, Witta SE, Freed CR, Prasad KN: Improvement of neurological deficits in 6-hydroxydopamine-lesioned rats after transplantation with allogeneic simian virus 40 large tumor antigen gene-induced immortalized dopamine cells. Proc Natl Acad Sci USA 1998, 95:1265–1270.View ArticlePubMedPubMed CentralGoogle Scholar
- McGuire SO, Ling ZD, Lipton JW, Sortwell CE, Collier TJ, Carvey PM: Tumor necrosis factor alpha is toxic to embryonic mesencephalic dopamine neurons. Exp Neurol 2001, 169:219–230.View ArticlePubMedGoogle Scholar
- Gordon R, Hogan CE, Neal ML, Anantharam V, Kanthasamy AG, Kanthasamy A: A simple magnetic separation method for high-yield isolation of pure primary microglia. J Neurosci Methods 2011, 194:287–296.View ArticlePubMedGoogle Scholar
- Sherer TB, Betarbet R, Stout AK, Lund S, Baptista M, Panov AV, Cookson MR, Greenamyre JT: An in vitro model of Parkinson’s disease: linking mitochondrial impairment to altered alpha-synuclein metabolism and oxidative damage. J Neurosci 2002, 22:7006–7015.PubMedGoogle Scholar
- Kaul S, Anantharam V, Yang Y, Choi CJ, Kanthasamy A, Kanthasamy AG: Tyrosine phosphorylation regulates the proteolytic activation of protein kinase Cdelta in dopaminergic neuronal cells. J Biol Chem 2005, 280:28721–28730.View ArticlePubMedGoogle Scholar
- Bradford MM: A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976, 72:248–254.View ArticlePubMedGoogle Scholar
- Yang Y, Kaul S, Zhang D, Anantharam V, Kanthasamy AG: Suppression of caspase-3-dependent proteolytic activation of protein kinase C delta by small interfering RNA prevents MPP + −induced dopaminergic degeneration. Mol Cell Neurosci 2004, 25:406–421.View ArticlePubMedGoogle Scholar
- Latchoumycandane C, Anantharam V, Kitazawa M, Yang Y, Kanthasamy A, Kanthasamy AG: Protein kinase Cdelta is a key downstream mediator of manganese-induced apoptosis in dopaminergic neuronal cells. J Pharmacol Exp Ther 2005, 313:46–55.View ArticlePubMedGoogle Scholar
- Liu B, Du L, Hong JS: Naloxone protects rat dopaminergic neurons against inflammatory damage through inhibition of microglia activation and superoxide generation. J Pharmacol Exp Ther 2000, 293:607–617.PubMedGoogle Scholar
- Gao HM, Hong JS, Zhang W, Liu B: Distinct role for microglia in rotenone-induced degeneration of dopaminergic neurons. J Neurosci 2002, 22:782–790.PubMedGoogle Scholar
- Saijo K, Winner B, Carson CT, Collier JG, Boyer L, Rosenfeld MG, Gage FH, Glass CK: A Nurr1/CoREST pathway in microglia and astrocytes protects dopaminergic neurons from inflammation-induced death. Cell 2009, 137:47–59.View ArticlePubMedPubMed CentralGoogle Scholar
- Sun F, Anantharam V, Zhang D, Latchoumycandane C, Kanthasamy A, Kanthasamy AG: Proteasome inhibitor MG-132 induces dopaminergic degeneration in cell culture and animal models. Neurotoxicology 2006, 27:807–815.View ArticlePubMedGoogle Scholar
- Hartmann A, Hunot S, Michel PP, Muriel MP, Vyas S, Faucheux BA, Mouatt-Prigent A, Turmel H, Srinivasan A, Ruberg M, Evan GI, Agid Y, Hirsch EC: Caspase-3: A vulnerability factor and final effector in apoptotic death of dopaminergic neurons in Parkinson’s disease. Proc Natl Acad Sci USA 2000, 97:2875–2880.View ArticlePubMedPubMed CentralGoogle Scholar
- Zhao X, Bausano B, Pike BR, Newcomb-Fernandez JK, Wang KK, Shohami E, Ringger NC, DeFord SM, Anderson DK, Hayes RL: TNF-alpha stimulates caspase-3 activation and apoptotic cell death in primary septo-hippocampal cultures. J Neurosci Res 2001, 64:121–131.View ArticlePubMedGoogle Scholar
- Mogi M, Harada M, Riederer P, Narabayashi H, Fujita K, Nagatsu T: Tumor necrosis factor-alpha (TNF-alpha) increases both in the brain and in the cerebrospinal fluid from parkinsonian patients. Neurosci Lett 1994, 165:208–210.View ArticlePubMedGoogle Scholar
- Le WD, Rowe DB, Jankovic J, Xie W, Appel SH: Effects of cerebrospinal fluid from patients with Parkinson disease on dopaminergic cells. Arch Neurol 1999, 56:194–200.View ArticlePubMedGoogle Scholar
- Dobbs RJ, Charlett A, Purkiss AG, Dobbs SM, Weller C, Peterson DW: Association of circulating TNF-alpha and IL-6 with ageing and parkinsonism. Acta Neurol Scand 1999, 100:34–41.View ArticlePubMedGoogle Scholar
- Zhang D, Kanthasamy A, Yang Y, Anantharam V: Protein kinase C delta negatively regulates tyrosine hydroxylase activity and dopamine synthesis by enhancing protein phosphatase-2A activity in dopaminergic neurons. J Neurosci 2007, 27:5349–5362.View ArticlePubMedPubMed CentralGoogle Scholar
- Apostolatos H, Apostolatos A, Vickers T, Watson JE, Song S, Vale F, Cooper DR, Sanchez-Ramos J, Patel NA: Vitamin A metabolite, all-trans-retinoic acid, mediates alternative splicing of protein kinase C deltaVIII (PKCdeltaVIII) isoform via splicing factor SC35. J Biol Chem 2010, 285:25987–25995.View ArticlePubMedPubMed CentralGoogle Scholar
- Jiang K, Apostolatos AH, Ghansah T, Watson JE, Vickers T, Cooper DR, Epling-Burnette PK, Patel NA: Identification of a novel antiapoptotic human protein kinase C delta isoform, PKCdeltaVIII in NT2 cells. Biochemistry 2008, 47:787–797.View ArticlePubMedGoogle Scholar
- Kanthasamy AG, Kitazawa M, Kanthasamy A, Anantharam V: Role of proteolytic activation of protein kinase Cdelta in oxidative stress-induced apoptosis. Antioxid Redox Signal 2003, 5:609–620.View ArticlePubMedGoogle Scholar
- DeVries TA, Neville MC, Reyland ME: Nuclear import of PKCdelta is required for apoptosis: identification of a novel nuclear import sequence. EMBO J 2002, 21:6050–6060.View ArticlePubMedPubMed CentralGoogle Scholar
- Ghayur T, Hugunin M, Talanian RV, Ratnofsky S, Quinlan C, Emoto Y, Pandey P, Datta R, Huang Y, Kharbanda S, Allen H, Kamen R, Wong W, Kufe D: Proteolytic activation of protein kinase C delta by an ICE/CED 3-like protease induces characteristics of apoptosis. J Exp Med 1996, 184:2399–2404.View ArticlePubMedPubMed CentralGoogle Scholar
- Humphries MJ, Ohm AM, Schaack J, Adwan TS, Reyland ME: Tyrosine phosphorylation regulates nuclear translocation of PKCdelta. Oncogene 2008, 27:3045–3053.View ArticlePubMedGoogle Scholar
- DeVries-Seimon TA, Ohm AM, Humphries MJ, Reyland ME: Induction of apoptosis is driven by nuclear retention of protein kinase C delta. J Biol Chem 2007, 282:22307–22314.View ArticlePubMedGoogle Scholar
- Herrera AJ, Castano A, Venero JL, Cano J, Machado A: The single intranigral injection of LPS as a new model for studying the selective effects of inflammatory reactions on dopaminergic system. Neurobiol Dis 2000, 7:429–447.View ArticlePubMedGoogle Scholar
- Liu B, Jiang JW, Wilson BC, Du L, Yang SN, Wang JY, Wu GC, Cao XD, Hong JS: Systemic infusion of naloxone reduces degeneration of rat substantia nigral dopaminergic neurons induced by intranigral injection of lipopolysaccharide. J Pharmacol Exp Ther 2000, 295:125–132.PubMedGoogle Scholar
- Block ML, Zecca L, Hong JS: Microglia-mediated neurotoxicity: uncovering the molecular mechanisms. Nat Rev Neurosci 2007, 8:57–69.View ArticlePubMedGoogle Scholar
- Whitton PS: Inflammation as a causative factor in the aetiology of Parkinson’s disease. Br J Pharmacol 2007, 150:963–976.View ArticlePubMedPubMed CentralGoogle Scholar
- Cunningham RL, Giuffrida A, Roberts JL: Androgens induce dopaminergic neurotoxicity via caspase-3-dependent activation of protein kinase Cdelta. Endocrinology 2009, 150:5539–5548.View ArticlePubMedPubMed CentralGoogle Scholar
- Kanthasamy AG, Anantharam V, Zhang D, Latchoumycandane C, Jin H, Kaul S, Kanthasamy A: A novel peptide inhibitor targeted to caspase-3 cleavage site of a proapoptotic kinase protein kinase C delta (PKCdelta) protects against dopaminergic neuronal degeneration in Parkinson’s disease models. Free Radic Biol Med 2006, 41:1578–1589.View ArticlePubMedGoogle Scholar
- Sun F, Kanthasamy A, Song C, Yang Y, Anantharam V, Kanthasamy AG: Proteasome inhibitor-induced apoptosis is mediated by positive feedback amplification of PKCdelta proteolytic activation and mitochondrial translocation. J Cell Mol Med 2008, 12:2467–2481.View ArticlePubMedPubMed CentralGoogle Scholar
- Gomel R, Xiang C, Finniss S, Lee HK, Lu W, Okhrimenko H, Brodie C: The localization of protein kinase Cdelta in different subcellular sites affects its proapoptotic and antiapoptotic functions and the activation of distinct downstream signaling pathways. Mol Cancer Res 2007, 5:627–639.View ArticlePubMedGoogle Scholar
- Steinberg SF: Distinctive activation mechanisms and functions for protein kinase Cdelta. Biochem J 2004, 384:449–459.View ArticlePubMedPubMed CentralGoogle Scholar
- Reyland ME: Protein kinase Cdelta and apoptosis. Biochem Soc Trans 2007, 35:1001–1004.View ArticlePubMedGoogle Scholar
- Mecklenbrauker I, Kalled SL, Leitges M, Mackay F, Tarakhovsky A: Regulation of B-cell survival by BAFF-dependent PKCdelta-mediated nuclear signalling. Nature 2004, 431:456–461.View ArticlePubMedGoogle Scholar
- Sriram K, Matheson JM, Benkovic SA, Miller DB, Luster MI, O’Callaghan JP: Deficiency of TNF receptors suppresses microglial activation and alters the susceptibility of brain regions to MPTP-induced neurotoxicity: role of TNF-alpha. FASEB J 2006, 20:670–682.View ArticlePubMedGoogle Scholar
- Qin L, Wu X, Block ML, Liu Y, Breese GR, Hong JS, Knapp DJ, Crews FT: Systemic LPS causes chronic neuroinflammation and progressive neurodegeneration. Glia 2007, 55:453–462.View ArticlePubMedPubMed CentralGoogle Scholar
- Sulzer D: Multiple hit hypotheses for dopamine neuron loss in Parkinson’s disease. Trends Neurosci 2007, 30:244–250.View ArticlePubMedGoogle Scholar
- Jin H, Kanthasamy A, Anantharam V, Rana A, Kanthasamy AG: Transcriptional regulation of pro-apoptotic protein kinase Cdelta: implications for oxidative stress-induced neuronal cell death. J Biol Chem 2011, 286:19840–19859.View ArticlePubMedPubMed CentralGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.