Proteolytic activation of proapoptotic kinase protein kinase Cδ by tumor necrosis factor α death receptor signaling in dopaminergic neurons during neuroinflammation

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).

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 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 % CO_2, 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 [27]. 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 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 [28]. 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.

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.

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.

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.

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 [30]. 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).

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 (PKCδ^D327A-CRM), stable N27 cell lines overexpressing either the mutant PKCδ^D327A protein or the β-galactosidase (Lac Z) protein as the vector control were prepared as described in our publications [32]. 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.

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.

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).

A single dose LPS injection model that was previously described [35] 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 [36]. 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.

To determine the downstream mechanisms underlying TNF stimulation in dopaminergic neuronal cells, we used the N27 dopaminergic neuronal cell model, which has been widely used by several laboratories to study dopaminergic degeneration related to PD [21, 24, 25]. First we examined the neurotoxic effect of TNF on N27 dopaminergic neuronal cells. The Sytox assay and the sensitive DNA fragmentation ELISA were used to quantify TNF cytotoxicity. TNF (30 ng/ml) induced significant increases in Sytox fluorescence and cell death morphology, which could be blocked using the TNF neutralizing drug etanercept (Figure 1A,B). Since TNF cytotoxicity can proceed through activation of caspases in various cell types, we examined caspase activation downstream of TNF signaling. High levels of the pro-caspase-8 protein were detected in N27 cells, and TNF treatment induced activation of caspase-8 at 3 h, with increased accumulation of the active p20 fragment (Figure 1C,D). Again, the activation of caspase-8 was attenuated by pretreatment with the TNF inhibitor etanercept. Since caspase-3 is an important effector caspase downstream of TNF signaling, and is known to be activated in PD models and in the human PD brain [37, 38], we studied caspase-3 activation in TNF-treated N27 cells. We found that TNF treatment for 6 h caused activation of caspase-3, which could be blocked by pretreatment with etanercept and the caspase-8 inhibitor IETD-fmk (Figure 1E). These results indicate that caspase-3 is activated in a caspase-8 dependent manner downstream of TNF death receptor signaling in dopaminergic neuronal cells. Next we used the sensitive DNA fragmentation ELISA assay to quantify TNF cell-death signaling. Exposure to TNF (30 ng/ml) for 16 h resulted in a threefold increase in DNA fragmentation compared to untreated controls (Figure 1F). Pretreatment with the TNF-neutralizing drug etanercept or a caspase-8 inhibitor (IETD-fmk) almost completely blocked TNF-induced DNA fragmentation, indicating that canonical TNF death receptor signaling through activation of caspase-8 is involved in the dopaminergic neurotoxicity caused by sustained exposure to TNF. In preliminary studies, we found that doses as low as 10 ng/ml TNF could induce detectable toxicity in N27 dopaminergic cells. We chose a 30 ng/ml dose of TNF based on previous in vitro studies [26] and because this concentration induced a consistent neurotoxic response in vitro. The dose range of TNF used in our mechanistic studies is high compared to the levels of circulating TNF in the serum and CSF of PD patients [39‐41] due to the fact that high doses are necessary to elicit a detectable response in cell culture models within a shorter timeframe. Also, the circulating TNF has a short half-life and therefore, it does not reflect the elevated levels of tissue TNF in the substantia nigra. The TNF concentration at 10 to 60 ng/ml used in our study is consistent with doses used in cell culture experiments by other researchers [17, 26].

We recently showed that PKCδ, a proapoptotic kinase belonging to the novel PKC isoform family, is highly expressed in dopaminergic neurons [21, 42]. Further, we demonstrated that PKCδ is activated by caspase-3 mediated proteolytic cleavage at its hinge region following mitochondrial oxidative stress, resulting in persistent activation of the kinase and amplification of apoptotic signals in dopaminergic neurons [21, 22, 24]. Since PKCδ activation by the extrinsic apoptotic pathway in dopaminergic cells or its role in neuroinflammation-induced dopaminergic neurotoxicity has not been examined, we tested if PKCδ can be activated by caspase-3 during TNF-induced death of dopaminergic neuronal cells and function as a downstream apoptotic effector. Interestingly, TNF treatment caused a time and dose dependent proteolytic activation of PKCδ that was detectable 3 h after treatment and increased at 6 h (Figure 2A,B). Dose response studies at the 6 h time point showed that doses as low as 10 ng/ml of TNF could induce detectable proteolytic activation of PKCδ, which increased substantially with 30 ng/ml TNF. Higher doses of TNF (60 ng/ml) did not significantly increase PKCδ cleavage further (Figure 2C,D). Since caspase-8 and caspase-3 were activated by TNF in these cells, we tested if PKCδ proteolytic activation was mediated by this signaling pathway using the specific caspase inhibitors DEVD-fmk and IETD-fmk. Pretreatment with the caspase-8 and caspase-3 inhibitors suppressed TNF-induced proteolytic activation of protein kinase Cδ (Figure 2E,F). Taken together, these results suggest that the proapoptotic kinase PKCδ could be activated by caspase-8 and caspase-3 dependent proteolytic cleavage during TNF toxicity in dopaminergic neuronal cells. The proteolytically cleaved PKCδ fragment appeared as a doublet band in Western blots in our experiments. Several alternate splice forms of PKCδ have been reported, including a caspase-3 cleavage-resistant form and a truncated form lacking the regulatory domain [43, 44]. Also, the C-terminal catalytic domain of PKCδ contains multiple phosphorylation sites. Because the antibody used in these studies recognizes the C-terminal catalytic domain, it is possible that the doublet band of the cleaved product represents a post-translational modification of the cleavage product or one of the truncated PKCδ alternate splice forms.

Unlike other proteins for which proteolytic cleavage can result in loss of function or inactivation, the proteolytic cleavage of PKCδ by caspase-3 during oxidative stress-induced dopaminergic cell death has been shown to persistently activate the kinase [21, 22, 24, 45]. We used IP-kinase assays and Western blotting to determine if TNF-induced proteolytic activation of PKCδ leads to sustained activation of the kinase in N27 dopaminergic cells. As shown in Figure 3A, treatment with TNF for 6 h significantly increased proteolytic cleavage of PKCδ, which was blocked by pretreatment with Etanercept or a TNFR1 receptor-neutralizing antibody. PKCδ IP-kinase assays performed in the absence of lipid cofactors showed a robust increase in PKCδ kinase activity in TNF-treated samples, which could be attenuated by pretreatment with etanercept or by TNFR1 receptor neutralization (Figure 3C). These results demonstrate for the first time that PKCδ can be proteolytically activated in dopaminergic cells by a TNFR1-dependent signaling pathway, resulting in sustained activation of the kinase during TNF-induced cell death.

Nuclear translocation of PKCδ has been shown to occur following proteolytic activation by apoptotic stimuli. A bipartite nuclear localization sequence (NLS) has been identified at the C-terminus (amino acids 611 to 623) of PKCδ [46, 47]. The NLS is required for nuclear localization of both the full-length and proteolytically-cleaved forms of PKCδ. However, previous studies have shown that proteolytic activation of PKCδ by caspase-3 facilitates unmasking of the NLS, resulting in increased nuclear translocation of the catalytic fragment during apoptosis [47‐49]. To further characterize the role of PKCδ in dopaminergic cell death induced by TNF, we used confocal microscopy to study the subcellular localization of PKCδ. N27 cells were treated with TNF for 6 h, the time point at which the highest level of cleaved PKCδ was detected, and confocal immunofluorescence was used to visualize the subcellular localization of PKCδ. We detected prominent nuclear localization of PKCδ after 6 h in TNF-treated cells, while in control cells PKCδ localized in the cytoplasm (Figure 4A). Nuclear translocation was also clearly evident in spatial fluorescence signal intensity plots (Figure 4B) for PKCδ (green channel) and the Hoechst nuclear stain (blue channel). Control cells had spatially distinct fluorescence signals for the nucleus and PKCδ, showing that PKCδ is largely localized outside the nucleus. However in TNF-treated cells, the spatial fluorescence signals had the same pattern along the XY plane. Interestingly, in addition to the overall increase in nuclear localization with TNF treatment, we also observed punctuate staining of PKCδ at distinct locations around the perinuclear region (shown in 60 × magnification and labeled with red arrows). The nuclear localization of PKCδ following TNF treatment further points to an important role for this kinase in dopaminergic degeneration during TNF toxicity.

To determine whether TNF-induced PKCδ activation contributes to the cell death process in dopaminergic cells, we used siRNA suppression of PKCδ and a caspase-3 cleavage-resistant mutant of PKCδ (PKCδ^D327A-CRM). First, N27 cells were transfected with siRNA to PKCδ or scramble siRNA and incubated for 24 h to allow for optimal suppression of PKCδ protein levels, which we validated by Western blotting (Figure 5B). The transfected cells were then treated with TNF for 16 h and processed for the DNA fragmentation assay. In N27 cells transfected with scramble siRNA, TNF caused a significant increase in DNA fragmentation, while the PKCδ siRNA transfected cells were protected from cell death (Figure 5A). These results indicate that PKCδ is required for TNF-induced apoptosis in dopaminergic cells and that genetic or pharmacological modulation of PKCδ may effectively attenuate TNF-induced neurotoxicity. To evaluate the significance of the PKCδ proteolytic cleavage events during proapoptotic TNF signaling, we used N27 cells overexpressing a mutant PKCδ protein resistant to proteolytic cleavage (PKCδ^D327A-CRM) by caspase-3 due to a point mutation at the cleavage site. N27 cells that stably express either the cleavage-resistant PKCδ mutant or the LacZ protein (Figure 5D) were established as described previously [32]. The cells were treated with TNF for 16 h and processed for DNA fragmentation. As shown in Figure 5C, TNF treatment caused a 2.5-fold increase in DNA fragmentation in LacZ cells, while cells expressing the PKCδ^D327A-CRM mutant were protected from TNF toxicity, indicating that proteolytic cleavage of PKCδ is crucial for TNF-induced cell death. Together, these results demonstrate that PKCδ functions as a proapoptotic kinase during TNF-induced dopaminergic neurotoxicity and that proteolytic cleavage of PKCδ by caspase-3 downstream of TNF signaling is required for cell death.

In order to extend our results from the previous mechanistic studies with the dopaminergic N27 clonal cell model to dopaminergic neurons, we used primary mouse ventral mesencephalic dopaminergic neuron cultures. We established primary mesencephalic neuronal (>95 % glial-free) cultures from PKCδ wild type (+/+) and knockout (−/−) mice to study the role of PKCδ in mediating TNF-induced dopaminergic neurodegeneration. Dopaminergic neurotoxicity was assessed by dopamine uptake assays and by TH-positive cell counting. As shown (Figure 6B,C), TNF exposure caused around 50 % decrease in dopamine uptake and TH-positive cell counts in cultures from PKCδ wild type (+/+) mice, consistent with previous reports of dopaminergic neurotoxicity in similar model systems [17, 26]. However, in primary neuron cultures obtained from PKCδ knockout (−/−) mice, TNF induced only around a 20 % reduction in dopamine uptake and TH-positive neuron counts, indicating that dopaminergic neurons from PKCδ knockout mice were resistant to TNF toxicity. In addition to functional measurement of TNF neurotoxicity by dopamine uptake, we also performed double labeling immunofluorescence morphometric analysis using TH as a marker for dopaminergic neurons and MAP-2 as a pan-neuronal marker. Untreated cultures from both PKCδ wild type (+/+) and knockout (−/−) mice had similar numbers of dopaminergic neurons and displayed extensive branched neurites and normal cell bodies (Figure 6A). In TNF-treated cultures, fewer TH-positive neurons with smaller cell bodies and a clear loss of neurites were evident in cultures from PKCδ wild type (+/+) mice. In contrast, dopaminergic neurons in cultures from PKCδ knockout (−/−) mice displayed visible branched neurites and more numerous cell bodies with normal morphology (Figure 6A lower panel), demonstrating significant protection against TNF toxicity in these cultures.

Next, we sought to extend the significance of our results obtained with the N27 dopaminergic cell culture model and primary neurons to an in vivo model of dopaminergic degeneration relevant to neuroinflammation. We selected the widely used stereotaxic LPS injection model of neuroinflammation-induced dopaminergic degeneration for our experiments [17, 35, 50, 51]. In this model, a single injection of LPS into the rodent substantia nigra elicits a sustained, localized neuroinflammatory response resulting in a delayed loss of dopaminergic neurons in the SNpc. We stereotaxically injected C57bl6 mice with a single dose of 5 μg LPS into the substantia nigra and sacrificed them 14 days later, the time at which significant dopaminergic degeneration occurs in this model [35]. The SN tissue was dissected and probed for proteolytic activation of PKCδ using Western blotting and IP-kinase assays. As shown in Figure 7A, mice that received saline injections did not show proteolytic activation of PKCδ in the SN, while LPS-injected mice showed strong proteolytic cleavage of PKCδ at 14 days. The PKCδ Western blots were reprobed for tyrosine hydroxylase to confirm dissection of the substantia nigra region. As expected, the LPS-injected mice had decreased TH protein levels (Figure 7A), indicative of increased dopaminergic degeneration in this model at 14 days [35, 50]. We also used PKCδ IP-kinase assays with a histone substrate on nigral tissue samples from LPS-injected and saline-injected mice. In nigral tissues from LPS-injected mice, we detected substantially higher PKCδ kinase activity compared to saline injected controls (Figure 7C), demonstrating that the proteolytic cleavage of PKCδ resulted in activation of the kinase in vivo. Equal protein loading and accurate dissection of the nigral tissue was confirmed by Western blotting for β-actin and TH, respectively. Together, these results demonstrate for the first time that protein kinase Cδ can be activated by proteolytic cleavage in the substantia nigra following LPS-induced neuroinflammation.