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Originally published In Press as doi:10.1074/jbc.M511560200 on December 16, 2005

J. Biol. Chem., Vol. 281, Issue 9, 5373-5382, March 3, 2006
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6-Hydroxydopamine-induced Apoptosis Is Mediated via Extracellular Auto-oxidation and Caspase 3-dependent Activation of Protein Kinase C{delta}*

Katharine Hanrott{ddagger}, Louise Gudmunsen{ddagger}, Michael J. O'Neill§, and Susan Wonnacott{ddagger}1

From the {ddagger}Department of Biology & Biochemistry, University of Bath, 4 South, Claverton Down, Bath BA2 7AY and §Eli Lilly & Co. Ltd., Windlesham, Surrey GU20 6PH, United Kingdom

Received for publication, October 25, 2005 , and in revised form, December 15, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
6-Hydroxydopamine is a neurotoxin commonly used to lesion dopaminergic pathways and generate experimental models for Parkinson disease, however, the cellular mechanism of 6-hydroxydopamine-induced neurodegeneration is not well defined. In this study we have explored how 6-hydroxydopamine neurotoxicity is initiated. We have also investigated downstream signaling pathways activated in response to 6-hydroxydopamine, using a neuronal-like, catecholaminergic cell line (PC12 cells) as an in vitro model system. We have shown that 6-hydroxydopamine neurotoxicity is initiated via extracellular auto-oxidation and the induction of oxidative stress from the oxidative products generated. Neurotoxicity is completely attenuated by preincubation with catalase, suggesting that hydrogen peroxide, at least in part, evokes neuronal cell death in this model. 6-Hydroxydopamine does not initiate toxicity by dopamine transporter-mediated uptake into PC12 cells, because both GBR-12909 and nisoxetine (inhibitors of dopamine and noradrenaline transporters, respectively) failed to reduce toxicity. 6-Hydroxydopamine has previously been shown to induce both apoptotic and necrotic cell-death mechanisms. In this study oxidative stress initiated by 6-hydroxydopamine caused mitochondrial dysfunction, activation of caspases 3/7, nuclear fragmentation, and apoptosis. We have shown that, in this model, proteolytic activation of the proapoptotic protein kinase C{delta} (PKC{delta}) is a key mediator of 6-hydroxydopamine-induced cell death. 6-Hydroxydopamine induces caspase 3-dependent cleavage of full-length PKC{delta} (79 kDa) to yield a catalytic fragment (41 kDa). Inhibition of PKC{delta} (with rottlerin or via RNA interference-mediated gene suppression) ameliorates the neurotoxicity evoked by 6-hydroxydopamine, implicating this kinase in 6-hydroxydopamine-induced neurotoxicity and Parkinsonian neurodegeneration.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Parkinson disease (PD)2 is a neurodegenerative disorder associated with a progressive loss of dopaminergic neurons of the substantia nigra pars compacta and associated depletion of dopamine in the terminal region, the caudate putamen. Although the neuropathological hallmarks of this disease are well described, the etiology is largely undefined. However, a number of biochemical processes and molecular mechanisms have been identified as mediators of neuronal cell death in PD. These include oxidative stress and mitochondrial dysfunction. Dopamine-rich areas of the brain are particularly vulnerable to oxidative stress, because metabolism of dopamine itself (both enzymatic and non-enzymatic) leads to the generation of reactive oxygen species (ROS), including hydrogen peroxide and hydroxyl radicals (1).

To elucidate the molecular pathways of neuronal death and to develop neuroprotective strategies, a number of in vitro and in vivo models have been characterized. Many of these utilize experimental neurotoxins, including 6-hydroxydopamine (6-OHDA) and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, which are thought to induce toxicity that mimics the neuropathological and biochemical characteristics of PD. 6-OHDA has been shown to induce toxicity in a wide range of neuronal in vitro models, including human neuroblastoma cell lines (2, 3), primary neuronal cultures (4, 5), and the rat adrenal pheochromocytoma cell line, PC12 cells (6–8). It is widely reported that 6-OHDA initiates cellular oxidative stress. However, the exact mechanism of ROS production and induction of toxicity is less clearly defined. It is traditionally thought that 6-OHDA enters neurons via dopamine transporters (DAT) (9) and initiates the activation of cell death pathways by generation of intracellular free radicals and mitochondrial inhibition (10). However, a number of recent studies have shown that 6-OHDA may not induce toxicity in this manner but, rather, via an extracellular mechanism (11, 12). This process has been explored in detail in this study, in which 6-OHDA is applied to the catecholaminergic rat pheochromocytoma cell line, PC12.

Both necrotic and apoptotic mechanisms of cell death occur in response to 6-OHDA (6, 13, 14). In an attempt to dissect which apoptotic pathways are activated, several studies have pinpointed a role for the mitochondrial-caspase cascade in 6-OHDA-induced apoptosis, which initiates the activation of the main effector caspases 3 and 7 (5, 6, 15–20). However, the crucial downstream targets of caspase 3 activation have not been clearly defined.

PKC{delta} is classified as a member of the novel PKC subfamily, because it is not activated in response to calcium but is activated by diacylglycerol (21). The most commonly studied mechanism of PKC{delta} activation is membrane translocation in response to lipid signaling, however, numerous studies over recent years have also identified a caspase 3-dependent proteolytic activation (22). The site of caspase 3 cleavage lies between the regulatory and catalytic domains, and proteolysis induces permanent dissociation of the two domains and constitutive activation of the catalytic domain (22, 23). This mechanism of activation has been shown to occur in response to a range of apoptotic stimuli, both in neuronal and non-neuronal cell models (22, 24–32). Several of these studies suggest that PKC{delta} is a redox-sensitive kinase and hence is activated in response to oxidative stress (29–32). In addition, PKC{delta} has been shown to be activated in response to a number of dopaminergic neurotoxins, including 1-methyl-4-phenylpyridinium (30, 33) and methylcyclopentadienyl manganese tricarbonyl (32) in the dopaminergic rat N27 cell line and dieldrin in PC12 cells (31). This may suggest that PKC{delta} plays a role in neuronal apoptosis in PD. The main purpose of this study was to elucidate the mechanisms by which 6-OHDA initiates oxidative stress and subsequent neurotoxicity in the rat neuronal-like, catecholaminergic cell line, PC12. The downstream effectors of mitochondrial dysfunction and caspase activation were investigated, and the possible role of caspase 3-dependent PKC{delta} activation in 6-OHDA-induced apoptosis was explored.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials
Dulbecco's modified Eagle's medium (DMEM), culture media supplements, poly-D-lysine, 6-OHDA, ascorbic acid, diethylenetriaminepentaacetic acid (DETAPAC), N-acetyl-L-cysteine (NAC), 3-[4,5-dimethylthiazol-2-y]-2,5-diphenyltetrazolium bromide (MTT), rottlerin, phenylmethylsulfonyl fluoride, aprotinin, sodium orthovanadate, and mouse anti-{alpha}-tubulin were purchased from Sigma-Aldrich. GBR-12909 and nisoxetine were obtained from Tocris Cookson, Inc. (Avonmouth, Bristol, UK). The CytoTox-ONETM Homogeneous Membrane Integrity assay and Apo-ONETM Homogeneous Caspase-3/7 assay were bought from Promega Corp. (Madison, WI). The Detergent Compatible Protein Assay was purchased from Bio-Rad Laboratories Ltd. (Hemel Hempstead, Herts, UK) and benzyloxycarbonyl-VAD-fluoromethylketone (z-VAD-FMK) and Ac-DEVD-CHO from Alexis Biochemicals (Lausen, Switzerland). The mammalian expression plasmids, pKD-PKC{delta}-v3 and pKD-NegCon-v1 were purchased from Upstate Cell Signaling Solutions (Lake Placid, NY), and the LipofectamineTM 2000 was from Invitrogen. Rabbit polyclonal PKC{delta} antibody was bought from Santa Cruz Biotechnology (Santa Cruz, CA), and monoclonal mouse anti-lamin B was from Zymed Laboratories Inc. Laboratories Inc. (San Francisco, CA). Hoechst 33258 was obtained from Molecular Probes. The enhanced chemiluminescence plus detection kit, horseradish peroxidase-conjugated anti-rabbit IgG secondary antibody, and [3H]dopamine (39 Ci/mmol) were purchased from Amersham Biosciences. All other chemicals used were of analytical grade and obtained from standard commercial sources.

Methods
Cell Culture and Drug Treatments—PC12 cells were cultured in DMEM, supplemented with 10% donor horse serum, 5% fetal calf serum, 2 mML-glutamine, 190 units/ml penicillin, and 0.2 mg/ml streptomycin, in 75-cm2 culture flasks. Cells were plated onto poly-L-lysine-coated 96-, 24-, or 6-well plates at a density of 3.5 x 104 cells/cm2, 48 h before 6-OHDA treatment. 6-OHDA preparation and administration were carried out as previously described (4). Briefly, vehicle solution containing 0.15% ascorbic acid and 10 mM DETAPAC was flushed with nitrogen gas for 30 min before addition of 6-OHDA. A solution of 6-OHDA at a concentration 10 times that of the desired final concentration (e.g. 1.5 mM 6-OHDA to give a final concentration of 150 µM) was applied to cells in the dark. Following 6-OHDA treatment for 15 min, cells were washed with DMEM and incubated for 0–24 h. Unless otherwise stated, inhibitors or antioxidants were preincubated with the cells 30 min before 6-OHDA treatment and were present for the duration of the experiment.

Neurotoxicity Assays—Lactate dehydrogenase (LDH) release was measured using the CytoTox-ONETM Homogeneous Membrane Integrity assay (Promega) according to the manufacturer's instructions. In brief, 0–24 h post 6-OHDA treatment, culture medium was removed from the cells and equilibrated to 22 °C and an equal volume of Cyto-Tox-ONETM reagent added for 10 min. Fluorescence (excitation, 560 nm; emission, 590 nm) was measured using a fluorescent plate reader. Data are expressed as a percentage of maximum LDH release (determined by incubation of cells with 9% Triton X-100) after subtraction of background fluorescence (determined by fluorescence from DMEM alone).

Mitochondrial function was determined by MTT reduction assay. Cells were incubated with MTT (2.5 mg/ml in DMEM) for 90 min at 37 °C. Excess MTT was removed, and remaining formazan crystals were dissolved in isopropanol and quantified by determining optical density (570 nm) using a colorimetric 96-well plate reader.

Spectrophotometric Assay of 6-OHDA Auto-oxidation—The autooxidation of 6-OHDA was measured spectrophotometrically by monitoring the formation of p-quinone at 490 nm (12). The assay was carried out in a cell-free system under conditions corresponding to cellular 6-OHDA treatments. DMEM alone, or DMEM containing NAC (5 mM) or catalase (300 units/ml), was thermostatically maintained at 37 °C during the experiment. 6-OHDA was prepared in vehicle solution, and the experiment was initiated by addition of 6-OHDA to give a final concentration of 150 µM. Absorbance at 490 nm was monitored at 10-s intervals for 10 min.

[3H]Dopamine Uptake—PC12 cells were grown in 24-well plates for 48 h. To assess the presence of DAT, cells were incubated with 20 nM [3H]dopamine for 15 min at 37 °C. Uptake was determined in buffer containing 120 mM NaCl, 4.7 mM KCl, 1.8 mM CaCl2, 1.2 mM MgSO4, 5.5 mM glucose, 16 mM NaH2PO4 and 16 mM Na2HPO4, 1.3 mM EDTA, 1 mM ascorbic acid, and 50 µM pargyline (pH 7.3) (34). Nonspecific uptake was determined in the presence of GBR-12909 (2 µM). Uptake was terminated by lysis of cells with 1% Triton X-100, and radioactivity was measured in a TRICARB liquid scintillation spectrometer.

Hoechst Staining—The fluorescent DNA and chromatin stain Hoechst 33258 was used to assess DNA fragmentation as a marker for apoptosis. PC12 cells were grown in 24-well plates on poly-L-lysine-coated coverslips. Cells were treated with 6-OHDA or vehicle alone (as described above) or staurosporine (1 µM) for 2 h and fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS; 120 mM NaCl, 19 mM Na2HPO4, 6 mM KH2PO4), pH 7.4, for 30 min. Cells were washed with PBS and permeabilized with 0.1% Triton X-100 in PBS for 10 min and washed again. Coverslips were incubated with Hoechst 33258 (60 ng/ml) for 10 min, washed in PBS, and mounted with Vectashield on glass slides prior to viewing under ultraviolet light. Apoptotic cells were distinguished by the presence of bright, fragmented nuclei. The percentage of apoptotic cells in relation to the total number of cells was determined from 10 random fields per slide, from 3 independent experiments.

Caspase 3/7 Fluorometric Assay—Activity of caspases 3/7 were assayed using the Apo-ONETM Homogeneous Caspase 3/7 assay (Promega) according to the manufacturer's instructions. Briefly, equal volumes of DMEM and Apo-ONETM caspase reagent (1:100 profluorescent substrate and lysis buffer) were added to cells, and the mixture was incubated for 5 h. Fluorescence (excitation, 485 nm; emission, 512 nm) was measured using a fluorescence plate reader. Background fluorescence was determined by fluorescence from DMEM alone and subtracted from all experimental values.

RNA Interference-mediated Gene Suppression of PKC{delta}—To reduce the expression of PKC{delta}, a commercially available mammalian expression plasmid that directs the transcription of an siRNA transcript for the PKC{delta} sequence (584-605, Upstate Cell Signaling Solutions) was used. The expression plasmid (designated pKD-PKC{delta}-v3) contains a sequence that when expressed forms a short-hairpin RNA, which is processed into a PKC{delta} siRNA. The expression of the short-hairpin RNA is under the control of the H1 RNA polymerase III promoter. The short-hairpin RNA showed no homology to other gene sequences when using BLAST. The same expression plasmid containing a negative control sequence (designated pKD-NegCon-v1), which is processed into a negative control siRNA, was used in parallel. Cells were transfected with pKD-PKC{delta}-v3 or pKD-NegCon-v1 using LipofectamineTM 2000. Characterization work carried out by Upstate Cell Signaling Solutions reported that transfection of pKD-PKC{delta}-v3 induced nearly 80% knockdown of PKC{delta} mRNA levels when compared with cells transfected with the pKD-NegCon-v1. To confirm PKC{delta} gene suppression in the PC12 cells used in this study, cells were transfected with pKD-PKC{delta}-v3 or pKD-NegCon-v1, and cell lysates were prepared 48, 72, and 96 h post-transfection as described below (whole cell lysis). Western blotting using rabbit anti-PKC{delta} was carried out as described below and equal loading confirmed using mouse anti-{alpha}-tubulin.

Cell Lysate Preparations
Whole Cell Lysis—Whole cell lysates were prepared for Western blotting by removal of cells from 6-well culture dishes using cell scrapers. Pellets were washed with ice-cold PBS (pH 7.4) and incubated with ice-cold whole cell lysis buffer (PBS, pH 7.4, containing 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 100 µg/ml phenylmethylsulfonyl fluoride, 33 µg/ml aprotinin, and 1 mM sodium orthovanadate) at 4 °C for 30 min. Cell lysates were centrifuged (15,000 x g for 20 min at 4 °C), and the supernatant was recovered.

Cytosolic and Nuclear Fractionation—Cells were removed from 6-well culture dishes as described above, and pellets were washed with ice-cold PBS (pH 7.4). Cellular fractionation was carried out using a protocol adapted from a previous study (28). Briefly, cell pellets were washed with ice-cold fractionation lysis buffer (10 mM HEPES, 10 mM KCl, 1.5 mM MgCl2, 0.5 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, pH 7.9) and lysed by incubation with fractionation lysis buffer containing 0.1% Nonidet P-40, for 10 min on ice. Cell suspensions were centrifuged (12,000 x g for 5 min at 4 °C). The supernatant fraction corresponded to the cytosolic fraction, whereas the pellet corresponded to the nuclear fraction. The nuclear pellet was washed with lysis buffer without Nonidet P-40, resuspended in extraction buffer (20 mM HEPES, 420 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 25% glycerol, 1% Nonidet P-40, 80 units/ml DNase I, pH 7.9), and incubated for 10–15 min at 4 °C. Following extraction, the suspension was centrifuged (12,000 x g for 5 min at 4 °C), and the supernatant was recovered as the purified nuclear fraction. The cytosolic fraction was further lysed by addition of whole cell lysis buffer and incubated for 15 min at 4 °C. The cytosolic suspension was centrifuged (12,000 x g for 5 min at 4 °C), and the supernatant was recovered as the purified cytosolic fraction.

Western Blotting
The protein concentration of lysates was determined by DC protein assay. Samples were diluted in appropriate lysis buffer and an equal volume of electrophoresis sample buffer (60 mM Tris (pH 6.8), 0.01% bromphenol blue, 2% SDS, 10% glycerol, 100 mM dithiothreitol). Lysates (containing 4 µg of protein) were boiled for 5 min and loaded onto a 9% SDS-polyacrylamide gel, which was run at 100 V. The gel was transferred to nitrocellulose membrane and blocked overnight in 4% nonfat milk powder in PBS, pH 7.4, at 4 °C. Membranes were incubated for 1 h with 0.2 µg/ml rabbit anti-PKC{delta}, 1.5 µg/ml mouse anti-{alpha}-tubulin, or 1 µg/ml mouse anti-lamin B in PBS containing 0.2% Tween 20 and 1% nonfat milk powder (blotto). After washing the membranes for 3 x 5 min with blotto, they were incubated for 1 h with horseradish peroxidase-conjugated anti-rabbit IgG or horseradish peroxidase-conjugated anti-mouse IgG; 1 µg/ml in blotto. Membranes were washed for 3 x 5 min in PBS, and immunoreactive protein bands were detected using the enhanced chemiluminescence technique. The intensity of bands was quantified by densitometry using Scion Image software.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
6-OHDA Induces Concentration- and Time-dependent Cell Death and Mitochondrial Dysfunction in PC12 Cells—In PC12 cells, preliminary experiments (data not shown) using a chronic 6-OHDA treatment regime (24 h) resulted in variable levels of toxicity. Therefore an acute mode of treatment (6-OHDA for 15 min) was used, with precautions taken to prevent auto-oxidation of 6-OHDA before application onto the cells (see "Methods") (4).

Following acute treatment of PC12 cells with 6-OHDA, cells were incubated for up to 24 h, and toxicity was assessed as an index of mitochondrial function (MTT reduction) and membrane integrity (release of LDH) (Fig. 1). 6-OHDA concentration dependently evoked loss of both mitochondrial function and membrane integrity with LD50 values of 186 µM and 158 µM, respectively (Fig. 1, A and C). Consequently, 6-OHDA was applied at 150 µM in all further experiments.

The time course of 6-OHDA-induced toxicity in PC12 cells (Fig. 1, B and D) indicated that changes in mitochondrial function preceded membrane damage. Mitochondrial function was immediately and dramatically reduced following 6-OHDA application to 51.3 ± 5.8% of control. However, this initial loss in mitochondrial function was reversed, with almost 30% recovery of function within 1 h. Sustained loss of mitochondrial function was not detected until 6 h post-treatment and remained constant between 6 and 24 h. In contrast, loss of membrane integrity reflecting ultimate cell death was not detected up to 10 h post 6-OHDA treatment. Between 10 and 18 h post treatment there was a gradual increase in cell death.

6-OHDA Induces Toxicity in PC12 Cells via Extracellular Auto-oxidation of 6-OHDA and Consequent Oxidative Stress—In vitro studies suggest that oxidative stress and disruption of mitochondrial function are the main mediators of 6-OHDA mediated cell death (10). To determine if 6-OHDA initiates oxidative stress in the PC12 cell model, experiments were conducted in the presence of the anti-oxidants NAC (Fig. 2, A and B) or catalase (Fig. 2, C and D). NAC (5 mM) and catalase (30 units/ml) provided complete protection against 6-OHDA-induced mitochondrial dysfunction (Fig. 2, A and C) and membrane damage (Fig. 2, B and D), and protection was concentration-dependent. Both NAC and catalase were ineffective when added immediately after the 15-min exposure to 6-OHDA, even when catalase was used at 10-fold higher concentration (data not shown). To assess the direct action of these anti-oxidants on 6-OHDA auto-oxidation, a cell-free system measuring the production of the 6-OHDA auto-oxidation product (p-quinone) was used (Fig. 2E). 6-OHDA (150 µM) was completely auto-oxidized within 5 min, and this was prevented by the thiol antioxidant NAC, suggesting a mechanism for the neuroprotection by this anti-oxidant in PC12 cells. Catalase (300 units/ml) had no effect on 6-OHDA auto-oxidation, suggesting that its neuroprotective effect is downstream of this initial event. Because the anti-oxidant properties of catalase are attributed to its ability to hydrolyze hydrogen peroxide, the neuroprotective effect of catalase therefore suggests that 6-OHDA is auto-oxidized to hydrogen peroxide (among other free radical species), and this initiates toxicity.


Figure 1
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  		FIGURE 1.
6-OHDA-induced mitochondrial dysfunction and cell death in PC12 cells. Mitochondrial function of PC12 cells was quantified by MTT reduction assay (A and B). Cell death was quantified by measuring release of LDH from PC12 cells into culture medium (C and D). Concentration dependence of 6-OHDA-evoked mitochondrial function (A) or cell death (C) was carried out by treating cells for 15 min with 6-OHDA (10–1000 µM). Assays were conducted following a 24-h post treatment incubation. LD50 values for 6-OHDA-induced loss of mitochondrial function (A) and membrane integrity (C) were 186 µM and 158 µM, respectively. The time course of 6-OHDA-induced mitochondrial damage (B) and cell death (D) was determined by treating cells for 15 min with 6-OHDA (150 µM). Measurements were made at intervals during the 24-h post-treatment incubation, as indicated in the scheme. Data represent the mean ± S.E. of five independent experiments, each carried out with eight (A and B) or four (C and D) replicates.

 
To confirm the hypothesis that 6-OHDA toxicity was elicited via extracellular auto-oxidation, the role of transporter-mediated uptake was investigated. Inhibition of DAT and noradrenaline transporters (by GBR-12909 and nisoxetine, respectively), separately or in combination, had no effect on 6-OHDA-induced toxicity in PC12 cells (Fig. 2F). The presence of functional DAT was confirmed using a [3H]dopamine uptake assay. PC12 cells accumulated 0.78 pmol/mg of protein/15 min of [3H]dopamine, comparable with previous reports (35); this uptake was completely blocked by GBR-12909 (2 µM) (data not shown).

6-OHDA-induced Oxidative Stress Activates Apoptotic Pathways—Morphological hallmarks that are characteristic of apoptosis include chromatin condensation and DNA fragmentation. The fluorescent chromatin and DNA stain Hoechst 33258 was used to stain the nuclei of 6-OHDA-treated and control PC12 cells. Fig. 3A shows a typical image of vehicle-treated cells with round intact nuclei. In contrast, cells treated with the apoptosis-inducing agent staurosporine (Fig. 3B) or with 6-OHDA (Fig. 3C) showed phase-bright nuclear fragmentation typical of apoptosis. Quantification of apoptotic induction (Fig. 3D) showed that 20.7 ± 2.1% of the total cell number was undergoing apoptosis 24 h following 6-OHDA (15 min and 150 µM) treatment.

Mitochondrial dysfunction initiated by 6-OHDA has been shown to induce release of cytochrome c, consequent activation of procaspase 9, formation of the apoptosome, and activation of caspases 3 and 7 in PC12 cells (6, 19). We confirmed that 6-OHDA concentration dependently induced activation of caspases 3/7 between 10 and 1000 µM in PC12 cells (Fig. 4A); activation was abolished by the pan-caspase inhibitor z-VAD-FMK and the specific caspase 3/7 inhibitor Ac-DEVD-CHO (Fig. 4A, inset). Activation of these caspases was also assessed over time (Fig. 4B). Following 6-OHDA treatment, levels of active caspase 3/7 increased significantly between 0 and 6 h and gradually decreased between 6 and 24 h.

Caspase 3/7-dependent Proteolytic Activation of PKC{delta} Mediates 6-OHDA-induced Apoptosis—Treatment of PC12 cells with 6-OHDA at 150 µM induced proteolytic cleavage of the redox-sensitive, proapoptotic PKC{delta} (79 kDa) to give a catalytic fragment (41 kDa, Fig. 5A). Proteolytic cleavage was monitored in whole cell lysates between 0 and 24 h post 6-OHDA treatment: activation significantly (p < 0.001) increased between 0 and 6 h but decreased thereafter (Fig. 5, A and B). The time of peak activation coincided with that of mitochondrial dysfunction and caspase 3/7 activation (Figs. 1B and 4B).

Previous studies (27, 28, 36–47) have shown that both the full-length and catalytic fragment of PKC{delta} may translocate to, or accumulate in, specific cellular compartments in response to apoptotic stimuli. Therefore, we investigated translocation/activation of PKC{delta} in both cytosolic and nuclear fractions of PC12 cells after 6-OHDA treatment. Purity of nuclear and cytosolic fractions was demonstrated using lamin B and {alpha}-tubulin, respectively (Fig. 5C). In vehicle-treated cells full-length PKC{delta} was present in both fractions. Following treatment with 6-OHDA (150 µM; 15 min, and 6-h post-treatment incubation), the catalytic fragment was detected in both fractions, suggesting proteolytic activation in both subcellular locations.

Rottlerin, a PKC{delta}-specific inhibitor (48) concentration dependently inhibited 6-OHDA-induced cleavage of PKC{delta} (Fig. 6A). PKC{delta} is a substrate for caspases 3/7 (22, 24–31, 31–33, 49, 50), and both the pan caspase inhibitor z-VAD-FMK and the specific caspase 3/7 inhibitor Ac-DEVD-CHO reduced 6-OHDA-induced PKC{delta} cleavage by >85% (determined by densitometry of the PKC{delta} catalytic fragment from three separate experiments; Fig. 6B), consistent with its activation via the caspase 3/7 cascade.


Figure 2
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  		FIGURE 2.
Extracellular auto-oxidation of 6-OHDA causes toxicity in PC12 cells. Mitochondrial function (A, C, and F) and cell death (B and D) were assessed via MTT reduction assay and LDH release assay, respectively. Cells were preincubated for 30 min with NAC (0.01–5 mM, A and B), catalase (1–30 units/ml, C and D), GBR-12909 (2 µM), or nisoxetine (10 µM, F), and drugs remained throughout the 6-OHDA treatment (150 µM, 15 min) and 24-h post-treatment incubation. Data represent the mean ± S.E. of at least four independent experiments, each carried out with four (B and D) or eight (A, C, and F) replicates. Significantly different from vehicle: #, p < 0.05; ##, p < 0.01; and ###, p < 0.001; significantly different from 6-OHDA (150 µM) alone: *, p < 0.05; ***, p < 0.001; one-way ANOVA and post-hoc Tukey's test. The auto-oxidation of 6-OHDA in cell-free conditions was measured spectrophotometrically by monitoring the formation of p-quinone at 490 nm (E). The assay was carried out in a cell-free system, but conditions corresponded to cellular 6-OHDA treatments. Readings were taken every 20 s for 10 min following the addition of 6-OHDA (final concentration, 150 µM) into DMEM alone or DMEM containing NAC (5 mM) or catalase (300 units/ml). Data represent the mean ± S.E. of three independent experiments. {square}, 6-OHDA (150 µM) plus catalase (300 units/ml); •, 6-OHDA (150 µM); and {blacktriangleup}, 6-OHDA (150 µM) plus NAC (5 mM).

 
We next examined if the activation of PKC{delta} contributes to 6-OHDA-mediated cell death in PC12 cells. The PKC{delta} inhibitor rottlerin (5 µM) significantly reduced 6-OHDA-induced LDH release by 41.5% (p < 0.001, Fig. 7B). A higher concentration of rottlerin (10 µM) did not afford protection; at this concentration rottlerin in the absence of 6-OHDA increased LDH release above that of vehicle alone (Fig. 7B), although this was not statistically significant. In contrast to its effect in ameliorating 6-OHDA-induced LDH release, 5 µM rottlerin did not significantly increase mitochondrial function in comparison to 6-OHDA alone (Fig. 7A), although there was a trend toward increased mitochondrial function at this concentration. The selective classic PKC inhibitor, Gö6976, did not affect 6-OHDA-induced changes in mitochondrial function or LDH release when used between 30 and 300 nM, concentrations that selectively inhibit the classic PKC isoforms ({alpha}, betaI, betaII, and {gamma} (51)) (Fig. 7, C and D). This suggests that the classic PKC isoforms are not involved in 6-OHDA-induced apoptosis.

A number of studies have questioned the specificity of rottlerin for PKC{delta} and suggested that rottlerin itself may induce toxicity (52, 53), which may explain the increase in LDH release observed at 10 µM rottlerin in this study (Fig. 7B). In light of this, siRNA-mediated gene suppression of PKC{delta} was also used to examine the contribution of PKC{delta} to 6-OHDA toxicity. Treatment of PC12 cells with the pKD-PKC{delta}-v3 expression plasmid reduced protein expression of PKC{delta} by 61.2 ± 4.1% 96 h post transfection, compared with cells transfected with the negative control plasmid (pKD-NegCon-v1, Fig. 8, A and B). The reduction in the protein level of PKC{delta} was accompanied by a significant attenuation of both loss of mitochondrial function and LDH release following treatment with 6-OHDA (Fig. 8, C and D), confirming the significant role of PKC{delta} in 6-OHDA-induced neurotoxicity in PC12 cells.


Figure 3
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  		FIGURE 3.
6-OHDA-induced apoptosis in PC12 cells. Apoptosis was assessed using Hoechst 33258. PC12 cells were treated with vehicle alone (A), vehicle containing staurosporine (1 µM, B), or vehicle containing 6-OHDA (150 µM, C) as described under "Methods." Nuclear staining was visualized by fluorescence microscopy. Numbers of apoptotic cells (distinguished by the presence of bright, fragmented nuclei) were determined in 10 random fields/slide from each of three independent experiments and expressed as a percentage of the total number of cells (D). Data represent the mean ± S.E. Significantly different from vehicle: #, p < 0.05; ##, p < 0.01; Student's unpaired t test.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The data presented in this study demonstrate that the commonly used Parkinsonian neurotoxin, 6-OHDA, induced neurotoxicity in PC12 cells via extracellular auto-oxidation and consequent oxidative stress. It is predicted that the oxidative products of 6-OHDA initiated intracellular oxidative stress, and the attenuation of this toxicity by catalase implicates hydrogen peroxide as a key mediator of 6-OHDA-induced cell death. A combination of both necrotic and apoptotic cell death mechanisms were triggered, including activation of the mitochondrial-caspase 3/7 apoptotic cascade. Caspase 3 induced the proteolytic cleavage of the redox-sensitive protein kinase, PKC{delta}, in both the cytosol and the nucleus, and inhibition of PKC{delta} reduced 6-OHDA-mediated neurotoxicity.

6-OHDA Toxicity Is Mediated by Extracellular Auto-oxidation in PC12 Cells—Oxidative stress is a key mediator of neurodegeneration in PD, and this is thought to be replicated by the neurotoxin 6-OHDA, which is used to generate both in vivo and in vitro models for PD. The specificity of 6-OHDA for dopaminergic neurons is assumed to arise from its structural similarity to dopamine and consequent uptake via the DAT and accumulation within dopaminergic cells (9), which is supported by the attenuation of 6-OHDA toxicity by DAT uptake inhibitors both in vivo and in vitro (4, 54, 55). However, in this study inhibition of both the DAT and noradrenaline transporter, alone or in combination, did not reduce toxicity mediated by 6-OHDA, suggesting an alternative mechanism for induction of toxicity. In agreement with a number of previous reports (11, 12) we have shown that 6-OHDA does induce oxidative stress in vitro but that this occurs due to extracellular autooxidation of 6-OHDA (Fig. 2). The complete auto-oxidation of 6-OHDA within 10 min in a cell-free system (Fig. 2E), and its reduction by the thiol anti-oxidant NAC, suggests a mechanism for the neuroprotective action of this anti-oxidant, which has been shown to attenuate 6-OHDA neurotoxicity both in vitro (11, 56, 57) and in vivo (12, 58).

Auto-oxidation of 6-OHDA generates p-quinones, but also an array of other free radical species, such as hydrogen peroxide, superoxide anions, and hydroxyl radicals (10). The second anti-oxidant used in this study, catalase, did not prevent the oxidation of 6-OHDA (Fig. 2E) but is known to catalyze the breakdown of hydrogen peroxide. Catalase completely attenuated 6-OHDA-induced mitochondrial loss of function and cell death (Fig. 2, C and D), suggesting that hydrogen peroxide is a key mediator of 6-OHDA toxicity. This is consistent with other reports (11, 56), although one study has failed to demonstrate this (57). The lack of effect of the anti-oxidants when applied after the 15-min 6-OHDA application (i.e. during the 24-h post-treatment incubation), suggests that extracellular auto-oxidation of 6-OHDA and the subsequent increase in hydrogen peroxide and other free radicals species during the 15-min treatment period is sufficient to induce intracellular oxidative stress. Neither of the anti-oxidants used in this study are cell-permeable, reinforcing the finding that 6-OHDA is oxidized extracellularly; we propose that toxicity is mediated, at least in part, by hydrogen peroxide.

The data presented in this study explicitly demonstrate that an acute application of 6-OHDA elicits toxicity via extracellular auto-oxidation in PC12 cells. However, PC12 cells, like other cell lines or primary culture systems (e.g. ventral mesencephalic cultures) that are used to study 6-OHDA toxicity, have a number of limitations. These cells are either immortal cell lines that are prone to changes in phenotype during culture or are isolated, immature cells. Therefore the direct relevance of mechanisms identified in culture systems to in vivo models should not be taken for granted. In addition, application of 6-OHDA onto cells in culture medium may hasten the auto-oxidation of the toxin and so may not directly reflect the in vivo situation (11, 59). Whether toxicity is induced via DAT uptake and accumulation or via extracellular ROS generation in vivo is yet to be clarified, however, toxicity could be the result of a combination of these mechanisms. This hypothesis is supported by a recent study in which the early effects of 6-OHDA were studied on substantia nigra pars compacta neurons in midbrain slices (60). Although this was an in vitro system, the neurons were intact and mature in phenotype, in slices not maintained in culture medium. Nomifensine only partially inhibited the neurotoxic effects of 6-OHDA, suggesting that toxicity is not induced via DAT uptake alone and extracellular generation of ROS and quinones could also contribute.

PKC{delta} Is Proteolytically Activated in Response to 6-OHDA-induced Caspase 3/7 Activation—6-OHDA induces a combination of both necrotic and apoptotic cell death (10); the toxin induces morphological changes in PC12 cells that are typical of apoptosis, such as, cell shrinkage, membrane blebbing, and DNA fragmentation (13) and rapid cell lysis that is characteristic of necrotic cell death (14). This is consistent with the 6-OHDA-induced DNA fragmentation (Fig. 3) and loss of membrane integrity (Fig. 1) observed in the present study.

We demonstrated that the mitochondrial-caspase cascade is activated in response to an acute application of 6-OHDA in PC12 cells, which is consistent with previous reports that show caspase 3/7 inhibitors prevent 6-OHDA-induced neurotoxicity (6, 15–17). The caspase 3 and 7 pathway is initiated by mitochondrial membrane degradation and consequent cytochrome c release. This may occur via free radical-induced loss of mitochondrial membrane potential (17) or in response to the activation of proapoptotic proteins, such as Bax (61), which have been shown to mediate the release of mitochondrial proteins. Release of cytochrome c in response to 6-OHDA (19) induces the proteolytic activation of procaspase 9 (18, 20) and downstream activation of caspases 3 and 7 (5, 6, 18, 19). In the present study, 6-OHDA-induced activation of caspases 3 and 7 was maximal at the same time point (6 h) as maximal mitochondrial dysfunction, consistent with loss of mitochondrial membrane potential, cytochrome c release, and consequent activation of caspases 3 and 7.


Figure 4
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  		FIGURE 4.
6-OHDA induced activation of caspases 3/7. Activation of caspases 3/7 was assayed by monitoring the cleavage of a fluorescent substrate (see "Methods"). PC12 cells were incubated with 6-OHDA (10–1000 µM) for 15 min and caspase activation measured after 24 h (A) or with 6-OHDA (150 µM) for 15 min, and caspase activation was measured at intervals between 0 and 24 h (B). Caspase inhibitors (Ac-DEVD-CHO (100 µM) and z-VAD-FMK (10 µM)) were added for 30 min prior to 6-OHDA and remained on the cells during the remainder of the experiment (A inset). Data represent the mean ± S.E. of at least four independent experiments, each carried out with four replicates. Significantly different from caspase 3/7 activity at 0 h: #, p < 0.05; ###, p < 0.001; significantly different from 6-OHDA (150 µM) alone: ***, p < 0.001; one-way ANOVA and post-hoc Tukey's test.

 


Figure 5
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  		FIGURE 5.
6-OHDA-induced proteolytic activation of PKC{delta}. Activation of PKC{delta} was measured following treatment of PC12 cells with 6-OHDA (150 µM; 15 min), by Western blotting to disclose proteolytic cleavage of full-length PKC{delta} (79 kDa) to give a catalytic subunit (PKC{delta}-CF; 41 kDa). Both PKC{delta} full-length and the catalytic fragment were visualized using a rabbit polyclonal PKC{delta} antibody (Santa Cruz Biotechnology) mapping to the catalytic end of the protein (A and C). Whole cell lysates were prepared between 0 and 24 h after 6-OHDA treatment. Densitometry of time-dependent activation of PKC{delta} was carried out, and data are expressed as a percentage of vehicle at the corresponding time point (B). Significantly different from vehicle: #, p < 0.05; ###, p < 0.001; one-way ANOVA and post-hoc Tukey's test. Data points represent the mean ± S.E. of at least three independent experiments. 6-OHDA-induced proteolysis in cytosolic and nuclear fractions was determined 6 h after treatment with 6-OHDA (C). Purity of cytosolic and nuclear fractions was determined using {alpha}-tubulin and lamin B, respectively. V, vehicle-treated cells; T, 6-OHDA-treated cells; C, cytosolic fraction; N, nuclear fraction.

 


Figure 6
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  		FIGURE 6.
Inhibition of 6-OHDA-induced PKC{delta} activation by rottlerin and caspase inhibitors. At the time of peak PKC{delta} activation (6 h, Fig. 5), PKC{delta} proteolysis was measured in the presence of the PKC{delta}-specific inhibitor rottlerin (5 and 10 µM)(A) or caspase inhibitors Ac-DEVD-CHO (100 µM) or z-VAD-FMK (10 µM)(B). Each inhibitor was added 30 min prior to 6-OHDA treatment and remained throughout the experiment. Whole cell lysates were prepared, and Western blotting was used to visualize both the full-length (79 kDa) and catalytic fragment (PKC{delta}-CF, 41 kDa) of PKC{delta}, using a PKC{delta} antibody (Santa Cruz Biotechnology) mapping to the catalytic end of the protein (representative Western blots are shown in A and B). Densitometry of the PKC{delta} catalytic fragment was carried out, and data are expressed as a % of vehicle-treated samples at the corresponding time point. All data points represent the mean ± S.E. of at least three independent experiments. Significantly different from vehicle: ###, p < 0.001; significantly different from 6-OHDA (150 µM) alone: *, p < 0.05; **, p < 0.01, one-way ANOVA and post-hoc Tukey's test.

 
Although it is well described that the mitochondrial-caspase pathway of apoptosis is activated in response to 6-OHDA, the molecular targets downstream of this pathway are not so well characterized. In this study we have demonstrated the involvement of the proapoptotic, serine threonine protein kinase PKC{delta}, which is known to be proteolytically activated by caspase 3. Caspase 3-dependent proteolysis of PKC{delta} occurred in response to 6-OHDA treatment in PC12 cells: PKC{delta} cleavage was detectable 4 h post 6-OHDA treatment and was maximal by 6 h (Fig. 5). This temporal pattern was concomitant with caspase 3/7 activation (Fig. 4), and inhibition of caspases 3 and 7 abolished PKC{delta} proteolysis (Fig. 6), suggesting a caspase 3-dependent mechanism of activation.


Figure 7
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  		FIGURE 7.
6-OHDA-induced toxicity is decreased by rottlerin inhibition of PKC{delta}. The effect of inhibiting PKC{delta} or cPKC isoforms, with rottlerin or Gö6976, respectively, on 6-OHDA-induced cell death and mitochondrial function was determined by MTT reduction assay (A and C) and LDH release assay (B and D). PC12 cells were preincubated with rottlerin (1–10 µM, A and B) or Gö6976 (30–300 nM, C and D) for 30 min prior to 6-OHDA treatment and remained throughout the experiment. Cells were treated with 6-OHDA (150 µM, 15 min) and incubated for 24 h. MTT (A and C) or LDH (B and D) assays were carried at 24 h post-treatment. Data represent the mean ± S.E. of at least five independent experiments, each carried out with eight (A and C) or four (B and D) replicates. Significantly different from vehicle: ###, p < 0.001; significantly different from 6-OHDA (150 µM) alone: ***, p < 0.001; one-way ANOVA and post-hoc Tukey's test.

 


Figure 8
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  		FIGURE 8.
6-OHDA-induced toxicity is decreased by RNA interference-mediated gene suppression of PKC{delta}. PC12 cells were transfected with the mammalian expression plasmid pKD-PKC{delta}-v3, which when expressed and following processing forms a PKC{delta} siRNA. Alternatively, PC12 cells were transfected with the negative control version of the plasmid, pKD-NegCon-v1, which forms a negative control siRNA. To confirm knockdown, cells were transfected, cell lysates were prepared at 48, 72, and 96 h post transfection, and protein levels were assessed via Western blotting with a PKC{delta} antibody (A). Densitometry was carried out for each time point, and data are expressed as a percentage of the PKC{delta} protein level of cells transfected with the negative control siRNA at the corresponding time point (B). The effect of inhibiting PKC{delta} with RNA-interference-mediated gene suppression on 6-OHDA-induced cell death and mitochondrial function was determined by MTT reduction assay (C) and LDH release assay (D). Cells were transfected with the pKD-PKC{delta}-v3 or pKD-NegCon-v1 expression plasmids for 72 h before being treated with 6-OHDA (150 µM, 15 min) and incubated for a further 24 h. MTT (C) or LDH (D) assays were carried at 24 h post 6-OHDA treatment. Data represent the mean ± S.E. of three (A and B) or four (C and D) independent experiments, each carried out with four (C and D) replicates. Significantly different from vehicle: ###, p < 0.001; significantly different from 6-OHDA (150 µM) alone: ***, p < 0.001, one-way ANOVA and post-hoc Tukey's test.

 
Caspase 3-dependent cleavage of PKC{delta} and release of the constitutively active catalytic fragment occurs in response to a variety of stimuli, including several dopaminergic neurotoxins (22, 24–33, 50). Overexpression of PKC{delta}, full-length or the catalytic fragment alone, is sufficient to induce apoptosis in the absence of stimuli (62). Similarly, overexpression of an inactive form of PKC{delta}, or knockdown of PKC{delta} expression, reduces DNA fragmentation and apoptosis in response to a range of stimuli and cell types (30, 32, 33, 44, 50). The proapoptotic effects of PKC{delta} have also been explored in PKC{delta} knock-out mice. Smooth muscle cells from PKC{delta}–/– aortae are resistant to cell death following treatment with several toxic stimuli and showed a reduction in cytochrome c release and caspase 3 activation (63).

The involvement of PKC{delta} proteolytic activation in 6-OHDA apoptosis was demonstrated by partial attenuation of 6-OHDA-induced neurotoxicity and prevention of caspase 3-dependent proteolysis of PKC{delta} by the PKC{delta} inhibitor rottlerin (48) (Fig. 7), consistent with its ability to reduce apoptosis following treatment with a variety of toxins in numerous cell lines (28, 30–32, 46, 50, 64). The mechanism by which rottlerin blocks PKC{delta} proteolysis and thereby inhibits PKC{delta}-dependent apoptosis is not clearly defined. Phosphorylation of tyrosine residues, including Tyr-311 and Tyr-332, is necessary for the apoptotic effect of PKC{delta} (28, 29, 65, 66), and inhibition of tyrosine phosphorylation has been implicated in the mode of action of rottlerin (52). However, there is some controversy surrounding the specificity of rottlerin for PKC{delta}, and several reports have suggested that rottlerin itself, rather than its effects on PKC{delta}, may affect cell viability (52, 53). This is compatible with the trend toward a decrease in mitochondrial function and membrane integrity observed when rottlerin was applied alone at the highest concentration tested (Fig. 7). However, RNA interference-mediated gene suppression of PKC{delta} confirmed the proapoptotic function of PKC{delta} in this model (Fig. 8), whereas inhibition of the classic PKC isoforms ({alpha}, betaI, betaII, and {gamma}) had no effect on 6-OHDA-induced toxicity (Fig. 7), showing that this effect is specific to the PKC{delta} isoform.

Upon activation, PKC{delta} has been shown to translocate to several subcellular locations, including the membrane (37, 45, 46), mitochondria (39, 42–44, 67), Golgi (41), and the nucleus (27, 28, 36, 38, 40). The localization of both the full-length and catalytic fragment of PKC{delta} to the nucleus of 6-OHDA-treated PC12 cells (Fig. 5) suggests that caspase 3-dependent activation of PKC{delta} occurs in both the cytosol and the nucleus. This activation may occur within the nucleus (full-length PKC{delta} is observed in the nucleus of non-treated cells), or PKC{delta} catalytic fragment may translocate to the nucleus following activation. The nuclear localization signal of PKC{delta} has previously been identified, and mutation within this region inhibited nuclear accumulation of PKC{delta} and induction of apoptosis (27). Tyrosine phosphorylation of PKC{delta} may be involved in translocation to the nucleus. Yuan et al. (36) have shown that, in response to ionizing radiation, the protein-tyrosine kinase c-Abl phosphorylates PKC{delta} and may induce translocation to the nucleus.

Localization of activated PKC{delta} within the nucleus suggests that it may play a role in the apoptotic disassembly of the nuclear structure and DNA fragmentation. Putative PKC{delta} substrates within the nucleus include DNA-protein kinase (67) and lamin B (40). Phosphorylation of DNA-protein kinase by the catalytic fragment of PKC{delta} inhibits its interaction with DNA and therefore its ability to repair double-stranded DNA (67). Phosphorylation of lamin B may facilitate proteolysis of lamin proteins and nuclear disassembly (40). In addition, the catalytic fragment of PKC{delta} has been shown to phosphorylate p73beta (a structural and functional homologue of p53), which regulates transcription of genes involved in apoptosis (68). PKC{delta} activated in the cytosolic fraction could be involved in the proapoptotic effects of PKC{delta} following translocation to the mitochondria, which has been reported in a number of studies (39, 42, 43). Several reports have shown that PKC{delta} has a positive feedback effect on caspase 3 activation, suggesting that PKC{delta} could induce mitochondrial dysfunction and consequent caspase 3 activation (30–32), perhaps via the phosphorylation of mitochondrial proteins and enhanced ROS production (47).


Figure 9
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  		FIGURE 9.
Model of 6-OHDA-mediated apoptosis in PC12 cells. 6-OHDA is extracellularly oxidized, and the oxidative products generated mediate its toxicity, in particular hydrogen peroxide. Toxicity is not elicited via uptake of 6-OHDA by monoamine transporters. Oxidative stress induces mitochondrial dysfunction, consequent release of cytochrome c, and activation of caspases 3/7. PKC{delta} is proteolytically activated by caspases 3/7 in both the cytosol and nucleus to produce a constitutively active catalytic fragment (PKC{delta}-CF). Inhibition of PKC{delta} by rottlerin or RNA interference-mediated gene suppression (siRNA) reduces 6-OHDA-induced apoptosis.

 
A model for 6-OHDA-induced activation of PKC{delta} in PC12 cells is presented in Fig. 9. 6-OHDA toxicity is elicited via extracellular autooxidation, which can be blocked by the thiol anti-oxidant NAC. 6-OHDA does not enter PC12 cells via dopamine transporter-mediated uptake in these cells. The oxidation products of 6-OHDA are membrane-permeable and so can enter the cell and induce oxidative stress. Oxidative stress can be blocked by catalase, suggesting that hydrogen peroxide is a key mediator of toxicity. Oxidative stress causes mitochondrial dysfunction, release of cytochrome c, and activation of caspases 3 and 7. Active caspases 3 and 7 proteolytically cleave PKC{delta} generating the constitutively active PKC{delta} catalytic fragment in both the nucleus and the cytosol. PKC{delta} can then phosphorylate nuclear substrates leading to breakdown of the nuclear structure and DNA fragmentation.

The finding that inhibition of PKC{delta} attenuates 6-OHDA toxicity suggests a major role for this kinase in 6-OHDA-induced neurodegeneration in vitro. The relevance of PKC{delta} activation in neurodegeneration in Parkinsonian in vivo models is yet to be determined; however PKC{delta} is highly expressed in the brain (69), particularly in dopaminergic regions, including the striatum and substantia nigra pars compacta (70). In addition, CNS expression of PKC{delta} increases with age (71). Both these factors indicate that PKC{delta} could be a mediator of Parkinsonian neurodegeneration in vivo.


   FOOTNOTES
 
* This work was supported by a Biological and Biotechnological Sciences Research Council Cooperative Award in Science and Engineering Studentship in conjunction with Eli Lilly & Co. (to K. H.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 To whom correspondence should be addressed: Tel.: 44-1225-385-870; Fax: 44-1225-386-779; E-mail: S.Wonnacott{at}bath.ac.uk.

2 The abbreviations used are: PD, Parkinson disease; ROS, reactive oxygen species; 6-OHDA, 6-hydroxydopamine; DAT, dopamine transporter; PKC, protein kinase C; DMEM, Dulbecco's modified Eagle's medium; NAC, N-acetyl-L-cysteine; MTT, 3-[4,5-dimethylthiazol-2-y]-2,5-diphenyltetrazolium bromide; z-VAD-FMK, benzyloxycarbonyl-VAD-fluoromethyl ketone; LDH, lactate dehydrogenase; PBS, phosphate-buffered saline; siRNA, small interference RNA; ANOVA, analysis of variance; DETAPAC, diethylenetriaminepentaacetic acid. Back



   REFERENCES
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 DISCUSSION
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