Caspase-3-Dependent Proteolytic Cleavage of Protein Kinase C? Is
Essential for Oxidative Stress-Mediated Dopaminergic Cell Death
after Exposure to Methylcyclopentadienyl Manganese Tricarbonyl
Vellareddy Anantharam, Masashi Kitazawa, Jarrad Wagner, Siddharth Kaul, and Anumantha G. Kanthasamy
Parkinson Disorders Research Program, Department of Biomedical Sciences, Iowa Sate University, Ames, Iowa 50011
In the present study, we characterized oxidative stress-dependent
cellular events in dopaminergic cells after exposure to an organic
form of manganese compound, methylcyclopentadienyl manga-
nese tricarbonyl (MMT). In pheochromocytoma cells, MMT expo-
sure resulted in rapid increase in generation of reactive oxygen
species (ROS) within 5–15 min, followed by release of mitochon-
drial cytochrome C into cytoplasm and subsequent activation of
caspase-3 (15- to 25-fold), but not caspase-8, in a time- and
dose-dependent manner. Interestingly, we also found that MMT
exposure induces a time- and dose-dependent proteolytic cleav-
age of native protein kinase C? (PKC?, 72–74 kDa) to yield 41 kDa
catalytically active and 38 kDa regulatory fragments. Pretreatment
with caspase inhibitors (Z-DEVD-FMK or Z-VAD-FMK) blocked
MMT-induced proteolytic cleavage of PKC?, indicating that cleav-
age is mediated by caspase-3. Furthermore, inhibition of PKC?
activity with a specific inhibitor, rottlerin, significantly inhibited
caspase-3 activation in a dose-dependent manner along with a
reduction in PKC? cleavage products, indicating a possible posi-
tive feedback activation of caspase-3 activity by PKC?. The pres-
ence of such a positive feedback loop was also confirmed by
delivering the catalytically active PKC? fragment. Attenuation of
ROS generation, caspase-3 activation, and PKC? activity before
MMT treatment almost completely suppressed DNA fragmenta-
PKC?K376R(dominant-negative mutant) prevented MMT-induced
apoptosis in immortalized mesencephalic dopaminergic cells. For
the first time, these data demonstrate that caspase-3-dependent
proteolytic activation of PKC? plays a key role in oxidative stress-
mediated apoptosis in dopaminergic cells after exposure to an
environmental neurotoxic agent.
Key words: apoptosis; oxidative stress; Parkinson’s disease;
environmental factors; manganese; dopaminergic degeneration
Parkinson’s disease (PD) is an idiopathic neurodegenerative dis-
order characterized by profound loss of dopaminergic neurons in
the nigrostriatal tract. Although debated, most studies have con-
cluded that aging, environmental neurotoxicant exposures, and
genetic alterations are potential risk factors in the development of
PD (Oertel and Kupsch, 1993; Langsten and Hill, 1998; Aschner,
2000; Simon et al., 2000). Recently, a study conducted on
thousands of twins concluded that genetic factors do not play a role
in the pathogenesis of geriatric onset of PD, which further supports
the view that environmental factors are dominant risk factors
in the etiology of PD (Tanner et al., 1999). Results of several
epidemiological studies conducted in rural areas have also sug-
gested that certain pesticides and other environmental factors,
including transition metals such as manganese, have a positive
association with increased incidences of PD (Seidler et al., 1996;
Liou et al., 1997; Gorell et al., 1999). Occupational exposure to
manganese during mining was shown to cause a Parkinson’s-like
syndrome known as Manganism (Mena et al., 1967; Barbeau, 1984;
Donaldson, 1987; Gorell et al., 1999). Furthermore, exposure to
manganese-containing compounds such as manganese ethylene-
bis-dithiocarbamate (a fungicide) and Bazooka (a cocaine-based
drug) among farm workers and abusers, respectively, has been
shown to result in adverse neurological defects (Roels et al., 1987;
Ferraz et al., 1988; Wang et al., 1989; Thiruchelvam et al., 2000).
Methylcyclopentadienyl manganese tricarbonyl (MMT) has
been used in Canada as an anti-knock gasoline agent and has
been recently legalized for use in the United States as a replace-
ment for tetraethyl lead [(CH3CH2)4Pb] in gasoline (Lynam et al.,
1999; Zayed et al., 1999). Because MMT is a manganese-
containing compound, its use has raised great a concern regarding
increased exposure to the public and its possible adverse health
effects (Frumkin and Solomon, 1997; Davis, 1998; Lynam et al.,
1999; Zayed et al., 1999). Exposure to MMT produces a pro-
longed and more pronounced accumulation of manganese in rat
brain as compared with manganese derived from an inorganic
source, for example, MnCl2(Zheng et al., 2000). Administration
of MMT produces seizures in mice (Fishman et al., 1987) and
also results in depletion of dopamine in the mouse striatum
(Gianutsos and Murray, 1982). Furthermore, MMT administra-
tion has been shown to be an effective inhibitor of complex I in
mitochondrial electron transport chain (Autissier et al., 1977), an
action similar to the pyridinium metabolite of 1-methyl-4-phenyl-
1,2,3,6-tetrahydropyridine (MPTP), a Parkinsonian toxin. Re-
cently, we demonstrated that MMT exposure induces reactive
oxygen species (ROS) generation, dopamine depletion, and cell
death in dopamine-producing rat pheochromocytoma (PC12)
Received Oct. 26, 2001; revised Dec. 7, 2001; accepted Dec. 12, 2001.
This work was supported by the National Institute of Environmental Health
Sciences Grant RO1-ES10586. We acknowledge Dr. Palur Gunasekar (Operational
Toxicology, Air Force Research Laboratories, Dayton, OH) for his initial assistance
in some experiments. We thank Dr. Michael L. Kirby, Dr. Arthi Kanthasamy, and
Mr. Siddharth Ranade in the preparation of this manuscript and Dr. Donghui Cheng
for help with flow cytometry.
Correspondence should be addressed to Dr. A. G. Kanthasamy, Parkinson
Disorders Research Laboratory, Department of Biomedical Sciences, 2062 Vet-
erinary Medicine Building, Iowa Sate University, Ames, IA 50011. E-mail:
J. Wagner’s present address: Department of Chemistry, California State Univer-
sity, Fresno, CA.
Copyright © 2002 Society for Neuroscience 0270-6474/02/221738-14$15.00/0
The Journal of Neuroscience, March 1, 2002, 22(5):1738–1751
cells, which can be protected by pretreatment with antioxidants
(Wagner et al., 2000). To further understand the cellular mech-
anism of MMT-mediated apoptosis, we investigated whether
oxidative stress induced by MMT can activate a series of cellular
factors associated with apoptotic pathways, which could subse-
quently lead to programmed cell death in dopaminergic cells.
Herein, we report that MMT exposure activates a novel apoptotic
pathway in dopaminergic cells through caspase-3-dependent pro-
teolytic cleavage of PKC?.
MATERIALS AND METHODS
Reagents. MMT was obtained from Sigma-Aldrich (St. Louis, MO);
rottlerin was purchased from Calbiochem (San Diego, CA); acetyl-Asp-
Glu-Val-Asp-aldehyde (Ac-DEVD-CHO), acetyl-Iso-Glu-Thr-Asp-7-
amino-4-methylcoumarin (Ac-IETD-AMC), acetyl-Leu-Glu-His-Asp-7-
amino-4-methylcoumarin (Ac-LEHD-AMC), and Z-Asp-Glu-Val-Asp-
fluoromethyl ketone (Z-DEVD-FMK) were obtained from Alexis
Biochemicals (San Diego, CA); Z-Val-Ala-Asp-fluoromethyl ketone (Z-
VAD-FMK) was obtained from Enzyme Systems (Livermore, CA).
was obtained from Bachem (King of Prussia, PA); fluorescein isothiocya-
nate conjugated to VAD-FMK (FITC-VAD-FMK) was purchased from
Promega (Madison, WI); antibodies to PKC?, PKC?, PKC?I, and PKC?II
were purchased from Santa Cruz Biotechnology (Santa Cruz, CA), cyto-
chrome C (mouse monoclonal) from PharMingen (San Diego, CA), green
fluorescent protein (GFP) (mouse monoclonal) from Clontech (Palo Alto,
CA), and ?-actin (mouse monoclonal) from Sigma (St. Louis, MO). ECL
chemiluminescence kit was purchased from Amersham Pharmacia Biotech
(Piscataway, NJ). PC12 cells were purchased from American Type Culture
Collection (ATCC) (Rockville, MD), and immortalized rat mesencephalic
dopaminergic neuronal cell line (1RB3AN27) was a kind gift of Dr. Kedar
N. Prasad (University of Colorado Health Sciences Center, Denver, CO).
Hydroethidine and Hoechst 33342 were purchased from Molecular Probes
(Eugene, OR). Cell Death Detection ELISA Plus assay kit was purchased
from Roche Molecular Biochemicals (Indianapolis, IN). PKC? catalytic
fragment, acridine orange, histone H1, ?-glycerophosphate, superoxide
dismutase (SOD), ATP, Protein-A-Sepharose, phosphatidylserine, and dio-
leoylglycerol were purchased from Sigma. Mn(III)tetrakis(4-Benzoic acid-
)porphyrin chloride (MnTBAP) was purchased from Oxis Health Products
(Portland, OR). [?-32P]ATP was purchased from NEN (Boston, MA).
Bradford protein assay kit was purchased from Bio-Rad (Hercules, CA).
Lipofectamine Plus reagent, Roswell Park Memorial Institute (RPMI)-
1640 medium, horse serum, fetal bovine serum, L-glutamine, penicillin,
streptomycin, and PCEP4 plasmid were purchased from Invitrogen (Gaith-
ersburg, MD). BioPORTER, protein delivery reagent was purchased from
Gene Therapy Systems (San Diego, CA), and plasmids PKC?K376-GFP
fusion protein and pEGFP-N1 were kind gifts of Dr. Stuart Yuspa (Na-
tional Cancer Institute, Bethesda, MD).
Cell culture. PC12 (ATCC CRL1721) cells were grown in RPMI
medium supplemented with 10% horse serum, 5% fetal bovine serum,
1% L-glutamine, penicillin (100 U/ml), and streptomycin (100 U/ml) and
maintained at 37°C in a humidified atmosphere of 5% CO2. Immortal-
ized rat mesencephalic cells (1RB3AN27) were grown in RPMI medium
supplemented with 10% fetal bovine serum, 1% L-glutamine, penicillin
(100 U/ml), and streptomycin (100 U/ml), maintained at 37°C in a
humidified atmosphere of 5% CO2(Prasad et al., 1998).
Stable transfection. Plasmid pPKC?K376R-GFP encodes protein kinase
C?-GFP fusion protein, the number K376R refers to the mutation of
lysine residue at position 376 to arginine in the catalytic site of PKC?
rendering it inactive (Li et al., 1999). Plasmid pEGFP-NI encodes the
green fluorescent protein alone and used as vector control. pEGFP-N1
and pPKC?K376R were transfected into 1RB3AN27cells using Lipo-
fectamine Plus reagent according to the procedure recommended by the
manufacturer. In brief, 8 ?g of DNA, 24 ?l of lipid, and 24 ?l of Plus
reagent were used to transfect 1RB3AN27cells in 100 mm tissue culture
dishes at 50% confluency in 4 ml of culture medium without serum. Fresh
medium containing serum was added 3 hr later. For stable cell lines, the
1RB3AN27cells were selected in 400 ?g/ml hygromycin, 48 hr after
cotransfection with PCEP4 plasmid, which confers hygromycin resis-
tance. Colonies were isolated with trypsin and glass cloning cylinders,
and they were then replated and grown to confluence in T75 flasks.
Subsequently, the stable cell lines were maintained in 200 ?g/ml
Treatment paradigm. After 2–4 d in culture, PC12 cells and 1RB3AN27
were harvested and resuspended in serum-free growth medium at a cell
density of 1–3 ? 106/ml. Cell suspensions were treated with varying
concentrations of MMT (30–500 ?M) over a period of 0.5–5 hr at 37°C.
In inhibitor studies SOD (ROS inhibitor, 100 U/ml), MnTBAP (ROS
inhibitor, 10 ?M), rottlerin (PKC? inhibitor, 5–20 ?M), Ac-DEVD-CHO
(caspase-3-specific inhibitor, 100–300 ?M), Z-DEVD-FMK (caspase-3-
specific inhibitor, 10–50 ?M), or Z-VAD-FMK (a broad spectrum
caspase inhibitor, 30–100 ?M) were added 30–90 min before the addition
of MMT. The reaction samples were removed at 0.25, 0.5, 1, 2, 3, and 5
hr, then spun at 200 ? g, and after 5 min, the cell pellets were used for
assessing cytochrome C release, caspase-3, caspase-8, and caspase-9
enzymatic activities, extent of PKC? cleavage, and DNA fragmentation.
Dimethylsulfoxide (DMSO) (0.5–1%) was used as a vehicle in control
Lactate dehydrogenase assay. Lactate dehydrogenase (LDH) activity in
the cell-free extracellular supernatant was quantified as an index of cell
death (Vassault, 1983). We modified the original method to a 96-well
format (Kitazawa et al., 2001). Briefly, PC12 cells were plated in 96-well
plate, and after treatment 10 ?l of the extracellular supernatant was
added to 200 ?l of 0.08 M Tris buffer, pH 7.2, containing 0.2 M NaCl, 0.2
mM NADH, and 1.6 mM sodium pyruvate. LDH activity was measured
continuously by monitoring the decrease in the rate of absorbance at 339
nm using a microplate reader (Molecular Devices, Sunnyvale, CA), and
the temperature was maintained at 37°C during reading. Changes in
absorbance per minute (?A/?T) were used to calculate LDH activity
(U/I), using the following equation: U/I ? (?A/?T) ? 9682 ? 0.66, where
9682 was a coefficient factor, and 0.66 was a correction factor at 37°C.
Detection of reactive oxygen species and lipid peroxidation by flow cytom-
etry. Flow cytometry analysis was performed on a Becton Dickinson (San
Francisco, CA) FACScan flow cytometer. Hydroethidine, a sodium
borohydride-reduced derivative of ethidium bromide, is used to detect
ROS produced specifically inside the cell (Narayanan et al., 1997). When
hydroethidine is loaded in the cells, it binds to cellular macromolecules.
and increases red fluorescence (620 nm). A 15 mW air-cooled argon–ion
laser was used as an excitation source for hydroethidine at 488 nm, and
the optical filter was 585/42 nm bandpass. Cells were detected and
distinguished from the background by forward-angle light scattering
and orthogonal light scattering characteristics. All the flow cytometric
data were analyzed by Cellquest data analysis software to determine the
significant increase or decrease of fluorescence intensity.
PC12 cells and engineered 1RB3AN27cells expressing kinase inactive
PKC? protein were resuspended with HBSS with 2 mM calcium at a
density of 0.5 ? 106cells/ml. Cells were then incubated with 10 ?M
hydroethidine for 15 min at 37°C in the dark to allow dye loading into the
cells. After incubation with dye, excess dye was removed, and the cells
were resuspended with HBSS. After addition of MMT (30–500 ?M) ROS
generation was measured at 0, 5, 15, 30, and 45 min after the exposure.
In inhibitor studies, cells were incubated with SOD (100 U/ml) and
MnTBAP (10 ?M) 10–30 min before MMT exposure.
Quantification of cytochrome C release. Cytochrome C release was
quantified using a recently developed ELISA kit developed by MBL
(Watertown, MA). This is a fast, highly sensitive and reliable assay for
the detection of early changes in cytochrome C levels. Briefly, after 2–4
d in culture, PC12 cells were harvested and resuspended in serum-free
growth medium at a cell density of 5 ? 106/ml. Cell suspensions were
exposed to 200 and 500 ?M MMT for 15–30 min at 37°C. After treatment
the cells were spun at 200 ? g, and after 5 min, washed once with 1?
ice-cold PBS and resuspended in 1 ml of ice-cold homogenization buffer
(10 mM Tris HCl, pH 7.5, 0.3 M sucrose, 1 mM phenylmethylsulfonyl
fluoride, 25 ?g/ml aprotinin, and 10 ?g/ml leupeptin) and homogenized
on ice. Cells were then centrifuged for 10,000 ? g for 60 min at 4°C. The
resulting supernatants were collected as cytoplasmic fraction and used
for cytochrome C release measurements. The MBL ELISA kit measures
cytochrome C by one-step sandwich ELISA. The assay uses affinity-
purified two polyclonal antibodies against cytochrome C. The cytoplas-
cytochrome C polyclonal antibody in the 96-well microtiter for 60 min at
room temperature (RT). After washing with buffer (provided with the
kit), the peroxidase substrate is mixed with the chromogen and allowed
to incubate for an additional 15 min. An acid solution provided with the
kit is then added to each well to terminate the enzyme reaction and to
stabilize the developed color. The optical density of each well is then
measured at 450 nm using a microplate reader. The concentration of
?is generated, it converts hydroethidine to ethidium bromide
Anantharam et al. • MMT Cleaves Protein Kinase C?
J. Neurosci., March 1, 2002, 22(5):1738–1751 1739
tively active PKC? fragment can promote loss of cellular regula-
tory function in many of its substrates, resulting in rapid apopto-
sis. Currently, our laboratory is focusing on identifying critical
cellular targets of PKC? that might contribute to apoptotic cell
death in dopaminergic cells.
In conclusion, we demonstrate for the first time that an envi-
ronmental neurotoxicant, MMT, induces dopaminergic degener-
ation by a novel oxidative stress-mediated apoptotic mechanism
in which caspase-3-dependent proteolytic cleavage of PKC? plays
a critical role (Fig. 13). Our data also demonstrate a positive
feedback amplification loop between PKC? and capsase-3, which
has a regulatory role in the promotion of apoptosis. Further
research into identifying molecules that participate in this loop
might provide very exciting information regarding cell signaling
and neuronal apoptosis. Finally, this study emphasizes the im-
portance of characterizing oxidative stress-induced cell signaling
molecules after neurotoxicant exposure to better understand the
role of environmental risk factors in the pathogenesis of Parkin-
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