TOXICOLOGICAL SCIENCES 86(1), 116–124 (2005)
doi:10.1093/toxsci/kfi164
Advance Access publication March 30, 2005
Enhancement of Cyanide-Induced Mitochondrial Dysfunction and
Cortical Cell Necrosis by Uncoupling Protein-2
L. Li,* K. Prabhakaran,* E. M. Mills,†,2 J. L. Borowitz,* and G. E. Isom*,1
*Department of Medicinal Chemistry & Molecular Pharmacology, Purdue University, West Lafayette, Indiana 47907–1333; †Cardiovascular Branch,
National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892
Received February 1, 2005; accepted March 28, 2005
Uncoupling protein 2 (UCP-2) is expressed in the inner
mitochondrial membrane and modulates mitochondrial function
by partially uncoupling oxidative phosphorylation, and it has been
reported to modulate cell death. Cyanide is a potent neurotoxin
that inhibits complex IV to alter mitochondrial function to induce neuronal death. In primary rat cortical cells KCN produced
an apoptotic death at 200–400 mM. Higher concentrations of
potassium cyanide (KCN) (500–600 mM) switched the mode of
death from apoptosis to necrosis. In necrotic cells, ATP levels were
severely depleted as compared to cortical cells undergoing
apoptosis. To determine if UCP-2 expression could alter KCNinduced cell death, cells were transiently transfected with fulllength human UCP-2 cDNA (UCP-2+). Overexpression switched
the mode of death produced by KCN (400 mM) from apoptosis to
necrosis. The change in cell death was mediated by impaired
mitochondrial function as reflected by a marked decrease of ATP
levels and reduction in mitochondrial membrane potential. RNA
interference or transfection with a dominant interfering mutant
blocked the necrotic response observed in UCP-2+ cells. Additionally, treatment of UCP-2+ cells with cyclosporin A blocked
necrosis, indicating the involvement of mitochondrial permeability pore transition in the necrotic death. These results show that
increased expression of UCP-2 alters the response to a potent
mitochondrial toxin by switching the mode of cell death from
apoptosis to necrosis. It is concluded that UCP-2 levels influence
cellular responses to cyanide-induced mitochondrial dysfunction.
Key Words: apoptosis; cyanide; necrosis; mitochondria; uncoupling protein.
INTRODUCTION
Uncoupling protein 2 (UCP-2) is a member of the mitochondrial anion carrier superfamily that is expressed in the
inner mitochondrial membrane (Busquets et al., 2001; Rousset
1
To whom correspondence should be addressed at Department of Medicinal
Chemistry & Molecular Pharmacology, Purdue University, West Lafayette, IN
47907–1333. Fax: (765) 404-1414. E-mail: [email protected].
2
Present address: University of Texas at Austin, Division of Pharmacology
and Toxicology, College of Pharmacy, Austin, TX 7812–1074.
et al., 2004). In the mitochondrion, UCP-2 partly dissipates the
proton electrochemical gradient by enhancing proton leakage
from the inner membrane into the matrix. The mild uncoupling
leads to a reduction of mitochondrial membrane potential
(DWm), a decrease of mitochondrial Ca2þ uptake, and a reduction of both cellular ATP production and reactive oxygen
species (ROS) generation. Uncoupling proteins are thought to
regulate energy metabolism and thermogenesis in peripheral
tissue. In brain, UCP-2 is constitutively expressed in hippocampus, amygdala, and limbic cortex, but its physiological
function is not known (Sullivan et al., 2003).
UCP-2 is both a death and survival factor, dependent on the
initiation stimulus and the death pathway executed (Duval
et al., 2002; Rashid et al., 1999). Hypoxic stress can induce
expression of UCP-2, which may be an adaptive mechanism to
conditions predisposing to oxidative stress (Pecqueur et al.,
2002). Activation or overexpression of UCP-2 reduces reactive
oxygen species (ROS) generation, thereby protecting against
oxidative stress generated in mitochondria (Negre-Salvayre
et al., 1997; Teshima et al., 2003). In contrast, decreased
expression of UCP-2 by antisense oligonucleotide treatment
increases ROS generation and enhances oxidative injury
(Duval et al., 2002). UCP-2 is neuroprotective by modulating
cellular redox signaling (de Bilbao et al., 2004) or inducing
mild mitochondrial uncoupling that prevents release of proapoptotic proteins (Mattiasson et al., 2003). Activation of
UCP-2 can decrease neuronal apoptosis that is mediated
through the caspase cascade (Bechmann et al., 2002). On the
other hand, high expression levels of UCP-2 can lead to a rapid
fall in DWm and reduction of both mitochondrial NADH and
ATP to selectively produce necrosis (Mills et al., 2002).
Expression of a dominant interfering mutant of UCP-2
conferred resistance to necrosis, but not apoptosis.
Cyanide is a rapid-acting mitochondrial toxicant that inhibits
cytochrome oxidase, thereby blocking the flow of electrons
through complex IV to block oxidative metabolism (Jones
et al., 2003) and to enhance ROS generation at complex III
(Chen et al., 2003). Depending on cell type and level of
oxidative stress, cyanide selectively produces either apoptosis or necrosis (Prabhakaran et al., 2002). Cyanide-induced
Ó The Author 2005. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved.
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UCP-2 AS A REGULATOR OF CELL DEATH
apoptosis and necrosis share common initiation stimuli, but the
death pathways are mediated by divergent intracellular cascades (Prabhakaran et al., 2002; Shou et al., 2003). The
apoptotic pathway is caspase-dependent and mediated by
mitochrondrial release of apoptogenic proteins (Shou et al.,
2004). Cells undergoing necrosis display a high level of
oxidative stress and rapid breakdown of mitochondrial function, characterized by onset of mitochondrial membrane
permeability transition and ATP depletion (Prabhakaran
et al., 2002). Because UCP-2 modulates mitochondrial function and has been linked to regulation of cell death, it was of
interest to determine the protein’s involvement in cyanideinduced cell death.
MATERIALS AND METHODS
Primary cell culture. Primary cortical cells (CX) were prepared from
embryonic day 16–17 Sprague-Dawley rats. In brief, cerebral cortices were
dissected and cells were dissociated in 0.025% trypsin at 37°C for 15 min.
Trypsin digestion was stopped by adding trypsin inhibitor and DNase I. Cells
were passed through a Pasteur pipette several times and plated at a density of
5 ± 105 cells/cm2 onto culture flasks or 6-well plates precoated with 10 lg/ml
poly-L-lysine. Cells were grown in Dulbecco’s modified Eagle’s medium
(DMEM) supplemented with 10% fetal bovine serum (FBS), 10% horse serum,
22 mM glucose, 2 mM glutamine, and 1 ml penicillin/streptomycin (10,000 U/ml)
at 37°C in an atmosphere of 5% CO2 and 95% air. For enriched cultures of postmitotic cortical neurons, cytosine arabinofuranoside (10 lM) was added at 6
days in vitro (DIV) to suppress proliferation of astrocytes. Medium was
changed twice a week until experiments were carried out at 12–14 DIV. For
treatment, cells were placed in 6-well plates and/or flasks, and potassium
cyanide (KCN) was added in a 10 ll volume so as to yield a 400 lM
concentration. Control cells were incubated under the same culture conditions
and times as treatment groups.
Evaluation of apoptosis (TUNEL staining). After transfections, cells
were treated with KCN (400 lM for 24 h) and/or pretreated with 1 lM
cyclosporin A (CsA) for 30 min before cyanide. Terminal deoxynucleotidyl
transferase (TdT)-mediated dUTP nick end-labeling (TUNEL) was performed
on paraformaldehyde (4% in phosphate buffered saline) fixed cells using the
Apoptag in situ apoptosis detection kit (Oncor, Gaithersburg, MD). Briefly,
cells were preincubated in equilibration buffer containing 0.1 M potassium
cacodylate (pH 7.2), 2 mM CaCl2, and 0.2 mM dithiothreitol for 10 min
at room temperature and then incubated in TUNEL reaction mixture at
pH 7.2 (containing 200 mM potassium cacodylate, 4 mM MgCl2, 2 mM 2mercaptoethanol, 30 lM biotin-16-2#-deoxyuridine-5#-triphosphate [dUTP],
and 300 U/ml TdT) in a humidified chamber at 37°C for 1 h. After incubation in
stop/wash buffer for 10 min, the elongated digoxigenin-labeled DNA fragments
were visualized using anti-digoxigenin peroxidase antibody solution, followed
by staining with DAB/H2O2 (0.2 mg/ml diaminobenzidine tetrachloride and
0.005% H2O2 in PBS, pH 7.4). Cells were then counterstained with
hematoxylin. In the microscopic field, the number of cells undergoing apoptosis
was determined by both TUNEL staining and characteristic apoptotic
morphology such as chromatin condensation and margination within the
nucleus and cell shrinkage. Based on morphology, astrocytic cells were
excluded from the counting.
The mode of cell death was confirmed by observation of the cell
morphology by electron microscopy. After treatment, cells were fixed by
replacing one half of the medium with 3% glutaraldehyde/PBS for 15 min. The
fixative–medium mixture was then replaced with fixative solution alone, and
cells were processed for transmission electron microscopy and photographed.
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Quantitation of necrotic cell death. Necrotic cell death was quantitated
using two DNA fluorescent dyes, SYTO-13 and propidium iodide (PI) by the
method of Ankarcrona et al. (1995). Both dyes bind DNA, but only SYTO-13 is
membrane permeable. Thus SYTO-13 stains normal cells with a green
fluorescence, whereas only cells with disrupted plasma membranes stain red
with PI. The percentage of non-astrocytic cells staining positive for PI was
determined as an estimate of necrosis. Microscopic examination showed these
cells exhibited necrotic morphology characterized by plasma membrane
damage and cytoplasmic vacuolization.
UCP-2 transient transfection and RNA interference. siRNA corresponding to the UCP-2 reporter gene was designed as recommended by Elbashir et al.
(2001) and synthesized by Ambion, Inc. (Austin, TX) with 5# phosphate,
3# hydroxyl, and two base overhangs on each strand. The gene-specific sequences
were used for UCP-2 interference: sense 5#-GAACGGGACACCUUUAGAGtt3# and antisense 5#-CUCUAAAGGUGUCCCGUUCtt-3#; annealing for duplex siRNA formation was performed as described by the manufacturer.
The full-length human UCP-2 cDNA (UCP-2þ) was subcloned into the
expression vector pCDNA3.1 as previously described (Mills et al., 2002).
Transient transfections of the UCP-2 plasmid and siRNA were performed with
Lipofectamine 2000 (Invitrogen, Carlsbad, CA) as described by Krichevsky
and Kosik (2002). Briefly, Lipofectamine diluted in Opti-MEM was applied to
the plasmids or siRNA and incubated for 45 min. To each microtiter plate well
containing cells, 2 lg of plasmid or 0.2 lg of 21-bp siRNA containing
Lipofectamine was applied in a final volume of 1 ml. The medium was changed
to regular CX cell culture medium 5 h after initiation of transfection, and cells
were treated with cyanide 24 h after transfection. The dominant interfering
mutant (UCP-2D212N) was a site-directed mutant of UCP-2 created by
substitution of position 212 (Asp-Asn) and was subcloned into the expression
vector pCDNA3.1 (Mills et al., 2002).
Measurement of mitochondrial membrane potential. DWm in cortical
cells was determined using the mitochondrial-specific fluorescent probe JC-1
based on the method of Reers et al. (1995). JC-1 exists as a green-fluorescent
monomer at low membrane potential (120 mV) and as a red fluorescent dimer
(J-aggregate) at membrane potential greater than 180 mV. Following excitation
at 485 nm, the ratio of red (595 nm emission) to green (525 nm emission)
fluorescence represents the ratio of high to low mitochondrial membrane
potential (Reers et al., 1995). After treatment CX cells were incubated with JC-1
(3.0 lM) for 30 min at 37°C in the dark and then washed twice with fresh PBS.
Fluorescence was measured with a plate reader at excitation 485 nm, emission
525 nm, and 595 nm.
Western blot analysis. After various treatments or transient transfection,
cells were washed with ice-cold PBS and harvested by scraping and then
centrifugation at 500 3 g for 5 min. Cell pellets were lysed in a buffer containing
220 mM mannitol; 68 mM sucrose; 20 mM HEPES, pH 7.4; 50 mM KCl; 5 mM
EGTA; 1 mM EDTA; 2 mM MgCl2; 1 mM dithiothreitol; 0.1% Triton-X100;
and protease inhibitors on ice for 15 min. After centrifugation, supernatants
were taken as whole cell protein extraction. The protein content in the
extractions was determined by the Bradford assay (Bio-Rad Laboratories,
Hercules, CA). The samples containing 30 lg protein were boiled in Laemmli
buffer for 5 min and subjected to electrophoresis in 12% sodium dodecyl sulfate
(SDS)-polyacrylamide gel, followed by transfer to a nitrocellulose membrane.
After blocking with Tris-buffered saline containing 5% nonfat dry milk and
0.1% Tween 20, the membrane was exposed to the primary UCP-2 antibody or
b-actin antibody for 3 h at room temperature on a shaker. The UCP-2 antibody
was a rabbit anti-mouse polyclonal antibody (diluted 1:2000) directed toward
the c-terminal domain of UCP-2 (Alpha Diagnostic International Inc., San
Antonio, TX). Antibody specificity was determined by using a 14 aa UCP-2
blocking peptide according to the manufacturer’s protocol. Reactions were
detected with a fluorescein-linked anti-mouse IgG (second antibody) conjugated to horseradish peroxidase using enhanced chemiluminescence.
Measurement of caspase-3 activity. Caspase-3 activity was determined by
measuring release of p-nitroanilide (pNA) after cleavage of the caspase
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tetrapeptide substrate Ac-DEVD-pNA following the protocol provided by the
manufacturer (BioVision Inc., Mountain View, CA) and as described by Liao
et al. (2003). Briefly, after treatment, cells grown in 6-well culture plates were
washed with PBS and lysed with 50 ll of lysis buffer (0.5% Triton X-100;
10 mM Tris-HCl, pH 7.5; 1 mM EDTA) on ice for 10 min. Lysate (50 ll) was
added to a reaction mixture containing 100 ll of reaction buffer, 40 ll of H2O,
and 5 ll of Ac-DEVD-pNA (final concentration 200 lM) and incubated for 1 h
at 37°C. Free pNA was measured at 405 nm in a microtiter plate reader, and
caspase activity was calculated after the protein content was normalized.
Measurement of cellular ATP. Cellular ATP content was determined
using a bioluminescence assay according to the manufacturer’s instructions
(Molecular Probes, Eugene, OR). Cells (2 3 105) were treated with cyanide,
and after the indicated times, cells were washed in PBS, lysed in 0.5% Triton X100, 10 mM Tris-HCl (pH 7.5), 1 mM EDTA, and incubated for 10 min on ice.
After removal of cell debris by centrifugation (10,000 3 g, 15 min, 4°C), the
ATP content was measured by the luciferin/luciferase method (Los et al.,
2002). The absolute values of ATP content were determined using an ATP
standard supplied by the manufacturer.
Statistics. Data were expressed as mean ± SEM, and statistical significance
was assessed by one-way analysis of variance (ANOVA), followed by the
Tukey-Kramer multiple range test. Differences were considered significant
at p < 0.05.
RESULTS
Cyanide-Induced Cell Death
We have shown that cyanide produces selective cell loss in
the CNS in which cortical brain areas undergo apoptotic
degeneration and mesencephalic cells undergo necrosis (Mills
et al., 1999). The response in primary cultured CX cells
parallels cytotoxicity observed in vivo; the concentrationresponse to cyanide in CX cells is illustrated in Figure 1A
and 1B. A concentration-related apoptotic death was observed
after 24-h exposure to KCN, with a switch to necrosis at higher
concentrations. The apoptosis correlated with a moderate decrease in ATP levels. For concentrations that produced
necrosis, cellular ATP declined by more than 35%, which is
a severe depletion for neuronal cells (Fig. 1C). It was
concluded that the mode of cell death switched from apoptosis
to necrosis when cellular energy reserves were depleted below
a critical threshold as described by Davey et al. (1998).
Altered UCP-2 Expression and Cell Death
To link UCP-2 expression to the mode of cell death, cells
were transfected with UCP-2 cDNA. Under the treatment
conditions, UCP-2 expression was increased twofold (Fig. 2A).
Electron micrographs of transfected CX cells exposed to 400 lM
KCN showed that UCP-2 overexpression produced a necroticlike morphology characterized by swollen mitochondria,
plasma membrane damage, and cytoplasmic vacuolization, as
opposed to a morphology characteristic of apoptosis in cells
treated with KCN alone, which shows chromatin condensation
and margination within the nucleus and cell shrinkage (Fig. 2B).
Increased expression of UCP-2 switched the mode of cell death
FIG. 1. Cyanide-induced apoptotic and necrotic responses in CX cells.
In all studies, cells were treated with KCN for 24 h before analysis.
A. Representative photomicrographs of cells exposed to KCN. For analysis of
apoptosis, cells were TUNEL stained (dark stain), and for necrosis, cells were
stained with SYTO-13 and propidium iodide (red stain ¼ necrosis). Magnification is 2003. B. The number of cells undergoing apoptosis (TUNEL þ) or
necrosis (PI þ) was counted, and the average percentage per total cells was
determined. Data represent mean ± SEM of four or more experiments. C. Cells
were treated for 24 h and then cellular ATP was determined. ATP levels in control
cells were 26.5 ± 1.6 nmol/mg protein. ATP levels are expressed as percent
control (non-treated cells), and each value is the mean of six or more experiments ± SEM. *Significantly different from the respective control, p < 0.05.
UCP-2 AS A REGULATOR OF CELL DEATH
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from apoptosis to necrosis at 400 lM cyanide (Fig. 2C). Thus
enhanced expression of UCP-2 predisposes to necrotic death.
To confirm the effect of transfection on cell death was due to
UCP-2 overexpression, UCP-2 expression was knocked down
with RNA interference (McManus and Sharp, 2002). Western
analysis of the constructs showed the UCP-2þ plasmid increased expression and RNAi blocked the enhanced expression
produced by the plasmid (Fig. 3A). Interestingly, constitutive
expression did not change after transfection with siRNA,
suggesting that UCP-2 undergoes a slow turnover in wild-type
cells. In cells treated with RNAi, the level of cyanide-induced
apoptosis was not significantly increased compared to wildtype cells treated with cyanide alone (Fig. 3B). In UCP-2þ
cells, RNAi blocked cyanide-induced necrosis, confirming that
overexpression of UCP-2 sensitizes the cells to the necrotic
effect of cyanide.
Transfection with the dominant interfering mutant UCP2D212N switched the necrotic response to apoptosis in UCP-2þ
cells (Fig. 4). Because previous studies have shown that UCP2D212N is a functional antagonist to UCP-2 (Mills et al., 2002),
it was concluded that activation of UCP-2 plays a pivotal role in
cyanide-induced necrosis.
UCP-2 Expression and Caspase Activation
The intrinsic apoptotic pathway is executed by activation of
caspase proteases and cellular caspase activity reflects commitment to this mode of death. Exposure to cyanide significantly increased cellular caspase-3 activity, correlating with
execution of apoptosis (Fig. 5). On the other hand, transfection
with UCP-2 cDNA decreased caspase activation, as expected if
the mode of cell death is switched to necrosis. Transfection with
UCP-2D212N or with siRNA increased caspase activation, which
may explain the blockade of necrosis by these treatments.
UCP-2 Expression and Mitochondrial Function
Mitochondrial dysfunction, characterized by a decline in
cellular ATP and loss of DWm, is central to execution of cell
death (Budd Haeberlein, 2004). In necrosis, cells experience
a catastrophic loss of mitochondrial function, whereas in
apoptosis, a minimal level of mitochondrial function is
FIG. 2. Overexpression of UCP-2 switches apoptosis to necrosis. All
studies were initiated 24 h after transfection with a UCP-2þ expression
plasmid. A. Cells were treated with KCN (400 lM) for 20 h, and then whole
cell lysates were subjected to SDS-PAGE and then immunoblotted. Densitometric analysis is the relative density of three or more assays ± SEM.
B. Representative transmission electron micrographs of (a) control cells, (b)
cells treated with KCN for 24 h, (c) UCP-2þ alone, and (d) UCP-2þ cells
treated with KCN for 24 h. Magnification, 60003. C. The number of cells
undergoing apoptosis (TUNEL þ) or necrosis (PI þ) was counted, and the
average percent per total cells was determined. The EV groups were transfected
with a plasmid containing an empty envelope (control). Data represent mean ±
SEM. *Significantly different from the KCN treatment group, p < 0.05.
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FIG. 4. The dominant interfering mutant UCP-2D212N decreases the
necrotic response. At 24 h after transfection, cells were treated with cyanide
(400 lM) for 24 h. The number of cells undergoing apoptosis (TUNEL þ) or
necrosis (PI þ) was counted. Data represent mean ± SEM of four experiments.
*Significantly different from the respective KCN alone group; #significantly
different from respective KCN þ UCP-2þ group, p < 0.05.
mitochondrial dysfunction and precede final execution of cell
death. In UCP-2þ cells, cyanide markedly lowered DWm,
correlating with necrotic death (Fig. 6B).
Opening of the permeability transition pore (PTP) is an
important mitochondrial event that mediates cell death.
FIG. 3. UCP-2 RNA interference blocks the necrotic response. All studies
were initiated 24 h after transfection with a UCP-2þ expression plasmid or cotransfected with UCP-2þ and UCP-2 siRNA (knockdown). A. Effect of
transfection on cellular UCP-2 levels. Then, 20 h after initiation of the
experiment, cell lysates were subjected to SDS-PAGE and then immunoblotted.
Densitometric analysis is the relative density of three or more assays ± SEM.
B. After transfection, cells were treated with cyanide (400 lM), and 24 h later
the number of cells undergoing apoptosis (TUNEL þ) or necrosis (PI þ) was
counted, and the average percent per total cells was determined. Data represent
mean ± SEM. *Significantly different from KCN alone group; #significantly
different from KCN þ UCP2þ group, p < 0.05.
necessary to execute cell death. In cells transfected with UCP2, cyanide markedly decreased cellular ATP, correlating with
the switch to necrosis (Fig. 6A). ATP levels were not
significantly altered by cyanide in cells transfected with the
dominant interfering mutant or siRNA, indicating a role for
UCP-2 in the response. Changes in DWm reflect the level of
FIG. 5. Effect of transfection with UCP-2 cDNA, siRNA or UCP-2D212N
on cellular caspase activity. At 24 h after transfection, cells were treated with
cyanide (400 lM) for 12 h, and cellular caspase activity was measured as the
change in absorbance of cleaved substrate at 405 nm. Data represent mean ±
SEM for six experiments. *Significantly different from control; #significantly
different from the KCN treatment group, p < 0.05.
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UCP-2 AS A REGULATOR OF CELL DEATH
FIG. 7. Blockade of mitochondrial membrane permeability pore transition
blocked cyanide-induced necrosis. At 24 h after transfection, cells were treated
with 1 lM cyclosporin A (CsA) for 30 min and then with cyanide (400 lM) for
24 h. The number of cells undergoing apoptosis (TUNEL þ) or necrosis (PI þ)
was counted. Data represent mean ± SEM of four experiments. þSignificantly
different from the respective control group; *significantly different from the
respective KCN group; #significantly different from the UCP-2þ þ KCN
group; p < 0.05.
the level of mitochondrial dysfunction, which in turn leads to
either apoptosis or necrosis.
DISCUSSION
FIG. 6. UCP-2 regulates cyanide-induced mitochondrial dysfunction. A.
After transfection (UCP-2, UCP-2D212N, or siRNA), cells were treated with
cyanide (400 lM) for 24 h, and then cellular ATP levels were determined. ATP
levels are expressed as percent control (non-treated cells), and each value is the
mean of six or more experiments ± SEM. *Significantly different from control;
#significantly different from KCN group, p < 0.05. B. Transfected cells were
treated with cyanide for 2 h and then DWm was determined by quantification of
JC-1 fluorescent intensity and the ratio of red/green was compared to untreated
controls that were set at l00%. *Significantly different from control;
#significantly different from KCN group, p < 0.05.
Inhibition of PTP opening with cyclosporin A (CsA) inhibits
caspase-independent death (Chang and Johnson, 2002). In
UCP2þ cells, pretreatment with CsA blocked the necrotic
response to cyanide, indicating that PTP opening is critical to
execution of necrosis (Fig. 7). Since CsA also inhibits
calcineurin, studies were conducted with bongkrekic acid,
a specific inhibitor of PTP opening. Pretreatment with bongkrekic acid produces responses parallel to those of CsA,
indicating that the CsA pretreatment blocks PTP opening. It
is concluded that the level of UCP-2 expression can influence
In a variety of injury paradigms UCP-2 mediates neuroprotection by uncoupling oxidative metabolism to decrease
ROS generation in mitochondria (Mattiasson et al., 2003;
Teshima et al., 2003). Similarly, in the present study, decreased
levels of UCP-2 increased caspase-mediated apoptotic CX cell
death. However, overexpression of UCP-2 potentiated cyanideinduced ATP depletion and loss of DWm, resulting in increased
cortical cell death by necrosis. In the presence of excessive
mitochondrial dysfunction, enhanced levels of UCP-2 appear
to contribute to execution of the necrotic pathway. These
observations have important implications by showing that
when overexpressed, UCP-2 can potentiate mitochondrial
dysfunction produced by a potent toxin and thus activate the
necrotic pathway of cell death.
In neuronal cells, cyanide produces two distinct modes of
death, apoptosis and necrosis, depending on the cell type and
level of oxidative insult (Prabhakaran et al., 2002). These
modes of cell death share common initiation stimuli, but
divergent intracellular cascades are activated to produce either
apoptosis or necrosis (Prabhakaran et al., 2004; Shou et al.,
2003). In both cell death modes, cyanide induces mitochondrial
dysfunction by inhibiting cytochrome oxidase (complex IV),
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leading to reduced oxidative metabolism and enhanced ROS
generation at complex III (Davey et al., 1998; Jones et al.,
2003). Apoptosis is caspase-dependent and mediated by release
of pro-apoptotic proteins from mitochondria. Cyanide-induced
apoptosis can be switched to necrosis by blocking caspase
activation, which is accompanied by marked reduction in both
DWm and cellular ATP levels (Prabhakaran et al., 2004). Cells
undergoing necrosis display an intense level of oxidative stress
and experience a rapid breakdown of mitochondrial function,
characterized by opening of the PTP and ATP depletion
(Prabhakaran et al., 2002).
Present results show that overexpression of UCP-2 switches
the mode of death from apoptosis to necrosis by potentiating
mitochondrial dysfunction. The enhanced necrosis was due to
a catastrophic drop in bioenergy production as reflected by
a decrease in DWm, ATP levels, and PTP. This is a specific
interaction between UPC-2 and cyanide since a UCP-2
dominant negative mutant and RNAi blocked the enhanced
necrosis, thus eliminating the possibility of nonspecific toxicity
associated with the transfection procedure.
Echtay et al. (2002) demonstrated that mitochondrial UCP-2
is activated indirectly by cyanide. In isolated mitochondria,
cyanide treatment generated superoxide in the mitochondrial
matrix to activate mitochondrial UCP-2, which then increased
uncoupling and caused progressive reduction of proton conductance. Apparently this action can also facilitate the
catastrophic energy collapse necessary for execution of
necrosis as observed in the present study. Other studies have
also linked increased UCP-2 expression to cell death. Sriram
et al. (2002) showed that two neurotoxicants (methamphetamine and kainic acid) increased UCP-2 expression in mouse
brain, and they therefore proposed that abnormal expression of
UCP-2 may cause the excessive energy depletion that leads to
mitochondrial dysfunction and necrosis. Mattson and Liu
(2003) proposed that uncoupling proteins are involved in
regulating mitochondrial-mediated cell death. The present
results are consistent with these studies.
To prevent apoptosis, UCP-2 may regulate production of
superoxide by mitochondria (Horvath et al., 2003; Mattiasson
et al., 2003; Sullivan et al., 2003; Teshima et al., 2003). Thus
overexpression of human UCP-2 in transgenic mice protected
against stroke and brain traumatic injury (Mattiasson et al.,
2003). Also, in a seizure model, transgenic mice displayed
a lower level of apoptotic hippocampal lesions in CA1
pyramidal cells (Diano et al., 2003). In brains of transgenic
mice expressing human UCP-2, decreased oxidative stress and
elevated ATP were observed, thus affording neuroprotection. In
UCP-2 knockout mice, lack of UCP-2 increased the mitochondrial complex I toxin MPTP-induced nigral dopamine cell
death, and overexpression decreased cell loss (Andrews et al.,
2005). Furthermore, in cultured cortical cells, UCP-2 inhibited
caspase activation and protected against oxygen/glucose
deprivation-induced death (Mattiasson et al., 2003). In other
studies, the influence of neonatal brain UCP-2 levels on
excitotoxic death was determined by altering dietary fat intake
(Sullivan et al., 2003). In immature brain with high basal
expression of UCP-2, reduced ROS generation and decreased
neuronal loss were observed following kainic acid-induced
seizures. Also, reduced UCP-2 increased vulnerability to
seizure-induced injury, thus supporting a neuroprotectant role
for UCP-2. It appears that UCP-2-mediated neuroprotection is
related to activation of cellular redox signaling or inhibition of
release of apoptogenic factors after mild mitochondrial uncoupling. Recently, de Bilbao et al. (2004) proposed that UCP-2
regulates the response of microglia to ischemia by modulating mitochondrial glutathione levels rather than ROS generation. Regardless of the mechanism of ROS production, the
level of ROS generated in mitochondria appears to determine
the mode of cortical cell death.
Despite reports that UCP-2 regulates apoptotic neuronal
death, a number of studies, including the present work, relate
UCP-2 to necrotic cell death. Over-expression of UCP-2
reduces DWm and decreases mitochondrial NADH and ATP
levels to produce necrosis (Mills et al., 2002). Studies in
hepatocytes have linked upregulation of UCP-2 to increased
vulnerability to necrotic death, possibly related to decreased
generation of ATP (Rashid et al., 1999; Uchino et al., 2004).
Overexpression of UCP-2 induced marked reductions in
DWm and ATP and produced apoptosis in cultured adipocytes
(3T3-L1 cells) (Sun and Zemel, 2004). Expression of a dominant interfering mutant of UCP-2 conferred resistance to
necrosis, but not apoptosis, in HeLa cells (Mills et al., 2002).
Overexpression of the anti-apoptotic protein Bcl-2 did not
influence the level of UCP-2–mediated cell death, suggesting
a non-apoptotic mode of death.
Clearly the present results show that overexpression of UCP-2
leads to enhanced cortical cell necrosis, although lowering
constitutive levels of UCP-2 reduced necrotic cell death and
slightly enhanced apoptosis. In mesencephalic cells, however,
reduction of UCP-2 levels markedly decreases cyanide-induced
necrosis (Prabhakaran et al., 2003). It is apparent that changes
in UCP-2 expression can regulate the response to a neurotoxin
in a cell-specific manner. Importantly, the constitutive expression level of UCP-2 may explain the selective vulnerability of
the CNS produced by cyanide (Mills et al., 1999). UCP-2
has region-specific expression patterns in the brain (Richard
et al., 2001). The present results suggest that higher levels of
UCP-2 expression may enhance the potential of mitochondrial
inhibitors to initiate necrotic death. It is also interesting to note
that, in this study, in the presence of cyanide, the expression
level of UCP-2 was greater after transfection. It is possible
that cyanide upregulates transcription of UCP-2, thus leading
to increased cellular expression. These results raise the possibility that cyanide may upregulate consititutive expression,
which in turn may alter the toxic response. Thus, changes in
UCP-2 constitutive expression may have important consequences with regard to neuronal sensitivity to mitochondrial active
compounds.
UCP-2 AS A REGULATOR OF CELL DEATH
It is concluded that UCP-2 can regulate mitochondriamediated cell death. Elevated levels of UCP-2 enhance nerve
cell vulnerability to mitochondrial complex IV inhibition by
resulting in a switch from the apoptotic to the necrotic mode of
neuronal cell death.
ACKNOWLEDGMENTS
This study was supported in part by National Institutes of Health (NIH)
grant ES 04140.
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