Journal of Neurochemistry, 2004, 88, 657–667
doi:10.1046/j.1471-4159.2003.02195.x
Endogenous mitochondrial oxidative stress: neurodegeneration,
proteomic analysis, specific respiratory chain defects, and
efficacious antioxidant therapy in superoxide dismutase 2 null mice
Douglas Hinerfeld,* Mathew D. Traini, Ron P. Weinberger, Bruce Cochran,* Susan R. Doctrow,à
Jenny Harry and Simon Melov*
*Buck Institute for Age Research, Novato, California, USA
Proteome Systems Inc., Sydney, New South Wales, Australia
àEukarion Inc., Bedford, Massachusetts, USA
Abstract
Oxidative stress and mitochondrial dysfunction have been
linked to neurodegenerative disorders such as Parkinson’s
and Alzheimer’s disease. However, it is not yet understood
how endogenous mitochondrial oxidative stress may result in
mitochondrial dysfunction. Most prior studies have tested
oxidative stress paradigms in mitochondria through either
chemical inhibition of specific components of the respiratory
chain, or adding an exogenous insult such as hydrogen peroxide or paraquat to directly damage mitochondria. In contrast, mice that lack mitochondrial superoxide dismutase
(SOD2 null mice) represent a model of endogenous oxidative
stress. SOD2 null mice develop a severe neurological phenotype that includes behavioral defects, a severe spongiform
encephalopathy, and a decrease in mitochondrial aconitase
activity. We tested the hypothesis that specific components of
the respiratory chain in the brain were differentially sensitive to
mitochondrial oxidative stress, and whether such sensitivity
would lead to neuronal cell death. We carried out proteomic
differential display and examined the activities of respiratory
chain complexes I, II, III, IV, V, and the tricarboxylic acid cycle
enzymes alpha-ketoglutarate dehydrogenase and citrate
synthase in SOD2 null mice in conjunction with efficacious
antioxidant treatment and observed differential sensitivities of
mitochondrial proteins to oxidative stress. In addition, we
observed a striking pattern of neuronal cell death as a result of
mitochondrial oxidative stress, and were able to significantly
reduce the loss of neurons via antioxidant treatment.
Keywords: antioxidant, mitochondria, neurodegeneration,
oxidative stress, proteomics, superoxide dismutase.
J. Neurochem. (2004) 88, 657–667.
Reactive oxygen species (ROS) have been implicated in the
pathogenesis of a number of neurodegenerative diseases
including Friedreich’s ataxia (FA) (Koutnikova et al. 1997;
Priller et al. 1997; Rotig et al. 1997), Alzheimer’s disease
(AD) (Kish et al. 1992; Mutisya et al. 1994), Parkinson’s
disease (PD) (Swerdlow et al. 1996) and amyotrophic lateral
sclerosis (ALS) (Brown 1997). Most studies examining the
consequences of oxidative stress on mitochondrial function
have been carried out in situ on preparations of isolated
mitochondria exposed to chemical insults, or alternatively
through use of chemical inhibitors of mitochondrial function
which result in increased ROS production (Boveris 1984;
Turrens et al. 1985; Martensson et al. 1991; Chinopoulos
et al. 1999; Gibson et al. 1999; Nulton-Persson and Szweda
2001). Hence, it is unclear what consequences to
mitochondrial function arise due to endogenously generated
ROS in the absence of external chemical insults. Genetic
models of oxidative stress are comparatively new, and
represent an alternate approach to examining the
Received July 28, 2003; revised manuscript received September 28,
2003; accepted October 1, 2003.
Address correspondence and reprint requests to Simon Melov, Buck
Institute for Age Research, 8001 Redwood Blvd., Novato, CA 94945,
USA. E-mail: [email protected]
Abbreviations used: AD, Alzheimer’s disease; ALS, amyotrophic
lateral sclerosis; BSA, bovine serum albumin; FA, Friedreich’s ataxia;
KGDH, a-ketoglutarate dehydrogenase; PD, Parkinson’s disease; ROS,
reactive oxygen species; SDH, succinate dehydrogenase; SDS–PAGE,
sodium dodecyl sulfate–polyacrylamide gel electrophoresis; TFA, trifluoroacetic acid.
2003 International Society for Neurochemistry, J. Neurochem. (2004) 88, 657–667
657
658 D. Hinerfeld et al.
consequences of oxidative stress to mitochondrial function
via the inactivation of key antioxidant proteins in the
mitochondria (Li et al. 1995; Jha et al. 2000; Higgins et al.
2002; Jung et al. 2002).
Mice on a CD-1 genetic background lacking SOD2
(sod2–/– mice) typically die within the first week of life
and display a heterogeneous phenotype including dilated
cardiomyopathy, hepatic lipid accumulation, metabolic
defects, mitochondrial enzyme defects, and oxidative damage to DNA (Li et al. 1995; Melov et al. 1999). Less than
10% of these mice survive beyond 2 weeks of age, and
those that do, display a severe neurological phenotype
(Melov et al. 1998). This behavioral phenotype, which has
also been recently reported in C57BL/6- DBA/2 J hybrid
backgrounds, includes ataxia and seizures (Huang et al.
2001). Histopathological analysis of the brains of CD1 SOD2 null mice reveals a profound focal spongiform
encephalopathy primarily within the frontal cortex and
brainstem. Mitochondrial aconitase activity, which is an
acutely sensitive marker of superoxide-mediated damage
(Gardner et al. 1995), is reduced in the brainstem, striatum,
cerebellum and cortex of the sod2–/– mice (Melov et al.
2001).
The pronounced neurological phenotype of SOD2 null
mice can be partially rescued in a dose-dependent manner
through the administration of the prototype low molecular
weight salen–manganese complex catalytic antioxidant
EUK-8 or either of its analogs EUK-134 and EUK-189
(Melov et al. 2001). These catalytic antioxidants are effective in many paradigms of oxidative stress (Malfroy et al.
1997; Baker et al. 1998; Rong et al. 1999; Melov et al.
2000; Pong et al. 2001). They have been shown to catalytically scavenge the reactive oxygen species superoxide
(Baudry et al. 1993; Baker et al. 1998) and hydrogen
peroxide (Baker et al. 1998) as well as reactive nitrogen
species (Sharpe et al. 2002), all potential contributors to
neurological injury in circumstances of oxidative stress.
In vitro studies demonstrate that EUK-8 and EUK-134
protect PC12 cells from superoxide and nitric oxide, and
EUK-134 attenuates staurosporine-induced apoptosis, oxidative stress and mitochondrial dysfunction in cultured neurons
(Pong et al. 2001). In vivo studies demonstrate that administration of EUK compounds significantly prolongs survival
and reduces oxidative stress in a mouse model of ALS (Jung
et al. 2001), prevents neuronal death and reduces oxidative
damage following kainic acid induced seizures in rats (Rong
et al. 1999), and extends lifespan in the nematode Caenorhabditis elegans (Melov et al. 2000). Administration of
1 mg/kg of EUK-8 is sufficient to extend the mean lifespan
of sod2–/– mice from 8 to 17 days of age, but not sufficient
to rescue the neurological phenotype encompassing behavioral abnormalities and neuropathology (Melov et al. 2001).
Administration of EUK-8 at 30 mg/kg extends the mean
lifespan to 24 days, completely rescues the spongiform
changes in the brain, and significantly diminishes behavioral
abnormalities (Melov et al. 2001). Collectively, these studies
illustrate the utility of this class of antioxidants in preventing
damage associated with oxidative stress at the level of
organelle, cell, tissue, and organism.
The purpose of this study was to gain further insight into
the specific targets of endogenously generated mitochondrial
oxidative stress in the brains of SOD2 null mice. Specifically,
we treated SOD2 null animals with either a high or low dose
of the antioxidant EUK-8 to allow differential brain pathology to develop (Melov et al. 2001) (untreated animals do not
survive long enough in sufficient numbers to be practically
useful), carried out mitochondrial proteomic differential
display, and then measured the activities of a number of
mitochondrial enzymes to test the hypothesis that specific
enzymes were differentially sensitive to endogenous oxidative stress. In addition, we tested the hypothesis that
endogenously generated mitochondrial oxidative stress
results in neuronal cell death, and whether or not antioxidant
treatment could either prevent or partially rescue neuronal
cell death in vivo. Our results have important implications for
the successful implementation of rational antioxidant therapy
in age-related neurodegenerative disease in which neuronal
cell loss is linked to mitochondrial dysfunction and oxidative
stress.
Materials and methods
Compound treatment and genotyping
As this is described in detail elsewhere (Melov et al. 1998; Melov
et al. 2001), a brief description of genotyping and compound
treatments follows. Mice were genotyped between 2 and 3 days of
age, and injected intraperitoneally with compound or vehicle daily
from 3 days of age at the indicated dose or an equivalent volume of
vehicle until killing. Wild types were also treated similarly to control
for compound specific effects. Mice were killed at 19–21 days of
age and tissues harvested. All animal procedures were carried out
under approved IACUC animal protocols at the Buck Institute,
which is ALAAC accredited.
Mitochondrial preparation and enzymology
Cortex was rapidly isolated and placed into 3 mL of ice cold Hbuffer (210 mM mannitol, 70 mM sucrose, 1 mM EGTA, 5 mM
Hepes, pH 7.2) (Trounce et al. 1996). Tissue was homogenized
with a Dounce homogenizer and centrifuged at 1000 g for 5 min.
The supernatant was transferred to a new tube and centrifuged at
8000 g for 10 min. The resulting supernatant was aspirated, and the
pellet resuspended in 100 lL H-buffer. Bovine serum albumin
(BSA) was added to a final concentration of 1 mg/mL. Electron
transport chain complexes I, II, III, and IV and citrate synthase
activities were determined as previously described (Trounce et al.
1996) except that for complex I dichloroindophenol was used as the
terminal electron acceptor. ATP synthase activity and a-ketoglutarate dehydrogenase (KGDH) activity were determined as previously
described (Catterall and Pedersen 1971; Nulton-Persson and Szweda
2003 International Society for Neurochemistry, J. Neurochem. (2004) 88, 657–667
Endogenous mitochondrial oxidative stress 659
2001) Statistical significance was determined by non-parametric ttests PRISM (Graphpad, San Diego).
Mitochondrial proteomics pH 4–7 isoelectric focusing
Mitochondrial sample was solubilized, reduced and alkylated for
one hour in a total of 220 lL extraction buffer containing 7 M urea,
2 M thiourea, 1% C7, 40 mM Tris, 5 mM tributylphosphine (TBP),
10 mM acrylamide and orange G. Samples were centrifuged at
21 000 g for 5 min, and the supernatants were applied to 11 cm
pH 4–7 (Amersham Pharmacia Biotech, Piscataway, NJ, USA)
immobilized pH gradient (IPG) strips and incubated for at least 6 h.
Samples were electrophoresed as follows; linear ramp to 10 000 V
over 5 h, 10 000 V for 5 h, 1000 V until stopped, maximum of
50 lA per IPG. Strips were then incubated for 15 min at room
temperature in equilibration buffer [2% sodium dodecyl sulfate
(SDS), 50 mM Tris-acetate, 6 M urea, 0.01% bromophenol blue], cut
in half and subjected to SDS–polyacrylamide electrophoresis
(PAGE) through two 4–12% gels at 100–125 V. Gels were fixed
for at least 30 min in 7% acetic acid/10% methanol and stained in
Sypro Ruby (Molecular Probes, Eugene, OR, USA) overnight. Gels
were destained in the same buffer as the fixation and imaged on a
UV light box at 302 nM. Image and statistical analysis was
performed using PDQuest (Bio-Rad Laboratories, Hercules, CA,
USA).
pH 6–11 isoelectric focusing pH 6–11 IPG strips were rehydrated in 180 lL extraction buffer without 40 mM Tris for at least
6 h. Mitochondrial samples were solubilized, reduced and alkylated for at least one hour in a total of 45 lL extraction buffer and
the reactions were centrifuged at 21 000 g for 5 min. The
supernatants were loaded into cup loaders at the acidic end of
the IPG and electrophoresed as follows: 300 V for 3 h, linear ramp
to 10 000 V over 12 h, 10 000 V until at least 80 000 V/h,
maximum of 50 lA per IPG. SDS–PAGE, staining, imaging, and
analysis was performed as described above except that the IPG
strip was cut 7 cm from the acidic end of the gel (no proteins
resolved beyond 7 cm), and that portion was electrophoresed onto
a single 7 cm 4–12% gel.
Protein identification
Protein spots were either manually excised or automatically
detected and excised using the Xcise apparatus (Shimadzu
Biotech, Japan). Gel pieces were washed twice with 150 lL
100 mM ammonium bicarbonate, pH 8.2, 50% v/v acetylnitrile
(ACN) and dried at 37C for 20 min. Trypsin in 50 mM
ammonium bicarbonate (20 lg/lL) was added to each gel piece
and incubated at 30C for 16 h. Peptides were extracted by
sonication in 20 lL 0.1% v/v trifluoroacetic acid (TFA), 50%
ACN. The peptide solution was either manually or automatically
desalted and concentrated using ZipTips from Millipore (Bedford, MA, USA) in the Xcise and spotted onto the Axima MALDI
target plate. Peptide mass fingerprints of tryptic peptides were
generated by matrix assisted laser desorption/ionisation-time of
flight-mass spectrometry (MALDI-TOF-MS) using an Axima CFR
(Kratos, Manchester, UK). Spectra were analyzed by the (BioinformatIQ) integrated suite of bioinformatics tools from Proteome
Systems (Sydney, Australia). Protein identifications were assigned
by comparing peak lists to databases containing theoretical tryptic
digests (SWISS-PROT and TrEMBL) using the Mascot software
(Matrix Science, London, UK). Criteria for the assignment of
identification were 100 p.p.m. or better peptide mass accuracy, a
Mowse score of at least 72 or greater indicating p ¼ 0.05, Mr and
pI matching theoretical values and percentage coverage of peptides
across theoretical protein sequence.
Western blot analysis
Equal amounts of mitochondrial protein (8 lg) was subjected to
SDS–PAGE on a 4–20% Criterion gel (Bio-Rad) and either stained
with colloidal blue (Invitrogen, Carlsbad, CA, USA) or transferred
to polyvinylidene difluoride membrane for western blot analysis.
Western blot analysis was performed using the ECF detection
system as recommended by the manufacturer (Amersham) probing
with rabbit polyclonal antibodies raised against the bovine FP
(70 kDa), IP (30 kDa) and CII-3 (13 kDa) subunits of complex II
(Gift of B. Ackrell). Specificity of these antibodies was previously
established (Birch-Machin et al. 2000) (data not shown). Probing of
all three antibodies was initially optimized singly to confirm
specificity, and then simultaneously together to facilitate simultaneous comparisons. Antibody dilutions were; FP 1 : 3000, IP
1 : 3000, CII-3 1, 2000. Western blot imaging was carried out
using a Typhoon 8600 and quantitated with ImageQuant Solutions
software (Amersham).
Histology
Mice were deeply anesthetized with sodium pentobarbital, and
perfused with phosphate-buffered saline. Brains were excised, and
then treated with 20% glycerol and 2% dimethylsulfoxide, embedded
in a gel matrix (Neuroscience Associates, Knoxville, TN, USA) and
frozen in a dry ice isopentane bath to ) 70C. They were then
sectioned throughout at a resolution of 50 lm. Every sixth section
was stained with amino cupric silver stain as previously described (de
Olmos et al. 1994). Silver staining is a well-established technique for
the enumeration of dying neurons, has been in use for over 30 years,
and is non-controversial (Fix et al. 1996; LaVaute et al. 2001). All
silver-stained neurons whose soma were contained within the section
were counted in all sections from the most rostral section containing
cortex until the first section containing the granular layer of the
dentate gyrus. This included at least 17 sections per brain. There was
no significant difference in the total number of sections containing
the region of cortex of interest between various treatment groups,
suggesting that treatment does not result in a change in cortical
volume. The number of silver-stained cells identified was multiplied
by six to estimate the total number of dying neurons per animal. We
went to such lengths due to the possibility that stereological methods
would be required in order to enumerate the number of dying cells.
However, due to the small number of positive cells, it was not
necessary to use intrasectional sampling.
Succinate dehydrogenase (SDH) histochemistry
Directly following killing, brains were harvested and frozen on dry
ice and sectioned at 16 lm at ) 25C. We evaluated succinate
dehydrogenase (SDH) levels via histochemical methods, a standard
technique in the evaluation of mitochondrial disease (Shoubridge
1994). SDH staining was performed as previously described on
frozen sections (Rifai et al. 1995), and results in a blue stain that can
be used quantitatively (Nakatani et al. 2000) or qualitatively
(Shoubridge 1994) to evaluate SDH activity.
2003 International Society for Neurochemistry, J. Neurochem. (2004) 88, 657–667
660 D. Hinerfeld et al.
(a)
Results
Sod2-/-
WT
E
Proteomic analysis of differentially expressed proteins in
cortical mitochondria of SOD2 null mice
To identify mitochondrial proteins whose concentration or
conformations via post-translational modifications might be
altered due to the lack of SOD2, we performed differential
proteomic profiling on mitochondrial protein from the
cortex of sod2–/– and wild-type mice treated with either
high or low dose EUK-8. SOD2 null mice that are not
treated with antioxidants do not survive long enough in
sufficient numbers to be of practical use (Melov et al.
1998). Mitochondria were subjected to two-dimensional
(2D) electrophoresis (n ¼ 5 for each group). Mitochondrial
protein was first separated on either pH 4–7 or 6–11
immobilized pH gradient (IPG) strips by isoelectric focusing
and then separated based on mass by SDS–PAGE. Image
analysis revealed approximately 1200 spots per animal (all
gels). After spot detection, normalization, and image
matching, matched spots between genotypes and treatment
groups were used to determine differential expression via a
non-parametric t-test. Proteins of interest were then excised
from the gel and identified by MALDI-TOF-MS. No
difference was seen between the expression profiles of
mitochondria from either high or low dose treated sod2–/–
mice. However, a number of notable differences were
observed in mitochondria derived from sod2–/– mice (high
or low dose treated), and wild-type mice. Regions of the
gels containing statistically significantly differentially
expressed proteins between low-dose-treated SOD2 null
and wild-type mice are shown in Fig. 1, and the identity,
percent coverage, and fold-difference of each of the
differentially expressed proteins is provided in Table 1.
Proteins involved in the tri-carboxylic acid (TCA) cycle,
electron-transport chain (ETC) function and maintenance of
a redox balance were identified as being differentially
expressed in sod2–/– mice in addition to the lack of SOD2
itself in the SOD2 null mice, a valuable confirmation of the
effectiveness of this approach. After using proteomic
profiling as a first-pass screen to identify candidate proteins
for follow-up characterization, we then tested the hypothesis
that specific mitochondrial enzymes were differentially
sensitive to mitochondrial oxidative stress, by measuring
mitochondrial enzyme activities including enzymes whose
subunits were identified in the initial proteomic analysis.
Mitochondrial enzyme activities in the cortex of sod2–/–
mice
Two of the proteins identified as being reduced in the
sod2–/– mice via our proteomic analysis were the
2-oxoglutarate dehydrogenase (E1k) subunit of the TCA
cycle enzyme complex KGDH, and two structural isoforms
of the flavoprotein (FP) subunit of the TCA cycle and ETC
H
E
H
(b)
Fig. 1 Regions of representative two-dimensional gels containing
differentially expressed proteins. (a) Expanded regions of the pH 6–11
gel from both wild-type and sod2–/– mice. (b) Expanded regions of the
basic end of the pH 4–7 gel from both wild-type and sod2–/– mice. The
labeled proteins refer to Table 1.
enzyme complex succinate dehydrogenase (SDH/complex
II). Both SDH and KGDH were assayed enzymatically to
complement and confirm the initial proteomic results. Our
previous studies demonstrated a highly significant decrease
in the catalytic activity of mitochondrial aconitase from
cortex of sod2–/– mice, which is partially rescued with
EUK-8 treatment (Melov et al. 2001). In order to evaluate
other mitochondrial enzymes for vulnerabilities to mitochondrial oxidative stress, we assessed enzyme activities of
ETC complexes I, II, III, IV, the ATP synthase (V), and the
TCA cycle enzymes citrate synthase (CS) and KGDH
(Figs 2a–f). For most mitochondrial assays, there was no
significant difference between the enzymatic activities of
ETC enzymes or TCA cycle enzymes from wild-type
control mice treated with either a high or low dose of EUK8, so these data were combined. Table 2 shows the
differential sensitivities of mitochondrial enzymes to endogenous oxidative stress, and the therapeutic benefit to specific
mitochondrial enzymes of increasing the dose of antioxidant
treatment. Complexes, I, II, III, and IV all appear to have
differential sensitivities to endogenous mitochondrial oxidative stress, in addition to citrate synthase and KGDH. These
enzymes also appear to be responsive to increased dosage of
antioxidant treatment, given the complete rescue of wildtype activities via EUK-8 treatment at 30 mg/kg (high dose)
(Table 2). However, complex II and KGDH did not benefit
from increased dosage of antioxidant, and complex V did
not have any detectable defects associated with lack of
SOD2 in cortical mitochondria. These data demonstrate that
2003 International Society for Neurochemistry, J. Neurochem. (2004) 88, 657–667
Endogenous mitochondrial oxidative stress 661
Table 1 Differentially expressed proteins in
the mitochondria identified by proteomic
profiling between sod2–/– (low dose) and
wild-type mice (n ¼ 5 per group). All proteins listed are statistically significantly
differentially expressed (p < 0.05), nonparametric t-test.
Fold Change
in –/–
%
Coverage
60
P99029
Q60597
Q9Z1Z4(TrEMBL)
Not detectable
in sod2–/–
+ 3.3
) 2.1
+ 2.3
+ 1.7
) 3.2
) 1.5
) 1.9
) 2.7
67
48
71
85
ND
42
ND
ND
Q9Z1Z4(TrEMBL)
) 2.5
ND
NP_619611(RefSeq)
) 1.8
43
Spot
Protein
SwissProt(other)
A
SOD2
P09671
B
C
D
E
F
G
H
I
Triosephosphate isomerase (TPI)
GST class-mu 1
GST class-mu 1
GST class-mu 1
unknown
Peroxiredoxin 5 (Prx 5)
2-Oxoglutarate dehydrogenase(E1k)
Succinate dehydrogenase flavoprotein
(FP)
Succinate dehydrogenase flavoprotein
(FP)
3-Mercaptopyruvate sulfurtransferase
(MST)
P17751
P10649
P10649
P10649
J
K
ND, not done.
Fig. 2 Mitochondrial enzyme assays for
respiratory chain complexes I, II, III, IV, and
the TCA cycle enzyme CS and KGDH. All
values are lmoles/min/mg of mitochondrial
protein. Mean values plus standard errors of
the means are shown from an n of 6–7 mice
per genotype per treatment. Complex V is
not shown, as there was no significant difference between genotypes or treatments.
Closed bars, low-dose (1 mg/kg) EUK-8
treated sod2–/– mice; striped bars, high
dose EUK-8 treated (30 mg/kg) sod2–/–
mice; hatched bars, high dose-treated
sod2+/+ animals in complex II assays; gray
bars, low dose EUK-8 treated sod2+/+ mice
in complex II assays; open bars, combined
high and low dose EUK-8 treated sod2+/+
mice. There was no significant difference
between sod2 wild-type mice treated with
either a high or low dose of drug for all
enzymes assayed except complex II (see
Results), as determined by a non-parametric t-test (PRISM), so the data were combined. For each condition n ¼ 6 or 7 animals,
and assays were performed in duplicate.
There is a statistically significant rescue of
the enzymatic activities of complexes I, II, III
and IV with high dose EUK-8 treatment, at
the indicated p-values.
specific enzymes of cortical mitochondria are differentially
sensitive to loss of SOD2 and by implication, mitochondrial
oxidative stress. As we identified a decrease in the
abundance of the Fp subunit of complex II, we wished to
determine if there was a corresponding decrease in other
subunits of complex II.
2003 International Society for Neurochemistry, J. Neurochem. (2004) 88, 657–667
662 D. Hinerfeld et al.
Table 2 Differential sensitivity of mitochondrial enzymes to endogenous oxidative stress, and therapeutic benefits of EUK-8 antioxidant
treatment. Values are given as a mean percent of the wild-type control
for the respective enzymes, and n ¼ 6–7 per group. Values which are
not statistically different from wild type are indicated by not significant
(NS) at p ¼ 0.05.
Mitochondrial enzyme
SOD2 –/– 1 mg/kg
SOD2 –/– 30 mg/kg
Complex I
Complex II
Complex III
Complex IV
Citrate synthase
KGDH
70%
12%
60%
88%
88%
35%
NS
32%
NS
NS
NS
36%
EUK-8 treatment, percentage of wild-type control activity.
Correlation of the abundance of complex II subunits with
enzyme activity
Complex II consists of four protein subunits; FP, an
iron-sulfur subunit (IP), and two small proteins that are
responsible for anchoring IP and FP to the membrane (CII-3
and CII-4) (Davis and Hatefi 1971; Capaldi et al. 1977).
Proteomic analysis revealed a reduction in the abundance of
the FP subunit of complex II in sod2–/– animals relative to
wild type (Table 1). Enzymological analysis of the corresponding samples revealed that the catalytic activity of
complex II could be statistically significantly increased
through high-dose treatment of sod2–/– mice compared with
low dose-treated sod2–/– mice. Having established by global
proteomic analysis that the FP subunit of complex II was
decreased in the mutant mice, and enzymological analysis
revealed that complex II was decreased in activity, we used
western blot analysis to quantitate levels of specific complex
II components (Fig. 3). Equal concentrations of mitochondrial protein from the same preparations used in the proteomic
and enzymology studies, were subjected to SDS–PAGE
(Fig. 3a) and probed with antibodies to FP, IP, and CII-3
(Fig. 3b). There is a significant reduction in the abundance of
the FP, IP, and CII-3 subunits in both high and low dosetreated sod2–/– mice compared with wild-type controls.
There was no difference in the abundance of subunits in the
wild-type mice treated with either a low dose or high dose of
EUK-8. There was a statistically significant increase in the
levels of the IP and CII-3 subunits between treatment groups
of the sod2–/– mice (Fig. 3c). This trend persists for the FP
subunit although it was not statistically significant (Fig. 3c).
The changes in the relative abundances of the subunits,
between genotypes and EUK-8 treatments, correlate with the
changes in complex II activity (r2 ‡ 0.92 for all subunits),
suggesting that the loss of enzyme activity in the sod2–/–
mice is due in major part to a reduction in the levels of these
subunits. Having established a number of specific proteomic
and mitochondrial enzymatic defects in SOD2 null mouse
Fig. 3 Western blot analysis of respiratory chain complex II subunits
FP, IP, and CII-3. Open bars, sod2–/– mice; closed bars, high dosetreated sod2+/+ mice; stippled bars, low dose-treated sod2–/– mice.
(a) Coomassie stain of SDS–PAGE demonstrating equal loading of
mitochondrial protein. (b) Western blot. The locations of the FP
(70 kDa), IP (30 kDa) and CII-3 (15 kDa) subunits are shown.
(c) Densitometric quantitation of complex II western blot results.
Because there was no difference in the concentration of subunits in
the high and low dose-treated wild-type mice, the data was combined.
Local background corrections were employed for each protein. All
subunits are significantly reduced in sod2–/– mice compared to wildtype mice. There is a significant reduction in the level of the IP and CII-3
subunits in the sod2–/– low dose treated mice compared to the
sod2–/– high dose treated mice (*p < 0.05), as determined by a nonparametric t-test (PRISM).
brain mitochondria, we now wished to determine if these
defects ultimately resulted in neuronal cell death.
Limited neuronal cell death accompanies the
mitochondrial defects and spongiform changes in the
cortex of sod2–/– mice and is partially rescued by EUK-8
treatment
Sod2–/– mice develop profound spongiform changes in the
brain that can be rescued by treatment with a high dose of
EUK-8 (Melov et al. 2001). Although the previously
described neuropathology is striking and severe, it was
unknown whether neuronal cell death accompanied the
spongiform pathology and enzymological defects in the
brains of the sod2–/– mice. Therefore, we used silverstaining
2003 International Society for Neurochemistry, J. Neurochem. (2004) 88, 657–667
Endogenous mitochondrial oxidative stress 663
(a)
(b)
Fig. 4 Rescue of neuronal cell death in the cortex of sod2–/– mice by
EUK-8. Closed bars, high-dose-treated sod2–/–. Stippled bars, lowdose-treated sod2–/–. (a) Representative sections of amino cupric
silver stained secondary motor cortex from a low dose-treated sod2–/–
mouse magnified at 50 · and 200 ·. Dark staining cells exhibit classic
morphology of neuronal cell death. (b) Quantitation of silver stained
positive neurons in the frontal cortex of high dose and low dose treated
mice. There is a 70% statistically significant reduction in the number of
silver stained positive neurons in the high dose treated sod2–/– mice
(*p < 0.05), as determined by a non-parametric t-test (PRISM). No
silver stained positive neurons were detected in wild-type mice, n ¼ 5
for all conditions.
(de Olmos 1994), a well established technique for detecting
dying cells to enumerate the total number of dying neurons
within the cortex of sod2–/– mice treated with either a high
or low dose of EUK-8. The amino-cupric silver stain
selectively impregnates degenerating neurons, and has been
used to evaluate neuronal cell death in toxicological and
genetic studies (Fix et al. 1996; LaVaute et al. 2001). Our
data clearly shows neuronal cell death accompanying the
spongiform changes seen in the low dose-treated mice and
this loss of neurons is statistically significantly reduced by
70%, with high dose EUK-8 treatment (Fig. 4). Wild-type
control brains did not contain any detectable silver stained
Fig. 5 Representative sections of SDH histochemistry of cortex.
Sections are at 50 · magnification of the secondary motor cortex. The
intensity of this colorimetric stain is reflective of the level of SDH
activity (Rifai et al. 1995). No difference in the intensity of staining was
evident between high and low dose-treated wild-type mice. However
positive neurons. In sod2–/– mice, neuronal cell death was
limited to the medial orbital, frontal association, primary
motor and secondary motor regions of the cortex. The
neuronal cell death observed in the high dose-treated SOD2
null mice corresponds with the persistence of the mild
behavioral phenotype as well as aconitase (Melov et al.
2001), SDH/complex II and KGDH defects. These data
indicate that neuronal cell death is limited to distinct regions
of the cortex at 3 weeks of age, is a feature of endogenous
mitochondrial oxidative stress, and appropriate antioxidant
treatment can attenuate the loss of neurons. Having demonstrated that neuronal cell death accompanies the enzymological defects, we wished to evaluate whether mitochondrial
defects were ubiquitous in the cortex, or restricted to the
regions of cell death.
SDH defects in the sod2–/– mice are ubiquitous within
the cortex
We performed in situ SDH histochemistry on frozen sections
of brains from low and high dose-treated sod2–/– and wildtype mice (Fig. 5). The brains from the sod2–/– mice showed
a dramatic reduction in SDH staining compared to controls,
consistent with the reduced enzymatic activities of SDH/
complex II described above. There was no difference between
the intensity of staining in the brains of treated controls (high
versus low dose). However, there was an increase in the SDH
stain intensity between the low and high dose-treated sod–/–
mice, consistent with our enzymology and western blot results
described above. The reduction in SDH staining was not
confined to the regions displaying cell death, but was evident
throughout all cortical regions analyzed (Fig. 5).
Discussion
There is substantial evidence that oxidative stress plays a role
in the progression of a constellation of neurological disorders, although these data are largely correlative in nature. The
neurological phenotype of mice lacking the mitochondrial
free radical-scavenging enzyme SOD2, supports the
there is a marked increase in the level of staining in the high
dose-treated sod2–/– mice compared to the low dose-treated mice
consistent with the increased activities observed via mitochondrial
enzymology, as well as proteomic profiling.
2003 International Society for Neurochemistry, J. Neurochem. (2004) 88, 657–667
664 D. Hinerfeld et al.
hypothesis that neurodegeneration and mitochondrial enzymological defects can be a direct consequence of endogenous
mitochondrial oxidative stress. The neurological phenotype
in the sod2–/– mice is partially rescued through treatment
with the catalytic antioxidants EUK-8, EUK-134 and EUK189, implying that it is the endogenous production of ROS
that ultimately results in neurodegeneration. Because of the
multifunctional specificity of these compounds (Doctrow
et al. 2002), it is not known which reactive oxygen species,
or reactive nitrogen species, are the most relevant targets of
EUK-8 action in sod2–/– mice. For example, with nitric
oxide synthase present in the mitochondria (Giulivi 2003),
the conversion of excess superoxide to peroxynitrite might be
occurring, and causing mitochondrial damage. It is, of
course, possible that the in vivo protective effects of EUK-8
involve mechanisms other than a direct antioxidant action.
However, there is ample evidence that EUK-8 and related
compounds inhibit protein, DNA and lipid oxidation in
tissues from treated animals exhibiting a variety of oxidative
injuries (Gonzalez et al. 1995; Rong et al. 1999; Jung et al.
2001; Liu et al. 2003). Also, evidence indicates that these
compounds suppress, rather than stimulate, stress-associated
transcriptional factors when administered in vivo (Rong et al.
1999). Finally, as discussed further below, in sod2–/– mice
EUK-8 protects mitochondrial enzymes, in particular, cisaconitase, that are known to be direct targets for reactive
oxygen species. Thus, it is our current hypothesis that EUK-8
protects sod2 –/– mice through a mitochondrial antioxidant
based mechanism.
Although prior studies have demonstrated defects in
specific respiratory chain complexes and TCA cycle enzymes
in response to exogenous chemical insults, it is not
immediately obvious which mitochondrial enzymes would
be particularly sensitive to endogenously produced superoxide in the absence of chemical inhibitors. In order to identify
potential consequences of mitochondrial oxidative stress in
an unbiased approach, we have analyzed the mitochondria of
the sod2–/– mice using a proteomic differential display
entailing differential 2D electrophoresis and MALDI-TOFMS. We detected seven proteins that underwent changes in
cortical mitochondria of sod2–/– mice, of which two had
multiple isoforms (Table 1). As a validation of this approach,
we detected SOD2 as being differentially displayed in our
proteomic screen, which we knew a priori was altered in
abundance between the SOD2 null mice and controls. Other
enzymes we detected which were differentially displayed
include the TCA cycle enzymes KGDH and SDH as well as
peroxiredoxin 5 (Prx5) and glutathione S transferase classmu 1 (GST class-mu 1), both of which are involved in
maintaining redox balance. Additional enzymes which were
altered in the sod2–/– mice are the cyanide-detoxifying
enzyme 3-mercaptosulfurtransferase and the glycolytic
enzyme triosephosphate isomerase (TPI), which is not
known to exist within the mitochondria, however, this
enzyme has been shown to copurify with mitochondria
(Taylor et al. 2003).
We further investigated the consequences of endogenous
mitochondrial oxidative stress by enzymatic assays, which
revealed that the electron transport chain complexes I, II (as
well as SDH), III, and IV, in addition to the previously
reported aconitase defect (Melov et al. 2001), are sensitive to
mitochondrial ROS (Fig. 2) in the cortex of sod2–/– mice.
The catalytic activities of complexes I, III, and IV are
restored to wild-type levels through treatment with a high
dose of EUK-8, demonstrating the potential for therapeutic
intervention where mitochondrial ROS are implicated in the
pathophysiology of neurological disease. Complex V appears
to be insensitive to endogenous oxidative stress in mitochondria isolated from the brain in SOD2 null mice. This
implies that not all enzymes are sensitive to ROS in the
mitochondria. Our data suggest that complex II is one of
the most sensitive enzymes to the loss of SOD2 activity, as
the activity was only 12% of wild-type levels. However, even
this amount of decrease was responsive to optimal antioxidant treatment as we were able to increase the activity by 2.7fold through high dose EUK-8 treatment (Table 2), although
this still was approximately only one-third the value of
untreated wild types. Complex I defects have previously
been detected in the hearts of sod2–/– mice along with
complex II defects in the heart and skeletal muscle (Melov
et al. 1999). What might underlie the sensitivity of these
mitochondrial enzymes to ROS produced during normal
respiration? Complexes I, II, and III and aconitase all contain
iron-sulfur clusters. The [4Fe–4S] cluster of aconitase has
previously been shown to be sensitive to superoxidemediated inactivation (Gardner et al. 1995), and the
[3Fe–4S] cluster of SDH has also been shown to be sensitive
to oxidation (Beinert et al. 1977). Therefore, these enzymes
may be particularly labile to mitochondrial-generated superoxide.
KGDH has been the focus of several studies (see (Gibson
et al. 1999) for a review) in relation to oxidative damage and
neurodegenerative disease. The detection of abundance
differences of KGDH in our initial proteomic differential
display in SOD2 null mitochondria compared to controls,
provides further evidence for the sensitivity of this enzyme to
mitochondrial oxidative stress. 2-Oxoglutarate dehydrogenase (E1k) is one of three protein subunits of KGDH and
exhibits a 1.9-fold reduction in abundance in the sod2–/–
mice (Fig. 1, Table 1). One of the other two subunits of
KGDH, dihydrolipoamide dehydrogenase (E2k) was not
altered in abundance (data not shown). To determine whether
the reduced level of E1k was limiting for the activity of
KGDH, the relative enzymatic activity of KGDH in both low
and high dose-treated sod2–/– and wild-type mice was
determined. The activity of KGDH was reduced by 65% in
the sod2–/– mice with no difference between high or low
dose treatments (Fig. 2, Table 2), confirming the mechanistic
2003 International Society for Neurochemistry, J. Neurochem. (2004) 88, 657–667
Endogenous mitochondrial oxidative stress 665
implications of our initial proteomic screen. It was interesting
to note that of all the enzymes we evaluated, KGDH was not
responsive to the increased dosage of EUK-8 treatment
(Fig. 2, Table 2), perhaps indicating its extreme sensitivity to
redox state within the mitochondria. KGDH is known to be
sensitive to ROS (Humphries and Szweda 1998; Chinopoulos et al. 1999). In humans, a deficiency in the E1k
component results in metabolic acidosis, hypotonia, and
hyperlactatemia leading to neurological deterioration and
death by 30 months of age (Gibson et al. 1999). KGDH
deficiencies are also associated with both AD and PD
(Gibson et al. 1999). Our data provides additional support
that this enzyme may be decreased in activity through
mitochondrial oxidative stress in these severe human disorders.
Western blot analysis of complex II in cortical mitochondria revealed that changes in the levels of subunits correlate
with changes in enzyme activity under the various experimental conditions used (Fig. 3). The IP subunit contains three
iron–sulfur clusters that are involved in the transfer of
electrons from succinate to ubiquinone (Ackrell et al. 1992).
Iron starvation studies have determined that only complexes
with their full complement of clusters are assembled in the
membrane (Ackrell et al. 1984). Therefore, it is possible that
in the sod2–/– mice the supply of iron–sulfur clusters is
limiting or that those in free IP are particularly sensitive to
oxidation such that assembly is restricted and free subunits
degraded. If subunits escape damage by ROS then a
functional complex may be formed that is resistant to further
oxidation. The data reported here support this hypothesis, in
as much as changes in enzyme activity are reflected in the
relative protein concentration of individual complex II
subunits. Our data do not allow us to rule out the formal
possibility that the lack of sod2 leads to a coordinated
repression of transcriptional or post-transcriptional synthesis
of complex II subunits (Ackrell et al. 1984). A decrease in the
activity of complex II alone is sufficient to cause neurological
disorders in humans (Ackrell 2002). Deleterious mutations in
the gene encoding the FP subunit (sdhA) can cause neurological defects that result in ataxia (Birch-Machin et al. 2000;
Baysal et al. 2001), and patients heterozygous for a mutation
in the gene encoding the FP subunit have 50% complex II
activity and display late-onset ataxia (Birch-Machin et al.
2000). Dramatic reductions in the TCA cycle enzymes
aconitase, SDH and KGDH could result in restricted production of reducing equivalents for the respiratory chain, which
could drastically impair energy production. Our proteomic
data indicate that the glycolytic enzyme TPI may be increased
in the cytosol in the sod2–/– mice (Table 1). This suggests
that glycolysis might be up-regulated to offset the potential
loss of mitochondrially produced ATP.
Our proteomic screening showed that lack of SOD2
resulted in changes in two enzymes associated with
maintaining a redox balance within the mitochondria. Three
different isoforms of the GST class-mu1 were differentially
expressed, with one being reduced and two forms increased
in the sod2–/– mice. Prx5 is a thioredoxin peroxidase that
reduces hydrogen peroxide and is able to inhibit intracellular
hydrogen peroxide accumulation by TNFa (Yamashita et al.
1999; Seo et al. 2000). Prx5 concentration is reduced 1.5fold in the mitochondria of the sod2–/– mice. Without SOD2
in the mitochondria, the concentration of hydrogen peroxide
may be significantly reduced, leading to a reduction in the
level of Prx5 in the mitochondria through an unknown
mechanism. Alternately, in the sod2–/– mice, Prx5 may be
converted into other undetected isoforms containing nonreduced intramolecular disulfide linkages that are necessary
for its peroxidase function, which would alter its mobility on
the gel such that any individual form could be too low an
abundance to detect. This general point may be true for other
proteins we detected as well.
We report here that neuronal cell death in defined regions
of the frontal cortex is a consequence of endogenous
mitochondrial oxidative stress. Notably, we can partially
rescue neuronal cell death by treatment with a high dose of
EUK-8 (Fig. 4). The amino-cupric silver stain detects
neurons that are in the process of degeneration (de Olmos
1994), i.e. this method provides a ‘snap shot’ of neuronal cell
death at the time of the animal’s death. The proteomics and
enzyme analyses were performed on mitochondria isolated
from the complete cortex, which includes the regions in
which neuronal cell death and spongiform changes had
occurred, as well as unaffected regions. In order to determine
if the complex II/SDH defects we measured in isolated
mitochondria from cortex were unique to the regions in
which neuronal cell death occurred, SDH staining was
performed on brains from high and low dose-treated sod2 –/–
and wild-type mice (Fig. 5). Our results demonstrate that
decreased complex II activity is not unique to the regions of
the brain in which cell death occurred and that the intensity
of SDH staining throughout the brain reflects the changes
seen in enzyme activity and subunit concentration under the
different conditions. This suggests that there are idiosyncratic
properties of affected cortex that make it particularly
sensitive to the loss of mitochondrial function. Treatment
with a high dose of EUK-8 completely rescues the spongiform pathology of the sod2 –/– mice in the cortex,
trigeminal motor nucleus and all other affected areas,
however, mild behavioral defects persist. The cause of this
behavioral defect in the high dose-treated mice may be the
incomplete prevention of neuronal cell death, possibly
arising due to the persistent defects in the TCA cycle and
electron transport chain.
We have shown that the SOD2 null mouse is a useful tool
for uncovering the vulnerability of specific mitochondrial
proteins to endogenous oxidative stress in the brain, and the
potential utility of catalytic antioxidants, such as EUK-8
and related compounds in therapeutically alleviating the
2003 International Society for Neurochemistry, J. Neurochem. (2004) 88, 657–667
666 D. Hinerfeld et al.
consequences of specific mitochondrial defects and neurodegeneration. By extension, such an approach may be of
potential benefit to patients with neurodegenerative diseases
in which oxidative stress is presumed to be involved.
Acknowledgements
We thank Dr Brian Ackrell for supplying the antibodies to complex
II and for helpful comments. This work was supported by a National
Institutes of Health Grant AG18679 awarded to SM.
References
Ackrell B. A. (2002) Cytopathies involving mitochondrial complex II.
Mol. Aspects Med. 23, 369–384.
Ackrell B. A., Maguire J. J., Dallman P. R. and Kearney E. B. (1984)
Effect of iron deficiency on succinate- and NADH-ubiquinone
oxidoreductases in skeletal muscle mitochondria. J. Biol. Chem.
259, 10053–10059.
Ackrell B. A. C., Johnson M. K., Gunsalus R. P. and Cecchini G.
(1992) Structure and function of succinate dehydrogenase and
fumarate reductase, in Chemistry and Biochemistry of Flavoenzymes, Vol. III (Muller, F., ed.), pp. 230–297. CRC Press, Boca
Raton.
Baker K., Marcus C. B., Huffman K., Kruk H., Malfroy B. and Doctrow
S. R. (1998) Synthetic combined superoxide dismutase/catalase
mimetics are protective as a delayed treatment in a rat stroke
model: a key role for reactive oxygen species in ischemic brain
injury. J. Pharmacol. Exp. Ther. 284, 215–221.
Baudry M., Etienne S., Bruce A., Palucki M., Jacobsen E. and Malfroy
B. (1993) Salen –manganese complexes are superoxide dismutasemimics. Biochem. Biophys. Res. Commun. 192, 964–968.
Baysal B. E., Rubinstein W. S. and Taschner P. E. (2001) Phenotypic
dichotomy in mitochondrial complex II genetic disorders. J. Mol.
Med. 79, 495–503.
Beinert H., Ackrell B. A., Vinogradov A. D., Kearney E. B. and Singer
T. P. (1977) Interrelations of reconstitution activity, reactions with
electron acceptors, and iron –sulfur centers in succinate dehydrogenase. Arch. Biochem. Biophys. 182, 95–106.
Birch-Machin M. A., Taylor R. W., Cochran B., Ackrell B. A. and
Turnbull D. M. (2000) Late-onset optic atrophy, ataxia, and myopathy associated with a mutation of a complex II gene. Ann.
Neurol. 48, 330–335.
Boveris A. (1984) Determination of the production of superoxide radicals and hydrogen peroxide in mitochondria. Methods Enzymol.
105, 429–435.
Brown R. H. (1997) Mitochondria and oxidative stress in amytrophic
lateral sclerosis, in Mitochondria and Free Radicals in Neurodegenerative Diseases (Beal, M. F., Howell, N. and Bodis-Wollner,
I., eds), pp. 309–318. Wiley-Liss, New York.
Capaldi R. A., Sweetland J. and Merli A. (1977) Polypeptides in the
succinate-coenzyme Q reductase segment of the respiratory chain.
Biochemistry 16, 5707–5710.
Catterall W. A. and Pedersen P. L. (1971) Adenosine triphosphatase from
rat liver mitochondria. I. Purification, homogeneity, and physical
properties. J. Biol. Chem. 246, 4987–4994.
Chinopoulos C., Tretter L. and Adam-Vizi V. (1999) Depolarization of in
situ mitochondria due to hydrogen peroxide-induced oxidative
stress in nerve terminals: inhibition of alpha-ketoglutarate dehydrogenase. J. Neurochem. 73, 220–228.
Davis K. A. and Hatefi Y. (1971) Succinate dehydrogenase. I. Purification, molecular properties, and substructure. Biochemistry 10,
2509–2516.
Doctrow S. R., Huffman K., Marcus C. B., Tocco G., Malfroy E.,
Adinolfi C. A., Kruk H. , Baker K. , Lazarowych N., Mascarenhas
J. and Malfroy B. (2002) Salen-manganese complexes as catalytic
scavengers of hydrogen peroxide and cytoprotective agents:
structure-activity relationship studies. J. Med. Chem. 45, 4549–
4558.
Fix A. S., Ross J. F., Stitzel S. R. and Switzer R. C. (1996) Integrated
evaluation of central nervous system lesions: stains for neurons,
astrocytes, and microglia reveal the spatial and temporal features of
MK-801-induced neuronal necrosis in the rat cerebral cortex.
Toxicol. Pathol. 24, 291–304.
Gardner P. R., Raineri I., Epstein L. B. and White C. W. (1995)
Superoxide radical and iron modulate aconitase activity in mammalian cells. J. Biol. Chem. 270, 13399–13405.
Gibson G. E., Park L. C., Sheu K. F., Blass J. P. and Calingasan N. Y.
(1999) The alpha-ketoglutarate dehydrogenase complex in neurodegeneration. Neurochem. Int. 36, 97–112.
Giulivi C. (2003) Characterization and function of mitochondrial nitricoxide synthase. Free Radic. Biol. Med. 34, 397–408.
Gonzalez P. K., Zhuang J., Doctrow S. R., Malfroy B., Benson P. F.,
Menconi M. J. and Fink M. P. (1995) EUK-8, a synthetic superoxide dismutase and catalase mimetic, ameliorates acute lung injury
in endotoxemic swine. J. Pharmacol. Exp. Ther. 275, 798–806.
Higgins C. M., Jung C., Ding H. and Xu Z. (2002) Mutant Cu, Zn
superoxide dismutase that causes motoneuron degeneration is
present in mitochondria in the CNS. J Neurosci. 22, RC215.
Huang T. T., Carlson E. J., Kozy H. M., Mantha S., Goodman S. I.,
Ursell P. C. and Epstein C. J. (2001) Genetic modification of
prenatal lethality and dilated cardiomyopathy in Mn superoxide
dismutase mutant mice. Free Radic. Biol. Med. 31, 1101–1110.
Humphries K. M. and Szweda L. I. (1998) Selective inactivation of
alpha-ketoglutarate dehydrogenase and pyruvate dehydrogenase:
reaction of lipoic acid with 4-hydroxy-2-nonenal. Biochemistry 37,
15835–15841.
Jha N., Jurma O., Lalli G., Liu Y., Pettus E. H., Greenamyre J. T., Liu R.
M., Forman H. J. and Andersen J. K. (2000) Glutathione depletion
in PC12 results in selective inhibition of mitochondrial complex I
activity. Implications for Parkinson’s disease. J. Biol. Chem. 275,
26096–26101.
Jung C., Higgins C. M. and Xu Z. (2002) Mitochondrial electron
transport chain complex dysfunction in a transgenic mouse model
for amyotrophic lateral sclerosis. J. Neurochem. 83, 535–545.
Jung C., Rong Y., Doctrow S., Baudry M., Malfroy B. and Xu Z. (2001)
Synthetic superoxide dismutase/catalase mimetics reduce oxidative
stress and prolong survival in a mouse amyotrophic lateral sclerosis
model. Neurosci. Lett. 304, 157–160.
Kish S. J., Bergeron C., Rajput A., Dozic S., Mastrogiacomo F., Chang
L. J., Wilson J. M., DiStefano L. M. and Nobrega J. N. (1992)
Brain cytochrome oxidase in Alzheimer’s disease. J. Neurochem.
59, 776–779.
Koutnikova H., Campuzano V., Foury F., Dolle P., Cazzalini O. and
Koenig M. (1997) Studies of human, mouse and yeast homologues
indicate a mitochondrial function for frataxin. Nat. Genet. 16, 345–
351.
LaVaute T., Smith S., Cooperman S., Iwai K., Land W., Meyron-Holtz
E., Drake S. K., Miller G., Abu-Asab M., Tsokos M. et al. (2001)
Targeted deletion of the gene encoding iron regulatory protein-2
causes misregulation of iron metabolism and neurodegenerative
disease in mice. Nat. Genet. 27, 209–214.
2003 International Society for Neurochemistry, J. Neurochem. (2004) 88, 657–667
Endogenous mitochondrial oxidative stress 667
Li Y., Huang T. T., Carlson E. J., Melov S., Ursell P. C., Olson J. L.,
Noble L. J., Yoshimura M. P., Berger C., Chan P. H. et al. (1995)
Dilated cardiomyopathy and neonatal lethality in mutant mice
lacking manganese superoxide dismutase. Nat. Genet. 11, 376–
381.
Liu R., Liu I. Y., Bi X., Thompson R. F., Doctrow S. R., Malfroy B. and
Baudry M. (2003) Reversal of age-related learning deficits and
brain oxidative stress in mice with superoxide dismutase/catalase
mimetics. Proc. Natl. Acad. Sci. USA 100, 8526–8531.
Malfroy B., Doctrow S. R., Orr P. L., Tocco G., Fedoseyeva E. V. and
Benichou G. (1997) Prevention and suppression of autoimmune
encephalomyelitis by EUK-8, a synthetic catalytic scavenger of
oxygen-reactive metabolites. Cell Immunol. 177, 62–68.
Martensson J., Jain A., Stole E., Frayer W., Auld P. A. and Meister A.
(1991) Inhibition of glutathione synthesis in the newborn rat: a
model for endogenously produced oxidative stress. Proc. Natl
Acad. Sci. USA 88, 9360–9364.
Melov S., Schneider J. A., Day B. J., Hinerfeld D., Coskun P., Mirra S.
S., Crapo J. D. and Wallace D. C. (1998) A novel neurological
phenotype in mice lacking mitochondrial manganese superoxide
dismutase. Nat. Genet. 18, 159–163.
Melov S., Coskun P., Patel M., Tuinstra R., Cottrell B., Jun A. S.,
Zastawny T. H., Dizdaroglu M., Goodman S. I., Huang T. T. et al.
(1999) Mitochondrial disease in superoxide dismutase 2 mutant
mice. Proc. Natl Acad. Sci. USA 96, 846–851.
Melov S., Ravenscroft J., Malik S., Gill M. S., Walker D. W., Clayton
P. E., Wallace D. C., Malfroy B., Doctrow S. R. and Lithgow G. J.
(2000) Extension of life-span with superoxide dismutase/catalase
mimetics. Science 289, 1567–1569.
Melov S., Doctrow S. R., Schneider J. A., Haberson J., Patel M., Coskun
P. E., Huffman K., Wallace D. C. and Malfroy B. (2001) Lifespan
extension and rescue of spongiform encephalopathy in superoxide
dismutase 2 nullizygous mice treated with superoxide dismutasecatalase mimetics. J. Neurosci. 21, 8348–8353.
Mutisya E. M., Bowling A. C. and Beal M. F. (1994) Cortical cytochrome oxidase activity is reduced in Alzheimer’s disease. J.
Neurochem. 63, 2179–2184.
Nakatani T., Nakashima T., Kita T., Hirofuji C., Itoh K., Itoh M. and
Ishihara A. (2000) Cell size and oxidative enzyme activity of
different types of fibers in different regions of the rat plantaris and
tibialis anterior muscles. Jpn J. Physiol. 50, 413–418.
Nulton-Persson A. C. and Szweda L. I. (2001) Modulation of mitochondrial function by hydrogen peroxide. J. Biol. Chem. 276, 23357–
23361.
de Olmos J. S., Beltramino C. A. and de Olmos de Lorenzo S. (1994)
Use of Amino-cupric-silver technique for the detection of early and
semiacute neuronal degeneration caused by neurotoxicants, hypoxia and physical trauma. Neurotoxicol. Teratol. 16, 545–561.
Pong K., Doctrow S. R., Huffman K., Adinolfi C. A. and Baudry M.
(2001) Attenuation of staurosporine-induced apoptosis, oxidative
stress, and mitochondrial dysfunction by synthetic superoxide
dismutase and catalase mimetics, in cultured cortical neurons. Exp.
Neurol. 171, 84–97.
Priller J., Scherzer C. R., Faber P. W., MacDonald M. E. and Young A.
B. (1997) Frataxin gene of Friedreich’s ataxia is targeted to mitochondria. Ann. Neurol. 42, 265–269.
Rifai Z., Welle S., Kamp C. and Thornton C. A. (1995) Ragged red
fibers in normal aging and inflammatory myopathy [see comments]. Ann. Neurol. 37, 24–29.
Rong Y., Doctrow S. R., Tocco G. and Baudry M. (1999) EUK-134, a
synthetic superoxide dismutase and catalase mimetic, prevents
oxidative stress and attenuates kainate-induced neuropathology.
Proc. Natl Acad. Sci. USA 96, 9897–9902.
Rotig A., de Lonlay P., Chretien D., Foury F., Koenig M., Sidi D.,
Munnich A. and Rustin P. (1997) Aconitase and mitochondrial
iron–sulphur protein deficiency in Friedreich ataxia. Nat. Genet.
17, 215–217.
Seo M. S., Kang S. W., Kim K., Baines I. C., Lee T. H. and Rhee S. G.
(2000) Identification of a new type of mammalian peroxiredoxin
that forms an intramolecular disulfide as a reaction intermediate. J.
Biol. Chem. 275, 20346–20354.
Sharpe M. A., Ollosson R., Stewart V. C. and Clark J. B. (2002) Oxidation of nitric oxide by oxomanganese–salen complexes: a new
mechanism for cellular protection by superoxide dismutase/catalase mimetics. Biochem. J. 366, 97–107.
Shoubridge E. A. (1994) Mitochondrial DNA diseases: histological and
cellular studies. J. Bioenerg. Biomembr. 26, 301–310.
Swerdlow R. H., Parks J. K., Miller S. W., Tuttle J. B., Trimmer P. A.,
Sheehan J. P., Bennett J. P. Jr, Davis R. E. and Parker W. D. Jr
(1996) Origin and functional consequences of the complex I defect
in Parkinson’s disease. Ann. Neurol. 40, 663–671.
Taylor S. W., Fahy E., Zhang B., Glenn G. M., Warnock D. E., Wiley S.,
Murphy S., Gaucher S. P., Capaldi R. A., Gicbson B. W. et al.
(2003) Characterization of the human mitochondrial proteome.
Nat. Biotechnol. 21, 281–286.
Trounce I. A., Kim Y. L., Jun A. S. and Wallace D. C. (1996) Assessment of mitochondrial oxidative phosphorylation in patient muscle
biopsies, lymphoblasts, and transmitochondrial cell lines. Methods
Enzymol. 264, 484–509.
Turrens J. F., Alexandre A. and Lehninger A. L. (1985) Ubisemiquinone
is the electron donor for superoxide formation by complex III of
heart mitochondria. Arch. Biochem. Biophys. 237, 408–414.
Yamashita H., Avraham S., Jiang S., London R., Van Veldhoven P. P.,
Subramani S., Rogers R. A. and Avraham H. (1999) Characterization of human and murine PMP20 peroxisomal proteins that
exhibit antioxidant activity in vitro. J. Biol. Chem. 274, 29897–
29904.
2003 International Society for Neurochemistry, J. Neurochem. (2004) 88, 657–667