Journal of Gerontology: BIOLOGICAL SCIENCES
Cite journal as: J Gerontol A Biol Sci Med Sci
2009. Vol. 64A, No. 5, 530–539
doi:10.1093/gerona/glp020
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Advance Access publication on March 12, 2009
SOD-1 Deletions in Caenorhabditis elegans Alter the
Localization of Intracellular Reactive Oxygen Species and
Show Molecular Compensation
Sumino Yanase,1,2 Akira Onodera,2 Patricia Tedesco,3 Thomas E. Johnson,3 and Naoaki Ishii2
1Department
of Health Science, Daito Bunka University School of Sports and Health Science, Saitama, Japan.
of Molecular Life Science, Tokai University School of Medicine, Kanagawa, Japan.
3Institute for Behavioral Genetics, University of Colorado at Boulder.
2Department
Superoxide dismutase (SOD) is an enzyme that catalytically removes the superoxide radical (⋅O −2 ) and protects organisms
from oxidative damage during normal aging. We demonstrate that not only the cytosolic ⋅O −2 level but also the mitochondrial ⋅O −2 level increases in the deletion mutants of sod-1 gene encoding Cu/Zn SOD in Caenorhabditis elegans (C. elegans).
Interestingly, this suggests that the activity of SOD-1, which so far has been thought to act mainly in cytoplasm, helps to
control the detoxification of ⋅O −2 also in the mitochondria. We also found functional compensation by other SODs, especially the sod-5 gene, which was induced several fold in the mutants. Therefore, the possibility exists that the compensative
expression of sod-5 gene in the sod-1 deficit is associated with the insulin/insulin-like growth factor-1 (Ins/IGF-1) signaling
pathway, which regulates longevity and stress resistance of C. elegans because the sod-5 gene may be a target of the pathway.
Key Words: Sod-1—Sod-5—Mitochondrial ROS production—Oxidative stress—Aging.
S
UPEROXIDE dismutase (SOD) was discovered in 1969
by McCord and Fridovich (1) as an enzyme that catalyti−
cally removes the superoxide radical (⋅O 2). It is known that
−
⋅O 2 is used in the leukocyte immune system and that it also
causes various degenerative diseases resulting from oxidative damage and normal aging. The free radical theory of
aging, proposed nearly 50 years ago by Harman (2), demonstrates how in the normal aging process, free radical damage
to cellular constituents inevitably accumulates with age.
Since the publication of this theory, the role of SODs as important endogenous antioxidants has become better understood. Although oxygen is essential for most metazoan life, a
variety of oxidative damage is produced in an aerobic envi−
ronment. ⋅O 2 plays a central role in the production of other
reactive oxygen species (ROS), hydrogen peroxide (H2O2)
and hydroxyl radical (OH˙). The generation of intracellular
⋅O −2 depends mainly on the mitochondrial respiratory chain
and it is estimated that around 2% of the total oxygen con−
sumed in the mitochondria is transformed into ⋅O 2 and re−
leased (3). ⋅O 2 is also released by other cellular sources such
as NAD(P)H oxidase–like activity in the leukocyte plasma
membrane, NAD(P)H cytochrome P-450 reductase in the
microsome, and xanthine oxidase in the cytoplasm (4).
These intracellular oxidants appear to be important in the
development of the senescent phenotype in various organisms.
For example, it is well known that life span is closely related
to the environmental oxygen concentration in the nematode
Caenorhabditis elegans (C. elegans). Exposing C. elegans to
hyperoxia accelerates its normal aging and reduces life span;
however, mature worms in hypoxia exhibit a prolonged life
span (5). The intracellular ROS levels increase in worms
continuously exposed to environmental hyperoxia, and then
530
endogenous antioxidant systems containing SOD, catalase,
and glutathione detoxify the ROS for the protection of cellular and extracellular biomolecules (lipids, proteins, and nucleic
acids) (6). However, both the defending and the respiratory electron transport systems in oxygen-utilizing organisms are not
perfect in the complete removal of ROS and usage of electron.
The intracellular ROS causes cumulative oxidative damage in
the cellular targets. Consequently, the intracellular and extracellular damages trigger the physiological decline, which is a characteristic of aging.
In these enzymatic antioxidants, we have taken notice of
association with SOD and normal aging. Because SOD has a
−
role in intracellular ⋅O 2 removal, which is produced in the
early process of ROS production in aerobic organisms, it is
thought that the antioxidant deficit may reflect a dramatic influence in normal aging. Several SOD isozymes reduce the
⋅O −2 in C. elegans; five genes encoding these SODs (sod-1 to
sod-5) have been identified in the genome (7–11). sod-1 and
sod-5 encode cytoplasmic Cu/Zn SODs, sod-2 and sod-3 encode Mn SODs containing a mitochondrial signal sequence,
and sod-4 encodes the homologue of the extracellular Cu/Zn
SOD in mammals. It has been strongly suggested that the expressions and activities of these SODs are related to the control of normal aging and help determine the longevity of
C. elegans (7,12). However, the role of each of these SOD
isozymes in C. elegans, and how they act collectively to affect
aging and longevity, has not been described. The problem involves the relation between expression level, localization, and
half-life of each SOD isozyme necessary to maintain proper
ROS levels in vivo. Particularly relevant is a previous publication indicating that decreased SOD activity during normal aging is probably due to increased protein carbonylation of SOD,
INTRACELLULAR ROS: SOD-1 BALANCE
a fact that may be very important in aging (13). Therefore, we
analyzed in detail the aging phenotypes of two sod-1 deletion
mutants obtained from the National Bioresource Project for the
Experimental Animal “Nematode C. elegans” (http://shigen.lab.
nig.ac.jp/c.elegans/index.jsp?lang=japanese). These deletion
mutants were isolated using a trimethyl psoralen/ultraviolet
(TMP/UV) method (14) and we report here that the intracellular
localization and quantitative balance of oxidative stress may
specify the rate of aging and longevity in C. elegans.
Furthermore, there is our recent finding that the insulin/insulin-like growth factor-1 (Ins/IGF-1) signaling pathway affects
expression control of some of these sod genes (15). The Ins/
IGF-1 signaling pathway in C. elegans was identified previously
as a key regulator of normal aging (16). According to identification of downstream targets of the forkhead box O (Fox O) transcription factor DAF-16, it is clear that the Ins/IGF-1 signaling
pathway participates closely in the control of antioxidants such
as SOD and catalase, and in the mitochondrial respiratory chain
systems (17). Analysis of a variant in the signaling pathway
suggests that resistance to ROS is associated with the life span
extension in many genetic mutants (17,18). We identified a predicted promoter region associated to the compensatory expression of sod-5 gene in the sod-1 deletion phenotype.
Methods
Nematode Strains and Maintenance
The wild-type strain (N2) of C. elegans var. Bristol and
age-1(hx546) strain (TJ1062) were obtained from the
Caenorhabditis Genetics Center at the University of Minnesota (Minneapolis, MN). The age-1(hx546) mutant was the
first long-lived mutant (19). Two sod-1 deletion mutants,
tm776 and tm783, were supplied by the National Bioresource
Project for the Experimental Animal “Nematode C. elegans”.
Both sod-1 deletion strains are homozygous viable and were
backcrossed with the Ishii lab wild-type strain three times to
remove unlinked mutations yielding two strains: tm776(kn86)
and tm783(kn87). The deleted regions were confirmed by genomic polymerase chain reaction (PCR) using the following
primers: 5'-AATGTCGAACCGTGCTGTC-3' and 5'GAAACTACAACCTTTATTTG-3' for the tm776 strain, and
5'-GTGGTTTTTAATCAGTGTCATAATC-3' and 5'-GGTCCACCATGAGTCTTTCC-3' for the tm783 strain. These
strains were kept frozen as described previously (20) until
used. Media and standard procedures for the culture of C. elegans were as reported previously (21). The worms were fed
Escherichia coli OP50, a uracil-requiring mutant, and grown
at 20°C on nematode growth medium (NGM) agar plates.
Northern Blots and Quantitative PCR for sod-1, sod-2,
sod-3, sod-4, and sod-5 Genes
The poly(A)+ RNA of animals was prepared and analyzed
as described previously (22). A total of 1 mg of poly(A)+ RNA
531
Figure 1. Structure of the sod-1 gene and the deletion sites in sod-1(tm776)
and sod-1(tm783) deletion strains of Caenorhabditis elegans. (A) The overall
structure of the sod-1 gene and the coding/transcript regions based on sequencing of the complementary DNAs. sod-1(tm776) contains a 612 base pair (bp)
deletion of exons 3 through 5, and sod-1(tm783) contains a 530 bp deletion of
exon 1 and the 5'-regulatory region of the gene, information from the National
Bioresource Project for the Experimental Animal “Nematode C. elegans.” (B)
The confirmation of the deletion size in the sod-1 deletion strains compared with
the wild-type (WT) N2 using genomic polymerase chain reaction (PCR). As
both deletions are homozygous viable, only a single short fragment was amplified by PCR. Using primers designed for tm776 amplification, the full-length
amplicon for N2 was 772 bp and 160 bp for tm776. Using primers for tm783, the
fragment size in N2 was 744 bp and 214 bp for the tm783 mutant. (C) The results
of the Northern blot analysis in the tm strains using the sod-1 gene as a probe.
per lane was separated by electrophoresis on a 1.2% agarose
gel containing 2.0 M formaldehyde and 0.02 M 3-morpholino
propane sulfonic acid (MOPS) (Wako Pure Chemical Co.,
Osaka, Japan) and transferred onto a nylon membrane (ZetaProbe GT Membrane; BIO-RAD, Hercules, CA) by a mild
alkaline transfer method. Northern hybridizations of the
transferred poly(A)+ RNAs were performed using PCR product probe containing sod-1a-coding and sod-1b-coding regions labeled with 32P-dCTP at 65°C overnight. We carried
out Northern blot analyses of the messenger RNAs (mRNAs)
for the sod-1 gene in the tm strains.
Poly (A)+ RNA of animals was prepared to conduct PCR,
and then complementary DNA (cDNA) synthesized as described previously (22,23) and used as a template for PCR.
We carried out the quantitative PCR (QPCR) for the sod
genes in 4-day-old animals. The target genes and primers
for QPCR were as follows: sod-1 (encoding a Cu/Zn SOD),
primers 5'-CGTAGGCGATCTAGGAAATGTG-3' and 5'AACAACCATAGATCGGCCAACG-3'; sod-2 (encoding
a Mn SOD), primers 5'-AGCTTTCGGCATCAACTGTC-3'
and 5'-AAGTCCAGTTGTTGCCTCAAGT-3'; sod-3 (encoding a Mn SOD), primers 5'-TTCAAAGGAGCTGATGGACACT-3' and 5'-AAGTGGGACCATTCCTTCCAA-3'; sod-4
532
YANASE ET AL.
Figure 2. Images of sod-1::egfp gene expressed in a young (4-day-old) and an older (9-day-old) adult wild-type animal. Panels A and B show, respectively, 4-day-old
and 9-day-old wild-type N2, carrying an extrachromosomal array of the sod-1::egfp construction. (C) The quantitative data of enhanced green fluorescent protein (GFP)
fluorescence intensity. Data are means ± SD for three or more independent transgenic animals. It is clear that the fluorescence intensities of 4-day-old and 9-day-old
animals are not statistically different by Student’s t test.
(encoding an extracellular Cu/Zn SOD), primers 5'GTTGTCTAAGTGCTGGTGG-3' and 5'-TTCCACATGCAAGTCGGCT-3'; sod-5 (encoding a Cu/Zn SOD), primers
5'-GCAAAATGAATCATGGAGGAAG-3' and 5'-AAGATCATCTCGATCGACGTGG-3'; and act-1 (encoding the body
wall and the pharyngeal muscle actin protein) for normalization, primers 5'-CCACGTCATCAAGGAGTCAT-3' and 5'GGAAGCGTAGAGGGAGAGGA-3'. A range of concentrations of worm cDNA was used as template for QPCR. SYBR
Green PCR Master Mix (Applied Biosystems Ltd., Foster City,
CA) was used and PCR carried out on the ABI 7000 Sequence
Detection System. Results were normalized to the transcript
level of act-1 using the wild-type strain N2 as the control.
Construction of SOD-1 Fusion Proteins With Enhanced
Green Fluorescent Protein and Transgenics
To amplify approximately 3 kb containing the putative
promoter region and the entire transcribed sequence of the
sod-1 gene, genomic PCR using sod-1 primers that included BamHI or HindIII restriction sites was performed.
The resulting sod-1 product was cleaved with the restriction enzymes (BamHI and HindIII) and cloned into the
pFXneEGFP vector (obtained from Dr. S. Mitani) to express an enhanced green fluorescent protein (EGFP). This
construct and one containing a dominant rol-6 marker were
co-injected into N2 and sod-1 mutant hermaphrodites;
roller lines incorporating the EGFP reporter and the rol-6
selectable marker into extrachromosomal arrays were isolated as individual transgenic strains: tm776(kn86);Ex[Psod1::sod-1::egfp] and tm783(kn87);Ex[Psod-1::sod-1::egfp].
Quantitative data of fluorescence intensity were obtained
using the Image Analysis Software “WinROOF” (MITANI
Co., Fukui, Japan).
Measurement of Life Span
Eggs were collected by washing gravid hermaphrodites from the NGM agar plates and dissolving them in
alkaline sodium hypochlorite. The released eggs were allowed to hatch through overnight incubation at 20°C
in S-buffer in absence of cholesterol to establish agesynchronous cultures (24). Life span of the hermaphrodites at 20°C was measured as described previously (25).
To prevent progeny production, 5-fluoro-2'-deoxyuridine
(Wako Pure Chemical Co., Osaka, Japan) was added to
the NGM agar plate at a final concentration of 40 mM,
after the animals had reached adulthood. For statistical
significance in both mean of life span and maximum of
life span, at least three independent experiments were
executed. A total of 100 worms were scored for each
experiment.
533
INTRACELLULAR ROS: SOD-1 BALANCE
various concentrations (0%, 60%, and 100%) of oxygen gas
in a closed plastic chamber (23). L1 larvae, compared with
adults, are more sensitive to various oxidants (25). Four
days later, survival was determined by counting the number
of late-stage larvae (L4 larvae) and adults. The L4 larvae
were considered to be survivors because we have observed
that such late-stage larvae usually attain adulthood.
To assess the resistance to various stressors, thermotolerance of the various strains was measured using young adults
at 35°C. Synchronously cultured animals were kept on
NGM plates with OP50 at 20°C until they reached youngadult stage. At the start of the thermotolerance assay, 20
worms from each strain were transferred onto a fresh NGM
plate and placed at 35°C. The number of surviving and dead
animals was determined every 2 hours (23,26).
Figure 3. Survival curves at 20°C in sod-1 deletion strains and transgenic
sod-1 deletion strains carrying an extrachromosomal array of the wild-type sod-1
gene. (A) Open circle shows wild-type N2, closed square shows tm776, and
closed triangle shows tm783. (B) Open circle shows N2;Ex[Psod-1::sod-1::egfp],
closed square shows tm776;Ex[Psod-1::sod-1::egfp], and closed triangle shows
tm783;Ex[Psod-1::sod-1::egfp]. Each value was indicated by at least three independent experiments. About 100 animals were used for each experiment. In these
two independent experiments, N2 life span seems to differ slightly; however, it is
clear from t test values that those variations are not statistically significant.
Assays of Resistance to Paraquat or Oxygen, and of
Thermotolerance
Eggs were collected from the NGM agar plates using alkaline sodium hypochlorite and allowed to hatch overnight at
20°C in S-buffer. The newly hatched L1 (first larval stage) larvae were then cultured on preseeded NGM agar plates. To
evaluate paraquat resistance, approximately 200 L1 larvae were
put on plates containing various concentrations (0–0.4 mM)
of paraquat dichloride (1,1'-dimethyl-4,4'-bipyridium dichloride; GmbH, Augsburg, Germany). To quantify oxygen
resistance, L1 worms were put on plates and exposed to
Measurement of SOD Activities
To confirm the intrinsic SOD activities in the sod-1 deletion mutants, 4-day-old animals were washed with phosphate-buffered saline (Ca2+ and Mg2+ free, pH 7.2) and 0.15 M
NaCl, 10 mM HEPES (pH 7.4) buffer. The animals, resuspended in an equal volume of HEPES buffer, were homogenized using a Teflon homogenizer. To fragment the cellular
membranes, each homogenized solution was treated three
times with ultrasonication for 3 minutes. To isolate the
cytosol and membrane fractions, each solution was ultracentrifuged at 100 kG for 30 minutes in a Beckman Optima
TL Ultracentrifuge (Beckman Instruments, Inc., Fullerton,
CA). The SOD activity was measured using the SOD Assay
Kit-WST (Dojindo Molecular Technologies, Inc., Kumamoto, Japan) and standardized with SOD from bovine
erythrocytes (Sigma Chemical Co., St. Louis, MO). The
BCA Protein Assay Kit was used to measure the total protein contents in the cytosol and membrane fractions for
normalization purpose.
−
Measurement of Mitochondrial and Cytosol ⋅O 2 Using
2-Methyl-6-p-methoxyphenylethynyl-imidazopyrazinone
For the isolation of mitochondria and cytosol fractions,
4-day-old animals washed with S-buffer and mannitol–
sucrose buffer were homogenized with 20–60 strokes at
Table 1. Mean and Maximum of Life Spans at 20°C in sod-1 Deletion Mutants With or Without the Extrachromosomal Arrays of Wild-Type
sod-1
Mean of Life Span (d)*
Strain
N2
tm776
tm783
N2;ExSOD-1::EGFP
tm776;ExSOD-1::EGFP
tm783;ExSOD-1::EGFP
27.9 ± 3.9
21.3 ± 3.1a
21.2 ± 3.4b
25.4 ± 3.8
26.5 ± 5.0
24.8 ± 3.3
32.1 ± 3.4
24.1 ± 3.4c
27.7 ± 2.9d
24.0 ± 2.5
26.8 ± 4.6g
26.5 ± 3.7h
Maximum of Life Span (d)†
26.5 ± 2.4
22.3 ± 3.2e
22.6 ± 3.3f
23.9 ± 3.1
25.9 ± 3.7i
26.4 ± 3.1j
39.3 ± 2.0
34.7 ± 1.2
36.0 ± 0.5
37.0 ± 2.6
48.7 ± 3.6k
45.0 ± 1.0l
Notes: * Results are indicated as means ± SD from three independent experiments.
are expressed as means ± SD of more than three determinations. p values by t test with different superscripts (N2 vs. each tm strain, transgenic N2 vs.
each transgenic tm strain) significantly differ as follows; a,bp < .001; c,dp < .001; e,fp < .001; gp < .001, hp < .002; ip < .05; jp < .01; kp < .05; and lp < .005.
† Results
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YANASE ET AL.
Statistical Analyses
All values in the figures and text are demonstrated as mean
± standard deviation or standard error of the mean. The significance was tested with paired Student’s t test. Differences
were considered significant when p was less than .05.
Results
Confirmation of Deletion Site in tm Strains
After outcrossing, we confirmed the deleted portions of
the sod-1 gene contained in tm776(kn86) and tm783(kn87).
Based on information provided by the National Bioresource
Project for the Experimental Animal “Nematode C. elegans,” these two strains are homozygous viable. The tm 776
mutant is a 612 base pair (bp) deletion in the 3'-region of
sod-1, and the tm 783 is a 530 bp deletion in the 5'-region
and has part of the promoter eliminated (Figure 1A). Deletions were confirmed by genomic PCR (Figure 1B). Both
deletions result in the absence of the full-size sod-1a
and sod-1b mRNAs, in contrast to the wild-type controls
(Figure 1C).
Figure 4. Images of sod-1::egfp gene expressed in sod-1 deletion mutants.
Panels A and B are images of tm776 and tm783 worms aged 4 days expressing
an extrachromosomal array of sod-1::egfp, respectively.
1,300 rpm on ice using a Teflon homogenizer, Eyela Mazela
Z (Tokyo Rikakikai Co., Ltd., Tokyo, Japan). The intact mitochondrial fraction was isolated by differential centrifugation at 500 and 25,000 G for 10 minutes, respectively, and
resuspended in Tris–ethylenediaminetetraacetic acid buffer.
The cytosol fraction was isolated as a supernatant of the
mitochondrial fraction after the first centrifugation. The
⋅O −2 production was measured using an ⋅O −2 specific chemiluminescent probe, 2-methyl-6-p-methoxyphenylethynylimidazopyrazinone (MPEC; ATTO Co., Tokyo, Japan) (27).
Forty micrograms of the intact mitochondria or cytosol fractions in 1 mL of assay buffer containing 0.7 mM MPEC were
placed in the photon counter of the Luminescencer-PSN AB2200 (ATTO) and luminescence per second recorded (28).
Survey of Predicted Promoter Region of sod-5 Gene
We searched the DAF-16 consensus binding element
(DBE) sequence (29) within the promoter region of sod-5
gene from the C. elegans database WormBase (http://www.
wormbase.org/) using the genetic information processing
software GENETYX-WIN (Software Development Co.,
Ltd., Tokyo, Japan). Furthermore, the predicted amino acids
sequence of SOD-5 was compared with one of SOD-1 using
homology and domain analyses of the software.
Localization of Wild-Type SOD-1::EGFP Fusion Protein
The sod-1 gene is predicted to encode a Cu/Zn SOD expressed in the cytosol (7). We constructed a sod-1::egfp
transgenic worm strain and confirmed through fluorescence
microscopy that the protein was ubiquitously expressed
throughout most tissues of the wild-type animals (Figure
2A and B). Significant changes in expression level and localization of EGFP between 4- and 9-day-old animals were
not observed (Figure 2C).
Comparison of Life Span and Resistance to Oxidative
Stress and Heat Stress in sod-1 Mutants
Life span of both sod-1(tm776)-carrying and sod-1(tm783)-carrying strains was shortened compared with the
wild type by approximately 4–6 days (Figure 3A). This
short–life span phenotype was completely rescued in sod-1
mutant transgenic animals carrying extrachromosomal arrays of the wild-type gene (Figure 3B). As a result of at least
three independent experiments, both mean and maximum
life spans of the transgenic tm strains were significantly rescued by the extrachromosomal arrays (Table 1).
The localization and expression levels of the fusion protein in both tm strains remained essentially unchanged compared with the expression of the SOD-1::EGFP in the wild
type (Figure 4A and B). Therefore, it appears that the expression systems of sod-1 are not disrupted by the sod-1
deletion mutants.
Compared with the wild type, both tm776 and tm783 mutant animals were not sensitive to oxygen but were sensitive
to paraquat (Figure 5A and B). The thermotolerance of
these tm strains was less than that of the wild type and the
INTRACELLULAR ROS: SOD-1 BALANCE
535
Figure 5. Sensitivities of the sod-1 deletion mutants to various stresses. Open circles indicate the wild-type N2, open diamonds are age-1(hx546), closed squares
are the tm776 mutant strain, and closed triangles are tm783. In these panels, error bars are based on at least three independent experiments. (A) Sensitivity to oxygen.
For N2 and tm strains, respectively, data are not statistically significant. (B) Sensitivity to paraquat. p values by t test (only N2 vs. tm783) significantly differed as
follows; p < .05 in 0.2–0.3 mM of paraquat. (C) Thermotolerance at 35°C. p values by t test (only N2 vs. tm783) significantly differed as follows; p < .005 in 6–10
hours of exposure time.
age-1 animals (Figure 5C). However, the decrease in the
thermotolerance of tm776 was less than that of tm783. The
mechanism that caused this difference between the two
strains is yet to be discovered.
Activities of Total SOD in sod-1 Deletion Strains
Differences in the total SOD activities in the sod-1 deletion mutants were observed compared with the wild-type
N2. SOD activities in the cytosol of tm776 and tm783 animals were reduced significantly compared with those in
wild-type animals. In contrast, differences of SOD activity
in the membrane fraction were not found in these strains
(Figure 6).
−
Changes in Mitochondrial and Cytosol ⋅O 2 Production
Levels in sod-1 Deletion Strains
−
We found that the ⋅O 2 production levels in not only the
mitochondria but also the cytosol of tm776 and tm783 mutants were greatly increased in comparison with the wild
−
type (Figure 7). Surprisingly, mitochondrial ⋅O 2 levels
were increased in both sod mutant strains despite the localization of SOD-1 to the cytoplasm of wild-type ani−
mals. Increases in the ⋅O 2 levels in the cytosol of these tm
strains were particularly significant compared with the
wild-type level.
Compensation for the sod-1 Deletion
sod-1 mRNA expression in tm776 and tm783 mutants could
not be detected by reverse trancribed (RT)-PCR (Figure 8A).
When we conducted RT-PCR on the other sod genes, we
found normal expression with the exception of sod-5, which
was higher. This increase in sod-5 mRNA levels was confirmed by QPCR (Figure 8B). QPCR also showed changes in
sod-3 and sod-4 in the sod-1 deletion mutant tm783.
In addition, we found that the sod-5 gene has one copy of
the DBE sequence at 192 bp upstream of its first exon (29).
In its amino acid sequence analysis, the percentage of
homology is approximately 69% compared with SOD-1
(Figure 9). We then examined the expression level of sod-5
in various mutants by RT-PCR and confirmed that the level
in a daf-16 null mutant is repressed (15).
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YANASE ET AL.
Figure 6. Intrinsic total superoxide dismutase (SOD) activities in sod-1 deletion mutants. The left-hand shaded column for each strain indicates the activity
of the enzyme in the cytosol fraction and the right-hand closed column indicates
the activity in the membrane fraction. Data are means ± SD of three independent
experiments. p values by t test are *p < .05 and **p < .001 compared with data
for the wild-type N2.
Discussion
It is well known that the total SOD activity in long-lived
C. elegans mutants is upregulated (7,12). However, this
leaves unanswered the question of how different SOD
genes contribute to increased longevity and stress resistance, if at all. The SOD-1::EGFP is ubiquitously expressed
in all tissues in the wild type, so we anticipated that a
SOD-1 deletion would have a strong effect on life span and
oxidative resistance. Contrary to this expectation, the reductions in life span of these sod-1 deletion mutants were
not very severe compared with the wild-type life span,
amounting to only about 25%. Moreover, these mutants are
homozygous viable and have normal rates of development
and normal fertility. Their responses to various stressors
were changed, although they were not more sensitive to
oxygen gas. The shorter life span of the deletion mutants is
likely due to the decreased resistance to various stressors
and the higher levels of cytoplasmic oxidants and was completely restored to normal by expression of the wild-type
SOD-1::EGFP reporter.
Several previous studies have focused on the Cu/Zn SOD
in both invertebrate and mammalian model systems (30–
37). Generally, the Cu/Zn SOD–deficient animals (SOD-1
knockout) tend to have a shortened life span with increased
oxidative damage such as 8-hydroxy-2'-deoxyguanosine
(35). Increase of oxidative stress resistance was observed in
the Cu/Zn SOD–overexpressing animals. However, cases
are reported in which the typical age-related phenotypes are
not observed, as in sod-1-overexpressing Drosophila melanogaster (31). It is conceivable that a molecular mechanism exists, in which a catalytic reaction of SOD affects
secondary ROS production such as H2O2. In particular, the
Figure 7. Superoxide radical (⋅O −2 ) production levels in mitochondria and
cytosol of sod-1 deletion strains using 2-methyl-6-p-methoxyphenylethynylimidazopyrazinone, a chemiluminescent superoxide probe. Data are means ±
SD of three independent experiments. Comparison of the means of mitochondrial ⋅O −2 production levels by t test reveals significant differences compared
with the wild type in the tm776 (p < .05) and tm783 (p < .05) strains. Likewise,
comparison of means of cytosol ⋅O −2 production was revealed by t test in tm776
strain (p < .05) and tm783 strain (p < .1).
influence of intracellular oxidative stress resulting from environmental stress is thought to be greater in C. elegans
because it contains three genes encoding the Cu/Zn-binding
SOD. This influence includes a subcellular distribution and
functional compensation of expression in each Cu/Zn SOD.
That is, changing of subcellular distribution and expression
of other compensating Cu/Zn SODs might be significant
for the longevity of C. elegans, rather than increasing
secondary ROS production due to the overexpression of
alternative SODs.
Based on the expression pattern of the SOD-1::EGFP fusion protein in C. elegans, the SOD-1 protein is ubiquitously
expressed in the cytoplasm of almost all tissues. Contrary to
what we expected, not only cytosolic levels of ROS increased but also ROS levels in mitochondria were increased
in the sod-1 deletion mutants. We showed that the SOD-1
activity levels in the cytoplasm also affect the ROS environment in the mitochondria, which is protected by a mitochondrion-specific Mn SOD activity, a remnant from its
prokaryotic origin (38). A detailed analysis conducted by
Okado-Matsumoto and colleagues on rat liver shows that
the intracellular locations of Cu/Zn SOD are not only in the
cytoplasm and lysosome but also in the intermembrane
space of mitochondria (39,40). Likewise, a report indicates
INTRACELLULAR ROS: SOD-1 BALANCE
Figure 8. Messenger RNA expression levels of sod genes in sod-1 deletion
strains using RT polymerase chain reaction (PCR). (A) Nonaltered photographs
of agarose gel electrophoresis of each fragment stained with ethidium bromide.
(B) Quantitative PCR data obtained with the ABI 7000 Sequence Detection System. Data are means ± SEM of three or more independent experiments. Expression
data are normalized to N2. p values were calculated using a two-tailed t test for
unpaired samples with unequal variances. Asterisks indicate that the fold change
is significantly different (p < .05) in comparison with that of the wild type.
that Cu/Zn SOD1 helps to protect mitochondria from intracellular oxidative stress in yeast (40). Thus, the expression
of cytosolic Cu/Zn SOD seems to be involved in defense of
the mitochondria against a ROS attack.
In addition to SOD-1, C. elegans is unusual in that it has
an extra Cu/Zn SOD, which is encoded by the sod-5 gene.
We observed that the sod-5 mRNA levels increased by 2
to 2.5-fold in the sod-1 deletion mutants; this genetic compensation may be reflecting either increased expression or
decreased mRNA turnover in the sod-5 gene. It is interesting to speculate whether it is the higher cytoplasmic oxidant levels that are responsible for the increased sod-5
mRNA in these mutants or, alternatively, the compensation may be dependent upon unknown transcriptional
feedback control mechanisms. The DBE sequence, which
is a consensus sequence in the 5'-untranslated region of
DAF-16 target genes, was confirmed recently in the promoter regions of not only sod-2 and sod-3 but also sod-5
(15). In fact, it was lately reported that the Ins/IGF-1 signaling pathway is involved in regulating expression of
sod-1, sod-3, and sod-5 via DAF-16 and that other pathways associated with life span extension (e.g., dietary restriction) show PHA-4-dependent expression of sod-1,
sod-2, sod-4, and sod-5 (41). Therefore, not only the Ins/
IGF-1 signaling pathway but also the Foxa/PHA-4 transcription factor might be activated in the sod-1 deletion
537
mutants under increased intracellular oxidative stress.
However, these compensation systems are not enough to
restore normal life span and stress resistance in the sod-1
deletion mutants. Presumably, the antioxidant system depending on the Cu/Zn SOD encoded by sod-1 plays an important role during normal aging.
An increase in steady-state cellular ROS production is
closely associated with the normal aging process (42). The
hypothesis that ROS generated by mitochondrial respiration plays the predominant role in normal aging has been
proposed for various organisms (43,44). The age-related
increase in activity of SOD during normal aging is not seen
in the wild type compared with the long-lived C. elegans
mutant (7). Consequently, it is possible that the age-related
increase in intracellular ROS depends on the suppression
of SOD activity. Thus, it is likely that the suppression of
SOD activity accelerates normal aging and reduces life
span in C. elegans. We also detected mitochondrial dysfunction in the sod-1 deletion mutants indicated by an ele−
vation of ⋅O 2 production. The intracellular ROS imbalance
induced by the inactivation of Cu/Zn SOD in the sod-1 deletion mutants might lead to accumulation of oxidative damage,
which then becomes a trigger for mitochondrial dysfunction.
Recently published reports investigating about the other
molecular mechanisms of intracellular signaling pathways
indicate that an age-related increase in ROS elevates p38
mitogen-activated protein kinase (MAPK) in the stress
response (45). Moreover, the activation of the p38 MAPK
pathway by NADH in mitochondrial electron transport
system (complex I) induces various cellular responses (e.g.,
apoptosis, differentiation, and stress response) in addition
to oxidative stress resistance (46). In C. elegans, oxidative
stress mediates the regulation of intracellular DAF-16
localization through the activation of the p38 MAPK and
Jun-N-terminal kinase (JNK) signaling pathways in parallel with the Ins/IGF-1 signaling pathway (47,48). Thus, it
shows a possibility that not only the Ins/IGF-1 signaling
pathway but also other signaling pathways such as the p38
MAPK pathway regulate the compensating SODs-dependent
recovery of the intracellular ROS imbalance due to the inactivation of cytoplasmic Cu/Zn SOD. The hypothesis might
give a hint of alternative solution to our view how the
molecular compensation of antioxidant systems acts in the
normal aging process.
Funding
This research was financially supported by grants-in-aid for scientific research from the Ministry of Education, Culture, Sports, Science and Technology of Japan to S.Y.; a special research grant from Daito Bunka University
to S.Y.; grants-in-aid for aging research from the Ministry of Health, Labor
and Welfare, Japan, to N.I.; and grants from the United States Public Health
Service (USPHS) to T.E.J.
Acknowledgments
We thank Dr. Shohei Mitani, Dr. Keiko Gengyo-Ando, and Dr. Mitani’s
lab members, Tokyo Women’s Medical University, for the supply of the tm
strains and the technical advice.
538
YANASE ET AL.
Figure 9. Characterization of sod-5 gene as related to the insulin/insulin-like growth factor-1 signaling pathway. (A) One copy of the DAF-16 consensus binding
element (DBE) sequence located upstream of the first exon of sod-5. The DBE region is indicated as a box, and capital letters indicate the first methionine. The DBE
sequence is located 192 base pair upstream of the first exon of the sod-5 gene. (B) The predicted amino acid sequence of SOD-5. SOD-5, as well as SOD-1, has a Cu/
Zn metal–binding domain. The amino acids binding to Cu/Zn metals in the homologous sequences with SOD-1 are underlined. One copper ion and one zinc ion bind
per subunit of SOD-1 or SOD-5. Two cysteines of the intrachain disulfide bridge in SOD-5 are black-boxed. (C) A schematic of the domain structure of SOD-5 and
its alignment with SOD-1 in C. elegans. The percentage of the similarity to SOD-1 is approximately 69%. Note: SOD = superoxide dismutase.
Correspondence
Address correspondence to Naoaki Ishii, PhD, Department of Molecular
Life Science, Tokai University School of Medicine, Bohseidai, Isehara,
Kanagawa 259-1193, Japan. Email: [email protected]
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Received May 11, 2008
Accepted July 8, 2008
Decision Editor: Huber R. Warner, PhD