Free Radical Research, August 2014; 48(8): 919–928 © 2014 Informa UK, Ltd. ISSN 1071-5762 print/ISSN 1029-2470 online DOI: 10.3109/10715762.2014.927063

ORIGINAL ARTICLE

Oxidation resistance 1 is essential for protection against oxidative stress and participates in the regulation of aging in Caenorhabditis elegans Y. Sanada, S. Asai, A. Ikemoto, T. Moriwaki, N. Nakamura, M. Miyaji & Q.-M. Zhang-Akiyama

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Department of Zoology, Graduate School of Science, Kyoto University, Kitashirakawa-Oiwakecho, Sakyo-ku, Kyoto, Japan Abstract Human oxidation resistance 1 (OXR1) functions in protection against oxidative damage and its homologs are highly conserved in eukaryotes examined so far, but its function still remains uncertain. In this study, we identified a homolog (LMD-3) of human OXR1 in the nematode Caenorhabditis elegans (C. elegans). The expressed LMD-3 was able to suppress the mutator phenotypes of E. coli mutMmutY and mutT mutants. Purified LMD-3 did not have enzymatic activity against 8-oxoG, superoxide dismutase (SOD), or catalase activities. Interestingly, the expression of LMD-3 was able to suppress the methyl viologen or menadione sodium bisulfite-induced expression of soxS and sodA genes in E. coli. The sensitivity of the C. elegans lmd-3 mutant to oxidative and heat stress was markedly higher than that of the wild-type strain N2. These results suggest that LMD-3 protects cells against oxidative stress. Furthermore, we found that the lifespan of the C. elegans lmd-3 mutant was significantly reduced compared with that of the N2, which was resulted from the acceleration of aging. We further examined the effects of deletions in other oxidative defense genes on the properties of the lmd-3 mutant. The deletion of sod-2 and sod-3, which are mitochondrial SODs, extended the lifespan of the lmd-3 mutant. These results indicate that, in cooperation with mitochondrial SODs, LMD-3 contributes to the protection against oxidative stress and aging in C. elegans. Keywords: LMD-3, OXR1, aging, nematode, oxidative stress Abbreviations: C. elegans, Caenorhabditis elegans; E. coli, Escherichia coli; ROS, reactive oxygen species; 8-oxoG, 8-oxo-7, 8-dihydroguanine; 8-oxo-dGTP, 8-oxo-7, 8-dihydrodeoxyguanosine-5’-triphosphate; 8-oxo-dGDP, 8-oxo-7, 8-dihydrodeoxyguanosine-5’diphosphate; BER, base excision repair; MV, methyl viologen; HPLC, high-performance liquid chromatography

Introduction Reactive oxygen species (ROS) are generated both endogenously (e.g., as a result of mitochondrial respiration) and exogenously (e.g., as a result of ionizing radiation and chemical treatment). ROS are highly reactive and can damage various cellular components such as DNA, proteins, and lipids [1–3]. Oxidative DNA damage can result in mutagenesis, carcinogenesis, and aging [1]. Cells protect themselves from ROS by preventing oxidative cellular damage, through the detoxification of ROS and repair of oxidative DNA damage [4,5]. For example, superoxide dismutase (SOD) converts superoxide anions into hydrogen peroxide (H2O2), while catalase detoxifies H2O2 by converting it into H2O and O2 [6–11]. To repair oxidized DNA, DNA glycosylases remove oxidized bases from DNA and initiate base excision repair (BER) pathway [12–14]. Thus, many enzymes that prevent oxidative stress and damage have been studied, but it remains possible that there are some other yet-unidentified enzymes. In a search for human genes that contribute to antioxidative defense systems, Volkert et al. identified OXR1

as a novel gene that suppressed spontaneous mutations in Escherichia coli nth mutH-deficient mutants [15]. The OXR1 gene is highly conserved among eukaryotes [15]. Human and yeast OXR1 genes are induced by oxidative stress and their protein products are targeted to mitochondria [16]. The OXR1 mutants are sensitive to H2O2 in Saccharomyces cerevisiae and OXR1 mutations are lethal in Drosophila melanogaster [15,17]. Mice lacking OXR1 display cerebellar neurodegeneration [18]. Jaramillo-Gutierrez et al. reported that the Anopheles gambiae (An. gambiae) OXR1 gene regulates the expression of ROS detoxification enzymes [19]. Knockdown of the An. gambiae OXR1 decreases endogenous catalase and glutathione peroxidase mRNAs [19]. Therefore, OXR1 is thought to play a role in antioxidant defense regulation. The level of OXR1 mRNA expression was raised by exposure to a high concentration of oxygen (75%) in mouse retina [20]. Although OXR1 appears to play an important role in protection against oxidative stress, its precise function is poorly understood. To clarify the function of OXR1 in multicellular organisms, we surveyed the activities of an OXR1 homolog (LMD-3) of Caenorhabditis elegans

Correspondence: Qiu-Mei Zhang-Akiyama, PhD, Department of Zoology, Laboratory of Stress Response Biology, Graduate School of Science, Kyoto University, Kitashirakawa-Oiwakecho, Sakyo-ku, Kyoto 606-8502, Japan. Tel: ⫹ 81-75-753-4097. Fax: ⫹ 81-75-753-4087. E-mail: [email protected]. (Received date: 7 April 2014; Accepted date: 19 May 2014; Published online: 27 June 2014)

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920 Y. Sanada et al. (C. elegans) and analyzed the biological phenotypes of the lmd-3 mutant. C. elegans is a useful model organism for aging studies because of the availability of its sequence and mutant strains, and because of its short lifespan (about 3 weeks). Studies on the function of OXR1 in vivo are expected to provide further knowledge about the relationship between oxidative stress and aging. Previously, we found that a lmd-3::GFP reporter assay revealed that the lmd-3 gene was induced by oxidative stress in C. elegans [21]. In this study, we showed that the C. elegans lmd-3 mutant exhibited sensitivity to oxidative stress compared with wild-type C. elegans strain N2. We found that the lifespan of the lmd-3 mutant was reduced compared with that of N2, and that shorter lifespan occurred as a result of an increase in the acceleration of mortality rate. These results suggest that LMD-3 participates in the regulation of aging through preventing oxidative stress in C. elegans.

Materials and methods Cloning of C. elegans lmd-3 (F52E1.13) We searched for proteins of C. elegans with homology to human OXR1 (hOXR1) using NCBI BLAST, and LMD-3 was detected as a homolog of hOXR1. According to Worm Base (http://www.wormbase.org/), there are four potential isoforms of LMD-3. The following primers were designed to amplify the cDNA of each isoform and of a modified isoform c (fragment c1-610) by PCR: forward primer for isoforms a and d: 5’- CAAATTCTCGAGATGGGCAGCTTCGGATCAG-3’ (with an XhoI site); forward primer for isoform b: 5’- CAAATTCTCGAGATGGGCGCCAACGAGTC-3’ (with an XhoI site); forward primer for isoform c and fragment c1-610: 5’- CAAATTCTCGAGATGTGGAGATCAAGGATTCCTC-3’ (with an XhoI site); reverse primer for isoforms a, b and c: 5’- AATCAATTGCGGCCGCACATTCTGAATCCATAAGCCTC-3’ (with a NotI site); reverse for isoform d: 5’- AATCAATTGCGGCCGCAGATTTTTGTGCATCGCAGC-3’ (with a NotI site); reverse primer for fragment c1-610: 5’- AATCAATTGCGGCCGCTTATCCAAGAGACTCGTCG-3’ (with a NotI site). The PCR products for isoforms b and c and fragment c1-610 were cloned into pTrc99A digested with appropriate restriction enzymes. For purification of LMD-3, expression plasmids of isoforms b and c and fragment c1-610 were generated using the following primers: forward primer for isoform b: 5’- CAAATTGGATCCATGGGCGCCAACGAGTC-3’; forward primer for isoform c and fragment c1-610: 5’-CAAATTGGATCCATGTGGAGATCAAGGATTCCTC-3’; reverse primer for isoforms b and c: 5’- AATCAATTGCGGCCGCACATTCTGAATCCATAAGCCTC-3’; reverse primer for fragment c1-610: 5’-AATCAATTGCGGCCGCTTATCCAAGAGACTCGTCG-3’. The PCR products were cloned into pGEX4T-3 digested with appropriate restriction enzymes. The sequence of the inserts was checked to verify that no mutations had been introduced by the PCR.

Assay for spontaneous mutations of Lac⫹ in E. coli E. coli CC104 mutMmutY and CC101 mutT cells were transformed with pTrc99A, pTrc99A/isoform b, pTrc99A/ isoform c, pTrc99A/fragment c1-610 or pTrc99A/hOXR1. Each strain was grown at 37°C to stationary phase in 5 ml of minimal medium containing 100 μg/ml ampicillin and glucose (0.2%). Appropriate dilutions of each culture were spread onto minimal plates containing glucose (0.2%) or lactose (0.2%), and then incubated at 37°C for 48 h. The number of Lac⫹ revertants was calculated as the number of colonies growing on lactose plates per 108 viable cells. Enzymatic assays To test whether LMD-3 has DNA-binding activity or nucleotide hydrolysis activity, the gel-shift assay and MutT assay were performed, respectively, with purified LMD-3 isoform b and c and fragment c1-610 as previously described [22,23]. Catalase activity was assayed by monitoring the H2O2 decay spectroscopically at 240 nm [24]. Reaction mixtures containing 9 mM H2O2, 45 mM phosphate buffer (pH 6.0), 50 mM Tris-HCl (pH 8.0), and 0.25 mM EDTA were incubated at 37°C for 10 min with purified LMD-3 protein. SOD activity was measured using purified LMD-3 protein and a SOD assay kit-WST (Doujin Chemical, Kumamoto, Japan) [25]. Colony formation assay E. coli GC4468 cells were transformed with pTrc99A, pTrc99A/isoform b, pTrc99A/isoform c, pTrc99A/fragment c1-610. Each strain was grown at 37°C to stationary phase in 5 ml of LB containing 100 μg/ml ampicillin. Appropriate dilutions of each culture were plated on LB plates containing methyl viologen (MV; Nacalai Tesque, Kyoto, Japan) at various concentrations. After incubation at 37°C for 24–48 h, the number of colonies was counted to estimate survival. Induction assay β-galactosidase activity was measured by a slight modification of Miller’s method [26]. The overnight cultures of E. coli QC772 (sodA::lacZ) and TN521(soxS::lacZ) cells expressing isoforms b and c and fragment c1-610 were added to fresh LB (1:100 dilution) containing 100 μg/ml ampicillin and 1 mM IPTG and grown at 37°C until logarithmic phase. The culture was further incubated with 50 μM MV or 1 mM menadione sodium bisulfite (Sigma-Aldrich, St. Louis) and OD600 and β-galactosidase activity were measured every 30 min. 0.2 ml of the culture was added to 1.8 ml of Z-buffer (0.1 M phosphate buffer [pH 7.0], 10 mM KCl, 1 mM MgCl2, 50 mM β-mercaptoethanol). Then, 100 μl of 1% SDS solution and 100 μl of chloroform were added to the mixture followed through vortex mixing. The enzyme reaction was initiated by the addition of 400 μl of o-nitrophenyl-β-galactopyranoside (4 mg/ml in 0.1 M phosphate buffer [pH 7.0]). The reaction was stopped

C. elegans OXR1 homolog delays aging

by adding 1 ml of 1 M Na2CO3 and the absorbance at OD420 and OD550 were measured. The β-galactosidase activity unit was calculated as previously described [26].

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C. elegans strains and culture conditions C. elegans strains N2, sod-2(gk257) and sod-3(gk235) strains were provided by the Caenorhabditis Genetics Center, University of Minnesota. lmd-3(tm2345) was provided by Dr. Shohei Mitani (the National BioResource Project for the Nematode, Japan). The double mutants lmd-3(tm2345);sod-2(gk257) and lmd-3(tm2345); sod-3(gk235) were generated for this work. Unless otherwise noted, the worms were cultured at 20°C on NGM agar plates (0.3% NaCl, 0.25% polypeptone, 0.002% cholesterol, 1 mM MgSO4, 1 mM CaCl2, 25 mM potassium phosphate [pH 6.0] and 0.17% agar) with a lawn of E. coli strain OP50. Assay for the sensitivity of C. elegans to oxidative stress and heat stress These assays were performed by a slight modification of previous methods [23,27,28]. To examine the sensitivity to oxidative stress, eggs were left to hatch in S buffer

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(100 mM NaCl, 50 mM potassium phosphate [pH 6.0]). After 24 h, some synchronized L1 worms were exposed to H2O2. The L1 worms were then placed on NGM plates. Other synchronized L1 worms were placed on NGM plates containing menadione sodium bisulfite at various concentrations. The L1 worms were counted. After incubation at 20°C for 4 days, L4-adult worms were counted and the number of L4 and adults/the number of L1 worms was determined. To examine the sensitivity to heat stress, young adult worms (30 each) were transferred to new NGM plates (10 worms per plate) with a lawn of E. coli OP50 and shifted to 35°C. The numbers of surviving and dead animals were scored every 1 h. Assay for lifespan These assay methods were performed as previously described [27]. To determine lifespan, the adult worms were transferred to fresh NGM plates (10–15 worms per plate) containing 40 μM of 5-fluoro-2’-deoxyuridine (FUdR). To determine the lifespan in the absence of FUdR, the adult worms were transferred to fresh NGM plates and then transferred to fresh NGM plates daily. The living worms were counted every 2 days. Worms failing

Figure 1. Expression of LMD-3 reduces spontaneous mutations in an E. coli mutMmutY and mutT mutant. (A) A scaled representation of C. elegans LMD-3(F52E1.13) compared with yeast OXR1 and human OXR1. The various conserved domains, including the LysM, GRAM and TLD domains are indicated. (B) PCR with isoform-specific primers. Arrows indicate the PCR product from cDNA library of C. elegans. (C and D) Complementation of the mutator phenotype in E. coli mutMmutY mutant (C) and mutT mutant (D) by expressing isoform b or c or fragment c1-610 of LMD-3 and human OXR1 (hOXR1). Mutation frequencies are determined using LacZ ⫹ reversion. The values represent the mean ⫾ standard deviation (n ⫽ 3). *p ⬍ 0.01; t test versus E. coli mutMmutY or mutT mutant harboring an empty vector.

922 Y. Sanada et al. to move were counted as dead. Age-specific mortality rates were calculated as previously described for each 2-day period [29,30]. At later 7 time-points except for the last day that approximated the straight line on a semilogarithmic graph (i.e., Days 12–24 for wild-type N2 and Days 8–20 for tm2345), the difference in the regression slopes were determined using analysis of covariance (ANCOVA).

Results

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Cloning of C. elegans lmd-3 (F52E1.13) To identify OXR1 homolog(s) in C. elegans, we searched for proteins with homology to human OXR1 (hOXR1). LMD-3(F52E1.13) was thereby detected as a homolog of hOXR1 using NCBI BLAST. According to WormBase (http://www.wormbase.org/), lmd-3 exists on chromosome V and there are four potential isoforms of LMD-3. OXR1 is a highly conserved eukaryotic protein that has the TLD domain in the C-terminal region (Figure 1A). Except in yeast, the N-terminal region of OXR1 homologs contains a LysM domain and a GRAM domain. LMD-3 isoform c possesses a conserved C-terminal TLD domain of approximately 200 amino acids that shares 28%/37% identity and 47%/51% similarity with yeast OXR1 and hOXR1, respectively. LMD-3 also shares homology over a 188 amino acid span (aa 7–194) with the corresponding region in hOXR1 (aa 95–277). We first tried to clone all the isoforms of LMD-3 from a cDNA library. However, PCR products produced using isoform a- and d-specific primers were not observed (Figure 1B). Thus, cDNA clones encoding isoforms b and c were amplified using PCR. Moreover, to investigate the function of the N-terminal region of isoform c, truncated isoform c (called fragment c1-610) lacking the TLD domain (see Figure 1A for a schematic representation) and cDNA clones encoding fragment c1-610 were also amplified. Each PCR fragment was inserted into a pTrc99A and pGEX4T-3 vector, and each of the plasmids was used in the following experiments. Complementation of spontaneous mutations in an E. coli mutMmutY and mutT mutant by LMD-3 isoforms b and c and fragment c1-610 Expression of hOXR1 has been shown to reduce the spontaneous mutation frequency in an E. coli mutMmutY mutant [31]. Recently, it was shown that the protein segment encoded by exon 8 of human OXR1 is responsible for the suppression of spontaneous mutations in the E. coli mutMmutY mutant [32]. MutM and MutY proteins are both involved in repair of an oxidized form of guanine, 8-oxo-7,8-dihydroguanine (8-oxoG). 8-oxoG can alter genetic information since it pairs with adenine and cytosine [33]. Disruption of both the mutM and mutY genes leads to a significant increase in spontaneous mutation frequency due to G:C to T:A transversions [33]. To exam-

ine whether isoform b and c and fragment c1-610 are able to complement the spontaneous mutator phenotype of an E. coli mutMmutY mutant, the mutation frequency was determined using LacZ⫹ reversion. As shown in Figure 1C, the frequency of mutation leading to reversion to LacZ⫹ in E. coli CC104 mutMmutY was potently reduced by expressing isoform b or c or fragment c1-610 compared with that of the mutMmutY mutant carrying an empty vector. MutT has an 8-oxo-dGTP pyrophosphatase activity that hydrolyzes 8-oxo-dGTP in the nucleotide pool and thus prevents its misincorporation into DNA [34]. Disruption of the mutT gene results in about an increase in spontaneous mutation frequency due to A:T to C:G transversions in E. coli [33]. We next tested whether isoforms b and c and fragment c1-610 are able to complement the spontaneous mutations in an E. coli mutT mutant. As shown in Figure 1D, the mutation frequency in E. coli CC101 mutT was also reduced by expressing isoform b or c, fragment c1-610 or hOXR1, compared with that of the mutT mutant carrying an empty vector. The expression of LMD-3 prevents the induction of soxS and sodA by MV and menadione sodium bisulfite in E. coli Expression of LMD-3 complemented the mutator phenotype of E. coli mutMmutY strain. This result prompted us to examine whether LMD-3 has DNA repair activity, especially toward 8-oxoG. We purified LMD-3 as a GST-fused protein and performed (1) a gel-shift assay to test whether LMD-3 has DNA-binding activity, like MutM and (2) the MutT assay to test whether LMD-3 has nucleotide hydrolysis activity. The gel-shift assay failed to show binding of LMD-3 to A/8-oxoG-containing DNA or to C/8-oxoGcontaining DNA (data not shown). In addition, the MutT assay using high-performance liquid chromatography failed to show hydrolysis of 8-oxo-dGTP or 8-oxo-dGDP by LMD-3 (data not shown). These results suggested that the decrease of spontaneous mutation frequencies of E. coli mutMmutY and mutT mutants by expression of LMD-3 is not due to DNA repair activity of LMD-3 toward 8-oxoG. Yeast OXR1 mutants are sensitive to H2O2 [15]. Therefore, we considered the possibility that LMD-3 is involved in elimination of H2O2 and examined whether LMD-3 possessed the catalase activity. However, purified LMD-3 did not display catalase activity (data not shown). We next examined the effect of expression of isoforms b and c and fragment c1-610 on the survival of E. coli exposed to MV, a superoxide-generating agent. E. coli GC4468 expressing isoform b or c or fragment c1-610 was more resistant to MV than GC4468 harboring the empty vector (Figure 2A). Based on this result, we hypothesized that LMD-3 eliminates the superoxide radical. SOD is one of the enzymes that scavenges superoxide, so an SOD assay was carried out with an SOD assay kit using WST-1. However, LMD-3 did not display SOD activity (data not shown). We next investigated whether LMD-3 is involved in transcriptional regulation in response to superoxide. E. coli soxS and sodA

C. elegans OXR1 homolog delays aging

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are superoxide-inducible genes. Therefore, we examined the regulation of soxS and sodA at the transcriptional level using operon fusions of lacZ to the soxS and sodA promoters. E. coli QC772 (sodA::lacZ) and TN521 (soxS::lacZ) were transformed to express isoform b or c or fragment c1-610 and the expression of the soxS::lacZ and sodA::lacZ fusion genes was assessed by measurement of β-galactosidase activity. Figure 2B and C shows that expression of isoform b or c or fragment c1-610 prevented induction of both soxS and sodA by MV. Moreover, the similar results were obtained from an induction assay using menadione sodium bisulfite, another superoxide-generating agent (data not shown). These results suggest that LMD-3 contributes to protection against oxidative stress by detoxifying superoxide radical. C. elegans lmd-3 mutant (tm2345) shows increased sensitivity to oxidative stress To examine the physiological roles of LMD-3 (F52E1.13) in C. elegans, we obtained a deletion mutant of F52E1.13 (tm2345). The tm2345 strain carries a 541-bp deletion

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spanning exons 3 and 4 in isoform c (Figure 3A and B). We used the tm2345 mutant as an lmd-3 mutant in the following analyses. We first examined the sensitivity of the lmd-3 mutant toward oxidative stress. We compared the sensitivity of lmd-3 worms to H2O2 by determining the percent of shortterm (⬍ 2 h) exposed L1 worms that developed to adults. As shown in Figure 3C, the lmd-3 mutant was more sensitive to H2O2 than wild-type N2. We next examined the effect of menadione sodium bisulfite by determining the percent of L1 worms that developed to adults after 4 days on plates containing menadione sodium bisulfite. As shown in Figure 3D, the long-term exposure to menadione sodium bisulfite resulted in a significant increase of the sensitivity of the lmd-3 mutant. It is thought that heat stress produces an oxidative stress originating from the electron transport chains of mitochondria [35]. We next compared the sensitivity of N2 and lmd-3 worms to heat stress. To test this, young adult worms were incubated under heat stress, and their survival was assessed by determining the percentage of live worms. As shown in Figure 3E, the exposure to heat

Figure 2. Expression of LMD-3 prevents the induction of soxS and sodA by methyl viologen in E. coli. (A) The sensitivity of E. coli GC4468, QC772 (sodA) expressing LMD-3 to MV. Overnight cultures are plated on LB agar plates containing MV and the number of colonies is counted to estimate survival after 24–48 h of incubation at 37°C. The values represent the mean⫾ standard deviation (n ⫽ 3). (♦), E. coli harboring pTrc99A; (■), E. coli harboring pTrc99A-isoform b; (▲), E. coli harboring pTrc99A-isoform c; (●), E. coli harboring pTrc99Afragment c1-610. (B and C) Prevention of the induction of the soxS::lacZ (B) and sodA::lacZ (C) fusion gene in E. coli following exposure to 50 μM MV. E. coli TN521 (soxS::lacZ) and QC772 (sodA::lacZ) expressing LMD-3 were cultured in LB medium and assayed for β-galactosidase (β-gal) activity. The values represent the mean⫾ standard deviation (n ⫽ 3). (♦), E. coli harboring pTrc99A; (■), E. coli harboring pTrc99A-isoform b; (▲), E. coli harboring pTrc99A-isoform c; (●), E. coli harboring pTrc99Afragment c1-610.

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Figure 3. C. elegans lmd-3 mutant (tm2345) is sensitive to oxidative stress. (A) Schematic of the gene structure of lmd-3 (F52E1.13). tm2345 carries a 541-bp deletion spanning exons 3 and 4 in isoform c. The position of the internal primers (arrows) used for PCR to detect the tm2345 deletion are shown. (B) PCR using specific primers (marked by arrows in (A)) for wild-type N2 and tm2345. Lower arrow indicates the PCR product obtained in tm2345 mutant. (C and D) The sensitivity of lmd-3 mutant to oxidative stress. (C) L1 stage worms are exposed to 0.5 mM H2O2 for the indicated time periods. (D) L1 stage worms are placed on NGM plates containing menadione sodium bisulfite at various concentrations. The values indicate the ratio of the number of viable L4 and adults/the number of L1. The values represent the mean standard deviation (n ⫽ 3). (E) The sensitivity of lmd-3 mutant to heat stress. Young adult stage worms (30 each) are transferred to a fresh NGM plates (10 worms per plate) with OP50 and shifted to 35°C. Numbers of surviving and dead animals are scored every 1 h. (♦), C. elegans wild-type N2; (■), lmd-3 mutant (tm2345).

stress resulted in lower survival of the lmd-3 mutant than of N2. C. elegans lmd-3 mutant exhibits a shorter lifespan Based on the oxidative stress theory of aging, the oxidative stress has been considered to be a major cause of aging [36]. As previously reported, the mev-1 mutant is sensitive to oxidative stress and exhibits a shorter lifespan than N2 [28,37]. However, the correlation between sensitivity to oxidative stress and lifespan is still controversial [38]. We compared the lifespan of the lmd-3 mutant with that of N2. As Figure 4A shows, similarly to the mev-1 mutant, lmd-3 mutant showed a shorter lifespan than those of N2. The mean and maximum lifespans of lmd-3 worms were shorter compared with N2 worms: mean (⫾ SEM) lifespans were 14.8 ⫾ 0.2 and 13.1 ⫾ 0.2 days and maximum lifespans were 26 and 22 days for N2 and lmd-3 worms, respectively. We next assessed the lifespan of the lmd-3 mutant at low (16°C) and high (25°C) temperature. At each temperature, the lmd-3 mutant exhibited a shorter lifespan than N2 as well (Figure 4B and C). FUdR is often used to prevent progeny development during lifespan experiments. FUdR inhibits DNA synthesis and can exert a significant effect on the lifespan of tub-1 mutants [39]. Although the effects of FUdR on lifespan

have been assessed in wild-type, it has not been confirmed whether FUdR has an effect on the lifespan of the lmd-3 mutant. Thus, lifespan was also assessed in the absence of FUdR. Figure 4D shows that lmd-3 mutant exhibited a shorter lifespan than N2 in the absence of FUdR. An increase in the age-specific mortality rate is one of the hallmarks of the aging process [30,40]. We calculated and plotted mortality rates of N2 and lmd-3 worms for each 2-day period on a semi-logarithmic graph. Figure 4E shows the age-specific mortality rates and regression lines of the mortality rates. The mortality rates of both N2 and lmd-3 worms accelerated exponentially with age. Moreover, there were significant differences in the slopes of these regression lines (ANCOVA, p ⬍ 0.05), indicating that lmd-3 worms showed an increase in the acceleration of mortality rate with age. Elimination of mitochondrial SOD extends lmd-3 mutant lifespan As described above, expression of LMD-3 prevented induction of soxS and sodA in E. coli. We analyzed the relationship between LMD-3 and SOD in C. elegans. Considering that SOD-2 and SOD-3 are localized in the mitochondria, a major site of superoxide production, we generated two double mutants, lmd-3;sod-2 and lmd-3;sod-3.

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Figure 4. C. elegans lmd-3 mutants (tm2345) have significantly shorter lifespan than wild-type worms. (A) The lifespan of the lmd-3 mutant on NGM plates containing FUdR at 20°C (A), 16°C (B) and 25°C (C). (D) The lifespan of the lmd-3 mutant at 20°C in the absence of FUdR. Young adult-stage worms are transferred to fresh NGM plates daily. The surviving population is counted every 2 days. (♦), wild-type N2 (n ⫽ 256 (A), 65 (B), 66 (C) and 58 (D)); (■), lmd-3 mutant (n ⫽ 241 (A), 64 (B), 66 (C) and 50 (D)). The experiment (A) is independently carried out four times. The experiment (B), (C) and (D) are carried out once. (E) Age-specific mortality rates of worms at 20°C (A) and regression lines of the mortality rates. The age-specific mortality rates are calculated for each 2-day period. (♦ and solid regression line), wild-type N2; (■ and dashed regression line), lmd-3 mutant (tm2345).

Lack of mitochondrial SOD activities results in a lethal phenotype in many other organisms [41,42]. On the other hand, in C. elegans, sod-2, and sod-3 mutants have been reported to actually exhibit a similar or longer lifespan compared to N2. This has generally been thought to be due to up-regulation of antioxidant genes by elimination of sod genes [38,43]. To investigate whether the relationship between lmd-3 and mitochondrial sod genes is involved in aging, we assessed the lifespan of lmd-3 mutant lacking each of the sod genes. We found that the lifespans of the sod-2 and lmd-3;sod-2 mutants were similar to that of N2 (Figure 5A). Deletion of sod-3 resulted in an increase in lifespan. The lifespan of the lmd-3;sod-3 mutant was slightly longer than that of the lmd-3 single mutant (Figure 5B).

Discussion ROS are thought to cause many diseases and aging [1]. Loss of the OXR1 gene causes sensitivity to oxidative stress, and the OXR1 expression level is altered by oxidative stress [15,16,44], suggesting that OXR1 plays a role in the resistance and response to oxidative stress. In this study, we analyzed the function of an OXR1 homolog of C. elegans (LMD-3). Among four potential isoforms of LMD-3, PCR products corresponding to isoforms a and d of LMD-3 were not obtained from C. elegans cDNA, suggesting that these two isoforms may hardly be transcribed. While the C-terminal TLD domain is a consensus sequence of the members of the OXR1 family and has been well

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Figure 5. C. elegans lmd-3;sod mutants live longer than lmd-3 mutants. The lifespan of worms on NGM plates containing FUdR at 20°C is measured. The surviving population is counted every 2 days. (♦), C. elegans wild-type N2 (n ⫽ 120); (■), lmd-3 mutant (n ⫽ 108); (▲), sod-2 (A) or sod-3 (B) mutant (n ⫽ 127 and 124, respectively); (●), lmd-3;sod-2 (A) or lmd-3;sod-3 (B) double mutant (n ⫽ 117 and 98, respectively). The experiments (A) and (B) are independently carried out twice.

studied, little is known about the N-terminal region of OXR1 in E. coli. To examine the region excluding the TLD domain, isoform c lacking the TLD domain was designated as fragment c1-610 (Figure 1A). Expression of human OXR1 suppresses the spontaneous mutation frequency in E. coli mutMmutY [31]. Likewise, expression of isoform b or c or fragment c1-610 of LMD-3 suppressed the spontaneous mutation frequencies in E. coli mutMmutY mutant (Figure 1C). Moreover, expression of isoform b or c or fragment c1-610 suppressed the spontaneous mutation frequency in E. coli mutT (Figure 1D). These results suggest that the N-terminal region of LMD-3 also plays a role in defense against oxidative DNA damage. Here, we considered two possibilities regarding the function of LMD-3: (1) Like MutM, MutY, and MutT, LMD-3 has DNA repair activity for oxidative lesions. (2) LMD-3 is involved in elimination of ROS and thereby prevents the production of 8-oxoG. We first surveyed whether LMD-3 protein has DNA repair activity and found that LMD-3 protein does not display the DNA-binding activity for 8-oxoG-containing DNA, or 8-oxo-dGTPase activity or 8-oxo-dGDPase activity (data not shown). These results strongly suggest that LMD-3 suppresses mutations induced by 8-oxoG by some mechanism other than DNA repair activity. ROS accumulation leads to a significant increase of 8-oxoG [12]. To scavenge ROS and to avoid oxidative damage, cells possess the antioxidant enzymes, including SOD and catalase. SOD and catalase directly scavenge superoxide radicals and H2O2, respectively [6]. Yeast OXR1 mutants are sensitive to H2O2 [15]. As shown in Figure 3C, C. elegans lmd-3 mutant worms were sensitive to H2O2. One might think that LMD-3 eliminates H2O2, but catalase activity of LMD-3 was not observed. Loss of LMD-3 might cause deficiency of the normal response to H2O2 stress. We next examined whether LMD-3 is involved in the elimination of superoxide. As shown in Figure 2A, E. coli expressing LMD-3 is resistant to MV. Moreover, expression of LMD-3 prevents induction of soxS and sodA, which are both superoxide-inducible genes (Figure 2B and C). The lmd-3 mutant was sensitive to menadione sodium

bisulfite (Figure 3D). These results suggest that LMD-3 might scavenge superoxide. However, SOD activity of LMD-3 was not observed, which suggest that LMD-3 is contributing to the defense against superoxide-mediated toxicity in a different manner than SOD. It is also possible that LMD-3 prevents an increase of the superoxide level. The C. elegans lmd-3 mutant showed a shorter lifespan than wild-type (Figure 4A–D). To further investigate this phenotype, we next focused on an increase in the agespecific mortality rate, one of the hallmarks of the aging process [30,40]. As shown in Figure 4E, the acceleration of the mortality rate for lmd-3 worms was significantly greater than that for N2 worms, suggesting that LMD-3 has an effect on the aging process. Among four potential isoforms of LMD-3, isoforms b and c seem to be most strongly expressed. Although isoform b seems not to be eliminated in the lmd-3 mutant, isoform c will be truncated in this mutant even if expressed (Figure 3A and B). This fact suggests that isoform c, at least, plays an essential role for protection against oxidative stress and the regulation of aging. As described above, LMD-3 is somehow contributing to the defense against superoxide-mediated toxicity. To investigate it further, here we considered the possibility that there is a relationship between LMD-3 and two mitochondrial SODs (SOD-2 and SOD-3), and assessed the effect of sod gene knockout on the lifespan of the lmd-3 mutant. Although the lmd-3 mutant had a shorter lifespan than wild-type, the lifespan of the lmd-3;sod-2 mutant was extended to the level of the wild-type N2 (Figure 5A). A similar result was observed in a lmd-3;sod-3 mutant (Figure 5B). Our interpretation of this is that superoxide is one cause of aging and LMD-3 prevents an increase of the superoxide level. In conclusion, the results from the mutation, sensitivity and lifespan assays in this study are consistent with the oxidative stress theory of aging [36], and suggest that LMD-3 participates in the regulation of aging through preventing oxidative stress in C. elegans. However, whether LMD-3 also suppresses mutator phenotypes of C. elegans is still unclear. Moreover, to evaluate more directly that shorter lifespan of the lmd-3 mutant result from aging, aging markers such as protein carbonyl, lipofuscin, or 8-oxoG in DNA

C. elegans OXR1 homolog delays aging

in the lmd-3 mutant should be compared with those in wildtype N2. We are currently investigating such problems using genetic and biochemical methods. A better understanding of the relationship between oxidative stress and lifespan will be achieved using genetic and biochemical methods by examining in detail the function of LMD-3.

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Declaration of interest The authors report no declarations of interest. The authors alone are responsible for the content and writing of the paper. This work was supported by Grants-in-Aid for Scientific Research (#24510071) from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan (to Q.-M.Z.-A.), and the Grants for Excellent Graduate Schools program (MEXT). The authors thank Dr. Elizabeth Nakajima for critically reading the manuscript. We are also grateful to the Shiseido Female Research Science Grant for supporting Q.-M.Z.-A. Some strains were provided by the CGC, which is funded by the NIH Office of Research Infrastructure Programs (P40 OD010440). The plasmid pTrc99A/hOXR1 was kindly provided by Dr. Kazunari Hashiguchi (National Institute of Biomedical Innovation).

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Oxidation resistance 1 is essential for protection against oxidative stress and participates in the regulation of aging in Caenorhabditis elegans.

Human oxidation resistance 1 (OXR1) functions in protection against oxidative damage and its homologs are highly conserved in eukaryotes examined so f...
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