A Complex II Defect Affects Mitochondrial Structure, Leading to ced-3- and ced-4-dependent Apoptosis and Aging

Abstract

The mev-1(kn1) mutation of Caenorhabditis elegans is in Cyt-1, which encodes a subunit of succinate-coenzyme Q oxidoreductase in the mitochondrial electron transport chain. Mutants are hypersensitive to oxidative stress and age precociously in part because of increased superoxide anion production. Here, we show that mev-1 mutants are defective in succinate-coenzyme Q oxidoreductase, possess ultrastructural mitochondrial abnormalities (especially in muscle cells), show a loss of membrane potential, have altered CED-9 and Cyt-1 protein levels under hyperoxia, and contain ced-3-and ced-4-dependent supernumerary apoptotic cells. These defects likely explain the failure of mev-1 to complete embryonic development under hyperoxia as well as its reduced life span. The mev-1(kn1) mutation of Caenorhabditis elegans is in Cyt-1, which encodes a subunit of succinate-coenzyme Q oxidoreductase in the mitochondrial electron transport chain. Mutants are hypersensitive to oxidative stress and age precociously in part because of increased superoxide anion production. Here, we show that mev-1 mutants are defective in succinate-coenzyme Q oxidoreductase, possess ultrastructural mitochondrial abnormalities (especially in muscle cells), show a loss of membrane potential, have altered CED-9 and Cyt-1 protein levels under hyperoxia, and contain ced-3-and ced-4-dependent supernumerary apoptotic cells. These defects likely explain the failure of mev-1 to complete embryonic development under hyperoxia as well as its reduced life span. The oxidative phosphorylation system or electron transport chain is located within the mitochondrial inner membrane and is intimately responsible for three important processes: (i) ATP production, (ii) generation of reactive oxygen species (ROS), 1The abbreviations used are: ROS, reactive oxygen species; CoQ, coenzyme Q; DIC, differential interference contrast.1The abbreviations used are: ROS, reactive oxygen species; CoQ, coenzyme Q; DIC, differential interference contrast. and (iii) regulation of programmed cell death or apoptosis. The proximal ROS is superoxide anion (O.2−), which derives from the Q cycle at rates estimated between 0.1 and 2% of the total electron flow (reviewed in Refs. 1Beckman K.B. Ames B.N. Physiol. Rev. 1999; 78: 547-581Crossref Scopus (3074) Google Scholar and 2Finkel T. Holbrook N.J. Nature. 2000; 408: 239-247Crossref PubMed Scopus (7117) Google Scholar). A variety of enzymatic and nonenzymatic defense mechanisms have evolved to either quench free radicals or ameliorate their resultant oxidative damage. Apoptosis (or programmed cell death) occurs by a mechanism that is conserved from nematodes to humans (reviewed in Refs. 3Liu Q.A. Hengartner M.C. Ann. N. Y. Acad. Sci. 1999; 887: 92-104Crossref PubMed Scopus (82) Google Scholar and 4Meier P. Finch A. Evan C. Nature. 2000; 407: 796-801Crossref PubMed Scopus (790) Google Scholar). In Caenorhabditis elegans, a largely invariant cell lineage (including 131 specific somatic cells destined to undergo apoptosis during hermaphroditic development) has provided fertile ground for the study of apoptosis. In other organisms, mitochondria are thought to provide a major initiation signal for apoptosis via the opening of the mitochondrial permeability transition pore, which leads to the release of a number of cell death-promoting factors, including cytochrome c. In C. elegans, normal, developmentally regulated apoptoses are not mediated by cytochrome c leakage from mitochondria. Mitochondrial oxidative phosphorylation system defects can cause a perplexing variety of diseases that exhibit a significant degree of clinical heterogeneity (reviewed in Refs. 5Morgan J.A. Hann M G. Biochim. Biophys. Acta. 1999; 1410: 125-145Crossref PubMed Scopus (50) Google Scholar and 6Wallace D.G. Science. 1999; 283: 1482-1488Crossref PubMed Scopus (2580) Google Scholar). For the most part, the pathophysiologies present themselves in tissues with high energy demands, such as heart and muscle. The interrelationship of the three activities described above (e.g. ATP generation, ROS production, and apoptosis initiation) may explain some of the perplexing pathophysiological features of mitochondrial diseases. We have previously described the mev-1(kn1) mutant of the nematode C. elegans as containing a point mutation in Cyt-1 (a large subunit of cytochrome b in complex II) (7Ishii N. Michikhiko F. Hartman P.S. Tsuda M. Yasuda K. Senoo-Matsuda N. Yanases S. Ayusawa D. Suzuki K. Nature. 1998; 394: 694-697Crossref PubMed Scopus (575) Google Scholar). Isolated by screening for hypersensitivity to oxidative damage (8Ishii N. Takahashi K. Tornita S. Keino T. Honda S. Yoshino K. Suzuki K. Mutat. Res. 1990; 237: 165-171Crossref PubMed Scopus (177) Google Scholar), mev-1 ages precociously at high oxygen concentrations and accumulates markers of aging (e.g. fluorescent materials and protein carbonyls) more rapidly compared with the wild type (9Honda S. Ishii N. Suzuki K. Matsuo M. J. Gerontol. 1993; 48: B57-B61Crossref PubMed Scopus (115) Google Scholar, 10Adachi H. Fujiwara Y. Ishii N. J. Gerontol. 1998; 53A: B240-B244Crossref Scopus (138) Google Scholar, 11Hosokawa H. Ishii N. Ishida H. Ichimori K. Nakazawa H. Suzuki K. Mech. Ageing Dev. 1994; 74: 161-170Crossref PubMed Scopus (89) Google Scholar). We have recently shown that there are two distinct pathophysiologies in mev-1 animals, viz. increased superoxide production from mitochondria and abnormal energy metabolism (12Senoo-Matsuda N. Yasuda K. Tsuda M. Ohkubo T. Yoshimura S. Nakazawa H. Hartman P.S. Ishii N. J. Biol. Chem. 2001; 276: 41553-41558Abstract Full Text Full Text PDF PubMed Scopus (218) Google Scholar). Here, we report the results from histochemical and ultrastructural examinations of mev-1 mutants that revealed severe defects in succinate-CoQ oxidoreductase and in the membrane potential (Δψm) of mitochondria. In addition, mev-1 was found to possess morphological abnormalities in muscle mitochondria, particularly under hyperoxia. CED-9 and Cyt-1 protein levels were also abnormal in the mev-1 mitochondrial membrane. This may explain our electron microscopic and Nomarski-DIC microscopic observations of supernumerary apoptosis in mev-1 embryos. The abnormal apoptosis was suppressed by mutations in either ced-3 or ced-4, indicating that the inappropriate signal in mev-1 embryos stimulates induction of the normal ced-9/ced-4/ced-3 apoptotic pathway. Furthermore, the mev-1,ced-3 double mutant lived longer than mev-1, suggesting that the supernumerary apoptoses contribute to the phenotype of life shortening in mev-1. General Methods and Strains—Culturing techniques have been described previously (13Brenner S. Genetics. 1974; 77: 71-94Crossref PubMed Google Scholar). PH126 (ced-3(n1286) mev-1(kn1) unc-50(e306)) was constructed by crossing MT3002 (ced-3(n1286)) hermaphrodites with mev-1 unc-50 heterozygous males and scoring for second generation uncoordinated animals, who gave rise exclusively to third generation embryos with no apoptotic cells. PH137 (ced-4(n1162) dpy-17(e164) mev-1(kn1) unc-50(e306)) was constructed by crossing MT2551 (ced-4(n1162) dpy-17(e164)) hermaphrodites with mev-1(kn1) unc-50(e306) heterozygous males and picking Dpy Unc second generation progeny. Age synchronous populations were prepared as described previously (9Honda S. Ishii N. Suzuki K. Matsuo M. J. Gerontol. 1993; 48: B57-B61Crossref PubMed Scopus (115) Google Scholar, 10Adachi H. Fujiwara Y. Ishii N. J. Gerontol. 1998; 53A: B240-B244Crossref Scopus (138) Google Scholar, 11Hosokawa H. Ishii N. Ishida H. Ichimori K. Nakazawa H. Suzuki K. Mech. Ageing Dev. 1994; 74: 161-170Crossref PubMed Scopus (89) Google Scholar). Hatching was determined as described previously (9Honda S. Ishii N. Suzuki K. Matsuo M. J. Gerontol. 1993; 48: B57-B61Crossref PubMed Scopus (115) Google Scholar, 10Adachi H. Fujiwara Y. Ishii N. J. Gerontol. 1998; 53A: B240-B244Crossref Scopus (138) Google Scholar, 11Hosokawa H. Ishii N. Ishida H. Ichimori K. Nakazawa H. Suzuki K. Mech. Ageing Dev. 1994; 74: 161-170Crossref PubMed Scopus (89) Google Scholar). The life spans of individual adult hermaphrodites were determined on agar plates. To prevent progeny production, 5-fluoro-2′-deoxyuridine (Sigma) was added to the nematode growth agar at a final concentration of 40 μm after animals had reached adulthood (14Mitchell D.H Stiles J.W. Satelli J. Sanadi D.R. J. Gerontol. 1979; 35: 28-36Crossref Scopus (177) Google Scholar). Histochemical Analysis of Succinate-CoQ Oxidoreductase—Young adults were embedded in Tissue-Tek O.C.T. compound (Miles Inc., Elkhart, IN) and stored at –80 °C until analyzed. Frozen cross-sections of 10 μm were cut on a cryostat at –20 °C; collected on glass slides; and incubated in 0.05 m sodium succinate, 0.05 m Tris-HCl (pH 7.4), and 0.5 mg/ml nitro blue tetrazolium chloride at 37 °C for 1 h to stain for succinate-CoQ oxidoreductase. The cross-sections were fixed in formaldehyde, dehydrated with a series of alcohols, and embedded in glycerol. Electron Microscopy—Embryos and young adults were fixed overnight in 2.5% glutaraldehyde and 0.05 m phosphate buffer (pH 7.4) at room temperature. The samples were then washed with 0.1 m phosphate buffer (pH 7.4) and post-fixed in 1% OsO4 and 0.05 m phosphate buffer (pH 7.4) for 1 h at 4 °C. After fixation, the samples were dehydrated with a series of alcohols and embedded in Quetol 812 (Nissin EM Ltd.). Thin sections were stained with 2% uranyl acetate and lead citrate and examined under a Joel 1200EX electron microscope operating at 80 kV. Assay of Δψm—JC-1 (5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide, catalog no. T-3168, Molecular Probes) was dissolved in Me2SO at a concentration of 1 mg/ml. The JC-1 solution was added to newly hatched larvae in S basal buffer at 1 μg/ml and incubated in the dark for 10 min. After washing three times with S basal buffer, the animals were observed under a fluorescence microscope. Images were analyzed by Adobe Photoshop for emission characteristic of JC-1 monomers and aggregates. Cell Corpse Assay—The mean numbers of cell corpses (mean ± S.D.) in comma stage embryos (∼400 min after the first cleavage) (15Sulston J.E. Schierenberg E. White J.G. Thomson N. Dev. Biol. 1983; 100: 64-119Crossref PubMed Scopus (2755) Google Scholar) of wild-type, mev-1, and mev-1,ced-3 animals were counted under Nomarski-DIC microscopy, examining for the signature raised appearance of apoptotic cells (18Evan G. Littlewood T. Science. 1998; 281: 1317-1322Crossref PubMed Scopus (1359) Google Scholar). Preparation of Monoclonal Antibodies against Cyt-1 and CED-9—To raise antibodies, peptides were first synthesized using an automated peptide synthesizer. The peptides consisted of either amino acids 40–54 of C. elegans Cyt-1 or amino acids 206–223 of C. elegans CED-9 with an additional cysteine residue in the COOH terminus. The synthesized peptides were then conjugated to the adjuvant keyhole limpet hemocyanin (Calbiochem). Five-week-old female BALB/c mice were immunized four times by injection with 100 μg of keyhole limpet hemocyaninconjugated Cyt-1 or CED-9 peptide. The serum antibody titers were determined by enzyme-linked immunosorbent assay. Spleens were removed 3 days after the final injection of the antigen, and 108 splenocytes were fused with 2 × 107 mouse myeloma P3U1 cells in the presence of polyethylene glycol 1000. Aliquots of the culture supernatants from the resultant hybridoma cells were screened by enzyme-linked immunosorbent assay and cloned twice by limited dilution to obtain monoclonal cell lines. Finally, stable clones showing anti-Cyt-1 (KM2408) or anti-CED-9 (KM2422) antibody activity were obtained. The hybridoma cells (5–20 × 106) were injected intraperitoneally into 8-week-old female BALB/c nude mice, and monoclonal antibodies were purified from the ascites fluid. Preparation of Mitochondrial Fractions for Western Blot Analysis— Young adults were harvested by centrifugation and washed five times with S basal buffer and once with isolation buffer (210 mm mannitol, 70 mm sucrose, 0.1 mm EDTA, and 5 mm Tris-HCl (pH 7.4)). The worms were homogenized (10%, w/v) in isolation buffer with a Teflon-glass homogenizer. Mitochondria were isolated by differential centrifugation at 600 × g to remove debris and then at 7200 × g to pellet mitochondria. Mitochondrial protein was measured using the BCA protein assay kit (Pierce). Western Blot Analysis—Mitochondrial and cytosolic fractions were boiled in sample buffer (50 mm Tris-HCl (pH 6.8), 2% SDS, 0.14 m 2-mercaptoethanol, 10% glycerol, and 0.001% bromphenol blue) and separated by electrophoresis on 10 or 12.5% SDS-polyacrylamide gels. Proteins were electroblotted onto Immobilon polyvinylidene difluoride membranes in buffer consisting of 48 mm Tris, 39 mm glycine, 0.036% SDS, and 20% methanol and probed with 1 μg/ml KM2408 or 3 μg/ml KM2422. Immunoreactive proteins were visualized using the enhanced chemiluminescence system (ECL, Amersham Biosciences). Succinate-CoQ Oxidoreductase Activity—Histochemical analysis of succinate-CoQ oxidoreductase activity was performed on frozen sections of wild-type and mev-1 young adults (Fig. 1). Enzyme activity was widespread throughout wild-type animals, including embryos, with a concentration in body wall muscles. Conversely, there was a complete absence of staining in mev-1 animals. This is consistent with the previous observation of reduced levels in mev-1 cell extracts (7Ishii N. Michikhiko F. Hartman P.S. Tsuda M. Yasuda K. Senoo-Matsuda N. Yanases S. Ayusawa D. Suzuki K. Nature. 1998; 394: 694-697Crossref PubMed Scopus (575) Google Scholar). Ultrastructural Abnormalities in mev-1—Electron micrographs of wild-type body wall muscles revealed normal mitochondrial morphologies, including extensive cristae, at 21% oxygen (Fig. 2A) as well as at 50 and 10% oxygen (Fig. 2, B and C). Conversely, the mitochondria from mev-1 body wall muscles had progressively disorganized cristae as oxygen was increased (Fig. 2, D–F). Many such mitochondria also contained electron-dense and amorphous inclusions at 21% oxygen (Fig. 2D), with additional paracrystalline inclusions apparent in animals reared under 50% oxygen (Fig. 2E). Similar ultrastructural abnormalities were present in hypodermal cells, including cuticles of mev-1 (but not wild-type) animals (data not shown). These abnormalities did not appear at 10% oxygen (Fig. 2F). Similar ultrastructural changes also characterized mev-1 intestinal cells. Although reasonably normal at 21% oxygen (Fig. 3C), mev-1 intestinal mitochondria were noticeably swollen and after animals were incubated under 50% oxygen (Fig. with mev-1 mitochondria in no were hyperoxia had no on the wild type (Fig. that the more ultrastructural abnormalities in mev-1 muscle intestinal the levels of oxidative phosphorylation in the of mitochondria to a membrane potential was by fluorescence after the This is by mitochondria as a and in mitochondrial This transition is by fluorescence for monomers and by fluorescence for In 4 and of the wild type and mev-1 with JC-1 are The of mev-1 a of there were indicating that was reduced in the mev-1 CED-9 and Cyt-1 in mev-1 antibodies raised against peptides that to by the and Western was performed using mitochondrial from wild-type and mev-1 animals at oxygen concentrations (Fig. revealed that the CED-9 and Cyt-1 protein levels were in the two (Fig. The of Cyt-1 in mev-1 mitochondria was reduced under hyperoxia. In hyperoxia CED-9 protein levels in wild-type mitochondria, a in mev-1 mitochondria. The levels of and in the wild type and mev-1 oxygen was between and 50% (data not the of CED-9 and Cyt-1 protein levels in mev-1 mitochondrial membranes may from a of mitochondria. and the of CED-9 in its abnormal in mev-1 that mutant have an altered of apoptosis. the that the mev-1 mutation supernumerary embryonic under hyperoxia. and mev-1 embryos were using Nomarski-DIC at the stage (∼400 min after the first cell for the presence of cells with the and raised and that apoptotic cells. The number of such cell corpses in mev-1 embryos was than in wild-type embryos (Fig. The that were supernumerary apoptoses is by the observation that mutations in either ced-3 or suppressed (Fig. These two are of the ced-9/ced-4/ced-3 apoptotic in C. elegans and are to the normal, developmentally regulated apoptoses for which nematode is well In to supernumerary we observed mev-1 embryos that contained of particularly under hyperoxia (data not shown). described previously (8Ishii N. Takahashi K. Tornita S. Keino T. Honda S. Yoshino K. Suzuki K. Mutat. Res. 1990; 237: 165-171Crossref PubMed Scopus (177) Google Scholar), the life of the mev-1 mutant was than that of the wild type (Fig. The of the mev-1,ced-3 double mutant was to that of the wild type during the first or of life and then that of mev-1 during the (Fig. This is by the that the times to to were in mev-1,ced-3 and the wild type which were longer than the days for mev-1. Conversely, the times to to 20% were in mev-1 and mev-1,ced-3 not in the wild type a of the in life was by a mutation that suggesting that the supernumerary apoptoses in mev-1 contribute to its life spans of the wild mev-1, and of the ced-3 which a of life in mev-1. This that the supernumerary apoptosis in mev-1 to its life the of wild-type animals embryonic development within h after and hatched to larvae (Fig. Conversely, hyperoxia in mev-1 embryos (Fig. of the in the has been to the mechanisms by which specific mutations themselves as This as to progressively of We have previously that mev-1(kn1) is a mutation in a complex subunit of the electron transport chain (7Ishii N. Michikhiko F. Hartman P.S. Tsuda M. Yasuda K. Senoo-Matsuda N. Yanases S. Ayusawa D. Suzuki K. Nature. 1998; 394: 694-697Crossref PubMed Scopus (575) Google Scholar). that a significant has been to for the by We present in the the that is the of is that the mev-1 mutation as three distinct (12Senoo-Matsuda N. Yasuda K. Tsuda M. Ohkubo T. Yoshimura S. Nakazawa H. Hartman P.S. Ishii N. J. Biol. Chem. 2001; 276: 41553-41558Abstract Full Text Full Text PDF PubMed Scopus (218) Google (i) ROS levels in mev-1 mutants superoxide anion levels in mitochondrial and were two times in mev-1 mutants than in the wild (ii) of the ROS in mev-1 animals, which mev-1 animals more to the increased ROS levels and (iii) a of to in mev-1 suggesting that a as occurs in animals. three the first in levels of oxidative stress in mev-1 animals, particularly in and mitochondria. oxidative stress leads to in mitochondrial that as ultrastructural particularly under 2 and These were more in body wall muscle than in intestinal cells, which with the rates of and the levels of oxidative Many electron-dense and amorphous inclusions at 21% oxygen (Fig. or additional paracrystalline inclusions apparent in animals reared under 50% oxygen (Fig. may mitochondrial T. Acad. Sci. S. A. 1994; PubMed Scopus Google Scholar). more was in mev-1 (Fig. This that the ultrastructural abnormalities lead to a loss of mitochondrial in mev-1 animals. is also that the was not to increased oxidative stress to abnormal of electron transport by the complex in mev-1. we have that CED-9 protein levels are altered in a mev-1 (Fig. the and of is a protein that apoptosis by and activity (reviewed in Refs. 3Liu Q.A. Hengartner M.C. Ann. N. Y. Acad. Sci. 1999; 887: 92-104Crossref PubMed Scopus (82) Google Scholar and 4Meier P. Finch A. Evan C. Nature. 2000; 407: 796-801Crossref PubMed Scopus (790) Google Scholar). CED-9 was in wild-type mitochondria by hyperoxia, induction was not observed in mev-1 mitochondria. the levels of CED-9 and Cyt-1 were the between the wild type and mev-1 some of is that are a of the increased oxidative stress that mev-1. may a more of the mev-1 mutation that to the mitochondrial membrane such that CED-9 The of CED-9 in mev-1 may in to against the oxidative damage by hyperoxia. is also that protein may in the levels were protein in mitochondria, not in the (data not shown). The in the CED-9 levels in the wild type may to to oxidative and mev-1 may not have the observation of supernumerary apoptosis in mev-1 embryos (Fig. is a and of the by mutations in either ced-3 or the is from that by C. elegans during the apoptotic is the of explain two important of the mev-1 The first was that mev-1 embryos to complete development under hyperoxia. oxidative abnormal mitochondrial and supernumerary apoptosis may have to The second of was the life by the mev-1 increased stress and abnormal mitochondrial may important in In addition, the of ced-3 to the life of mev-1 to that of the wild type that at a of the reduced life of mev-1 animals is to abnormal apoptosis. In is to the with the mev-1 mutation into a that levels of Finally, is that three distinct of apoptosis have been described in C. The first and most is the developmentally regulated in which specific cells are to undergo programmed cell death in C. 131 specific somatic cells are destined for apoptosis during normal development of A. S. S. J. Hengartner 2000; Full Text Full Text PDF PubMed Scopus Google Scholar) have described widespread apoptosis that can in the C. elegans in to damage. we have of supernumerary apoptosis in somatic cells of mev-1 embryos 5 and a likely of the increased oxidative stress by are a number of between three of apoptosis. are the apoptotic cells appear under Nomarski-DIC three of apoptosis are using the are the ced-9/ced-4/ced-3 are also some between the three of apoptosis. The first type of apoptosis is developmentally regulated and is to specific cells in the is not by oxidative evolved as a characteristic to specific cells in the C. elegans Conversely, the two are and are to damage. are likely more to that in in which apoptosis as a mechanism to cells that the (reviewed in G. Littlewood T. Science. 1998; 281: 1317-1322Crossref PubMed Scopus (1359) Google Scholar). is likely that the apoptosis and the apoptosis the or mechanisms as by to cells. in to the that somatic cells and the other a exhibit the apoptosis and apoptoses not in and mutants A. S. S. J. Hengartner 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). The and mutants are not hypersensitive to oxidative as is mev-1. S. In addition, mutant supernumerary apoptoses in embryos. S. that the that two of apoptosis are from more and between three of apoptosis. in the in and a previous report (12Senoo-Matsuda N. Yasuda K. Tsuda M. Ohkubo T. Yoshimura S. Nakazawa H. Hartman P.S. Ishii N. J. Biol. Chem. 2001; 276: 41553-41558Abstract Full Text Full Text PDF PubMed Scopus (218) Google Scholar) a the of the mev-1 We are to K. and A. for antibodies against the CED-9 and Cyt-1