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• W2023147388 abstract "Although there is a consensus that mitochondrial function is somehow linked to the aging process, the exact role played by mitochondria in this process remains unresolved. The discovery that reduced activity of the mitochondrial enzyme CLK-1/MCLK1 (also known as COQ7) extends lifespan in both Caenorhabditis elegans and mice has provided a genetic model to test mitochondrial theories of aging. We have recently shown that the mitochondria of young, long-lived, Mclk1+/− mice are dysfunctional, exhibiting reduced energy metabolism and a substantial increase in oxidative stress. Here we demonstrate that this altered mitochondrial condition in young animals paradoxically results in an almost complete protection from the age-de pend ent loss of mitochondrial function as well as in a significant attenuation of the rate of development of oxidative biomarkers of aging. Moreover, we show that reduction in MCLK1 levels can also gradually prevent the deterioration of mitochondrial function and associated increase of global oxidative stress that is normally observed in Sod2+/− mutants. We hypothesize that the mitochondrial dysfunction observed in young Mclk1+/− mutants induces a physiological state that ultimately allows for their slow rate of aging. Thus, our study provides for a unique vertebrate model in which an initial alteration in a specific mitochondrial function is linked to long term beneficial effects on biomarkers of aging and, furthermore, provides for new evidence which indicates that mitochondrial oxidative stress is not causal to aging. Although there is a consensus that mitochondrial function is somehow linked to the aging process, the exact role played by mitochondria in this process remains unresolved. The discovery that reduced activity of the mitochondrial enzyme CLK-1/MCLK1 (also known as COQ7) extends lifespan in both Caenorhabditis elegans and mice has provided a genetic model to test mitochondrial theories of aging. We have recently shown that the mitochondria of young, long-lived, Mclk1+/− mice are dysfunctional, exhibiting reduced energy metabolism and a substantial increase in oxidative stress. Here we demonstrate that this altered mitochondrial condition in young animals paradoxically results in an almost complete protection from the age-de pend ent loss of mitochondrial function as well as in a significant attenuation of the rate of development of oxidative biomarkers of aging. Moreover, we show that reduction in MCLK1 levels can also gradually prevent the deterioration of mitochondrial function and associated increase of global oxidative stress that is normally observed in Sod2+/− mutants. We hypothesize that the mitochondrial dysfunction observed in young Mclk1+/− mutants induces a physiological state that ultimately allows for their slow rate of aging. Thus, our study provides for a unique vertebrate model in which an initial alteration in a specific mitochondrial function is linked to long term beneficial effects on biomarkers of aging and, furthermore, provides for new evidence which indicates that mitochondrial oxidative stress is not causal to aging. Because it is well known that the aging process is characterized by declines in basal metabolic rate and in the general performance of energy-dependent processes, many aging studies have focused on mitochondria because of their central role in producing chemical energy (ATP) by oxidative phosphorylation (1Navarro A. Boveris A. Am. J. Physiol. Cell Physiol. 2007; 292: C670-686Crossref PubMed Scopus (549) Google Scholar). Among the various theories of aging that have been proposed, the mitochondrial oxidative stress theory of aging is the most widely acknowledged and studied (2Harman D. J. Am. Geriatr. Soc. 1972; 20: 145-147Crossref PubMed Scopus (1540) Google Scholar, 3Balaban R.S. Nemoto S. Finkel T. Cell. 2005; 120: 483-495Abstract Full Text Full Text PDF PubMed Scopus (3311) Google Scholar, 4Muller F.L. Lustgarten M.S. Jang Y. Richardson A. Van Remmen H. Free Radic. Biol. Med. 2007; 43: 477-503Crossref PubMed Scopus (848) Google Scholar). It is based on the observation that mitochondrial energy metabolism produces reactive oxygen species (ROS), 2The abbreviations used are: ROSreactive oxygen speciesUQcoenzyme Q8-OHdG8-hydroxy-2-deoxyguanosineHPLChigh performance liquid chromatographyMDAmalondialdehyde. that mitochondrial components are damaged by ROS, that mitochondrial function is progressively lost during aging, and that the progressive accumulation of global oxidative damage is strongly correlated with the aged phenotype. However, the crucial question of whether these facts mean that mitochondrial dysfunction and the related ROS production cause aging remains unproven (5Austad S. Aging Cell. 2008; 7: 119-124Crossref PubMed Scopus (10) Google Scholar, 6Lambert A.J. Brand M.D. Aging Cell. 2007; 6: 417-420Crossref PubMed Scopus (36) Google Scholar, 7Sohal R.S. Mockett R.J. Orr W.C. Free Radic. Biol. Med. 2002; 33: 575-586Crossref PubMed Scopus (524) Google Scholar). Furthermore, recent observations made in various species, including mammals, have begun to directly challenge this hypothesis, notably by relating oxidative stress to long (8Andziak B. O'Connor T.P. Qi W. DeWaal E.M. Pierce A. Chaudhuri A.R. Van Remmen H. Buffenstein R. Aging Cell. 2006; 5: 463-471Crossref PubMed Scopus (284) Google Scholar) or increased (9Schulz T.J. Zarse K. Voigt A. Urban N. Birringer M. Ristow M. Cell Metab. 2007; 6: 280-293Abstract Full Text Full Text PDF PubMed Scopus (907) Google Scholar) lifespans, by demonstrating that overexpression of the main antioxidant enzymes does not extend lifespan (10Pérez V.I. Van Remmen H. Bokov A. Epstein C.J. Vijg J. Richardson A. Aging Cell. 2009; 8: 73-75Crossref PubMed Scopus (261) Google Scholar) as well as by showing that mitochondrial dysfunction could protect against age-related diseases (11Pospisilik J.A. Knauf C. Joza N. Benit P. Orthofer M. Cani P.D. Ebersberger I. Nakashima T. Sarao R. Neely G. Esterbauer H. Kozlov A. Kahn C.R. Kroemer G. Rustin P. Burcelin R. Penninger J.M. Cell. 2007; 131: 476-491Abstract Full Text Full Text PDF PubMed Scopus (333) Google Scholar). reactive oxygen species coenzyme Q 8-hydroxy-2-deoxyguanosine high performance liquid chromatography malondialdehyde. A direct and powerful approach to attempt to clarify this major question and to test the theory is to characterize the mitochondrial function of long-lived mutants (12Hekimi S. Nat. Genet. 2006; 38: 985-991Crossref PubMed Scopus (50) Google Scholar). CLK-1/MCLK1 is an evolutionary conserved protein (13Ewbank J.J. Barnes T.M. Lakowski B. Lussier M. Bussey H. Hekimi S. Science. 1997; 275: 980-983Crossref PubMed Scopus (264) Google Scholar) and has been found to be located in the mitochondria of yeast (14Jonassen T. Larsen P.L. Clarke C.F. Proc. Natl. Acad. Sci. U.S.A. 2001; 98: 421-426Crossref PubMed Scopus (166) Google Scholar), worms (15Felkai S. Ewbank J.J. Lemieux J. Labbé J.C. Brown G.G. Hekimi S. EMBO J. 1999; 18: 1783-1792Crossref PubMed Scopus (223) Google Scholar), and mice (16Jiang N. Levavasseur F. McCright B. Shoubridge E.A. Hekimi S. J. Biol. Chem. 2001; 276: 29218-29225Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). The inactivation of the Caenorhabditis elegans gene clk-1 substantially increases lifespan (17Lakowski B. Hekimi S. Science. 1996; 272: 1010-1013Crossref PubMed Scopus (422) Google Scholar). Moreover, the elimination of one functional allele of its murine orthologue also resulted in an extended longevity for Mclk1+/− mice in three distinct genetic backgrounds (18Liu X. Jiang N. Hughes B. Bigras E. Shoubridge E. Hekimi S. Genes Dev. 2005; 19: 2424-2434Crossref PubMed Scopus (287) Google Scholar). These findings have provided for an evolutionarily conserved pathways of animal aging that is affected by the function of a mitochondrial protein (19Hekimi S. Guarente L. Science. 2003; 299: 1351-1354Crossref PubMed Scopus (377) Google Scholar, 20Stepanyan Z. Hughes B. Cliche D.O. Camp D. Hekimi S. Exp. Gerontol. 2006; 41: 940-951Crossref PubMed Scopus (30) Google Scholar). In mitochondria CLK1/MCLK1 acts as an hydroxylase and is implicated in the biosynthesis of ubiquinone (coenzyme Q or UQ), a lipid-like molecule primarily known as an electron carrier in the mitochondrial respiratory chain and as a membrane antioxidant but which is also associated with an increasing number of different aspects of cellular metabolism (20Stepanyan Z. Hughes B. Cliche D.O. Camp D. Hekimi S. Exp. Gerontol. 2006; 41: 940-951Crossref PubMed Scopus (30) Google Scholar, 21Turunen M. Olsson J. Dallner G. Biochim. Biophys. Acta. 2004; 1660: 171-199Crossref PubMed Scopus (818) Google Scholar). Taken together, these observations indicate that the long-lived Mclk1+/− mouse is a model of choice for the understanding of the links between mitochondrial energy metabolism, oxidative stress, and the aging process in mammals. Previous analysis of Mclk1+/− mice, which show the expected reduction of MCLK1 protein levels (22Levavasseur F. Miyadera H. Sirois J. Tremblay M.L. Kita K. Shoubridge E. Hekimi S. J. Biol. Chem. 2001; 276: 46160-46164Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar), have revealed that their tissues as well as their mitochondria contain normal levels of UQ at 3 months of age (23Lapointe J. Hekimi S. J. Biol. Chem. 2008; 283: 26217-26227Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar). Yet the same study also revealed a host of phenotypes induced by Mclk1 heterozygosity (see below). Thus, it appears that MCLK1 has an additional function that is unrelated to UQ biosynthesis but responsible for the phenotypes observed in young Mclk1+/− mutants. This is consistent with several results from nematodes which also strongly suggest that CLK-1 has other functions (24Branicky R. Nguyen P.A. Hekimi S. Mol. Cell. Biol. 2006; 26: 3976-3985Crossref PubMed Scopus (22) Google Scholar, 25Hihi A.K. Kebir H. Hekimi S. J. Biol. Chem. 2003; 278: 41013-41018Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). In depth characterization of the phenotype of young Mclk1+/− mutants has revealed that the reduction of MCLK1 levels in these animals profoundly alters their mitochondrial function despite the fact that UQ production is unaffected (23Lapointe J. Hekimi S. J. Biol. Chem. 2008; 283: 26217-26227Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar). In fact, we have shown that Mclk1 heterozygosity induces a severe impairment of mitochondrial energy metabolism as revealed by a reduction in the rates of mitochondrial electron transport and oxygen consumption as well as in ATP synthesis and ATP levels in both the mitochondria and the whole cell. ATP levels in several organs were surprisingly strongly affected with, for example, a 50% reduction of overall cellular ATP levels in the livers of Mclk1+/− mutants (23Lapointe J. Hekimi S. J. Biol. Chem. 2008; 283: 26217-26227Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar). Moreover, we have found that the Mclk1+/− mice sustain high mitochondrial oxidative stress by a variety of measurements, including aconitase activity, protein carbonylation, and ROS production (23Lapointe J. Hekimi S. J. Biol. Chem. 2008; 283: 26217-26227Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar). Additionally, we have shown that this early mitochondrial dysfunction is associated with a reduction in some aspects of cytosolic oxidative damage and global oxidative stress that can be measured via recognized plasma biomarkers such as 8-isoprostanes and 8-hydroxy-2-deoxyguanosine (8-OHdG). Considering that the accumulation of global oxidative damage is known to be tightly linked to the aging process (26Sohal R.S. Weindruch R. Science. 1996; 273: 59-63Crossref PubMed Scopus (2631) Google Scholar), this latter result suggests that the anti-aging effect triggered by low MCLK1 levels might already act at a young age. To further investigate the clk-1/Mclk1-dependent mechanism of aging as well as to try to elucidate the still unclear relation between mitochondrial dysfunction, oxidative stress, and aging, we have now carefully analyzed the evolution of the phenotype of Mclk1+/− mutants over time. We have also studied the effects of reduced MCLK1 levels on the phenotype of mice heterozygous for the mitochondrial superoxide dismutase (Sod2), which represent a well known model of mitochondrial oxidative stress (27Williams M.D. Van Remmen H. Conrad C.C. Huang T.T. Epstein C.J. Richardson A. J. Biol. Chem. 1998; 273: 28510-28515Abstract Full Text Full Text PDF PubMed Scopus (403) Google Scholar). In addition of confirming the long lifespan phenotype of the Mclk1+/− mutants in a mixed background (129S6 x BALB/c), we also report here a study of mutants and controls on a completely isogenic background where we find that the condition of Mclk1+/− mutants unexpectedly results in protection against the age-dependent loss of mitochondrial function. Moreover, we found that the mutants are characterized by a significant attenuation of the age-associated increase in global oxidative stress normally observed in mammals. We also show that the Mclk1+/− condition can gradually reverse the deterioration of mitochondrial function and the associated increase of global oxidative stress that is normally observed in Sod2+/− mutants. Thus, this study provides for a unique vertebrate model in which reduced levels of a specific mitochondrial protein causes early mitochondrial dysfunction but has long term beneficial effects that slow down the rate of aging, as established with appropriate biomarkers, and can ultimately prolong lifespan in mice. Furthermore, in line with recent studies that have raised doubts about the validity of the mitochondrial oxidative stress theory of aging (4Muller F.L. Lustgarten M.S. Jang Y. Richardson A. Van Remmen H. Free Radic. Biol. Med. 2007; 43: 477-503Crossref PubMed Scopus (848) Google Scholar, 8Andziak B. O'Connor T.P. Qi W. DeWaal E.M. Pierce A. Chaudhuri A.R. Van Remmen H. Buffenstein R. Aging Cell. 2006; 5: 463-471Crossref PubMed Scopus (284) Google Scholar, 10Pérez V.I. Van Remmen H. Bokov A. Epstein C.J. Vijg J. Richardson A. Aging Cell. 2009; 8: 73-75Crossref PubMed Scopus (261) Google Scholar), our results, which relate to a recognized long-lived mice model, represent a novel and crucial indication that mitochondrial oxidative stress might not by itself be causal to aging. All of the mice were housed in a pathogen-free animal facility at McGill University and were given a standard rodent diet and water ad libitum. At the time of analysis animals were anesthetized, sacrificed by cervical dislocation, and perfused with phosphate buffer. Tissues were then rapidly removed, rinsed, and placed in ice-cold mitochondrial isolation buffer or immediately frozen in liquid nitrogen. All procedures were approved by McGill's Animal Care and Ethics committees. The mice were separated from their mother at 21 days of age and housed 3–5 per cage, with both genotypes present in each cage. Lifespan was determined by recording the age of spontaneous death or when one of the following criteria was met: unresponsiveness to touch, slow respiration, coldness to touch, a hunched up position with matted fur, sudden weight loss, or the presence of a tumor large enough to inhibit the animal's normal behavior. All of the mice used in the experiments comparing animals of different ages (3, 12, and 23 months) were F1 hybrid progeny generated by crossing mice of two different pure inbred strains. Indeed, these animals were produced by mating Mclk1+/− males from the original knock-out background (129S6) to females on a pure BALB/c background. These animals were, therefore, all genetically identical (isogenic) except at the Mclk1 locus. In contrast, all mice used in the survival curves analysis were from a mixed background derived at an earlier time (18Liu X. Jiang N. Hughes B. Bigras E. Shoubridge E. Hekimi S. Genes Dev. 2005; 19: 2424-2434Crossref PubMed Scopus (287) Google Scholar) from the same two backgrounds that were used to produce the isogenic F1 mice. Note that the original knock-out background was previous described as 129/SvJ (22Levavasseur F. Miyadera H. Sirois J. Tremblay M.L. Kita K. Shoubridge E. Hekimi S. J. Biol. Chem. 2001; 276: 46160-46164Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). However recent changes in nomenclature mean that it should now more correctly be called 129S6/SvEvTac, which can be abbreviated to 129S6. The F1 animals of the mutant and wild-type groups were siblings and were co-housed immediately after weaning. Because homozygous Sod2−/− mice have the longest survival in the DBA/2J/B6 F1 background (29Huang T.T. Johnson M.S. Figueroa-Colon R. Dwyer J.H. Goran M.I. Obes. Res. 2001; 9: 283-289Crossref PubMed Scopus (108) Google Scholar), we generated the Sod2+/− Mclk1+/− double mutant mice used in the present study by mating Sod2+/− Mclk1+/− mice in the C57BL/6J background to DBA/2J wild-type animals to create a mixed background. The extraction of quinones as well as their quantification by high performance liquid chromatography (HPLC) were performed as described previously (18Liu X. Jiang N. Hughes B. Bigras E. Shoubridge E. Hekimi S. Genes Dev. 2005; 19: 2424-2434Crossref PubMed Scopus (287) Google Scholar). The total amount of quinone was normalized to the amount of protein. On the day of the experiment fresh livers were homogenized in 10 volumes (w/v) of an homogenization buffer consisting of 0.25 m sucrose, 10 mm Hepes buffer, pH 7.4, 1 mm EDTA. Liver mitochondria were then isolated by standard differential centrifugation according to detailed procedures described elsewhere (30Sohal R.S. Free Radic. Biol. Med. 1993; 14: 583-588Crossref PubMed Scopus (126) Google Scholar). The purity of the mitochondrial as well as cytosolic preparations obtained was then checked with antisera against porin and α-tubulin as reported previously (23Lapointe J. Hekimi S. J. Biol. Chem. 2008; 283: 26217-26227Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar). Oxygen consumption of isolated mitochondria was measured polarographically with a Clark-type oxygen electrode connected to a suitable recorder (Yellow Springs Instrument Co.) by following published procedures (23Lapointe J. Hekimi S. J. Biol. Chem. 2008; 283: 26217-26227Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar). Mitochondrial oxygen consumption in the presence of substrates and ADP (0.8 mm) is reported as state 3, whereas the state corresponding to the period after all added ADP has been converted into ATP or in the presence of 1.25 mg/ml oligomycin is defined as state 4 respiration (Table 1). The respiratory control ratio (state 3/state 4) and the adenosine diphosphate-to-oxygen ratio (ADP/O), which are indicators of the intactness of the inner mitochondrial membrane and of the level of coupling of mitochondrial respiration, have been measured for all the animals used in the aging study (Table 1). Also, the quality of mitochondria preparation was confirmed by at least a 3-fold increase in respiration rate in the presence of the uncoupler.TABLE 1Mitochondrial oxygen consumption parameters3 months12 months23 monthsMalesFemalesMalesFemalesMalesFemalesMclk1+/+Mclk1+/−Mclk1+/+Mclk1+/−Mclk1+/+Mclk1+/−Mclk1+/+Mclk1+/−Mclk1+/+Mclk1+/−Mclk1+/+Mclk1+/−O2 consumption state 4 (glutamate) (nmol/mg/min)31.8 ± 3.1aData are the means ± S.E. of 8–12 samples.27.8 ± 1.728.9 ± 2.127.5 ± 0.534 ± 2.732 ± 1.431 ± 2.434 ± 1.929.2 ± 1.531.1 ± 1.828.4 ± 0.627.8 ± 2.9O2 consumption state 4 (succinate) (nmol/mg/min)102 ± 7.8101.1 ± 693.5 ± 3.497.9 ± 3.4104 ± 3.799.6 ± 4.999.8 ± 3.895.6 ± 488.5 ± 3.390.8 ± 4.796.7 ± 4.3103.3 ± 5RCR (glutamate)7.3 ± 0.76.9 ± 0.57.5 ± 0.46.1 ± 0.4bSignificantly different from the controls (Mclk1+/+) at p < 0.05.6 ± 0.45.6 ± 0.3bSignificantly different from the controls (Mclk1+/+) at p < 0.05.6.3 ± 0.65.5 ± 0.46.1 ± 0.56.4 ± 0.55.1 ± 0.35.9 ± 0.6RCR (succinate)3.8 ± 0.23.5 ± 0.14.1 ± 0.23.2 ± 0.2bSignificantly different from the controls (Mclk1+/+) at p < 0.05.3.1 ± 0.22.7 ± 0.23.8 ± 0.23.4 ± 0.23.1 ± 0.23.6 ± 0.23.2 ± 0.23.3 ± 0.2ADP/O (glutamate)3 ± 0.13 ± 0.12.6 ± 0.22.8 ± 0.23 ± 0.082.9 ± 0.062.8 ± 0.072.9 ± 0.23.1 ± 0.063.1 ± 0.052.7 ± 0.12.9 ± 0.1ADP/O (succinate)2 ± 0.12.1 ± 0.12 ± 0.22 ± 0.11.9 ± 0.082 ± 0.072 ± 0.11.9 ± 0.071.8 ± 0.051.7 ± 0.061.7 ± 0.11.8 ± 0.07a Data are the means ± S.E. of 8–12 samples.b Significantly different from the controls (Mclk1+/+) at p < 0.05. Open table in a new tab Lipid peroxidation was determined in cytosolic and mitochondrial fractions of liver by the indirect measurement of free malondialdehyde (MDA) using a standard published method with some modifications (31Gonzales S. Polizio A.H. Erario M.A. Tomaro M.L. World J. Gastroenterol. 2005; 11: 3533-3538Crossref PubMed Scopus (29) Google Scholar). Briefly, one volume of cytosolic or mitochondrial fraction (0.1 ml of sample and 0.4 ml of 50 mm Tris-HCl, pH 7.3) was mixed with a 0.5 volume of trichloroacetic acid (150 mg/ml) and centrifuged at 2000 rpm for 10 min. The resulting supernatant was mixed with 0.5 ml of thiobarbituric acid (0.7 mg/ml) and boiled for 15 min. After cooling, absorbance was measured at 535 nm on a Beckman DU 640 spectrophotometer. MDA concentration was calculated using an extinction coefficient of 1.56 × 105 mol/liter·cm. Results were expressed in nmol of MDA/mg of protein (determined using Bio-Rad Bradford kit). The level of protein carbonyl contents in liver tissues was determined with the Protein Carbonyl Assay kit (Cayman Chemical) according to the manufacturer's instructions. Plasma free and esterified 8-isoprostanes were quantified with an 8-isoprostanes enzyme immunoassay kit (Cayman Chemical) according to the provided protocol. The plasma levels of 8-OHdG, a biomarker for oxidative damage to DNA, were determined using an enzyme-linked immunosorbent assay kit according to the manufacturer's protocol (Stressgen). Aconitase activity in liver mitochondrial and cytosolic extracts was determined as described elsewhere (32Nulton-Persson A.C. Szweda L.I. J. Biol. Chem. 2001; 276: 23357-23361Abstract Full Text Full Text PDF PubMed Scopus (336) Google Scholar). SOD2 activity was assessed in presence of 1 mm KCN using a commercially available kit according to the manufacturer's instructions (Cayman Chemical). All activities were normalized to the quantity of proteins used in the assays. Comparisons between Mclk1+/+ and Mclk1+/− as well as between Sod2+/− and Sod2+/− Mclk1+/− animals were performed using an unpaired two-tailed Student's t test, and differences between the two genotypes were considered to be significant when p was <0.05. Statistical comparisons between control Sod2+/+ Mclk1+/+ mice and each of the three other genotypes individually have been performed by using a one-way analysis of variance followed by the Dunnett's post-hoc test, and the differences were considered to be significant at p < 0.05. For evaluating survival data, the log-rank (Mandel-Cox) test was performed using GraphPad Prism Version 5.00 for Windows, GraphPad Software, San Diego. The Gompertz equation, Rm = R0eat, where Rm is the mortality rate as a function of time or age (t), R0 is the nonexponential factor in mortality, and a is the exponential (Gompertz) mortality rate coefficient, used to model the aging process. The linear regression was obtained from ln(Rm) = ln(R0) + αt and the mortality rate doubling time, a measure of the rate of aging, was estimated from the slope as described (33de Magalhães J.P. Cabral J.A. Magalhães D. Genetics. 2005; 169: 265-274Crossref PubMed Scopus (92) Google Scholar). In a previous study (18Liu X. Jiang N. Hughes B. Bigras E. Shoubridge E. Hekimi S. Genes Dev. 2005; 19: 2424-2434Crossref PubMed Scopus (287) Google Scholar) in which the effect of Mclk1 heterozygosity on three genetic backgrounds was examined, we found that Mclk1+/− mutants from a mixed background (129S6 x BALB/c) showed the greatest increase in lifespan (31% longer on average). However, because the sample sizes examined were relatively small and contained both sexes in unequal ratios, we have carried out a new study with larger cohorts to verify that Mclk1 heterozygosity could indeed increase the average and maximum lifespan, as the earlier results suggested. We scored the lifespan of 14 Mclk1+/+ mice, of which 8 were males, and of 54 Mclk1+/− mutants, of which 24 were males. The two survival curves (not shown) were different by the log-rank (Mantel-Cox) test (p = 0.0231). There was no significant difference of survival between the sexes within each genotype. As there was also no significant difference for each genotype between the previous data and the new data, we pooled the data from the two experiments for the best possible visualization of the difference of survival between the genotypes in this background. In Fig. 1A, males are identified with red symbols, and the symbols corresponding to animals from the previous dataset are marked with an asterisk. The median survival for the pooled samples was 764 days with a maximum of 915 days for Mclk1+/+ animals and 900 days with a maximum of 1180 for the Mclk1+/− mutants. The survival curves were different by the log-rank (Mantel-Cox) test (p = 0.0008). Visually, the two curves are strikingly different, with the wild-type animals all dying within a relatively short period, but the mutants showing a much greater diversity of survival, including animals that died substantially before any wild-type animal and animals that lived considerably longer than any wild-type animal. Furthermore, we used the data from the survival curve in the Gompertz model, which is commonly used to evaluate the intrinsic rate of mortality in different populations (34Riggs J.E. Millecchia R.J. Mech. Ageing Dev. 1992; 62: 191-199Crossref PubMed Scopus (35) Google Scholar) (see “Experimental Procedures”). From the Gompertz analysis we obtained the mortality rate doubling time, a recognized measure of the rate of aging (33de Magalhães J.P. Cabral J.A. Magalhães D. Genetics. 2005; 169: 265-274Crossref PubMed Scopus (92) Google Scholar), and we found that it was significantly different between the cohorts (0.23 year for the Mclk1+/+ animals and 0.41 year for the Mclk1+/− mutants). This suggests that the Mclk1+/− mutants have indeed a slower rate of aging. We have shown previously that various tissues from 3-month-old Mclk1+/− mice in several genetic backgrounds exhibit the expected decrease in Mclk1 mRNA and MCLK1 protein levels but that whole tissue homogenates as well as mitochondrial extracts from the same organs display no reduction in either UQ9 or UQ10 levels (22Levavasseur F. Miyadera H. Sirois J. Tremblay M.L. Kita K. Shoubridge E. Hekimi S. J. Biol. Chem. 2001; 276: 46160-46164Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar, 23Lapointe J. Hekimi S. J. Biol. Chem. 2008; 283: 26217-26227Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar). The very same mice, however, displayed numerous mitochondrial phenotypes (23Lapointe J. Hekimi S. J. Biol. Chem. 2008; 283: 26217-26227Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar). This has revealed that MCLK1 has an additional activity unrelated to UQ biosynthesis but necessary for normal mitochondrial function. Here, we have used HPLC to determine the levels of quinones in liver mitochondria of young isogenic animals from an additional genetic background, 129S6 x BALB/c, in both 3- and 23-month-old F1 animals to determine whether quinone content changes with age. As in the previously investigated backgrounds, the UQ9 levels were unaffected by Mclk1 heterozygosity in 3-month-old mice (Fig. 1B). In addition, we also found no differences in UQ9 levels between Mclk1+/− and Mclk1+/+ mice at 23 months of age (Fig. 1C). We have analyzed mitochondrial function in young (3 months), middle-aged (12 months), and old (23 months) F1 males and females from the 129S6 x BALB/c cross. Mitochondria isolated from both genotypes are well coupled as revealed by standard respiratory control ratio and ADP/O ratio values, and no significant differences between the genotypes was observed for state 4 respiration (Table 1). In wild-type Mclk1+/+ controls from both sexes, oxygen consumption of isolated mitochondria measured in the presence of ADP was found to decrease with increasing age (Fig. 2), as has been found in other studies (1Navarro A. Boveris A. Am. J. Physiol. Cell Physiol. 2007; 292: C670-686Crossref PubMed Scopus (549) Google Scholar, 35Shigenaga M.K. Hagen T.M. Ames B.N. Proc. Natl. Acad. Sci. U.S.A. 1994; 91: 10771-10778Crossref PubMed Scopus (1836) Google Scholar). We have recently reported that the rate of oxygen consumption from intact isolated liver mitochondria was reduced with both complex I substrates (glutamate in combination with malate) and a complex II substrate (succinate) in 3-month-old Mclk1+/− males from the BALB/c and the C57BL/6J backgrounds and that these defects in oxidative phosphorylation are accompanied by reduced ATP production (23Lapointe J. Hekimi S. J. Biol. Chem. 2008; 283: 26217-26227Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar). We show here that this phenotype of low oxygen consumption with both types of substrates is also observe in young males and females originating from the 129S6 x BALB/c cross (Fig. 2). Furthermore, we found that this difference between both genotypes persist in 12-month animals as the mitochondria isolated from Mclk1+/− mutants also consumed less oxygen than the controls with all studied substrates. However, after 23 months, the age-dependent decrease in oxygen consumption in Mcl" @default.
• W2023147388 created "2016-06-24" @default.
• W2023147388 creator A5003410121 @default.
• W2023147388 creator A5030298374 @default.
• W2023147388 creator A5071988641 @default.
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• W2023147388 date "2009-07-01" @default.
• W2023147388 modified "2023-10-14" @default.
• W2023147388 title "Reversal of the Mitochondrial Phenotype and Slow Development of Oxidative Biomarkers of Aging in Long-lived Mclk1+/− Mice" @default.
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