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Ubiquinone (UQ, Coenzyme Q, CoQ) transfers electrons from complexes I and II to complex III in the mitochondrial electron transport chain. Depending on the degree of reduction, UQ can act as either a pro-or an antioxidant. Mutations disrupting ubiquinone synthesis increase lifespan in both the nematode (clk-1) and the mouse (mclk-1). The mutated nematodes survive using exogenous ubiquinone from bacteria, which has a shorter isoprenyl tail length (UQ8) than the endogenous nematode ubiquinone (UQ9).
The mechanism underlying clk-1’s increased longevity is not clear. Here we directly measure the effect of different exogenous ubiquinones on clk-1 lifespan and mitochondrial function. We fed clk-1 engineered bacteria that produced UQ6, UQ7, UQ8, UQ9 or UQ10, and measured clk-1’s lifespan, mitochondrial respiration, ROS production, and accumulated ROS damage to mitochondrial protein. Regardless of dietary UQ, clk-1 animals have increased lifespan, decreased mitochondrial respiration, and decreased ROS damage to mitochondrial protein than N2. However clk-1 mitochondria did not produce less ROS than N2. The simplest explanation of our results is that clk-1 mitochondria scavenge ROS more effectively than wildtype due to the presence of DMQ9. Moreover, when compared to other dietary quinones, UQ10 further decreased mitochondrial oxidative damage and extended adult lifespan in clk-1.
Ubiquinone (UQ, CoenzymeQ, CoQ) is a redox-active lipid primarily found in the mitochondrial inner membrane (Battino et al., 1990; Tran and Clarke, 2007). UQ functions in multiple ways within the cell. It is an essential link in mitochondrial electron transport from complex I and II to complex III in the mitochondrial electron transport chain. The reduced form of UQ can serve as a free radical scavenger to protect cells from oxidative damage (Kagan et al., 1990; Lenaz et al., 2002; Genova et al., 2003), while the partially oxidized semiquinone generates the superoxide free radical (Lass et al., 1997).
UQ consists of a modified benzoquinone ring which can be reversibly reduced and oxidized, and a hydrophobic isoprenyl tail which contains 6 to 10 isoprenyl units. The isoprenyl tail length of UQ is species-specific, being ten (UQ10) in humans, nine (UQ9) in rodents and Caenorhabditis elegans (C. elegans), and eight (UQ8) in Escherichia coli (E. coli). It has been suggested that different isoprenyl tail lengths of UQs may correlate with the lifespan of different species (Olgun et al., 2003). UQ analogues with shorter isoprenyl tails enhance superoxide formation more than UQs with longer tails (Lenaz et al., 2002; Genova et al., 2003). UQ can promote superoxide formation by mediating electron transfer from the iron-sulfur cluster N2 of complex I to oxygen, instead of transferring electrons directly to complex III.
In C. elegans, clk-1 mutants lack the evolutionarily conserved enzyme necessary for UQ biosynthesis (Miyadera et al., 2001; Jiang et al., 2001). C. elegans clk-1 mutants accumulate demethoxyquinone-9 (DMQ9) instead of UQ9 (Miyadera et al., 2001). For survival the animals rely on both endogenous DMQ9 and exogenous UQ8 from the bacterial food source (Jonassen et al 2001). With dietary UQ8 from standard nematode bacteria OP50 or K12, clk-1 mutants display the abnormal “clock” (Clk) phenotype. This includes slow and desynchronized embryonic and postembryonic development, decreased brood size, slower egg production rate, and most interestingly, a prolonged lifespan as well as less oxidative damage to their mitochondrial proteins (Wong et al., 1995) (Kayser et al., 2004a). However, the underlying cause for the increased lifespan of clk-1 remains a point of debate.
Several studies have indirectly investigated whether the large amount of DMQ in clk-1 mutants supports mitochondrial function and is the cause of their extended lifespan. In yeast, clk-1 mutants have been constructed which contain only DMQ6 (Coq7/Cat5). Without added exogenous UQ6, these cells grew slowly, failed to maintain respiratory activity, and lost antioxidant activity (Padilla et al., 2004). C. elegans clk-1 mutants also require UQ8 from their diet to become fertile adults; clk-1 fed UQ-less bacteria arrested as L2 larvae (Jonassen et al., 2001; Jonassen et al., 2002). Mouse clk-1 null mutants (mclk1-/-) arrest development around embryonic day 10, but partial loss of activity extends lifespan in mice (Jiang et al., 2001; Liu et al., 2005). Interestingly, in embryonic stem cells from mclk1-/-, both NADH-and succinate-cytochrome c reductase activity were significantly decreased (Levavasseur et al., 2001; Liu et al., 2005; Stepanyan et al., 2006), suggesting that DMQ is a relatively poor electron carrier compared to UQ. Using direct measurements of mitochondrial function, we found that clk-1 mitochondria display a specific defect in complex I-dependent respiration, whereas complex II-dependent respiration remains normal (Kayser et al., 2004b). More recently, it was shown that only UQ9, and not DMQ9, can be reduced by NADH in the plasma membrane of C. elegans (Arroyo et al., 2006). These studies suggest that DMQ is not able to substitute for UQ function completely, and the UQ profile is an important factor in establishing the clk-1 mutant phenotype.
Two groups have shown that brood size, gonad morphology and rate of development of clk-1 animals are sensitive to the length of the side chain of exogenous UQ (Hihi et al. 2003, Jonassen et al., 2003). However, neither group studied the effect of the ubiquinone species on lifespan or mitochondrial function. We hypothesized that differences in UQ isoprenyl tail length are functionally important in vivo, and that variation in UQ tail length would affect lifespan in the nematode. Changes in lifespan may directly result from changes in generation or scavenging of reactive oxygen species (ROS), as well as from altered rates of mitochondrial respiration. The role of scavengers in determining the lifespan of clk-1 has been questioned (Yang et al., 2007, Rea et al., 2007). However, in neither of these studies were mitochondrial respiration, ROS production, or oxidative damage to proteins directly studied.
We directly studied mitochondrial function in clk-1(qm30) fed different UQ species to understand the mechanism underlying its increased lifespan, and to test the effects of different exogenous UQs on lifespan. The results suggest that the lifespan extension caused by different tail-lengths of UQ is not caused by differences in ROS generation, but by variations in ROS scavenging.
N2 (Bristol) and clk-1 (qm30) were obtained from the Caenorhabditis Genetics Center (Minneapolis, MN, USA). clk-1 (qm30) is the canonical null allele. Worms were grown on nematode growth media (NGM) agar with a lawn of the desired E. coli as food source and maintained at 20°C. The E. coli strains (Okada et al., 1997) used in this study were the kind gift of M. Kawamukai, Shimane University, Japan. Normally worms are fed either E. coli K12 (growth in liquid culture) or E. coli OP50 (growth on agar plates), each of which produce UQ8.
Surface-sterilized eggs were obtained by dissolving gravid qm30 or N2 adults in hypochlorite/NaOH solution (Wood, 1988). Eggs were washed three times with S-basal and transferred to plates containing a lawn of bacteria producing either UQ6, UQ7, UQ8, UQ9 or UQ10, or the standard bacteria strain OP50 (producing UQ8) and kept at 20°C. Worm lifespans were determined by recording the number of alive or dead worms over time; day 0 was defined as the day the worms started to lay eggs. Worms were scored as dead if they did not move after touch with a platinum wire. Worms that crawled off the plate, died because of bagging or protrusion of the gonad through the vulva were discarded from the initial data set. Each lifespan study was performed with over 100 worms. Animals were raised at 20°C. No FUDR was used for lifespan study. Lifespans were reported as the day that 50% of the animals had died.
Worms were grown for mitochondrial isolation as previously published (Kayser et al., 2001), with the exception that the different bacterial strains in Table 1 were used as food. clk-1 animals lay very few eggs when they have been grown on UQ6 or UQ7 from hatching. To obtain large quantities of UQ6 or UQ7 fed clk-1 adults, clk-1 were first grown in S-basal with K12 until dauer larvae were obtained. Dauers were isolated with 2% SDS treatment and washed several times with S-basal. clk-1 dauers were collected and allowed them to exit dauer stage in S-basal with UQ6 or UQ7. Mitochondria preparation and assays of oxidative phosphorylation (OXPHOS) were performed as previously described. (Kayser et al., 2004b)
Mitochondrial protein concentration was determined by the Lowry assay (Lowry et al., 1951). Western blots of 30ug of mitochondrial protein from clk-1 fed with wildtype bacteria K12 (producing UQ8), or bacteria producing UQ6, UQ7, UQ8, UQ9 or UQ10 were performed by electrophoresis on 15% SDS polyacrylamide tris-glycine gels. Proteins were then transferred electrophoretically to 0.45-μm polyvinylidine difluoride membranes (Francis and Waterston, 1985). Membranes were blocked overnight in 5% milk-PBST and incubated for 2 h with primary antibodies. Anti-HNE (Calbiochem, San Diego, CA) was used at a 1:5000 dilution. Anti-complex I subunit NDUFS3 (Mitosciences, Eugene, Oregon) was used to ensure equal loading. After three washes with PBS-0.05% Tween 20, the membranes were incubated for 1 h with horseradish peroxidase-conjugated secondary antibody (Santa Cruz Biotech, Santa Cruz, CA) diluted in 5% milk-PBST. This secondary antibody was detected using SuperSignal West Pico Chemiluminescent Substrate (Pierce, Rockford, IL).
ROS were measured as H2O2 by the Amplex Red assay (Invitrogen, Carlsbad, CA, USA) as described in Chen et al. (Chen et al., 2003). The concentration of fluorogenic indicator amplex red and horseradish peroxidase used were 50uM and 0.5 unit/ul, respectively.
Samples were prepared by adding a known fixed amount of the internal standard di-propoxy-UQ10 (diPrQ10, a generous gift from Iain P. Hargreaves, Institute of Child Heath, London, United Kingdom) and 1-propanol (0.5mL for 400μg mitochondrial protein). 20μL of 2mg/mL p-benzoquinone were added to ensure that all the quinones were in the oxidized form. All the quinones; ubiquinones (UQ), demethoxyquinones (DMQ) and rhodoquinones (RQ), were ionized by Atmospheric Pressure Chemical Ionization (APCI) and analyzed using a triple quadrupole mass spectrometer. Specific multiple reaction monitoring mode (MRM) transitions were performed, corresponding to m/z 864→197 (UQ10), 920→253 (diPrQ10), 795.5→197 (UQ9), 765.5→166.9 (DMQ9), 780.9→181.9 (RQ9), 727.5→197 (UQ8), 659.5→197 (UQ7) and 591.5→197 (UQ6), respectively. The samples were delivered to the API 4000 tandem mass spec (Applied Biosystems, Foster City, California, USA) using an Agilent 1200 series HPLC binary pump (Agilent technologies, Waldbronn, Germany) delivering 50% methanol:isopropanol at 0.5mL/min in flow injection analysis (FIA) mode. Samples were quantified against a 6 concentration levels calibration curve made with known amounts of UQ6, UQ9 and UQ10 and the internal standard (diPrQ10) spanning the concentration range found in the samples. UQ7 was quantified assuming the same response in the detector as UQ6. UQ8, DMQ9 and RQ9 were quantified assuming the same response in the detector as UQ9.
All data are presented as the mean of n experiments ± SD, with n ≥ 3. Comparison of lifespan data were performed with logrank. All other comparisons were first analyzed by ANOVA, using Prism software. If ANOVA indicated a statistical difference, data sets were individually compared using a students t-test. Two sets of data were considered as statistically different when p < 0.05.
First we showed that all bacteria primarily contained their designated quinone (Figure S1), consistent with the results previously reported (Jonassen et al., 2001). After feeding clk-1(qm30) with UQ8, UQ9, or UQ10 for 3 or more generations, we isolated mitochondria protein from them to verify whether dietary UQ accumulated in the mitochondria. clk-1 fed with UQ6 or UQ7 is nearly sterile and will not grow in sufficient quantities to isolate mitochondria. Therefore, for studies of mitochondria containing UQ6 or UQ7, liquid cultures of clk-1 were harvested as dauers and fed with UQ6 or UQ7 to let them exit dauer stage. Synchronous cultures of young adults which had been fed UQ6 or UQ7 for approximately 7 days were harvested for mitochondrial isolation. The endogenous UQ which did accumulate in clk-1 mitochondria was enriched by the primary species produced in the dietary bacteria (Figure 1). The amount of different UQs from dietary bacteria delivered to clk-1 mitochondria was similar for all UQs, ranging from 40 pmol/mg to 65 pmol/mg mitochondrial protein. The amount of DMQ9 species was similar in all clk-1 cultures and significantly increased from that of N2 (p<0.01 for each clk-1 culture compared to N2, Figure 1) though our measurements were lower than those reported previously (Jonassen et al., 2001).
clk-1 mutants reach maturity later and lay fewer viable offspring than N2, regardless of which ubiquinone is provided (Figure S2). In order to interpret clk-1 lifespan in the face of developmental delay, we examined the lifespan data by defining day 0 as the day the worms reached adulthood (began to lay eggs). In addition, we wanted to correlate these results to measurements of mitochondrial function (see below). clk-1 animals grown on UQ6 and UQ7 did not have enough offspring to allow isolation of mitochondria. For these UQ6 and UQ7 lifespans (Figure 2), to correlate the data to the mitochondrial assays, we transferred clk-1 animals grown on UQ8 as dauers to UQ6 and UQ7 bacteria.
clk-1 mutants lived longer than wild-type (N2) animals, regardless of which ubiquinone they were fed. As shown in Figure 2, the overall and adult lifespan of clk-1 fed with OP50 and UQ8 were similar, indicating the animal’s lifespan is not influenced by the different parental strain of bacteria. The adult lifespans of clk-1 fed with OP50, UQ6, UQ7, UQ8 and UQ9 are similar (16 ± 0.7, 15 ± 0.5, 16 ± 0.7, 17 ± 0.3 days and 17 ± 0.4 days, respectively). UQ10 fed worms show a longer adult lifespan of 19 ± 0.4 days (p<0.05).
Different UQs may affect C. elegans lifespan by changing mitochondrial respiration. We examined mitochondrial respiratory activity, since UQ is an essential link of mitochondrial electron transport. Mitochondria were isolated from clk-1 fed with UQ6, UQ7, UQ8, UQ9, UQ10 or K12 (producing UQ8), and N2 fed with K12. Oxidative phosphorylation studies were performed to evaluate the integrated mitochondrial function of intact mitochondria. The state 3 respiration rates shown reflect the near-maximal rate of oxygen consumption and the capacity for electron flux through the respiratory chain. The complex I dependent respiration rate was significantly decreased in clk-1 regardless of the species of dietary quinone (Figure 3A) when compared with N2 (p<0.05). clk-1 fed with UQ6, UQ7, or UQ10 each displayed a more decreased complex I-dependent respiration rate when compared to clk-1 fed with UQ8 or UQ9 (p < 0.05). When using the complex II specific electron donor succinate, the respiration rates of clk-1 fed with engineered bacteria expressing UQ6~10 also decreased when compared to the wildtype worm, N2 (p < 0.05) (Figure 3B), but not as severely as complex I dependent respiration rates. clk-1 worms fed with UQ7 or UQ10 showed lower complex II-dependent respiration rates than those fed K12, UQ6 or UQ8 (p < 0.05). clk-1 worms fed with UQ7 also showed lower complex II-dependent respiration rates than those fed with UQ9 (p < 0.05), in agreement with our earlier report (21).
ROS are byproducts of mitochondrial electron transport and are proposed to be a major cause of aging (Balaban et al., 2005). Since there were differences in lifespan between N2 and clk-1 fed with different UQs, we examined whether there were differences in accumulated ROS damage between these animals. The toxic aldehyde, 4-hydroxynonenal (HNE), is produced by lipid peroxidation triggered by ROS (Esterbauer et al., 1991). HNE is capable of damaging multiple cell components. We examined mitochondrial proteins for the presence of the HNE-adduct (Tsai et al., 1998; Kayser et al., 2004a) as a proxy for overall cumulative ROS -induced damage to mitochondria. clk-1 exhibits much less damage to mitochondrial protein than controls, regardless of its UQ species (Figure 4). However, clk-1 fed with UQ10 showed a further reduction in accumulated damage to mitochondrial protein. The differences seen in specific bands in the clk-1 cultures (e.g. compare 55 kDa and 32 kDa bands in UQ7 and UQ8 cultures), were not uniformly observed in every experiment, though the overall decrease in HNE staining was always observed.
ROS damage is likely dependent on the rate of ROS production from the respiratory chain (Adam-Vizi and Chinopoulos, 2006). We therefore quantified the amount of H2O2 released from intact mitochondria from clk-1 fed with bacteria producing UQ6~10, or wildtype bacteria K12. We compared those amounts to each other and to that released from mitochondria from N2 fed with K12. In the absence of inhibitors, clk-1 and N2 mitochondria generate similar amounts of H2O2 with either malate (Figure 5A) or succinate as a substrate (Figure 5B). Since ROS released into the mitochondrial matrix could be scavenged and not measured externally from intact mitochondria, we also measured ROS production from submitochondrial particles from N2 and clk-1 fed K12 or UQ10. We limited our studies to three cultures due to the difficulty in isolating enough mitochondria to study as SMPs. The three cultures studied represent the three groups with differing amounts of ROS damage seen on Western blot and thus give information about the underlying cause for these differences. The amounts of ROS production by clk-1 SMPs stimulated with NADH was increased compared to N2 (Figure 5C). With succinate, SMPs from clk-1 generated the same or more H2O2 than N2 (Figure 5D). In no case was the ROS production lower in clk-1 mitochondria than in N2 mitochondria. The sites of potential ROS release from the electron transport chain were similar to those previously reported in mammalian mitochondria (Chen et al., 2003) (Figures S3, S4).
To understand how UQ tail lengths affect lifespan, we directly tested the effects of different UQ tail lengths on: 1) C. elegans lifespan, 2) respiration and electron transport from complex I and II to complex III in the mitochondrial respiratory chain, 3) free radical damage to mitochondrial proteins, 4) generation of reactive oxygen species (ROS).
Regardless of which UQ species clk-1 animals ingest, they live longer than do N2 animals. However, animals fed UQ10 live the longest. Saiki et al. recently reported that the lifespan extension in wildtype C. elegans fed on Q-less bacteria was not influenced by the addition of a water-soluble UQ10 (Saiki et al., 2008). They concluded that the respiratory deficiency of the Q-less E. coli extended the lifespan of the nematode, rather than the precise nature of the UQ ingested. It is likely that a Q-less diet will alter metabolism in a way not seen in the bacteria used in the present study, and may independently affect lifespan. In addition, they did not study the effects of the water soluble UQ10 on clk-1 lifespan. Clearly our studies show that different exogenous UQs from otherwise genetically identical bacteria can have an effect on lifespan in clk-1.
Complex I displayed more marked UQ-specific differences in respiratory capacity when compared with complex II, with the effect of UQ10 the most notable. This agrees with our previous study of clk-1 fed wildtype E. coli (Kayser et al., 2004a). Lenaz and colleagues proposed that the length of isoprenyl tails of UQs relates to their location and diffusion in the membrane (Lenaz et al., 2002; Genova et al., 2003). UQs with shorter isoprenyl tails are hypothesized to be relatively more exposed to the surface of the inner mitochondrial membrane and the intermembrane space. Consequently, they may be less favorably positioned to receive electrons from complex I. In addition, since virtually all complex I appears to exist in supercomplexes in the nematode (Suthammarak et al., J Biol Chem, In Press), it may be that less supplemental UQ is accessible to complex I, even with multiple generations of feeding. We saw no correlation between the length of isoprenyl tails and the rate of mitochondrial respiration. Decreased (Lee et al., 2003) or increased (Bonawitz et al., 2007) mitochondrial activity have both been shown to extend lifespan. We found no simple correlation between mitochondrial respiratory capacities and lifespan; e.g. the effects of UQ species on ROS damage seem to outweigh the effects on respiration per se (see below).
Strikingly, clk-1 mitochondrial protein had much lower levels of oxidative damage than N2, regardless of which UQ had been fed to the animal. The decreases in ROS damage in the clk-1 animals as a group correlated with their longer lifespans, as compared to N2. Protein from clk-1 mitochondria with UQ10 was consistently the least labeled by HNE. The remarkable lack of ROS damage to clk-1 animals fed UQ10 is accompanied by a further, significant lengthening of clk-1’s already prolonged lifespan. We allowed the animals to all reach young adulthood before isolating the mitochondria. The similar developmental stage should indicate similar total amounts of metabolic flux during development.
Since animals with varied UQs differ in their accumulation of ROS damage, we hypothesized that different UQ species cause a decrease in either ROS production or scavenging. No significant differences in ROS released from intact mitochondria were seen when either malate or succinate were used as substrates. Interestingly, the amounts of H2O2 produced from clk-1 SMPs with K12 (producing UQ8) or UQ10 were both higher than that in N2 using NADH as a substrate. Thus, the decrease in ROS damage in the clk-1 mitochondrial proteins is seen despite equal to higher rates of ROS production. The rates of ROS production did not correlate either with lifespan or with ROS damage, as measured by HNE staining. The simplest interpretation of these data is that ROS are more effectively scavenged in clk-1 animals than in N2. This results in less accumulated oxidative damage to mitochondrial proteins, and prolongs lifespan.
Recently, Yang et al. reported that knock down of the cytoplasmic scavenger sod-1, or the two mitochondrial scavengers, sod-2 and sod-3, by RNAi does not shorten the lifespan of clk-1 (Yang et al., 2007). In addition, Van Raamsdonk and Heikimi show that deletion of sod-2 actually extends lifespan of clk-1 animals (Van Raamsdonk and Hekimi, 2009). Thus, these ROS scavengers are unlikely to be important in scavenging ROS in clk-1 animals or ROS are not important in determining lifespan. The roles of other potential ROS scavengers such as glutathione S-transferase or catalase have not been studied. It would be of interest to see which, if any, of the ROS scavengers are upregulated in clk-1.
How do large amounts of DMQ9 in clk-1 mitochondria affect our results? When the electrochemical properties of UQ2 and DMQ2 were evaluated by Miyadera and colleagues, they concluded that UQ2 is a stronger catalyst than DMQ2 in terms of the O2 reduction into O2•- (Miyadera et al., 2002). Given that the total amount of ubiquinone species (DMQ plus UQ) between clk-1 and N2, one candidate for the increased scavenging across all the clk-1 groups is DMQ9. The large amount of DMQ9 as a potential ROS scavenger may explain why all clk-1 groups had lower oxidative damage and longer lifespans than N2. Hekimi and colleagues have isolated tRNA suppressor mutations which reverse many of the phenotypes of clk-1 (including longevity) but which do not change DMQ levels (Branicky et al., 2006). This would argue that DMQ9 is not an important scavenger in clk-1. However, no report of the lifespans of the suppressors alone was given nor was of ROS production measured. Clearly further studies are necessary to resolve this issue. As noted above, other scavengers may be of primary importance in clk-1 as well.
However, the increased DMQ9 levels do not explain the differences between clk-1 fed UQ10 and clk-1 fed UQ6, UQ7, UQ8 or UQ9. Since UQ10 fed animals should have similar DMQ9 levels as the clk-1 animals fed other UQ species, our results may indicate that UQ10 provides stronger protection against oxidative damage than the other UQs used in this study.
Though mitochondria have been implicated as the major ROS production site, studies have questioned whether lifespan, oxidative damage and mitochondrial activity are independently regulated (Rea et al., 2007; Yang et al., 2007). Recently, Rea et al. (Rea et al., 2007) blocked mitochondrial function by knocking down expression of subunit of F0F1 ATPase atp-3 to different levels. There was no correlation between degree of lifespan extension and level of oxidative damage to total worm protein, which appeared to differ by less than 50% from controls for any level of RNAi treatment. However, the results of Rea et al. blocked metabolism in a way that does not necessarily alter ROS production or scavenging. In their study, lifespan may have been extended by specifically affecting development. In addition, our studies measure oxidative damage to mitochondrial proteins, not total worm protein, which may be a better predictor of lifespan.
Based on our data, UQ6, UQ7, and UQ10 fed clk-1 animals shared low respiration rates but were diverse in lifespan. clk-1 animals fed with UQ10 have longer adult lifespans than those fed with UQ6-9. By comparing adult lifespan, we intended to eliminate effects of UQs on developmental delay. The decreased oxidative damage in mitochondria with UQ10 makes us speculate that UQ10 is a better ROS scavenger than other UQ homologues used in this study. These results demonstrated that, in these animals, oxidative damage is an important factor in lifespan determination. In support of this conclusion, the C. elegans gas-1 mutant, which has a defective mitochondrial complex I subunit, also displays a low complex-I specific mitochondrial respiratory capacity, similar to clk-1. However, it accumulates intense oxidative damage in mitochondrial protein and has a very short lifespan (Kayser et al., 2001; Kayser et al., 2004a). By virtue of its role as a key subunit of complex I itself, it may have effects on ROS production that are not seen in clk-1 mutants. Studies to understand the differences between clk-1 and gas-1 are in progress.
In all cases, the distributions of UQ species were broader in clk-1 mitochondria than were the distributions in the bacteria on which they were raised. In the cases of clk-1 raised on UQ6 and UQ7, this was primarily the result of methodology. As stated in the Results, clk-1 animals raised on UQ6 or UQ7 alone have very few offspring which precludes isolation of their mitochondria. To obtain enough nematodes for those studies, we allowed animals raised on K12 (which produce UQ8) to reach dauer stage and then fed them bacteria producing UQ6 or UQ7. As a result, the animals were exposed to UQ6 or UQ7 for only one period of mitochondrial proliferation (dauer to young adult). However, even clk-1 animals grown several generations on bacteria producing almost entirely UQ9 or UQ10 had distributions of quinones more broad than those of the bacteria. Earlier studies found that null allele strains of clk-1 accumulated UQ9 from OP50 bacteria, despite the finding that such bacteria made no measureable UQ9 (Arroyo et al., 2006). Thus, the distribution of quinones in clk-1 mitochondria may result from a “leakiness” in the production of UQ9 or UQ10 species in the animal and from unequal uptake of the different quinones in their food.
Exogenous UQ10 and related quinone supplementation has been shown to support and improve mitochondrial oxidative phosphorylation (Marriage et al., 2004; Yen et al., 2005), supply protective benefits in oxidative stress (Matthews et al., 1998) and apoptosis (Moon et al., 2005). Our findings, which show that supplemental UQ10 increases lifespan and minimizes oxidative damage to mitochondrial proteins, may substantiate therapies that use it to treat mitochondrial and aging related diseases.
Many studies in worms make conclusions about lifespan as it relates to mitochondrial function and ROS production. These are seldom accompanied by measurements of either. We report the first in vivo characterization of the effects of different UQs on lifespan, and relate dietary supplementation of UQ10 to mitochondrial function in an experimental model. Moreover, we provide evidence linking oxidative damage of mitochondrial proteins to lifespan, and support the roles of DMQ9 and UQ10 as antioxidants.
We extend appreciation to Dr. Ernst-Bernhard Kayser, Dr. Qun Chen and Dr. Charles Hoppel for giving us important suggestions and critical interpretations of our data. We would also like to thank generous help from Dr. Bruce Barshop for identification of quinone species. MMS, Y-YY and PGM were supported by NIH grants GM58881 and AG026273.
Author Contributions YY performed or helped with all experiments, analyzed the data and contributed to writing the manuscript. JAG contributed and performed a unique protocol and assisted in writing the manuscript. PGM and MMS designed the studies, oversaw all parts of the project, analyzed the data and contributed to writing the manuscripts,
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