Mitochondrial ROS production correlates with, but does not directly regulate lifespan in Drosophila

Abstract

The Mitochondrial Free Radical Theory of Aging (MFRTA) is currently one of the most widely accepted theories used to explain aging. From MFRTA three basic predictions can be made: long-lived individuals or species should produce fewer mitochondrial Reactive Oxygen Species (mtROS) than short-lived individuals or species; a decrease in mtROS production will increase lifespan; and an increase in mtROS production will decrease lifespan. It is possible to add a further fourth prediction: if ROS is controlling longevity separating these parameters through selection would be impossible. These predictions have been tested in Drosophila melanogaster. Firstly, we studied levels of mtROS production and lifespan of three wild-type strains of Drosophila, Oregon R, Canton S and Dahomey. Oregon R flies live the longest and produce significantly fewer mtROS than both Canton S and Dahomey. These results are therefore in accordance with the first prediction. A new transgenic Drosophila model expressing the Ciona intestinalis Alternative Oxidase (AOX) was used to test the second prediction. In fungi and plants, AOX expression regulates both free radical production and lifespan. In Drosophila, AOX expression decreases mtROS production, but does not increase lifespan. This result contradicts the second prediction of MFRTA. The third prediction was tested in flies mutant for the gene dj-1beta. These flies are characterized by an age-associated decline in locomotor function and increased levels of mtROS production. Nevertheless, dj-1beta mutant flies do not display decreased lifespan, which again is in contradiction with MFRTA. In our final experiment we utilized flies with DAH mitochondrial DNA in an OR nuclear background, and OR mitochondrial DNA in DAH nuclear background. From this, Mitochondrial DNA does not control free radical production, but it does determine longevity of females independently of mtROS production. In summary, these results do not systematically support the predictions of the MFRTA. Accordingly, MFRTA should be revised to accommodate these findings.

Figure 1.. Mitochondrial ROS production versus lifespan in three wild type strains of Drosophila melanogaster.

(A) Rate of mtROS production (assayed as H₂O₂, mean + SEM). a, b, c and d indicate statistically significant differences between groups (ANOVA, p < 0.05, n = 5-9 samples per group), m: male, f: female. (B) Survival curves. Combined data from two independent experiments using 100 flies per group per experiment. Mean, maximum lifespans (d) were: DAH males [39,49]; CS males (46, 53); OR males (63, 74); DAH females (79, 84); CS females (69, 81); OR females (86, 91).

Figure 2.. Effects of AOX expression on resistance to respiratory chain inhibitors.

(A) Survival after exposure to 3 mM rotenone or 3 mM antimycin A, of flies of strains and sexes indicated (AOX / -, flies transgenic for UAS-AOX in absence of GAL4 driver; AOX / da-GAL4, flies transgenic for AOX in presence of da-GAL4 driver). (B) Representative oxygraph traces of mitochondrial suspensions (0.5 mg/ml in state 3) in presence of inhibitors shown. Inferred oxygen consumption rates (nmol/min) as indicated. Pyruvate+proline was used as substrate in all experiments. (C) mtROS production (mean + SEM) in presence of inhibitors (at least 4 independent samples per experiment, a, b denote significantly different groups, ANOVA, p < 0.05).

Figure 3.. Effect of AOX expression on mtROS production and lifespan.

(A) mtROS production (mean + SEM). a, b: statistically significant differences between groups (ANOVA, p < 0.05, n=4-8 samples per group) m: male, f: female. (B) Survival curves for wild type (wt), AOX non-expressing (AOX / -), AOX expressing (AOX / da-GAL4 +), and driver only (- / da-GAL4) flies, all in the DAH (w^-) background. Flies of AOX transgenic lines F6 and F24 as indicated. Combined data from two independent experiments using 200 flies per group per experiment. Mean, maximum life spans (d) were: wt males (42,51); wt females (75, 82); - / daGAL4 males (44, 54); - / da-GAL4 females (75, 81), F6 AOX / - females (77, 82); F6 AOX / - males (42,51); F6 AOX / da-GAL4 males (40,47); F6 AOX / da-GAL4 females (82, 51); F24 AOX / - males (42, 54); F24 AOX / - females (77, 81); F24 F24 AOX / da-GAL4 males (42, 54); AOX / da-GAL4 females (73, 80).

Figure 4.. Effect of AOX on mitochondrial bioenergetics and mtROS during aging.

Oxygen consumption in state 3 (% of that in the young group) in 30 d old males (A) and 50 d old females. (B) AOX expression is not able to compensate the decrease in oxygen consumption associated with aging. mtROS generation (nmol H₂O₂/min.mg.prot) in 30 d old males (C) and 50 d old females (D). AOX expression diminishes mtROS production in both young and aged flies, and compensates for the age-associated increase. a, b and c denote statistically significant differences between groups (ANOVA, p < 0.05, n = 4-10 samples per group). Pyruvate+proline was used as substrate in all experiments. Plotted data are means ± SEM.

Figure 5.. Effects of the dj-1β mutation on mtROS production and lifespan in Drosophila melanogaster.

(A) mtROS production, assayed as H₂O₂, (mean + SEM). a, b and c denote statistically significant differences between groups (ANOVA, p<0.05, n = 4-6 samples per group), m: male, f: female. (B) Survival curves. Combined data from two independent experiments using 100 flies per group per experiment. Mean, maximum lifespans (d) were: DAH males (43, 58); DAH females (75, 79); dj-1β mutant males (67, 75); dj-1β mutant females (79, 86).

Figure 6.. Effects of the changes on mtDNA content on mtROS production and lifespan in new wild type strains of Drosophila melanogaster (DAHOR and ORDAH).

(A) mtROS production, assayed as H₂O₂, (mean + SEM). a and b denote statistically significant differences between groups (ANOVA, p <0.05, n = 4-5 samples per group), m: male, f: female. (B) Survival curves. Combined data from two independent experiments using between 80-100 flies per group per experiment. Mean, maximum lifespans (d) were: ORDAH males (68, 76); ORDAH FEMALES (78, 87); DAHOR males (47,59); DAHOR females (87,92).