Enhanced catabolism of mitochondrial superoxide/hydrogen peroxide and aging in transgenic Drosophila

Abstract

The generation of superoxide anion radicals (O2*-) and hydrogen peroxide (H2O2) during mitochondrial respiration has been widely postulated to be causally linked to the aging process. The hypothesis that a specific enhancement of mitochondrial O2*-/H2O2 catabolism would delay age-associated physiological changes and extend the lifespan was tested by simultaneous overexpression of MnSOD (manganese superoxide dismutase) and catalase, ectopically targeted to the mitochondrial matrix of transgenic Drosophila melanogaster. The increased activities of these antioxidative enzymes resulted in a decrease of mitochondrial H2O2 release and enhancement of free methionine content. The MnSOD/mitochondrial catalase transgenic flies displayed an enhanced resistance to experimental oxidative stress, induced by dietary H2O2 administration or by exposure to 100% ambient oxygen. However, the lifespan of the flies was decreased, by up to 43%, and this effect coincided with (i) an overall decrease in physical fitness, as measured by the speed of walking, and (ii) an age-related decrease in mitochondrial state 3 (ADP-stimulated) respiration. These findings support the notion that mitochondrial O2*-/H2O2 production at physiological levels is essential for normal biological processes leading to the attainment of a normal lifespan.

Figure 1. Cumulative H₂O₂ release

Results are presented as percentage of Dm4_Control H₂O₂ accumulation at 3 min for thoracic mitochondria obtained from 2-day-old Dm4_Control flies (C1–C3, open symbols) and Dm4_SOD/OCAT flies (E1–E3, closed symbols). P<0.0005 for cumulative H₂O₂ release from 3 to 8 min.

Figure 2. Lifespans at 25 °C

(A) Pooled survivorship curves of nine Dm2_Control and 14 Dm2_SOD/OCAT transgenic lines. Approximately 100 flies were used for each line. Results are shown for experiment 1 (P=0.004) and are representative of two independent experiments. Mean lifespans of the individual lines are presented in Table 1. (B) Pooled survivorship curves of six Dm4_Control and six Dm4_SOD/OCAT combinations. Approximately 100 flies were used for each combination. Results are shown for experiment 1 (P=0.0007) and are representative of two independent experiments. Mean lifespans of the individual combinations are indicated in Table 2.

Figure 3. Maximum walking speed

Individual flies were confined in 5 ml graduated pipettes and gently tapped to the base, and the maximum height reached by each fly after 10 s was recorded. The maximum walking speed (cm/s) was determined on the basis of the greatest height attained in three trials, using 15 different flies for each of three Dm4_Control groups (C1–C3) and three Dm4_SOD/OCAT groups (E1–E3), at each age. Results are pooled data and are expressed as means±S.E.M. for the three groups of Dm4_Control flies (white bars) and Dm4_SOD/OCAT flies (black bars). The Dm4_SOD/OCAT flies were significantly slower than the control flies (P<0.0005) and the walking speed decreased with advancing age (P<0.0005).

Figure 4. Mitochondrial respiration as a function of age

Mitochondrial respiration rates (states 3 and 4) of Dm4_Control C3 and Dm4_SOD/OCAT E1 flies were measured using pyruvate+proline as substrates, and expressed as nmol of O₂·min⁻¹·mg of protein⁻¹. Results are means±S.D. for three independent experiments at each age. (A) State 3 respiration (in the presence of excess substrate and ADP) was the same in both groups at 10 days of age, but it was lower in experimental versus control flies at 20 and 40 days of age. (B) State 4 respiration (in the presence of excess substrate after exhaustion of ADP) was well maintained at all ages tested.

Figure 5. Methionine content

Results are presented as means±S.D. of the data for three Dm4_Control (C1–C3; □) and three Dm4_SOD/OCAT combinations (E1–E3, ■) and are expressed as nmol/ml of initial preparation [5% (w/v) homogenates in 5% (w/v) metaphosphoric acid]. Results for each combination, at each age, were obtained from four independent preparations of 50 flies each. The methionine content was significantly higher in Dm4_SOD/OCAT flies than in Dm4_Control flies (P<0.0005) and decreased as a function of age (P<0.0005).

Figure 6. Resistance to H₂O₂ exposure

H₂O₂ was administered to control flies (□) and experimental flies (■) in 1% sucrose solutions. Survival times (mean±S.D.) were pooled for three Dm4_Control groups (C1–C3) versus three Dm4_SOD/OCAT groups (E1–E3) (P<0.001).