Hypersensitivity to oxygen and shortened lifespan in a Drosophila mitochondrial complex II mutant

Abstract

Oxidative stress is implicated as a major cause of aging and age-related diseases, such as Parkinson's and Alzheimer's, as well as ischemia-reperfusion injury in stroke. The mitochondrial electron transport chain is the principal source of reactive oxygen species within cells. Despite considerable medical interest, the molecular mechanisms that regulate reactive oxygen species formation within the mitochondrion remain poorly understood. Here, we report the isolation and characterization of a Drosophila mutant with a defect in subunit b of succinate dehydrogenase (SDH; mitochondrial complex II). The sdhB mutant is hypersensitive to oxygen and displays hallmarks of a progeroid syndrome, including early-onset mortality and age-related behavioral decay. Pathological analysis of the flight muscle, which is amongst the most highly energetic tissues in the animal kingdom, reveals structural abnormalities in the mitochondria. Biochemical analysis shows that, in the mutant, there is a complex II-specific respiratory defect and impaired complex II-mediated electron transport, although the other respiratory complexes remain functionally intact. The complex II defect is associated with an increased level of mitochondrial hydrogen peroxide production, suggesting a possible mechanism for the observed sensitivity to elevated oxygen concentration and the decreased lifespan of the mutant fly.

Fig. 1.

Drosophila sdhB mutant is dramatically sensitive to hyperoxia. (A) Survival curves (±SEM) of sdhB^EY12081 and the wild-type strain Canton-S (C-S) under hyperoxia (100% O₂). n > 25 flies; log-rank test; P < 0.0001. (B) Structure of the sdhB gene, with triangle indicating the insertion site of the 10.9-kb P-element (EY12081). Open and hatched blocks indicate untranslated and coding sequences, respectively. (C) Transcription of the sdhB gene is greatly reduced in the mutant. RT-PCR experiments were performed by using total RNA from wild-type (C-S) (lanes 1 and 2) and sdhB mutant (lanes 3 and 4) flies. Shown are RT-PCR products from 10-fold dilutions of sample. Actin served as an internal control.

Fig. 2.

Genetic rescue of the sdhB mutant hypersensitivity to oxygen. (A) Precise excision (sdhB^ex29) of the P-element returns the oxygen resistance to normal. Seven additional, independent, precise excision alleles also fully reverted the mutant phenotype. sdhB^ex66, a mutant allele, generated by imprecise excision of the P-element, retains the oxygen sensitivity (n > 25; log-rank test; P < 0.0001). The same was true for four independent, imprecise excision alleles. (B) Genomic rescue of the sdhB mutant phenotype. Survival curves (±SE) under hyperoxia (100% O₂) of sdhB^EY12081, the wild-type strain (C-S), and sdhB^EY12081 flies carrying a transgenic genomic DNA rescue fragment (P-rescue-sdhB). This fragment consisted of the sdhB transcription unit plus 5 kb of upstream genomic DNA. The genomic rescue construct is sufficient to rescue the sdhB^EY12081 mutation (n > 25; log-rank test; P < 0.0001). These data confirm that the mutant phenotype is due to a defect in the sdhB gene.

Fig. 3.

Hyperoxia causes mitochondrial ultrastructural abnormalities in sdhB mutants. Electron micrographs of flight muscle of 7-day-old wild-type (C-S) with (C) and without (A) 15-h hyperoxia, show normal mitochondrial ultrastructure. Seven-day-old sdhB^EY12081 under normoxia (B) show normal mitochondrial ultrastructure, but, after 15 h under hyperoxia (D), the majority of mitochondria are severely damaged, their cristae in disarray. (E and F) Higher magnification, showing abnormal pattern of cristae in the mutant. (Scale bars, 1 μm.) M, mitochondrion; F, myofibril; n, nucleus. The damage in individual mitochondria appears to be a largely all-or-none phenomenon. Note also that the outer membranes of the affected mitochondria are still intact, in contrast to the mitochondrial lesions seen in the mutant hyperswirl (15).

Fig. 4.

Under normoxia, the sdhB mutant displays reduced lifespan and an age-related decline in climbing ability. (A) Survival curves (±SEM) of sdhB^EY12081 mutants and wild-type (C-S). Flies were maintained at 25°C, transferred every 2–4 days, and scored for survival. Even under normoxia, the sdhB^EY12081 mutants were short-lived (n > 300 flies; log-rank test, P < 0.0001). (B) Climbing ability score. At 2 days of age, sdhB^EY12081 mutant flies performed normally (n > 60 flies; P = 0.6), but, with age, there was a rapid decay to 46% by 8 days (n > 50 flies; P < 0.001) and 23% by 12 days (n > 50 flies; P < 0.001).

Fig. 5.

sdhB mutant fly mitochondria display a complex II-specific respiratory defect, reduced complex II electron transport activity and increased H₂O₂ production. (A) Oxygen consumption compared in mitochondria isolated from wild-type (C-S) and sdhB^EY12081 mutant flies by using an oxygraph and complex-specific substrates and inhibitors. The mutant showed a 56% decrease in complex II-specific respiration (n = 5, P < 0.0001). In contrast, complex I-specific respiration, as well as complex IV-specific respiration, showed no significant differences (n = 5, P = 0.32 and n = 5, P = 0.09, respectively). Complex III was not measured. (B) Complex II-mediated electron transport activity, assayed by malonate-sensitive succinate-cytochrome c reductase activity on mitochondria from wild-type (C-S) and mutant flies. Activity was assayed per milligram of protein. Activity in sdhB^EY12081 was reduced by 40% (n = 3, P < 0.0001). (C) Hydrogen peroxide production was measured by using the Amplex Red hydrogen peroxide assay kit (Molecular Probes). Activity was assayed per milligram of protein. Mitochondria from sdhB^EY12081 mutant animals showed a 32% increase in hydrogen peroxide production, as compared with controls from wild-type (C-S) (n = 12, P = 0.02).