Defects in mitochondrial OXPHOS are associated with diverse and mostly intractable human disorders. The single-subunit alternative oxidase (AOX) found in many eukaryotes, but not in arthropods or vertebrates, offers a potential bypass of the OXPHOS cytochrome chain under conditions of pathological OXPHOS inhibition. We have engineered Ciona intestinalis AOX for conditional expression in Drosophila melanogaster. Ubiquitous AOX expression produced no detrimental phenotype in wild-type flies. However, mitochondrial suspensions from AOX-expressing flies exhibited a significant cyanide-resistant substrate oxidation, and the flies were partially resistant to both cyanide and antimycin. AOX expression was able to complement the semilethality of partial knockdown of both cyclope (COXVIc) and the complex IV assembly factor Surf1. It also rescued the locomotor defect and excess mitochondrial ROS production of flies mutated in dj-1beta, a Drosophila homolog of the human Parkinson's disease gene DJ1. AOX appears to offer promise as a wide-spectrum therapeutic tool in OXPHOS disorders.

Mutations in mitochondrial oxidative phosphorylation complex I are associated with multiple pathologies, and complex I has been proposed as a crucial regulator of animal longevity. In yeast, the single-subunit NADH dehydrogenase Ndi1 serves as a non-proton-translocating alternative enzyme that replaces complex I, bringing about the reoxidation of intramitochondrial NADH. We have created transgenic strains of Drosophila that express yeast NDI1 ubiquitously. Mitochondrial extracts from NDI1-expressing flies displayed a rotenone-insensitive NADH dehydrogenase activity, and functionality of the enzyme in vivo was confirmed by the rescue of lethality resulting from RNAi knockdown of complex I. NDI1 expression increased median, mean, and maximum lifespan independently of dietary restriction, and with no change in sirtuin activity. NDI1 expression mitigated the aging associated decline in respiratory capacity and the accompanying increase in mitochondrial reactive oxygen species production, and resulted in decreased accumulation of markers of oxidative damage in aged flies. Our results support a central role of mitochondrial oxidative phosphorylation complex I in influencing longevity via oxidative stress, independently of pathways connected to nutrition and growth signaling.aging | mitochondria | respiratory chain | free radicals M itochondria are key metabolic organelles whose oxidative phosphorylation (OXPHOS) system is considered to be one of the most efficient producers of bioenergy. When OX-PHOS function is compromized (e.g., by mutations or toxins), bioenergy supply and cellular homeostasis are seriously disrupted, which can be lethal.OXPHOS complex I plays a central role in the regulation of ATP production, intermediary metabolism, and apoptosis (1, 2), and mutations affecting it cause many human pathologies (3). It has also been proposed as a pacemaker of the aging process (4). Treatments inferred to decrease the production of reactive oxygen species (ROS) at the level of complex I can prolong lifespan in Drosophila (5). All these characteristics make it essential to understand better the role of complex I in vivo and its involvement in aging.Many organisms possess alternative enzymes that can bypass or replace the proton-translocating complexes of the mitochondrial respiratory chain. These include the alternative oxidases (AOX) and the NADH dehydrogenases of the Ndi and Nde families. Together these enzymes provide an alternative respiratory chain that potentially allows the maintenance of redox homeostasis and intermediary metabolism under conditions where flux through the "standard" respiratory chain is limited by high ATP levels, the action of toxins or other physiological restraints (6, 7). AOX acts as a bypass of complexes III and IV, whereas Nde or Ndi can bypass complex I.In previous studies these bypass enzymes were shown to be active when introduced into the mitochondria of higher metazoans such as mammals (8-12), arthropods (13), or nematodes (14), all of which lack endogenous alternative enzymes. Fu...

Mitochondrial dysfunction is a significant factor in human disease, ranging from systemic disorders of childhood to cardiomyopathy, ischaemia and neurodegeneration. Cytochrome oxidase, the terminal enzyme of the mitochondrial respiratory chain, is a frequent target. Lower eukaryotes possess alternative respiratory-chain enzymes that provide non-proton-translocating bypasses for respiratory complexes I (single-subunit reduced nicotinamide adenine dinucleotide dehydrogenases, e.g. Ndi1 from yeast) or III + IV [alternative oxidase (AOX)], under conditions of respiratory stress or overload. In previous studies, it was shown that transfer of yeast Ndi1 or Ciona intestinalis AOX to Drosophila was able to overcome the lethality produced by toxins or partial knockdown of complex I or IV. Here, we show that AOX can provide a complete or substantial rescue of a range of phenotypes induced by global or tissue-specific knockdown of different cIV subunits, including integral subunits required for catalysis, as well as peripheral subunits required for multimerization and assembly. AOX was also able to overcome the pupal lethality produced by muscle-specific knockdown of subunit CoVb, although the rescued flies were short lived and had a motility defect. cIV knockdown in neurons was not lethal during development but produced a rapidly progressing locomotor and seizure-sensitivity phenotype, which was substantially alleviated by AOX. Expression of Ndi1 exacerbated the neuronal phenotype produced by cIV knockdown. Ndi1 expressed in place of essential cI subunits produced a distinct residual phenotype of delayed development, bang sensitivity and male sterility. These findings confirm the potential utility of alternative respiratory chain enzymes as tools to combat mitochondrial disease, while indicating important limitations thereof.

Mitochondrial disorders are nowadays recognized as impinging on most areas of medicine. They include specific and widespread organ involvement, including both tissue degeneration and tumour formation. Despite the spectacular progresses made in the identification of their underlying molecular basis, effective therapy remains a distant goal. Our still rudimentary understanding of the pathophysiological mechanisms by which these diseases arise constitutes an obstacle to developing any rational treatments. In this context, the idea of using a heterologous gene, encoding a supplemental oxidase otherwise absent from mammals, potentially bypassing the defective portion of the respiratory chain, was proposed more than 10 years ago. The recent progress made in the expression of the alternative oxidase in a wide range of biological systems and disease conditions reveals great potential benefit, considering the broad impact of mitochondrial diseases. This review addresses the state of the art and the perspectives that can be now envisaged by using this strategy.

A point mutation [technical knockout25t (tko25t)] in the Drosophila gene coding for mitoribosomal protein S12 generates a phenotype of developmental delay and bang sensitivity. tko25t has been intensively studied as an animal model for human mitochondrial diseases associated with deficiency of mitochondrial protein synthesis and consequent multiple respiratory chain defects. Transgenic expression in Drosophila of the alternative oxidase (AOX) derived from Ciona intestinalis has previously been shown to mitigate the toxicity of respiratory chain inhibitors and to rescue mutant and knockdown phenotypes associated with cytochrome oxidase deficiency. We therefore tested whether AOX expression could compensate the mutant phenotype of tko25t using the GeneSwitch system to activate expression at different times in development. The developmental delay of tko25t was not mitigated by expression of AOX throughout development. AOX expression for 1 d after eclosion, or continuously throughout development, had no effect on the bang sensitivity of tko25t adults, and continued expression in adults older than 30 d also produced no amelioration of the phenotype. In contrast, transgenic expression of the yeast alternative NADH dehydrogenase Ndi1 was synthetically semi-lethal with tko25t and was lethal when combined with both AOX and tko25t. We conclude that AOX does not rescue tko25t and that the mutant phenotype is not solely due to limitations on electron flow in the respiratory chain, but rather to a more complex metabolic defect. The future therapeutic use of AOX in disorders of mitochondrial translation may thus be of limited value.