Expression of alternative oxidase in Drosophila ameliorates diverse phenotypes due to cytochrome oxidase deficiency

Abstract

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.

Ciona intestinalis NADH dehydrogenase NDX confers stress-resistance and extended lifespan on Drosophila

“…Third, most likely as a consequence of constitutive activity, yeast Ndi1 expression can also be deleterious, under conditions where the ATP supply is limiting. This was found to be the case in two Drosophila models of mitochondrial disease, created by the knockdown of complex IV subunits [26] or the tko 25t mutation affecting mitochondrial protein synthesis [32]. Finally, whereas animals such as C. intestinalis have a single NADH dehydrogenase, yeast has three and plants typically several more.…”

“…However, the properties of these alternative NADH dehydrogenases remain uninvestigated, despite an accumulating literature on the alternative respiratory chain of some animals and protozoans, based on our own studies and those of others [25][26][27][28][29][30][31]. Our laboratory has, for example, exploited the alternative oxidase (AOX) from the tunicate Ciona intestinalis to by-pass defects in complexes III and IV, by expressing it in fruit fly [25,26], mouse [27] and human cell [28][29][30] models.…”

Emerging concepts in the therapy of mitochondrial disease

“…AOX is an alternative electron transport system present in lower eukaryotes, plants and several invertebrates that by-passes the complex III + IV segment of the respiratory chain. Expression of these proteins is well tolerated in mammalian cells [154], flies and mice [155] and has successfully been exploited to by-pass complex I or complex III/IV defects in human cells [156,157] and Drosophila models [158][159][160]. The therapeutic mechanism is based on the capacity of these enzymes to restore the electron flow through the quinone pool, thus preventing accumulation of reduced intermediates and oxidative damage [161].…”

Keep the fire burning: Current avenues in the quest of treating mitochondrial disorders

“…AOX was targeted to mitochondria and enzymatically active only upon blockade of complex IV: AOX expression enabled bypassing cytochrome c oxidase blockade after cyanide administration without showing deleterious side effects (El-Khoury et al, 2013). In flies, it was shown that AOX can provide a complete or substantial rescue of a range of phenotypes induced by global or tissue-specific knockdown of different complex IV subunits and assembly factors (Kemppainen et al, 2014).…”

“…El-Khoury, R., Kemppainen, K.K., Dufour, E., Szibor, M., Jacobs, H.T., and Rustin, P. (2014). Engineering the alternative oxidase gene to better understand and counteract mitochondrial defects: state of the art and perspectives.…”