The Alternative Oxidase AOX Does Not Rescue the Phenotype oftko25tMutant Flies

Abstract

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.

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

“…Yeast Ndi1 expression was shown previously to complement the lethality (at 25°C) of knockdown of subunits of cI [9] in Drosophila, whilst leaving a residual phenotype [32]. We therefore tested whether NDX was able to act similarly.…”

“…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.…”

Shortened lifespan and other age-related defects in bang sensitive mutants ofDrosophila melanogaster

“…Our findings are supported by another study which show that mitochondrial mutants have been shown to compensate for their energetic losses (Celotto et al 2011). In addition, Kemppainen et al (2014) found that tko is not rescued by an alternative oxidase, which should alleviate cytochrome oxidase insufficiency. This suggests that tko is a complex mutant with multiple metabolic effects.…”

Xenotopic expression of alternative electron transport enzymes in animal mitochondria and their impact in health and disease

“…This is the case of the tko 25t mutant of D. melanogaster , which harbors a defective mitoribosomal protein S12, leading to deficiency of OXPHOS subunits encoded by the mtDNA, manifesting as developmental delay and several neurological defects that may resemble seizures and hearing loss in human patients. Unexpectedly, the expression of either AOX or Ndi1 in tko 25t mutant flies was unable to rescue these harmful phenotypes (Kemppainen et al, ). Even more unexpected was the fact that bypassing complex I, which has 7 of the 13 proteins encoded by mtDNA, using Ndi1, caused a 50% decrease in the mutant viability, and when AOX was co‐expressed with Ndi1, the phenotype was 100% lethality.…”