Developmental arrest in Drosophila melanogaster caused by mitochondrial DNA replication defects cannot be rescued by the alternative oxidase

Abstract

The xenotopic expression of the alternative oxidase AOX from the tunicate Ciona intestinalis in diverse models of human disease partially alleviates the phenotypic effects of mitochondrial respiratory chain defects. AOX is a non-proton pumping, mitochondrial inner membrane-bound, single-subunit enzyme that can bypass electron transport through the cytochrome segment, providing an additional site for ubiquinone reoxidation and oxygen reduction upon respiratory chain overload. We set out to investigate whether AOX expression in Drosophila could counteract the effects of mitochondrial DNA (mtDNA) replication defects caused by disturbances in the mtDNA helicase or DNA polymerase γ. We observed that the developmental arrest imposed by either the expression of mutant forms of these enzymes or their knockdown was not rescued by AOX. Considering also the inability of AOX to ameliorate the phenotype of tko25t, a fly mutant with mitochondrial translation deficiency, we infer that this alternative enzyme may not be applicable to cases of mitochondrial gene expression defects. Finding the limitations of AOX applicability will help establish the parameters for the future putative use of this enzyme in gene therapies for human mitochondrial diseases.

Coenzyme Q redox signalling and longevity

“…AOX (Figure 2) has been shown to by-pass chemical inhibition of CIII and CIV in human cell culture, flies and mice [86,90,91], depletion of CIV subunits in flies [92], mutation of the CIII assembly factor BCS1L [93], increase survival in mice injected with Lipopolysaccharides (LPS) [75], rescue developmental defects caused by interruption of Jun N-terminal Kinase and restore mobility [94] in dj1β mutant flies (orthologue of the human PARK7) [90]. However, AOX expression in flies did not rescue mutations in tko (fly orthologue of the human MRPS12) [95], sesB (orthologue of the Adenine Nucleotide Translocator, ANT 1) [96], mtDNA-helicase (orthologue of Twinkle) or tamas [97]. Furthermore, AOX lowers survival of mice carrying a muscle-specific mutation of COX15 [98], aggravates symptoms associated with cardiac arrest, despite preventing ROS-RET, in mouse models where heart circulation is interrupted [99] and severely shortens lifespan of flies under thermal stress, hypoxia and hyperoxia [42].…”

Alternative oxidase encoded by sequence-optimized and chemically-modified RNA transfected into mammalian cells is catalytically active

“…Notably, AOX by-passes two proton pumps of the ETC, namely cIII and cIV [6,7], and represents a metabolic rescue mechanism that is absent in vertebrates [3]. Notwithstanding its natural absence, AOX can be xenotopically expressed in a catalytically active form in human cells with primary respiratory chain dysfunction [8] or mouse mtDNA-depleted cells [9], in drosophila disease models [10][11][12][13][14][15], and in the mouse [7,16] by using an AOX cDNA transiently expressed or integrated into specific sites in the genome. Remarkably, in most cases this has been achieved without producing adverse phenotypic effects, at least under standard physiological conditions, i.e., in the absence of metabolic stress signaling related to respiratory disruption [7].…”

The DNA polymerases of Drosophila melanogaster

“…Mutants die during late larval stages, exhibiting impaired mtDNA replication and decreased mtDNA content [107][108][109]. Ubiquitous RNAi-mediated knock-down of PolG1 depletes mtDNA, decreases mitochondrial respiratory activity and results in lethality, while neuronal-specific knock-down causes progressive behavioral deficits [110,111]. Other studies have utilized sophisticated genetic techniques to engineer flies to express either polymerase-or exonuclease-deficient PolG1the former resulted in mtDNA depletion, whereas the latter led to accumulation of mutations/deletions of mitochondrial DNA [112,113].…”

“…Mutations in human POLG and POLG2 are associated with several diseases including mtDNA depletion syndromes, such as Alpers syndrome, and mtDNA deletion disorders, such as progressive external ophthalmoplegia [121]. Significantly, several studies in the last decade have manipulated the orthologous D. melanogaster genes to provide important insights into the etiology of, and possible therapies for, these diseases [108][109][110][111][112][113]122]. For example, two studies generated flies expressing exonuclease-or polymerase-deficient versions of PolG1 in order to investigate how defects in these two different activities of the enzyme may contribute to pathophysiology [112,113].…”