No abstract

The mitochondrial respiratory chain in vertebrates and arthropods is different from that of most other eukaryotes because they lack alternative enzymes that provide electron transfer pathways additional to the oxidative phosphorylation (OXPHOS) system. However, the use of diverse experimental models, such as human cells in culture, Drosophila melanogaster and the mouse, has demonstrated that the transgenic expression of these alternative enzymes can impact positively many phenotypes associated with human mitochondrial and other cellular dysfunction, including those typically presented in complex IV deficiencies, Parkinson's, and Alzheimer's. In addition, these enzymes have recently provided extremely valuable data on how, when, and where reactive oxygen species, considered by many as “by‐products” of OXPHOS, can contribute to animal longevity. It has also been shown that the expression of the alternative enzymes is thermogenic in cultured cells, causes reproductive defects in flies, and enhances the deleterious phenotype of some mitochondrial disease models. Therefore, all the reported beneficial effects must be considered with caution, as these enzymes have been proposed to be deployed in putative gene therapies to treat human diseases. Here, we present a brief review of the scientific data accumulated over the past decade that show the benefits and the risks of introducing alternative branches of the electron transport into mammalian and insect mitochondria, and we provide a perspective on the future of this research field.

The expression of the mitochondrial alternative oxidase AOX from Ciona intestinalis (Tunicata: Ascidiacea) has provided clear beneficial effects in a variety of mammalian and insect mitochondrial disease models. Because of its non‐proton pumping terminal oxidase activity, AOX can bypass the cytochrome c segment of the respiratory chain (complexes III and IV), and alleviate the possible overload of electrons that occurs upon oxidative phosphorylation dysfunction, not contributing, though, to the proton‐motive force needed for mitochondrial ATP synthesis. Significant detrimental outcomes have also been reported upon AOX expression, raising concerns regarding its putative deployment as a therapy enzyme for human diseases. In Drosophila, AOX expression is developmentally advantageous at low temperatures when the flies are cultured on a standard, rich diet, but it dramatically compromises adult eclosion when the flies are cultured on a low‐nutrient diet (LN), at 25ºC or above. Here, we applied transcriptomics and metabolomics analyses to show that the interaction between LN and AOX expression causes a general alteration of larval amino acid metabolism, leading to an almost 40% decrease in biomass at the pre‐metamorphosis stage. This reduced nutrient storage impairs development at the late pupa stage with a clear signature for starvation and an overall downregulation of mitochondrial metabolism. Interestingly, lactate dehydrogenase, lactate and 2‐hydroxyglutarate are elevated in AOX‐expressing flies, irrespective of diet. We have also identified that the addition of very low levels of ethanol to LN is sufficient to rescue the lethal phenotype imposed to the flies by the LN‐AOX interaction. We are currently exploring how alcohol dehydrogenase and other enzymes in the ethanol catabolic pathway participate in this rescue phenomenon. Nevertheless, our data points to important roles for two key redox‐regulating enzymes, lactate dehydrogenase and alcohol dehydrogenase, in adjusting the physiological changes induced by AOX function in larvae. As diet is one of the most important external factors that influence metabolism, our work provides important insights into how AOX expression could be safely accomplished, with minimal impact for higher animals.

Despite the beneficial effects shown when the mitochondrial alternative oxidase AOX from Ciona intestinalis (Tunicata: Ascidiacea) is xenotopically expressed in mammalian and insect models, important detrimental outcomes have also been reported, raising concerns regarding its envisioned deployment as a therapy enzyme for human mitochondrial and related diseases. Because of its non-proton pumping terminal oxidase activity, AOX can bypass the cytochrome c segment of the respiratory chain and alleviate the possible overload of electrons that occurs upon oxidative phosphorylation (OXPHOS) dysfunction, not contributing though to the proton-motive force needed for mitochondrial ATP synthesis. We have shown previously that AOX-expressing flies present a dramatic drop in pupal viability when the larvae are cultured on a low nutrient diet, indicating that AOX interferes with normal developmental metabolism. Here, we applied combined omics analyses to show that the interaction between low nutrient diet and AOX expression causes a general alteration of larval amino acid metabolism and lipid accumulation, which are associated with functional and morphological alterations of the larval digestive tract and with a drastic decrease in larval biomass accumulation. Pupae at the pre-lethality stage present a general downregulation of mitochondrial metabolism and a signature for starvation and deregulated signaling processes. This AOX-induced lethality is partially rescued when the low nutrient diet is supplemented with tryptophan and/or methionine. The developmental dependence on these amino acids, associated with elevated levels of lactate dehydrogenase, lactate, 2-hydroxyglutarate, choline-containing metabolites and breakdown products of membrane phospholipids, indicates that AOX expression promotes tissue proliferation and growth of the Drosophila larvae, but this is ultimately limited by energy dissipation via mitochondrial uncoupling. We speculate that the combination of diet and AOX expression may be used for the metabolic regulation of proliferative tissues, such as tumors.