Engineering the alternative oxidase gene to better understand and counteract mitochondrial defects: state of the art and perspectives

Abstract

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.

Keywords: allospecific gene expression; alternative oxidase; mitochondrial diseases.

Figure 1

A schematized view of the mitochondrial respiratory chain of the animal kingdom. Electron flow (green line) through the chain is coupled to proton extrusion (open arrows) by the respirasome, which associates various proportions of respiratory complexes I, III and IV (CI, CIII, CIV). Protons released in the intermembrane space are subsequently used by complex V (CV; ATP synthase) to phosphorylate ADP (imported by the adenylate carrier; Ant) to ATP, making use of the inorganic phosphate imported by the phosphate carrier (Pic). Build-up of the proton gradient (ΔμH⁺) in case of impairment of ATP synthase function exerts a negative feedback control on electron flow in the respirasome. Under these conditions, quinones (Q) are essentially converted to reduced forms (QH₂), with formation of highly unstable semi-quinones (Q°) prone to react with oxygen to produce superoxides (O₂°). The scheme additionally features the two allotopic bypasses that have been successfully introduced in human cultured cells, Drosophila, and rodents, namely the internal NADH dehydrogenase from yeast (Ndi1; see Yagi et al., for a review) and the AOX. Site of action (red dash) of inhibitors is indicated for nPG, Aa (antimycin A) and cyanide (CN⁻).

Figure 2

State of the art and prospect for allotopic AOX expression. Upper left sector: first successful expression of the AOX gene in human cultured cells (cells), Drosophila melanogaster (flies) and Mus musculus (CD-1/B6 MitAOX mice). Upper right sector: first complementation of respiratory chain defect in human COX15-mutant fibroblasts, with siRNA-down-regulated COX10 HEK cells, in cyclope, Surf1 and dj-1β mutant D. melanogaster. Lower sector (left): a partial list of mouse models which should be informative to cross with the MitAOX mouse, either to establish/rule out the pathological involvement of mitochondrial superoxides, and/or of components from the mitochondrial cytochrome pathway, or to demonstrate the ability of AOX to complement genetic defects of the mitochondrial cytochrome pathway (CIV, CIII). One on the right-hand side: potential use of adeno-associated virus (AAV) constructs containing the AOX gene as a therapeutic strategy to target affected organs, for example, the eyes in cases of mutations affecting the cytochrome pathway.