Expression of the alternative oxidase complements cytochrome c oxidase deficiency in human cells

Abstract

Cytochrome c oxidase (COX) deficiency is associated with a wide spectrum of clinical conditions, ranging from early onset devastating encephalomyopathy and cardiomyopathy, to neurological diseases in adulthood and in the elderly. No method of compensating successfully for COX deficiency has been reported so far. In vitro, COX-deficient human cells require additional glucose, pyruvate and uridine for normal growth and are specifically sensitive to oxidative stress. Here, we have tested whether the expression of a mitochondrially targeted, cyanide-resistant, alternative oxidase (AOX) from Ciona intestinalis could alleviate the metabolic abnormalities of COX-deficient human cells either from a patient harbouring a COX15 pathological mutation or rendered deficient by silencing the COX10 gene using shRNA. We demonstrate that the expression of the AOX, well-tolerated by the cells, compensates for both the growth defect and the pronounced oxidant-sensitivity of COX-deficient human cells.

Figure 1. Alternative oxidase (AOX) bypasses the cyt segment of the respiratory chain

1. The cyt pathway bypass provided by AOX and the targets of different respiratory chain inhibitors. Aa, antimycin; c, cyt c; CI–V, the various respiratory chain complexes; im, inner membrane; UQ, ubiquinone.

2. The effect of AOX expression on respiration of HEK-293 cells treated with various OXPHOS inhibitors.

Figure 2. AOX compensates for the effects COX deficiency caused by COX10 knockdown in HEK-293 cells

1. COX activity in control (AOX-transgenic) and HEK-293 cells expressing COX10-targeted shRNA (clone selected after transfection with pSM2C-COX10 construct). Values are means of triplicates performed on three different clones. Inset shows immunoblot of 100 µg total cell lysates. Specificity of the anti-COX10 shRNA was established by using a scrambled shRNA which did not affect COX10 level or COX activity (data not shown).

2. Cell growth of either control (AOX-transgenic) or COX10⁻ HEK-293 cells, with or without doxycyclin induction of AOX expression, in non-selective medium. Cells were seeded at 10,000 cells/cm².

3. Number of cells after 7 days of growth (end of the exponential growth phase) of either control (AOX-transgenic) or COX10⁻ cells, with or without doxycycline induction of AOX expression, in selective medium (i.e. 1 g/l glucose).

4. Respiration of intact HEK control and COX10⁻ cells ± AOX. KCN was 300 µM, n-propyl gallate (n-PG) 50 µM. Numbers along the traces represent nmol O₂/min per mg protein (mean values from three experiments).

Figure 3. AOX compensates for COX deficiency in immortalized fibroblasts derived from a patient harbouring a pathological mutation in the COX15 gene

1. COX and SCCR activity ratios in immortalized control, COX15⁻ and COX15⁻: AOX fibroblasts.

2. Respiration of intact control, COX15⁻ and COX15⁻: AOX cells measured using a Clark electrode. KCN was 300 µM, n-PG 50µM. Numbers along the traces represent nmol O₂/min per mg protein (mean values from three experiments).

3. Immunoblot of 25 µg whole cell lysates from COX15-mutant fibroblasts transduced (or not) with AOX, probed with antibodies against lily AOX (lanes 1 and 2), and actin (lanes 3 and 4).

4. AOX mitochondrial localization assessed by AOX antibody. Scale bar, 20 µm.

5. Numbers of cells after 7 day growth in selective or non-selective media, expressed as percentage of cell number obtained under non-selective condition. Cells seeded at 15,000 cells/cm².

Figure 4. AOX compensates for the hypersensitivity of COX15-mutant fibroblasts to oxidative insult

1. Growth of AOX-expressing or non-expressing cells (3 d) in low glucose medium in the presence or absence of 5 µM antimycin ±1 mM TEMPOL (4-hydroxy-2,2,6,6-tetramethylpiperidinyloxy) (percentage of cells in the absence of antimycin). Cells seeded at 15,000 cells/cm².

2. Fluorescence microscopy images using MitoSOX™ probe, with or without 5 µM antimycin (30 min). Panels 1 and 2, COX15⁻ cells (1 untreated; 2 antimycin treated), Panels 3 and 4, COX15⁻ : AOX cells (3 untreated; 4 antimycin treated) (scale bars represent 40 µm).

3. Quantification of MitoSOX™ staining (Fig 3B panels 1–4) expressed as a percentage of the labelling measured in control COX15⁻ cells (panel 1).