Knockdown of APOPT1/COA8 Causes Cytochrome c Oxidase Deficiency, Neuromuscular Impairment, and Reduced Resistance to Oxidative Stress in Drosophila melanogaster

Abstract

Cytochrome c oxidase (COX) deficiency is the biochemical hallmark of several mitochondrial disorders, including subjects affected by mutations in apoptogenic-1 (APOPT1), recently renamed as COA8 (HGNC:20492). Loss-of-function mutations are responsible for a specific infantile or childhood-onset mitochondrial leukoencephalopathy with a chronic clinical course. Patients deficient in COA8 show specific COX deficiency with distinctive neuroimaging features, i.e., cavitating leukodystrophy. In human cells, COA8 is rapidly degraded by the ubiquitin-proteasome system, but oxidative stress stabilizes the protein, which is then involved in COX assembly, possibly by protecting the complex from oxidative damage. However, its precise function remains unknown. The CG14806 gene (dCOA8) is the Drosophila melanogaster ortholog of human COA8 encoding a highly conserved COA8 protein. We report that dCOA8 knockdown (KD) flies show locomotor defects, and other signs of neurological impairment, reduced COX enzymatic activity, and reduced lifespan under oxidative stress conditions. Our data indicate that KD of dCOA8 in Drosophila phenocopies several features of the human disease, thus being a suitable model to characterize the molecular function/s of this protein in vivo and the pathogenic mechanisms associated with its defects.

Keywords: APOPT1; Drosophila melanogaster; cytochrome c oxidase deficiency; knockdown models; mitochondrial disease; resistance to oxidative stress.

Figure 1

Identification and characterization of the D. melanogaster ortholog of H. sapiens COA8 (APOPT1). (A) Amino acid sequence alignment of C. elegans, D. melanogaster, D. rerio, H. sapiens, M. musculus, and R. norvegicus COA8 orthologs. The shown alignment was performed using the Multiple Sequence Alignment Tool Clustal Omega and it highlights identical residues (*) and similar ones (. and:), conserved patterns and motifs (red lines) and the point mutations found in patients (orange stars). (B) Subcellular localization of dCOA8. Drosophila cells were transiently transfected with dCOA8-GFP (green), incubated with MitoTracker dye (red), and analyzed by confocal microscopy. Overlay of images (yellow) confirmed the mitochondrial localization of dCOA8. (C) Enzymatic activities of MRC complexes (I–IV) were measured in parental controls (act5C-Gal4>+ and UAS-CG14806-IR>+) and in ubiquitous KD flies (act5C-Gal4>UAS-CG14806-IR, dark gray column). Activities of complexes I–IV were normalized to the activity of citrate synthase (CS). For each genotype, three biological replicates of mitochondrial preparations (100 flies for each biological replicate) were analyzed. For each, enzymatic activities from at least 3 replicate reactions were performed. Data plotted are mean ± S.D. (one-way ANOVA with Sidak’s multiple comparisons test *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001). (D) 1D-BNGE analysis of MRC complexes and quantification of the relative intensity (RI) of CIV₁ bands normalized to the R.I. of CV band of the same sample (one-way ANOVA with Sidak’s multiple comparisons test **p ≤ 0.01). Isolated mitochondria from flies of the indicated genotypes were solubilized and ran in native conditions. (E) In gel activity of complex I, complex II, and complex IV after short (10 min) and long (30 min) incubation times and quantification of the relative intensity (RI) of CI and CIV₁ bands normalized to the R.I. of CII band of the same sample after 10 min of reaction (one-way ANOVA with Sidak’s multiple comparisons test *p ≤ 0.05, **p ≤ 0.01). Genotypes are [(1) act5C-Gal4>+, (2) UAS-CG14806-IR, (3) act5C-Gal4>UAS-CG14806-IR]. (F) Spontaneous locomotor activity was measured in ubiquitous (act5C-Gal4>UAS-CG14806-IR) and pan-neuronal (elav-Gal4 > UAS-CG14806-IR) dCOA8 KD flies with respect to parental controls (UAS-CG14806-IR > +, act5C-Gal4>+, and elav-Gal4>+) for 2 days (48 h). Data plotted are mean ± S.D. (one-way ANOVA with Sidak’s multiple comparisons test *p ≤ 0.05, **p ≤ 0.01). (G) The climbing assay was performed in ubiquitous (act5C-Gal4 > UAS-CG14806-IR) and pan-neuronal (elav-Gal4 > UAS-CG14806-IR) dCOA8 KD flies with respect to parental controls (UAS-CG14806-IR>+, act5C-Gal4>+, and elav-Gal4>+). Charts show mean and 95% CI, n = 60 animals. Statistical analysis used one-way ANOVA with Dunn’s multiple comparisons test (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001).

Figure 2

Functional characterization of dCOA8. (A) Cell death analysis. dCOA8-GFP over-expressing and control S2R+ cells (transfected with an empty vector expressing a cytosolic GFP) were double-stained with Annexin V and propidium iodide (PI). Percentage of early apoptotic (blue bars) and necrotic (red bars) cells was measured 48, 72, and 96 h post-transfection. Data plotted are mean ± S.D. (n = 3 biological replicates, two-way ANOVA with Sidak’s multiple comparisons test not significant). (B) Representative lifespan curves (Kaplan-Meier) of ubiquitous (act5C-Gal4 > UAS-CG14806-IR) and pan-neuronal (elav-Gal4 > UAS-CG14806-IR) dCOA8 KD flies with respect to parental controls (UAS-CG14806-IR>+, act5C-Gal4>+, and elav-Gal4>+) under oxidative stress at 23°C. Flies were treated with 20 mM Paraquat in standard food. Statistical analysis was performed with log-rank (Mantel-Cox). All the comparisons between the two KD lines with respect to parental controls are statistically significant (****p ≤ 0.0001).