Bulk autophagy, but not mitophagy, is increased in cellular model of mitochondrial disease.
Oxidative phosphorylation system (OXPHOS) deficiencies are rare diseases but constitute the most frequent inborn errors of metabolism. We analyzed the autophagy route in 11 skin fibroblast cultures derived from patients with well characterized and distinct OXPHOS defects. Mitochondrial membrane potential determination revealed a tendency to decrease in 5 patients' cells but reached statistical significance only in 2 of them. The remaining cells showed either no change or a slight increase in this parameter. Colocalization analysis of mitochondria and autophagosomes failed to show evidence of increased selective elimination of mitochondria but revealed more intense autophagosome staining in patients' fibroblasts compared with controls. Despite the absence of increased mitophagy, Parkin recruitment to mitochondria was detected in both controls' and patients' cells and was slightly higher in cells harboring complex I defects. Western blot analysis of the autophagosome marker LC3B, confirmed significantly higher levels of the protein bound to autophagosomes, LC3B-II, in patients' cells, suggesting an increased bulk autophagy in OXPHOS defective fibroblasts. Inhibition of lysosomal proteases caused significant accumulation of LC3B-II in control cells, whereas in patients' cells this phenomenon was less pronounced. Electron microscopy studies showed higher content of late autophagic vacuoles and lysosomes in OXPHOS defective cells, accompanied by higher levels of the lysosomal marker LAMP-1. Our findings suggest that in OXPHOS deficient fibroblasts autophagic flux could be partially hampered leading to an accumulation of autophagic vacuoles and lysosomes.
Mitochondrial and Neuromuscular Diseases Laboratory, Hospital Universitario 12 de Octubre Research Institute (i+12), Madrid, Spain; Spanish Network for Biomedical Research in Rare Diseases (CIBERER), U723, Spain. Electronic address: mmoran@h12o.es.
María
M
Morán
Mitochondrial and Neuromuscular Diseases Laboratory, Hospital Universitario 12 de Octubre Research Institute (i+12), Madrid, Spain; Spanish Network for Biomedical Research in Rare Diseases (CIBERER), U723, Spain.
Aitor
A
Delmiro
Mitochondrial and Neuromuscular Diseases Laboratory, Hospital Universitario 12 de Octubre Research Institute (i+12), Madrid, Spain; Spanish Network for Biomedical Research in Rare Diseases (CIBERER), U723, Spain.
Alberto
A
Blázquez
Mitochondrial and Neuromuscular Diseases Laboratory, Hospital Universitario 12 de Octubre Research Institute (i+12), Madrid, Spain; Spanish Network for Biomedical Research in Rare Diseases (CIBERER), U723, Spain.
Cristina
C
Ugalde
Mitochondrial and Neuromuscular Diseases Laboratory, Hospital Universitario 12 de Octubre Research Institute (i+12), Madrid, Spain.
Joaquín
J
Arenas
Mitochondrial and Neuromuscular Diseases Laboratory, Hospital Universitario 12 de Octubre Research Institute (i+12), Madrid, Spain; Spanish Network for Biomedical Research in Rare Diseases (CIBERER), U723, Spain.
Miguel A
MA
Martín
D000255:Adenosine Triphosphate / Q000378:metabolism; D001343:Autophagy / Q000502:physiology*; D002478:Cells, Cultured; D005347:Fibroblasts / Q000378:metabolism / Q000502:physiology; D006801:Humans; D051907:Lysosomal Membrane Proteins / Q000378:metabolism; D008247:Lysosomes / Q000378:metabolism / Q000502:physiology; D053078:Membrane Potential, Mitochondrial / Q000502:physiology; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D008928:Mitochondria / Q000378:metabolism / Q000502:physiology*; D028361:Mitochondrial Diseases / Q000378:metabolism / Q000473:pathology* / Q000503:physiopathology; D063306:Mitophagy / Q000502:physiology*; D010588:Phagosomes / Q000378:metabolism / Q000502:physiology; D052118:Lysosomal-Associated Membrane Protein 1
D000255:Adenosine Triphosphate; D051907:Lysosomal Membrane Proteins; D008869:Microtubule-Associated Proteins; C489116:LAMP1 protein, human; D052118:Lysosomal-Associated Membrane Protein 1; C502070:MAP1LC3B protein, human
Autophagosome; Autophagy; Lysosome; Mitophagy; OXPHOS deficiency; Parkin