title: Allotopic mRNA localization to the mitochondrial surface rescues respiratory chain defects in fibroblasts harboring mitochondrial DNA mutations affecting complex I or v subunits.
abstract: The possibility of synthesizing mitochondrial DNA (mtDNA)-coded proteins in the cytosolic compartment, called allotopic expression, provides an attractive option for genetic treatment of human diseases caused by mutations of the corresponding genes. However, it is now appreciated that the high hydrophobicity of proteins encoded by the mitochondrial genome represents a strong limitation on their mitochondrial import when translated in the cytosol. Recently, we optimized the allotopic expression of a recoded ATP6 gene in human cells, by forcing its mRNA to localize to the mitochondrial surface. In this study, we show that this approach leads to a long-lasting and complete rescue of mitochondrial dysfunction of fibroblasts harboring the neurogenic muscle weakness, ataxia and retinitis Pigmentosa T8993G ATP6 mutation or the Leber hereditary optic neuropathy G11778A ND4 mutation. The recoded ATP6 gene was associated with the cis-acting elements of SOD2, while the ND4 gene was associated with the cis-acting elements of COX10. Both ATP6 and ND4 gene products were efficiently translocated into the mitochondria and functional within their respective respiratory chain complexes. Indeed, the abilities to grow in galactose and to produce adenosine triphosphate (ATP) in vitro were both completely restored in fibroblasts allotopically expressing either ATP6 or ND4. Notably, in fibroblasts harboring the ATP6 mutation, allotopic expression of ATP6 led to the recovery of complex V enzymatic activity. Therefore, mRNA sorting to the mitochondrial surface represents a powerful strategy that could ultimately be applied in human therapy and become available for an array of devastating disorders caused by mtDNA mutations.
authors.affiliation: Laboratoire de Physiopathologie Cellulaire et Moléculaire de la Rétine, INSERM U592, Université Pierre et Marie Curie (UPMC-Paris6), Hôpital St. Antoine-Bât. Kourilsky, 184 rue du Faubourg Saint-Antoine, 75571 Paris Cedex 12, France.
authors.forename: Crystel
authors.initials: C
authors.lastname: Bonnet
authors.forename: Valérie
authors.initials: V
authors.lastname: Kaltimbacher
authors.forename: Sami
authors.initials: S
authors.lastname: Ellouze
authors.forename: Sébastien
authors.initials: S
authors.lastname: Augustin
authors.forename: Paule
authors.initials: P
authors.lastname: Bénit
authors.forename: Valérie
authors.initials: V
authors.lastname: Forster
authors.forename: Pierre
authors.initials: P
authors.lastname: Rustin
authors.forename: José-Alain
authors.initials: JA
authors.lastname: Sahel
authors.forename: Marisol
authors.initials: M
authors.lastname: Corral-Debrinski
mesh_terms: D000251:Adenosine Triphosphatases / Q000235:genetics*; D002352:Carrier Proteins / Q000235:genetics*; D002455:Cell Division / Q000187:drug effects / Q000502:physiology; D002478:Cells, Cultured; D003470:Culture Media / Q000494:pharmacology; D003600:Cytosol / Q000378:metabolism; D004272:DNA, Mitochondrial / Q000235:genetics; D004579:Electron Transport / Q000235:genetics; D042967:Electron Transport Complex I / Q000235:genetics*; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D005690:Galactose / Q000494:pharmacology; D015316:Genetic Therapy / Q000379:methods*; D006801:Humans; D007223:Infant; D007888:Leigh Disease / Q000235:genetics / Q000378:metabolism / Q000628:therapy*; D008565:Membrane Proteins / Q000235:genetics*; D008928:Mitochondria / Q000235:genetics*; D025261:Mitochondrial Proton-Translocating ATPases / Q000235:genetics; D009154:Mutation; D009245:NADH Dehydrogenase / Q000235:genetics; D010085:Oxidative Phosphorylation; D010957:Plasmids / Q000493:pharmacokinetics; D012333:RNA, Messenger / Q000493:pharmacokinetics*; D014162:Transfection / Q000379:methods
chemical_list: D002352:Carrier Proteins; D003470:Culture Media; D004272:DNA, Mitochondrial; C501005:MT-ATP6 protein, human; D008565:Membrane Proteins; C098483:NADH dehydrogenase subunit 4; D012333:RNA, Messenger; D009245:NADH Dehydrogenase; D000251:Adenosine Triphosphatases; D025261:Mitochondrial Proton-Translocating ATPases; D042967:Electron Transport Complex I; C020439:oligomycin sensitivity-conferring protein; D005690:Galactose