Successful Exogenous Expression of ATP8, a Mitochondrial Encoded Protein, From the Nucleus In Vivo

Replicative errors, inefficient repair, and proximity to reactive oxygen species production sites make the mitochondrial DNA (mtDNA) susceptible to damage with time. mtDNA mutations accumulate with age and accompany a progressive decline in organelle function. We lack molecular biology tools to manipulate mtDNA, thus we explore the possibility in vivo of utilizing allotopic expression, or the re-engineering mitochondrial genes and expressing them from the nucleus, as an approach to rescue defects arising from mtDNA mutations. This study uses a mouse model with a mutation in the mitochondrial ATP8 gene that encodes a protein subunit of the ATP synthase. We generated a transgenic mouse with an epitope-tagged recoded and mitochondrial-targeted ATP8 gene expressed from the nucleus. Our results show that the allotopically expressed ATP8 protein in the transgenic mice is robustly expressed across all tested tissues, successfully transported into the mitochondria, and incorporated into ATP synthase. We are currently evaluating if allotopic expression of ATP8 will functionally rescue the behavioral and bioenergetic defects in ATP8 mutant mice. Translating allotopic expression technology into a mammal and demonstrating systemic functional rescue will lend credence to utilizing allotopic expression as a gene therapy in humans to repair physiological consequences of mtDNA defects that may accumulate with age.

Treatment for mitochondrial diseases

Mitochondrial diseases are predominantly caused by mutations of mitochondrial or nuclear DNA, resulting in multisystem defects. Current treatments are largely supportive, and the disorders progress relentlessly. Nutritional supplements, pharmacological agents and physical therapies have been used in different clinical trials, but the efficacy of these interventions need to be further evaluated. Several recent reviews discussed some of the interventions but ignored bias in those trials. This review was conducted to discover new studies and grade the original studies for potential bias with revised Cochrane Collaboration guidelines. We focused on seven published studies and three unpublished studies; eight of these studies showed improvement in outcome measurements. In particular, two of the interventions have been tested in studies with strict design, which we believe deserve further clinical trials with a large sample. Additionally, allotopic expression of the ND4 subunit seemed to be an effective new treatment for patients with Leber hereditary optic neuropathy.

Key within-membrane residues and precursor dosage impact the allotopic expression of yeast subunit II of cytochrome c oxidase

Experimentally relocating mitochondrial genes to the nucleus for functional expression (allotopic expression) is a challenging process. The high hydrophobicity of mitochondria-encoded proteins seems to be one of the main factors preventing this allotopic expression. We focused on subunit II of cytochrome c oxidase (Cox2) to study which modifications may enable or improve its allotopic expression in yeast. Cox2 can be imported from the cytosol into mitochondria in the presence of the W56R substitution, which decreases the protein hydrophobicity and allows partial respiratory rescue of a cox2-null strain. We show that the inclusion of a positive charge is more favorable than substitutions that only decrease the hydrophobicity. We also searched for other determinants enabling allotopic expression in yeast by examining the COX2 gene in organisms where it was transferred to the nucleus during evolution. We found that naturally occurring variations at within-membrane residues in the legume Glycine max Cox2 could enable yeast COX2 allotopic expression. We also evidence that directing high doses of allotopically synthesized Cox2 to mitochondria seems to be counterproductive because the subunit aggregates at the mitochondrial surface. Our findings are relevant to the design of allotopic expression strategies and contribute to the understanding of gene retention in organellar genomes.

Mitochondrial versus nuclear gene expression and membrane protein assembly: the case of subunit 2 of yeast cytochrome c oxidase

Deletion of the yeast mitochondrial gene COX2, encoding subunit 2 (mtCox2) of cytochrome c oxidase (CcO), results in a respiratory-incompetent Δcox2 strain. For a cytosol-synthesized Cox2 to restore respiratory growth, it must carry the W56R mutation (cCox2W56R). Nevertheless, only a fraction of cCox2W56R is matured in mitochondria, allowing ∼60% steady-state accumulation of CcO. This can be attributed either to the point mutation or to an inefficient biogenesis of cCox2W56R. We generated a strain expressing the mutant protein mtCox2W56R inside mitochondria which should follow the canonical biogenesis of mitochondria-encoded Cox2. This strain exhibited growth rates, CcO steady-state levels, and CcO activity similar to those of the wild type; therefore, the efficiency of Cox2 biogenesis is the limiting step for successful allotopic expression. Upon coexpression of cCox2W56R and mtCox2, each protein assembled into CcO independently from its genetic origin, resulting in a mixed population of CcO with most complexes containing the mtCox2 version. Notably, the presence of the mtCox2 enhances cCox2W56R incorporation. We provide proof of principle that an allotopically expressed Cox2 may complement a phenotype due to a mutant mitochondrial COX2 gene. These results are relevant to developing a rational design of genes for allotopic expression intended to treat human mitochondrial diseases.