• Boominathan A, Vanhoozer S, Basisty N, Powers K, Crampton AL, Wang X, Friedricks N, Schilling B, Brand MD, O'Connor MS. Stable nuclear expression of ATP8 and ATP6 genes rescues a mtDNA Complex V null mutant. Nucleic Acids Res. 2016 Sep 4. PubMed: 27596602. Categories: MitoSENS

    Stable nuclear expression of ATP8 and ATP6 genes rescues a mtDNA Complex V null mutant.

    Nucleic Acids Res. 2016 Sep 4.

    Stable nuclear expression of ATP8 and ATP6 genes rescues a mtDNA Complex V null mutant.

    Boominathan A, Vanhoozer S, Basisty N, Powers K, Crampton AL, Wang X, Friedricks N, Schilling B, Brand MD, O'Connor MS.

    Abstract

    Abstract:

    We explore the possibility of re-engineering mitochondrial genes and expressing them from the nucleus as an approach to rescue defects arising from mitochondrial DNA mutations. We have used a patient cybrid cell line with a single point mutation in the overlap region of the ATP8 and ATP6 genes of the human mitochondrial genome. These cells are null for the ATP8 protein, have significantly lowered ATP6 protein levels and no Complex V function. Nuclear expression of only the ATP8 gene with the ATP5G1 mitochondrial targeting sequence appended restored viability on Krebs cycle substrates and ATP synthesis capabilities but, failed to restore ATP hydrolysis and was insensitive to various inhibitors of oxidative phosphorylation. Co-expressing both ATP8 and ATP6 genes under similar conditions resulted in stable protein expression leading to successful integration into Complex V of the oxidative phosphorylation machinery. Tests for ATP hydrolysis / synthesis, oxygen consumption, glycolytic metabolism and viability all indicate a significant functional rescue of the mutant phenotype (including re-assembly of Complex V) following stable co-expression of ATP8 and ATP6. Thus, we report the stable allotopic expression, import and function of two mitochondria encoded genes, ATP8 and ATP6, resulting in simultaneous rescue of the loss of both mitochondrial proteins.

  • Wiley CD, Velarde MC, Lecot P, Liu S, Sarnoski EA, Freund A, Shirakawa K, Lim HW, Davis SS, Ramanathan A, Gerencser AA, Verdin E, Campisi J. Mitochondrial Dysfunction Induces Senescence with a Distinct Secretory Phenotype. Cell Metab. 23(2):303-14. PubMed: 26686024. Categories: ApoptoSENS, MitoSENS

    Mitochondrial Dysfunction Induces Senescence with a Distinct Secretory Phenotype.

    Cell Metab. 23(2):303-14.

    Mitochondrial Dysfunction Induces Senescence with a Distinct Secretory Phenotype.

    Wiley CD, Velarde MC, Lecot P, Liu S, Sarnoski EA, Freund A, Shirakawa K, Lim HW, Davis SS, Ramanathan A, Gerencser AA, Verdin E, Campisi J.

    Abstract

    Abstract:

    Cellular senescence permanently arrests cell proliferation, often accompanied by a multi-faceted senescence-associated secretory phenotype (SASP). Loss of mitochondrial function can drive age-related declines in the function of many post-mitotic tissues, but little is known about how mitochondrial dysfunction affects mitotic tissues. We show here that several manipulations that compromise mitochondrial function in proliferating human cells induce a senescence growth arrest with a modified SASP that lacks the IL-1-dependent inflammatory arm. Cells that underwent mitochondrial dysfunction-associated senescence (MiDAS) had lower NAD+/NADH ratios, which caused both the growth arrest and prevented the IL-1-associated SASP through AMPK-mediated p53 activation. Progeroid mice that rapidly accrue mtDNA mutations accumulated senescent cells with a MiDAS SASP in vivo, which suppressed adipogenesis and stimulated keratinocyte differentiation in cell culture. Our data identify a distinct senescence response and provide a mechanism by which mitochondrial dysfunction can drive aging phenotypes.

  • Ellouze S, Augustin S, Bouaita A, Bonnet C, Simonutti M, Forster V, Picaud S, Sahel JA, Corral-Debrinski M. Optimized allotopic expression of the human mitochondrial ND4 prevents blindness in a rat model of mitochondrial dysfunction. Am J Hum Genet. 2008 Sep;83(3):373-87. doi: 10.1016/j.ajhg.2008.08.013. PubMed: 18771762. Categories: MitoSENS

    Optimized allotopic expression of the human mitochondrial ND4 prevents blindness in a rat model of mitochondrial dysfunction.

    Am J Hum Genet. 2008 Sep;83(3):373-87. doi: 10.1016/j.ajhg.2008.08.013.

    Optimized allotopic expression of the human mitochondrial ND4 prevents blindness in a rat model of mitochondrial dysfunction.

    Ellouze S, Augustin S, Bouaita A, Bonnet C, Simonutti M, Forster V, Picaud S, Sahel JA, Corral-Debrinski M.

    Abstract

    Abstract:

    Mitochondrial diseases due to mutations in mitochondrial DNA can no longer be ignored in most medical areas. With prevalence certainly higher than one in 6000, they probably represent the most common form of metabolic disorders. Despite progress in identification of their molecular mechanisms, little has been done with regard to therapy. We have recently optimized the allotopic expression for the mitochondrial genes ATP6, ND1, and ND4 and obtained a complete and long-lasting rescue of mitochondrial dysfunction in the human fibroblasts in which these genes were mutated. However, biosafety and benefit to mitochondrial function must be validated in animal models prior to clinical applications. To create an animal model of Leber Hereditary Optic Neuropathy (LHON), we introduced the human ND4 gene harboring the G11778A mutation, responsible of 60% of LHON cases, to rat eyes by in vivo electroporation. The treatment induced the degeneration of retinal ganglion cells (RGCs), which were 40% less abundant in treated eyes than in control eyes. This deleterious effect was also confirmed in primary cell culture, in which both RGC survival and neurite outgrowth were compromised. Importantly, RGC loss was clearly associated with a decline in visual performance. A subsequent electroporation with wild-type ND4 prevented both RGC loss and the impairment of visual function. Hence, these data provide the proof-of-principle that optimized allotopic expression can be an effective treatment for LHON, and they open the way to clinical studies on other devastating mitochondrial disorders.

  • Bonnet C, Kaltimbacher V, Ellouze S, Augustin S, Bénit P, Forster V, Rustin P, Sahel JA, Corral-Debrinski M. Allotopic mRNA localization to the mitochondrial surface rescues respiratory chain defects in fibroblasts harboring mitochondrial DNA mutations affecting complex I or V subunits. Rejuvenation Res 2007;10(2):127-144. PubMed: 17518546. Categories: MitoSENS

    Allotopic mRNA localization to the mitochondrial surface rescues respiratory chain defects in fibroblasts harboring mitochondrial DNA mutations affecting complex I or V subunits.

    Rejuvenation Res 2007;10(2):127-144.

    Allotopic mRNA localization to the mitochondrial surface rescues respiratory chain defects in fibroblasts harboring mitochondrial DNA mutations affecting complex I or V subunits.

    Bonnet C, Kaltimbacher V, Ellouze S, Augustin S, Bénit P, Forster V, Rustin P, Sahel JA, Corral-Debrinski M.

    Abstract

    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.

  • de Grey ADNJ. Forces maintaining organellar genomes: is any as strong as genetic code disparity or hydrophobicity? BioEssays 2005;27(4):436-446. PubMed: 15770678. Categories: MitoSENS

    Forces maintaining organellar genomes: is any as strong as genetic code disparity or hydrophobicity?

    BioEssays 2005;27(4):436-446.

    Forces maintaining organellar genomes: is any as strong as genetic code disparity or hydrophobicity?

    de Grey ADNJ.

    Abstract

    Abstract:

    It remains controversial why mitochondria and chloroplasts retain the genes encoding a small subset of their constituent proteins, despite the transfer of so many other genes to the nucleus. Two candidate obstacles to gene transfer, suggested long ago, are that the genetic code of some mitochondrial genomes differs from the standard nuclear code, such that a transferred gene would encode an incorrect amino acid sequence, and that the proteins most frequently encoded in mitochondria are generally very hydrophobic, which may impede their import after synthesis in the cytosol. More recently it has been suggested that both these interpretations suffer from serious "false positives" and "false negatives": genes that they predict should be readily transferred but which have never (or seldom) been, and genes whose transfer has occurred often or early, even though this is predicted to be very difficult. Here I consider the full known range of ostensibly problematic such genes, with particular reference to the sequences of events that could have led to their present location. I show that this detailed analysis of these cases reveals that they are in fact wholly consistent with the hypothesis that code disparity and hydrophobicity are much more powerful barriers to functional gene transfer than any other. The popularity of the contrary view has led to the search for other barriers that might retain genes in organelles even more powerfully than code disparity or hydrophobicity; one proposal, concerning the role of proteins in redox processes, has received widespread support. I conclude that this abandonment of the original explanations for the retention of organellar genomes has been premature. Several other, relatively minor, obstacles to gene transfer certainly exist, contributing to the retention of relatively many organellar genes in most lineages compared to animal mtDNA, but there is no evidence for obstacles as severe as code disparity or hydrophobicity. One corollary of this conclusion is that there is currently no reason to suppose that engineering nuclear versions of the remaining mammalian mitochondrial genes, a feat that may have widespread biomedical relevance, should require anything other than sequence alterations obviating code disparity and causing modest reductions in hydrophobicity without loss of enzymatic function.

  • de Grey ADNJ. Mitochondrial gene therapy: an arena for the biomedical use of inteins. Trends Biotechnol 2000;18(9):394-399. PubMed: 10942964. Categories: MitoSENS

    Mitochondrial gene therapy: an arena for the biomedical use of inteins.

    Trends Biotechnol 2000;18(9):394-399.

    Mitochondrial gene therapy: an arena for the biomedical use of inteins.

    de Grey ADNJ.

    Abstract

    Abstract:

    Mitochondrial DNA (mtDNA) mutations underlie many rare diseases and might also contribute to human ageing. Gene therapy is a tempting future possibility for intervening in mitochondriopathies. Expression of the 13 mtDNA-encoded proteins from nuclear transgenes (allotopic expression) might be the most effective gene-therapy strategy. Its only confirmed difficulty is the extreme hydrophobicity of these proteins, which prevents their import into mitochondria from the cytosol. Inteins (self-splicing 'protein introns') might offer a solution to this problem: their insertion into such transgenes could greatly reduce the encoded proteins' hydrophobicity, enabling import, with post-import excision restoring the natural amino acid sequence.