• 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.

  • Haendeler J, Dröse S, Büchner N, Jakob S, Altschmied J, Goy C, Spyridopoulos I, Zeiher AM, Brandt U, Dimmeler S. Mitochondrial telomerase reverse transcriptase binds to and protects mitochondrial DNA and function from damage. Arterioscler Thromb Vasc Biol. 2009 Jun;29(6):929-35. PubMed: 19265030. Categories: MitoSENS, OncoSENS

    Mitochondrial telomerase reverse transcriptase binds to and protects mitochondrial DNA and function from damage.

    Arterioscler Thromb Vasc Biol. 2009 Jun;29(6):929-35.

    Mitochondrial telomerase reverse transcriptase binds to and protects mitochondrial DNA and function from damage.

    Haendeler J, Dröse S, Büchner N, Jakob S, Altschmied J, Goy C, Spyridopoulos I, Zeiher AM, Brandt U, Dimmeler S.

    Abstract

    Abstract:

    OBJECTIVE: The enzyme telomerase and its catalytic subunit the telomerase reverse transcriptase (TERT) are important for maintenance of telomere length in the nucleus. Recent studies provided evidence for a mitochondrial localization of TERT. Therefore, we investigated the exact localization of TERT within the mitochondria and its function. METHODS AND RESULTS: Here, we demonstrate that TERT is localized in the matrix of the mitochondria. TERT binds to mitochondrial DNA at the coding regions for ND1 and ND2. Binding of TERT to mitochondrial DNA protects against ethidium bromide-induced damage. TERT increases overall respiratory chain activity, which is most pronounced at complex I and dependent on the reverse transcriptase activity of the enzyme. Moreover, mitochondrial reactive oxygen species are increased after genetic ablation of TERT by shRNA. Mitochondrially targeted TERT and not wild-type TERT revealed the most prominent protective effect on H(2)O(2)-induced apoptosis. Lung fibroblasts from 6-month-old TERT(-/-) mice (F2 generation) showed increased sensitivity toward UVB radiation and heart mitochondria exhibited significantly reduced respiratory chain activity already under basal conditions, demonstrating the protective function of TERT in vivo. CONCLUSIONS: Mitochondrial TERT exerts a novel protective function by binding to mitochondrial DNA, increasing respiratory chain activity and protecting against oxidative stress-induced damage.

  • 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.

  • Dufour E, Terzioglu M, Hansson FS, Sörensen L, Galter D, Olson L, Wilbertz J, Larsson NG. Age-associated mosaic respiratory chain deficiency causes trans-neuronal degeneration. Hum Mol Genet 2008;17(10):1418-26. PubMed: 18245781. Categories: MitoSENS

    Age-associated mosaic respiratory chain deficiency causes trans-neuronal degeneration.

    Hum Mol Genet 2008;17(10):1418-26.

    Age-associated mosaic respiratory chain deficiency causes trans-neuronal degeneration.

    Dufour E, Terzioglu M, Hansson FS, Sörensen L, Galter D, Olson L, Wilbertz J, Larsson NG.

    Abstract

    Abstract:

    Heteroplasmic mitochondrial DNA (mtDNA) mutations (mutations present only in a subset of cellular mtDNA copies) arise de novo during the normal ageing process or may be maternally inherited in pedigrees with mitochondrial disease syndromes. A pathogenic mtDNA mutation causes respiratory chain deficiency only if the fraction of mutated mtDNA exceeds a certain threshold level. These mutations often undergo apparently random mitotic segregation and the levels of normal and mutated mtDNA can vary considerably between cells of the same tissue. In human ageing, segregation of somatic mtDNA mutations leads to mosaic respiratory chain deficiency in a variety of tissues, such as brain, heart and skeletal muscle. A similar pattern of mutation segregation with mosaic respiratory chain deficiency is seen in patients with mitochondrial disease syndromes caused by inherited pathogenic mtDNA mutations. We have experimentally addressed the role of mosaic respiratory chain deficiency in ageing and mitochondrial disease by creating mouse chimeras with a mixture of normal and respiratory chain-deficient neurons in cerebral cortex. We report here that a low proportion (>20%) of respiratory chain-deficient neurons in the forebrain are sufficient to cause symptoms, whereas premature death of the animal occurs only if the proportion is high (>60-80%). The presence of neurons with normal respiratory chain function does not only prevent mortality but also delays the age at which onset of disease symptoms occur. Unexpectedly, respiratory chain-deficient neurons have adverse effect on normal adjacent neurons and induce trans-neuronal degeneration. In summary, our study defines the minimal threshold level of respiratory chain-deficient neurons needed to cause symptoms and also demonstrate that neurons with normal respiratory chain function ameliorate disease progression. Finally, we show that respiratory chain-deficient neurons induce death of normal neurons by a trans-neuronal degeneration mechanism. These findings provide novel insights into the pathogenesis of mosaic respiratory chain deficiency in ageing and mitochondrial disease.

  • Vermulst M, Wanagat J, Kujoth GC, Bielas JH, Rabinovitch PS, Prolla TA, Loeb LA. DNA deletions and clonal mutations drive premature aging in mitochondrial mutator mice. Nat Genet 2008;40(4):392-394. PubMed: 18311139. Categories: MitoSENS

    DNA deletions and clonal mutations drive premature aging in mitochondrial mutator mice.

    Nat Genet 2008;40(4):392-394.

    DNA deletions and clonal mutations drive premature aging in mitochondrial mutator mice.

    Vermulst M, Wanagat J, Kujoth GC, Bielas JH, Rabinovitch PS, Prolla TA, Loeb LA.

    Abstract

    Abstract:

    Mitochondrial DNA (mtDNA) mutations are thought to have a causal role in many age-related pathologies. Here we identify mtDNA deletions as a driving force behind the premature aging phenotype of mitochondrial mutator mice, and provide evidence for a homology-directed DNA repair mechanism in mitochondria that is directly linked to the formation of mtDNA deletions. In addition, our results demonstrate that the rate at which mtDNA mutations reach phenotypic expression differs markedly among tissues, which may be an important factor in determining the tolerance of a tissue to random mitochondrial mutagenesis.

  • 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.

  • Ellouze S, Bonnet C, Augustin S, Kaltimbacher V, Forster V, Simonutti M, Sahel JA, Corral-Debrinski M. Allotopic mRNA localization to the mitochondrial surface: a tool for rescuing respiration deficiencies. Rejuvenation Res 2007;10(Suppl 1):S24. Categories: MitoSENS

    Allotopic mRNA localization to the mitochondrial surface: a tool for rescuing respiration deficiencies.

    Rejuvenation Res 2007;10(Suppl 1):S24.

    Allotopic mRNA localization to the mitochondrial surface: a tool for rescuing respiration deficiencies.

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

    Abstract

    Abstract:

    No abstract available.

  • Qi X, Sun L, Lewin AS, Hauswirth WW, Guy J. The mutant human ND4 subunit of complex I induces optic neuropathy in the mouse. Invest Ophthalmol Vis Sci. 2007 Jan;48(1):1-10. PubMed: 17197509. Categories: MitoSENS

    The mutant human ND4 subunit of complex I induces optic neuropathy in the mouse.

    Invest Ophthalmol Vis Sci. 2007 Jan;48(1):1-10.

    The mutant human ND4 subunit of complex I induces optic neuropathy in the mouse.

    Qi X, Sun L, Lewin AS, Hauswirth WW, Guy J.

    Abstract

    Abstract:

    PURPOSE: To produce a mouse model of Leber hereditary optic neuropathy. METHODS: A mutant ND4 subunit made compatible with the universal genetic code and containing an arginine-to-histidine substitution at residue 340, or a synthetic normal human ND4 gene was delivered to the mouse visual system. The expression and effects of the mutant ND4 gene on the optic nerve and cultured retinal ganglion cells was assessed by magnetic resonance imaging, immunohistochemistry, and light and transmission electron microscopy. RESULTS: The ATPc mitochondrial targeting sequence directed the allotopically expressed mutant human R340H and wild-type ND4FLAG polypeptides into mitochondria. Expression of normal human ND4 in murine mitochondria posed no ocular toxicity. In contrast, the mutant ND4 disrupted mitochondrial cytoarchitecture, elevated reactive oxygen species, induced swelling of the optic nerve head, and induced apoptosis, with a progressive demise of ganglion cells in the retina and their axons comprising the optic nerve. CONCLUSIONS: Allotopic expression of the mutant human R340H ND4 subunit of complex I replicated the hallmarks of human mitochondrial disease in the mouse. In contrast, ocular expression of the wild-type human ND4 subunit in lower mammals appears safe, suggesting that it may be useful for treatment of patients with Leber hereditary optic neuropathy.

  • Mukherjee S, Mahata B, Mahato B, Adhya S. Use of a parasite-derived protein complex to modulate the function of mitochondria in human cells. Rejuvenation Res 2007;10(Suppl 1):S19. Categories: MitoSENS

    Use of a parasite-derived protein complex to modulate the function of mitochondria in human cells.

    Rejuvenation Res 2007;10(Suppl 1):S19.

    Use of a parasite-derived protein complex to modulate the function of mitochondria in human cells.

    Mukherjee S, Mahata B, Mahato B, Adhya S.

    Abstract

    Abstract:

    No abstract available.

  • Katrangi E, D'Souza G, Boddapati SV, Kulawiec M, Singh KK, Bigger B, Weissig V. Xenogenic transfer of isolated murine mitochondria into human rho0 cells can improve respiratory function. Rejuvenation Res 2007;10(4):561-570. PubMed: 18069915. Categories: MitoSENS

    Xenogenic transfer of isolated murine mitochondria into human rho0 cells can improve respiratory function.

    Rejuvenation Res 2007;10(4):561-570.

    Xenogenic transfer of isolated murine mitochondria into human rho0 cells can improve respiratory function.

    Katrangi E, D'Souza G, Boddapati SV, Kulawiec M, Singh KK, Bigger B, Weissig V.

    Abstract

    Abstract:

    Mitochondrial DNA mutations are the direct cause of several physiological disorders and are also associated with the aging process. The modest progress made over the past two decades towards manipulating the mitochondrial genome and understanding its function within living mammalian cells means that cures for mitochondrial DNA mutations are still elusive. Here, we report that transformed mammalian cells internalize exogenous isolated mitochondria upon simple co-incubation. We first demonstrate the physical presence of internalized mitochondria within recipient cells using fluorescence microscopy. Second, we show that xenogenic transfer of murine mitochondria into human cells lacking functional mitochondria can functionally restore respiration in cells lacking mtDNA. Third, utilizing the natural competence of isolated mitochondria to take up linear DNA molecules, we demonstrate the feasibility of using cellular internalization of isolated exogenous mitochondria as a potential tool for studying mitochondrial genetics in living mammalian cells.

  • de Grey ADNJ. Free radicals in aging: causal complexity and its biomedical implications. Free Radic Res. 2006 Dec;40(12):1244-9. PubMed: 17090413. Categories: MitoSENS

    Free radicals in aging: causal complexity and its biomedical implications.

    Free Radic Res. 2006 Dec;40(12):1244-9.

    Free radicals in aging: causal complexity and its biomedical implications.

    de Grey ADNJ.

    Abstract

    Abstract:

    Superoxide generated adventitiously by the mitochondrial respiratory chain can give rise to much more reactive radicals, resulting in random oxidation of all classes of macromolecules. Harman's 1956 suggestion that this process might drive aging has been a leading strand of biogerontological thinking since the discovery of superoxide dismutase. However, it has become apparent that the many downstream consequences of free radical damage can also be caused by processes not involving oxidation. Moreover, free radicals have been put to use by evolution to such an extent that their wholesale elimination would certainly be fatal. This multiplicity of parallel pathways and side-effects illustrates why attempts to postpone aging by "cleaning up" metabolism will surely fail for the foreseeable future: we simply understand metabolism too poorly. This has led me to pursue the alternative, "repair and maintenance" approach that sidesteps our ignorance of metabolism and may be feasible relatively soon.

  • Corral-Debrinski M. mRNA specific subcellular localization represents a crucial step for fine-tuning of gene expression in mammalian cells. Biochim Biophys Acta. 2007 Apr;1773(4):473-5. Epub 2006 Jul 1. PubMed: 17292980. Categories: MitoSENS

    mRNA specific subcellular localization represents a crucial step for fine-tuning of gene expression in mammalian cells.

    Biochim Biophys Acta. 2007 Apr;1773(4):473-5. Epub 2006 Jul 1.

    mRNA specific subcellular localization represents a crucial step for fine-tuning of gene expression in mammalian cells.

    Corral-Debrinski M.

    Abstract

    Abstract:

    mRNA subcellular distribution and translational control are key player mechanisms for post-transcriptional gene expression regulation. In the last decade it has become increasingly clear that these processes are associated with various human diseases. Understanding the interconnected multistep process of mRNA localization and its involvement in organelle biogenesis and in the overall spatial structure of eukaryotic cells will be an important step towards the long-term goal of curing individual molecular defects. In a recent issue, Russo et al. [The 3'-untranslated region directs ribosomal protein-encoding mRNAs to specific cytoplasmic regions, Biochim. Biophys. Acta, Mol. Cell Res. 1763 (8) (2006) 833-843] reported interesting findings on the mechanisms that direct mRNAs encoding different ribosomal proteins to specific cytoplasmic regions in human cells.

  • Schriner SE, Linford NJ. Extension of mouse lifespan by overexpression of catalase. AGE 2006;28(2):209-218. PubMed: 19943142. Categories: MitoSENS

    Extension of mouse lifespan by overexpression of catalase.

    AGE 2006;28(2):209-218.

    Extension of mouse lifespan by overexpression of catalase.

    Schriner SE, Linford NJ.

    Abstract

    Abstract:

    The free radical theory of aging was originally proposed 50 years ago, and is arguably the most popular mechanism explaining the aging process. According to this theory, aging results from the progressive decline in organ function due to the damage generated by reactive oxygen species (ROS). These chemical species are a normal part of metabolism, and a group of enzymes exists to protect cells against their toxic effects. One of these species is hydrogen peroxide (H(2)O(2)), which can be degraded by catalase. To determine the role of hydrogen peroxide in aging and its importance in different subcellular compartments, transgenic mice were developed with increased catalase activities localized to the peroxisome (PCAT), nucleus (NCAT), or mitochondrion (MCAT). The largest effect on lifespan was found in MCAT animals, with a 20% increase in median lifespan and a 10% increase in the maximum lifespan. A more modest effect was seen in PCAT animals, and no significant change was found in NCAT animals. Upon further examination of the MCAT mice, it was found that H(2)O(2) production and H(2)O(2)-induced aconitase inactivation were attenuated, oxidative damage and the development of mitochondrial deletions were reduced, and cardiac pathology and cataract development were delayed. These results are consistent with a role of H(2)O(2) in the development of pathology and in the limitation of mouse lifespan. They also demonstrate the importance of mitochondria as a source, and possible target, of ROS.

  • Kaltimbacher V, Bonnet C, Lecoeuvre G, Forster V, Sahel JA, Corral-Debrinski M. mRNA localization to the mitochondrial surface allows the efficient translocation inside the organelle of a nuclear recoded ATP6 protein. RNA 2006;12(7):1408-1417. PubMed: 16751614. Categories: MitoSENS

    mRNA localization to the mitochondrial surface allows the efficient translocation inside the organelle of a nuclear recoded ATP6 protein.

    RNA 2006;12(7):1408-1417.

    mRNA localization to the mitochondrial surface allows the efficient translocation inside the organelle of a nuclear recoded ATP6 protein.

    Kaltimbacher V, Bonnet C, Lecoeuvre G, Forster V, Sahel JA, Corral-Debrinski M.

    Abstract

    Abstract:

    As previously established in yeast, two sequences within mRNAs are responsible for their specific localization to the mitochondrial surface-the region coding for the mitochondrial targeting sequence and the 3'UTR. This phenomenon is conserved in human cells. Therefore, we decided to use mRNA localization as a tool to address to mitochondria, a protein that is not normally imported. For this purpose, we associated a nuclear recoded ATP6 gene with the mitochondrial targeting sequence and the 3'UTR of the nuclear SOD2 gene, which mRNA exclusively localizes to the mitochondrial surface in HeLa cells. The ATP6 gene is naturally located into the organelle and encodes a highly hydrophobic protein of the respiratory chain complex V. In this study, we demonstrated that hybrid ATP6 mRNAs, as the endogenous SOD2 mRNA, localize to the mitochondrial surface in human cells. Remarkably, fusion proteins localize to mitochondria in vivo. Indeed, ATP6 precursors synthesized in the cytoplasm were imported into mitochondria in a highly efficient way, especially when both the MTS and the 3'UTR of the SOD2 gene were associated with the re-engineered ATP6 gene. Hence, these data indicate that mRNA targeting to the mitochondrial surface represents an attractive strategy for allowing the mitochondrial import of proteins originally encoded by the mitochondrial genome without any amino acid change in the protein that could interfere with its biologic activity.

  • Mahata B, Mukherjee S, Mishra S, Bandyopadhyay A, Adhya S. Functional delivery of a cytosolic tRNA into mutant mitochondria of human cells. Science 2006;314(5798):471-474. PubMed: 17053148. Categories: MitoSENS

    Functional delivery of a cytosolic tRNA into mutant mitochondria of human cells.

    Science 2006;314(5798):471-474.

    Functional delivery of a cytosolic tRNA into mutant mitochondria of human cells.

    Mahata B, Mukherjee S, Mishra S, Bandyopadhyay A, Adhya S.

    Abstract

    Abstract:

    Many maternally inherited and incurable neuromyopathies are caused by mutations in mitochondrial (mt) transfer RNA (tRNA) genes. Kinetoplastid protozoa, including Leishmania, have evolved specialized systems for importing nucleus-encoded tRNAs into mitochondria. We found that the Leishmania RNA import complex (RIC) could enter human cells by a caveolin-1-dependent pathway, where it induced import of endogenous cytosolic tRNAs, including tRNA(Lys), and restored mitochondrial function in a cybrid harboring a mutant mt tRNA(Lys) (MT-TK) gene. The use of protein complexes to modulate mitochondrial function may help in the management of such genetic disorders.

  • Spees JL, Olson SD, Whitney MJ, Prockop DJ. Mitochondrial transfer between cells can rescue aerobic respiration. Proc Natl Acad Sci USA 2006;103(5):1283-1288. PubMed: 16432190. Categories: MitoSENS

    Mitochondrial transfer between cells can rescue aerobic respiration.

    Proc Natl Acad Sci USA 2006;103(5):1283-1288.

    Mitochondrial transfer between cells can rescue aerobic respiration.

    Spees JL, Olson SD, Whitney MJ, Prockop DJ.

    Abstract

    Abstract:

    Current theory indicates that mitochondria were obtained 1.5 billion years ago from an ancient prokaryote. The mitochondria provided the capacity for aerobic respiration, the creation of the eukaryotic cell, and eventually complex multicellular organisms. Recent reports have found that mitochondria play essential roles in aging and determining lifespan. A variety of heritable and acquired diseases are linked to mitochondrial dysfunction. We report here that mitochondria are more dynamic than previously considered: mitochondria or mtDNA can move between cells. The active transfer from adult stem cells and somatic cells can rescue aerobic respiration in mammalian cells with nonfunctional mitochondria.

  • Zullo SJ, Parks WT, Chloupkova M, Wei B, Weiner H, Fenton WA, Eisenstadt JM, Merril CR. Stable transformation of CHO cells and human NARP cybrids confers oligomycin resistance (olir) following transfer of a mitochondrial DNA-encoded olir ATPase6 gene to the nuclear genome: a model system for mtDNA gene therapy. Rejuvenation Res 2005;8(1):18-28. PubMed: 15798371. Categories: MitoSENS

    Stable transformation of CHO cells and human NARP cybrids confers oligomycin resistance (olir) following transfer of a mitochondrial DNA-encoded olir ATPase6 gene to the nuclear genome: a model system for mtDNA gene therapy.

    Rejuvenation Res 2005;8(1):18-28.

    Stable transformation of CHO cells and human NARP cybrids confers oligomycin resistance (olir) following transfer of a mitochondrial DNA-encoded olir ATPase6 gene to the nuclear genome: a model system for mtDNA gene therapy.

    Zullo SJ, Parks WT, Chloupkova M, Wei B, Weiner H, Fenton WA, Eisenstadt JM, Merril CR.

    Abstract

    Abstract:

    Point and deletion mutations and a general depletion of mammalian mitochondrial DNA (mtDNA) give rise to a wide variety of medical syndromes that are refractory to treatment, possibly including aging itself. While gene therapy directed at correcting such deficits in the mitochondrial genome may offer some therapeutic benefits, there are inherent problems associated with a direct approach. These problems are primarily due to the high mitochondrial genome copy number in each cell and the mitochondrial genome being "protected" inside the double-membrane mitochondrial organelle. In an alternative approach there is evidence that genes normally present in the mitochondrial genome can be incorporated into the nuclear genome. To extend such studies, we modified the Chinese Hamster Ovary (CHO) mtDNA-located ATPase6 gene (possessing a mutation which confers oligomycin resistance- oli(r)) by altering the mtDNA code to the universal code (U-code) to permit the correct translation of its mRNA in the cytoplasm. The U-code construct was inserted into the nuclear genome (nucDNA) of a wild type CHO cell. The expressed transgene products enabled the transformed CHO cell lines to grow in up to 1000 ng mL(-1) oligomycin, while untransformed sensitive CHO cells were eliminated in 1 ng mL(-1) oligomycin. This approach, termed allotopic expression, provides a model that may make possible the transfer of all 13 mtDNA mammalian protein-encoding genes to the nucDNA, for treatments of mtDNA disorders. The CHO mtATPase6 protein is 85% identical to both the mouse and human mtATPase6 protein; these proteins are highly conserved in the region of the oligomycin resistance mutation. They are also well conserved in the regions of the oligomycin resistance mutation of the mouse, and in the region of a mutation found in Leigh's syndrome (T8993G), also called NARP (neurogenic weakness, ataxia, retinitis pigmentosum). It is likely that the CHO oli(r) mtATPase6 Ucode construct could impart oligomycin-resistance in human and mouse cells, as well as function in place of the mutant ATPase subunit in a NARP cell line. Preliminary experiments on human cybrids homoplasmic for the NARP mutation (kindly supplied by D.C. Wallace), transformed with our construct, display an increased oligomycin resistance that supports these suppositions.

  • Schriner SE, Linford NJ, Martin GM, Treuting P, Ogburn CE, Emond M, Coskun PE, Ladiges W, Wolf N, Van Remmen H, Wallace DC, Rabinovitch PS. Extension of murine life span by overexpression of catalase targeted to mitochondria. Science 2005;308(5730):1909-1911. PubMed: 15879174. Categories: MitoSENS

    Extension of murine life span by overexpression of catalase targeted to mitochondria.

    Science 2005;308(5730):1909-1911.

    Extension of murine life span by overexpression of catalase targeted to mitochondria.

    Schriner SE, Linford NJ, Martin GM, Treuting P, Ogburn CE, Emond M, Coskun PE, Ladiges W, Wolf N, Van Remmen H, Wallace DC, Rabinovitch PS.

    Abstract

    Abstract:

    To determine the role of reactive oxygen species in mammalian longevity, we generated transgenic mice that overexpress human catalase localized to the peroxisome, the nucleus, or mitochondria (MCAT). Median and maximum life spans were maximally increased (averages of 5 months and 5.5 months, respectively) in MCAT animals. Cardiac pathology and cataract development were delayed, oxidative damage was reduced, H2O2 production and H2O2-induced aconitase inactivation were attenuated, and the development of mitochondrial deletions was reduced. These results support the free radical theory of aging and reinforce the importance of mitochondria as a source of these radicals.

  • 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. Reactive oxygen species production in the mitochondrial matrix: implications for the mechanism of mitochondrial mutation accumulation. Rejuvenation Res. 2005 Spring;8(1):13-7. PubMed: 15798370. Categories: MitoSENS

    Reactive oxygen species production in the mitochondrial matrix: implications for the mechanism of mitochondrial mutation accumulation.

    Rejuvenation Res. 2005 Spring;8(1):13-7.

    Reactive oxygen species production in the mitochondrial matrix: implications for the mechanism of mitochondrial mutation accumulation.

    de Grey ADNJ.

    Abstract

    Abstract:

    The vicious cycle theory postulates that typical mitochondrial DNA (mtDNA) mutations cause their host mitochondria to generate more superoxide and other reactive oxygen species (ROS) than do normal mitochondria, thereby promoting the occurrence of additional mtDNA mutations at an ever-accelerating rate. However, nearly all the loss-of-function mtDNA mutations seen in vivo are large deletions, which (as the original statement of the theory indeed noted, though this has been widely overlooked) should not trigger a vicious cycle because they will prevent the assembly of the potentially superoxide-generating enzyme complexes. Consistent with this is the observation that each cell exhibiting loss of mtDNA-encoded function in vivo contains copies of a single, evidently clonally expanded, mutant mtDNA species, whereas the vicious cycle theory predicts a spectrum of mutant forms in each cell. Two recent papers, however, unveil a way in which mtDNA mutations could indeed promote ROS production of their host mitochondria. MtDNA mutations probably shift the intramitochondrial NAD(+)/NADH redox couple towards NADH, and this is now shown in vitro to cause ROS production by alpha-ketoglutarate dehydrogenase, an essential enzyme of the TCA cycle. This does not revive the vicious cycle theory, but it has complex implications for the two most plausible more recent theories, known as "survival of the slowest" and "crippled mitochondria." It may also prove to explain other recent observations in mitochondrially mutant cells in vivo.

  • de Grey ADNJ. The plasma membrane redox system: a candidate source of aging-related oxidative stress. AGE J Am Aging Assoc 2005; 27(2):129-138. Categories: MitoSENS

    The plasma membrane redox system: a candidate source of aging-related oxidative stress.

    AGE J Am Aging Assoc 2005; 27(2):129-138.

    The plasma membrane redox system: a candidate source of aging-related oxidative stress.

    de Grey ADNJ.

    Abstract

    Abstract:

    The plasma membrane redox system (PMRS) is an electron transport chain in the plasma membrane that transfers electrons from either intra- or extracellular donors to extracellular acceptors. Unlike the superoxide-generating NADPH oxidase of phagocytes and the homologous (but much less active) enzymes found in some other cells, the PMRS is still incompletely characterised at the molecular level. Much is known, however, concerning its function and affinity for both physiological and nonphysiological substrates. A role for it in aging, the “reductive hotspot hypothesis” (RHH), was proposed in 1998 as part of an explanation for the apparently indefinite survival in vivo of cells that have entirely lost mitochondrial respiratory capacity as a result of the accumulation of mitochondrial mutations. Stimulation of the PMRS might allow the cell to maintain redox homeostasis even while continuing to operate the Krebs cycle, which may be advantageous in many ways. However, the PMRS may, like the mitochondrial respiratory chain, be prone to generate superoxide when thus dysregulated—and in this case superoxide would be generated outside the cell, where antioxidant defences are more limited than inside the cell and where much highly oxidisable material is present. Cascades of peroxidation chain reactions initiated by this process may greatly amplify the oxidative stress on the organism that is caused by rare mitochondrially mutant cells. Since such cells increase in abundance with aging (though remaining rare), this is an economical hypothesis to explain the rise in oxidative stress seen in (and generally believed to contribute substantially to) mammalian aging. In an extension of previously published accounts of RHH, I propose here that the lysosomal toxicity of oxidised cholesterol derivatives (oxysterols) may contribute to the toxicity of mitochondrial mutations by affecting lysosomal function in many cell types in the same way as they have been proposed to do in arterial macrophages.

  • de Grey ADNJ. Inter-species therapeutic cloning: the looming problem of mitochondrial DNA and two possible solutions. Rejuvenation Res 2004;7(2):95-98. PubMed: 15312296. Categories: MitoSENS, RepleniSENS

    Inter-species therapeutic cloning: the looming problem of mitochondrial DNA and two possible solutions.

    Rejuvenation Res 2004;7(2):95-98.

    Inter-species therapeutic cloning: the looming problem of mitochondrial DNA and two possible solutions.

    de Grey ADNJ.

    Abstract

    Abstract:

    No abstract available.

  • de Grey ADNJ. Mitochondrial mutations in mammalian aging: an over-hasty about-turn? Rejuvenation Res. 2004 Fall;7(3):171-4. PubMed: 15588517. Categories: MitoSENS

    Mitochondrial mutations in mammalian aging: an over-hasty about-turn?

    Rejuvenation Res. 2004 Fall;7(3):171-4.

    Mitochondrial mutations in mammalian aging: an over-hasty about-turn?

    de Grey ADNJ.

    Abstract

    Abstract:

    The very low abundance of mitochondrial DNA (mtDNA) mutations in nearly all mammalian tissues even in old age has led most mitochondriologists to reject the idea that such mutations might have a causal role in aging, despite (1) the strong circumstantial (e. g., interspecies) evidence that they do have such a role, (2) the promulgation since 1998 of two detailed mechanisms whereby low levels of mtDNA mutations could be harmful, and (3) the report of a transgenic mouse with cardiomyopathy apparently caused by artificially high levels of mtDNA mutations in the heart. A recent report of a mouse with ubiquitously accelerated accumulation of mtDNA mutations and an array of phenotypes reminiscent of aging has abruptly overturned this consensus, with not only the authors but also many other expert commentators suggesting that the mtDNA mutation theory of aging has risen from the ashes. However, there are compelling reasons to doubt the relevance of this mouse to normal mammalian aging, and thus to seek further testing of specific mechanistic hypotheses for how mtDNA mutations could cause age-related dysfunction.

  • de Grey ADNJ. Mitochondria in homeotherm aging: will detailed mechanisms consistent with the evidence now receive attention? Aging Cell. 2004 Apr;3(2):77 PubMed: 15038822. Categories: MitoSENS

    Mitochondria in homeotherm aging: will detailed mechanisms consistent with the evidence now receive attention?

    Aging Cell. 2004 Apr;3(2):77

    Mitochondria in homeotherm aging: will detailed mechanisms consistent with the evidence now receive attention?

    de Grey ADNJ.

    Abstract

    Abstract:

    No abstract available.

  • Khan SM, Bennett JP. Development of mitochondrial gene replacement therapy. J Bioenerg Biomembr 2004;36(4):387-393. PubMed: 15377877. Categories: MitoSENS

    Development of mitochondrial gene replacement therapy.

    J Bioenerg Biomembr 2004;36(4):387-393.

    Development of mitochondrial gene replacement therapy.

    Khan SM, Bennett JP.

    Abstract

    Abstract:

    Many "classic" mitochondrial diseases have been described that arise from single homoplasmic mutations in mitochondrial DNA (mtDNA). These diseases typically affect nonmitotic tissues (brain, retina, muscle), present with variable phenotypes, can appear sporadically, and are untreatable. Evolving evidence implicates mtDNA abnormalities in diseases such as Alzheimer's, Parkinson's, and type II diabetes, but specific causal mutations for these conditions remain to be defined. Understanding the mtDNA genotype-phenotype relationships and developing specific treatment for mtDNA-based diseases is hampered by inability to manipulate the mitochondrial genome. We present a novel protein transduction technology ("protofection") that allows insertion and expression of the human mitochondrial genome into mitochondria of living cells. With protofection, the mitochondrial genotype can be altered, or exogenous genes can be introduced to be expressed and either retained in mitochondria or be directed to other organelles. Protofection also delivers mtDNA in vivo, opening the way to rational development of mitochondrial gene replacement therapy of mtDNA-based diseases.

  • Kissova I, Deffieu M, Manon S, Camougrand N. Uth1p is involved in the autophagic degradation of mitochondria. J Biol Chem 2004;279(37):39068-39074. PubMed: 15247238. Categories: MitoSENS

    Uth1p is involved in the autophagic degradation of mitochondria.

    J Biol Chem 2004;279(37):39068-39074.

    Uth1p is involved in the autophagic degradation of mitochondria.

    Kissova I, Deffieu M, Manon S, Camougrand N.

    Abstract

    Abstract:

    The absence of the outer mitochondrial membrane protein Uth1p was found to induce resistance to rapamycin treatment and starvation, two conditions that induce the autophagic process. Biochemical studies showed the onset of a fully active autophagic activity both in wild-type and Deltauth1 strains. On the other hand, the disorganization of the mitochondrial network induced by rapamycin treatment or 15 h of nitrogen starvation was followed in cells expressing mitochondria-targeted green fluorescent protein; a rapid colocalization of green fluorescent protein fluorescence with vacuole-selective FM4-64 labeling was observed in the wild-type but not in the Deltauth1 strain. Degradation of mitochondrial proteins, followed by Western blot analysis, did not occur in mutant strains carrying null mutations of the vacuolar protease Pep4p, the autophagy-specific protein Atg5p, and Uth1p. These data show that, although the autophagic machinery was fully functional in the absence of Uth1p, this protein is involved in the autophagic degradation of mitochondria.

  • Ozawa T, Sako Y, Sato M, Kitamura T, Umezawa Y. A genetic approach to identifying mitochondrial proteins. Nat Biotechnol 2003;21(3):287-293. PubMed: 12577068. Categories: MitoSENS

    A genetic approach to identifying mitochondrial proteins.

    Nat Biotechnol 2003;21(3):287-293.

    A genetic approach to identifying mitochondrial proteins.

    Ozawa T, Sako Y, Sato M, Kitamura T, Umezawa Y.

    Abstract

    Abstract:

    The control of intricate networks within eukaryotic cells relies on differential compartmentalization of proteins. We have developed a method that allows rapid identification of novel proteins compartmentalized in mitochondria by screening large-scale cDNA libraries. The principle is based on reconstitution of split-enhanced green fluorescent protein (EGFP) by protein splicing of DnaE derived from Synechocystis sp. PCC6803. The cDNA libraries are expressed in mammalian cells following infection with retrovirus. If a test protein contains a functional mitochondrial targeting signal (MTS), it translocates into the mitochondrial matrix, where EGFP is then formed by protein splicing. The cells harboring this reconstituted EGFP are screened rapidly by fluorescence-activated cell sorting, and the cDNAs are isolated and identified from the cells. The analysis of 258 cDNAs revealed various MTSs, among which we identified new transcripts corresponding to mitochondrial proteins. This method should provide a means to map proteins distributed within intracellular organelles in a broad range of different tissues and disease states.

  • de Grey ADNJ. Mechanisms underlying the age-related accumulation of mutant mitochondrial DNA. In: Genetics of mitochondrial diseases (I.J. Holt, ed.), Oxford University Press, 2003, pp. 247-275. Categories: MitoSENS

    Mechanisms underlying the age-related accumulation of mutant mitochondrial DNA.

    In: Genetics of mitochondrial diseases (I.J. Holt, ed.), Oxford University Press, 2003, pp. 247-275.

    Mechanisms underlying the age-related accumulation of mutant mitochondrial DNA.

    de Grey ADNJ.

    Abstract

    Abstract:

    No abstract available.

  • Jacobs HT. The mitochondrial theory of aging: dead or alive? Aging Cell 2003;2(1):11-17. PubMed: 12882330. Categories: MitoSENS

    The mitochondrial theory of aging: dead or alive?

    Aging Cell 2003;2(1):11-17.

    The mitochondrial theory of aging: dead or alive?

    Jacobs HT.

    Abstract

    Abstract:

    The mitochondrial theory of aging is based around the idea of a vicious cycle, in which somatic mutation of mtDNA engenders respiratory chain dysfunction, enhancing the production of DNA-damaging oxygen radicals. In turn, this is proposed to result in the accumulation of further mtDNA mutations. Finally, a bioenergetic crisis leads to overt tissue dysfunction and degeneration. A substantial body of circumstantial evidence seems to support this idea. However, the extent of detectable mtDNA mutation is far less than can easily be reconciled to this hypothesis, unless it is assumed that a subset of cells with much higher than average mtDNA mutation load is systematically lost by apoptosis. A rigorous test of the hypothesis remains to be undertaken, but would require a direct manipulation of the rate of mtDNA mutagenesis, to test whether this could alter the kinetics of aging.

  • Guy J, Qi X, Pallotti F, Schon EA, Manfredi G, Carelli V, Martinuzzi A, Hauswirth WW, Lewin AS. Rescue of a mitochondrial deficiency causing Leber Hereditary Optic Neuropathy. Ann Neurol 2002;52(5):534-542. PubMed: 12402249. Categories: MitoSENS

    Rescue of a mitochondrial deficiency causing Leber Hereditary Optic Neuropathy.

    Ann Neurol 2002;52(5):534-542.

    Rescue of a mitochondrial deficiency causing Leber Hereditary Optic Neuropathy.

    Guy J, Qi X, Pallotti F, Schon EA, Manfredi G, Carelli V, Martinuzzi A, Hauswirth WW, Lewin AS.

    Abstract

    Abstract:

    A G to A transition at nucleotide 11778 in the ND4 subunit gene of complex I was the first point mutation in the mitochondrial genome linked to a human disease. It causes Leber Hereditary Optic Neuropathy, a disorder with oxidative phosphorylation deficiency. To overcome this defect, we made a synthetic ND4 subunit compatible with the "universal" genetic code and imported it into mitochondria by adding a mitochondrial targeting sequence. For detection we added a FLAG tag. This gene was inserted in an adeno-associated viral vector. The ND4FLAG protein was imported into the mitochondria of cybrids harboring the G11778A mutation, where it increased their survival rate threefold, under restrictive conditions that forced the cells to rely predominantly on oxidative phosphorylation to produce ATP. Since assays of complex I activity were normal in G11778A cybrids we focused on changes in ATP synthesis using complex I substrates. The G11778A cybrids showed a 60% reduction in the rate of ATP synthesis. Relative to mock-transfected G11778A cybrids, complemented G11778A cybrids showed a threefold increase in ATP synthesis, to a level indistinguishable from that in cybrids containing normal mitochondrial DNA. Restoration of respiration by allotopic expression opens the door for gene therapy of Leber Hereditary Optic Neuropathy.

  • Barja G. Rate of generation of oxidative stress-related damage and animal longevity. Free Radic Biol Med 2002;33(9):1167-1172. PubMed: 12398924. Categories: MitoSENS

    Rate of generation of oxidative stress-related damage and animal longevity.

    Free Radic Biol Med 2002;33(9):1167-1172.

    Rate of generation of oxidative stress-related damage and animal longevity.

    Barja G.

    Abstract

    Abstract:

    Comparative studies about the relationship between endogenous antioxidant and pro-oxidant factors and maximum longevity of different animal species are reviewed. The majority of studies on antioxidant supplementation indicate that it can increase mean survival without changing maximum longevity. On the other hand, endogenous antioxidants are negatively correlated with maximum longevity. The same is true for the rates of mitochondrial oxygen radical generation, oxidative damage to mitochondrial DNA, and the degree of fatty acid unsaturation of cellular membranes in postmitotic tissues. The lower rate of mitochondrial oxygen radical generation of long-lived animals in relation to that of short-lived ones can be a primary cause of their slow aging rate. This is secondarily complemented in long-lived animals with low rates of lipid peroxidation due to their low degrees of fatty acid unsaturation. These two traits suggest that the rate of generation of endogenous oxidative damage determines, at least in part, the rate of aging in animals.

  • Brunk UT, Terman A. The mitochondrial-lysosomal axis theory of aging: accumulation of damaged mitochondria as a result of imperfect autophagocytosis. Eur J Biochem 2002;269(8):1996-2002. PubMed: 11985575. Categories: LysoSENS, MitoSENS

    The mitochondrial-lysosomal axis theory of aging: accumulation of damaged mitochondria as a result of imperfect autophagocytosis.

    Eur J Biochem 2002;269(8):1996-2002.

    The mitochondrial-lysosomal axis theory of aging: accumulation of damaged mitochondria as a result of imperfect autophagocytosis.

    Brunk UT, Terman A.

    Abstract

    Abstract:

    Cellular manifestations of aging are most pronounced in postmitotic cells, such as neurons and cardiac myocytes. Alterations of these cells, which are responsible for essential functions of brain and heart, are particularly important contributors to the overall aging process. Mitochondria and lysosomes of postmitotic cells suffer the most remarkable age-related alterations of all cellular organelles. Many mitochondria undergo enlargement and structural disorganization, while lysosomes, which are normally responsible for mitochondrial turnover, gradually accumulate an undegradable, polymeric, autofluorescent material called lipofuscin, or age pigment. We believe that these changes occur not only due to continuous oxidative stress (causing oxidation of mitochondrial constituents and autophagocytosed material), but also because of the inherent inability of cells to completely remove oxidatively damaged structures (biological 'garbage'). A possible factor limiting the effectiveness of mitochondial turnover is the enlargement of mitochondria which may reflect their impaired fission. Non-autophagocytosed mitochondria undergo further oxidative damage, resulting in decreasing energy production and increasing generation of reactive oxygen species. Damaged, enlarged and functionally disabled mitochondria gradually displace normal ones, which cannot replicate indefinitely because of limited cell volume. Although lipofuscin-loaded lysosomes continue to receive newly synthesized lysosomal enzymes, the pigment is undegradable. Therefore, advanced lipofuscin accumulation may greatly diminish lysosomal degradative capacity by preventing lysosomal enzymes from targeting to functional autophagosomes, further limiting mitochondrial recycling. This interrelated mitochondrial and lysosomal damage irreversibly leads to functional decay and death of postmitotic cells.

  • de Grey ADNJ. The reductive hotspot hypothesis of mammalian aging: membrane metabolism magnifies mutant mitochondrial mischief. Eur J Biochem 2002; 269(8):2003-2009. PubMed: 11985576. Categories: MitoSENS

    The reductive hotspot hypothesis of mammalian aging: membrane metabolism magnifies mutant mitochondrial mischief.

    Eur J Biochem 2002; 269(8):2003-2009.

    The reductive hotspot hypothesis of mammalian aging: membrane metabolism magnifies mutant mitochondrial mischief.

    de Grey ADNJ.

    Abstract

    Abstract:

    A severe challenge to the idea that mitochondrial DNA mutations play a major role in the aging process in mammals is that clear loss-of-function mutations accumulate only to very low levels (under 1% of total) in almost any tissue, even by very old age. Their accumulation is punctate: some cells become nearly devoid of wild-type mitochondrial DNA and exhibit no activity for the partly mitochondrially encoded enzyme cytochrome c oxidase. Such cells accumulate in number with aging, suggesting that they survive indefinitely, which is itself paradoxical. The reductive hotspot hypothesis suggests that these cells adjust their metabolism to use plasma membrane electron transport as a substitute for the mitochondrial electron transport chain in the reoxidation of reduced dinucleotides, and that, like mitochondrial electron transport, this process is imperfect and generates superoxide as a side-effect. This superoxide, generated on the outside of the cell, can potentially initiate classical free radical chemistry including lipid peroxidation chain reactions in circulating material such as lipoproteins. These, in turn, can be toxic to mitochondrially nonmutant cells that import them to satisfy their cholesterol requirements. Thus, the relatively few cells that have lost oxidative phosphorylation capacity may be toxic to the rest of the body. In this minireview, recent results relevant to this hypothesis are surveyed and approaches to intervening in the proposed process are discussed.

  • de Grey ADNJ. Mitochondrial mutations in vertebrate aging. In: Oxidative stress and aging: advances in basic science, diagnostics, and intervention (R.G. Cutler and H. Rodriguez, eds.), World Scientific Publishing, 2002, pp. 437-451. Categories: MitoSENS

    Mitochondrial mutations in vertebrate aging.

    In: Oxidative stress and aging: advances in basic science, diagnostics, and intervention (R.G. Cutler and H. Rodriguez, eds.), World Scientific Publishing, 2002, pp. 437-451.

    Mitochondrial mutations in vertebrate aging.

    de Grey ADNJ.

    Abstract

    Abstract:

    No abstract available.

  • Ojaimi J, Pan J, Santra S, Snell WJ, Schon EA. An algal nucleus-encoded subunit of mitochondrial ATP synthase rescues a defect in the analogous human mitochondrial-encoded subunit. Mol Biol Cell 2002;13(11):3836-3844. PubMed: 12429828. Categories: MitoSENS

    An algal nucleus-encoded subunit of mitochondrial ATP synthase rescues a defect in the analogous human mitochondrial-encoded subunit.

    Mol Biol Cell 2002;13(11):3836-3844.

    An algal nucleus-encoded subunit of mitochondrial ATP synthase rescues a defect in the analogous human mitochondrial-encoded subunit.

    Ojaimi J, Pan J, Santra S, Snell WJ, Schon EA.

    Abstract

    Abstract:

    Unlike most organisms, the mitochondrial DNA (mtDNA) of Chlamydomonas reinhardtii, a green alga, does not encode subunit 6 of F(0)F(1)-ATP synthase. We hypothesized that C. reinhardtii ATPase 6 is nucleus encoded and identified cDNAs and a single-copy nuclear gene specifying this subunit (CrATP6, with eight exons, four of which encode a mitochondrial targeting signal). Although the algal and human ATP6 genes are in different subcellular compartments and the encoded polypeptides are highly diverged, their secondary structures are remarkably similar. When CrATP6 was expressed in human cells, a significant amount of the precursor polypeptide was targeted to mitochondria, the mitochondrial targeting signal was cleaved within the organelle, and the mature polypeptide was assembled into human ATP synthase. In spite of the evolutionary distance between algae and mammals, C. reinhardtii ATPase 6 functioned in human cells, because deficiencies in both cell viability and ATP synthesis in transmitochondrial cell lines harboring a pathogenic mutation in the human mtDNA-encoded ATP6 gene were overcome by expression of CrATP6. The ability to express a nucleus-encoded version of a mammalian mtDNA-encoded protein may provide a way to import other highly hydrophobic proteins into mitochondria and could serve as the basis for a gene therapy approach to treat human mitochondrial diseases.

  • Daley DO, Clifton R, Whelan J. Intracellular gene transfer: reduced hydrophobicity facilitates gene transfer for subunit 2 of cytochrome c oxidase. Proc Natl Acad Sci USA 2002;99(16):10510-10515. PubMed: 12142462. Categories: MitoSENS

    Intracellular gene transfer: reduced hydrophobicity facilitates gene transfer for subunit 2 of cytochrome c oxidase.

    Proc Natl Acad Sci USA 2002;99(16):10510-10515.

    Intracellular gene transfer: reduced hydrophobicity facilitates gene transfer for subunit 2 of cytochrome c oxidase.

    Daley DO, Clifton R, Whelan J.

    Abstract

    Abstract:

    Subunit 2 of cytochrome c oxidase (Cox2) in legumes offers a rare opportunity to investigate factors necessary for successful gene transfer of a hydrophobic protein that is usually mitochondrial-encoded. We found that changes in local hydrophobicity were necessary to allow import of this nuclear-encoded protein into mitochondria. All legume species containing both a mitochondrial and nuclear encoded Cox2 displayed a similar pattern, with a large decrease in hydrophobicity evident in the first transmembrane region of the nuclear encoded protein compared with the organelle-encoded protein. Mitochondrial-encoded Cox2 could not be imported into mitochondria under the direction of the mitochondrial targeting sequence that readily supports the import of nuclear encoded Cox2. Removal of the first transmembrane region promotes import ability of the mitochondrial-encoded Cox2. Changing just two amino acids in the first transmembrane region of mitochondrial-encoded Cox2 to the corresponding amino acids in the nuclear encoded Cox2 also promotes import ability, whereas changing the same two amino acids in the nuclear encoded Cox2 to what they are in the mitochondrial-encoded copy prevents import. Therefore, changes in amino acids in the mature protein were necessary and sufficient for gene transfer to allow import under the direction of an appropriate signal to achieve the functional topology of Cox2.

  • Manfredi G, Fu J, Ojaimi J, Sadlock JE, Kwong JQ, Guy J, Schon EA. Rescue of a deficiency in ATP synthesis by transfer of MTATP6, a mitochondrial DNA-encoded gene, to the nucleus. Nat Genet 2002;30(4):394-399. PubMed: 11925565. Categories: MitoSENS

    Rescue of a deficiency in ATP synthesis by transfer of MTATP6, a mitochondrial DNA-encoded gene, to the nucleus.

    Nat Genet 2002;30(4):394-399.

    Rescue of a deficiency in ATP synthesis by transfer of MTATP6, a mitochondrial DNA-encoded gene, to the nucleus.

    Manfredi G, Fu J, Ojaimi J, Sadlock JE, Kwong JQ, Guy J, Schon EA.

    Abstract

    Abstract:

    A T-->G transversion at nt 8993 in mitochondrial DNA of MTATP6 (encoding ATPase 6 of complex V of the respiratory chain) causes impaired mitochondrial ATP synthesis in two related mitochondrial disorders: neuropathy, ataxia and retinitis pigmentosa and maternally inherited Leigh syndrome. To overcome the biochemical defect, we expressed wildtype ATPase 6 protein allotopically from nucleus-transfected constructs encoding an amino-terminal mitochondrial targeting signal appended to a recoded ATPase 6 gene (made compatible with the universal genetic code) that also contained a carboxy-terminal FLAG epitope tag. After transfection of human cells, the precursor polypeptide was expressed, imported into and processed within mitochondria, and incorporated into complex V. Allotopic expression of stably transfected constructs in cytoplasmic hybrids (cybrids) homoplasmic with respect to the 8993T-->G mutation showed a significantly improved recovery after growth in selective medium as well as a significant increase in ATP synthesis. This is the first successful demonstration of allotopic expression of an mtDNA-encoded polypeptide in mammalian cells and could form the basis of a genetic approach to treat a number of human mitochondrial disorders.

  • de Grey ADNJ. Response to "approaches and limitations to gene therapy for mitochondrial diseases," Antioxid. Redox Signal. 2001;3:451-460. Antioxid Redox Signal. 2001 Dec;3(6):1153-5. PubMed: 11813989. Categories: MitoSENS

    Response to "approaches and limitations to gene therapy for mitochondrial diseases," Antioxid. Redox Signal. 2001;3:451-460.

    Antioxid Redox Signal. 2001 Dec;3(6):1153-5.

    Response to "approaches and limitations to gene therapy for mitochondrial diseases," Antioxid. Redox Signal. 2001;3:451-460.

    de Grey ADNJ.

    Abstract

    Abstract:

    No abstract available.

  • Elmore SP, Qian T, Grissom SF, Lemasters JJ. The mitochondrial permeability transition initiates autophagy in rat hepatocytes. FASEB J 2001;15(12):2286-2287. PubMed: 11511528. Categories: MitoSENS

    The mitochondrial permeability transition initiates autophagy in rat hepatocytes.

    FASEB J 2001;15(12):2286-2287.

    The mitochondrial permeability transition initiates autophagy in rat hepatocytes.

    Elmore SP, Qian T, Grissom SF, Lemasters JJ.

    Abstract

    Abstract:

    Cells degrade excess and effete organelles by the process of autophagy. Autophagic stimulation of rat hepatocytes by serum deprivation and glucagon (1 M) caused a fivefold increase of spontaneously depolarizing mitochondria to about 1.5% of total mitochondria after 90 min. Cyclosporin A (CsA, 5 M), an immunosuppressant that blocks the mitochondrial permeability transition (MPT), prevented this depolarization. Depolarized mitochondria moved into acidic vacuoles labeled by LysoTracker Red. These autophagosomes also increased several-fold after autophagic stimulation. CsA blocked autophagosomal proliferation, whereas tacrolimus, an immunosuppressant that does not block the MPT, did not. In conclusion, the MPT initiates mitochondrial depolarization after autophagic stimulation and the subsequent sequestration of mitochondria into autophagosomes.

  • de Grey ADNJ. The non-correlation between maximum longevity and enzymatic antioxidant levels among homeotherms; implications for retarding human aging. J Anti-Aging Med 2000; 3(1):25-36. Categories: MitoSENS

    The non-correlation between maximum longevity and enzymatic antioxidant levels among homeotherms; implications for retarding human aging.

    J Anti-Aging Med 2000; 3(1):25-36.

    The non-correlation between maximum longevity and enzymatic antioxidant levels among homeotherms; implications for retarding human aging.

    de Grey ADNJ.

    Abstract

    Abstract:

    No abstract available.

  • de Grey ADNJ. The reductive hotspot hypothesis: an update. Arch Biochem Biophys 2000; 373(1):295-301. PubMed: 10620352. Categories: MitoSENS

    The reductive hotspot hypothesis: an update.

    Arch Biochem Biophys 2000; 373(1):295-301.

    The reductive hotspot hypothesis: an update.

    de Grey ADNJ.

    Abstract

    Abstract:

    The mitochondrial free radical theory of aging is seriously challenged by the finding that mutant mtDNA never becomes abundant in vivo, a result disputed only in experiments using novel PCR variants whose quantitative accuracy is widely doubted. However, evidence continues to mount that mitochondria are the crucial site of free radical damage in vivo, most notably that mice lacking the nonmitochondrial isoforms of superoxide dismutase are healthy. It is thus important to determine whether a low level of mutant mtDNA could have serious systemic effects. This possibility exists because of the observed mosaic distribution of mutant mtDNA: some cells (or muscle fiber segments) lack any aerobic respiration. Such cells are presumed to satisfy their ATP needs by glycolysis. In vitro, however, NADH recycling by transmembrane pyruvate/lactate exchange does not suffice: cells only survive if they can up-regulate the plasma membrane oxidoreductase (PMOR). The PMOR's physiological electron acceptor is unknown. It was proposed recently (de Grey, A. D. N. J. (1998) J. Anti-Aging Med. 1(1), 53-66) that a prominent in vivo acceptor from these mitochondrially mutant cells may be oxygen, forming extracellular superoxide. The mosaic ("hotspot") distribution of this superoxide would limit its dismutation by extracellular superoxide dismutase; it may thus reduce transition metals leading to oxidation of circulating material, such as LDL. This would raise systemic oxidative stress, greatly amplifying the damage done by the originating mitochondrially mutant cells. This model, now known as the "reductive hotspot hypothesis," has recently gained much indirect experimental support; several direct tests of it are also feasible.

  • 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.

  • de Grey ADNJ. Are those 13 proteins really unimportable? In: From Symbiosis to Eukaryotism - Endocytobiology VII (E. Wagner et al., eds.), Geneva University Press, 1999, pp. 489-502. Categories: MitoSENS

    Are those 13 proteins really unimportable?

    In: From Symbiosis to Eukaryotism - Endocytobiology VII (E. Wagner et al., eds.), Geneva University Press, 1999, pp. 489-502.

    Are those 13 proteins really unimportable?

    de Grey ADNJ.

    Abstract

    Abstract:

    No abstract available.

  • de Grey ADNJ. The mitochondrial free radical theory of aging. Austin, TX: Landes Bioscience, 1999, 212pp, hardcover (ISBN 1-57059-564-X). Read on external site. Categories: MitoSENS

    The mitochondrial free radical theory of aging.

    Austin, TX: Landes Bioscience, 1999, 212pp, hardcover (ISBN 1-57059-564-X).

    The mitochondrial free radical theory of aging.

    de Grey ADNJ.

    Abstract

    Abstract:

    No abstract available.

  • de Grey ADNJ. A Mechanism Proposed to Explain the Rise in Oxidative Stress During Aging. J Anti-Aging Med. 1998 Spring. 1(1): 53-66. doi:10.1089/rej.1.1998.1.53. Read on external site. Categories: MitoSENS

    A Mechanism Proposed to Explain the Rise in Oxidative Stress During Aging.

    J Anti-Aging Med. 1998 Spring. 1(1): 53-66. doi:10.1089/rej.1.1998.1.53.

    A Mechanism Proposed to Explain the Rise in Oxidative Stress During Aging.

    de Grey ADNJ.

    Abstract

    Abstract:

    Most phenotypes of aging in vertebrates may be caused by a progressive decline in the ability of antioxidant defences to maintain cellular and systemic homeostasis. This is due both to a diminished efficacy of those defences and to an enhanced level of pro-oxidant toxicity; the imbalance between the two has been termed oxidative stress. However, the cause of this increasing imbalance remains obscure. This article proposes a mechanism by which spontaneously mutant mitochondrial DNA (mtDNA), despite being present only in very small quantities in the body, may be the main generator of oxidative stress. Mutant mtDNA is distributed very unevenly within a tissue: some cells apparently contain no wild-type mtDNA whatever. Those cells must rely on glycolysis for ATP production; furthermore, they require a system to stabilize their NAD+/NADH ratio. This can only be achieved by an efflux of electrons from the cell, most probably mediated by the plasma membrane oxidoreductase (PMOR). It is proposed that the required rate of electron efflux from these anaerobic cells exceeds the local electron-accepting capacity of "safe" acceptors in plasma such as dehydroascorbate, with the result that reactive species, such as Superoxide, are formed. This leads to increased oxidation of lipids in the plasma, notably of low-density lipoprotein (LDL) particles, which are subsequently imported into mitochondrially healthy cells. This oxidized lipoprotein must be destroyed by the recipient cells' antioxidant defences. That task diverts the cell from the degradation of pro-oxidants that it is itself generating; thus, it imposes oxidative stress on the cell. As the number of anaerobic cells in the body rises, so does oxidative stress in all cells. The consistency of this hypothesis with known facts is discussed, and technically feasible tests are suggested both of the proposed mechanism and of its overall contribution to mammalian aging, including plausible interventions to retard the process.

  • de Grey ADNJ. A proposed refinement of the mitochondrial free radical theory of aging. BioEssays 1997; 19(2):161-166. PubMed: 9046246. Categories: MitoSENS

    A proposed refinement of the mitochondrial free radical theory of aging.

    BioEssays 1997; 19(2):161-166.

    A proposed refinement of the mitochondrial free radical theory of aging.

    de Grey ADNJ.

    Abstract

    Abstract:

    No abstract available.

  • Shigenaga MK, Hagen TM, Ames BN. Oxidative damage and mitochondrial decay in aging. Proc Natl Acad Sci USA 1994;91(23):10771-10778. PubMed: 7971961. Categories: MitoSENS

    Oxidative damage and mitochondrial decay in aging.

    Proc Natl Acad Sci USA 1994;91(23):10771-10778.

    Oxidative damage and mitochondrial decay in aging.

    Shigenaga MK, Hagen TM, Ames BN.

    Abstract

    Abstract:

    We argue for the critical role of oxidative damage in causing the mitochondrial dysfunction of aging. Oxidants generated by mitochondria appear to be the major source of the oxidative lesions that accumulate with age. Several mitochondrial functions decline with age. The contributing factors include the intrinsic rate of proton leakage across the inner mitochondrial membrane (a correlate of oxidant formation), decreased membrane fluidity, and decreased levels and function of cardiolipin, which supports the function of many of the proteins of the inner mitochondrial membrane. Acetyl-L-carnitine, a high-energy mitochondrial substrate, appears to reverse many age-associated deficits in cellular function, in part by increasing cellular ATP production. Such evidence supports the suggestion that age-associated accumulation of mitochondrial deficits due to oxidative damage is likely to be a major contributor to cellular, tissue, and organismal aging.

  • Bandy B, Davison AJ. Mitochondrial mutations may increase oxidative stress: implications for carcinogenesis and aging? Free Radic Biol Med 1990;8(6):523-39. PubMed: 2193852. Categories: MitoSENS

    Mitochondrial mutations may increase oxidative stress: implications for carcinogenesis and aging?

    Free Radic Biol Med 1990;8(6):523-39.

    Mitochondrial mutations may increase oxidative stress: implications for carcinogenesis and aging?

    Bandy B, Davison AJ.

    Abstract

    Abstract:

    The sensitivity of mitochondrial DNA to damage by mutagens predisposes mitochondria to injury on exposure of cells to genotoxins or oxidative stress. Damage to the mitochondrial genome causing mutations or loss of mitochondrial gene products, or to some nuclear genes encoding mitochondrial membrane proteins, may accelerate release of reactive species of oxygen. Such aberrant mitochondria may contribute to cellular aging and promotion of cancer.

  • van Zutphen H, Cornwell DG. Some studies on lipid peroxidation in monomolecular and bimolecular lipid films. J Membr Biol 1973;13:79-88. PubMed: 4796220. Categories: MitoSENS

    Some studies on lipid peroxidation in monomolecular and bimolecular lipid films.

    J Membr Biol 1973;13:79-88.

    Some studies on lipid peroxidation in monomolecular and bimolecular lipid films.

    van Zutphen H, Cornwell DG.

    Abstract

    Abstract:

    No abstract available.

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