X-Message-Number: 26279 Date: Fri, 3 Jun 2005 20:27:37 -0700 (PDT) From: Doug Skrecky <> Subject: first success at engineered negligible senescence [Quote from reference #2: "A single culture of mutant ex1 has been growing in the laboratory on cornmeal agar plates for over 12 years without signs of senescence." Note that the maximum lifespan of wild type podospora is 50 days, and this can be extended to 130 days by the addition to the medium of 30 micromole of the copper chelator bathocuproinedisulfonic acid.] Reference #1: Biogerontology. 2002;3(3):143-53 Respiration, copper availability and SOD activity in P. anserina strains with different lifespan. P. anserina mutants with impairments in complex IV (COX) of the respiratory chain are characterized by an increase in lifespan. Examples are the nuclear grisea mutant with a moderate lifespan extension (60%) and the immortal extranuclear ex1 mutant. Here we report data demonstrating that in mutant ex1 the level of the alternative oxidase (PaAOX) is significantly higher than in mutant grisea. PaAOX levels appear to be reversely dependent on COX activity. The activity profile of superoxide dismutases in the ex1 mutant resembles the profile in senescent wild-type cultures with a high cytoplasmic copper/zinc superoxide dismutase (PaSOD1) and a low mitochondrial manganese superoxide dismutase (PaSOD2) activity. In the grisea mutant, PaSOD1 activity is only detectable in cultures grown in copper-supplemented medium. The two copper-regulated genes PaCtr3 (coding for a high affinity copper transporter) and PaSod2 are not expressed in the two mutants grown in standard medium. The repression of these genes as well as the activity of PaSOD1 is dependent on the availability of cellular copper, which appears to be high in COX-deficient strains such as mutant ex1 and in the senescent wild-type strain. In the wild-type, changes in the cellular localization of copper and in the delivery of this metal to different proteins appear to occur during senescence. Collectively, the data explain the characteristic lifespan of the investigated strains as the result of differences in energy transduction and in the machinery protecting against oxidative stress. Reference #2: Mol Cell Biol. 2001 Jan;21(2):390-9. Copper-modulated gene expression and senescence in the filamentous fungus Podospora anserina. We have previously shown that the control of cellular copper homeostasis by the copper-modulated transcription factor GRISEA has an important impact on the phenotype and lifespan of Podospora anserina. Here we demonstrate that copper depletion leads to the induction of an alternative respiratory pathway and to an increase in lifespan. This response compensates mitochondrial dysfunctions via the expression of PaAox, a nuclear gene coding for an alternative oxidase. It resembles the retrograde response in Saccharomyces cerevisiae. In P. anserina, this pathway appears to be induced by specific impairments of the copper-dependent cytochrome c oxidase. It is not induced as the result of a general decline of mitochondrial functions during senescence. We cloned and characterized PaAox. Transcript levels are decreased when cellular copper, superoxide, and hydrogen peroxide levels are raised. Copper also controls transcript levels of PaSod2, the gene encoding the mitochondrial manganese superoxide dismutase (PaSOD2). PaSod2 is a target of transcription factor GRISEA. During the senescence of wild-type strain s, the activity of PaSOD2 decreases, whereas the activity of the cytoplasmic copper/zinc superoxide dismutase (PaSOD1) increases. Collectively, the data explain the postponed senescence of mutant grisea as a defined consequence of copper depletion, ultimately leading to a reduction of oxidative stress. Moreover, they suggest that during senescence of the wild-type strain, copper is released from mitochondria. The involved mechanism is unknown. However, it is striking that the permeability of mitochondrial membranes in animal systems changes during apoptosis and that mitochondrial proteins with an important impact on this type of cellular death are released. Free full text for reference #2: http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pubmed&pubmedid=11134328 Reference #3 Mech Ageing Dev. 1992 Sep;65(2-3):277-88 Oxidative stress and ageing in the fungus Podospora anserina. The ageing phenomenon exhibited by the ascomycetous fungus Podospora anserina can be either delayed or induced by either different carbon sources or effectors. As these effects seem to have analogy to catabolite-repression of respiratory genes, experiments concerning respiratory functions have been carried out. Ageing is parallelled by switching from cytochrome c-oxidase-mediated respiration to alternative, cyanide-resistant respiration for reasons still unknown. The latter is always accompanied by appearance of the phenol oxidizing enzyme laccase (EC 1.10.3.2), which seems to act as an alternative oxidase. The existence of a second, non-mitochondrially encoded respiratory pathway relieves the selective pressure on mitochondria leading to disintegrated, non-functional mtDNA and thereby whole mitochondria which accumulate in the hyphal cells. Mutants lacking cytochrome c-oxidase aa3 or laccase have stable mitochondrial populations and live eternally. Rate This Message: http://www.cryonet.org/cgi-bin/rate.cgi?msg=26279