Reducing mitochondrial fission results in increased life span and fitness of two fungal ageing models.

Ageing of biological systems is accompanied by alterations in mitochondrial morphology, including a transformation from networks and filaments to punctuate units. The significance of these alterations with regard to ageing is not known. Here, we demonstrate that the dynamin-related protein 1 (Dnm1p), a mitochondrial fission protein conserved from yeast to humans, affects ageing in the two model systems we studied, Podospora anserina and Saccharomyces cerevisiae. Deletion of the Dnm1 gene delays the transformation of filamentous to punctuate mitochondria and retards ageing without impairing fitness and fertility typically observed in long-lived mutants. Our data further suggest that reduced mitochondrial fission extends life span by increasing cellular resistance to the induction of apoptosis and links mitochondrial dynamics, apoptosis and life-span control.

MiMage: a Pan-European project on the role of mitochondria in aging.

The new European research project MiMage, supported by the European Community's Sixth Framework for Research and Technological Development, focuses on elucidating the role of mitochondria in conserved mechanisms of aging. Expertise in different research areas, including biochemistry, cell biology, genetics, molecular biology, and physiology, is provided by twelve research teams from seven European countries, together with one associated team from Canada and the United States. This report provides an introduction to the participating laboratories and the topics that will be addressed within the project, together with a concise report on the first symposium on the role of mitochondria in aging.

Metabolism and aging in the filamentous fungus Podospora anserina.

In Podospora anserina, lifespan is under the control of environmental and genetic factors. Both suggest an important impact of metabolism on lifespan and aging. Environmental changes of temperature, of the carbon source in the growth medium, or the addition of specific inhibitors to the growth medium are some of the investigated factors. Genetic approaches underscore the significance of metabolism. In particular, the mitochondrial electron transport plays a major role. As a by-product of a cytochrome oxidase (COX) dependent energy transduction, reactive oxygen species (ROS) are generated and lead to damage of cellular biomolecules. Damaged mitochondria, compromised at complex IV (COX) of the respiratory chain, signal to the nucleus and induce a nuclear gene, PaAox, encoding an alternative oxidase (AOX). This pathway resembles the retrograde response that, at least in yeast, is induced by dysfunctional mitochondria. ROS generation is lowered when electrons are transferred via an alternative pathway utilizing the AOX. As a consequence, lifespan of the corresponding strains is increased. Cellular copper levels were found to play a significant role not only in the generation of ROS but also have an impact on the cytoplasmic and the mitochondrial superoxide dismutase (SOD). In addition, copper is involved in the control of mitochondrial DNA rearrangements and affects the ability of the system to remodel damaged mitochondria. All these different components and pathways are part of the complex molecular network involved in lifespan control of this aging model.

Molecular control of copper homeostasis in filamentous fungi: increased expression of a metallothionein gene during aging of Podospora anserina.

The lifespan of the ascomycete Podospora anserina was previously demonstrated to be significantly increased in a copper-uptake mutant, suggesting that copper is a potential stressor involved in degenerative processes. In order to determine whether changes in copper stress occur in the cells during normal aging of cultures, we cloned and characterized a gene coding for a component of the molecular machinery involved in the control of copper homeostasis. This gene, PaMt1, is a single-copy gene that encodes a metallothionein of 26 amino acids. The coding sequence of PaMt1 is interrupted by a single intron. The deduced amino acid sequence shows a high degree of sequence identity to metallothioneins of the filamentous ascomycete Neurospora crassa and the basidiomycete Agaricus bisporus, and to the N-terminal portion of mammalian metallothioneins. Levels of PaMt1 transcript increase in response to elevated amounts of copper in the growth medium and during aging of wild-type cultures. In contrast, in the long-lived mutant grisea, transcript levels first increase but then decrease again. The ability of wild-type cultures to respond to exogenous copper stress via the induction of PaMt1 transcription is not affected as they grow older.

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.

Yeti--a degenerate gypsy-like LTR retrotransposon in the filamentous ascomycete Podospora anserina.

In the filamentous ascomycete Podospora anserina a 6,935-bp retrotransposon, Yeti, has been identified and characterized. It is flanked by a 5-bp target site duplication and contains long terminal repeats (LTRs) 354 bp in length. The LTRs show a high degree of identity to the previously reported repetitive element repa, a sequence suggested to represent a solo-LTR element of an unknown transposon. In the investigated Podospora strains, the number of complete Yeti copies is significantly lower than the number of repa elements, with up to 25 copies. Yeti appears to be inactive: it is highly degenerate and no transcripts of the element have been detected even in Podospora cultures grown under elevated stress conditions. The amino acid sequences deduced from Yeti display significant homology, particularly in the reverse transcriptase region, to those of other fungal retrotransposons, indicating that it is a member of the gypsy family. As suggested by the unusual dinucleotide content, degeneration of Yeti appears to be the result of a molecular mechanism resembling repeat-induced point mutation in Neurospora crassa.

Cellular copper homeostasis, mitochondrial DNA instabilities, and lifespan control in the filamentous fungus Podospora anserina.

In the fungal aging model Podospora anserina, lifespan is controlled by mitochondrial and nuclear genetic traits. Different nuclear genes are known to affect the integrity of the mitochondrial DNA (mtDNA). One gene of this type is Grisea encoding a copper-modulated transcription factor involved in the control of cellular copper homeostasis. The characterization of a long-lived mutant with a loss-of-function mutation in this gene revealed that the last step in the pathway, homologous recombination, leading to the characteristic age-related mtDNA reorganizations is copper-dependent. In growing parts of the culture, the stabilization of the mtDNA has an important impact on the biogenesis of functional mitochondria, on their capacity to remodel damaged respiratory chains and on longevity.

The degenerate DNA transposon Pat and repeat-induced point mutation (RIP) in Podospora anserina.

A degenerate DNA transposon, Pat, was identified in the genomes of various wild-type strains of the filamentous fungus Podospora anserina. In these strains, the number (approximately 20-25 copies per genome) and location of Pat sequences appear to be conserved. Two copies of Pat, one complete and one partial, were cloned and characterized. The sequence of the complete element is 1856 bp long and contains imperfect inverted terminal repeats (ITRs) of 53 bp. The target site duplication comprises the sequence TA. The amino acid sequence derived from one reading frame of Pat shows significant homology to members of the Fot1 family of transposons. However, this reading frame is interrupted by numerous stop codons. Since no transcripts of Pat were identified in different P. anserina strains grown under standard conditions and under increased stress, we conclude that none of the copies of Pat is active in the strains analyzed, under the environmental conditions investigated. Comparison of the sequences of the two cloned Pat sequences revealed 89% (589/747 nucleotides) identity. Most of the differences (82%, 129/158) can be attributed to transitions preferentially at CpA:TpG and CpT:ApG dinucleotides. The dinucleotide ratios in Pat are similar to those in a Neurospora crassa transposon which was subject to repeat-induced mutation (RIP), but differ significantly from those found in single-copy genes of P. anserina and in fungal DNA transposons not modified by this mechanism. Molecular analysis of the progeny of a cross between the wild-type strain and a transgenic strain in which a nuclear gene was duplicated by transformation yielded the first clear evidence that a RIP-like process is active in P. anserina.

Mitochondrial oxidative stress and aging in the filamentous fungus Podospora anserina.

In the filamentous fungus Podospora anserina, mitochondrial oxidative stress is a major contributor to aging. Reactive oxygen species (ROS) generated as a result of electron leakage during respiration lead to damage of components of the electron transport chain. In aging wild-type cultures, damaged proteins cannot be replaced because the mitochondrial genes encoding some of the corresponding subunits gradually become deleted from the mitochondrial DNA (mtDNA). Consequently, these defects result in an increased generation of reactive oxygen species and respiration deficits leading to cell death. Analyses of wild-type strains and of different long-lived mutants of P. anserina provide strong evidence that molecular mechanisms controlling aging processes in this fungus are complex and act at different levels. A basic mechanism (e.g., damage by ROS) appears to be overlaid by prominent instabilities of the mtDNA.

Identification and characterization of PaMTH1, a putative O-methyltransferase accumulating during senescence of Podospora anserina cultures.

A differential protein display screen resulted in the identification of a 27-kDa protein which strongly accumulates during the senescence of Podospora anserina cultures grown under standard conditions. After partial determination of the amino-acid sequence by mass-spectrometry analysis of trypsin-generated fragments, pairs of degenerated primers were deduced and used to amplify parts of the sequence coding for the protein. These PCR products were utilized to select specific cDNA and genomic clones from DNA libraries of P. anserina. A subsequent DNA-sequence analysis revealed that the 27-kDa protein is encoded by a discontinuous gene, PaMth1, capable of coding for 240 amino acids. The first three amino-terminal residues appear to be removed post-translationally. The deduced amino-acid sequence shows significant homology to S-adenosylmethionine (SAM)-dependent methyltransferases. We hypothesize that the 27-kDa protein, PaMTH1, is involved in age-related methylation reactions protecting aging cultures against increasing oxidative stress.

Copper-dependence of mitochondrial DNA rearrangements in Podospora anserina.

Rearrangements of the mitochondrial DNA (mtDNA) are a hallmark of senescence in wild-type strains of the ascomycete P. anserina. These rearrangements include the systematic amplification of the first intron (p1-intron) of the cytochrome oxidase subunit-I gene (CoI) as a circular DNA molecule (p1DNA). In addition, deletions and amplifications of other regions of the mtDNA occur. The molecular basis of the underlying processes is not understood in detail. A comparative analysis of the wild-type strain and of the long-lived mutant grisea, affected in the uptake of copper, revealed that mtDNA instabilities are dependent on the availability of cellular copper. In the mutant, the first steps in the corresponding pathway, including the transcription of the CoI gene, the splicing of the p1-intron and the transposition of this mobile element, are not impaired. In contrast, recombination processes between short direct repeats, as well as rearrangements between two tandem intron copies leading to the formation of p1DNA, appear to be affected. Additional copper in the growth medium rescues this molecular phenotype. We suggest that copper is a cofactor of a component of the molecular machinery leading to the characteristic age-related mtDNA rearrangements.

Modulation of gene expression by (CA)n microsatellites in the filamentous ascomycete Podospora anserina.

A microsatellite consisting of the alternating pyrimidine-purine sequence (CA)n. (TG)n is found to occur in very conserved form in the genome of various races of the filamentous ascomycete Podospora anserina. Screening of a cDNA library revealed that this sequence is frequently transcribed. In this study, we focused our attention on a short (CA)5 microsatellite located in the 5' untranslated sequence of the glyceraldehyde-3-phosphate dehydrogenase (gpd) gene of P. anserina. Specifically, we investigated whether or not the number of repeat units present in the microsatellite affects the expression of the beta-D-glucuronidase (gusA) reporter gene introduced on an autonomously replicating plasmid into fungal protoplasts. The results show that an increase in the number of microsatellite repeat units positively affects reporter gene expression.

PaGrg1, a glucose-repressible gene of Podospora anserina that is differentially expressed during lifespan.

A loss-of-function mutation in Grisea, a gene of Podospora anserina that was previously shown to code for a copper-modulated transcription factor, leads to a significant increase in lifespan. In an attempt to identify and to isolate potential target genes controlled by GRISEA, a RT-differential-display screen was performed. This approach resulted in the identification of a gene, PaGrg1, that is differentially expressed in the wild-type and in the long-lived grisea mutant. In the mutant, transcript levels of PaGrg1 were found to be much lower than in the wild-type even if copper was added to the growth medium in amounts that revert the phenotype of this copper-uptake mutant to wild-type characteristics. PaGrg1 is a discontinuous gene with a single intron and encodes a protein of 71 amino acids sharing a high degree of sequence identity (65%) with the developmentally regulated, catabolite-repressed grg-1 gene of Neurospora crassa. Transcription of PaGrg1 increases upon carbon starvation indicating that PaGRG1 represents a putative stress protein. Transcript levels of PaGrg1 were found to increase during aging in both the wild-type strain and the long-lived mutant. However, even in the senescent stage of grisea, they are much lower than in juvenile cultures of the wild-type strain. The data suggest that threshold transcript levels of PaGrg1, and/ or additional unidentified genes which are controlled by GRISEA and which are subject to catabolite repression, are significantly involved in lifespan control. This conclusion is supported by the finding that, in contrast to the wild-type, the lifespan of grisea does not increase when cultures are grown on non-repressible carbon sources.

Mitochondrial-nuclear interactions and lifespan control in fungi.

In fungi, mitochondrial-nuclear interactions are part of a complex molecular network involved in the control of aging processes. The generation of reactive oxygen species at the mitochondrial respiratory chain plays a major role in this network. Mitochondrial DNA instabilities, which are under the control of nuclear genes, affect the generation of reactive oxygen species and modulate the rate of aging. As mitochondria become dysfunctional, they transduce signals to the nucleus and induce the expression of a set of nuclear genes, a process termed retrograde regulation. Molecular data are emerging which suggest that retrograde regulation is involved in lifespan control.

Genome analysis of filamentous fungi: identification and characterization of an unusual GT-rich minisatellite in the ascomycete Podospora anserina.

A genomic DNA fragment, PaGT7-5, of Podospora anserina was cloned that contains three different repetitive sequence motifs including a minisatellite with an unusual structure. This element, PaMin1, consists of ten copies of a 16 bp repeat unit with five GT dinucleotide repeats. Adjacent to PaMin1, a short poly(GT) stretch and four repeats of a 12 bp sequence were identified. Six alleles which differ in the number of the minisatellite unit were demonstrated in 18 different P. anserina strains. The flanking sequences of PaMin1 did not display any sequence alterations. A PCR analysis of total DNA from cultures of different age revealed no sequence changes, indicating that this repetitive DNA remains stable during aging. Southern-blot hybridization experiments of DNA from different strains detected only minor fragment length polymorphisms in the immediate vicinity of PaMin1. In contrast, major polymorphisms were observed at a greater distance from the locus indicating that this locus can be used as an informative probe to discriminate between different P. anserina strains.

The dynamics of pAL2-1 homologous linear plasmids in Podospora anserina.

A natural population of recently isolated Podospora anserina strains was screened for homologues of the linear longevity-inducing plasmid pAL2-1. Of the 78 wild-type isolates, 14 hybridised with a pAL2-1 specific probe, half of which contained a single plasmid and the other half multiple plasmid copies (plasmid family). All strains except one plasmid-containing strain, senesced normally. However, no inserted plasmid sequences were detected in the mitochondrial DNA, as was the case for the longevity-inducing pAL2-1 plasmid. Occasional loss of plasmids and of repeated plasmid sequences occurred during sexual transfer. Plasmid transmission was equally efficient for mono- and dikaryotic spores and was independent of the genetic background of the strains. Furthermore, horizontal transfer experiments showed that the linear plasmid could easily infect plasmid-free strains. Horizontal transfer was even observed between strains showing a clear vegetative incompatibility response (barrage). The linear plasmids are inherited maternally; however, paternal transmission was observed in crosses between confronted vegetative-incompatible strains. Paternal transmission of the plasmid was never observed using isolated spermatia for fertilisation, showing that mitochondrial plasmids can only gain access to maternal sexual reproductive structures following horizontal transfer. These findings have implications for both the function of vegetative incompatibility in fungi and for the mechanism of maintenance of linear plasmids.

GRISEA, a copper-modulated transcription factor from Podospora anserina involved in senescence and morphogenesis, is an ortholog of MAC1 in Saccharomyces cerevisiae.

The initial characterization of Grisea suggested that this gene codes for a transcription factor involved in the genetic control of cellular copper homeostasis in Podospora anserina. Here we demonstrate that GRISEA activates in vivo gene expression in Saccharomyces cerevisiae and is characterized by a modular organization. The DNA-binding domain was mapped to the first 168 N-terminal amino acids and the transactivation domain to the C-terminal half of the protein. Increased levels of copper in the growth medium lead to repression of the transactivation function possibly via intramolecular interactions between parts of the DNA-binding domain and the transactivation domain. The wild-type copy of Grisea was found to complement the phenotype of the mac1-1 mutant of S. cerevisiae. GRISEA is able to bind to the promoter of CTR1, a MAC1 target gene that encodes a high-affinity copper transporter. Taken together, the data reported here and in earlier investigations indicate that GRISEA is an ortholog of the yeast transcription factor MAC1 and suggest at least a partial conservation of the molecular machinery involved in the control of cellular copper homeostasis in eukaryotes. Remarkably, in P. anserina, the spectrum of phenotypes affected by this regulatory protein is much broader than that known in yeast and includes morphogenetic traits as well as lifespan and senescence.

Genetic regulation of aging.

Aging of biological systems is a complex process that is controlled by both environmental factors and the genetic constitution of the individual. Although the molecular mechanisms have not been elucidated for any system in detail, it is clear that various genetic traits are involved in the modulation of life span. In particular, the genetic information located in the mitochondria has been identified as a major genetic component. Instabilities of the mitochondrial DNA (mtDNA) lead to mitochondrial dysfunction and increased oxidative stress. In some cases mtDNA instabilities are related to the activity of mobile genetic elements. In addition, nuclear genes appear to be crucially involved in mtDNA maintenance. Furthermore, the initial analysis of a few cloned nuclear genes affecting life span suggests a cellular machinery dealing with various stress situations as a major component involved in the genetic control of aging. This conclusion may hold true for all biological systems and be related to a unified mechanism of aging. However, in the various lineages this mechanism may be superimposed by other species or lineage-specific mechanisms.

Mitochondrial DNA rearrangements of Podospora anserina are under the control of the nuclear gene grisea.

Podospora anserina is a filamentous fungus with a limited life span. Life span is controlled by nuclear and extranuclear genetic traits. Herein we report the nature of four alterations in the nuclear gene grisea that lead to an altered morphology, a defect in the formation of female gametangia, and an increased life span. Three sequence changes are located in the 5' upstream region of the grisea ORF. One mutation is a G --> A transition at the 5' splice site of the single intron of the gene, leading to a RNA splicing defect. This loss-of-function affects the amplification of the first intron of the mitochondrial cytochrome c oxidase subunit I gene (COI) and the specific mitochondrial DNA rearrangements that occur during senescence of wild-type strains. Our results indicate that the nuclear gene grisea is part of a molecular machinery involved in the control of mitochondrial DNA reorganizations. These DNA instabilities accelerate but are not a prerequisite for the aging of P. anserina cultures.