Mitochondrial Phospholipid Homeostasis Is Regulated by the i-AAA Protease PaIAP and Affects Organismic Aging.

Mitochondria are ubiquitous organelles of eukaryotic organisms with a number of essential functions, including synthesis of iron-sulfur clusters, amino acids, lipids, and adenosine triphosphate (ATP). During aging of the fungal aging model Podospora anserina, the inner mitochondrial membrane (IMM) undergoes prominent morphological alterations, ultimately resulting in functional impairments. Since phospholipids (PLs) are key components of biological membranes, maintenance of membrane plasticity and integrity via regulation of PL biosynthesis is indispensable. Here, we report results from a lipidomic analysis of isolated mitochondria from P. anserina that revealed an age-related reorganization of the mitochondrial PL profile and the involvement of the i-AAA protease PaIAP in proteolytic regulation of PL metabolism. The absence of PaIAP enhances biosynthesis of characteristic mitochondrial PLs, leads to significant alterations in the acyl composition of the mitochondrial signature PL cardiolipin (CL), and induces mitophagy. These alterations presumably cause the lifespan increase of the PaIap deletion mutant under standard growth conditions. However, PaIAP is required at elevated temperatures and for degradation of superfluous CL synthase PaCRD1 during glycolytic growth. Overall, our study uncovers a prominent role of PaIAP in the regulation of PL homeostasis in order to adapt membrane plasticity to fluctuating environmental conditions as they occur in nature.

Identification of autophagy as a longevity-assurance mechanism in the aging model Podospora anserina.

The filamentous ascomycete Podospora anserina is a well-established aging model in which a variety of different pathways, including those involved in the control of respiration, ROS generation and scavenging, DNA maintenance, proteostasis, mitochondrial dynamics, and programmed cell death have previously been demonstrated to affect aging and life span. Here we address a potential role of autophagy. We provide data demonstrating high basal autophagy levels even in strains cultivated under noninduced conditions. By monitoring an N-terminal fusion of EGFP to the fungal LC3 homolog PaATG8 over the lifetime of the fungus on medium with and without nitrogen supplementation, respectively, we identified a significant increase of GFP puncta in older and in nitrogen-starved cultures suggesting an induction of autophagy during aging. This conclusion is supported by the demonstration of an age-related and autophagy-dependent degradation of a PaSOD1-GFP reporter protein. The deletion of Paatg1, which leads to the lack of the PaATG1 serine/threonine kinase active in early stages of autophagy induction, impairs ascospore germination and development and shortens life span. Under nitrogen-depleted conditions, life span of the wild type is increased almost 4-fold. In contrast, this effect is annihilated in the Paatg1 deletion strain, suggesting that the ability to induce autophagy is beneficial for this fungus. Collectively, our data identify autophagy as a longevity-assurance mechanism in P. anserina and as another surveillance pathway in the complex network of pathways affecting aging and development. These findings provide perspectives for the elucidation of the mechanisms involved in the regulation of individual pathways and their interactions.

The transcriptional response to nonself in the fungus Podospora anserina.

In fungi, heterokaryon incompatibility is a nonself recognition process occurring when filaments of different isolates of the same species fuse. Compatibility is controlled by so-called het loci and fusion of strains of unlike het genotype triggers a complex incompatibility reaction that leads to the death of the fusion cell. Herein, we analyze the transcriptional changes during the incompatibility reaction in Podospora anserina. The incompatibility response was found to be associated with a massive transcriptional reprogramming: 2231 genes were up-regulated by a factor 2 or more during incompatibility. In turn, 2441 genes were down-regulated. HET, NACHT, and HeLo domains previously found to be involved in the control of heterokaryon incompatibility were enriched in the up-regulated gene set. In addition, incompatibility was characterized by an up-regulation of proteolytic and other hydrolytic activities, of secondary metabolism clusters and toxins and effector-like proteins. The up-regulated set was found to be enriched for proteins lacking orthologs in other species and chromosomal distribution of the up-regulated genes was uneven with up-regulated genes residing preferentially in genomic islands and on chromosomes IV and V. There was a significant overlap between regulated genes during incompatibility in P. anserina and Neurospora crassa, indicating similarities in the incompatibility responses in these two species. Globally, this study illustrates that the expression changes occurring during cell fusion incompatibility in P. anserina are in several aspects reminiscent of those described in host-pathogen or symbiotic interactions in other fungal species.

A differential genome-wide transcriptome analysis: impact of cellular copper on complex biological processes like aging and development.

The regulation of cellular copper homeostasis is crucial in biology. Impairments lead to severe dysfunctions and are known to affect aging and development. Previously, a loss-of-function mutation in the gene encoding the copper-sensing and copper-regulated transcription factor GRISEA of the filamentous fungus Podospora anserina was reported to lead to cellular copper depletion and a pleiotropic phenotype with hypopigmentation of the mycelium and the ascospores, affected fertility and increased lifespan by approximately 60% when compared to the wild type. This phenotype is linked to a switch from a copper-dependent standard to an alternative respiration leading to both a reduced generation of reactive oxygen species (ROS) and of adenosine triphosphate (ATP). We performed a genome-wide comparative transcriptome analysis of a wild-type strain and the copper-depleted grisea mutant. We unambiguously assigned 9,700 sequences of the transcriptome in both strains to the more than 10,600 predicted and annotated open reading frames of the P. anserina genome indicating 90% coverage of the transcriptome. 4,752 of the transcripts differed significantly in abundance with 1,156 transcripts differing at least 3-fold. Selected genes were investigated by qRT-PCR analyses. Apart from this general characterization we analyzed the data with special emphasis on molecular pathways related to the grisea mutation taking advantage of the available complete genomic sequence of P. anserina. This analysis verified but also corrected conclusions from earlier data obtained by single gene analysis, identified new candidates of factors as part of the cellular copper homeostasis system including target genes of transcription factor GRISEA, and provides a rich reference source of quantitative data for further in detail investigations. Overall, the present study demonstrates the importance of systems biology approaches also in cases were mutations in single genes are analyzed to explain the underlying mechanisms controlling complex biological processes like aging and development.

Discovery of novel inhibitors of amyloid ß-peptide 1-42 aggregation.

Alzheimer's disease, characterized by deposits of amyloid ß-peptide (Aß), is the most common neurodegenerative disease, but it still lacks a specific treatment. We have discovered five chemically unrelated inhibitors of the in vitro aggregation of the Aß17-40 peptide by screening two commercial chemical libraries. Four of them (1-4) exhibit relatively low MCCs toward HeLa cells (17-184 µM). The usefulness of compounds 1-4 to inhibit the in vivo aggregation of Aß1-42 has been demonstrated using two fungi models, Saccharomyces cerevisiae and Podospora anserina, previously transformed to express Aß1-42. Estimated IC(50)s are around 1-2 µM. Interestingly, addition of any of the four compounds to sonicated preformed P. anserina aggregates completely inhibited the appearance of SDS-resistant oligomers. This combination of HTP in vitro screening with validation in fungi models provides an efficient way to identify novel inhibitory compounds of Aß1-42 aggregation for subsequent testing in animal models.

Reactive oxygen species target specific tryptophan site in the mitochondrial ATP synthase.

The release of reactive oxygen species (ROS) as side products of aerobic metabolism in the mitochondria is an unavoidable consequence. As the capacity of organisms to deal with this exposure declines with age, accumulation of molecular damage caused by ROS has been defined as one of the central events during the ageing process in biological systems as well as in numerous diseases such as Alzheimer's and Parkinson's Dementia. In the filamentous fungus Podospora anserina, an ageing model with a clear defined mitochondrial etiology of ageing, in addition to the mitochondrial aconitase the ATP synthase alpha subunit was defined recently as a hot spot for oxidative modifications induced by ROS. In this report we show, that this reactivity is not randomly distributed over the ATP Synthase, but is channeled to a single tryptophan residue 503. This residue serves as an intra-molecular quencher for oxidative species and might also be involved in the metabolic perception of oxidative stress or regulation of enzyme activity. A putative metal binding site in the proximity of this tryptophan residue appears to be crucial for the molecular mechanism for the selective targeting of oxidative damage.

PaCATB, a secreted catalase protecting Podospora anserina against exogenous oxidative stress.

A differential mass spectrometry analysis of secreted proteins from juvenile and senescent Podospora anserina cultures revealed age-related differences in protein profiles. Among other proteins with decreased abundance in the secretome of senescent cultures a catalase, termed PaCATB, was identified. Genetic modulation of the abundance of PaCATB identified differential effects on the phenotype of the corresponding strains. Deletion of PaCatB resulted in decreased resistance, over-expression in increased resistance against hydrogen peroxide. While the lifespan of the genetically modified strains was found to be unaffected under standard growth conditions, increased exogenous hydrogen peroxide stress in the growth medium markedly reduced the lifespan of the PaCatB deletion strain but extended the lifespan of PaCatB over-expressors. Overall our data identify a component of the secretome of P. anserina as a new effective factor to cope with environmental stress, stress that under natural conditions is constantly applied on organisms and influences aging processes.

Sensitivities to DMI fungicides in populations of Podosphaera fusca in south central Spain.

BACKGROUND: Cucurbit powdery mildew elicited by Podosphaera fusca (Fr.) U Braun & N Shishkoff limits crop production in Spain. Disease control is largely dependent on fungicides such as sterol demethylation inhibitors (DMIs). Fungicide resistance is an increasing problem in this pathogen. To overcome such risk, it is necessary to design rational control programmes based upon knowledge of field resistance. The aim of this study was to investigate the state of DMI sensitivity of Spanish P. fusca populations and provide tools for improved disease management. RESULTS: Using a leaf-disc assay, sensitivity to fenarimol, myclobutanil and triadimenol of 50 isolates of P. fusca was analysed to determine discriminatory concentrations between sensitive and resistant isolates. As no clearly different groups of isolates could be identified, discriminatory concentrations were established on the basis of maximum fungicide field application rate, 100 mg L(-1) for the three fungicides tested. Subsequently, a survey of DMI resistance was carried out in different provinces located in the south central area of Spain during the cucurbit growing seasons in 2002, 2003 and 2004. Examination of a collection of 250 isolates revealed that 23% were resistant to fenarimol and 7% to triadimenol, the provinces of Almería, Badajoz and Murcia being the locations with the highest frequencies of resistance. By contrast, no resistance to myclobutanil was found. CONCLUSION: Results show that fenarimol and, to a lesser extent, triadimenol have become less efficient for controlling cucurbit powdery mildew in Spain. These are important observations that should lead to reconsideration of the current disease management programmes.

Increasing mitochondrial superoxide dismutase abundance leads to impairments in protein quality control and ROS scavenging systems and to lifespan shortening.

The fungal aging model Podospora anserina contains three superoxide dismutases (SODs) in different cellular compartments. While PaSOD1 represents the Cu/Zn isoform located in the cytoplasm and in the mitochondrial inter-membrane space, PaSOD2 localizes to the perinuclear ER. PaSOD3, a protein with a manganese binding domain and a mitochondrial targeting sequence (MTS) is the mitochondrial SOD. Over-expression of PaSod3 leads to lifespan reduction and increased sensitivity against paraquat and hydrogen peroxide. The negative effects of PaSod3 over-expression correlate with a strong reduction in the abundance of mitochondrial peroxiredoxin, PaPRX1, and the matrix protease PaCLPP disclosing impairments of mitochondrial quality control and ROS scavenging pathways in PaSod3 over-expressors. Deletion of PaSod3 leads to increased paraquat sensitivity while hydrogen peroxide sensitivity and lifespan are not significantly changed when compared to the wild-type strain. These latter characteristics are unexpected and challenge the 'mitochondrial free radical theory of aging'.

Increasing organismal healthspan by enhancing mitochondrial protein quality control.

Degradation of damaged proteins by members of the protein quality control system is of fundamental importance in maintaining cellular homeostasis. In mitochondria, organelles which both generate and are targets of reactive oxygen species (ROS), a number of membrane bound and soluble proteases are essential components of this system. Here we describe the regulation of Podospora anserina LON (PaLON) levels, an AAA(+) family serine protease localized in the matrix fraction of mitochondria. Constitutive overexpression of PaLon results in transgenic strains of the fungal ageing model P. anserina showing increased ATP-dependent serine protease activity. These strains display lower levels of carbonylated (aconitase) and carboxymethylated proteins, reduced secretion of hydrogen peroxide and a higher resistance against exogenous oxidative stress. Moreover, they are characterized by an extended lifespan without impairment of vital functions such as respiration, growth and fertility. The reported genetic manipulation proved to be a successful intervention in organismal ageing and it led to an increase in the healthy lifespan, the healthspan, of P. anserina.

Mitochondria-targeted plastoquinone derivatives as tools to interrupt execution of the aging program. 5. SkQ1 prolongs lifespan and prevents development of traits of senescence.

Very low (nano- and subnanomolar) concentrations of 10-(6'-plastoquinonyl) decyltriphenylphosphonium (SkQ1) were found to prolong lifespan of a fungus (Podospora anserina), a crustacean (Ceriodaphnia affinis), an insect (Drosophila melanogaster), and a mammal (mouse). In the latter case, median lifespan is doubled if animals live in a non-sterile vivarium. The lifespan increase is accompanied by rectangularization of the survival curves (an increase in survival is much larger at early than at late ages) and disappearance of typical traits of senescence or retardation of their development. Data summarized here and in the preceding papers of this series suggest that mitochondria-targeted antioxidant SkQ1 is competent in slowing down execution of an aging program responsible for development of age-related senescence.

Deletion of putative apoptosis factors leads to lifespan extension in the fungal ageing model Podospora anserina.

Podospora anserina is a filamentous fungus with a limited lifespan. After a strain-specific period of growth, cultures turn to senescence and ultimately die. Here we provide evidence that the last step in the ageing of P. anserina is not accidental but programmed. In this study, PaAMID1, a homologue of a mammalian 'AIF-homologous mitochondrion-associated inducer of death', was analysed as a putative member of a caspase-independent signalling pathway. In addition, two metacaspases, PaMCA1 and PaMCA2, were investigated. While deletion of PaAmid1 as well as of PaMca2 was found to result in a moderate lifespan extension (59% and 78%, respectively), a 148% increase in lifespan was observed after deletion of PaMca1. Measurement of arginine-specific protease activity demonstrates a metacaspase-dependent activity in senescent but not in juvenile cultures, pointing to an activation of these proteases in the senescent stage of the life cycle. Moreover, treatment of juvenile wild-type cultures with hydrogen peroxide leads to a PaMCA1-dependent activity. The presented data strongly suggest that death of senescent wild-type cultures is triggered by an apoptotic programme induced by an age-dependent increase of reactive oxygen species during ageing of cultures and is executed after metacaspase activation.

A non-Q/N-rich prion domain of a foreign prion, [Het-s], can propagate as a prion in yeast.

Prions are self-propagating, infectious aggregates of misfolded proteins. The mammalian prion, PrP(Sc), causes fatal neurodegenerative disorders. Fungi also have prions. While yeast prions depend upon glutamine/asparagine (Q/N)-rich regions, the Podospora anserina HET-s and PrP prion proteins lack such sequences. Nonetheless, we show that the HET-s prion domain fused to GFP propagates as a prion in yeast. Analogously to native yeast prions, transient overexpression of the HET-s fusion induces ring-like aggregates that propagate in daughter cells as cytoplasmically inherited, detergent-resistant dot aggregates. Efficient dot propagation, but not ring formation, is dependent upon the Hsp104 chaperone. The yeast prion [PIN(+)] enhances HET-s ring formation, suggesting that prions with and without Q/N-rich regions interact. Finally, HET-s aggregates propagated in yeast are infectious when introduced into Podospora. Taken together, these results demonstrate prion propagation in a truly foreign host. Since yeast can host non-Q/N-rich prions, such native yeast prions may exist.

Impact of ROS on ageing of two fungal model systems: Saccharomyces cerevisiae and Podospora anserina.

To provide a foundation for the development of effective interventions to counteract various age-related diseases in humans, ageing processes have been extensively studied in various model organisms and systems. However, the mechanisms underlying ageing are still not unravelled in detail in any system including rather simple organisms. In this article, we review some of the molecular mechanisms that were found to affect ageing in two fungal models, the unicellular ascomycete Saccharomyces cerevisiae and the filamentous ascomycete Podospora anserina. A selection of issues like retrograde response, genomic instability, caloric restriction, mtDNA reorganisation and apoptosis is presented and discussed with special emphasis on the role reactive oxygen species (ROS) play in these diverse molecular pathways.

Podospora anserina target of rapamycin.

We have isolated the Podospora anserina TOR gene. The PaTOR protein displayed strong identities with TOR proteins from other eukaryotes especially in the FRB domain and the kinase domain. Genome analysis suggests that a single TOR gene exists in Podospora. The serine residue known to be one site of missense mutations conferring rapamycin resistance in other organisms is conserved in the PaTOR protein (S1895). A PaTOR-S1895R mutated allele has been constructed and introduced in the wild-type strain, as expected strains expressing the PaTOR-S1895R gene become resistant to rapamycin. The dominance of the PaTOR-S1895R allele indicates that apparently the mutation does not impair the kinase activity. We confirm that all cytological modifications associated with rapamycin treatment in Podospora are indeed mediated by PaTOR. We conclude that the PaTOR gene is likely to be essential and that rapamycin treatment might be useful to further investigate rapamycin-sensitive TOR functions in Podospora and especially newly identified rapamycin-sensitive functions such as the autophagy-independent control of vacuole remodeling and septation.