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Find video protocols related to scientific articles indexed in Pubmed.
The role of mitochondria in fungal aging.
Curr. Opin. Microbiol.
PUBLISHED: 06-25-2014
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Time-dependent impairments of mitochondrial function play a key role in biological aging. Work on fungal aging models has been instrumental in unraveling basic mechanisms leading to mitochondrial dysfunction and the identification of different pathways active in keeping mitochondria 'healthy' over time. Pathways including those involved in reactive oxygen scavenging, repair of damage, proteostasis, mitochondrial dynamics, and biogenesis, are interconnected and part of a complex quality control system. The individual components of this network are limited in capacity. However, if the capacity of one pathway is overwhelmed, another one may be activated. The mechanisms controlling the underlying cross-talk are poorly understood and subject of intensive investigation.
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Identification of autophagy as a longevity-assurance mechanism in the aging model Podospora anserina.
Autophagy
PUBLISHED: 02-27-2014
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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.
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Proteomic analysis of mitochondria from senescent Podospora anserina casts new light on ROS dependent aging mechanisms.
Exp. Gerontol.
PUBLISHED: 02-04-2014
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The mitochondrial free radical theory of aging (MFRTA) states that reactive oxygen species (ROS) generated at the respiratory electron transport chain are active in causing age-related damage of biomolecules like lipids, nucleic acids and proteins. Accumulation of this kind of damage results in functional impairments, aging and death of biological systems. Here we report data of an analysis to monitor the age-related quantitative protein composition of the mitochondria of the fungal aging model Podospora anserina. The impact of senescence on mitochondrial protein composition was analyzed by LC-MS. In an untargeted proteomic approach, we identified 795 proteins in samples from juvenile and senescent wild-type cultures and obtained quantitative information for 226 of these proteins by spectral counting. Despite the broad coverage of the proteome, no substantial changes in known age-related pathways could be observed. For a more detailed analysis, a targeted proteome analysis was applied focusing on 15 proteins from respiratory, ROS-scavenging and quality control pathways. Analyzing six distinct age-stages from juvenile to senescent P. anserina cultures revealed low, but statistically significant changes for the mitochondrial respiratory complexes. A P. anserina PaSod3 over-expression mutant with a phenotype of mitochondrial ROS over-production was used for biological evaluation of changes observed during aging. LC-MS analysis of the mutant revealed severe changes to the mitochondrial proteome--substantially larger than observed during senescence. Interestingly the amount of ATP synthase subunit g, involved in cristae formation is significantly decreased in the mutant implicating ROS-induced impairments in ATP synthase dimer and cristae formation. The difference between protein-profiles of aging wild type and ROS stressed mutant suggests that oxidative stress within the mitochondria is not the dominating mechanism for the aging process in P. anserina. Collectively, while our data do not exclude an effect of ROS on specific proteins and in signaling and control of pathways which are governing aging of P. anserina, it contradicts increasing ROS as a cause of a gross general and non-selective accumulation of damaged proteins during senescence. Instead, ROS may be effective by controlling specific regulators of mitochondrial function.
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Loss of mitochondrial peptidase Clpp leads to infertility, hearing loss plus growth retardation via accumulation of CLPX, mtDNA and inflammatory factors.
Hum. Mol. Genet.
PUBLISHED: 07-12-2013
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The caseinolytic peptidase P (CLPP) is conserved from bacteria to humans. In the mitochondrial matrix, it multimerizes and forms a macromolecular proteasome-like cylinder together with the chaperone CLPX. In spite of a known relevance for the mitochondrial unfolded protein response, its substrates and tissue-specific roles are unclear in mammals. Recessive CLPP mutations were recently observed in the human Perrault variant of ovarian failure and sensorineural hearing loss. Here, a first characterization of CLPP null mice demonstrated complete female and male infertility and auditory deficits. Disrupted spermatogenesis already at the spermatid stage and ovarian follicular differentiation failure were evident. Reduced pre-/post-natal survival and marked ubiquitous growth retardation contrasted with only light impairment of movement and respiratory activities. Interestingly, the mice showed resistance to ulcerative dermatitis. Systematic expression studies detected up-regulation of other mitochondrial chaperones, accumulation of CLPX and mtDNA as well as inflammatory factors throughout tissues. T-lymphocytes in the spleen were activated. Thus, murine Clpp deletion represents a faithful Perrault model. The disease mechanism probably involves deficient clearance of mitochondrial components and inflammatory tissue destruction.
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Age-related changes in the mitochondrial proteome of the fungus Podospora anserina analyzed by 2D-DIGE and LC-MS/MS.
J Proteomics
PUBLISHED: 05-15-2013
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Many questions concerning the molecular processes during biological aging remain unanswered. Since mitochondria are central players in aging, we applied quantitative two-dimensional difference gel electrophoresis (2D-DIGE) coupled to protein identification by mass spectrometry to study the age-dependent changes in the mitochondrial proteome of the fungus Podospora anserina - a well-established aging model. 67 gel spots exhibited significant, but remarkably moderate intensity changes. While typically the observed changes in protein abundance occurred progressively with age, for several proteins a pronounced change was observed at late age, sometimes inverting the trend observed at younger age. The identified proteins were assigned to a wide range of metabolic pathways including several implicated previously in biological aging. An overall decrease for subunits of complexes I and V of oxidative phosphorylation was confirmed by Western blot analysis and blue-native electrophoresis. Changes in several groups of proteins suggested a general increase in protein biosynthesis possibly reflecting a compensatory mechanism for increased quality control-related protein degradation at later age. Age-related augmentation in abundance of proteins involved in biosynthesis, folding, and protein degradation pathways sustain these observations. Furthermore, a significant decrease of two enzymes involved in the degradation of ?-aminobutyrate (GABA) supported its previously suggested involvement in biological aging.
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Human CLPP reverts the longevity phenotype of a fungal ClpP deletion strain.
Nat Commun
PUBLISHED: 01-31-2013
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Mitochondrial maintenance crucially depends on the quality control of proteins by various chaperones, proteases and repair enzymes. While most of the involved components have been studied in some detail, little is known on the biological role of the CLPXP protease complex located in the mitochondrial matrix. Here we show that deletion of PaClpP, encoding the CLP protease proteolytic subunit CLPP, leads to an unexpected healthy phenotype and increased lifespan of the fungal ageing model organism Podospora anserina. This phenotype can be reverted by expression of human ClpP in the fungal deletion background, demonstrating functional conservation of human and fungal CLPP. Our results show that the biological role of eukaryotic CLP proteases can be studied in an experimentally accessible model organism.
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Assessing organismal aging in the filamentous fungus Podospora anserina.
Methods Mol. Biol.
PUBLISHED: 01-09-2013
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Podospora anserina is an extensively studied model organism to unravel the mechanism of organismal aging. This filamentous fungus is short-lived and accessible to experimentation. Aging and lifespan are controlled by genetic and environmental traits and, in this model, have a strong mitochondrial etiology. Here, we describe methods and protocols to manipulate and study the aging process in P. anserina at different levels including biochemistry, cell biology, genetics, and physiology.
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A Genome-Wide Longitudinal Transcriptome Analysis of the Aging Model Podospora anserine.
PLoS ONE
PUBLISHED: 01-01-2013
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Aging of biological systems is controlled by various processes which have a potential impact on gene expression. Here we report a genome-wide transcriptome analysis of the fungal aging model Podospora anserina. Total RNA of three individuals of defined age were pooled and analyzed by SuperSAGE (serial analysis of gene expression). A bioinformatics analysis identified different molecular pathways to be affected during aging. While the abundance of transcripts linked to ribosomes and to the proteasome quality control system were found to decrease during aging, those associated with autophagy increase, suggesting that autophagy may act as a compensatory quality control pathway. Transcript profiles associated with the energy metabolism including mitochondrial functions were identified to fluctuate during aging. Comparison of wild-type transcripts, which are continuously down-regulated during aging, with those down-regulated in the long-lived, copper-uptake mutant grisea, validated the relevance of age-related changes in cellular copper metabolism. Overall, we (i) present a unique age-related data set of a longitudinal study of the experimental aging model P. anserina which represents a reference resource for future investigations in a variety of organisms, (ii) suggest autophagy to be a key quality control pathway that becomes active once other pathways fail, and (iii) present testable predictions for subsequent experimental investigations.
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Reactive oxygen species target specific tryptophan site in the mitochondrial ATP synthase.
Biochim. Biophys. Acta
PUBLISHED: 09-12-2011
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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 Alzheimers and Parkinsons 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.
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Cyclophilin D links programmed cell death and organismal aging in Podospora anserina.
Aging Cell
PUBLISHED: 08-15-2010
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Cyclophilin D (CYPD) is a mitochondrial peptidyl prolyl-cis,trans-isomerase involved in opening of the mitochondrial permeability transition pore (mPTP). CYPD abundance increases during aging in mammalian tissues and in the aging model organism Podospora anserina. Here, we show that treatment of the P. anserina wild-type with low concentrations of the cyclophilin inhibitor cyclosporin A (CSA) extends lifespan. Transgenic strains overexpressing PaCypD are characterized by reduced stress tolerance, suffer from pronounced mitochondrial dysfunction and are characterized by accelerated aging and induction of cell death. Treatment with CSA leads to correction of mitochondrial function and lifespan to that of the wild-type. In contrast, PaCypD deletion strains are not affected by CSA within the investigated concentration range and show increased resistance against inducers of oxidative stress and cell death. Our data provide a mechanistic link between programmed cell death (PCD) and organismal aging and bear implications for the potential use of CSA to intervene into biologic aging.
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Overexpression of PaParp encoding the poly(ADP-ribose) polymerase of Podospora anserina affects organismal aging.
Mech. Ageing Dev.
PUBLISHED: 07-23-2010
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Poly(ADP-ribose) polymerases (PARPs) are a diverse group of proteins present in all multicellular eukaryotes. They catalyze the NAD(+)-dependent modification of proteins with poly(ADP-ribose). Poly(ADP-ribosyl)ation plays a key role in a plethora of processes including DNA repair, tumor progression and aging. Here we report that PaPARP, the single protein with a PARP catalytic domain, in the fungal aging model Podospora anserina, indeed displays a NAD(+)-dependent poly(ADP-ribose) polymerase activity. While unable to select a PaParp deletion strain, we succeeded in the generation of PaParp overexpressors. Biochemically these strains are characterized by reduced mitochondrial membrane potential and a lowered ATP content. They show an increased sensitivity against different stressors including the DNA damaging agent phleomycin, the reactive oxygen generator paraquat, and the apoptosis inducer farnesol. PaParp overexpressors are impaired in growth, in pigmentation and fertility, and have a shortened lifespan. Our results demonstrate the relevance of poly(ADP-ribose) metabolism for aging and development in P. anserina. With a single PARP this metabolism is less complex than in higher eukaryotes and thus P. anserina appears to be a promising system to connect basic PARP functions with the well established network of pathways relevant for organismal aging.
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Mitochondrial pathways governing stress resistance, life, and death in the fungal aging model Podospora anserina.
Ann. N. Y. Acad. Sci.
PUBLISHED: 06-12-2010
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Work from more than 50 years of research has unraveled a number of molecular pathways that are involved in controlling aging of the fungal model system Podospora anserina. Early research revealed that wild-type strain aging is linked to gross reorganization of the mitochondrial DNA. Later it was shown that aging of P. anserina does also take place, although at a slower pace, when the wild-type specific mitochondrial DNA rearrangements do not occur. Now it is clear that a network of different pathways is involved in the control of aging. Branches of these pathways appear to be connected and constitute a hierarchical system of responses. Although cross talk between the individual pathways seems to be fundamental in the coordination of the overall system, the precise underlying interactions remain to be unraveled. Such a systematic approach aims at a holistic understanding of the process of biological aging, the ultimate goal of modern systems biology.
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Deletion of PaAif2 and PaAmid2, two genes encoding mitochondrial AIF-like oxidoreductases of Podospora anserina, leads to increased stress tolerance and lifespan extension.
Curr. Genet.
PUBLISHED: 03-02-2010
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Wild-type strains of the ascomycete Podospora anserina are characterized by a limited lifespan. Mitochondria play a central role in this ageing process raising the question of whether apoptosis-like processes, which are also connected to mitochondrial function, are involved in the control of the final stage in the fungal life cycle. While a role of two metacaspases in apoptosis and lifespan control was recently demonstrated in P. anserina, virtually nothing is known about the function of the protein family of apoptosis-inducing factors (AIFs). Here we report data about proteins belonging to this family. We demonstrate that the cytosolic members PaAIF1 and PaAMID1 do not affect lifespan. In contrast, loss of PaAIF2 and PaAMID2, which both were localized to mitochondria, are characterized by a significantly increased ROS tolerance and a prolonged lifespan. In addition, deletion of PaAmid2 severely affects sporogenesis. These data identify components of a caspase-independent molecular pathway to be involved in developmental processes and in the induction of programmed cell death in the senescent stage of P. anserina.
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Increasing mitochondrial superoxide dismutase abundance leads to impairments in protein quality control and ROS scavenging systems and to lifespan shortening.
Exp. Gerontol.
PUBLISHED: 01-08-2010
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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.
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A differential genome-wide transcriptome analysis: impact of cellular copper on complex biological processes like aging and development.
PLoS ONE
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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.
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A systems biological analysis links ROS metabolism to mitochondrial protein quality control.
Mech. Ageing Dev.
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The analyses of previously generated Podospora anserina strains in which the mitochondrial superoxide dismutase, PaSOD3, is increased in abundance, revealed unexpected results, which, at first glance, are contradictory to the free radical theory of aging (FRTA). To re-analyze these results, we performed additional experiments and developed a mathematical model consisting of a set of differential equations describing the time course of various ROS (reactive oxygen species), components of the cellular antioxidant system (PaSOD3 and mitochondrial peroxiredoxin, PaPRX1), and PaCLPP, a mitochondrial matrix protease involved in protein quality control. Incorporating these components we could identify a positive feed-back loop and demonstrate that the role of superoxide as the primary ROS responsible for age-related molecular damage is more complicated than originally stated by the FRTA. Our study is a first step towards the integration of the various pathways known to be involved in the control of biological aging.
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Mitochondrial quality control: an integrated network of pathways.
Trends Biochem. Sci.
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Mitochondria are organelles of eukaryotic cells with various functions. Best known is their role in energy transduction leading to the formation of ATP. As byproducts of this process, reactive oxygen species (ROS) are formed that can damage different types of molecules leading to mitochondrial dysfunction. Different quality control (QC) mechanisms keep mitochondria functional. Although several components involved in mitochondrial QC have been characterized in some detail, others remain to be integrated into what is currently emerging as a hierarchical network of interacting pathways. The elucidation of this network holds the key to the understanding of complex biological processes such as aging and the development of age-related diseases.
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