The caseinolytic peptidase P (CLPP) is conserved from bacteria to humans. In the mitochondrial ma... more 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.
<p>(a) Common down-regulated genes within the age-dependent transcriptome of the wild-type ... more <p>(a) Common down-regulated genes within the age-dependent transcriptome of the wild-type ‘s’ and the long-lived mutant grisea of <i>P. anserina</i>. 556 genes of the grisea transcriptome were identified to be down-regulated with a factor of at least 3 and a p-value of ≤0.01 for differential expression <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0083109#pone.0083109-Servos1" target="_blank">[22]</a>. The 1,202 genes, identified as being down-regulated during aging in this study, have 89 genes in common. (b) 10,000 simulated experiments with two sets of randomly picked gene names, each set of the same size as the two sets (wild type, grisea), were applied. The box plot shows that the 89 common genes were extreme outliers. (c) The results of the random experiments are normally distributed with a mean of μ≈66.05 and a standard deviation of σ≈7.39. Our determined value of 89 is greater than three times sigma, indicating that the corresponding genes do not occur randomly. (d) All enriched GO terms in the set of the 89 common genes with a p-value ≤0.01 are depicted. Copper associated terms can be found on the very top. (e) Four genes already identified as putative target genes of transcription factor GRISEA are found within these 89 common genes. Due to its irregular expression profile, the gene, encoding the putative copper transporter PaCTR1, is not among the 89 common genes.</p
<p>The figure depicts the enrichment map for the corresponding GO enrichment analysis. Anal... more <p>The figure depicts the enrichment map for the corresponding GO enrichment analysis. Analogous to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0083109#pone-0083109-g004" target="_blank">Figure 4</a>, each node represents one GO term and node colors correspond to the group and the degree of significance. Edge thickness indicates the amount of genes shared by two terms and node sizes are proportional to the number of genes assigned to the corresponding term. Striking groups were subsequently circled and labelled.</p
Function of mitochondria largely depends on a characteristic ultrastructure with typical invagina... more Function of mitochondria largely depends on a characteristic ultrastructure with typical invaginations, namely the cristae of the inner mitochondrial membrane. The mitochondrial signature phospholipid cardiolipin (CL), the F1Fo-ATP-synthase, and the ‘mitochondrial contact site and cristae organizing system’ (MICOS) complex are involved in this process. Previous studies with Podospora anserina demonstrated that manipulation of MICOS leads to altered cristae structure and prolongs lifespan. While longevity of Mic10-subcomplex mutants is induced by mitohormesis, the underlying mechanism in the Mic60-subcomplex deletion mutants was unclear. Since several studies indicated a connection between MICOS and phospholipid composition, we now analyzed the impact of MICOS on mitochondrial phospholipid metabolism. Data from lipidomic analysis identified alterations in phospholipid profile and acyl composition of CL in Mic60-subcomplex mutants. These changes appear to have beneficial effects on me...
A global depletion of cellular copper as the result of a deficiency in high-affinity copper uptak... more A global depletion of cellular copper as the result of a deficiency in high-affinity copper uptake was previously shown to affect the phenotype and life span of the filamentous fungusPodospora anserina. We report here the construction of a strain in which the delivery of copper to complex IV of the mitochondrial respiratory chain is affected. This strain, PaCox17::ble, is aPaCox17-null mutant that does not synthesize the molecular chaperone targeting copper to cytochromecoxidase subunit II. PaCox17::ble is characterized by a decreased growth rate, a reduction in aerial hyphae formation, reduced female fertility, and a dramatic increase in life span. The mutant respires via a cyanide-resistant alternative pathway, displays superoxide dismutase (SOD) activity profiles significantly differing from those of the wild-type strain and is characterized by a stabilization of the mitochondrial DNA. Collectively, the presented data define individual components of a molecular network effective ...
The maintenance of cellular homeostasis over time is essential to avoid the degeneration of biolo... more The maintenance of cellular homeostasis over time is essential to avoid the degeneration of biological systems leading to aging and disease. Several interconnected pathways are active in this kind of quality control. One of them is autophagy, the vacuolar degradation of cellular components. The absence of the sorting nexin PaATG24 (SNX4 in other organisms) has been demonstrated to result in impairments in different types of autophagy and lead to a shortened lifespan. In addition, the growth rate and the size of vacuoles are strongly reduced. Here, we report how an oleic acid diet leads to longevity of the wild type and a PaAtg24 deletion mutant (ΔPaAtg24). The lifespan extension is linked to altered membrane trafficking, which abrogates the observed autophagy defects in ΔPaAtg24 by restoring vacuole size and the proper localization of SNARE protein PaSNC1. In addition, an oleic acid diet leads to an altered use of the mitochondrial respiratory chain: complex I and II are bypassed, l...
Centre for Molecular and Biomolecular Informatics, and Department of Biochemistry (286), Radboud ... more Centre for Molecular and Biomolecular Informatics, and Department of Biochemistry (286), Radboud Institute for Molecular Life Sciences, Radboud University Medical Centre, 6500 HB Nijmegen, The Netherlands Department of Clinical Genetics, Unit Clinical Genomics, Maastricht University Medical Centre, 6200 MD Maastricht, The Netherlands Faculty for Biosciences and Cluster of Excellence ‘Macromolecular Complexes’, Goethe University, Molecular Developmental Biology, 60438 Frankfurt am Main, Germany
Mitochondria are organelles of eukaryotic cells with various functions. Best known is their role ... more 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.
The regulation of cellular copper homeostasis is crucial in biology. Impairments lead to severe d... more 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.
aging process. The success of such a systems biology approach is strongly dependent on the develo... more aging process. The success of such a systems biology approach is strongly dependent on the development of methods for data mining and an efficient analysis and modeling of the huge data sets that are raised.
able or can be generated by specific genetic manipulation. Already in the late 70’s and early 80’... more able or can be generated by specific genetic manipulation. Already in the late 70’s and early 80’s of last century, clear evidence for mitochondrial pathways involved in lifespan extension has been provided from for example fungi like Podospora anserina [6-7], Neurospora crassa or baker’s yeast Saccharomyces cerevisiae. Later on, flies [8] and the worm Caenorhabditis elegans [9] turned out to be very attractive systems to elucidate molecular pathways effective in governing lifespan and aging. The current view about the value of these systems is that, although less complex and shortlived, the comparison of mechanisms active in these systems can be expected to unravel the most basic molecular pathways which have been conserved during evolution from simple to complex organisms. Thus, unraveling these mechanisms in simple models provides a key Editorial: Mitochondria in aging and age-related diseases
Aging of biological systems is a fundamental process controlled by a complex network of molecular... more Aging of biological systems is a fundamental process controlled by a complex network of molecular pathways. In the filamentous fungus Podospora anserina, a model in which organismal aging can conveniently be analysed, mitochondria play a central role. A wide range of relevant pathways were identified that contribute to the maintenance of a population of functional mitochondria. These pathways act in a hierarchical manner, but all the pathways are limited in capacity. At the end of the life cycle, when the various surveillance pathways are overwhelmed and damage has passed certain thresholds, programmed cell death brings the life of individual P. anserina to an end.
The two metacaspases MCA1 andMCA2 of the fungal aging model organism Podospora anserina (PaMCA1 a... more The two metacaspases MCA1 andMCA2 of the fungal aging model organism Podospora anserina (PaMCA1 and PaMCA2, re-spectively) have previously been demonstrated to be involved in the control of programmed cell death (PCD) and life span. In order to identify specific pathways and components which are controlled by the activity of these enzymes, we set out to charac-terize them further. Heterologous overexpression in Escherichia coli of the two metacaspase genes resulted in the production of proteins which aggregate and form inclusion bodies fromwhich the active protein has been recovered via refolding in appropri-ate buffers. The renaturated proteins are characterized by an arginine-specific activity and are active in caspase-like self-matura-tion leading to the generation of characteristic small protein fragments. Both activities are dependent on the presence of calcium. Incubation of the twometacaspases with recombinant poly(ADP-ribose) polymerase (PARP), a known substrate of mammalian...
The caseinolytic peptidase P (CLPP) is conserved from bacteria to humans. In the mitochondrial ma... more 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.
<p>(a) Common down-regulated genes within the age-dependent transcriptome of the wild-type ... more <p>(a) Common down-regulated genes within the age-dependent transcriptome of the wild-type ‘s’ and the long-lived mutant grisea of <i>P. anserina</i>. 556 genes of the grisea transcriptome were identified to be down-regulated with a factor of at least 3 and a p-value of ≤0.01 for differential expression <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0083109#pone.0083109-Servos1" target="_blank">[22]</a>. The 1,202 genes, identified as being down-regulated during aging in this study, have 89 genes in common. (b) 10,000 simulated experiments with two sets of randomly picked gene names, each set of the same size as the two sets (wild type, grisea), were applied. The box plot shows that the 89 common genes were extreme outliers. (c) The results of the random experiments are normally distributed with a mean of μ≈66.05 and a standard deviation of σ≈7.39. Our determined value of 89 is greater than three times sigma, indicating that the corresponding genes do not occur randomly. (d) All enriched GO terms in the set of the 89 common genes with a p-value ≤0.01 are depicted. Copper associated terms can be found on the very top. (e) Four genes already identified as putative target genes of transcription factor GRISEA are found within these 89 common genes. Due to its irregular expression profile, the gene, encoding the putative copper transporter PaCTR1, is not among the 89 common genes.</p
<p>The figure depicts the enrichment map for the corresponding GO enrichment analysis. Anal... more <p>The figure depicts the enrichment map for the corresponding GO enrichment analysis. Analogous to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0083109#pone-0083109-g004" target="_blank">Figure 4</a>, each node represents one GO term and node colors correspond to the group and the degree of significance. Edge thickness indicates the amount of genes shared by two terms and node sizes are proportional to the number of genes assigned to the corresponding term. Striking groups were subsequently circled and labelled.</p
Function of mitochondria largely depends on a characteristic ultrastructure with typical invagina... more Function of mitochondria largely depends on a characteristic ultrastructure with typical invaginations, namely the cristae of the inner mitochondrial membrane. The mitochondrial signature phospholipid cardiolipin (CL), the F1Fo-ATP-synthase, and the ‘mitochondrial contact site and cristae organizing system’ (MICOS) complex are involved in this process. Previous studies with Podospora anserina demonstrated that manipulation of MICOS leads to altered cristae structure and prolongs lifespan. While longevity of Mic10-subcomplex mutants is induced by mitohormesis, the underlying mechanism in the Mic60-subcomplex deletion mutants was unclear. Since several studies indicated a connection between MICOS and phospholipid composition, we now analyzed the impact of MICOS on mitochondrial phospholipid metabolism. Data from lipidomic analysis identified alterations in phospholipid profile and acyl composition of CL in Mic60-subcomplex mutants. These changes appear to have beneficial effects on me...
A global depletion of cellular copper as the result of a deficiency in high-affinity copper uptak... more A global depletion of cellular copper as the result of a deficiency in high-affinity copper uptake was previously shown to affect the phenotype and life span of the filamentous fungusPodospora anserina. We report here the construction of a strain in which the delivery of copper to complex IV of the mitochondrial respiratory chain is affected. This strain, PaCox17::ble, is aPaCox17-null mutant that does not synthesize the molecular chaperone targeting copper to cytochromecoxidase subunit II. PaCox17::ble is characterized by a decreased growth rate, a reduction in aerial hyphae formation, reduced female fertility, and a dramatic increase in life span. The mutant respires via a cyanide-resistant alternative pathway, displays superoxide dismutase (SOD) activity profiles significantly differing from those of the wild-type strain and is characterized by a stabilization of the mitochondrial DNA. Collectively, the presented data define individual components of a molecular network effective ...
The maintenance of cellular homeostasis over time is essential to avoid the degeneration of biolo... more The maintenance of cellular homeostasis over time is essential to avoid the degeneration of biological systems leading to aging and disease. Several interconnected pathways are active in this kind of quality control. One of them is autophagy, the vacuolar degradation of cellular components. The absence of the sorting nexin PaATG24 (SNX4 in other organisms) has been demonstrated to result in impairments in different types of autophagy and lead to a shortened lifespan. In addition, the growth rate and the size of vacuoles are strongly reduced. Here, we report how an oleic acid diet leads to longevity of the wild type and a PaAtg24 deletion mutant (ΔPaAtg24). The lifespan extension is linked to altered membrane trafficking, which abrogates the observed autophagy defects in ΔPaAtg24 by restoring vacuole size and the proper localization of SNARE protein PaSNC1. In addition, an oleic acid diet leads to an altered use of the mitochondrial respiratory chain: complex I and II are bypassed, l...
Centre for Molecular and Biomolecular Informatics, and Department of Biochemistry (286), Radboud ... more Centre for Molecular and Biomolecular Informatics, and Department of Biochemistry (286), Radboud Institute for Molecular Life Sciences, Radboud University Medical Centre, 6500 HB Nijmegen, The Netherlands Department of Clinical Genetics, Unit Clinical Genomics, Maastricht University Medical Centre, 6200 MD Maastricht, The Netherlands Faculty for Biosciences and Cluster of Excellence ‘Macromolecular Complexes’, Goethe University, Molecular Developmental Biology, 60438 Frankfurt am Main, Germany
Mitochondria are organelles of eukaryotic cells with various functions. Best known is their role ... more 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.
The regulation of cellular copper homeostasis is crucial in biology. Impairments lead to severe d... more 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.
aging process. The success of such a systems biology approach is strongly dependent on the develo... more aging process. The success of such a systems biology approach is strongly dependent on the development of methods for data mining and an efficient analysis and modeling of the huge data sets that are raised.
able or can be generated by specific genetic manipulation. Already in the late 70’s and early 80’... more able or can be generated by specific genetic manipulation. Already in the late 70’s and early 80’s of last century, clear evidence for mitochondrial pathways involved in lifespan extension has been provided from for example fungi like Podospora anserina [6-7], Neurospora crassa or baker’s yeast Saccharomyces cerevisiae. Later on, flies [8] and the worm Caenorhabditis elegans [9] turned out to be very attractive systems to elucidate molecular pathways effective in governing lifespan and aging. The current view about the value of these systems is that, although less complex and shortlived, the comparison of mechanisms active in these systems can be expected to unravel the most basic molecular pathways which have been conserved during evolution from simple to complex organisms. Thus, unraveling these mechanisms in simple models provides a key Editorial: Mitochondria in aging and age-related diseases
Aging of biological systems is a fundamental process controlled by a complex network of molecular... more Aging of biological systems is a fundamental process controlled by a complex network of molecular pathways. In the filamentous fungus Podospora anserina, a model in which organismal aging can conveniently be analysed, mitochondria play a central role. A wide range of relevant pathways were identified that contribute to the maintenance of a population of functional mitochondria. These pathways act in a hierarchical manner, but all the pathways are limited in capacity. At the end of the life cycle, when the various surveillance pathways are overwhelmed and damage has passed certain thresholds, programmed cell death brings the life of individual P. anserina to an end.
The two metacaspases MCA1 andMCA2 of the fungal aging model organism Podospora anserina (PaMCA1 a... more The two metacaspases MCA1 andMCA2 of the fungal aging model organism Podospora anserina (PaMCA1 and PaMCA2, re-spectively) have previously been demonstrated to be involved in the control of programmed cell death (PCD) and life span. In order to identify specific pathways and components which are controlled by the activity of these enzymes, we set out to charac-terize them further. Heterologous overexpression in Escherichia coli of the two metacaspase genes resulted in the production of proteins which aggregate and form inclusion bodies fromwhich the active protein has been recovered via refolding in appropri-ate buffers. The renaturated proteins are characterized by an arginine-specific activity and are active in caspase-like self-matura-tion leading to the generation of characteristic small protein fragments. Both activities are dependent on the presence of calcium. Incubation of the twometacaspases with recombinant poly(ADP-ribose) polymerase (PARP), a known substrate of mammalian...
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