Disruption of the MICOS complex leads to an aberrant cristae structure and an unexpected, pronounced lifespan extension in Podospora anserina.

Mitochondria are dynamic eukaryotic organelles involved in a variety of essential cellular processes including the generation of adenosine triphosphate (ATP) and reactive oxygen species as well as in the control of apoptosis and autophagy. Impairments of mitochondrial functions lead to aging and disease. Previous work with the ascomycete Podospora anserina demonstrated that mitochondrial morphotype as well as mitochondrial ultrastructure change during aging. The latter goes along with an age-dependent reorganization of the inner mitochondrial membrane leading to a change from lamellar cristae to vesicular structures. Particularly from studies with yeast, it is known that besides the F1 Fo -ATP-synthase and the phospholipid cardiolipin also the "mitochondrial contact site and cristae organizing system" (MICOS) complex, existing of the Mic60- and Mic10-subcomplex, is essential for proper cristae formation. In the present study, we aimed to understand the mechanistic basis of age-related changes in the mitochondrial ultrastructure. We observed that MICOS subunits are coregulated at the posttranscriptional level. This regulation partially depends on the mitochondrial iAAA-protease PaIAP. Most surprisingly, we made the counterintuitive observation that, despite the loss of lamellar cristae and of mitochondrial impairments, the ablation of MICOS subunits (except for PaMIC12) leads to a pronounced lifespan extension. Moreover, simultaneous ablation of subunits of both MICOS subcomplexes synergistically increases lifespan, providing formal genetic evidence that both subcomplexes affect lifespan by different and at least partially independent pathways. At the molecular level, we found that ablation of Mic10-subcomplex components leads to a mitohormesis-induced lifespan extension, while lifespan extension of Mic60-subcomplex mutants seems to be controlled by pathways involved in the control of phospholipid homeostasis. Overall, our data demonstrate that both MICOS subcomplexes have different functions and play distinct roles in the aging process of P. anserina.

Lifespan Extension of Podospora anserina Mic60-Subcomplex Mutants Depends on Cardiolipin Remodeling.

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 membrane properties and promote longevity. Impairments of CL remodeling in a PaMIC60 ablated mutant lead to a complete abrogation of longevity. This effect is reversed by supplementation of the growth medium with linoleic acid, a fatty acid which allows the formation of tetra-octadecanoyl CL. In the PaMic60 deletion mutant, this CL species appears to lead to longevity. Overall, our data demonstrate a tight connection between MICOS, the regulation of mitochondrial phospholipid homeostasis, and aging of P. anserina.

Loss of the selective autophagy receptor p62 impairs murine myeloid leukemia progression and mitophagy.

Autophagy maintains hematopoietic stem cell integrity and prevents malignant transformation. In addition to bulk degradation, selective autophagy serves as an intracellular quality control mechanism and requires autophagy receptors, such as p62 (SQSTM1), to specifically bridge the ubiquitinated cargos into autophagosomes. Here, we investigated the function of p62 in acute myeloid leukemia (AML) in vitro and in murine in vivo models of AML. Loss of p62 impaired expansion and colony-forming ability of leukemia cells and prolonged latency of leukemia development in mice. High p62 expression was associated with poor prognosis in human AML. Using quantitative mass spectrometry, we identified enrichment of mitochondrial proteins upon immunoprecipitation of p62. Loss of p62 significantly delayed removal of dysfunctional mitochondria, increased mitochondrial superoxide levels, and impaired mitochondrial respiration. Moreover, we demonstrated that the autophagy-dependent function of p62 is essential for cell growth and effective mitochondrial degradation by mitophagy. Our results highlight the prominent role of selective autophagy in leukemia progression, and specifically, the importance of mitophagy to maintain mitochondrial integrity.

Global Protein Oxidation Profiling Suggests Efficient Mitochondrial Proteome Homeostasis During Aging.

The free radical theory of aging is based on the idea that reactive oxygen species (ROS) may lead to the accumulation of age-related protein oxidation. Because themajority of cellular ROS is generated at the respiratory electron transport chain, this study focuses on the mitochondrial proteome of the aging model Podospora anserina as target for ROS-induced damage. To ensure the detection of even low abundant modified peptides, separation by long gradient nLC-ESI-MS/MS and an appropriate statistical workflow for iTRAQ quantification was developed. Artificial protein oxidation was minimized by establishing gel-free sample preparation in the presence of reducing and iron-chelating agents. This first large scale, oxidative modification-centric study for P. anserina allowed the comprehensive quantification of 22 different oxidative amino acid modifications, and notably the quantitative comparison of oxidized and nonoxidized protein species. In total 2341 proteins were quantified. For 746 both protein species (unmodified and oxidatively modified) were detected and the modification sites determined. The data revealed that methionine residues are preferably oxidized. Further prominent identified modifications in decreasing order of occurrence were carbonylation as well as formation of N-formylkynurenine and pyrrolidinone. Interestingly, for the majority of proteins a positive correlation of changes in protein amount and oxidative damage were noticed, and a general decrease in protein amounts at late age. However, it was discovered that few proteins changed in oxidative damage in accordance with former reports. Our data suggest that P. anserina is efficiently capable to counteract ROS-induced protein damage during aging as long as protein de novo synthesis is functioning, ultimately leading to an overall constant relationship between damaged and undamaged protein species. These findings contradict a massive increase in protein oxidation during aging and rather suggest a protein damage homeostasis mechanism even at late age.

The autophagy interaction network of the aging model Podospora anserina.

BACKGROUND: Autophagy is a conserved molecular pathway involved in the degradation and recycling of cellular components. It is active either as response to starvation or molecular damage. Evidence is emerging that autophagy plays a key role in the degradation of damaged cellular components and thereby affects aging and lifespan control. In earlier studies, it was found that autophagy in the aging model Podospora anserina acts as a longevity assurance mechanism. However, only little is known about the individual components controlling autophagy in this aging model. Here, we report a biochemical and bioinformatics study to detect the protein-protein interaction (PPI) network of P. anserina combining experimental and theoretical methods. RESULTS: We constructed the PPI network of autophagy in P. anserina based on the corresponding networks of yeast and human. We integrated PaATG8 interaction partners identified in an own yeast two-hybrid analysis using ATG8 of P. anserina as bait. Additionally, we included age-dependent transcriptome data. The resulting network consists of 89 proteins involved in 186 interactions. We applied bioinformatics approaches to analyze the network topology and to prove that the network is not random, but exhibits biologically meaningful properties. We identified hub proteins which play an essential role in the network as well as seven putative sub-pathways, and interactions which are likely to be evolutionary conserved amongst species. We confirmed that autophagy-associated genes are significantly often up-regulated and co-expressed during aging of P. anserina. CONCLUSIONS: With the present study, we provide a comprehensive biological network of the autophagy pathway in P. anserina comprising PPI and gene expression data. It is based on computational prediction as well as experimental data. We identified sub-pathways, important hub proteins, and evolutionary conserved interactions. The network clearly illustrates the relation of autophagy to aging processes and enables further specific studies to understand autophagy and aging in P. anserina as well as in other systems.

Path2PPI: an R package to predict protein-protein interaction networks for a set of proteins.

UNLABELLED: : We introduce Path2PPI, a new R package to identify protein-protein interaction (PPI) networks for fully sequenced organisms for which nearly none PPI are known. Path2PPI predicts PPI networks based on sets of proteins from well-established model organisms, providing an intuitive visualization and usability. It can be used to combine and transfer information of a certain pathway or biological process from several reference organisms to one target organism. AVAILABILITY AND IMPLEMENTATION: Path2PPI is an open-source tool implemented in R. It can be obtained from the Bioconductor project: http://bioconductor.org/packages/Path2PPI/ CONTACT: : ina.koch@bioinformatik.uni-frankfurt.de SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Mitochondrial quality control: an integrated network of pathways.

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.

Structure and Biophysical Characterization of the S-Adenosylmethionine-dependent O-Methyltransferase PaMTH1, a Putative Enzyme Accumulating during Senescence of Podospora anserina.

Low levels of reactive oxygen species (ROS) act as important signaling molecules, but in excess they can damage biomolecules. ROS regulation is therefore of key importance. Several polyphenols in general and flavonoids in particular have the potential to generate hydroxyl radicals, the most hazardous among all ROS. However, the generation of a hydroxyl radical and subsequent ROS formation can be prevented by methylation of the hydroxyl group of the flavonoids. O-Methylation is performed by O-methyltransferases, members of the S-adenosyl-l-methionine (SAM)-dependent O-methyltransferase superfamily involved in the secondary metabolism of many species across all kingdoms. In the filamentous fungus Podospora anserina, a well established aging model, the O-methyltransferase (PaMTH1) was reported to accumulate in total and mitochondrial protein extracts during aging. In vitro functional studies revealed flavonoids and in particular myricetin as its potential substrate. The molecular architecture of PaMTH1 and the mechanism of the methyl transfer reaction remain unknown. Here, we report the crystal structures of PaMTH1 apoenzyme, PaMTH1-SAM (co-factor), and PaMTH1-S-adenosyl homocysteine (by-product) co-complexes refined to 2.0, 1.9, and 1.9 Å, respectively. PaMTH1 forms a tight dimer through swapping of the N termini. Each monomer adopts the Rossmann fold typical for many SAM-binding methyltransferases. Structural comparisons between different O-methyltransferases reveal a strikingly similar co-factor binding pocket but differences in the substrate binding pocket, indicating specific molecular determinants required for substrate selection. Furthermore, using NMR, mass spectrometry, and site-directed active site mutagenesis, we show that PaMTH1 catalyzes the transfer of the methyl group from SAM to one hydroxyl group of the myricetin in a cation-dependent manner.

Age-dependent dissociation of ATP synthase dimers and loss of inner-membrane cristae in mitochondria.

Aging is one of the most fundamental, yet least understood biological processes that affect all forms of eukaryotic life. Mitochondria are intimately involved in aging, but the underlying molecular mechanisms are largely unknown. Electron cryotomography of whole mitochondria from the aging model organism Podospora anserina revealed profound age-dependent changes in membrane architecture. With increasing age, the typical cristae disappear and the inner membrane vesiculates. The ATP synthase dimers that form rows at the cristae tips dissociate into monomers in inner-membrane vesicles, and the membrane curvature at the ATP synthase inverts. Dissociation of the ATP synthase dimer may involve the peptidyl prolyl isomerase cyclophilin D. Finally, the outer membrane ruptures near large contact-site complexes, releasing apoptogens into the cytoplasm. Inner-membrane vesiculation and dissociation of ATP synthase dimers would impair the ability of mitochondria to supply the cell with sufficient ATP to maintain essential cellular functions.

Loss of mitochondrial peptidase Clpp leads to infertility, hearing loss plus growth retardation via accumulation of CLPX, mtDNA and inflammatory factors.

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.

Overexpression of Pa_1_10620 encoding a mitochondrial Podospora anserina protein with homology to superoxide dismutases and ribosomal proteins leads to lifespan extension.

In biological systems, reactive oxygen species (ROS) represent 'double edged swords': as signaling molecules they are essential for proper development, as reactive agents they cause molecular damage and adverse effects like degeneration and aging. A well-coordinated control of ROS is therefore of key importance. Superoxide dismutases (SODs) are enzymes active in the detoxification of superoxide. The number of isoforms of these proteins varies among species. Here we report the characterization of the putative protein encoded by Pa_1_10620 that has been previously annotated to code for a mitochondrial ribosomal protein but shares also sequence domains with SODs. We report that the gene is transcribed in P. anserina cultures of all ages and that the encoded protein localizes to mitochondria. In strains overexpressing Pa_1_10620 in a genetic background in which PaSod3, the mitochondrial MnSOD of P. anserina, is deleted, no SOD activity could be identified in isolated mitochondria. However, overexpression of the gene leads to lifespan extension suggesting a pro-survival function of the protein in P. anserina.

Quality control of mitochondria during aging: is there a good and a bad side of mitochondrial dynamics?

Maintenance of functional mitochondria is essential in order to prevent degenerative processes leading to disease and aging. Mitochondrial dynamics plays a crucial role in ensuring mitochondrial quality but may also generate and spread molecular damage through a population of mitochondria. Computational simulations suggest that this dynamics is advantageous when mitochondria are not or only marginally damaged. In contrast, at a higher degree of damage, mitochondrial dynamics may be disadvantageous. Deceleration of fusion-fission cycles could be one way to adapt to this situation and to delay a further decline in mitochondrial quality. However, this adaptive response makes the mitochondrial network more vulnerable to additional molecular damage. The "mitochondrial infectious damage adaptation" (MIDA) model explains a number of inconsistent and counterintuitive data such as the "clonal expansion" of mutant mitochondrial DNA. We propose that mitochondrial dynamics is a double-edged sword and suggest ways to test this experimentally.

Poly(ADP-ribose) polymerase is a substrate recognized by two metacaspases of Podospora anserina.

The two metacaspases MCA1 and MCA2 of the fungal aging model organism Podospora anserina (PaMCA1 and PaMCA2, respectively) 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 characterize them further. Heterologous overexpression in Escherichia coli of the two metacaspase genes resulted in the production of proteins which aggregate and form inclusion bodies from which the active protein has been recovered via refolding in appropriate buffers. The renaturated proteins are characterized by an arginine-specific activity and are active in caspase-like self-maturation leading to the generation of characteristic small protein fragments. Both activities are dependent on the presence of calcium. Incubation of the two metacaspases with recombinant poly(ADP-ribose) polymerase (PARP), a known substrate of mammalian caspases, led to the identification of PARP as a substrate of the two P. anserina proteases. Using double mutants in which P. anserina Parp (PaParp) is overexpressed and PaMca1 is either overexpressed or deleted, we provide evidence for in vivo degradation of PaPARP by PaMCA1. These results support the idea that the substrate profiles of caspases and metacaspases are at least partially overlapping. Moreover, they link PCD and DNA maintenance in the complex network of molecular pathways involved in aging and life span control.

Macromolecular organization of ATP synthase and complex I in whole mitochondria.

We used electron cryotomography to study the molecular arrangement of large respiratory chain complexes in mitochondria from bovine heart, potato, and three types of fungi. Long rows of ATP synthase dimers were observed in intact mitochondria and cristae membrane fragments of all species that were examined. The dimer rows were found exclusively on tightly curved cristae edges. The distance between dimers along the rows varied, but within the dimer the distance between F(1) heads was constant. The angle between monomers in the dimer was 70° or above. Complex I appeared as L-shaped densities in tomograms of reconstituted proteoliposomes. Similar densities were observed in flat membrane regions of mitochondrial membranes from all species except Saccharomyces cerevisiae and identified as complex I by quantum-dot labeling. The arrangement of respiratory chain proton pumps on flat cristae membranes and ATP synthase dimer rows along cristae edges was conserved in all species investigated. We propose that the supramolecular organization of respiratory chain complexes as proton sources and ATP synthase rows as proton sinks in the mitochondrial cristae ensures optimal conditions for efficient ATP synthesis.

Impact of Mitochondrial Architecture, Function, Redox Homeostasis, and Quality Control on Organismic Aging: Lessons from a Fungal Model System.

Significance: Mitochondria are eukaryotic organelles with various essential functions. They are both the source and the targets of reactive oxygen species (ROS). Different branches of a mitochondrial quality control system (mQCS), such as ROS balancing, degradation of damaged proteins, or whole mitochondria, can mitigate the adverse effects of ROS stress. However, the capacity of mQCS is limited. Overwhelming this capacity leads to dysfunctions and aging. Strategies to interfere into mitochondria-dependent human aging with the aim to increase the healthy period of life, the health span, rely on the precise knowledge of mitochondrial functions. Experimental models such as Podospora anserina, a filamentous fungus with a clear mitochondrial aging etiology, proved to be instrumental to reach this goal. Recent Advances: Investigations of the P. anserina mQCS revealed that it is constituted by a complex network of different branches. Moreover, mitochondrial architecture and lipid homeostasis emerged to affect aging. Critical Issues: The regulation of the mQCS is only incompletely understood. Details about the involved signaling molecules and interacting pathways remain to be elucidated. Moreover, most of the currently generated experimental data were generated in well-controlled experiments that do not reflect the constantly changing natural life conditions and bear the danger to miss relevant aspects leading to incorrect conclusions. Future Directions: In P. anserina, the precise impact of redox signaling as well as of molecular damaging for aging remains to be defined. Moreover, natural fluctuation of environmental conditions needs to be considered to generate a realistic picture of aging mechanisms as they developed during evolution.

The impact of biomembranes and their dynamics on organismic aging: insights from a fungal aging model.

Biomembranes fulfill several essential functions. They delimitate cells and control the exchange of compounds between cells and the environment. They generate specialized cellular reaction spaces, house functional units such as the respiratory chain (RC), and are involved in content trafficking. Biomembranes are dynamic and able to adjust their properties to changing conditions and requirements. An example is the inner mitochondrial membrane (IMM), which houses the RC involved in the formation of adenosine triphosphate (ATP) and the superoxide anion as a reactive oxygen species (ROS). The IMM forms a characteristic ultrastructure that can adapt to changing physiological situations. In the fungal aging model Podospora anserina, characteristic age-related changes of the mitochondrial ultrastructure occur. More recently, the impact of membranes on aging was extended to membranes involved in autophagy, an important pathway involved in cellular quality control (QC). Moreover, the effect of oleic acid on the lifespan was linked to basic biochemical processes and the function of membranes, providing perspectives for the elucidation of the mechanistic effects of this nutritional component, which positively affects human health and aging.

CLPP-Null Eukaryotes with Excess Heme Biosynthesis Show Reduced L-arginine Levels, Probably via CLPX-Mediated OAT Activation.

The serine peptidase CLPP is conserved among bacteria, chloroplasts, and mitochondria. In humans and mice, its loss causes Perrault syndrome, which presents with growth deficits, infertility, deafness, and ataxia. In the filamentous fungus Podospora anserina, CLPP loss leads to longevity. CLPP substrates are selected by CLPX, an AAA+ unfoldase. CLPX is known to target delta-aminolevulinic acid synthase (ALAS) to promote pyridoxal phosphate (PLP) binding. CLPX may also influence cofactor association with other enzymes. Here, the evaluation of P. anserina metabolomics highlighted a reduction in arginine/histidine levels. In Mus musculus cerebellum, reductions in arginine/histidine and citrulline occurred with a concomitant accumulation of the heme precursor protoporphyrin IX. This suggests that the increased biosynthesis of 5-carbon (C5) chain deltaALA consumes not only C4 succinyl-CoA and C1 glycine but also specific C5 delta amino acids. As enzymes responsible for these effects, the elevated abundance of CLPX and ALAS is paralleled by increased OAT (PLP-dependent, ornithine delta-aminotransferase) levels. Possibly as a consequence of altered C1 metabolism, the proteome profiles of P. anserina CLPP-null cells showed strong accumulation of a methyltransferase and two mitoribosomal large subunit factors. The reduced histidine levels may explain the previously observed metal interaction problems. As the main nitrogen-storing metabolite, a deficiency in arginine would affect the urea cycle and polyamine synthesis. Supplementation of arginine and histidine might rescue the growth deficits of CLPP-mutant patients.

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.

Deceleration of fusion-fission cycles improves mitochondrial quality control during aging.

Mitochondrial dynamics and mitophagy play a key role in ensuring mitochondrial quality control. Impairment thereof was proposed to be causative to neurodegenerative diseases, diabetes, and cancer. Accumulation of mitochondrial dysfunction was further linked to aging. Here we applied a probabilistic modeling approach integrating our current knowledge on mitochondrial biology allowing us to simulate mitochondrial function and quality control during aging in silico. We demonstrate that cycles of fusion and fission and mitophagy indeed are essential for ensuring a high average quality of mitochondria, even under conditions in which random molecular damage is present. Prompted by earlier observations that mitochondrial fission itself can cause a partial drop in mitochondrial membrane potential, we tested the consequences of mitochondrial dynamics being harmful on its own. Next to directly impairing mitochondrial function, pre-existing molecular damage may be propagated and enhanced across the mitochondrial population by content mixing. In this situation, such an infection-like phenomenon impairs mitochondrial quality control progressively. However, when imposing an age-dependent deceleration of cycles of fusion and fission, we observe a delay in the loss of average quality of mitochondria. This provides a rational why fusion and fission rates are reduced during aging and why loss of a mitochondrial fission factor can extend life span in fungi. We propose the 'mitochondrial infectious damage adaptation' (MIDA) model according to which a deceleration of fusion-fission cycles reflects a systemic adaptation increasing life span.