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Research on Podospora anserina unraveled a network of molecular pathways affecting biological aging. In particular, a number of pathways active in the control of mitochondria were identified on different levels. A long-known key process active during aging of P. anserina is the age- related reorganization of the mitochondrial DNA (mtDNA). Mechanisms involved in the stabilization of the mtDNA lead to lifespan extension. Another critical issue is to balance mitochondrial levels of reactive oxygen species (ROS). This is important because ROS are essential signaling molecules, but at increased levels cause molecular damage. At a higher level of the network, mechanisms are active in the repair of damaged compounds. However, if damage passes critical limits, the corresponding pathways are overwhelmed and impaired molecules as well as those present in excess are degraded by specific enzymes or via different forms of autophagy. Subsequently, degraded units need to be replaced by novel functional ones. The corresponding processes are dependent on the availability of intact genetic information. Although a number of different pathways involved in the control of cellular homeostasis were uncovered in the past, certainly many more exist. In addition, the signaling pathways involved in the control and coordination of the underlying pathways are only initially understood. In some cases, like the induction of autophagy, ROS are active. Additionally, sensing and signaling the energetic status of the organism plays a key role. The precise mechanisms involved are elusive and remain to be elucidated.
Die im Rahmen dieser Arbeit durchgeführten Untersuchungen führten zu folgenden Ergebnissen: 1. Eindimensionale Gelelektrophoresen Die Analyse mitochondrialer Proteine aus juvenilen und seneszenten P. anserina-Wildstämmen mit Hilfe von eindimensionalen SDS- und eindimensionalen Blau-Nativen-Gelelektrophoresen zeigt keine deutlichen, seneszenzspezifischen Unterschiede. Im Gegensatz dazu werden in initialen Versuchen der nicht-radioaktiven 2D-PAGE differentiell gebildete Proteine visualisiert. 2. 2D-PAGE mit radioaktiv-markierten, mitochondrialen Proteinen aus jungen und alten P. anserina-Wildstämmen In der ungerichteten Proteomanalyse wurden 29 differentiell-gebildete Proteine identifiziert und zusätzlich zahlreiche Isoformen einiger Proteine gezeigt. Von der ß-ATPase wurden modifizierte Isoformen gefunden. Außerdem wurde eine seneszenspezifisch verringerte Bildung von ROS-Abwehr-Proteinen in den Mitochondrien detektiert. Im Gegensatz dazu wurde eine größere Menge eines Chaperons gefunden, das bei der Proteinsynthese eine Rolle spielt: eine Protein-Disulfid-Isomerase, die die Umlagerung und Neubildung von Di-Sulfid-Brücken bei der Faltung von Proteinen katalysiert. Zusätzlich wurde eine erhöhte Menge des Proteins SSC1 identifiziert. Dieses gehört zur Hsp70-Hitzeschock-Proteinfamilie. Es wurde ebenfalls eine erhöhte Menge des Apoptosefaktors Cyclophilin D in den mitochondrialen Proben aus den seneszenten Wildstämmen identifiziert. Die Identifizierung dieses Proteins in Mitochondrien von P. anserina stellt neben der Charakterisierung der Metacaspasen (Hamann et al., 2007) einen weiteren Ansatzpunkt für die Apoptoseforschung in P. anserina dar. Die molekularbiologische Analyse dieses Proteins wurde aufgrund dieser Proteomanalyse im Arbeitskreis aufgenommen (Dissertation D. Brust). Ein weiteres Protein, das in stark erhöhter Menge in den Proteinisolaten identifiziert wurde, ist PaMTH1. Im Rahmen der vorliegenden Arbeit wurden die Struktur und die Funktion dieser neu identifizierten differentiell-gebildeten Methyltransferase während der Alterung in P. anserina mit Hilfe molekularbiologischer, biochemischer und physiologischen Analysen untersucht. 3. Charakterisierung von PaMTH1 Im Rahmen von Northernblot-Analysen wurde gezeigt, dass die PaMth1-Transkriptmenge in drei unabhängigen alten Wildstämmen im Vergleich zu den entsprechenden jungen Wildtsämmen deutlich erhöht ist. In einer Westernblot-Analyse von Gesamtproteinen und Mitochondrien aus jungen und seneszenten Wildstämmen wird der seneszenzspezifische Anstieg der Proteinmenge verifiziert. Die genauere Einordnung von PaMTH1 in die Klasse I der Methyltransferasen und die Ergebnisse der Analyse der Substratspezifizität geben einen Hinweis auf eine Schutzfunktion durch die Verhinderung einer ROS-Entstehung unter der Beteiligung von Substanzen mit einer Catecholgruppe. Die Ergebnisse der Analyse der Modulation der PaMth1-Expression in P. anserina deuten ebenfalls auf eine Schutzwirkung von PaMTH1 hin: PaMth1-Überexpressionsstämme zeigen eine verbesserte Wuchsrate auf stress-induzierenden Medien, weniger carbonylierte Proteine und vor allem eine verlängerte Lebensspanne ohne physiologische Nachteile im Vergleich zum Wildstamm. Dagegen lebt die PaMth1-Deletionsmutante kürzer und wächst schlechter auf ROS-induzierenden Medien, sie zeigt allerdings keine erhöhte Menge von carbonylierten Proteinen im eindimensionalen „Oxyblot“. Die beobachtete Lebensspannenverkürzung der PaMth1-Deletionsmutante wird jedoch durch die Reversion dieser Stämme wieder aufgehoben, sodass die Hypothese des Schutzes vor der ROS-Generierung durch die Methylierung von Dihydroxylgruppen anhand der erhaltenen Daten unterstützt wird.
Synaptic release sites are characterized by exocytosis-competent synaptic vesicles tightly anchored to the presynaptic active zone (PAZ) whose proteome orchestrates the fast signaling events involved in synaptic vesicle cycle and plasticity. Allocation of the amyloid precursor protein (APP) to the PAZ proteome implicated a functional impact of APP in neuronal communication. In this study, we combined state-of-the-art proteomics, electrophysiology and bioinformatics to address protein abundance and functional changes at the native hippocampal PAZ in young and old APP-KO mice. We evaluated if APP deletion has an impact on the metabolic activity of presynaptic mitochondria. Furthermore, we quantified differences in the phosphorylation status after long-term-potentiation (LTP) induction at the purified native PAZ. We observed an increase in the phosphorylation of the signaling enzyme calmodulin-dependent kinase II (CaMKII) only in old APP-KO mice. During aging APP deletion is accompanied by a severe decrease in metabolic activity and hyperphosphorylation of CaMKII. This attributes an essential functional role to APP at hippocampal PAZ and putative molecular mechanisms underlying the age-dependent impairments in learning and memory in APP-KO mice.
The degradation of nonfunctional mitochondrial proteins is of fundamental relevance for maintenance of cellular homeostasis. The heteromeric CLPXP protein complex in the mitochondrial matrix is part of this process. In the fungal aging model Podospora anserina, ablation of CLPXP leads to an increase in healthy lifespan. Here, we report that this counterintuitive increase depends on a functional autophagy machinery. In PaClpXP mutants, autophagy is involved in energy conservation and the compensation of impairments in respiration. Strikingly, despite the impact on mitochondrial function, it is not mitophagy but general autophagy that is constitutively induced and required for longevity. In contrast, in another long-lived mutant ablated for the mitochondrial PaIAP protease, autophagy is neither induced nor required for lifespan extension. Our data provide novel mechanistic insights into the capacity of different forms of autophagy to compensate impairments of specific components of the complex mitochondrial quality control network and about the biological role of mitochondrial CLPXP in the control of cellular energy metabolism.
Dissecting the complexities of mammalian heart development and regenerative capacity require thorough understanding of the underlying molecular mechanisms through the expression pattern of proteins and post-translational modifications. To obtain insights intoactivated signaling pathways that control the cellular phenotype during postnatal heart development, we generated a comprehensive map of phosphorylation sites. In total we identified 21,261 phosphorylation sites and 8985 proteins in developing mouse hearts by mass spectrometry. The in-vivo SILAC (stable isotope labeling of amino acids in cell culture) approach allowed robust quantification of phosphorylation sites and proteins, which are regulated during heart development. We found several activated pathways involved in cell cycle regulation and detected numerous kinases and transcription factors to be regulated on protein and phosphopeptide level. Most strikingly, we identified a novel mitochondrial protein, known previously as Perm1, as a highly phosphorylated factor regulated during heart development. We renamed Perm1 as MICOS complex subunit Mic85 since it shows robust physical interaction with MICOS complex subunits, including Mitofilin (Mic60), Chchd3 (Mic19), Chchd6 (Mic25) and the outer membrane protein Samm50. Moreover, Mic85 is localized to the mitochondrial inner membrane facing the intermembrane space and the dynamics of Mic85 protein expression is regulated by the ubiquitin-proteasomal system through phosphorylation of casein kinase 2 on its PEST motif. Silencing of Mic85 in cultured neonatal cardiomyocytes impairs mitochondrial morphology and compromises oxidative capacity. Our findings support a clear role for Mic85 in the maintenance of mitochondrial architecture and in its contribution to enhanced energetics during developing and adult mouse cardiomyocytes. The transgenic Mic85 knockout mouse generated with a GFP knock-in will support future in vivo investigations on the integrity of mitochondria and the function of Mic85 in cardiac development.
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 F1Fo-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.
Impact of F1Fo-ATP-synthase dimer assembly factors on mitochondrial function and organismic aging
(2018)
In aerobic organisms, mitochondrial F1Fo-ATP-synthase is the major site of ATP production. Beside this fundamental role, the protein complex is involved in shaping and maintenance of cristae. Previous electron microscopic studies identified the dissociation of F1Fo-ATP-synthase dimers and oligomers during organismic aging correlating with a massive remodeling of the mitochondrial inner membrane. Here we report results aimed to experimentally proof this impact and to obtain further insights into the control of these processes. We focused on the role of the two dimer assembly factors PaATPE and PaATPG of the aging model Podospora anserina. Ablation of either protein strongly affects mitochondrial function and leads to an accumulation of senescence markers demonstrating that the inhibition of dimer formation negatively influences vital functions and accelerates organismic aging. Our data validate a model that links mitochondrial membrane remodeling to aging and identify specific molecular components triggering this process.
Lifespan Extension of Podospora anserina Mic60-Subcomplex Mutants Depends on Cardiolipin Remodeling
(2022)
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.
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, leading to reduced reactive oxygen species (ROS) production. Overall, our study uncovers multiple effects of an oleic acid diet, which extends the lifespan of P. anserina and provides perspectives to explain the positive nutritional effects on human aging.