Stress profiling of longevity mutants identifies Afg3 as a mitochondrial determinant of cytoplasmic mRNA translation and aging

Although environmental stress likely plays a significant role in promoting aging, the relationship remains poorly understood. To characterize this interaction in a more comprehensive manner, we examined the stress response profiles for 46 long‐lived yeast mutant strains across four different stress conditions (oxidative, ER, DNA damage, and thermal), grouping genes based on their associated stress response profiles. Unexpectedly, cells lacking the mitochondrial AAA protease gene AFG3 clustered strongly with long‐lived strains lacking cytosolic ribosomal proteins of the large subunit. Similar to these ribosomal protein mutants, afg3Δ cells show reduced cytoplasmic mRNA translation, enhanced resistance to tunicamycin that is independent of the ER unfolded protein response, and Sir2‐independent but Gcn4‐dependent lifespan extension. These data demonstrate an unexpected link between a mitochondrial protease, cytoplasmic mRNA translation, and aging.     

Mitochondrial metabolites extend lifespan

Disruption of mitochondrial respiration in the nematode Caenorhabditis elegans can extend lifespan. We previously showed that long‐lived respiratory mutants generate elevated amounts of α‐ketoacids. These compounds are structurally related to α‐ketoglutarate, suggesting they may be biologically relevant. Here, we show that provision of several such metabolites to wild‐type worms is sufficient to extend their life. At least one mode of action is through stabilization of hypoxia‐inducible factor‐1 (HIF‐1). We also find that an α‐ketoglutarate mimetic, 2,4‐pyridinedicarboxylic acid (2,4‐PDA), is alone sufficient to increase the lifespan of wild‐type worms and this effect is blocked by removal of HIF‐1. HIF‐1 is constitutively active in isp‐1(qm150) Mit mutants, and accordingly, 2,4‐PDA does not further increase their lifespan. Incubation of mouse 3T3‐L1 fibroblasts with life‐prolonging α‐ketoacids also results in HIF‐1α stabilization. We propose that metabolites that build up following mitochondrial respiratory dysfunction form a novel mode of cell signaling that acts to regulate lifespan.     

Lethal mitochondrial genotypes in Podospora anserina: A model for senescence

Summary Crosses between spg1 and spg2, two mitochondrial mutants of Podospora anserina, yield a new type of strain, called pseudo wild-type (PSW), in addition to wild-type recombinants. PSW strains are characterized by a variable phenotype for germination of ascospores and a variable longevity. By autofecondation, PSW strains yield early lethal strains (which die soon after the germination of the spores and so cannot be used for further studies), short-lived strains (which stop their vegetative growth after several centimeters) and long-lived strains (which grow longer than 16 cm). Genetic analysis of the last two categories shows that the PSW phenotype corresponds to a new mitochondrial genotype resulting from the interaction of the two parental mitochondrial genomes.Variability in the longevity of PSW strains is interpretated as the result of a high rate of mutation of their mitochondrial genome into a lethal and suppressive genome, similar to that of the mitochondrial rho ^- suppressive mutant of yeast. Furthermore, on the basis of the striking similarities observed between short-lived PSW strains and senescent cultures of Podospora anserina, we propose that commitment and development of senescence in wild-type strains of Podospora anserina would result, in a similar way, of spontaneous suppressive rho ^--like mitochondrial mutations.     

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

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

Inhibition of peroxisome fission,but not mitochondrial fission,increases yeast chronological lifespan

Mitochondria are key players in aging and cell death. It has been suggested that mitochondrial fragmentation, mediated by the Dnm1/Fis1 organelle fission machinery, stimulates aging and cell death. This was based on the observation that Saccharomyces cerevisiae Δdnm1 and Δfis1 mutants show an enhanced lifespan and increased resistance to cell death inducers. However, the Dnm1/Fis1 fission machinery is also required for peroxisome division. Here we analyzed the significance of peroxisome fission in yeast chronological lifespan, using yeast strains in which fission of mitochondria was selectively blocked. Our data indicate that the lifespan extension caused by deletion of FIS1 is mainly due to a defect in peroxisome fission and not caused by a block in mitochondrial fragmentation. These observations are underlined by our observation that deletion of FIS1 does not lead to lifespan extension in yeast peroxisome deficient mutant cells.     

Evidence that mutation accumulation does not cause aging in Saccharomyces cerevisiae

The concept that mutations cause aging phenotypes could not be directly tested previously due to inability to identify age‐related mutations in somatic cells and determine their impact on organismal aging. Here, we subjected Saccharomyces cerevisiae to multiple rounds of replicative aging and assessed de novo mutations in daughters of mothers of different age. Mutations did increase with age, but their low numbers, < 1 per lifespan, excluded their causal role in aging. Structural genome changes also had no role. A mutant lacking thiol peroxidases had the mutation rate well above that of wild‐type cells, but this did not correspond to the aging pattern, as old wild‐type cells with few or no mutations were dying, whereas young mutant cells with many more mutations continued dividing. In addition, wild‐type cells lost mitochondrial DNA during aging, whereas shorter‐lived mutant cells preserved it, excluding a causal role of mitochondrial mutations in aging. Thus, DNA mutations do not cause aging in yeast. These findings may apply to other damage types, suggesting a causal role of cumulative damage, as opposed to individual damage types, in organismal aging.     

Does autophagy mediate age-dependent effect of dietary restriction responses in the filamentous fungus Podospora anserina?

Autophagy is a well-conserved catabolic process, involving the degradation of a cell''s own components through the lysosomal/vacuolar machinery. Autophagy is typically induced by nutrient starvation and has a role in nutrient recycling, cellular differentiation, degradation and programmed cell death. Another common response in eukaryotes is the extension of lifespan through dietary restriction (DR). We studied a link between DR and autophagy in the filamentous fungus Podospora anserina, a multicellular model organism for ageing studies and mitochondrial deterioration. While both carbon and nitrogen restriction extends lifespan in P. anserina, the size of the effect varied with the amount and type of restricted nutrient. Natural genetic variation for the DR response exists. Whereas a switch to carbon restriction up to halfway through the lifetime resulted in extreme lifespan extension for wild-type P. anserina, all autophagy-deficient strains had a shorter time window in which ageing could be delayed by DR. Under nitrogen limitation, only PaAtg1 and PaAtg8 mediate the effect of lifespan extension; the other autophagy-deficient mutants PaPspA and PaUth1 had a similar response as wild-type. Our results thus show that the ageing process impinges on the DR response and that this at least in part involves the genetic regulation of autophagy.     

Collagenase‐resistant collagen promotes mouse aging and vascular cell senescence

Collagen fibrils become resistant to cleavage over time. We hypothesized that resistance to type I collagen proteolysis not only marks biological aging but also drives it. To test this, we followed mice with a targeted mutation (Col1a1^r/r) that yields collagenase‐resistant type I collagen. Compared with wild‐type littermates, Col1a1^r/r mice had a shortened lifespan and developed features of premature aging including kyphosis, weight loss, decreased bone mineral density, and hypertension. We also found that vascular smooth muscle cells (SMCs) in the aortic wall of Col1a1^r/r mice were susceptible to stress‐induced senescence, displaying senescence‐associated ß‐galactosidase (SA‐ßGal) activity and upregulated p16^INK4A in response to angiotensin II infusion. To elucidate the basis of this pro‐aging effect, vascular SMCs from twelve patients undergoing coronary artery bypass surgery were cultured on collagen derived from Col1a1^r/r or wild‐type mice. This revealed that mutant collagen directly reduced replicative lifespan and increased stress‐induced SA‐ßGal activity, p16^INK4A expression, and p21^CIP1 expression. The pro‐senescence effect of mutant collagen was blocked by vitronectin, a ligand for αvß3 integrin that is presented by denatured but not native collagen. Moreover, inhibition of αvß3 with echistatin or with αvß3‐blocking antibody increased senescence of SMCs on wild‐type collagen. These findings reveal a novel aging cascade whereby resistance to collagen cleavage accelerates cellular aging. This interplay between extracellular and cellular compartments could hasten mammalian aging and the progression of aging‐related diseases.     

Autophagy compensates impaired energy metabolism in CLPXP‐deficient Podospora anserina strains and extends healthspan

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.     

The ceramide-activated protein phosphatase Sit4p controls lifespan,mitochondrial function and cell cycle progression by regulating hexokinase 2 phosphorylation

Sit4p is the catalytic subunit of a ceramide-activated PP2A-like phosphatase that regulates cell cycle, mitochondrial function, oxidative stress resistance and chronological lifespan in yeast. In this study, we show that hexokinase 2 (Hxk2p) is hyperphosphorylated in sit4Δ mutants grown in glucose medium by a Snf1p-independent mechanism and Hxk2p-S15A mutation suppresses phenotypes associated with SIT4 deletion, namely growth arrest at G1 phase, derepression of mitochondrial respiration, H₂O₂ resistance and lifespan extension. Consistently, the activation of Sit4p in isc1Δ mutants, which has been associated with premature aging, leads to Hxk2p hypophosphorylation, and the expression of Hxk2p-S15E increases the lifespan of isc1Δ cells. The overall results suggest that Hxk2p functions downstream of Sit4p in the control of cell cycle, mitochondrial function, oxidative stress resistance and chronological lifespan.     

Unmasking a temperature-dependent effect of the P. anserina i-AAA protease on aging and development

Different molecular pathways involved in maintaining mitochondrial function are of fundamental importance to control cellular homeostasis. Mitochondrial i-AAA protease is part of such a surveillance system, and PaIAP is the putative ortholog in the fungal aging model Podospora anserina. Here, we investigate the role of PaIAP in aging and development. Deletion of the gene encoding PaIAP resulted in a specific phenotype. When incubated at 27°C, spore germination and fruiting body formation are not different from that of the corresponding wild-type strain. Unexpectedly, the lifespan of the deletion strain is strongly increased. In contrast, cultivation at an elevated temperature of 37°C leads to impairments in spore germination and fruiting body formation and to a reduced lifespan. The higher PaIAP abundance in wild-type strains of the fungus grown at elevated temperature and the phenotype of the deletion strain unmasks a temperature-related role of the protein. The protease appears to be part of a molecular system that has evolved to allow survival under changing temperatures, as they characteristically occur in nature.Key words: Podospora anserina, aging, mitochondria, protein quality control, temperature, i-AAA protease     

Unmasking a temperature-dependent effect of the P. anserina i-AAA protease on aging and development

Different molecular pathways involved in maintaining mitochondrial function are of fundamental importance to control cellular homeostasis. Mitochondrial i-AAA protease is part of such a surveillance system and PaIAP is the putative ortholog in the fungal aging model Podospora anserina. Here we investigated the role of PaIAP in aging and development. Deletion of the gene encoding PaIAP resulted in a specific phenotype. When incubated at 27°C, spore germination and fruiting body formation are not different from that of the corresponding wild-type strain. Unexpectedly, the lifespan of the deletion strain is strongly increased. In contrast, cultivation at an elevated temperature of 37°C leads to impairments in spore germination and fruiting body formation, and to a reduced lifespan. The higher PaIAP abundance in wild-type strains of the fungus grown at elevated temperature and the phenotype of the deletion strain unmasks a temperaturerelated role of the protein. The protease appears to be part of a molecular system that has evolved to allow survival under changing temperatures as they characteristically occur in nature.     

Chemical genetic screen in fission yeast reveals roles for vacuolar acidification,mitochondrial fission,and cellular GMP levels in lifespan extension

The discovery that genetic mutations in several cellular pathways can increase lifespan has lent support to the notion that pharmacological inhibition of aging pathways can be used to extend lifespan and to slow the onset of age‐related diseases. However, so far, only few compounds with such activities have been described. Here, we have conducted a chemical genetic screen for compounds that cause the extension of chronological lifespan of Schizosaccharomyces pombe. We have characterized eight natural products with such activities, which has allowed us to uncover so far unknown anti‐aging pathways in S. pombe. The ionophores monensin and nigericin extended lifespan by affecting vacuolar acidification, and this effect depended on the presence of the vacuolar ATPase (V‐ATPase) subunits Vma1 and Vma3. Furthermore, prostaglandin J₂ displayed anti‐aging properties due to the inhibition of mitochondrial fission, and its effect on longevity required the mitochondrial fission protein Dnm1 as well as the G‐protein‐coupled glucose receptor Git3. Also, two compounds that inhibit guanosine monophosphate (GMP) synthesis, mycophenolic acid (MPA) and acivicin, caused lifespan extension, indicating that an imbalance in guanine nucleotide levels impinges upon longevity. We furthermore have identified diindolylmethane (DIM), tschimganine, and the compound mixture mangosteen as inhibiting aging. Taken together, these results reveal unanticipated anti‐aging activities for several phytochemicals and open up opportunities for the development of novel anti‐aging therapies.     

Endogenous reactive oxygen species cause astrocyte defects and neuronal dysfunctions in the hippocampus: a new model for aging brain

The etiology of astrocyte dysfunction is not well understood even though neuronal defects have been extensively studied in a variety of neuronal degenerative diseases. Astrocyte defects could be triggered by the oxidative stress that occurs during physiological aging. Here, we provide evidence that intracellular or mitochondrial reactive oxygen species (ROS) at physiological levels can cause hippocampal (neuronal) dysfunctions. Specifically, we demonstrate that astrocyte defects occur in the hippocampal area of middle‐aged Tet‐mev‐1 mice with the SDHC^V69E mutation. These mice are characterized by chronic oxidative stress. Even though both young adult and middle‐aged Tet‐mev‐1 mice overproduced MitoSOX Red‐detectable mitochondrial ROS compared to age‐matched wild‐type C57BL/6J mice, only young adult Tet‐mev‐1 mice upregulated manganese and copper/zinc superoxide dismutase (Mn‐ and Cu/Zn‐SODs) activities to eliminate the MitoSOX Red‐detectable mitochondrial ROS. In contrast, middle‐aged Tet‐mev‐1 mice accumulated both MitoSOX Red‐detectable mitochondrial ROS and CM‐H₂DCFDA‐detectable intracellular ROS. These ROS levels appeared to be in the physiological range as shown by normal thiol and glutathione disulfide/glutathione concentrations in both young adult and middle‐aged Tet‐mev‐1 mice relative to age‐matched wild‐type C57BL/6J mice. Furthermore, only middle‐aged Tet‐mev‐1 mice showed JNK/SAPK activation and Ca²⁺ overload, particularly in astrocytes. This led to decreasing levels of glial fibrillary acidic protein and S100β in the hippocampal area. Significantly, there were no pathological features such as apoptosis, amyloidosis, and lactic acidosis in neurons and astrocytes. Our findings suggest that the age‐dependent physiologically relevant chronic oxidative stress caused astrocyte defects in mice with impaired mitochondrial electron transport chain functionality.