Accelerated chronological aging of a mutant fission yeast deficient in both glutathione and superoxide dismutase having cu and zn as cofactors and its enhancement by sir2 deficiency

A mutant of Schizosaccharomyces pombe deficient in both superoxide dismutase with copper and zinc as cofactors and glutathione was hypersensitive to menadione, which intracellularly generates superoxide radicals, and showed short chronological lifespan with more oxidation of proteins. Disruption of the sir2 gene in the double mutant enhanced the short chronological lifespan without more enhanced protein oxidation.     

Extension of Yeast Chronological Lifespan by Methylamine

Background

Chronological aging of yeast cells is commonly used as a model for aging of human post-mitotic cells. The yeast Saccharomyces cerevisiae grown on glucose in the presence of ammonium sulphate is mainly used in yeast aging research. We have analyzed chronological aging of the yeast Hansenula polymorpha grown at conditions that require primary peroxisome metabolism for growth.

Methodology/Principal Findings

The chronological lifespan of H. polymorpha is strongly enhanced when cells are grown on methanol or ethanol, metabolized by peroxisome enzymes, relative to growth on glucose that does not require peroxisomes. The short lifespan of H. polymorpha on glucose is mainly due to medium acidification, whereas most likely ROS do not play an important role. Growth of cells on methanol/methylamine instead of methanol/ammonium sulphate resulted in further lifespan enhancement. This was unrelated to medium acidification. We show that oxidation of methylamine by peroxisomal amine oxidase at carbon starvation conditions is responsible for lifespan extension. The methylamine oxidation product formaldehyde is further oxidized resulting in NADH generation, which contributes to increased ATP generation and reduction of ROS levels in the stationary phase.

Conclusion/Significance

We conclude that primary peroxisome metabolism enhanced chronological lifespan of H. polymorpha. Moreover, the possibility to generate NADH at carbon starvation conditions by an organic nitrogen source supports further extension of the lifespan of the cell. Consequently, the interpretation of CLS analyses in yeast should include possible effects on the energy status of the cell.     

The significance of peroxisome function in chronological aging of Saccharomyces cerevisiae

We studied the chronological lifespan of glucose‐grown Saccharomyces cerevisiae in relation to the function of intact peroxisomes. We analyzed four different peroxisome‐deficient (pex) phenotypes. These included Δpex3 cells that lack peroxisomal membranes and in which all peroxisomal proteins are mislocalized together with Δpex6 in which all matrix proteins are mislocalized to the cytosol, whereas membrane proteins are still correctly sorted to peroxisomal ghosts. In addition, we analyzed two mutants in which the peroxisomal location of the β‐oxidation machinery is in part disturbed. We analyzed Δpex7 cells that contain virtually normal peroxisomes, except that all matrix proteins that contain a peroxisomal targeting signal type 2 (PTS2, also including thiolase), are mislocalized to the cytosol. In Δpex5 cells, peroxisomes only contain matrix proteins with a PTS2 in conjunction with all proteins containing a peroxisomal targeting signal type 1 (PTS1, including all β‐oxidation enzymes except thiolase) are mislocalized to the cytosol. We show that intact peroxisomes are an important factor in yeast chronological aging because all pex mutants showed a reduced chronological lifespan. The strongest reduction was observed in Δpex5 cells. Our data indicate that this is related to the complete inactivation of the peroxisomal β‐oxidation pathway in these cells due to the mislocalization of thiolase. Our studies suggest that during chronological aging, peroxisomal β‐oxidation contributes to energy generation by the oxidation of fatty acids that are released by degradation of storage materials and recycled cellular components during carbon starvation conditions.     

The Fission Yeast php2 Mutant Displays a Lengthened Chronological Lifespan

The Schizosaccharomyces pombe php2 ⁺ gene encodes a subunit of the CCAAT-binding factor complex. We found that disruption of the php2 ⁺ gene extended the chronological lifespan of the fission yeast. Moreover, the lifespan of the Δphp2 mutant was barely extended under calorie restricted (CR) conditions. Many other phenotypes of the Δphp2 mutant resembled those of wild-type cells grown under CR conditions, suggesting that the Δphp2 mutant might undergo CR. The mutant also showed low respiratory activity concomitant with decreased expression of the cyc1 ⁺ and rip1 ⁺ genes, both of which are involved in mitochondrial electron transport. On the basis of a chromatin immunoprecipitation assay, we determined that Php2 binds to a DNA region upstream of cyc1 ⁺ and rip1 ⁺ in S. pombe. Here we discuss the possible mechanisms by which the chronological lifespan of Δphp2 mutant is extended.     

lac‐1 and lag‐1 with ras‐1 affect aging and the biological clock in Neurospora crassa

Using an automated cell counting technique developed previously (Case et al., Ecology and Evolution 2014; 4: 3494), we explore the lifespan effects of lac‐1, a ceramide synthase gene paralogous to lag‐1 in Neurospora crassa in conjunction with the band bd (ras‐1) gene. We find that the replicative lifespan of a lac‐1^KO bd double mutants is short, about one race tube cycle, and this double mutant lacks a strong ~21‐hr clock cycle as shown by race tube and fluorometer analysis of fluorescent strains including lac‐1^KO. This short replicative lifespan phenotype is contrasted with a very long estimated chronological lifespan for lac‐1^KO bd double mutants from 247 to 462 days based on our regression analyses on log viability, and for the single mutant lac‐1^KO, 161 days. Both of these estimated lifespans are much higher than that of previously studied WT and bd single mutant strains. In a lac‐1 rescue and induction experiment, the expression of lac‐1⁺ as driven by a quinic acid‐dependent promoter actually decreases the median chronological lifespan of cells down to only 7 days, much lower than the 34‐day median lifespan found in control bd conidia also grown on quinic acid media, which we interpret as an effect of balancing selection acting on ceramide levels based on previous findings from the literature. Prior work has shown phytoceramides can act as a signal for apoptosis in stressed N. crassa cells. To test this hypothesis of balancing selection on phytoceramide levels, we examine the viability of WT, lag‐1^KO bd, and lac‐1^KO bd strains following the dual stresses of heat and glycolysis inhibition, along with phytoceramide treatments of different dosages. We find that the phytoceramide dosage–response curve is altered in the lag‐1^KO bd mutant, but not in the lac‐1^KO bd mutant. We conclude that phytoceramide production is responsible for the previously reported longevity effects in the lag‐1^KO bd mutant, but a different ceramide may be responsible for the longevity effect observed in the lac‐1^KO bd mutant.     

SKO1 deficiency extends chronological lifespan in Saccharomyces cerevisiae

ABSTRACT

Sko1 plays a key role in the control of gene expression by osmotic and oxidative stress in yeast. We demonstrate that the decrease in chronological lifespan (CLS) of hog1Δ cells was suppressed by SKO1 deletion. sko1Δ single mutant cells were shown to have a longer CLS, thus implicating Sko1 in the regulation of their CLS.     

Acacetin promotes healthy aging by altering stress response in Caenorhabditis elegans

The progression in lifespan has been associated with elevated intracellular reactive oxygen species (ROS) and oxidative stress level which contributes to development of age related disorders. The discovery of lifespan modulating phytomolecules may promote development of natural therapies against age related afflictions. Acacetin (5,7-dihydroxy-4-methoxyflavone), is a naturally occurring flavonoid known to possess therapeutic properties. To this end, the present study evaluates effect of acacetin (AC) on lifespan, stress and neurotoxicity for the first time by using well-established free living, multicellular Caenorhabditis elegans model system. The 25?μM dose of AC significantly prolonged the mean lifespan of worms by 27.31% in comparison to untreated control and other tested doses of AC. Additionally, AC enhanced stress resistance against oxidative and thermal stress in worms. Furthermore, AC attenuated age related intracellular ROS level, aggregation of age pigment lipofuscin and increased the mean survival in stress hypersensitive mev-1 mutant by 40.5%. AC supplementation also reduced the alpha synuclein aggregation in transgenic worm model of Parkinson’s disease. The enhanced stress resistance, lifespan and alleviation of age related pathology can be attributed to increment in stress modulatory enzymes like superoxide dismutase (SOD) and catalase (CAT) level. Altogether the results suggest AC exposure maintains stress level, health span and extends mean lifespan of C. elegans. The longevity promoting and neuromodulatory effects of AC are mediated by up regulation of the stress response genes sod-3 and gst-4. The present finding gives new insights of natural remedies and their future prospects in developing therapeutic interventions for managing age related diseases.     

Isolation of mutations synthetic-lethal to prohibitin 2 null mutants of Saccharomyces cerevisiae

Prohibitins are ubiquitous, abundant proteins found in a wide range of organisms and that have a high degree of sequence conservation. In yeast it has previously been demonstrated that prohibitin proteins form a complex and are involved in maintaining the morphological and functional integrity of mitochondria. We have used a colony-sectoring assay as a screen for mutants that are dependent upon the presence of functional Phb2p in the cell. Two classes of prohibitin dependent mutation (pbd1 and pbd2) were isolated and characterised. The effect of these mutations on replicative lifespan was determined, demonstrating that the pbd1 mutant slightly extended lifespan, whereas in contrast, the pbd2 mutation resulted in a shortening in both the mean- and the maximum-lifespan. The pbd1 mutation was also found to reduce chronological lifespan. Reducing the expression of the PHB2 gene in the pbd mutants was found to retard the rate of growth and to affect replicative lifespan. As the two mutants behave in a different manner they probably affect different aspects of prohibitin function.     

Unopposed mitochondrial fission leads to severe lifespan shortening

Mitochondrial morphology is controlled by the opposing processes of fusion and fission. Previously, in baker’s yeast it was shown that reduced mitochondrial fission leads to a network-like morphology, decreased sensitivity for the induction of apoptosis and a remarkable extension of both replicative and chronological lifespan. However, the effects of reduced mitochondrial fusion on aging are so far unknown and complicated by the fact that deletion of genes encoding components of mitochondrial fusion are often lethal to higher organisms. This is also true for the mammalian OPA1 protein, which is a key regulator of mitochondrial inner membrane fusion. Baker’s yeast contains an OPA1 ortholog, Mgm1p. Deletion of Mgm1 is possible in yeast due to the fact that mitochondrial function is not essential for growth on glucose-containing media. In this study, we report that absence of mitochondrial fusion in the Δmgm1 mutant leads to a striking reduction of both replicative and chronological lifespan. Concomitantly, sensitivity to apoptosis elicitation via the reactive oxygen species hydrogen peroxide is substantially increased. These results demonstrate that the unopposed mitochondrial fission as displayed by the Δmgm1 mutant strongly affects organismal aging. Moreover, our results bear important clues for translational research to intervene into age-related degenerative processes also in multicellular organisms including humans.     

Interaction between sexes and between different circadian phenotypes affects lifespan in the fruit fly Drosophila melanogaster

Circadian clocks regulate the daily temporal structure of physiological and behavioural functions. In the fruit fly Drosophila melanogaster Meigen, disruption of daily rhythms is suggested to reduce the fly's lifespan. In the present study, because pairs of mixed‐sex flies are known to show an activity pattern different from that of individual flies, this hypothesis is tested by measuring the lifespan of flies housed same‐sexually or mixed‐sexually under an LD 12 : 12 h photocycle at a constant temperature of 25 °C. The effect of housing wild‐type (Canton‐S) flies with period (per) circadian clock mutant flies is also examined because the mutant flies have different daily activity patterns. When males and females of wild‐type flies are housed together, their lifespan is substantially lengthened (males) or shortened (females) compared with same‐sex housed flies. The shortening of the lifespan in females is significantly enhanced when mated with per mutant males. The shortening effects are significantly reduced when the mixed‐sex interaction is limited for the first 5 days after emergence. A slight elongation in lifespan, rather than a reduction, occurs when wild‐type females are housed same‐sexually with per⁰ or per^L mutant flies. In male flies, the elongation of lifespan occurs not only when wild‐type males are housed with wild‐type, per⁰ or per^L females, but also when housed with per⁰ or per^S mutant males. Mixed‐sex couples always show altered daily locomotor rhythms with an enhanced night‐time activity, whereas same‐sex couples show daily behavioural profiles slightly altered but essentially similar to a sum of the respective two flies. No significant correlation is found between the lifespan and reproductive capacity. These results suggest that the alteration of daily activity rhythms and sexual interaction may have significant impact on the fly's lifespan.     

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.     

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.     

Mitochondrial dysfunction leads to reduced chronological lifespan and increased apoptosis in yeast

We previously isolated a Saccharomyces cerevisiae mutant (HsTnII), which displays 40% reduced chronological lifespan as compared to the wild type (WT). In this study, we found HsTnII cultures to be characterized by fragmented and dysfunctional mitochondria, and by increased initiation of apoptosis during chronological aging as compared to WT. Expression of genes encoding subunits of mitochondrial electron transport chain and ATP synthase is significantly downregulated in HsTnII, and as a consequence, HsTnII is not able to respire ethanol. All these data confirm the importance of functional mitochondria and respiration in determining yeast chronological lifespan and apoptosis.     

Screening for long-lived genes identifies Oga1, a guanine-quadruplex associated protein that affects the chronological lifespan of the fission yeast Schizosaccharomyces pombe

Schizosaccharomyces pombe and Saccharomyces cerevisiae are excellent model organisms to study lifespan. We conducted screening to identify novel genes that, when overexpressed, extended the chronological lifespan of fission yeast. We identified seven genes, among which we focused on SPBC16A3.08c. The gene product showed similarity to Ylr150w of S. cerevisiae, which has affinity for guanine-quadruplex nucleic acids (G4). The SPBC16A3.08c product associated with G4 in vitro and complemented the phenotype of an S. cerevisiae Ylr150w deletion mutant. From these results, we proposed that SPBC16A3.08c encoded for a functional homolog of Ylr150w, which we designated ortholog of G4-associated protein (oga1 ⁺). oga1 ⁺ overexpression extended the chronological lifespan and also decreased mating efficiency and caused both high and low temperature-sensitive growth. Deleting oga1 ⁺ resulted in caffeine-sensitive and canavanine-resistant phenotypes. Based on these results, we discuss the function of Oga1 on the chronological lifespan of fission yeast.     

A Microarray-Based Genetic Screen for Yeast Chronological Aging Factors

Model organisms have played an important role in the elucidation of multiple genes and cellular processes that regulate aging. In this study we utilized the budding yeast, Saccharomyces cerevisiae, in a large-scale screen for genes that function in the regulation of chronological lifespan, which is defined by the number of days that non-dividing cells remain viable. A pooled collection of viable haploid gene deletion mutants, each tagged with unique identifying DNA “bar-code” sequences was chronologically aged in liquid culture. Viable mutants in the aging population were selected at several time points and then detected using a microarray DNA hybridization technique that quantifies abundance of the barcode tags. Multiple short- and long-lived mutants were identified using this approach. Among the confirmed short-lived mutants were those defective for autophagy, indicating a key requirement for the recycling of cellular organelles in longevity. Defects in autophagy also prevented lifespan extension induced by limitation of amino acids in the growth media. Among the confirmed long-lived mutants were those defective in the highly conserved de novo purine biosynthesis pathway (the ADE genes), which ultimately produces IMP and AMP. Blocking this pathway extended lifespan to the same degree as calorie (glucose) restriction. A recently discovered cell-extrinsic mechanism of chronological aging involving acetic acid secretion and toxicity was suppressed in a long-lived ade4Δ mutant and exacerbated by a short-lived atg16Δ autophagy mutant. The identification of multiple novel effectors of yeast chronological lifespan will greatly aid in the elucidation of mechanisms that cells and organisms utilize in slowing down the aging process.     

Mitochondrial and Cytoplasmic ROS Have Opposing Effects on Lifespan

Reactive oxygen species (ROS) are highly reactive, oxygen-containing molecules that can cause molecular damage within the cell. While the accumulation of ROS-mediated damage is widely believed to be one of the main causes of aging, ROS also act in signaling pathways. Recent work has demonstrated that increasing levels of superoxide, one form of ROS, through treatment with paraquat, results in increased lifespan. Interestingly, treatment with paraquat robustly increases the already long lifespan of the clk-1 mitochondrial mutant, but not other long-lived mitochondrial mutants such as isp-1 or nuo-6. To genetically dissect the subcellular compartment in which elevated ROS act to increase lifespan, we deleted individual superoxide dismutase (sod) genes in clk-1 mutants, which are sensitized to ROS. We find that only deletion of the primary mitochondrial sod gene, sod-2 results in increased lifespan in clk-1 worms. In contrast, deletion of either of the two cytoplasmic sod genes, sod-1 or sod-5, significantly decreases the lifespan of clk-1 worms. Further, we show that increasing mitochondrial superoxide levels through deletion of sod-2 or treatment with paraquat can still increase lifespan in clk-1;sod-1 double mutants, which live shorter than clk-1 worms. The fact that mitochondrial superoxide can increase lifespan in worms with a detrimental level of cytoplasmic superoxide demonstrates that ROS have a compartment specific effect on lifespan – elevated ROS in the mitochondria acts to increase lifespan, while elevated ROS in the cytoplasm decreases lifespan. This work also suggests that both ROS-dependent and ROS-independent mechanisms contribute to the longevity of clk-1 worms.     

Osh6 overexpression extends the lifespan of yeast by increasing vacuole fusion

In yeast cells, the vacuole divides and fuses in each round of cell cycle. While mutants defective in vacuole fusion are “wild type” for vegetative growth, most have shortened replicative lifespans under caloric restriction (CR) condition, a manipulation that extends lifespan in wild type cells. To explore whether vacuole fusion extends lifespan, we screened for genes that can complement the fusion defect of selected mutants (erg6Δ, a sterol mutant; nyv1Δ, a mutant involved in the vacuolar SNARE complex and vac8Δ, a vacuolar membrane protein mutant). This screen revealed that Osh6, a member of the oxysterol-binding protein family, can complement the vacuole fusion defect of nyv1Δ, but not erg6Δ or vac8Δ, suggesting that Osh6’s function in vacuole fusion is partly dependent on membrane ergosterol and Vac8. To measure the effect of OSH6 on lifespan, we replaced the endogenous promoter of OSH6 with a shorter version of the ERG6 promoter to obtain P_ERG6-OSH6. This mutant construct significantly extended the replicative lifespan in a wild type background and in a nyv1Δ mutant. Interestingly, P_ERG6-OSH6 cells were more sensitive to drugs that inhibit the activity of the TOR complex 1 (TORC1) than wild type cells. Moreover, a P_ERG6-OSH6 tor1Δ double mutant demonstrated a greatly shortened lifespan, suggesting a genetic interaction between Osh6 and Tor1. Since active TORC1 stimulates vacuole scission and CR downregulates TORC1, Osh6 may link these two pathways by adjusting vacuolar membrane organization to extend lifespan.