Sir2-independent life span extension by calorie restriction in yeast

Calorie restriction slows aging and increases life span in many organisms. In yeast, a mechanistic explanation has been proposed whereby calorie restriction slows aging by activating Sir2. Here we report the identification of a Sir2-independent pathway responsible for a majority of the longevity benefit associated with calorie restriction. Deletion of FOB1 and overexpression of SIR2 have been previously found to increase life span by reducing the levels of toxic rDNA circles in aged mother cells. We find that combining calorie restriction with either of these genetic interventions dramatically enhances longevity, resulting in the longest-lived yeast strain reported thus far. Further, calorie restriction results in a greater life span extension in cells lacking both Sir2 and Fob1 than in cells where Sir2 is present. These findings indicate that Sir2 and calorie restriction act in parallel pathways to promote longevity in yeast and, perhaps, higher eukaryotes.     

High osmolarity extends life span in Saccharomyces cerevisiae by a mechanism related to calorie restriction

Calorie restriction (CR) extends life span in many different organisms, including mammals. We describe here a novel pathway that extends the life span of Saccharomyces cerevisiae mother cells but does not involve a reduction in caloric content of the media, i.e., there is growth of yeast cells in the presence of a high concentration of external osmolytes. Like CR, this longevity-promoting response to high osmolarity requires SIR2, suggesting a common mechanism of life span regulation. Genetic and microarray analysis indicates that high osmolarity extends the life span by activating Hog1p, leading to an increase in the biosynthesis of glycerol from glycolytic intermediates. This metabolic shift likely increases NAD levels, thereby activating Sir2p and promoting longevity.     

Calorie restriction--the SIR2 connection

A nutritious diet low in calories improves the health and extends the life span of rodents. Recent studies identified a gene, SIR2, which encodes an NAD-dependent deacetylase and may mediate the effects of calorie restriction. In this review, we discuss SIR2 genes and calorie restriction in the lower organisms yeast and Drosophila. We then describe the physiological changes in mammals during calorie restriction and how they may lead to the observed health benefits. We summarize the roles of mammalian Sirt1 in mediating these changes in tissues and endocrine systems and propose that Sirt1 regulates calorie restriction by sensing low calories and triggering physiological changes linked to health and longevity.     

The dihydrolipoamide acetyltransferase is a novel metabolic longevity factor and is required for calorie restriction-mediated life span extension

Calorie restriction (CR) extends life span in a wide variety of species. Recent studies suggest that an increase in mitochondrial metabolism mediates CR-induced life span extension. Here we present evidence that Lat1 (dihydrolipoamide acetyltransferase), the E2 component of the mitochondrial pyruvate dehydrogenase complex, is a novel metabolic longevity factor in the CR pathway. Deleting the LAT1 gene abolishes life span extension induced by CR. Overexpressing Lat1 extends life span, and this life span extension is not further increased by CR. Similar to CR, life span extension by Lat1 overexpression largely requires mitochondrial respiration, indicating that mitochondrial metabolism plays an important role in CR. Interestingly, Lat1 overexpression does not require the Sir2 family to extend life span, suggesting that Lat1 mediates a branch of the CR pathway that functions in parallel to the Sir2 family. Lat1 is also a limiting longevity factor in nondividing cells in that overexpressing Lat1 extends cell survival during prolonged culture at stationary phase. Our studies suggest that Lat1 overexpression extends life span by increasing metabolic fitness of the cell. CR may therefore also extend life span and ameliorate age-associated diseases by increasing metabolic fitness through regulating central metabolic enzymes.     

Implication of Ca2+ in the regulation of replicative life span of budding yeast

In eukaryotic cells, Ca(2+)-triggered signaling pathways are used to regulate a wide variety of cellular processes. Calcineurin, a highly conserved Ca(2+)/calmodulin-dependent protein phosphatase, plays key roles in the regulation of diverse biological processes in organisms ranging from yeast to humans. We isolated a mutant of the SIR3 gene, implicated in the regulation of life span, as a suppressor of the Ca(2+) sensitivity of zds1Δ cells in the budding yeast Saccharomyces cerevisiae. Therefore, we investigated a relationship between Ca(2+) signaling and life span in yeast. Here we show that Ca(2+) affected the replicative life span (RLS) of yeast. Increased external and intracellular Ca(2+) levels caused a reduction in their RLS. Consistently, the increase in calcineurin activity by either the zds1 deletion or the constitutively activated calcineurin reduced RLS. Indeed, the shortened RLS of zds1Δ cells was suppressed by the calcineurin deletion. Further, the calcineurin deletion per se promoted aging without impairing the gene silencing typically observed in short-lived sir mutants, indicating that calcineurin plays an important role in a regulation of RLS even under normal growth condition. Thus, our results indicate that Ca(2+) homeostasis/Ca(2+) signaling are required to regulate longevity in budding yeast.     

SIR2 calls upon the ER

Resveratrol induces longevity in C. elegans through the action of SIR2. Recently published work shows that resveratrol induces genes of the unfolded protein stress response of the endoplasmic reticulum. Paradoxically, these stress genes are repressed by SIR2, suggesting that resveratrol increases life span by inhibiting this SIR2 action.     

Longevity regulation in Saccharomyces cerevisiae: linking metabolism, genome stability, and heterochromatin.

When it was first proposed that the budding yeast Saccharomyces cerevisiae might serve as a model for human aging in 1959, the suggestion was met with considerable skepticism. Although yeast had proved a valuable model for understanding basic cellular processes in humans, it was difficult to accept that such a simple unicellular organism could provide information about human aging, one of the most complex of biological phenomena. While it is true that causes of aging are likely to be multifarious, there is a growing realization that all eukaryotes possess surprisingly conserved longevity pathways that govern the pace of aging. This realization has come, in part, from studies of S. cerevisiae, which has emerged as a highly informative and respected model for the study of life span regulation. Genomic instability has been identified as a major cause of aging, and over a dozen longevity genes have now been identified that suppress it. Here we present the key discoveries in the yeast-aging field, regarding both the replicative and chronological measures of life span in this organism. We discuss the implications of these findings not only for mammalian longevity but also for other key aspects of cell biology, including cell survival, the relationship between chromatin structure and genome stability, and the effect of internal and external environments on cellular defense pathways. We focus on the regulation of replicative life span, since recent findings have shed considerable light on the mechanisms controlling this process. We also present the specific methods used to study aging and longevity regulation in S. cerevisiae.     

Longevity Regulation in Saccharomyces cerevisiae: Linking Metabolism, Genome Stability, and Heterochromatin

When it was first proposed that the budding yeast Saccharomyces cerevisiae might serve as a model for human aging in 1959, the suggestion was met with considerable skepticism. Although yeast had proved a valuable model for understanding basic cellular processes in humans, it was difficult to accept that such a simple unicellular organism could provide information about human aging, one of the most complex of biological phenomena. While it is true that causes of aging are likely to be multifarious, there is a growing realization that all eukaryotes possess surprisingly conserved longevity pathways that govern the pace of aging. This realization has come, in part, from studies of S. cerevisiae, which has emerged as a highly informative and respected model for the study of life span regulation. Genomic instability has been identified as a major cause of aging, and over a dozen longevity genes have now been identified that suppress it. Here we present the key discoveries in the yeast-aging field, regarding both the replicative and chronological measures of life span in this organism. We discuss the implications of these findings not only for mammalian longevity but also for other key aspects of cell biology, including cell survival, the relationship between chromatin structure and genome stability, and the effect of internal and external environments on cellular defense pathways. We focus on the regulation of replicative life span, since recent findings have shed considerable light on the mechanisms controlling this process. We also present the specific methods used to study aging and longevity regulation in S. cerevisiae.     

Ssd1 is required for thermotolerance and Hsp104-mediated protein disaggregation in Saccharomyces cerevisiae

In the budding yeast Saccharomyces cerevisiae, the Hsp104-mediated disaggregation of protein aggregates is essential for thermotolerance and to facilitate the maintenance of prions. In humans, protein aggregation is associated with neuronal death and dysfunction in many neurodegenerative diseases. Mechanisms of aggregation surveillance that regulate protein disaggregation are likely to play a major role in cell survival after acute stress. However, such mechanisms have not been studied. In a screen using the yeast gene deletion library for mutants unable to survive an aggregation-inducing heat stress, we find that SSD1 is required for Hsp104-mediated protein disaggregation. SSD1 is a polymorphic gene that plays a role in cellular integrity, longevity, and pathogenicity in yeast. Allelic variants of SSD1 regulate the level of thermotolerance and cell wall remodeling. We have shown that Ssd1 influences the ability of Hsp104 to hexamerize, to interact with the cochaperone Sti1, and to bind protein aggregates. These results provide a paradigm for linking Ssd1-mediated cellular integrity and Hsp104-mediated disaggregation to ensure the survival of cells with fewer aggregates.     

Localization of Sir2p: the nucleolus as a compartment for silent information regulators.

In wild-type budding yeast strains, the proteins encoded by SIR3, SIR4 and RAP1 co-localize with telomeric DNA in a limited number of foci in interphase nuclei. Immunostaining of Sir2p shows that in addition to a punctate staining that coincides with Rap1 foci, Sir2p localizes to a subdomain of the nucleolus. The presence of Sir2p at both the spacer of the rDNA repeat and at telomeres is confirmed by formaldehyde cross-linking and immunoprecipitation with anti-Sir2p antibodies. In strains lacking Sir4p, Sir3p becomes concentrated in the nucleolus, by a pathway requiring SIR2 and UTH4, a gene that regulates life span in yeast. The unexpected nucleolar localization of Sir2p and Sir3p correlates with observed effects of sir mutations on rDNA stability and yeast longevity, defining a new site of action for silent information regulatory factors.     

Heat Stress-Induced Life Span Extension in Yeast

The yeastSaccharomyces cerevisiaehas a limited life span that can be measured by the number of times individual cells divide. Several genetic manipulations have been shown to prolong the yeast life span. However, environmental effects that extend longevity have been largely ignored. We have found that mild, nonlethal heat stress extended yeast life span when it was administered transiently early in life. The increased longevity was due to a reduction in the mortality rate that persisted over many cell divisions (generations) but was not permanent. The genesRAS1andRAS2were necessary to observe this effect of heat stress. TheRAS2gene is consistently required for maintenance of life span when heat stress is chronic or in its extension when heat stress is transient or absent altogether.RAS1,on the other hand, appears to have a role in signaling life extension induced by transient, mild heat stress, which is distinct from its life-span-curtailing effect in the absence of stress and its lack of involvement in the response to chronic heat stress. This distinction between theRASgenes may be partially related to their different effects on growth-promoting genes and stress-responsive genes. Theras2mutation clearly hindered resumption of growth and recovery from stress, while theras1mutation did not. TheHSP104gene, which is largely responsible for induced thermotolerance in yeast, was necessary for life extension induced by transient heat stress. An interaction between mitochondrial petite mutations and heat stress was found, suggesting that mitochondria may be necessary for life extension by transient heat stress. The results raise the possibility that theRASgenes and mitochondria may play a role in the epigenetic inheritance of reduced mortality rate afforded by transient, mild heat stress.     

Autophagy is required for dietary restriction-mediated life span extension in C. elegans

Dietary restriction extends life span in diverse species including Caenorhabditis elegans. However, the downstream cellular targets regulated by dietary restriction are largely unknown. Autophagy, an evolutionary conserved lysosomal degradation pathway, is induced under starvation conditions and regulates life span in insulin signaling C. elegans mutants. We now report that two essential autophagy genes (bec-1 and Ce-atg7) are required for the longevity phenotype of the C. elegans dietary restriction mutant (eat-2(ad1113) animals. Thus, we propose that autophagy mediates the effect, not only of insulin signaling, but also of dietary restriction on the regulation of C. elegans life span. Since autophagy and longevity control are highly conserved from C. elegans to mammals, a similar role for autophagy in dietary restriction-mediated life span extension may also exist in mammals.     

Manipulation of a nuclear NAD+ salvage pathway delays aging without altering steady-state NAD+ levels

Yeast deprived of nutrients exhibit a marked life span extension that requires the activity of the NAD(+)-dependent histone deacetylase, Sir2p. Here we show that increased dosage of NPT1, encoding a nicotinate phosphoribosyltransferase critical for the NAD(+) salvage pathway, increases Sir2-dependent silencing, stabilizes the rDNA locus, and extends yeast replicative life span by up to 60%. Both NPT1 and SIR2 provide resistance against heat shock, demonstrating that these genes act in a more general manner to promote cell survival. We show that Npt1 and a previously uncharacterized salvage pathway enzyme, Nma2, are both concentrated in the nucleus, indicating that a significant amount of NAD(+) is regenerated in this organelle. Additional copies of the salvage pathway genes, PNC1, NMA1, and NMA2, increase telomeric and rDNA silencing, implying that multiple steps affect the rate of the pathway. Although SIR2-dependent processes are enhanced by additional NPT1, steady-state NAD(+) levels and NAD(+)/NADH ratios remain unaltered. This finding suggests that yeast life span extension may be facilitated by an increase in the availability of NAD(+) to Sir2, although not through a simple increase in steady-state levels. We propose a model in which increased flux through the NAD(+) salvage pathway is responsible for the Sir2-dependent extension of life span.     

Role ofRAS2in Recovery from Chronic Stress: Effect on Yeast Life Span

The replicative life span ofSaccharomyces cerevisiaewas previously shown to be modulated by the homologous signal transducers Ras1p and Ras2p in a reciprocal manner. We have used thermal stress as a life span modulator in order to uncover functional differences between theRASgenes that may contribute to their divergent effects on life span. Chronic exposure of cells throughout life to recurring heat shocks at sublethal temperatures decreased their replicative life span.ras2mutants, however, suffered the largest decrease compared to wild-type andras1mutant cells. The decrease was correlated with a substantial delay in resumption of budding upon recovery from these heat shocks, indicating an impaired renewal of cell cycling. Detailed analysis of gene expression showed that, during recovery,ras2mutants were selectively impaired in down-regulation of stress-responsive genes and up-regulation of growth-promoting genes. Our results suggest that one of the functions ofRAS2in maintaining life span, for whichRAS1does not substitute, is to ensure renewal of growth and cell division after bouts of stress that cells encounter during their life. This activity ofRAS2is effected by the cyclic AMP pathway. Overexpression ofRAS2,but notRAS2^ser42which is deficient in the activation of adenylate cyclase, completely reversed the effect of chronic stress on life span. Thus,RAS2is limiting for longevity in the face of chronic stress. SinceRAS2is known to down-regulate stress responses, this demonstrates that for longevity the ability to recover from stress is at least as important as the ability to mount a stress response.     

A chromosomal SIR2 homologue with both histone NAD-dependent ADP-ribosyltransferase and deacetylase activities is involved in DNA repair in Trypanosoma brucei

SIR2-like proteins have been implicated in a wide range of cellular events including chromosome silencing, chromosome segregation, DNA recombination and the determination of life span. We report here the molecular and functional characterization of a SIR2-related protein from the protozoan parasite Trypanosoma brucei, which we termed TbSIR2RP1. This protein is a chromosome-associated NAD-dependent enzyme which, in contrast to other known proteins of this family, catalyses both ADP-ribosylation and deacetylation of histones, particulary H2A and H2B. Under- or overexpression of TbSIR2RP1 decreased or increased, respectively, cellular resistance to DNA damage. Treatment of trypanosomal nuclei with a DNA alkylating agent resulted in a significant increase in the level of histone ADP-ribosylation and a concomitant increase in chromatin sensitivity to micrococcal nuclease. Both of these responses correlated with the level of TbSIR2RP1 expression. We propose that histone modification by TbSIR2RP1 is involved in DNA repair.     

Cell resilience in species life spans: a link to inflammation?

Species differences in life span have been attributed to cellular survival during various stressors, designated here as ‘cell resilience’. In primary fibroblast cultures, cell resilience during exposure to free radicals, hypoglycemia, hyperthermia, and various toxins has shown generally consistent correlations with the species characteristic life spans of birds and mammals. However, the mechanistic links of cell resilience in fibroblast cultures to different species life spans are poorly understood. We propose that certain experimental stressors are relevant to somatic damage in vivo during inflammatory responses of innate immunity, particularly, resistance to reactive oxygen species (ROS), low glucose, and hyperthermia. According to this hypothesis, somatic cell resilience determines species differences in longevity during repeated infections and traumatic injuries in the natural environment. Infections and injury expose local fibroblasts and other cells to ROS generated by macrophages and to local temperature elevations. Systemically, acute phase immune reactions cause hypoglycemia and hyperthermia. We propose that cell resilience to somatic stressors incurred in inflammation is important in the evolution of longevity and that longer‐lived species are specifically more resistant to immune‐related stressors. This hypothesis further specifies Kirkwood’s disposable soma theory. We suggest expanding the battery of stressors and markers used for comparative studies to additional cell types and additional parameters relevant to host defense and to their ecological specificities.