A genomic analysis of chronological longevity factors in budding yeast

Chronological life span (CLS) has been studied as an aging paradigm in yeast. A few conserved aging genes have been identified that modulate both chronological and replicative longevity in yeast as well as longevity in the nematode Caenorhabditis elegans; however, a comprehensive analysis of the relationship between genetic control of chronological longevity and aging in other model systems has yet to be reported. To address this question, we performed a functional genomic analysis of chronological longevity for 550 single-gene deletion strains, which accounts for approximately 12% of the viable homozygous diploid deletion strains in the yeast ORF deletion collection. This study identified 33 previously unknown determinants of CLS. We found no significant enrichment for enhanced CLS among deletions corresponding to yeast orthologs of worm aging genes or among replicatively long-lived deletion strains, although a trend toward overlap was noted. In contrast, a subset of gene deletions identified from a screen for reduced acidification of culture media during growth to stationary phase was enriched for increased CLS. These results suggest that genetic control of CLS under the most commonly utilized assay conditions does not strongly overlap with longevity determinants in C. elegans, with the existing confined to a small number of genetic pathways. These data also further support the model that acidification of the culture medium plays an important role in survival during chronological aging in synthetic medium, and suggest that chronological aging studies using alternate medium conditions may be more informative with regard to aging of multicellular eukaryotes.Key words: aging, genomic, screen, lifespan, yeast, C. elegans, pH, chronological, replicative     

An Energy-Independent Pro-longevity Function of Triacylglycerol in Yeast

Intracellular triacylglycerol (TAG) is a ubiquitous energy storage lipid also involved in lipid homeostasis and signaling. Comparatively, little is known about TAG’s role in other cellular functions. Here we show a pro-longevity function of TAG in the budding yeast Saccharomyces cerevisiae. In yeast strains derived from natural and laboratory environments a correlation between high levels of TAG and longer chronological lifespan was observed. Increased TAG abundance through the deletion of TAG lipases prolonged chronological lifespan of laboratory strains, while diminishing TAG biosynthesis shortened lifespan without apparently affecting vegetative growth. TAG-mediated lifespan extension was independent of several other known stress response factors involved in chronological aging. Because both lifespan regulation and TAG metabolism are conserved, this cellular pro-longevity function of TAG may extend to other organisms.     

A genomic analysis of chronological longevity factors in budding yeast

Chronological life span (CLS) has been studied as an aging paradigm in yeast. A few conserved aging genes have been identified that modulate both chronological and replicative longevity in yeast as well as longevity in the nematode Caenorhabditis elegans; however, a comprehensive analysis of the relationship between genetic control of chronological longevity and aging in other model systems has yet to be reported. To address this question, we performed a functional genomic analysis of chronological longevity for 550 single-gene deletion strains, which accounts for approximately 12% of the viable homozygous diploid deletion strains in the yeast ORF deletion collection. This study identified 33 previously unknown determinants of CLS. We found no significant enrichment for enhanced CLS among deletions corresponding to yeast orthologs of worm aging genes or among replicatively long-lived deletion strains, although a trend toward overlap was noted. In contrast, a subset of gene deletions identified from a screen for reduced acidification of culture media during growth to stationary phase was enriched for increased CLS. These results suggest that genetic control of CLS under the most commonly utilized assay conditions does not strongly overlap with longevity determinants in C. elegans, with the existing confined to a small number of genetic pathways. These data also further support the model that acidification of the culture medium plays an important role in survival during chronological aging in synthetic medium, and suggest that chronological aging studies using alternate medium conditions may be more informative with regard to aging of multicellular eukaryotes.     

High-Resolution Profiling of Stationary-Phase Survival Reveals Yeast Longevity Factors and Their Genetic Interactions

Lifespan is influenced by a large number of conserved proteins and gene-regulatory pathways. Here, we introduce a strategy for systematically finding such longevity factors in Saccharomyces cerevisiae and scoring the genetic interactions (epistasis) among these factors. Specifically, we developed an automated competition-based assay for chronological lifespan, defined as stationary-phase survival of yeast populations, and used it to phenotype over 5,600 single- or double-gene knockouts at unprecedented quantitative resolution. We found that 14% of the viable yeast mutant strains were affected in their stationary-phase survival; the extent of true-positive chronological lifespan factors was estimated by accounting for the effects of culture aeration and adaptive regrowth. We show that lifespan extension by dietary restriction depends on the Swr1 histone-exchange complex and that a functional link between autophagy and the lipid-homeostasis factor Arv1 has an impact on cellular lifespan. Importantly, we describe the first genetic interaction network based on aging phenotypes, which successfully recapitulated the core-autophagy machinery and confirmed a role of the human tumor suppressor PTEN homologue in yeast lifespan and phosphatidylinositol phosphate metabolism. Our quantitative analysis of longevity factors and their genetic interactions provides insights into the gene-network interactions of aging cells.     

miR-223促进小鼠胚胎成纤维细胞复制性衰老

微小RNA（MicroRNAs（或miRNAs）是作为强大的基因表达调控子,广泛参与多种生命过程,在细胞衰老进程中的作用也日益受到关注。miR-223是一个典型的抑癌基因,可显著抑制细胞增殖能力。此外,miR-223与阿尔茨海默症、心血管疾病以及类风湿性关节炎等衰老相关疾病的发生发展密切相关。尽管如此,miR-223在细胞衰老进程中的作用及其分子机制尚未见报道。本研究通过连续传代建立了小鼠胚胎成纤维细胞（MEF细胞）的复制性衰老模型,并利用荧光定量qRT-PCR检测发现,miR-223在衰老MEF细胞中的表达水平显著上调。随后,通过转染miR-223模拟物Agomir-223在MEF细胞中过表达miR-223,结果显示过表达miR-223可显著促进MEF细胞的衰老表型并抑制其增殖能力,而抑制miR-223的表达可延缓MEF细胞的复制性衰老进程。进一步利用生物信息学方法预测获得多个miR-223的候选衰老相关靶基因,包括Rasa1、Ddit4和Smad1等。然而双萤光素酶报告系统结果显示,miR-223并不显著影响其萤光强度,表明它们很可能并不是miR-223的下游靶基因。综上所述,miR-223可显著促进MEF细胞复制性衰老,然而其调节细胞衰老进程的分子机制依然有待深入研究。     

Molecular phenotyping of aging in single yeast cells using a novel microfluidic device

Budding yeast has served as an important model organism for aging research, and previous genetic studies have led to the discovery of conserved genes/pathways that regulate lifespan across species. However, the molecular causes of aging and death remain elusive, because it is very difficult to directly observe the cellular and molecular events accompanying aging in single yeast cells by the traditional approach based on micromanipulation. We have developed a microfluidic system to track individual mother cells throughout their lifespan, allowing automated lifespan measurement and direct observation of cell cycle dynamics, cell/organelle morphologies, and various molecular markers. We found that aging of the wild-type cells is characterized by an increased general stress and a progressive lengthening of the cell cycle for the last few cell divisions; these features are much less apparent in the long-lived FOB1 deletion mutant. Following the fate of individual cells revealed that there are different forms of cell death that are characterized by different terminal cell morphologies, and associated with different levels of stress and lifespan. We have identified a molecular marker - the level of the expression of Hsp104, as a good predictor for the lifespan of individual cells. Our approach allows detailed molecular phenotyping of single cells in the process of aging and thus provides new insight into its mechanism.     

Genome-Wide Screen in Saccharomyces cerevisiae Identifies Vacuolar Protein Sorting,Autophagy, Biosynthetic,and tRNA Methylation Genes Involved in Life Span Regulation

The study of the chronological life span of Saccharomyces cerevisiae, which measures the survival of populations of non-dividing yeast, has resulted in the identification of homologous genes and pathways that promote aging in organisms ranging from yeast to mammals. Using a competitive genome-wide approach, we performed a screen of a complete set of approximately 4,800 viable deletion mutants to identify genes that either increase or decrease chronological life span. Half of the putative short-/long-lived mutants retested from the primary screen were confirmed, demonstrating the utility of our approach. Deletion of genes involved in vacuolar protein sorting, autophagy, and mitochondrial function shortened life span, confirming that respiration and degradation processes are essential for long-term survival. Among the genes whose deletion significantly extended life span are ACB1, CKA2, and TRM9, implicated in fatty acid transport and biosynthesis, cell signaling, and tRNA methylation, respectively. Deletion of these genes conferred heat-shock resistance, supporting the link between life span extension and cellular protection observed in several model organisms. The high degree of conservation of these novel yeast longevity determinants in other species raises the possibility that their role in senescence might be conserved.     

Regulation of chronological aging in Schizosaccharomyces pombe by the protein kinases Pka1 and Sck2

Budding yeast shows a progressive decline in viability after entering stationary phase, a phenomenon known as chronological aging. We show here that the fission yeast Schizosaccharomyces pombe also undergoes chronological aging and that the process is regulated by genes controlling two related nutrient signalling pathways. The first pathway includes the serine/threonine cAMP-activated protein kinase Pka1 and the second pathway comprises the serine/threonine kinase Sck2, a homologue of Saccharomyces cerevisiae SCH9. A double mutant for pka1 and sck2 displayed an additive effect on prolonging the fission yeast lifespan, suggesting that these genes regulate related but independent pathways. These long-lived mutants also accumulated less reactive oxygen species and had a delayed initiation of apoptosis compared with wild-type cells. We also found that strains carrying pka1 deletion but not those with sck2 deletion gained resistance to oxidative stress due to exposure to H(2)O(2) or menadione. On the other hand, the additional increase in lifespan shown by the Deltapka1Deltasck2 double-mutant strain correlated with an increased resistance to both oxidative stress and heat shock. These results underscore the importance of nutrient signalling pathways and reactive oxygen species on organismal lifespan and establish S. pombe as a new model organism to study the molecular mechanisms underlying aging.     

A role for the actin cytoskeleton in cell death and aging in yeast

Several determinants of aging, including metabolic capacity and genetic stability, are recognized in both yeast and humans. However, many aspects of the pathways leading to cell death remain to be elucidated. Here we report a role for the actin cytoskeleton both in cell death and in promoting longevity. We have analyzed yeast strains expressing mutants with either increased or decreased actin dynamics. We show that decreased actin dynamics causes depolarization of the mitochondrial membrane and an increase in reactive oxygen species (ROS) production, resulting in cell death. Important, however, is the demonstration that increasing actin dynamics, either by a specific actin allele or by deletion of a gene encoding the actin-bundling protein Scp1p, can increase lifespan by over 65%. Increased longevity appears to be due to these cells producing lower than wild-type levels of ROS. Homology between Scp1p and mammalian SM22/transgelin, which itself has been isolated in senescence screens, suggests a conserved mechanism linking aging to actin stability.     

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.     

Genes encoding longevity: from model organisms to humans

Ample evidence from model organisms has indicated that subtle variation in genes can dramatically influence lifespan. The key genes and molecular pathways that have been identified so far encode for metabolism, maintenance and repair mechanisms that minimize age-related accumulation of permanent damage. Here, we describe the evolutionary conserved genes that are involved in lifespan regulation of model organisms and humans, and explore the reasons of discrepancies that exist between the results found in the various species. In general, the accumulated data have revealed that when moving up the evolutionary ladder, together with an increase of genome complexity, the impact of candidate genes on lifespan becomes smaller. The presence of genetic networks makes it more likely to expect impact of variation in several interacting genes to affect lifespan in humans. Extrapolation of findings from experimental models to humans is further complicated as phenotypes are critically dependent on the setting in which genes are expressed, while laboratory conditions and modern environments are markedly dissimilar. Finally, currently used methodologies may have only little power and validity to reveal genetic variation in the population. In conclusion, although the study of model organisms has revealed potential candidate genetic mechanisms determining aging and lifespan, to what extent they explain variation in human populations is still uncertain.     

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.     

The DrugAge database of aging‐related drugs

Aging is a major worldwide medical challenge. Not surprisingly, identifying drugs and compounds that extend lifespan in model organisms is a growing research area. Here, we present DrugAge ( http://genomics.senescence.info/drugs/ ), a curated database of lifespan‐extending drugs and compounds. At the time of writing, DrugAge contains 1316 entries featuring 418 different compounds from studies across 27 model organisms, including worms, flies, yeast and mice. Data were manually curated from 324 publications. Using drug–gene interaction data, we also performed a functional enrichment analysis of targets of lifespan‐extending drugs. Enriched terms include various functional categories related to glutathione and antioxidant activity, ion transport and metabolic processes. In addition, we found a modest but significant overlap between targets of lifespan‐extending drugs and known aging‐related genes, suggesting that some but not most aging‐related pathways have been targeted pharmacologically in longevity studies. DrugAge is freely available online for the scientific community and will be an important resource for biogerontologists.     

Lifespan regulation by evolutionarily conserved genes essential for viability

Evolutionarily conserved mechanisms that control aging are predicted to have prereproductive functions in order to be subject to natural selection. Genes that are essential for growth and development are highly conserved in evolution, but their role in longevity has not previously been assessed. We screened 2,700 genes essential for Caenorhabditis elegans development and identified 64 genes that extend lifespan when inactivated postdevelopmentally. These candidate lifespan regulators are highly conserved from yeast to humans. Classification of the candidate lifespan regulators into functional groups identified the expected insulin and metabolic pathways but also revealed enrichment for translation, RNA, and chromatin factors. Many of these essential gene inactivations extend lifespan as much as the strongest known regulators of aging. Early gene inactivations of these essential genes caused growth arrest at larval stages, and some of these arrested animals live much longer than wild-type adults. daf-16 is required for the enhanced survival of arrested larvae, suggesting that the increased longevity is a physiological response to the essential gene inactivation. These results suggest that insulin-signaling pathways play a role in regulation of aging at any stage in life.     

pH neutralization protects against reduction in replicative lifespan following chronological aging in yeast

Chronological and replicative aging have been studied in yeast as alternative paradigms for post-mitotic and mitotic aging, respectively. It has been known for more than a decade that cells of the S288C background aged chronologically in rich medium have reduced replicative lifespan relative to chronologically young cells. Here we report replication of this observation in the diploid BY4743 strain background. We further show that the reduction in replicative lifespan from chronological aging is accelerated when cells are chronologically aged under standard conditions in synthetic complete medium rather than rich medium. The loss of replicative potential with chronological age is attenuated by buffering the pH of the chronological aging medium to 6.0, an intervention that we have previously shown can extend chronological lifespan. These data demonstrate that extracellular acidification of the culture medium can cause intracellular damage in the chronologically aging population that is asymmetrically segregated by the mother cell to limit subsequent replicative lifespan.     

pH neutralization protects against reduction in replicative lifespan following chronological aging in yeast

Chronological and replicative aging have been studied in yeast as alternative paradigms for post-mitotic and mitotic aging, respectively. It has been known for more than a decade that cells of the S288C background aged chronologically in rich medium have reduced replicative lifespan relative to chronologically young cells. Here we report replication of this observation in the diploid BY4743 strain background. We further show that the reduction in replicative lifespan from chronological aging is accelerated when cells are chronologically aged under standard conditions in synthetic complete medium rather than rich medium. The loss of replicative potential with chronological age is attenuated by buffering the pH of the chronological aging medium to 6.0, an intervention that we have previously shown can extend chronological lifespan. These data demonstrate that extracellular acidification of the culture medium can cause intracellular damage in the chronologically aging population that is asymmetrically segregated by the mother cell to limit subsequent replicative lifespan.     

A genome‐wide screen identifies genes that suppress the accumulation of spontaneous mutations in young and aged yeast cells

To ensure proper transmission of genetic information, cells need to preserve and faithfully replicate their genome, and failure to do so leads to genome instability, a hallmark of both cancer and aging. Defects in genes involved in guarding genome stability cause several human progeroid syndromes, and an age‐dependent accumulation of mutations has been observed in different organisms, from yeast to mammals. However, it is unclear whether the spontaneous mutation rate changes during aging and whether specific pathways are important for genome maintenance in old cells. We developed a high‐throughput replica‐pinning approach to screen for genes important to suppress the accumulation of spontaneous mutations during yeast replicative aging. We found 13 known mutation suppression genes, and 31 genes that had no previous link to spontaneous mutagenesis, and all acted independently of age. Importantly, we identified PEX19, encoding an evolutionarily conserved peroxisome biogenesis factor, as an age‐specific mutation suppression gene. While wild‐type and pex19Δ young cells have similar spontaneous mutation rates, aged cells lacking PEX19 display an elevated mutation rate. This finding suggests that functional peroxisomes may be important to preserve genome integrity specifically in old cells.     

Yeast chronological lifespan and proteotoxic stress: is autophagy good or bad?

Autophagy, a highly conserved proteolytic mechanism of quality control, is essential for the maintenance of metabolic and cellular homoeostasis and for an efficient cellular response to stress. Autophagy declines with aging and is believed to contribute to different aspects of the aging phenotype. The nutrient-sensing pathways PKA (protein kinase A), Sch9 and TOR (target of rapamycin), involved in the regulation of yeast lifespan, also converge on a common targeted process: autophagy. The molecular mechanisms underlying the regulation of autophagy and aging by these signalling pathways in yeast, with special attention to the TOR pathway, are discussed in the present paper. The question of whether or not autophagy could contribute to yeast cell death occurring during CLS (chronological lifespan) is discussed in the light of our findings obtained after autophagy activation promoted by proteotoxic stress. Autophagy progressively increases in cells expressing the aggregation-prone protein α-synuclein and seems to participate in the early cell death and shortening of CLS under these conditions, highlighting that autophagic activity should be maintained below physiological levels to exert its promising anti-aging effects.     

Lifespan Extension by Methionine Restriction Requires Autophagy-Dependent Vacuolar Acidification

Reduced supply of the amino acid methionine increases longevity across species through an as yet elusive mechanism. Here, we report that methionine restriction (MetR) extends yeast chronological lifespan in an autophagy-dependent manner. Single deletion of several genes essential for autophagy (ATG5, ATG7 or ATG8) fully abolished the longevity-enhancing capacity of MetR. While pharmacological or genetic inhibition of TOR1 increased lifespan in methionine-prototroph yeast, TOR1 suppression failed to extend the longevity of methionine-restricted yeast cells. Notably, vacuole-acidity was specifically enhanced by MetR, a phenotype that essentially required autophagy. Overexpression of vacuolar ATPase components (Vma1p or Vph2p) suffices to increase chronological lifespan of methionine-prototrophic yeast. In contrast, lifespan extension upon MetR was prevented by inhibition of vacuolar acidity upon disruption of the vacuolar ATPase. In conclusion, autophagy promotes lifespan extension upon MetR and requires the subsequent stimulation of vacuolar acidification, while it is epistatic to the equally autophagy-dependent anti-aging pathway triggered by TOR1 inhibition or deletion.     

Lithocholic acid extends longevity of chronologically aging yeast only if added at certain critical periods of their lifespan

Our studies revealed that LCA (lithocholic bile acid) extends yeast chronological lifespan if added to growth medium at the time of cell inoculation. We also demonstrated that longevity in chronologically aging yeast is programmed by the level of metabolic capacity and organelle organization that they developed before entering a quiescent state and, thus, that chronological aging in yeast is likely to be the final step of a developmental program progressing through at least one checkpoint prior to entry into quiescence. Here, we investigate how LCA influences longevity and several longevity-defining cellular processes in chronologically aging yeast if added to growth medium at different periods of the lifespan. We found that LCA can extend longevity of yeast under CR (caloric restriction) conditions only if added at either of two lifespan periods. One of them includes logarithmic and diauxic growth phases, whereas the other period exists in early stationary phase. Our findings suggest a mechanism linking the ability of LCA to increase the lifespan of CR yeast only if added at either of the two periods to its differential effects on various longevity-defining processes. In this mechanism, LCA controls these processes at three checkpoints that exist in logarithmic/diauxic, post-diauxic and early stationary phases. We therefore hypothesize that a biomolecular longevity network progresses through a series of checkpoints, at each of which (1) genetic, dietary and pharmacological anti-aging interventions modulate a distinct set of longevity-defining processes comprising the network; and (2) checkpoint-specific master regulators monitor and govern the functional states of these processes.