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The budding yeast Saccharomyces cerevisiae (S. cerevisiae) has relatively short lifespan and is genetically tractable, making it a widely used model organism in aging research. Here, we carried out a systematic and quantitative investigation of yeast aging with single-cell resolution through transcriptomic sequencing. We optimized a single-cell RNA sequencing (scRNA-seq) protocol to quantitatively study the whole transcriptome profiles of single yeast cells at different ages, finding increased cell-to-cell transcriptional variability during aging. The single-cell transcriptome analysis also highlighted key biological processes or cellular components, including oxidation-reduction process, oxidative stress response (OSR), translation, ribosome biogenesis and mitochondrion that underlie aging in yeast. We uncovered a molecular marker of FIT3, indicating the early heterogeneity during aging in yeast. We also analyzed the regulation of transcription factors and further characterized the distinctive temporal regulation of the OSR by YAP1 and proteasome activity by RPN4 during aging in yeast. Overall, our data profoundly reveal early heterogeneity during aging in yeast and shed light on the aging dynamics at the single cell level.

Summary In the coming decades, a massive shift in the aging segment of the population will have major social and economic consequences around the world. One way to offset this increase is to expedite the development of geroprotectors, substances that slow aging, repair age‐associated damage and extend healthy lifespan, or healthspan. While over 200 geroprotectors are now reported in model organisms and some are in human use for specific disease indications, the path toward determining whether they affect aging in humans remains obscure. Translation to the clinic is hampered by multiple issues including absence of a common set of criteria to define, select, and classify these substances, given the complexity of the aging process and their enormous diversity in mechanism of action. Translational research efforts would benefit from the formation of a scientific consensus on the following: the definition of ‘geroprotector’, the selection criteria for geroprotectors, a comprehensive classification system, and an analytical model. Here, we review current approaches to selection and put forth our own suggested selection criteria. Standardizing selection of geroprotectors will streamline discovery and analysis of new candidates, saving time and cost involved in translation to clinic.

In mammals, extended periods of fasting leads to the accumulation of blood ketone bodies including acetoacetate. Here we show that similar to the conversion of leucine to acetoacetate in fasting mammals, starvation conditions induced ketone body-like acetic acid generation from leucine in S. cerevisiae. Whereas wild-type and ras2Δ cells accumulated acetic acid, long-lived tor1Δ and sch9Δ mutants rapidly depleted it through a mitochondrial acetate CoA transferase-dependent mechanism, which was essential for lifespan extension. The sch9Δ-dependent utilization of acetic acid also required coenzyme Q biosynthetic genes and promoted the accumulation of intracellular trehalose. These results indicate that Tor-Sch9 deficiency extends longevity by switching cells to an alternative metabolic mode, in which acetic acid can be utilized for the storage of stress resistance carbon sources. These effects are reminiscent of those described for ketone bodies in fasting mammals and raise the possibility that the lifespan extension caused by Tor-S6K inhibition may also involve analogous metabolic changes in higher eukaryotes.

Summary 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.

Summary β-adrenoceptors are the common pharmacological targets for the treatment of cardiovascular diseases and asthma. Genetic modifications of β-adrenergic system in engineered mice affect their lifespan. Here, we tested whether genes encoding for key components of the β-adrenergic signaling pathway are associated with human longevity. We performed a 10-year follow-up study of the Chinese longitudinal healthy longevity survey. The Han Chinese population in this study consisted of 963 long-lived and 1028 geography-matched young individuals. Sixteen SNPs from ADRB1, ADRB2, ADCY5, ADCY6, and MAPK1 were selected and genotyped. Two SNPs, rs1042718 (C/A) and rs1042719 (G/C), of ADRB2 in linkage disequilibrium (D' = 1.0; r2 = 0.67) were found to be associated with enhanced longevity in men in two geographically isolated populations. Bonferroni-corrected P-values in a combined analysis were 0.00053–0.010. Men with haplotype A-C showed an increased probability to become centenarians (the frequency of A-C in long-lived and young individuals are 0.332 and 0.250, respectively, OR = 1.49, CI 95% = 1.17–1.88, P = 0.0007), in contrast to those with haplotype C-G (the frequency of C-G in long-lived and young individuals are 0.523 and 0.635, respectively, OR = 0.63, CI 95% = 0.51–0.78, P = 0.000018). The permuted P-values were 0.00005 and 0.0009, respectively. ADRB2 encodes the β2-adrenergic receptor; the haplotype A-C markedly reduced its translational efficiency compared with C-G (P = 0.002) in transfected HEK293 cells. Thus, our data indicate that enhanced production of β2-adrenergic receptors caused by genetic variants is inversely associated with human lifespan.

In this, the fourth installment of our annual Hot Topics review on mRNA translation and aging, we have decided to expand our scope to include recent findings related to the role of TOR signaling in aging. As new data emerge, it is clear that TOR signaling acts upstream of mRNA translation, as well as a variety of other cellular processes, to modulate longevity and healthspan in evolutionarily diverse species. This Hot Topics review will cover important new findings in this area that have occurred over the past year. These include the demonstration that the TOR substrate ribosomal S6 kinase modulates longevity in mammals, the potential for TOR inhibitors as therapeutic treatments for Alzheimer's disease, and further studies emphasizing the importance of differential translation of specific mRNAs for healthy aging and enhanced longevity.

Summary Although AIMP3 ⁄p18 is normally associated with the macromolecular tRNA synthetase complex, recent reports have revealed a new role of AIMP3 in tumor suppression. In this study, we generated a transgenic mouse that overexpresses AIMP3 and characterized the associated phenotype in vivo and in vitro. Surprisingly, the AIMP3 transgenic mouse exhibited a progeroid phenotype, and the cells that overexpressed AIMP3 showed accelerated senescence and defects in nuclear morphology. We found that overexpression of AIMP3 resulted in proteasomedependent degradation of mature lamin A, but not of lamin C, prelamin A, or progerin. The resulting imbalance in the protein levels of lamin A isoforms, namely altered stoichiometry of prelamin A and progerin to lamin A, appeared to be responsible for a phenotype that resembled progeria. An increase in the level of endogenous AIMP3 has been observed in aged human tissues and cells. The findings in this report suggest that AIMP3 is a specific regulator of mature lamin A and imply that enhanced expression of AIMP3 might be a factor driving cellular and ⁄or organismal aging.

The important role that regulation of protein translation plays in determining longevity in invertebrate organisms became widely appreciated in 2007, with the publication of several papers discussed in last year's review. During 2008, several studies have further strengthened the idea that regulation of translation is one component of a highly evolutionarily conserved pathway that modifies longevity. Importantly, studies published this year also began to provide insights into specific mechanisms by which altered mRNA translation does (and in some cases does not) slow aging in invertebrate model organisms.

Dietary restriction increases lifespan and slows the onset of age-associated disease in organisms from yeast to mammals. In humans, several age-related diseases are associated with aberrant protein folding or aggregation, including neurodegenerative disorders such as Alzheimer's, Parkinson's, and Huntington's diseases. We report here that dietary restriction dramatically suppresses age-associated paralysis in three nematode models of proteotoxicity. Similar to its longevity-enhancing properties, dietary restriction protects against proteotoxicity by a mechanism distinct from reduced insulin/IGF-1-like signaling. Instead, the heat shock transcription factor, hsf-1, is required for enhanced thermotolerance, suppression of proteotoxicity, and lifespan extension by dietary restriction. These findings demonstrate that dietary restriction confers a general protective effect against proteotoxicity and promotes longevity by a mechanism involving hsf-1.

Intense effort has been directed at understanding pathways modulating ageing in invertebrate model organisms. Prior to this decade, several longevity genes had been identified in flies, worms and yeast. More recently, with the development of RNAi libraries in worms and the yeast open reading frame (ORF) deletion collection, it has become routine to perform genome-wide screens for phenotypes of interest. A number of worm screens have now been performed to identify genes whose reduced expression leads to longer lifespan, and two ORF deletion longevity screens have been performed in yeast. Interestingly, these screens have linked previously unidentified cellular pathways to invertebrate ageing. More surprising, however, is the sheer number of longevity genes in worms and yeast. In this review, I discuss data from genome-wide screens in the context of evolutionary theories of ageing and raise issues regarding the increasing complexity associated with the genetics of longevity.

Translation of RNA to protein is essential for life. It should perhaps not be surprising, therefore, that appropriate regulation of translation plays a key role in determining longevity. This Hot Topic article discusses papers published in the last year related to the importance of translation and its regulation by signaling through the target of rapamycin kinase, in modulating aging and age-associated diseases.

Summary Two models have been proposed for how calorie restriction (CR) enhances replicative longevity in yeast: (i) suppression of rDNA recombination through activation of the sirtuin protein deacetylase Sir2 or (ii) decreased activity of the nutrient-responsive kinases Sch9 and TOR. We report here that CR increases lifespan independently of all Sir2-family proteins in yeast. Furthermore, we demonstrate that nicotinamide, an inhibitor of Sir2-mediated deacetylation, interferes with lifespan extension from CR, but does so independent of Sir2, Hst1, Hst2, and Hst4. We also find that 5 mm nicotinamide, a concentration sufficient to inhibit other sirtuins, does not phenocopy deletion of HST3. Thus, we propose that lifespan extension by CR is independent of sirtuins and that nicotinamide has sirtuin-independent effects on lifespan extension by CR.

A partial reduction in food intake has been found to increase lifespan in many different organisms. We report here a new dietary restriction regimen in the nematode Caenorhabditis elegans, based on the standard agar plate lifespan assay, in which adult worms are maintained in the absence of a bacterial food source. These findings represent the first report in any organism of lifespan extension in response to prolonged starvation. Removal of bacterial food increases lifespan to a greater extent than partial reduction of food through a mechanism that is distinct from insulin/IGF-like signaling and the Sir2-family deacetylase, SIR-2.1. Removal of bacterial food also increases lifespan when initiated in postreproductive adults, suggesting that dietary restriction started during middle age can result in a substantial longevity benefit that is independent of reproduction.

Combining two or more longevity-altering interventions and determining the resulting effect on lifespan is a common method for examining the relationship between such interventions. An important subset of this type of analysis occurs when one of the factors under study promotes longevity, such as daf-16 in Caenorhabditis elegans or SIR2 in Saccharomyces cerevisiae. For both of these genes, several studies have combined a lifespan shortening null allele with an intervention that extends lifespan. A resulting lifespan similar to that of the short-lived single mutant has generally been interpreted as suggesting that the factors act in the same pathway. In contrast, an intervention extending the lifespan of the short-lived mutant has been interpreted as suggesting that the factors act in genetically distinct pathways. Specific examples of this type of comparison are studies in which DR fails to extend lifespan in yeast (Lin et al. 2000), invertebrates (Rogina & Helfand 2004; Wang & Tissenbaum 2006), and mice (Li et al. 2008) when Sir2-orthologs are mutated. These data have been, and continue to be, interpreted by some to support a model in which DR promotes longevity and healthspan through activation of sirtuins (Baur et al. 2010). It has been previously reported that deletion of SIR2 blocks RLS extension from DR by reduction of glucose and in strains lacking GPA2 or HXK2, two genetic mimics of DR, but not in a strain lacking the rDNA replication fork block protein, FOB1 (Kaeberlein et al. 2004). In order to examine the influence of deleting SIR2 on RLS extension more generally, we generated 30 additional double mutant strains in which a RLS extending deletion was combined with deletion of SIR2. We also tested three additional methods of DR involving growth on alternative carbon sources (ethanol, glycerol, or raffinose). Strikingly, none of these interventions resulted in a significant RLS extension relative to sir2Δ cells (Figure 1; Figure S2; Table S1). Figure 1 Single-gene deletions that extend RLS in wild-type cells do not extend RLS of sir2Δ cells One possible interpretation of these data is that each of the RLS-extending interventions acts upstream of Sir2, perhaps by promoting Sir2 activity. Two observations are inconsistent with this model. First, at least eight single-gene deletions that increase wild type RLS, and all four forms of DR, significantly extend the RLS of sir2Δ fob1Δ cells (Figure S1A; Figure S2; Table S1), demonstrating that SIR2 is not absolutely required for RLS extension in these cases. Second, at least five long-lived deletion mutants show no indication of enhanced Sir2 activity in vivo, as measured by rDNA recombination or rDNA silencing (Figure S3). A similar lack of increased Sir2 activity has been previously reported in cells subjected to DR (Kaeberlein et al. 2005; Riesen & Morgan 2009; Smith et al. 2009). Interestingly, deletion of TOR1 caused a significant decrease in rDNA recombination, but this effect was independent of SIR2 (Figure S3A). An alternative explanation for these data is that loss of SIR2 alters aging such that molecular processes that do not limit RLS in wild-type cells become limiting in sir2Δ cells. Sir2 has multiple functions, including repression of extrachromosomal rDNA circle formation (Kaeberlein et al. 1999), enhancing global rDNA stability and silencing (Gottlieb & Esposito 1989; Smith & Boeke 1997), promoting asymmetric inheritance of damaged proteins (Aguilaniu et al. 2003), and maintaining telomeric chromatin during aging (Dang et al. 2009). Our observation that only deletion of FOB1 is sufficient to suppress the short RLS of sir2Δ cells suggests that (1) the primary RLS-limiting defect in sir2Δ cells is likely related to rDNA instability and (2) none of the 32 deletions tested that slow aging in wild-type cells is able to overcome this defect. One prior study reported that overexpression of Hsp104 could also suppress the short RLS of sir2Δ cells (Erjavec et al. 2007), raising the possibility that accumulation of damaged proteins in sir2Δ mother cells may also contribute to the reduced longevity. While it is likely that many of the genes examined in this study do not require Sir2 for their effect on RLS, we do not believe that all of the 32 long-lived single gene deletion mutants examined here necessarily act via Sir2-independent mechanisms. For example, deletion of SAS2, a histone acetyltransferase known to antagonize Sir2 effects on chromatin (Dang et al. 2009), extends wild-type RLS but fails to extend the RLS of sir2Δ fob1Δ cells (FigureS2b). Thus, both functional and genetic evidence suggest that Sas1 likely acts in the same longevity pathway as Sir2. This study provides a clear demonstration of the challenges associated with interpreting longevity epistasis data. In particular, the failure of a longevity-intervention to extend lifespan in a short-lived background may not be informative regarding the mechanism of lifespan extension in the wild-type context. In the absence of strong evidence indicating that the lifespan shortening is caused by acceleration of the wild-type aging process, caution is warranted when interpreting these types of data.

Recent studies suggest that the initial expression of adrenal phenylethanolamine N-methyltransferase (PNMT) and epinephrine (E) are dependent upon stimulation of adrenal glucocorticoid receptors. However, evidence suggests that the expression of heart and brain PNMT is independent of glucocorticoids. We measured PNMT activity and E levels in adrenal, heart and head over the latter half of gestation in rat fetuses treated chronically with glucocorticoids, and in normal controls. Chronic glucocorticoid treatment ending on embryonic day (e)12 did not affect heart, head or trunk PNMT activity or E levels. In contrast, chronic glucocorticoid exposure ending e19 or e20 resulted in marked increases in both PNMT and E in adrenal, heart and head tissues. The elevation of E in all three tissues was unaffected by maternal adrenalectomy, indicating enhanced fetal E synthesis. In the absence of exogenous glucocorticoid treatment heart PNMT activity peaked on e12, prior to the earliest reported appearance of glucocorticoid receptors. We conclude that expression of PNMT in all three tissues is glucocorticoid independent until the latter part of gestation when it is readily enhanced by glucocorticoids.

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.

In the February issue of Aging Cell , Yang et al . (2007) describe resveratrol derivatives with improved stability and report that they increase yeast replicative lifespan. The authors refer to these drugs as ‘sirtuin activating compounds’, implying that lifespan is increased due to activation of the Sir2 histone deacetylase. We applaud the development of more stable and potent resveratrol analogs. We wish to point out, however, that whether resveratrol and related compounds activate Sir2 in vivo remains unknown and further experimental validation is necessary. The Yang et al . (2007) study as well as a prior report (Howitz et al ., 2003) are complicated by the use of the PSY316AT yeast strain background. Among the strains commonly used in yeast aging research, PSY316AT is the only known case where overexpression of Sir2 fails to increase lifespan (Kaeberlein et al ., 2004, 2005b). While it is possible that a sirtuin agonist could increase Sir2 activity in a manner not achieved by overexpression of the enzyme, it also needs to be considered that resveratrol (and its analogs) could affect longevity in a Sir2-independent manner. Resveratrol is known to interact with a variety of proteins that have yeast homologs, such adenosine monophosphate (AMP) kinase, protein kinase C and mitochondrial adenosine triphosphate (ATP) synthase, among others (Kaeberlein & Rabinovitch, 2006). Any of these proteins could potentially mediate effects of resveratrol on yeast cells or in other organisms. Another complicating factor is the use of replicative lifespan as the sole measure of drug activity. While longevity is of clear interest, increased lifespan does not imply increased Sir2 activity, as several single-gene mutations are known to increase lifespan in a Sir2-independent manner (Kaeberlein et al ., 2004, 2005c). Sir2 activity can be measured more directly, for example, by monitoring transcription of marker genes at telomeres, silent mating loci and rDNA (Smith & Boeke, 1997; Roy & Runge, 2000; Sandmeier et al ., 2002; Kaeberlein et al ., 2005a). In fact, the PSY316AT strain used by Yang et al . was engineered for such a purpose and contains a subtelomerically integrated ADE2 marker. Activation of Sir2 by overexpression of the enzyme results in decreased ADE2 transcription, which in turn causes accumulation of a red pigment in this strain (Kaeberlein et al ., 2005b). Visual inspection of the colonies in the lifespan assays would reveal this pigmentation and it would have been of interest for Yang et al . to comment whether the resveratrol derivatives increased Sir2 activity by this assay. It would also be of great interest to assess the effect of resveratrol and related compounds on Sir2-dependent silencing and histone acetylation at each of the loci Sir2 is known to silence. If locus-specific effects are observed, they could be compared between drug treatments and Sir2 overexpression, which would provide additional evidence of whether Sir2 enzymatic activity is enhanced in vivo by these compounds. The utility of these experiments extends beyond simple verification of these compounds as Sir2 agonists. In a prior study (Howitz et al ., 2003), it was suggested that resveratrol activated Sir2 in a selective fashion, decreasing rDNA recombination without increasing rDNA silencing. Increased expression of Sir2, in contrast, enhances rDNA silencing (Fritze et al ., 1997; Gallo et al ., 2004). Thus, the reported locus-specific activity of resveratrol remains unexplained, but is of great interest when considering potential therapeutic applications in humans. In vitro assays have, however, also produced ambiguous results: while resveratrol activates Sir2 to deacetylate a fluorescent peptide substrate, it does not activate either Sir2 or the human ortholog SIRT1 towards more native peptide substrates (Borra et al ., 2005; Kaeberlein et al ., 2005b). We greet the development of more stable resveratrol analogs with optimism. Thus far, attempts to replicate the reported effects of resveratrol on yeast replicative lifespan (Howitz et al ., 2003) or rDNA recombination have been unsuccessful (Kaeberlein et al ., 2005b). One possible explanation is that resveratrol may be unstable over the time course required to carry out these assays. Fortunately, if resveratrol and related compounds do activate Sir2 in vivo , this activation should be easily detected and independently verified using the more stable derivatives, and most importantly, more specific assays for Sir2 activity. It is our hope that a more thorough examination of the in vivo effects of resveratrol and related compounds in yeast will resolve these interesting questions.