A methionine-restricted diet robustly improves healthspan in key model organisms. For example, methionine restriction reduces age-related pathologies and extends lifespan up to 45% in rodents. However, the mechanisms underlying these benefits remain largely unknown. We tested whether the yeast chronological aging assay could model the benefits of methionine restriction, and found that this intervention extends lifespan when enforced by either dietary or genetic approaches, and furthermore, that the observed lifespan extension is due primarily to reduced acid accumulation. In addition, methionine restriction-induced lifespan extension requires the activity of the retrograde response, which regulates nuclear gene expression in response to changes in mitochondrial function. Consistent with an involvement of stress-responsive retrograde signaling, we also found that methionine-restricted yeast are more stress tolerant than control cells. Prompted by these findings in yeast, we tested the effects of genetic methionine restriction on the stress tolerance and replicative lifespans of cultured mouse and human fibroblasts. We found that such methionine-restricted mammalian cells are resistant to numerous cytotoxic stresses, and are substantially longer-lived than control cells. In addition, similar to yeast, the extended lifespan of methionine-restricted mammalian cells is associated with NFκB-mediated retrograde signaling. Overall, our data suggest that improved stress tolerance and extension of replicative lifespan may contribute to the improved healthspan observed in methionine-restricted rodents, and also support the possibility that manipulation of the pathways engaged by methionine restriction may improve healthspan in humans.

To study the underlying basis of lifespan extension by Meth-R, we developed genetically tractable cell-based model systems. The first of these, the yeast chronological aging assay, assesses the length of time that yeast cells remain viable in a non-dividing state, and is considered to model the aging of quiescent cells in higher organisms [15] . Using this assay, studies have demonstrated interventions, genetic and otherwise, that regulate lifespan not only in yeast, but also in higher organisms, including mammals. For example, calorie restriction (CR) extends yeast chronological lifespan and has been shown to increase lifespan by up to 40% in mice, while impairment of the conserved insulin/IGF-1-like and TOR pathways produces similar gains in both organisms [7] , [8] , [16] , [17] . The second model system, the replicative lifespan of mammalian cells in culture, reflects the propensity of cells to senesce in vivo due to the accumulation of genotoxic damage, as well as other types of cellular stress. Such cells accumulate with age in several tissues [18] – [21] and can contribute to age-related pathology [22] . Here we show that two manipulations (genetic and dietary) aimed at producing a methionine-restricted state robustly extend the chronological lifespan of yeast cells. During the initial preparation of this manuscript, Wu et al. also demonstrated that dietary Meth-R extends yeast chronological lifespan [23] , consistent with our findings, but here we also describe several of the underlying mechanisms. Moreover, we also show that genetic Meth-R confers stress tolerance to both yeast and mammalian cells, engages the retrograde response, and dramatically extends the replicative lifespan of cultured mouse and human fibroblasts.

A clue to the mechanistic basis of Meth-R might be found, however, in the observation that cellular stress resistance tends to correlate positively with cellular and organismal longevity. For example, similar to Meth-R, rapamycin treatment robustly extends lifespan in mammals [7] , [8] , and TOR (‘Target Of Rapamycin’, which is inhibited by rapamycin) negatively affects stress tolerance [9] – [11] . In addition, skin-derived fibroblasts from long-lived mouse strains are resistant to a number of cytotoxic stresses [12] – [14] . Collectively, such findings raise the possibility that interventions that confer organismal lifespan extension, like Meth-R, might do so by improving cellular stress tolerance.

It is well documented in rodents that a diet with a normal caloric content, but containing limiting amounts of methionine, robustly improves healthy lifespan. Rats fed such a diet are up to 45% longer-lived than control rats [1] , [2] . Methionine-restricted mice benefit from a less robust, but still significant extension of lifespan and show a marked amelioration of various age-related pathologies as compared with mice fed a normal diet [3] . While the mechanistic basis of this benefit remains largely unknown, it has been suggested that methionine restriction (Meth-R) might act through mechanisms as diverse as reducing the rate of translation, altering gene expression through hypomethylation of nucleic acids, inducing stress hormesis, modulating the levels of glutathione or endocrine factors like IGF-1, or limiting the production of reactive oxygen species (ROS) [2] – [6] .

Results

Genetic methionine restriction extends yeast chronological lifespan To test the possibility that lifespan extension by Meth-R could be modeled in a highly genetically tractable organism, we made use of the yeast chronological aging assay, which measures the length of time that yeast cells remain viable in a non-dividing state. As mentioned above, a recent study has demonstrated that dietary Meth-R robustly extends yeast chronological lifespan [23]. Our aim, however, was to develop cell-based model systems wherein the methionine-restricted state is produced by genetic means, which has the benefit of allowing all aging experiments to be performed using a single media preparation. Although the intent in preparing matched normal and methionine-limited media is to hold the concentrations of all other components constant, in practice, small differences between media preparations may somewhat confound interpretation of studies utilizing dietary Meth-R. To test whether genetic interventions that abrogate methionine biosynthesis extend lifespan in yeast, we assessed the chronological lifespans of yeast deleted for either of two genes involved in methionine production (MET2 and MET15). We found these mutants to be significantly longer-lived than wild-type (p<0.0001) (Fig. 1A). To determine the extent to which this intervention (“genetic Meth-R”, as we term it) recapitulates dietary Meth-R in our strain background, we measured the survival of wild-type haploid yeast cells aged in either normal media or media lacking both methionine and cysteine, the latter of which can be converted into methionine via a salvage pathway. We found that cells grown in methionine-restricted media showed a robust extension of lifespan (p<0.0001), to a similar extent as observed for genetic Meth-R (Fig. 1A–B). This suggests that genetic Meth-R is at least as efficient as dietary methionine limitation in producing the methionine-restricted state. For subsequent experiments characterizing genetic Meth-R in yeast, we chose to use the met15Δ deletion (as opposed to met2Δ), owing to the fact that cells of the haploid BY4741 Yeast Knockout Collection already lack MET15, thus facilitating study of the effects of additional mutations on lifespan extension by MET15 deletion. PPT PowerPoint slide

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larger image TIFF original image Download: Figure 1. Extension of yeast chronological lifespan (CLS) by methionine restriction (Meth-R). (A) Effects of methionine biosynthetic deficiencies (genetic Meth-R) on CLS, (B) Effect of dietary Meth-R on CLS, (C) Effect of pH-buffering (BUF) on CLS, (D) Survival of wild-type and met15Δ cells following treatment with increasing amounts of acetic acid, (E) Requirement of the retrograde response for CLS extension by genetic Meth-R. For all panels, bars denote standard error of the mean (SEM). https://doi.org/10.1371/journal.pone.0097729.g001 While we found that Meth-R in yeast recapitulated the benefits of this manipulation to rodents, it was unclear whether the benefit to yeast was due to methionine limitation, specifically, or merely the consequence of reducing the cellular pool of any single amino acid. It was previously demonstrated that limitation of total amino acids extends yeast chronological lifespan, and a subsequent study revealed that removal of either asparagine or glutamate from culture media results in a moderate extension of median lifespan [24], [25]. The latter finding, however, is difficult to reconcile with data from another group indicating that cells aged in media containing 20-fold higher levels of glutamate than normal are also long-lived [23]. What is clear, however, is that amino acid availability can have profound consequences for the stationary phase survival of yeast. To determine whether the simple removal of any one amino acid was sufficient to extend chronological lifespan, we aged wild-type yeast in normal media, as well as four other media formulations, each lacking a randomly selected amino acid (lysine, valine, isoleucine or threonine). We observed no lifespan extension for cells aged under these conditions (Fig. S1A). In addition, we found that genetic restriction of lysine, through deletion of the LYS2 gene, was similarly incapable of extending chronological lifespan (Fig. S1B). These data therefore indicate that the mere limitation of any particular amino acid is insufficent to extend chronological lifespan. Rather, the Meth-R-responsive pathway(s) that confer extended lifespan in yeast do so in response to manipulations that specifically restrict methionine.

Improved longevity of methionine-restricted yeast is due to reduced acid accumulation Cells undergoing chronological aging are subjected to multiple stresses, including nutrient-limited conditions and the accumulation of acetic acid. In fact, acetic acid toxicity has been reported to be the major cause of death during yeast chronological aging, and dietary and genetic interventions that extend chronological lifespan result in reduced acetic acid accumulation and/or improved acid tolerance as compared with controls [26]. In addition, it was demonstrated that high acetate levels do not confer toxicity at a neutral pH. To test whether the lifespan extension of methionine-restricted yeast might be due to altered acid metabolism, we determined the lifespans of cells grown in media pH-buffered using 2-(N-morpholino)ethanesulfonic acid (MES, pH 6.0). Under such conditions, aging cells are incapable of acidifying the culture medium below a pH of 5.5, and acetate remains in its conjugate base form, which is non-toxic. In MES-buffered media, the lifespans of wild-type cultures were extended to that of met15Δ (Fig. 1C), with no significant difference in overall longevity (p = 0.5598). Similar results were observed by Wu et al. when assessing the effect of pH buffering on the lifespan of yeast chemically restricted for methionine [23]. Therefore, the benefit of Meth-R to yeast chronological lifespan is either reduced acid accumulation, resistance to acetic acid-induced death, or some combination thereof. To discriminate between these possibilities, we tested whether acid accumulation was affected in methionine-restricted cultures by measuring the pH of normally aged cultures (i.e., unbuffered) at varying intervals. We found that there was a direct correlation between age-related pH and lifespan, with cultures of long-lived cells genetically restricted for methionine (met15Δ) demonstrating higher pH values. Measurements of WT cultures revealed a pH of 3.5 after 13 days of aging, whereas met15Δ cultures were less acidic even 12 days later (Day 25, pH 3.75). While this finding might superficially appear to be contrary to a previous report that pH is not altered between normal and methionine-restricted yeast during chronological aging [23], the authors of the aforementioned study assessed pH only at one timepoint, after 2 days of aging, and were therefore unable to report on subsequent pH changes. To determine whether genetic Meth-R might also render cells more tolerant to acetic acid-induced death, we assessed the survival of both wild-type and met15Δ cells after treatment with an extrinsic acetic acid source at a concentration similar to that typically achieved during chronological aging (10 mM), as well as at higher concentrations (100–200 mM), in order to offset the transient nature of the treatment (acetic acid represents a persistent or chronic stress during chronological aging). All cells were similarly sensitive to acetic acid-induced death, regardless of genotype (Fig. 1D), indicating that genetic Meth-R does not also confer acetic acid tolerance. Our data are therefore consistent with genetic Meth-R extending yeast chronological lifespan primarily by reducing acid accumulation.

Extension of yeast chronological lifespan by methionine restriction requires the retrograde response As methionine-restricted growth resulted in reduced acidification of yeast culture media, we hypothesized that Meth-R might alter the expression of factors involved in cellular metabolism. Because the so-called retrograde response pathway regulates nuclear gene expression in response to nutritional stress and mitochondrial dysfunction [27], [28], we considered the possibility that this pathway might be upregulated in methionine-restricted cells. The key mediator of retrograde signaling is the translocation of the Rtg1/3 transcription factor complex to the nucleus, where it alters expression of a number of genes, enriched for factors involved in metabolism, chromatin remodeling and genome stability. Activation of retrograde signaling has been shown to extend the replicative lifespan of yeast mother cells [29]. Furthermore, it is known that TOR signaling inhibits the retrograde response, and that inhibition of TOR extends both replicative and chronological lifespan [30]–[32]. To explore putative connections between Meth-R and the retrograde response, we asked whether the altered transcriptional program of methionine-restricted cells required RTG3 (which is indispensible for retrograde signaling in yeast [27], [28]). Towards this end, we performed expression profiling of aged (Day 3) wild-type, met15Δ, and met15Δ rtg3Δ cells. We chose the Day 3 timepoint as it corresponds approximately to the timing of the diauxic shift, when important changes in the metabolic program take place, and hypothesized that the modulation of such changes may underlie the decreased acid accumulation and extended lifespan of met15Δ cells. Of 1625 probes revealing differential gene expression in met15Δ cells as compared with wild-type, the altered expression of 313 (19%) was either blunted or absent in met15Δ rtg3Δ cells lacking a functional retrograde response (Tables 1 and S1). Using the DAVID functional annotation tool, [33], we found that 222 functional categories were enriched within the identified probe sets (88 for upregulated genes, 134 for downregulated genes; Table S2). Furthermore, several of the enriched categories (particularly those associated with upregulated genes) correspond to gene groupings with functions in metabolism, e.g., sulfur metabolic process (p = 0.00011), acetyl-CoA metabolic process (p = 0.00792), tricarboxylic acid cycle (p = 0.00328), glycolysis/gluconeogenesis (p = 0.03555), and generation of precursor metabolites and energy (p = 0.02101) (see Table S2 for complete list). Differentially-expressed genes were also enriched for factors involved in protein turnover (p = 0.01162) and proteasome function (p = 0.04398), which is intriguing given that protein quality control mechanisms have been implicated in the regulation of longevity [34]. Consistent with our model of Meth-R-mediated yeast lifespan extension, it is likely that perturbation of one or more of these processes contributes to the reduced acid accumulation and extended chronological lifespan of methionine-restricted cells. PPT PowerPoint slide

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larger image TIFF original image Download: Table 1. List of genes differentially-expressed in aged (Day 3) yeast by genetic Meth-R, dependent on the retrograde response. https://doi.org/10.1371/journal.pone.0097729.t001 In order to directly test whether the extended lifespan of methionine-restricted yeast cells required the retrograde response, we assessed the chronological lifespan of met15Δ cells that also lacked RTG3. We found that lifespan extension by genetic Meth-R was indeed dependent upon RTG3 (Fig. 1E), as cultures of met15Δ rtg3Δ double mutant cells failed to persist longer than wild-type (p = 0.4405). Furthermore, the impairment of genetic Meth-R-dependent lifespan extension in cells lacking the retrograde response was not due merely to non-specific sickness, as we found that rtg3Δ single mutant cells were not shorter-lived than wild-type. Collectively, these experiments have revealed an integral role for retrograde signaling in the extension of yeast chronological lifespan by genetic Meth-R.

Altered tRNA metabolism and extension of yeast chronological lifespan Studies have demonstrated that growth in media lacking methionine results in hypomethylation of tRNAs [35], [36], raising the possibility that the extension of yeast lifespan observed upon Meth-R is mediated by altered tRNA metabolism. Prompted by this, as well as a report that cells lacking the Trm9 tRNA methyltransferase show extended longevity in the chronological aging assay [37], we determined the chronological lifespans of several yeast strains, each lacking one of several different tRNA methyltransferase gene (not shown). Of these, only cells lacking the NCL1 gene were found to be long-lived, showing a dramatic increase in lifespan as compared with wild-type (p<0.0001) (Fig. 2A). Notably, Ncl1 is responsible for all m5c methylation of tRNAs [38], [39]. Similar to the results for Meth-R, the lifespan of long-lived ncl1Δ cells was inversely proportional to the extent to which they acidified their culture media (Day 23, pH 3.75 and Day 13, pH 3.5; ncl1Δ and WT, respectively). Additionally, the buffering of media (MES, pH 6.0) used for aging of wild-type and ncl1Δ cells removed the benefit of NCL1 deletion to the relative longevity of these cells, rendering all cells long-lived (not shown). To further explore connections between Meth-R and tRNA metabolism, we used qRT-PCR to determine the relative abundance of three randomly selected tRNA species (tRNA-elongator-Met, tRNA-Cys, and tRNA-Ser) in aged wild-type cells, ncl1Δ cells and cells restricted for methionine. As a positive control for the sensitivity of our assay, we were able to detect elevated tRNA levels in cells lacking the Rny1 ribonuclease, which cleaves tRNAs under conditions of stress. Importantly, we found that all long-lived cells showed elevated accumulation of the interrogated tRNAs as compared with wild-type (∼2-fold for each tRNA in methionine-restricted or ncl1Δ cells; p = 0.0085–0.0339) (Fig. S2). These studies therefore suggest that tRNA metabolism may indeed play an important role in propagating the effects of Meth-R. PPT PowerPoint slide

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larger image TIFF original image Download: Figure 2. Extension of yeast CLS by mutations predicted to phenocopy the cellular consequences of Meth-R. (A) Effect of tRNA hypomethylation on CLS, (B) Effect of cytochrome C deficiency on CLS, dependent on the retrograde response, (C) Effect of cytochrome C overexpression on CLS, as compared with control cells (vector only), (D) Deletion of yeast metacaspase does not extend CLS. For all panels, bars denote SEM. https://doi.org/10.1371/journal.pone.0097729.g002

Evidence for involvement of cytochrome C in the methionine restriction–induced activation of the retrograde response Data from two published studies suggested to us a putative mechanism by which Meth-R might alter tRNA metabolism to promote longevity. First, a recent study in mice has shown that tRNA binds to cytochrome C and inhibits its ability to activate caspase [40]. Second, activation of the retrograde response in yeast is induced by mitochondrial dysfunction, nutritional stress and defects in metabolism [27], [28]. As cytochrome C supports oxidative phosphorylation and functions in mitochondria, we were intrigued by the possibility that elevated tRNA levels in methionine-restricted yeast might inhibit some aspect of cytochrome C function, thereby activating the retrograde response. We thus hypothesized that a reduction in the levels of cytochrome C, by deletion of a gene encoding one of its isoforms (CYC1), might mimic a partial inhibition of cytochrome C function, thereby activating the retrograde response and extending yeast chronological lifespan. As predicted, we found that cyc1Δ cells were long-lived (p = 0.0195) (Fig. 2B). In addition, and similar to the results for Meth-R, we found a requirement for retrograde signaling in extension of lifespan by loss of Cyc1, as cyc1Δ rtg3Δ cells were not longer-lived than wild-type (p = 0.538). To further explore the role of cytochrome C in regulating yeast chronological lifespan, we introduced into wild-type yeast an overexpression construct encoding cytochrome C. Analysis of the resulting strain revealed that forced expression of cytochrome C negatively impacted chronological lifespan as compared with a vector-only control (9 days vs. 13 days; p<0.0001) (Fig. 2C). To insure that the observed reduction in lifespan was not due simply to non-specific toxicity associated with elevated levels of cytochrome C, we confirmed that Cyc1 overexpression did not impair cell growth (not shown). These results are therefore consistent with a model wherein a threshold level of functional cytochrome C precludes activation of the retrograde response, this in turn having negative implications for cellular longevity. While the requirement for the retrograde response, and extension of lifespan by reduced acid accumulation are sufficient to explain the increased longevity of methionine-restricted cells, impairment of cytochrome C is a putative step in this extension, and given the role of Cyc1 in yeast programmed cell death (PCD), we sought to determine whether a blunting of PCD might partially underlie the Meth-R phenotype. Therefore, we tested whether deletion of the yeast metacaspase (Yca1), a key mediator of acetic acid-induced PCD [41], [42], might phenocopy the extended longevity of Meth-R. Although Yca1-independent acetic acid-induced PCD pathways also exist in yeast, PCD caused by Cyc1 release requires Yca1 [43]. Similar to a previous study showing only a very modest improvement in chronological lifespan by deletion of YCA1 [44], we saw no benefit of yca1Δ to yeast longevity (Fig. 2D), indicating that inhibition of Cyc1-dependent PCD does not contribute significantly to the extended lifespan of methionine-restricted yeast. In total, however, our genetic studies exploring the mechanism(s) underlying Meth-R in yeast suggest that this intervention may extend yeast chronological lifespan, at least in part, by causing the accumulation of tRNAs, which, through effects on cytochrome C, alters nuclear gene expression and acid metabolism via the retrograde response.

Methionine restriction of yeast confers resistance to multiple cellular stresses As the benefits of Meth-R may be conferred, at least partially, through stress-responsive retrograde signaling, we wondered whether improved stress tolerance might be associated with Meth-R-dependent longevity. Consistent with this idea, genetic Meth-R (met15Δ) was previously shown to confer resistance to oxidative (diamide) and heavy metal stresses (methyl mercury and cadmium) [45]–[47]. Further, as met15Δ cells, and those chemically restricted for methionine (Fig. 1A–B) [23], are long-lived in the chronological aging assay, these cells can thus be considered to be resistant to the nutritional stresses encountered during aging. These findings suggested to us that Meth-R might promote generalized resistance to cytotoxic insults. We therefore tested whether methionine-restricted yeast might also be resistant to heat stress. Specifically, we assessed the survival of wild-type and met15Δ cells subjected to heat shock at 55°C for 5 mins as compared with those incubated at the standard temperature of 30°C. Survival of met15Δ cells following heat shock was found to be nearly 2-fold greater than that of wild-type control cells (Fig. 3A). PPT PowerPoint slide

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larger image TIFF original image Download: Figure 3. Stress tolerance of methionine-restricted yeast. (A) Relative survival (compared with cells grown at the permissive temperature) of yeast strains, as indicated, to 55°C heat shock, (B) Relative survival (compared with cells grown in normal, non-toxic medium) of cells incubated in medium containing the toxic compound 1,10-phenanthroline (10 mM). Values are normalized to survival of met15Δ cells. For both panels, bars denote SEM. https://doi.org/10.1371/journal.pone.0097729.g003 1,10-phenanthroline chelates divalent metal cations and is highly toxic to yeast cells. To determine whether the apparent generalized stress tolerance conferred by Meth-R might also protect cells from the toxic effects of this agent, we incubated both wild-type and met15Δ cells at 30°C in SC medium containing 10 mM 1,10-phenanthroline and assessed their survival after 24 hrs. Cells undergoing genetic Meth-R demonstrated nearly 4-fold greater survival than wild-type cells (Fig. 3B). Therefore, in addition to oxidative damage and heavy metal stress, genetic Meth-R also protects against nutrient-limited conditions, temperature stress and 1,10-phenanthroline toxicity.