Identification of compounds that delay aging in S. pombe
We sought to identify compounds that increase CLS in S. pombe by performing a chemical genetic screen. In order to facilitate high-throughput screening, we adopted the method of lifespan determination by counting the number of colony forming units (CFUs) within an aging yeast culture (Roux et al., 2010) to the format of a 96-well microtiter plate (Fig. 1A). Using this assay, we were able to recapitulate the well-documented effect of DR on lifespan extension in S. pombe (Chen & Runge, 2009). When cells were grown in the standard glucose concentration of 3%, cell survival decreased by day 7, and hardly any cells survived beyond day 10 (Fig. 1A). Conversely, when grown under conditions of DR (0.5% glucose), cells readily survived up to day 10 and beyond. Furthermore, an increased glucose concentration (5%) shortened their survival (Fig. 1A).
Figure 1. Chemical genetic screen for lifespan-extending compounds in Schizosaccharomyces pombe. (A) Establishment of a microtiter assay for lifespan measurement that recapitulates the effect of dietary restriction on aging in fission yeast. S. pombe wild-type (wt, AEP57) cells were cultured in wells of a microtiter plate in supplemented SD medium containing 0.5, 3, or 5% glucose (one row of wells contains the same glucose concentration), and aliquots were transferred to agar plates using a replica tool at days 3, 5, 7, etc., of cultivation. Plates were incubated for 2 days at 30°C and documented. (B) Wortmannin, an inhibitor of phosphoinositide 3-kinases and TOR kinases, caused lifespan extension (2 μg mL−1) as measured with the microtiter assay presented in (A). Dimethyl sulfoxide (DMSO) was used as a solvent control. (C) Lifespan extension was induced by 20 μg mL−1 acivicin, 4 μg mL−1 monensin, 2 μg mL−1 nigericin, 20 μg mL−1 prostaglandin J2 (PGJ2), 50 μg mL−1 mangosteen, 20 μg mL−1 diindolylmethane (DIM), 4 μg mL−1 tschimganine, and 20 μg mL−1 mycophenolic acid (MPA). DMSO served as a solvent control. (D) Lifespan-extending effect of the indicated compounds in the standard CLS assay. Viability at day 0 (=reaching stationary phase, approximately 30 h after inoculation) was set to 100%, and subsequent survival in the aging cultures was normalized to day 0. Lifespan extension of an sck2∆ strain (AEP62) is shown for comparison. Compound concentrations are as in C. Gray curves represent compound-treated/sck2∆ cells, while the black curves display the controls (DMSO/wt). The black bars represent the standard deviation. (E) Lifespan extension by PGJ2. Cells were cultured in liquid medium and exposed to 20 μg mL−1 PGJ2 or the solvent control (DMSO). Aliquots were sampled at the indicated times, serially diluted and spotted on complete medium. Plates were grown for 2 days at 30°C.
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We subsequently used this assay to screen a small, commercially available library of 522 natural products (Table S1) for substances that increased the CLS of S. pombe. Compounds were added at day 0 (initial inoculation of the microculture) to the growth medium (3% glucose) at a concentration of 4 μg mL−1 or 20 μg mL−1. Because the compounds in the library have different molecular weights, these concentrations corresponded to an average of 10 and 50 μm, respectively, which are concentrations frequently chosen for substance screens (Bedalov et al., 2001). Thus, the cells were exposed to the substance during the whole course of the aging experiment. Cells were transferred back to agar plates after days 3, 5, 7, 10, and 12, etc., and the formation of cell spots after incubation of the plates was documented. In an initial screen, candidate compounds were identified that prolonged cell viability in aging cultures as measured by this assay. Their effect on lifespan extension was subsequently validated using a range of compound concentrations (see below), which led us to identify 19 compounds that increased CLS in S. pombe (Table 1).
Table 1. Compounds extending chronological lifespan in Schizosaccharomyces pombea
|CAS No.||Common name||Concentration (μg mL−1) b||Effectc|
Among these compounds was wortmannin, a known inhibitor of phosphoinositide 3-kinases and TOR kinases (Fig. 1B). As the inhibition of TOR kinases in S. cerevisiae and other organisms extends lifespan (Fontana et al., 2010), we surmise that wortmannin causes lifespan extension in S. pombe due to its inhibitory effect on these kinases, and we therefore did not pursue it further.
Here, we concentrated on the further analysis of eight life-extending compounds that showed strong (++) or very strong (+++) CLS extension in the microtiter dish assay (Table 1): acivicin, 3,3′-diindolylmethane (DIM), mangosteen (a compound mixture, here referred to as a compound for simplicity), monensin, mycophenolic acid (MPA), nigericin, prostaglandin J2 (PGJ2), and tschimganine (Fig. S1). We have not pursued the eleven other lifespan-extending compounds (Table 1), because their effect on aging was mild. Nicotine only increased lifespan at a relatively high dose (1 mg mL−1) and for this reason was not further investigated.
We tested several concentrations of the eight compounds for CLS extension and used the lowest possible concentration that did not cause a growth defect in subsequent experiments (Fig. S2). For instance, tschimganine showed the most significant lifespan extension at a concentration of 4 μg mL−1, but caused a growth delay at 20 μg mL−1 (Fig. S2). Thus, all eight compounds caused a marked increase in longevity in the microtiter aging assay (Fig. 1C). To our knowledge, these eight compounds have no apparent structural similarity to known life-extending molecules (Fig. S1), thus suggesting that they may modulate longevity by so far unknown mechanisms.
Because the compounds cause an effect on aging, we surmise that they are able to cross the cell wall and plasma membrane and affect an intracellular target. Furthermore, none of the compounds affected aging by changing the pH of the growth medium (data not shown). Of note, previous studies have shown that aging in S. pombe, unlike in S. cerevisiae (Longo et al., 2012), is insensitive to acetic acid in the medium (Zuin et al., 2010).
We further sought to validate the effect of seven of these compounds on lifespan extension by determining their effect on the viability of S. pombe cell cultures as determined by measuring CFUs (the effect of PGJ2 was measured using a modified assay because of limited compound availability). In this assay, all seven compounds caused an increased longevity of the S. pombe strain that was comparable to that of the absence of Sck2 (Fig. 1D), thus confirming their effect on lifespan extension in an independent assay. A modified version of this assay was employed for PGJ2 (Fig. 1E, see Supplementary Information for experimental procedure), which confirmed its effect on longevity.
Because DR induces longevity in many organisms, it was possible that the lifespan-extending compounds effectively were mimetics of DR conditions. All eight compounds were able to increase the lifespan of cells under DR (1% glucose) (Fig. 2A), indicating that they functioned in pathways that were parallel to or partially overlapping with those of DR. All compounds also increased lifespan of cells with overnutrition (5% glucose, Fig. 2B), showing that they were able to counteract the negative effects of a high-caloric diet in yeast.
Figure 2. The lifespan-extending compounds acted independently of dietary restriction and Sck2 and partially depended on Pka1. (A) Effect of the indicated compounds on aging under dietary restriction (1% glucose) in a wt strain (AEP57). Concentrations of substances are as in Fig. 1C. (B) Effect of the compounds in overnutrition conditions (5% glucose). Compound concentrations as in Fig. 1C. (C) Lifespan-extending effect of the indicated compounds (concentrations as in Fig. 1C) in an sck2∆ strain (AEP62). Aging of a wt strain (AEP60) is shown for comparison. (D) Effect of lifespan-extending compounds in a pka1∆ strain (AEP117) under overnutrition conditions (5% glucose).
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Aging in S. pombe is delayed in the absence of Sck2 and Pka1 (Roux et al., 2006). Notably, while sck2∆ caused an extended lifespan as compared to wild-type (wt) cells, administration of the compounds even further increased the lifespan of the sck2∆ strain (Fig. 2C), which suggested that none of the compounds acted in aging by inhibiting Sck2. The effect in the pka1∆ strain background was less homogeneous. Tschimganine and nigericin caused lifespan extension of pka1∆ cells (Fig. 2D), indicating that their effect on aging was independent of Pka1. Furthermore, pka1∆ itself causes a slight growth defect as compared to wt strains, and four substances, DIM, mangosteen, acivicin, and MPA, exacerbated this growth defect, indicating that they acted in pathways parallel to Pka1. Furthermore, monensin and PGJ2 were unable to further increase longevity of the pka1∆ strain, suggesting that their effect in aging was epistatic to the Git3/PKA nutrient signaling pathway.
Many long-lived mutants in S. cerevisiae cause increased resistance to heat shock and oxidative stress (Fabrizio et al., 2010), and suppression of DNA damage may cause longevity. We therefore tested whether the anti-aging compounds affected the response of cells to these treatments as well as to DNA damage by methyl-methane sulfonate (MMS). Notably, with the exception of monensin and tschimganine, all compounds caused increased resistance to oxidative stress (Fig. 3). In contrast, only DIM, mangosteen, and tschimganine caused heat-shock resistance, whereas monensin, nigericin, MPA, and acivicin had the contrary effect and resulted in increased sensitivity to heat shock and additionally led to increased sensitivity to MMS (Fig. 3). Therefore, as has been reported in earlier studies for genetic mutations (Fabrizio et al., 2010), there was little concordance between the effect of the compounds on lifespan extension and on stress response or DNA damage response.
Figure 3. Effect of anti-aging compounds on sensitivity to oxidative stress, heat shock, and DNA damage. A wt strain (AEP57) was treated with H2O2 (150 mm) for 1 h, heat shocked (HS) at 48°C for 1 h or treated with methyl-methane sulfonate (MMS), 0.2%] for 45 min. Serial dilutions were spotted onto full medium plates and grown for 2 days at 30°C.
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In summary, the above results showed that two compounds, monensin and PGJ2, may influence aging by inhibiting the PKA signaling pathway, whereas the other compounds acted in pathways parallel to the kinases Pka1 and Sck2 as well as to DR, suggesting that they functioned by novel anti-aging mechanisms. We next sought to gain more mechanistic insight into the aging pathways affected by these compounds.
Anti-aging effects of monensin and nigericin reveal a role for vacuolar acidification in aging
Comparison of the chemical structures of the lifespan-extending compounds showed that two of them, monensin and nigericin, have similar structures (Fig. S1). Both compounds are ionophores that mediate Na+/H+ and K+/H+ exchange. As a consequence, they are able to disturb the membrane potential and thus the function of several intracellular compartments, including Golgi and the vacuole (Dinter & Berger, 1998). Notably, treatment with the Golgi-disturbing agent brefeldin A had no effect on lifespan (data not shown), indicating that endoplasmic reticulum (ER) stress alone was not sufficient to extend lifespan. Importantly, in S. cerevisiae, monensin treatment is epistatic to the function of the vacuolar H+ ATPase (V-ATPase) (Gustavsson et al., 2008), indicating that its most prominent effect in yeast is its interference with the maintenance of an acidic pH in the vacuole.
In S. pombe, the genes vma1+ and vma3+ code for subunits A and C of the V-ATPase (Iwaki et al., 2004), which is required for vacuolar acidification, vacuole morphology, and endocytosis. If monensin and nigericin influence lifespan by disturbing the pH of the vacuole, then one might expect that disturbance of vacuolar acidification may similarly affect lifespan and that this effect should be epistatic to the ionophores. Interestingly, we observed that vma1∆ and vma3∆ both caused a shortening of the S. pombe lifespan (Fig. 4A), indicating that the absence of an acidic pH in the vacuole or changes in vacuolar morphology enhanced aging. This was in agreement with observations in S. cerevisiae that the deletion of genes implicated in protein targeting to the vacuole (VPS genes) caused a shortening of lifespan (Fabrizio et al., 2010). Significantly, in these vacuolar mutants, both monensin and nigericin were no longer able to cause a prolonged lifespan (Fig. 4A). This showed that the effect of these compounds on aging required the activity of the V-ATPase.
Figure 4. Pharmacological or genetic disturbance of vacuolar acidification caused lifespan extension. (A) Lifespan extension by monensin and nigericin required a functional V-ATPase. Aging assays with wt (AEP67), vma1∆ (AEP68), and vma3∆ (AEP69) strains were performed in the presence of 4 μg mL−1 monensin or 2 μg mL−1 nigericin or with the solvent control (DMSO). vma1∆ caused lifespan shortening. (B) Overexpression of vma1+ prolonged lifespan and was not further extended by treatment with monensin. A wt strain (AEP57) carrying an empty vector (pAE1429) or a plasmid with vma1+ under control of the nmt1+ promoter (pAE1958) was used in an aging assay with 4 μg mL−1 monensin or DMSO as a control. (C) Vacuolar fragmentation in aged cells was counteracted by treatment with monensin or nigericin, or by overexpression of vma1+. Acidic compartments of young cells (exponential growth phase) or aged cells (day 3) were stained with quinacrine to visualize vacuoles. Monensin (4 μg mL−1) or nigericin-treated cells (2 μg mL−1) were compared with control cells treated with DMSO.
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As the absence of V-ATPase accelerated S. pombe aging, this suggested that the opposite, namely increased V-ATPase function, may have the reverse effect and cause lifespan extension. Indeed, overexpression of vma1+ from the heterologous nmt1+ promoter attenuated aging (Fig. 4B), and as above, the presence of monensin was unable to further increase lifespan in this context.
We further sought to verify the effect of monensin and nigericin on vacuolar acidification by measuring the accumulation of the fluorescent dye quinacrine in acidic compartments. Untreated young wt cells showed maximal vacuolar fluorescence, and no further increase was observable in young cells treated with the compounds (Fig. 4C). In contrast, as expected, vma3∆ cells lacking the V-ATPase showed no quinacrine staining. Interestingly, aged untreated cells showed a weaker quinacrine staining that was more dispersed and punctate, suggesting that the vacuoles became fragmented and that vacuolar acidity decreased with increasing age in S. pombe cells. Strikingly, aged cells treated with monensin or nigericin showed a distinct quinacrine staining in that the cells were strongly and uniformly stained and lacked the fragmentation of acidic compartments observed in untreated cells. Furthermore, the overexpression of vma1+ showed the same staining pattern as monensin or nigericin treatment in aged cells (Fig. 4C). This indicated that the two compounds or vma1+ overexpression counteracted the age-associated fragmentation of acidic compartments and the loss of acidity during aging.
In summary, our results showed that maintenance of the acidic pH of the vacuole and suppression of vacuolar fragmentation, either using the V-ATPase or by pharmacological intervention using monensin and nigericin, were able to extend lifespan in S. pombe cells, thus showing an unanticipated role for vacuolar pH homeostasis in aging.
Prostaglandin J2 uncovers a role for mitochondrial fission in lifespan extension
In our screen, we identified the class 2 prostaglandin PGJ2 to promote longevity in S. pombe. Of note, yeast cells do not produce prostaglandins. We also tested other prostaglandins (PGA1, PGB1, PGE1, PGE2, and PGF2α) for an effect on aging, but none of them caused lifespan extension (data not shown). Importantly, 15d-PGJ2 has previously been shown to induce mitochondrial elongation in mammalian cells (Mishra et al., 2010), and we therefore asked whether the lifespan-extending effect in yeast was related to this function.
Mitochondria are dynamic organelles that continuously fuse and divide. In mammals, the key molecules for mitochondrial fission are dynamin-related protein 1 (Drp1) and Fis1 (Scorrano, 2007). The reciprocal process, mitochondrial fusion, is controlled by mitofusin (Mfn) 1/2 and OPA1 (Scorrano, 2007). Accordingly, the induction of mitochondrial elongation by 15d-PGJ2 in mammalian cells results from the inhibition of mitochondrial fission by covalent modification and inactivation of the fission protein Drp1 (Mishra et al., 2010). In fission yeast, homologs of Drp1 and OPA1 are called Dnm1 and Msp1.
In order to test whether there is a relationship between the potential inactivation of the mitochondrial fission protein Dnm1 by PGJ2 and lifespan extension, the CLS of dnm1∆ and msp1∆ cells was determined. Notably, the CLS of dnm1∆ cells was comparable to that of wt cells, while msp1∆ cells displayed a slightly reduced lifespan (Fig. 5A), indicating that impaired mitochondrial fusion accelerated aging. This was in agreement with the observation in S. cerevisiae that the absence of the OPA1/Msp1 homolog Mgm1 reduced lifespan (Scheckhuber et al., 2011). Importantly, treatment with PGJ2 resulted in increased lifespan of wt and msp1∆ cells, whereas the CLS of dnm1∆ cells was unchanged by PGJ2 (Fig. 5B). These results indicated that lifespan extension by PGJ2 required Dnm1 and that it thus acted in the Dnm1-dependent mitochondrial division pathway. However, dnm1∆ cells, which have a defect in mitochondrial fission, did not display an increased CLS compared to wt cells, suggesting that PGJ2 affected other anti-aging pathways apart from mitochondrial fission.
Figure 5. Prostaglandin J2 caused lifespan extension by inhibition of mitochondrial fission and the glucose receptor Git3. (A) The longevity-promoting effect of PGJ2 required the mitochondrial fission protein Dnm1, but not the fusion protein Msp1. Aging assays were performed with wt (AEP57), dnm1∆ (AEP176), and msp1∆ (AEP177) in the presence of 20 μg mL−1 PGJ2 or DMSO. (B) Treatment with PGJ2 caused mitochondrial elongation. Young and aged wt cells (AEP57, at exponential growth phase or at day 1) treated with DMSO or 20 μg mL−1 PGJ2 were stained with MitoTracker Orange to visualize mitochondria. (C) Induction of longevity by PGJ2 required the G-protein-coupled glucose receptor Git3, but none of the other G-protein-coupled proteins in Schizosaccharomyces pombe. The indicated strains were used for aging assays in the presence or absence of PGJ2 as indicated in (A).
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The above results suggested that PGJ2 acted in aging by inhibiting mitochondrial fission. To further test this, we monitored mitochondrial morphology in young and aged cells in the presence or absence of PGJ2. In young cells, a punctate pattern of mitochondrial staining was observed, regardless of whether cells were treated or not (Fig. 5B). In aged untreated cells, mitochondrial staining was weak and showed an altered distribution. Importantly, aged cells that were treated with PGJ2 contained elongated, tubular mitochondria that were not observed in untreated cells. This demonstrated that PGJ2 caused mitochondrial elongation, most likely by inhibiting fission.
Notably, prostaglandins in human cells have been reported to inhibit G-protein-coupled receptors (GPCR) (Narumiya et al., 1999), indicating that PGJ2 may also inhibit such (a) receptor(s) in yeast. Therefore, we analyzed the CLS of strains carrying a deletion of genes encoding GPCRs or proteins involved in G-protein signaling (Xue et al., 2008). One such protein is the glucose receptor Git3 whose absence has previously been shown to increase CLS (Roux et al., 2010). In our experiment, git3∆ cells were the only cells whose lifespan was increased compared to that of wt (Fig. 5C). Furthermore, PGJ2 treatment caused no further lifespan extension in git3∆ cells, while it increased CLS in all other GPCR deletion strains (Fig. 5C). This suggested that PGJ2 extended the CLS of fission yeast by inhibiting Git3 in addition to its effect on mitochondrial fission. Because Git3 signals via Pka1 (Roux et al., 2010), this was in agreement with our earlier observation of a Pka1 dependence of lifespan extension by PGJ2 (Fig. 2D).
Anti-aging properties of diindolylmethane, mangosteen, and tschimganine
We further considered the effects of the three remaining anti-aging compounds identified here, DIM, mangosteen, and tschimganine. DIM influences several different pathways that are linked to a delay of aging (Aggarwal & Ichikawa, 2005), and DIM is being used in clinical trials against multiple forms of cancer. Notably, it has been reported to increase the production of ROS, but also to protect against oxidative stress (Fan et al., 2009). To test for an effect of DIM on ROS production in yeast cells, we stained DIM-treated wt cells with dihydrorhodamine 123 (DHR123), a cell-permeable fluorogenic probe that can be used to detect ROS (Roux et al., 2006). Interestingly, DIM-treated cells in the exponential phase of growth showed increased ROS levels as compared to control cells, whereas ROS levels were decreased in stationary-phase cells (Fig. 6A), an effect that was not observed for any of the other lifespan-extending compounds reported here (data not shown).
Figure 6. Anti-aging properties of diindolylmethane (DIM), mangosteen, and tschimganine. (A) DIM induced the production of reactive oxygen species (ROS) in exponentially growing cells, and 20 μg mL−1 DIM and 50 μg mL−1 mangosteen caused a reduction in cells in stationary phase. wt cells (AEP57) in the respective growth phases were stained with DHR123. Numbers above the bars indicate the number of cells evaluated. (B) Chemical structure of tschimganine, tschimgine, and tschimganidine. (C) Tschimganidine, but not tschimgine, increased lifespan in fission yeast. The aging assay was performed as in Fig. 1C using the indicated compound concentrations.
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Similar to DIM, mangosteen also has known anti-oxidative properties (Obolskiy et al., 2009). It is a compound mixture extracted from the exocarp of the mangosteen fruit (Garcinia mangostana). Accordingly, like DIM, mangosteen caused a decrease in ROS levels in stationary-phase cells (Fig. 6A). However, in contrast to DIM, mangosteen did not lead to increased ROS levels in exponentially growing cells. We furthermore tested the individual effects of the four xanthones that are the main constituents of mangosteen, namely γ-mangostin, α-mangostin, gartanin, and 8-desoxygartanin (Obolskiy et al., 2009), but none of them altered the CLS of S. pombe cells (data not shown), suggesting that another unknown constituent of mangosteen, or the interplay of single compounds, led to lifespan extension.
Tschimganine was first isolated from the roots of Ferula tschimganica. Except for an estrogenic activity (Ikeda et al., 2002), little is known about this compound. Tschimgine and tschimganidine are two related compounds (Fig. 6B), and we therefore tested their effect on aging in S. pombe. Interestingly, tschimganidine caused lifespan extension, although to a lesser extent than tschimganine and at a higher compound concentration, whereas tschimgine showed no effect on lifespan (Fig. 6C). Because the only difference between tschimganine and tschimgine is the presence of a methoxy group in the former, this indicates that this chemical group contributes to the anti-aging effect of tschimganine. This group is contained in tschimganidine, but the bornyl substituent is replaced in tschimganidine by a more complex structure. One possibility is that the bornyl moiety enhances membrane permeability, which may explain why tschimganine has more potent anti-aging properties than tschimganidine.