Sphingolipids Control Lifespan Figure 7. Myriocin enhances CLS by mechanisms that are independent of Sch9 and caloric restriction. (A) CLS of sch9D cells (PF102) +/2 Myr treatment. Data represent the mean 6 SEM of survival (* p,0.05, ** p,0.01, No Myr vs 300 ng/ml Myr). (B) CLS of WT (DBY746) cells grown in SDC medium (2% glucose, pH 4.5, 36 iron) +/2 Myr for 3 days was measured. After 72 hrs of incubation (CLS day 1) the medium in half of the cultures was switched to water (E-CR) while the other half were not switched to CR (NR). Water was replaced every third day with fresh water to remove nutrients released from dead cells. Data represent the mean 6 SEM of viable cells in triplicate cultures (** p,0.01, No Myr vs 300 or 600 ng/ ml Myr, E-CR cultures). doi:10.1371/journal.pgen.1002493.g007 This should in turn lower Sit4 activity and increase CLS just like deletion of SIT4 (Figure 1A). Finally, other studies implicate complex sphingolipids in CLS, since ipt1D and skn1D cells, which lack the most complex sphingolipid M(IP)2C, have a slight increase in CLS and deletion of both genes yields an additive increase in CLS [49]. It remains to be determined how a reduction in M(IP)2C increases CLS, but this effect is probably real as skn1D cells ranked high in a survey of lifespan in the yeast single-deletion strain set [1]. Sphingolipids have been implicated also in RLS. The first S. cerevisiae gene found to regulate RLS, LAG1 (Longevity-Assurance Gene 1) [50], was later shown to encode a component of ceramide synthase which catalyzes ceramide synthesis from LCBs and fatty acids (Figure S1 in Text S1) [51,52]. Deleting LAG1 increases RLS but has no effect on CLS [1]. Since the mechanism for increased RLS without increased CLS is unknown, it is difficult to relate these data to our CLS data. In cases where a decrease in LCBs has perturbed a cellular process in yeast it has been possible to restore the process by adding LCBs to the culture medium [53–55]. This strategy suggests that exogenous PHS should prevent the increase in CLS produced by Dox treatment of tetO7-LCB1 cells. In actuality, low levels of PHS (10–500 nM) did not thwart the increase in CLS and higher doses (10 mM) actually enhanced CLS. Exogenous PHS is likely to enhance CLS by inducing the general stress response via activation of genes with stress response elements (STREs) in their promoter, as previously reported [53,56]. In addition, exogenous PHS is known to down-regulate amino acid and other nutrient transporters in yeast [57,58] and this is likely to increase lifespan since amino acid or glucose deprivation enhance yeast lifespan [1,59]. Lifespan regulation by myriocin and CR Because CR is the standard for judging interventions that extend lifespan, we determined if myriocin enhances lifespan in CR-treated cells. We found that myriocin treatment does enhance CLS in cells grown under moderate (0.5% glucose, Figure S3 in Text S1) or extreme CR (water) conditions (Figure 7B). These results are similar to the recent finding that rapamycin treatment increases the lifespan of caloric restricted Drosophila [3] and they have implications for human health. For example, it may be possible to reduce disease incidence and increase human lifespan by combining a drug with low level CR or with a CR mimetic such as rapamycin. Furthermore, a combination regime may reduce disease incidence in genotypic backgrounds which are unrespon- sive to single treatment regimes. Sphingolipids and lifespan in multicellular eukaryotes Increasing lifespan by using a pharmacological agent to down- regulate sphingolipid synthesis may be applicable to other organisms in addition to yeasts much as rapamycin treatment has been used to down-regulate the TOR pathway and increase lifespan in yeast, worms, flies and mice [1–3,10,18]. The sphingolipids that regulate lifespan in yeast may not be present in mammals and, thus, lifespan extension may require modulating another type of mammalian sphingolipid [11]. Ceramides and sphingosine-1-phosphate are the most widely appreciated sphin- golipid signaling molecules in mammals [11,12]. Ceramides are thought to promote age-related diseases since their concentration increases with age in many mammalian cells and tissues where they regulate processes that may control lifespan [60–64]. Recently long-chain hexosylceramides and lactosylceramides have been implicate in aging and lifespan in mice and cultured human cells [65]. The challenge will be to determine if mammalian lifespan can be extended by preventing the increase in ceramides or whether up or down-modulating the concentration of other sphingolipids will extend lifespan. There is, however, precedent for extending lifespan in multicellular eukaryotes through modulation of sphingolipids: inactivation of the Dacer gene, which encodes an PLoS Genetics | www.plosgenetics.org 12 February 2012 | Volume 8 | Issue 2 | e1002493