Author Summary Studies with rats in the 1930s showed a surprising increase in lifespan when the diet contained 30%–40% fewer calories than normal. This experiment has been repeated on many organisms and is the gold standard for extending lifespan. While we are beginning to understand how calorie restriction regulates lifespan, the mechanisms are complex and much remains to be learned. In the work presented here, we demonstrate a novel way to increase lifespan in the budding yeast Saccharomyces cerevisiae. Our strategy is to lower the rate of sphingolipid synthesis either by reducing the synthesis of the first enzyme in the biosynthesis pathway or by using a drug to reduce enzyme activity. This strategy works in part by lowering the activity of a protein kinase, termed Sch9 in yeast and S6K in mammals, both of which are involved in the processes affected by calorie restriction and that control lifespan. In addition, we find that reducing sphingolipid synthesis increases lifespan in ways that are independent of Sch9 and calorie restriction. Since sphingolipids are found in all eukaryotes, our results suggest that pharmacological down-regulation of one or more sphingolipids may provide a means to reduce age-related diseases and increase lifespan in other eukaryotes. including endocytosis, cell wall integrity, growth, actin dynamics and stress resistance [15,16,24,25]. Sphingolipids are known activators of Pkh1/2 [15,16,26,27] and we posit that they ought to regulate lifespan by controlling the activity of the Pkh1/2-Sch9 pathway. By lowering the rate of sphingolipid biosynthesis, we demonstrate enhancement of chronological lifespan (CLS), a measure of how long non-dividing cells survive in stationary phase. The increase in lifespan is due to Sch9-dependent and independent processes that are also con- trolled by sphingolipids. Results Genetic strategy for down-regulating sphingolipid synthesis In one strategy to determine if sphingolipids regulate CLS we used a tetracycline-repressible promoter cassette (tetO7) to regulate gene transcription [28]. The utility of this strategy for studying CLS was verified by using cells having transcription of the SCH9 gene (tetO7-SCH9) or the RIM15 gene (tetO7-RIM15) controlled by the tetO7 promoter. Based on the roles of Sch9 and Rim15 in lifespan [7], we expected CLS to increase in doxycycline (Dox)- treated tetO7-SCH9 cells and to decrease in Dox-treated tetO7- RIM15 cells. We find that a low concentration of Dox (100 ng/ml) significantly increases the CLS of tetO7-SCH9 cells (Figure 1B) while a higher dose of Dox (1000 ng/ml) is needed to decrease the CLS of tetO7-RIM15 cells (Figure 1C). In control experiments we find that Dox treatment has no effect on the CLS of the parental strain R1158 (Figure 1B and 1C, WT). Thus, this experimental strategy is able to recapitulate the CLS phenotype of sch9D and rim15D mutant cells. It also recapitulates the increased oxidative stress resistance of sch9D cells and the reduced resistance of rim15D cells [7] (Figure 1D). Down-regulating tetO7-LCB1 and tetO7-LCB2 increases CLS The essential LCB1 and LCB2 genes encode subunits of serine palmitoyltransferase (SPT), the first and rate-limiting enzyme in Sphingolipids Control Lifespan sphingolipid biosynthesis (Figure S1 in Text S1) [15] and we expected that by lowering expression of LCB1 or LCB2 it should be possible to lower the level of sphingolipids and extend lifespan via reduction of the sphingolipid-controlled Pkh1/2-Sch9 path- way. We identified concentrations of Dox that reduce the initial rate of growth but not the final cell density of strains with tetO7- LCB1 or tetO7-LCB2 alleles, consistent with a lowered rate of sphingolipid synthesis but not low enough to affect the final cell density or mass yield (Figure 2A and 2B). Under these conditions Dox-treated and untreated cells should be in a similar metabolic state upon entering stationary phase and any effect of Dox on lifespan should be due to down-regulation of LCB1 or LCB2 rather than to off target effects. We find that the CLS of tetO7-LCB1 or tetO7-LCB2 cells treated with 100 or 300 ng/ml of Dox increases significantly (Figure 2C and 2D). Control experiments demonstrate that this increase in CLS is due to down-regulation of LCB1 or LCB2 and not to off target effects of Dox. First, Dox treatment has no effect on the CLS of wild-type R1158 cells, the parent of tetO7-LCB1 and tetO7- LCB2 cells (Figure 1B and 1C). Next, we reasoned that introduction of a plasmid-borne wild-type LCB1 gene into tetO7- LCB1 cells should preclude Dox from increasing CLS, since the wild-type LCB1 allele will not respond to Dox and SPT activity should be normal. As expected, the CLS of Dox-treated tetO7- LCB1 cells transformed with a single-copy vector carrying the LCB1 gene is reduced compared to cells transformed with just the vector and is similar to cells not treated with Dox (Figure 2E). Analogous experiments with tetO7-LCB2 cells give similar results (Figure 2F). We conclude from these data that Dox treatment of cells carrying a tetO7-LCB1 or tetO7-LCB2 allele increases CLS because expression of the LCB1 or LCB2 gene is reduced. Finally, most experimental manipulations enhance lifespan in part by increasing stress resistance. Thus we anticipated that down-regulating the tetO7-LCB1 or tetO7-LCB2 genes by Dox treatment would increase stress resistance and, indeed, resistance to heat and hydrogen peroxide stress did increase (Figure 2G). Myriocin treatment increases CLS A second strategy for determining if sphingolipids regulate CLS uses myriocin, an inhibitor of SPT, to down-regulate enzyme activity. We examined the effect of low doses of myriocin on three strains: DBY746, used extensively to study CLS [7], BY4741, the parent of one of the non-essential yeast gene deletion sets which is often used to study lifespan [8], and a derivative of BY4741, R1158, the parent strain for making tetO7-regulated genes [28]. The initial rate of R1158 cell growth is the least impaired by myriocin treatment while growth of DBY746 and BY4741 cells is more impaired (Figure 3A, 3C and 3E). The highest concentration of myriocin used for each strain causes a delay in initial cell growth and increases the time it takes for DBY746 and BY4741 cells to enter stationary phase. However, the final cell density is either the same as or within 20% of the cell density found in the absence of drug, which argues that differences in final cell density or failure to enter stationary phase are not likely to explain the effect of myriocin on CLS. Using these same drug concentrations, there is a robust, dose-dependent and statistically significant increase in the CLS of each strain (Figure 3B, 3D and 3F) with DBY746 cells showing the largest increase (Figure 3B). DBY746 cells treated with 400 ng/ml of myriocin grow slowly and, unlike the other cells, do not stop growing at 72 hrs (CLS day 1), but keep growing up to about the 120 hr time point. This causes an increase in viable cells on CLS days 3 and 6 (Figure 3B, solid red line) and overstates the increase in CLS. To compensate for this extended growth phase, we set the 120 hr time point as CLS day 1 PLoS Genetics | www.plosgenetics.org 2 February 2012 | Volume 8 | Issue 2 | e1002493