Sphingolipids Control Lifespan Figure 3. Myriocin treatment extends CLS. (A) Growth rate of DBY746 cells +/2 myriocin (Myr) treatment. Myriocin (300 ng/ml) treatment reduces cell size and there are fewer cells/A600 nm in the treated than in the untreated cultures (1.66107 cells/A600 nm in treated cultures verses 1.86107 cells/A600 nm in untreated cultures). (B) CLS of DBY746 cells treated as described in (A). Data represent the mean 6 SEM of surviving cells (* p,0.05, ** p,0.01, No Myr vs 200 and 400 ng/ml Myr). To compensate for the slow growth and extended growth phase of cells treated with 400 ng/ ml of myriocin (solid red line), the viability at the 120 hr time point, CLS day 3, was set as CLS day 1 (dashed red line). (C) Growth rate of R1158 cells +/2 myriocin (Myr) treatment. Cells/A600 nm unit are similar to those described in (A). (D) CLS of R1158 cells treated as described in (C). Data represent the mean 6 SEM of surviving cells (* p,0.05, ** p,0.01, two-tailed t-test for No Myr vs 400 ng/ml Myr). (E) Growth rate of BY4741 cells +/2 myriocin (Myr) treatment. (F) CLS of BY4741 cells incubated as described in (E). Data represent the mean 6 SEM of surviving cells (* p,0.05, ** p,0.01, No Myr vs 600 ng/ml Myr). (G) Resistance of myriocin-treated DBY746 cells to heat (55uC) or hydrogen peroxide (H2O2) stress. Photographs show a ten-fold dilution series of cells (from left to right). (H) CLS of DBY746 cells grown in SDC medium buffered to pH 6 or 4.5 +/2 myriocin (Myr) treatment. Data represent the mean 6 SEM of surviving cells (* p,0.05, ** p,0.01, No Myr vs 300 ng/ml Myr). doi:10.1371/journal.pgen.1002493.g003 support the hypothesis that down-regulating sphingolipid levels by genetically or pharmacologically lowering SPT activity reduces T570 phosphorylation and consequently Sch9 activity thereby promoting increased CLS. In all cases examined (Figure 5E, 5G and 5H), the total level of Sch9 protein is not changed by drug treatment. However, drug treatment does cause a shift of the Sch9 band to a slightly faster migrating form, similar to what is seen when phosphorylation of Sch9 is reduced [20]. In the case where Pkh activity was reduced by deleting PKH2 rather than by down-regulating sphingolipid synthesis, there was no shift in the mobility of Sch9 to a faster migrating form even though phosphorylation of T570 decreased (Figure 5B). Thus, the band shift we observe suggests that a reduction in sphingolipids triggers dephosphorylation of other Sch9 amino acid residues in addition to T570 and further work will be required to identify the residues involved. The effect of myriocin treatment on the phosphorylation level of the Sch9 substrate Dot6 was also examined. Sch9 regulates ribosome biogenesis in part by phosphorylating transcriptional repressor proteins including Dot6, Tod6 and Stb3 [35,36]. Dot6 controls transcription of ribosome biogenesis [37] genes that are essential for ribosome formation. We find that Dot6-5HA in myriocin-treated cells migrates faster on a denaturing gel than it does in untreated cells (Figure 5I), indicating reduced phosphor- ylation by Sch9 [36]. To directly demonstrate reduced Sch9 activity in these myriocin-treated cells, the blot shown in Figure 5I was reprobed for T570 phosphorylation. As found in other myriocin-treated cells, there was a reduction in T570 phosphor- ylation and an increase in Sch9 mobility, both indicative of reduced Sch9 activity (Figure 5J). Together, the data shown in Figure 5 demonstrate that down-regulating sphingolipid synthesis by treatment of cells with myriocin or Dox reduces Sch9 activity. Reduced activity is at least partly responsible for the increase in CLS we observe in drug-treated cells (Figure 2, Figure 3, and Figure 7). Down-regulating sphingolipid synthesis enhances CLS by Sch9-dependent and -independent mechanisms Part of the mechanism for increased CLS in sch9D cells is greater resistance to oxidative, heat and acetic acid stress [7,31]. If sphingolipids are regulating CLS by Sch9-dependent mechanisms, then reducing the rate of sphingolipid synthesis should increase stress resistance. Indeed, resistance to heat and oxidative stress do increase (Figure 2G and Figure 3G) as does resistance to acetic acid when tetO7-LCB1 cells are treated with Dox, and resistance is nearly as robust as in sch9D cells (Figure 6A). Likewise, treating DBY746 cells with myriocin increases resistance to acetic acid albeit resistance is not enhanced as much as in sch9D cells (Figure 6B). These data support the hypothesis that sphingolipids regulate CLS by Sch9-dependent mechanisms. Deletion of SCH9 is known to extend CLS by enhancing respiration [38,39]. To determine if myriocin treatment also extends CLS by inducing respiration we measured oxygen consumption. We find a more than 2-fold, statistically significant increase in oxygen consumption in myriocin-treated cells com- pared to untreated cells (Figure 6C). In contrast, there is no statistically significant stimulation of oxygen consumption in sch9D cells treated with myriocin, indicating that Sch9 mediates most if not all of the effect of myriocin on oxygen consumption (Figure 6C). Sch9 also affects aging and lifespan by controlling genome stability [40,41]. For example, sgs1D cells display premature age- dependent genome instability that is prevented by deletion of SCH9 [42]. Sgs1 is a member of the RecQ helicase family of DNA unwinding proteins which maintain genome stability. Sgs1 is related to human RecQ helicases WRN, defective in the premature aging disorder Werner’s syndrome, and BLM, defective in the cancer-prone disorder Blooms’ syndrome [42]. If sphingo- lipids control Sch9 activity, then age-dependent genome instability in a sgs1D strain should be prevented by treating cells with myriocin. Genome instability was quantified by measuring the frequency of Canr mutations. As in previous studies [42], we find Canr mutations accumulating at a faster rate in sgs1D cells during chronological aging and by day 12 they are 4-fold more abundant than in wild-type cells (Figure 6D). Deletion of SCH9 reverses the effect of the sgs1 mutation since cultures of sgs1D sch9D cells have fewer Canr mutants than the wild-type cultures. Notably, treating sgs1D cells with myriocin reduces the frequency of Canr mutants to the wild-type level at all times during a CLS assay (Figure 6D). In a second assay of genome instability the frequency of gross chromosomal rearrangements (GCRs) was measured [42]. By day 12 there are five times more GCRs in sgs1D cells than in wild-type or sgs1D sch9D cells (Figure 6E). Again, myriocin treatment reduces the frequency of GCRs about 75% at day 12 in sgs1D cells, very similar to the reduction obtained by deleting SCH9 (sgs1D sch9D cells, Figure 6E). In summary, the data presented in Figure 6 support the hypothesis that sphingolipids regulate CLS by Sch9- dependent mechanisms. Finally, if myriocin enhances CLS entirely by mechanisms requiring Sch9, then myriocin should not enhance CLS in sch9D cells. We find that a dose of 25 or 100 ng/ml myriocin has no effect on the CLS of sch9D cells, but a higher dose of 300 ng/ml produces a statistically significant increase in CLS starting around day 22 (Figure 7A). Thus, myriocin most likely extends CLS in the early and middle time-frame of a CLS assay by Sch9-dependent mechanisms, but at later times the enhancement in lifespan is independent of Sch9. Possible mechanisms for the Sch9-indepen- dent effects on CLS are presented in the Discussion. Down-regulating sphingolipid synthesis extends CLS in caloric-restricted cells Since calorie restriction increases the CLS of sch9D cells [30], we determined if myriocin produces an effect similar to CR. CR PLoS Genetics | www.plosgenetics.org 7 February 2012 | Volume 8 | Issue 2 | e1002493