Sphingolipids Control Lifespan Figure 5. Evidence for the sphingolipidRPkh1/2RSch9 pathway. (A) CLS of pkh2D cells (RCD1051) compared to parental PKH2 cells (RCD956). Average survival values and statistical significance for eight replicate cultures (total cultures from two different assays) are shown. Statistical significance is indicated for specific time points. These data are nearly identical to published data obtained using a different strain background and culture conditions [34]. (B and C) Immunoblot analysis of Sch9 and P-T570 in the strains used (A) and grown from a starting A600 nm of 0.005 to a value of 1. For each lane the Sch9 signal was normalized to the signal for the Vma2 internal standard (value shown below each lane) and then the P-T570 signal (value shown below the T-570 band) was normalized to this number. Average values are shown in parentheses. (D) Dilution assay (10-fold serial dilution from left to right) for myriocin sensitivity of WT (15Dau/pRS315/pRS316), pkh1ts pkh2D (INA106-3B/pRS316) and INA106-3B cells transformed with pRS316-PKH1 or pRS316-PKH2. (E) Sch9 and P-T570 were quantified by immunoblotting of extracts made from four cultures of DBY746 cells treated or not treated with myriocin (Myr, 400 ng/ml) while growing from a starting A600 nm of 0.005 to a value of 1. Normalized values were calculated as described in (B). (F) The average of the P-T570 signals from (E) is plotted along with the statistical significance. (G) Sch9 and P-T570 were measured in myriocin-treated or untreated WT (R1158) and tetO7-LCB1 (RCD956) cells as described in (B). (H) Sch9 and P-T570 were measured in Dox- treated or untreated WT (R1158) and tetO7-LCB1 (RCD956) cells as described in (B). (I) Myriocin treatment reduces Sch9-mediated phosphorylation of Dot6-5HA. RCD1052 cells transformed with the indicated plasmids were grown as described in (B) and immunoblots of extracts were probed with anti-HA antibody or with antibody for the internal standard Vph1 protein. (J) Same blot as shown in panel (I) but reprobed for Sch9 T570 phosphorylation. The asterisk indicates a not specific band. doi:10.1371/journal.pgen.1002493.g005 was implemented by switching cells on day 3 from culture medium to water. This extreme form of CR was used because it prevents adaptive regrowth and eliminates regrowth as an explanation for myriocin-induced CLS extension [40]. We find that myriocin produces a dose-dependent increase in the CLS of cells subjected to extreme CR (Figure 7B). Thus, myriocin extends CLS in sch9D cells similar to CR treatment, extends CLS even under conditions where adaptive regrowth does not occur, and programs cells for an extended lifespan prior to the end of the diauxic shift (prior to shifting cells to water). In addition, these data further support the conclusion that myriocin does not simply extend CLS by protecting cells from acetic acid killing, since replacing the culture medium with water and changing the water every third day eliminates acetic acid accumulation. Finally, we find that myriocin also enhances CLS when CR is applied by reducing the concentration of glucose from 2 to 0.5% (Figure S3 in Text S1). Discussion The desire for a long and healthy life is universal, but most of us will succumb sooner than hoped for due to age-related disorders, particularly cancer, cardiovascular disease and diabetes. Fortu- nately, strategies that enhance lifespan in model organisms work to a large degree by delaying the onset of these diseases. A striking example of this relationship is seen in monkeys during CR where the incidence of age-related diseases decreases while lifespan increases [43,44]. CR is not a practical strategy to extend lifespan in humans, but pharmacological agents are, and the rapamycin- type compounds seem quite promising [4–6]. As promising as rapamycin and related drugs are for improving human health and extending lifespan, they may have unforeseen drawbacks and be suitable only for individuals with specific genotypes. Hence, other strategies are needed. Sphingolipids regulate yeast lifespan Our data show that down-regulating sphingolipid synthesis produces an increase in the CLS of S. cerevisiae cells (Figure 2, Figure 3, and Figure 7) and that part of this increase depends on the Sch9 protein kinase (Figure S2 in Text S1, Figure 5, Figure 6, and Figure 7A), an established regulator of lifespan. We show that CLS can be increased by lowering sphingolipid synthesis via manipulation of SPT, the initial enzyme in sphingolipid biosynthesis (Figure 2, Figure 3, and Figure 7). We propose that lowering SPT activity increases CLS, at least in part, by reducing the concentration of LCBs thereby lowering the activity of Pkh1/2 and Sch9 which programs cells for an extended lifespan (Figure 1A). This scenario is supported by data showing that myriocin treatment or down-regulation of tetO7-LCB1 gene expression diminishes Pkh1/2-dependent phosphorylation of Sch9 residue T570, essential for activity (Figure 5) and reduces phosphorylation of the Sch9 substrate Dot6 (Figure 5I). In addition, as a surrogate for Sch9 activity in vivo, we measured processes that play key roles in lifespan and are regulated by Sch9. All processes including resistance to heat, oxidative and acetic acid stress (Figure 2, Figure 3, Figure 6A and 6B), cell size (Figure S2 in Text S1), mitochondrial oxygen consumption (Figure 6C), and genome stability (Figure 6D and 6E) responded to down- regulation of sphingolipid synthesis in a manner indicative of reduced Sch9 activity. Deletion of PKH2 was recently shown to extend CLS [34]. We verified this finding (Figure 5A) and show that phosphorylation of Sch9 T570 is reduced in pkh2D cells (Figure 5B). Finally, we show that cells with low Pkh kinase activity are much more sensitive to growth inhibition by myriocin or Dox than are cells with higher Pkh activity (Figure 5D and Figure S4 in Text S1). Together these data add further support for a signaling pathway comprised of sphingolipidsRPkh1/2RSch9RCLS. While most of our data suggest that down-regulating sphingo- lipid synthesis increases lifespan by Sch9-dependent mechanisms, the finding that myriocin causes a mobility shift in Sch9, indicative of reduced phosphorylation, and increases the CLS of sch9D cells argues for some aspects of CLS extension operating by Sch9- independent mechanisms. Down-regulation of the protein kinase A [45] pathway is the most likely way for sphingolipids to affect lifespan independently of Sch9, since these two pathways have been known to interact ever since the initial discovery of Sch9 [46]. Also, the pathways are known to interact in a way that extends CLS, since ras2D sch9D double mutant cells have a longer lifespan than the single mutants [30]. This interaction phenotype is similar to what we find with myriocin treatment of sch9D cells (Figure 7A). Recent publications have detected biochemical links between the Pkh1/2-Sch9 and PKA pathways [24,25,47] and these may explain how myriocin increases the lifespan of sch9D cells, but further effort is needed to determine if this is actually the case. In addition to LCBs, complex sphingolipids (IPCs and perhaps MIPCs) may be regulating CLS, since their concentration is lowered along with LCBs when SPT activity is down-regulated (Figure 4C, 4D, and 4E). A multi-membrane spanning protein Nce102 has been proposed to sense complex sphingolipids and negatively regulate Pkh1/2 [26]. According to this proposal, a reduction in complex sphingolipids should increase Pkh1/2 activity by releasing Nce102-mediated down-regulation. As a consequence, Sch9 activity should increase; however, this would not enhance CLS and is contrary to our finding that reducing sphingolipids decreases Sch9 activity. Thus, Nce102 is unlikely to be mediating the increase in CLS that we observe. PLoS Genetics | www.plosgenetics.org 10 February 2012 | Volume 8 | Issue 2 | e1002493