Down-Regulating Sphingolipid Synthesis Increases Yeast Lifespan Xinhe Huang1, Jun Liu1,2, Robert C. Dickson 1 * 1 Department of Molecular and Cellular Biochemistry and the Lucille Markey Cancer Center, University of Kentucky College of Medicine, Lexington, Kentucky, United States of America, 2 Key Laboratory of Bio-Resources and Eco-Environment of Ministry of Education, College of Life Science, Sichuan University, Chengdu, China Abstract Knowledge of the mechanisms for regulating lifespan is advancing rapidly, but lifespan is a complex phenotype and new features are likely to be identified. Here we reveal a novel approach for regulating lifespan. Using a genetic or a pharmacological strategy to lower the rate of sphingolipid synthesis, we show that Saccharomyces cerevisiae cells live longer. The longer lifespan is due in part to a reduction in Sch9 protein kinase activity and a consequent reduction in chromosomal mutations and rearrangements and increased stress resistance. Longer lifespan also arises in ways that are independent of Sch9 or caloric restriction, and we speculate on ways that sphingolipids might mediate these aspects of increased lifespan. Sch9 and its mammalian homolog S6 kinase work downstream of the target of rapamycin, TOR1, protein kinase, and play evolutionarily conserved roles in regulating lifespan. Our data establish Sch9 as a focal point for regulating lifespan by integrating nutrient signals from TOR1 with growth and stress signals from sphingolipids. Sphingolipids are found in all eukaryotes and 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. Citation: Huang X, Liu J, Dickson RC (2012) Down-Regulating Sphingolipid Synthesis Increases Yeast Lifespan. PLoS Genet 8(2): e1002493. doi:10.1371/ journal.pgen.1002493 Editor: Stuart K. Kim, Stanford University Medical Center, United States of America Received August 25, 2011; Accepted December 7, 2011; Published February 2, 2012 Copyright: 2012 Huang et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: JL was sponsored by the China Scholarship Council. This research was supported by grant AG024377 from the National Institutes of Health. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: bobd@uky.edu Introduction Most of us dream of a long life and wonder if we can increase our time on earth. Except for caloric restriction (CR), there has been little prospect for increasing lifespan. But recent studies offer hope for developing drugs that increase lifespan by reducing the incidence of age-related diseases that kill most humans. The immunosuppressant drug rapamycin has been shown to reduce age-related diseases and increase lifespan in yeasts, flies and mice [1–3] and these results have stimulated searches for new ways to enhance lifespan [4–6]. Here we introduce a novel strategy to extend the lifespan of Saccharomyces cerevisiae cells that involves reducing the rate of sphingolipid synthesis. This strategy increases lifespan at least partly by reducing the activity of the Sch9 protein kinase, a homolog of mammalian ribosomal S6 kinase, both of which regulate lifespan and function downstream of the target of rapamycin (TOR) protein kinase, a well established and evolutionarily conserved regulator of lifespan [7–10]. Sphingolipids are recognized as structural components of eukaryotic membranes and as signaling molecules for regulating cell growth and migration, differentiation, apoptosis, senescence and inflammation [11–13]. This wide range of functions arises from the variation in carbon chain length, degree and location of unsaturation and hydroxylation along with other modifications of the long-chain bases (LCBs) and fatty acids that are amide-linked to each other to form ceramides, to which polar groups are attached to form complex sphingolipids [14]. Variation in polar head groups further expands the types of sphingolipids found in nature and implies that many more functions await discovery. Although yeast sphingolipids (Figure S1 in Text S1) lack the complexity of those in mammals, S. cerevisiae has, nevertheless, been vital in identifying sphingolipid metabolic genes and in understanding sphingolipid functions [15–17]. Studies in yeast, worms, flies and mice establish the TOR pathway as a vital regulator of aging and lifespan [1,2,8,10,18,19]. Eukaryotes have two types of TOR complexes and TOR complex 1 (TORC1) regulates lifespan. Yeast TORC1 phosphorylates serine and threonine residues in the C-terminus of Sch9 (Figure 1A) [20] while an additional residue, T570, in the activation loop of the kinase domain must be phosphorylated also for Sch9 to be active [20,21]. Residue T570 is phosphorylated by the redundant Pkh1 and Pkh2 protein kinases, homologs of mammalian phosphoinosi- tide-dependent protein kinase 1 (PDK1) [20–22]. Dual regulation of Sch9 provides a way to monitor environmen- tal and cellular cues and integrate them with multiple biological processes necessary for growth and survival. TORC1 senses and is activated by nutrients, particularly amino acids, and growth factors to promote biosynthetic processes such as protein synthesis [10,23]. In contrast, nutrient depletion or environmental stresses reduce TORC1 activity and biosynthetic processes become less active while catabolic processes, such as autophagy, become more active in order to salvage nutrients and reduce their consumption. Pkh1/2 are also involved in nutrient and stress signaling and activate not only Sch9 (Figure 1A), but also the Ypk1 and Ypk2 protein kinases (homologs of mammalian serum and glucocorticoid-inducible kinase, SGK), Pkc1 and PKA thereby controlling many cellular processes PLoS Genetics | www.plosgenetics.org 1 February 2012 | Volume 8 | Issue 2 | e1002493