Drug Synergy Drives Conserved Pathways to Increase Fission Yeast Lifespan

March Drug Synergy Drives Conserved Pathways to Increase Fission Yeast Lifespan Xinhe Huang 0 1 2 Markos Leggas 0 1 2 Robert C. Dickson 0 1 2 0 Current Address: School of Life Science and Engineering, Southwest Jiaotong University , Chengdu , China 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 Department of Pharmaceutical Sciences and the Lucille Markey Cancer Center, College of Pharmacy, University of Kentucky , Lexington, Kentucky , United States of America 2 Academic Editor: Juan Mata, University of Cambridge, UNITED KINGDOM Aging occurs over time with gradual and progressive loss of physiological function. Strategies to reduce the rate of functional loss and mitigate the subsequent onset of deadly agerelated diseases are being sought. We demonstrated previously that a combination of rapamycin and myriocin reduces age-related functional loss in the Baker's yeast Saccharomyces cerevisiae and produces a synergistic increase in lifespan. Here we show that the same drug combination also produces a synergistic increase in the lifespan of the fission yeast Schizosaccharomyces pombe and does so by controlling signal transduction pathways conserved across a wide evolutionary time span ranging from yeasts to mammals. Pathways include the target of rapamycin complex 1 (TORC1) protein kinase, the protein kinase A (PKA) and a stress response pathway, which in fission yeasts contains the Sty1 protein kinase, an ortholog of the mammalian p38 MAP kinase, a type of Stress Activated Protein Kinase (SAPK). These results along with previous studies in S. cerevisiae support the premise that the combination of rapamycin and myriocin enhances lifespan by regulating signaling pathways that couple nutrient and environmental conditions to cellular processes that fine-tune growth and stress protection in ways that foster long term survival. The molecular mechanisms for fine-tuning are probably species-specific, but since they are driven by conserved nutrient and stress sensing pathways, the drug combination may enhance survival in other organisms. - Data Availability Statement: Relevant data are within the paper and its Supporting Information files. Funding: This study was funded by the NIH/NIA grant AG024377 to RCD and by the National Institute of General Medical Sciences grant P20GM103486 to Louis Hersh. The funders had no role in the study design, data collection and analysis, decisions to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Most organisms show signs of aging, characterized by the gradual but progressive loss of physiological functions over time. As humans enter their fifties and sixties the consequences of such functional losses manifest as increased incidence of cancer, type 2 diabetes, neurodegeneration, cardiovascular disease and immune dysfunction, the age-related diseases. Current research seeks to reduce the incidence and severity of these diseases to improve health in the elderly. One promising avenue of research centers on the target of rapamycin complex 1 (TORC1) protein kinase [13]. TORC1 is a central regulator of aging and longevity, since down-regulating its activity by treatment with the natural product rapamycin or related synthetic compounds, reduces signs of aging and extends lifespan in model organisms ranging from yeast to mice [48]. These seminal results have spawned an array of TOR-related research including work from our laboratory which identified a strategy that produces a synergistic increase in the chronological lifespan (CLS) of the Bakers yeast Saccharomyces cerevisiae [9]. This being the first example of a true synergistic increase in lifespan produced by drugs. Our strategy uses a low dose of rapamycin to lower (not inhibit) TORC1 activity and another natural product, myriocin, to lower (not inhibit) the activity of serine palmitoyltransferase (SPT), the first enzyme in the sphingolipid biosynthesis pathway. The conserved nature of TORC1 and SPT in eukaryotes suggested that the combination drug treatment (ComboDT) might enhance lifespan in other eukaryotes. Here we verify this prediction by using the fission yeast Schizosaccharomyces pombe, a distant and divergent relative of budding yeast. Most eukaryotes have two TOR complexes with TORC1 primarily responsible for sensing nutrients, stresses, growth factors, and energy status and coupling these to growth and survival including lifespan whereas TORC2 responds to growth factors and controls cytoskeletal functions but has no known role in lifespan [1, 2, 10]. Rapamycin primarily inhibits TORC1 and at high doses it inhibits growth. However, it does effect TORC2 in some organisms such as mammals [1]. Rapamycin does not inhibit growth in fission yeasts probably because of incomplete inhibition of TORC1 activity, but combining rapamycin with caffeine completely inhibits enzyme activity and growth [11]. Despite incomplete inhibition of TORC1, rapamycin treatment extends CLS in fission yeast as does treatment with caffeine [12], similar to the effect of caffeine on budding yeast [13]. In contrast to ComboDT, rapamycin and caffeine in combination are no more effective in increasing fission yeast lifespan CLS than are the single drugs. Sphingolipids have many functions in eukaryotes including roles in age-related human diseases [1417]. In S. cerevisiae they can be modulated to increase both CLS, a measure of survival in non-dividing cells, and replicative lifespan (RLS), a measure of how many times a cell can divide [reviewed in [18]]. For example, treatment with a low dose of myriocin to reduce SPT activity increases CLS in S. cerevisiae [19] and treatment with even lower doses of myriocin in combination with low doses of rapamycin produce a synergistic increase in CLS [9]. It is not clear how such modulation of sphingolipids increases CLS or RLS, but it is likely to be complex and involve changes in sphingolipids that act as second messengers as well as effects on cellular processes connected to membranes including membrane trafficking and the functionality of membrane-bound proteins. What we do know is that myriocin and ComboDT increase budding yeast CLS by controlling conserved nutrient sensing and stress response signaling pathways including, besides TORC1, the protein kinase A (PKA) and AMP kinase (AMPK) pathways [9, 19]. In addition, we know that myriocin [20] and ComboDT (unpublished data) modulate gene expression and cellular processes (GO terms) in ways that are similar to what is found with rapamycin treatment and dietary or calorie restriction (CR), the gold standards for slowing aging and enhancing lifespan. With this information along with current knowledge of S. pombe aging and lifespan as background, we examined the effect of ComboDT on fission yeast and show that the (...truncated)