Sir2-Independent Life Span Extension by Calorie Restriction in Yeast

Received May Sir2-Independent Life Span Extension by Calorie Restriction in Yeast Matt Kaeberlein 0 1 Kathryn T. Kirkland 0 1 Stanley Fields 0 1 Brian K. Kennedy 0 1 Andy Dillin, Salk Institute 0 Abbreviations: CR , calorie restriction; ERC, extrachromosomal rDNA circle; RLS, replicative life span 1 1 Departments of Genome Sciences and Medicine, University of Washington, Seattle, Washington, United States of America, 2 Department of Biochemistry, University of Washington, Seattle, Washington, United States of America, 3 Howard Hughes Medical Institute, University of Washington , Seattle, Washington , United States of America Calorie restriction slows aging and increases life span in many organisms. In yeast, a mechanistic explanation has been proposed whereby calorie restriction slows aging by activating Sir2. Here we report the identification of a Sir2independent pathway responsible for a majority of the longevity benefit associated with calorie restriction. Deletion of FOB1 and overexpression of SIR2 have been previously found to increase life span by reducing the levels of toxic rDNA circles in aged mother cells. We find that combining calorie restriction with either of these genetic interventions dramatically enhances longevity, resulting in the longest-lived yeast strain reported thus far. Further, calorie restriction results in a greater life span extension in cells lacking both Sir2 and Fob1 than in cells where Sir2 is present. These findings indicate that Sir2 and calorie restriction act in parallel pathways to promote longevity in yeast and, perhaps, higher eukaryotes. - The budding yeast Saccharomyces cerevisiae has served as a useful model for aging research, leading to the identification of new longevity genes and pathways whose counterparts can be examined in higher eukaryotes (Kaeberlein et al. 2001). One measure of aging in yeast is the finite replicative life span (RLS) of mother cells, defined as the number of mitotic cycles completed prior to senescence (Mortimer and Johnston 1959). Alternatively, the survival of nondividing yeast cells over time can be monitored and has been termed chronological aging (Fabrizio and Longo 2003). It has been proposed that replicative aging in yeast may be a suitable model for the aging of dividing cells in mammals, such as germ cells; whereas, chronological aging of yeast may be related to the aging of postmitotic tissues. Replicative aging of yeast can be caused by the accumulation of extrachromosomal rDNA circles (ERCs) in the mother cell nucleus (Sinclair and Guarente 1997), and mutations that decrease ERC formation correlate with increased life span. One example of such a mutation is deletion of the gene encoding the rDNA replication fork barrier protein Fob1, which results in a dramatic decrease in ERC levels accompanied by a 30%40% increase in mean and maximum RLS (Defossez et al. 1999). In addition to Fob1, the Sir2 protein has also been found to affect longevity by regulating the rate at which ERCs are formed (Kaeberlein et al. 1999). Sir2 is an NAD-dependent histone deacetylase (Imai et al. 2000; Landry et al. 2000; Tanner et al. 2000) necessary for transcriptional silencing near telomeres (Aparicio et al. 1991), HM loci (Ivy et al. 1986; Rine and Herskowitz 1987), and rDNA (Bryk et al. 1997; Smith and Boeke 1997). Deletion of Sir2 increases both rDNA recombination (Gottlieb and Esposito 1989) and ERC formation, while shortening life span by approximately 50% (Kaeberlein et al. 1999). Conversely, overexpression of Sir2 increases life span by 30%40%. Overexpression of Sir2 in the context of FOB1 deletion fails to further extend life span, consistent with the idea that Sir2 and Fob1 both impact aging by regulating ERC levels (Kaeberlein et al. 1999). Calorie restriction (CR) of yeast cells can be accomplished by a reduction in the glucose concentration of growth media from 2% to 0.5% (or lower) and results in a 30%40% increase in life span (Lin et al. 2000). Several genetic models of CR have also been described. In one model, deletion of the HXK2 gene, coding for hexokinase, reduces the availability of glucose for glycolysis; while in the others, deletion of other genes, including gpa2D and gpr1D, decreases cAMP-dependent protein kinase activity (Lin et al. 2000). Growth in low glucose and the various genetic models of CR have been treated as experimentally interchangeable. While they are clearly not identical, evidence to date suggests that they behave in a similar manner with respect to yeast aging (Lin et al. 2000, 2002, 2004; Kaeberlein et al. 2002, 2004). Several reports have suggested a link between the enhanced longevity associated with CR and increased activity of Sir2 (Koubova and Guarente 2003). In one genetic model of CR, cdc25-10 is reported to decrease both rDNA recombination and ERC levels (Lin et al. 2000). In addition, deletion of Sir2 has been shown to prevent life span extension by cdc25-10 and low glucose (Lin et al. 2000, 2002). These data have been used to support a model whereby CR activates Sir2, thus causing decreased ERC accumulation and increased life span. It was initially proposed that life span extension by CR is the consequence of a metabolic shift resulting in increased cellular NAD available as a substrate for Sir2-dependent histone deacetylation (Lin et al. 2002). More recently, this theory has been supplanted by two competing models for activation of Sir2 by CR: (1) a decrease in cellular nicotinamide (a product inhibitor of Sir2) via upregulation of PNC1 (Anderson et al. 2003a), and (2) a decrease in cellular NADH (a competitive inhibitor of Sir2) (Lin et al. 2004). We present here evidence that CR and Sir2 act in different genetic pathways to promote longevity and show that Sir2 is not required for full life span extension in response to CR. In addition, we offer data suggesting that previous experiments were misinterpreted. Finally, we propose a model that reconciles our findings with earlier reports and suggests a greater level of conservation between aging in yeast and higher eukaryotes. We recently carried out a large-scale study of more than 40 single-gene deletions reported to affect aging in yeast (unpublished data). This analysis was performed in the BY4742 genetic background, which has a mean life span significantly longer than most other yeast strains commonly used for aging research (Table 1). Included in this analysis were three genetic models of CR (hxk2D, gpa2D, and gpr1D) and fob1D. As previously reported for shorter-lived strain backgrounds (Defossez et al. 1999; Lin et al. 2000), each of these single-gene deletions resulted in a 30%40% increase in life span in BY4742 (Figure 1A). Since both CR and deletion of FOB1 increased life span individually in BY4742, we examined the effect of CR combined with deletion of FOB1. It is notable that this experiment has not to our knowledge been previously reported. We constructed a fob1D hxk2D double mutant and (...truncated)