Reducing signs of aging and increasing lifespan by drug synergy

Abstract

Disease incidence rises rapidly with age and increases both human suffering and economic hardship while shortening life. Advances in understanding the signaling pathways and cellular processes that influence aging support the possibility of reducing the incidence of age-related diseases and increasing lifespan by pharmacological intervention. Here, we demonstrate a novel pharmacological strategy that both reduces signs of aging in the budding yeast Saccharomyces cerevisiae and generates a synergistic increase in lifespan. By combining a low dose of rapamycin, to reduce activity of the target of rapamycin complex 1 (TORC1) protein kinase, and myriocin, to reduce sphingolipid synthesis, we show enhancement of autophagy, genomic stability, mitochondrial function, and AMP kinase pathway activity. These processes are controlled by evolutionarily conserved signal transduction pathways that are vital for maintaining a healthy state and promoting a long life. Thus, our data show that it ought to be possible to find pharmacological approaches to generate a synergistic reduction in the incidence of human age-related diseases to improve health quality in the elderly and enhance lifespan.

Keywords: S6 Kinase; TORC1; aging; autophagy; genomic stability; longevity.

Fig. 1

Combination drug treatment enhances CLS and stress resistance. (A) Outline of the signaling pathways (colored ovals and lines), transcription factors (gray boxes) and cellular processes (tan boxes) that are modulated by combination drug treatment. NPD: Nitrogen Discrimination Pathway. (B) The viability of DBY746 cells incubated in SDC medium is shown as a function of days of incubation (day 1 = 72 hrs). Cells were treated with no drug, 45 ng/ml Myriocin (Myr, 112 nM), 450 pg/ml Rapamycin (Rap. 0.49 nM), 45 ng/ml Myr plus 450 pg/ml Rap in these and all other experiments, unless indicated otherwise. Data are for the mean ± SEM of viable cells in triplicate cultures in these and all other CLS experiments. The dotted straight line with an arrowhead indicates an increase in the CLS of cells treated with both drugs that is greater than the additive effect on CLS of each drug treatment compared to untreated cells (additive effect is indicated by a dashed survival curve). The p value for lifespan increase in panels B to E is computed using the area under the viability curves. (C) CLS of DBY746 cells switched to water after 72 hrs (CLS day 1) of incubation in SDC. (D) CLS of BY4743 cells grown in SDC medium. (E) CLS of BY4743 cells switched to water on CLS day 1. (F and G) Resistance of DBY746 or BY4743 cells on CLS day 1 to heat (55°C) or hydrogen peroxide (H₂O₂) stress. Photographs show a 10-fold dilution series (from left to right).

Fig. 2

Combination drug treatment enhances autophagy and genome stability. (A) The effect of drug treatment on the level of autophagy, as measured by cleavage of GFP from GFP-Atg8, in log phase (A_{600nm =} 1.0) wild-type (DBY746) or atg1Δ cells is shown in these immunoblots. The Vma2 protein is a loading control and the asterisks indicate antibody reaction with non-specific proteins. (B) CLS of wild-type or atg1Δ cells switched to water at CLS day 1. (C) The frequency of small chromosomal changes was evaluated during a CLS assay by measuring the frequency of canavanine-resistant mutants (Can^r). Cells carrying a sgs1Δ allele were used to increase mutation frequency and improve quantification of drug effects. (D) The frequency of gross chromosomal rearrangements (GCRs) during a CLS assay is shown.

Fig. 3

Mitochondrial function and ROS levels are remodeled by combination drug treatment. (A) The rate of oxygen consumption is higher in log (A_{600nm =} 1.0) and stationary phase cells (CLS day1) treated with both myriocin and rapamycin than in cells treated with a single drug or with no drug. (B) Combination drug treatment does not affect the concentration of superoxide anions in log phase cells, but does lower the concentration in stationary phase cells. (C) Dual drug treatment increases the concentration of hydrogen peroxide in log phase cells but reduces it in stationary phase cells.

Fig. 4

Combination drug treatment regulates signaling pathways that modulate aging and lifespan. (A) Phosphorylation of residue T570 of Sch9 in log phase (A_600nm=1.0) DBY746 cells was monitored by using a phosphor-specific antibody (upper panel, Sch9-T570-P) whereas total Sch9 protein was measured by using a different antibody (middle panel). The ratio of phosphorylated to unphosphorylated T570 is shown below each lane in the top panel and below that are the average values, normalized to the no drug treatment. Total Sch9 protein was quantified for each lane (middle row of blots), averaged, and the average was normalized to the Vma2 loading control. (B) Phosphorylation of C-terminal residues in Sch9 by TORC1 is greatly reduced by combination drug treatment in log phase DBY746 cells producing HA-tagged Sch9. The ratio of phosphorylated (+P) to dephosphorylated (−P) is shown below the top panel. A high dose of rapamycin was used as a control to down-regulate TORC1 activity and cause nearly complete dephosphorylation of C-terminal residues (lane 5). (C) Combination drug treatment lowers TORC1 pathway activity. TORC1 activity was evaluated in log phase cells by measuring β-galactosidase activity in DBY746 cells carrying a MEP2-lacZ gene. (D) Dual drug treatment greatly reduces PKA activity as shown by decreased phosphorylation of the Atg13 protein. The panels show immunoblots made by using HA-tagged Atg13 protein immunoprecipitated from log phase (A_600nm=1.0) DBY746 cells. The upper panel was probed with an antibody specific for PKA phosphorylation sites (Atg13-P) and the lower panel for total HA-tagged Atg13 protein (Atg13-3HA). The numerical ratio of the PKA-specific phosphorylation to total Atg13-3HA protein is shown below the panels (Atg13-P/Atg13). (E) Combination drug treatment activates the Snf1/AMPK pathway. Snf1/AMPK pathway activity was quantified by measuring β-galactosidase activity in log (Insert, A_600nm=1.0, 16 hrs of incubation), post-diauxic shift (40 hrs of incubation) or stationary phase (70 hrs of incubation) DBY746 cells carrying pBGM18-ADH2/lacZ.