Extension of chronological life span in yeast by decreased TOR pathway signaling - PubMed (original) (raw)

Extension of chronological life span in yeast by decreased TOR pathway signaling

R Wilson Powers 3rd et al. Genes Dev. 2006.

Abstract

Chronological life span (CLS) in Saccharomyces cerevisiae, defined as the time cells in a stationary phase culture remain viable, has been proposed as a model for the aging of post-mitotic tissues in mammals. We developed a high-throughput assay to determine CLS for approximately 4800 single-gene deletion strains of yeast, and identified long-lived strains carrying mutations in the conserved TOR pathway. TOR signaling regulates multiple cellular processes in response to nutrients, especially amino acids, raising the possibility that decreased TOR signaling mediates life span extension by calorie restriction. In support of this possibility, removal of either asparagine or glutamate from the media significantly increased stationary phase survival. Pharmacological inhibition of TOR signaling by methionine sulfoximine or rapamycin also increased CLS. Decreased TOR activity also promoted increased accumulation of storage carbohydrates and enhanced stress resistance and nuclear relocalization of the stress-related transcription factor Msn2. We propose that up-regulation of a highly conserved response to starvation-induced stress is important for life span extension by decreased TOR signaling in yeast and higher eukaryotes.

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Figures

Figure 1.

Figure 1.

A genome-wide deletion screen for mutations that increase CLS. (A) A schematic diagram of high-throughput CLS measurement. Cells are aged in a 96-well plate and, at serial time points, aliquots are inoculated into a second 96-well plate containing rich media. After a fixed period of incubation, the OD of each well in the second plate is measured, and this OD corresponds to the number of viable cells originally pinned into the well. (B) Validation of the high-throughput screening method. Serial dilutions of yeast were inoculated into rich media, and OD measurements were taken at several time points. Observed OD is highly correlated to the number of viable cells inoculated into the media. (C) Relative survival of ∼4800 single-gene deletion strains over a 7-wk experiment. Ranked survival values (see Materials and Methods) were log2 normalized and projected as a heat map. Longer-lived strains appear red. (D) Deletion of TOR-regulated nitrogen acquisition genes extends life span. The integrals under the life span curves are _gln3_Δ = 6.55, _lys12_Δ = 5.39, _mep3_Δ = 3.95, _agp1_Δ = 3.65, and _mep2_Δ = 3.41, compared with the parental strain = 2.75 (P < 0.001 for all deletion strain life spans compared with the parental strain).

Figure 2.

Figure 2.

Multiple means of TOR pathway inhibition extend life span. (A) Removal of preferred amino acids from the media extends life span in proportion to the nutrient value of the eliminated amino acid, even though total nitrogen content is held constant. Removal of the preferred amino acid asparagine (integral = 3.24) or of the intermediately favored glutamate (integral = 2.56) confers increased survival compared with cells grown in synthetic complete media (integral = 1.99) (P < 0.001 for each amino acid drop out compared with synthetic complete). (_B_) Direct inhibition of the TOR pathway by low doses of rapamycin extends life span in a dose-responsive manner. The integrals under the life span curves are drug vehicle = 2.93, 100 pg/mL = 3.01, 300 pg/mL = 4.23, 600 pg/mL = 4.49, and 1 ng/mL = 4.51 (_P_ < 0.001 for each dose >100 pg/mL vs. drug vehicle). (C) Inhibition of glutamine synthetase by the drug MSX extends life span by reducing intracellular glutamine, which reduces TOR signaling. The integrals under the life span curves are drug vehicle = 2.88, 30 μM = 2.95, 100 μM = 3.57, 200 μM = 4.11, 300 μM = 4.26, and 400 μM = 4.53 (P < 0.001 for each dose >30μM vs. drug vehicle).

Figure 2.

Figure 2.

Multiple means of TOR pathway inhibition extend life span. (A) Removal of preferred amino acids from the media extends life span in proportion to the nutrient value of the eliminated amino acid, even though total nitrogen content is held constant. Removal of the preferred amino acid asparagine (integral = 3.24) or of the intermediately favored glutamate (integral = 2.56) confers increased survival compared with cells grown in synthetic complete media (integral = 1.99) (P < 0.001 for each amino acid drop out compared with synthetic complete). (_B_) Direct inhibition of the TOR pathway by low doses of rapamycin extends life span in a dose-responsive manner. The integrals under the life span curves are drug vehicle = 2.93, 100 pg/mL = 3.01, 300 pg/mL = 4.23, 600 pg/mL = 4.49, and 1 ng/mL = 4.51 (_P_ < 0.001 for each dose >100 pg/mL vs. drug vehicle). (C) Inhibition of glutamine synthetase by the drug MSX extends life span by reducing intracellular glutamine, which reduces TOR signaling. The integrals under the life span curves are drug vehicle = 2.88, 30 μM = 2.95, 100 μM = 3.57, 200 μM = 4.11, 300 μM = 4.26, and 400 μM = 4.53 (P < 0.001 for each dose >30μM vs. drug vehicle).

Figure 3.

Figure 3.

Increased CLS correlates with increased starvation response. (A) Long-lived deletion strains accumulate glycogen on rich media as determined by an iodine vapor assay, which stains intracellular glycogen dark brown. (B) Rapamycin causes glycogen accumulation in a dose-dependent manner. (C) MSX treatment causes glycogen accumulation. For all groups, n = 9, bars are mean ± SEM; all test groups were significantly different than appropriate control (P < 0.001), and representative colonies are shown.

Figure 3.

Figure 3.

Increased CLS correlates with increased starvation response. (A) Long-lived deletion strains accumulate glycogen on rich media as determined by an iodine vapor assay, which stains intracellular glycogen dark brown. (B) Rapamycin causes glycogen accumulation in a dose-dependent manner. (C) MSX treatment causes glycogen accumulation. For all groups, n = 9, bars are mean ± SEM; all test groups were significantly different than appropriate control (P < 0.001), and representative colonies are shown.

Figure 4.

Figure 4.

Long-lived strains are resistant to lethal heat-stress or oxidative insult. (A) Mid-log cells were treated with a 2-min, 55°C heat shock, and survival was calculated relative to untreated isogenic strains. The _mep2_Δ and _gln3_Δ strains were significantly protected compared with the parental strain (P < 0.0159 for _mep2_Δ and P < 0.0001 for _gln3_Δ). (B) Mid-log cells were inoculated into media containing 1 mM of the free-radical-generating drug paraquat, and growth was compared with untreated isogenic strains after 15 h. Deletion of MEP2 or GLN3 affords significant protection from paraquat (P = 0.002 for _mep2_Δ and P < 0.0001 for _gln3_Δ). For all groups, n = 6, and bars are mean ± SEM.

Figure 5.

Figure 5.

Partial inhibition of TORC1 activity causes precocious nuclear localization of Msn2. (A) Eight hours after strains were inoculated into SC media, an Msn2-GFP fusion protein accumulates in the nucleus of _gln3_Δ cells and cells treated with 600 pg/mL rapamycin or 200 μM MSX, but not in untreated parental cells. The frequency of Msn2-GFP nuclear localization is 56% of _gln3_Δ cells, 52% of rapamycin-treated cells, and 57% of MSX-treated cells, but only 12% of untreated parental control cells. Approximately 100 cells were examined per group. (B) Msn2 and Msn4 are required for the full life span extension of the _gln3_Δ strain. We measured viability 4 wk post-inoculation and observed that transcriptional activity of Msn2 and Msn4 is crucial for maximal CLS. However, the _gln3_Δ mutation activates an Msn2- and Msn4-independent pathway that can extend life span relative to wild type cells. (C) An integrated model linking long-lived deletion strains, reduced amino acid levels, and rapamycin treatment to increased stress response and longevity.

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