Altered Expression of Mitochondrial NAD+ Carriers Influences Yeast Chronological Lifespan by Modulating Cytosolic and Mitochondrial Metabolism

Abstract

Nicotinamide adenine dinucleotide (NAD⁺) represents an essential cofactor in sustaining cellular bioenergetics and maintaining cellular fitness, and has emerged as a therapeutic target to counteract aging and age-related diseases. Besides NAD⁺ involvement in multiple redox reactions, it is also required as co-substrate for the activity of Sirtuins, a family of evolutionary conserved NAD⁺-dependent deacetylases that regulate both metabolism and aging. The founding member of this family is Sir2 of Saccharomyces cerevisiae, a well-established model system for studying aging of post-mitotic mammalian cells. In this context, it refers to chronological aging, in which the chronological lifespan (CLS) is measured. In this paper, we investigated the effects of changes in the cellular content of NAD⁺ on CLS by altering the expression of mitochondrial NAD⁺ carriers, namely Ndt1 and Ndt2. We found that the deletion or overexpression of these carriers alters the intracellular levels of NAD⁺ with opposite outcomes on CLS. In particular, lack of both carriers decreases NAD⁺ content and extends CLS, whereas NDT1 overexpression increases NAD⁺ content and reduces CLS. This correlates with opposite cytosolic and mitochondrial metabolic assets shown by the two types of mutants. In the former, an increase in the efficiency of oxidative phosphorylation is observed together with an enhancement of a pro-longevity anabolic metabolism toward gluconeogenesis and trehalose storage. On the contrary, NDT1 overexpression brings about on the one hand, a decrease in the respiratory efficiency generating harmful superoxide anions, and on the other, a decrease in gluconeogenesis and trehalose stores: all this is reflected into a time-dependent loss of mitochondrial functionality during chronological aging.

Keywords: NAD+; Ndt1; Ndt2; Saccharomyces cerevisiae; chronological lifespan.

Figure 1

The NDT1-over mutant and the ndt1Δndt2Δ one have a short-lived and a long-lived phenotype, respectively. The indicated strains were grown in minimal medium/2% glucose and the required supplements in excess (see section Materials and Methods) and followed up to stationary phase. (A,B) CLS was determined by assessing clonogenicity on YEPD plates. 72 h after the diauxic shift (Day 3) was considered the first age-point (100% survival). Day 0, diauxic shift. In parallel, intracellular NAD⁺ (C), NADH (D) and extracellular ethanol (E,F) concentrations were determined at the indicated time-points. Exp, exponential growth phase. All data refer to mean values of three independent experiments with three technical replicates each. Standard deviation (SD) is indicated. Statistical significance as assessed by one-way ANOVA test is indicated (^*P ≤ 0.05 and ^**P ≤ 0.01).

Figure 2

Altered expression of mitochondrial NAD⁺ carriers influences Ald enzymatic activities. In the context of CLS experiments of Figure 1, total Ald (A), Ald4/5 (B), and Ald6 (C) enzymatic activities were measured at the indicated time-points. Day 0, diauxic shift. All data are the mean ± SD obtained from three independent experiments with three technical replicates each. Statistical significance as in Figure 1 (^**P ≤ 0.01).

Figure 3

Altered expression of mitochondrial NAD⁺ carriers influences gluconeogenesis and threalose levels. Bar charts of Icl1 (A) and Pck1 (B) enzymatic activities and intracellular trehalose concentrations (C) determined for the indicated strains grown as in Figure 1. Day 0, diauxic shift. All data refer to mean values of three independent experiments with three technical replicates each. SD is indicated (^**P ≤ 0.01).

Figure 4

The ndt1Δndt2Δ mutant displays decreased levels of TCA intermediates. The indicated strains were grown as in Figure 1 and intracellular concentrations of citrate (A), succinate (B) and malate (C) were measured. Day 0, diauxic shift. Bar charts show the mean values determined in three independent experiments with three technical replicates each. SD is indicated (^**P ≤ 0.01).

Figure 5

Lack of Ndt1 and Ndt2 has a positive effect on mitochondrial functionality during chronological aging. Cells were grown as in Figure 1. Mitochondrial NAD⁺ (A) and NADH (B) contents were determined at the indicated time-points. Day 0, diauxic shift. Bar charts show the mean values determined in three independent experiments with three technical replicates each. SD is indicated (^**P ≤ 0.01). (C) Bar charts of the percentage of fluorescent/superoxide positive cells assessed by the superoxide-driven conversion of non-fluorescent dihydroethidium into fluorescent ethidium (Eth). Day 0, diauxic shift. About 1,000 cells for each sample (three technical replicates) in three independent experiments were examined. SD is indicated (^*P ≤ 0.05 and ^**P ≤ 0.01). (D) Starting from the diauxic shift (Day 0), at indicated time-points aliquots of wt, NDT1-over, and ndt1Δndt2Δ cultures were serially diluted and plated onto YEPD and YEPG plates in order to determine the index of respiratory competence (IRC). SD is indicated.

Figure 6

Simplified scheme summarizing the effects of the altered expression of mitochondrial NAD⁺ carriers during chronological aging.