Spermidine toxicity in Saccharomyces cerevisiae due to mitochondrial complex III deficiency

Abstract

Spermidine is a naturally occurring polyamine present in all cells and is necessary for viability in eukaryotic cells. The cellular levels of spermidine decline as an organism ages, and its supplementation has been found to extend lifespan in yeast, worms, flies, mice, and human cultured cells. The lifespan extending effect of spermidine is thought to be due to its ability to induce autophagy, a turnover of cellular components. Mitochondrial dysfunction is believed to be a major driver of the aging process. We asked whether spermidine could rescue mitochondrial dysfunction using the yeast Saccharomyces cerevisiae lacking mtDNA (ρ⁰ cells) as a model. Not only was spermidine unable to rescue survival in ρ⁰ cells, but it appeared to exhibit toxicity resulting in a shortened lifespan. This toxicity appears to not be due to the loss of mitochondrial respiration, elevated oxidative stress, or depleted ATP. Spermidine toxicity could be recapitulated by the genetic or pharmacological inactivation of mitochondrial complex III. It can also be prevented by the impairment of autophagy, through the inactivation of ATG8, or by impairment of mitochondrial complex II through the inactivation of SDH2. Spermidine toxicity in ρ⁰ cells was present in yeast strains BY4741 and W303, but not D273-10B, demonstrating genetic variance in the phenotype. Thus, caution may be suggested regarding the use of spermidine to alleviate aging in humans. Depending on the genotype of the individual, spermidine could potentially harm the very individuals it is intended to help.

Keywords: Aging; Mitochondrial DNA; Mitochondrial dysfunction; Spermidine; Yeast.

Fig. 1

The effect of SPD on A survival of BY4741 ρ⁰ cells, B 99% mortality lifespan of BY4741, S288C, W303, and D273-10B ρ⁰ cells, and C survival of BY4741 ρ⁰ cells with hap1 repaired to wildtype (HAP1R). N = 6 independent cultures for all groups except for later data points in the survival assays where individual cultures gradually reach their end point. *P < 0.05, **P < 0.01, ****P < 0.0001, unpaired t test vs. respective control

Fig. 2

Expression levels and effect on survival of autophagy related genes. A Down-regulation of ATG8 by EtBr, P < 0.05, two-way ANOVA B ATG33 expression unaffected by either SPD or EtBr P > 0.05, two-way ANOVA. C Loss of ATG8 had no effect on lifespan with regard to SPD, whereas D SPD still shortened lifespan in cells deficient in ATG33, P < 0.005 for median lifespan, t test. N = 6 independent cultures for all groups except for later data points in the survival assays where individual cultures gradually die off

Fig. 3

Steady-state yeast ATP levels. A ATP levels were impacted by both SPD (P < 0.05) and yeast strain (P < 0.005), three-way ANOVA. Impairment of autophagy increases ATP levels in B ATG8 deficient cells (P < 0.001, t test vs. KO) and radically increases ATP in C ATG33 deficient cells (P < 0.00001, t test vs. KO). Note: The Y axis for panel C is on a log scale. n = 6 for each group

Fig. 4

The effect of SPD on mitochondrial content and overall metabolism. A When given SPD, D273-10B ρ⁰ cells acidify media to a greater extent to D273-10B ρ⁺ cells (P < 0.005) or BY4741 ρ⁺ or ρ⁰ cells (P < 0.05), Tukey’s multiple comparison test. n = 5–7 for mtDNA content n = 6 for all other groups. B Mitochondrial content via measurement of citrate synthase was markedly higher in both ρ⁺ and ρ⁰ D273-10B cells relative BY4741 cells. However, SPD had no effect on either parameter in either strain (P = 0.92, three-way ANOVA for SPD). C D273-10B cells had a much higher mtDNA content relative to BY4741 cells (P < 0.0001, two-way ANOVA), but again, SPD had no effect (P = 0.96, two-way ANOVA)

Fig. 5

Resistance to PQ by SPD supplementation. A PQ tolerance in control and SPD fed ρ⁺ and ρ⁰ BY4741 cells. B PQ tolerance in control and SPD fed ρ⁺ and ρ⁰ D273-10B cells

Fig. 6

Lifespan 90% mortality for single/double gene knockouts of nuclear encoded respiratory chain complex subunits. SPD increased lifespan when fed to A decreased lifespan in cells lacking CYT1 (P < 0.005). SPD increased lifespan in cells deficient in either B COX6 (P < 0.0001) or C ATP1 (P < 0.01), D cells deficient in both NDE1 and NDE2 (P < 0.001) and E NDI1 (P < 0.0001). SPD had F no effect on cells lacking SDH2 (P = 0.78) and t test. N = 6 independent cultures for all groups except for later data points in the survival assays where individual cultures gradually die off

Fig. 7

The effect of antimycin A (ANT) in BY4741 ρ⁺ cells on A survival and B 90% mortality lifespan. *P < 0.05, t test. N = 6 independent cultures for both groups except for later data points in the survival assays where individual cultures gradually die off