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.

MCART1/SLC25A51 is required for mitochondrial NAD transport

“…NAD plays a critical role in cellular and mitochondrial metabolism beyond its role as an enzymatic cofactor in redox reactions as a cosubstrate of sirtuins and poly-ADP-ribose-polymerases. Three sirtuin homologs, SIRT3 to SIRT5, serving as signaling factors connecting metabolism to cell state are present in mitochondria, and their activity is likely coupled to mitochondrial NAD levels ( 36 , 42 ). As NAD levels decline in aging and recent efforts are aimed at boosting cellular NAD levels to delay the onset of aging and age-related diseases ( 1 , 2 , 43 ), MCART1 emerges as a tool to study the role of the mitochondrial NAD pool in the regulation of these processes and perhaps as an interesting target to modulate life span.…”

“…The wild-type yeast strain derived from CEN.PK 113-7D ( MATa MAL2-8c SUC2 ) ( 44 ). In the ndt1 Δ ndt2 Δ strain ( 36 ), NDT1 and NDT2 genes were replaced with the KanMX3 and HphMX4 cassettes by homologous recombination, conferring resistance to G418 and hygromycin B, respectively. For yeast complementation experiments, MCART1 and MCART1K91A sequences were codon-optimized for expression in S. cerevisiae and cloned into a centromeric expression plasmid with 5′ and 3′ untranslated regions (UTRs) of NDT1 (see Table 1 ).…”

MCART1 is required for mitochondrial NAD transport

“…NAD plays a critical role in cellular and mitochondrial metabolism beyond its role as an enzymatic co-factor in redox reactions as a co-substrate of sirtuins and poly-ADPribose-polymerases. Three sirtuin homologs, SIRT3-5, serving as signaling factors connecting metabolism to cell state are present in mitochondria and their activity is likely coupled to mitochondrial NAD levels (36,42). As NAD levels decline in aging and recent efforts are aimed at boosting cellular NAD levels to delay the onset of aging and agerelated diseases (1, 2, 43), MCART1 emerges as a tool to study the role of the mitochondrial NAD pool in the regulation of these processes and perhaps as an interesting target to modulate life span.…”

“…While a mammalian mitochondrial NAD transporter has not been identified, NDT1 and NDT2 are well known as the mitochondrial NAD + transporters in yeast and plants (5,6,36,37). Like MCART1, NDT1 and 2 are part of the SLC25 family of mitochondrial transporters, but they are not closely related to MCART1 in sequence or predicted structure and sequence analysis has failed to reveal the mitochondrial NAD transporter in higher eukaryotes (38).…”

Nicotinamide, Nicotinamide Riboside and Nicotinic Acid—Emerging Roles in Replicative and Chronological Aging in Yeast

“…Thus, the possible effects of NAD + precursors on chronological aging can involve, on the one hand, the mechanisms that face the physiological reduction of NAD + levels observed as cells age [35,86] and, on the other, the metabolic pathways that affect CLS.…”