Chronological Lifespan Research Articles

Lipid Hydrolase Enzymes: Pragmatic Prolongevity Targets for Improved Human Healthspan?

Compelling evidence suggests that lipid metabolism, which plays critical roles in fat storage, cell membrane maintenance, and cell signaling, is intricately linked to aging. Lipid hydrolases are important enzymes that catalyze the hydrolysis of more complex lipids into simpler lipids. Diverse interventions targeting lipid hydrolases can prolong or shorten life in model organisms. For example, the genetic removal of or RNAi knockdown against a phospholipase can reduce lifespan in Caenorhabditis elegans, Drosophila melanogaster, and Mus musculus. The removal of lysosomal acid lipase results in premature death in mice, while its overexpression in nematodes generates lean, long-lived individuals. The overexpression or inhibition of diacylglycerol lipase leads to enhanced or reduced longevity, respectively, in both worms and flies. Lifespan can also be extended by knocking down triacylglycerol lipases in yeast, overexpressing fatty acid amide hydrolase in worms, or removing hepatic lipase in a mouse model of coronary disease. Conversely, flies lacking the triacylglycerol lipase Brummer are obese and short lived. Linking sphingolipids and aging, removing the sphingomyelinase inositol phosphosphingolipid phospholipase shortens chronological lifespan in Saccharomyces cerevisiae, while inhibiting an acid sphingomyelinase in worms or inactivating alkaline ceramidase in flies extends lifespan. The clinical potential of manipulating these enzymes is highlighted by the FDA-approved obesity drug orlistat, which is an inhibitor of pancreatic and hepatic lipases that induces weight loss and improves insulin/glucose homeostasis. Additional research is warranted to better understand how these lipid hydrolases impact aging and to determine if clinical interventions targeting them are capable of improving human healthspan.

Monitoring changes in the cellular content of biomolecules during ageing with FTIR spectroscopy

Dietary regimens have proven to promote longevity in several eukaryotic model organisms including the budding yeast Saccharomyces cerevisiae. These interventions are effective strategies for preventing ageing and diseases and many of them are linked to amino acid and protein levels. The aim of this work was to better understand how the age-related TOR1 and SCH9 genes and the presence of amino acids affect cell metabolomes and to establish their impact on the ageing process. Cellular metabolic profiles were determined by FTIR spectroscopy. We demonstrated that metabolic signatures of cells deficient in SCH9, the major TORC1 effector, were very different to those of wild type and TOR1 deficient cells. In cells lacking Sch9 we also observed changes in other processes related to ageing such as endoplasmic reticulum stress and autophagy. We identified several anti-ageing biomarkers being the most relevant the intracellular content of pyruvate, glucose, ribose/deoxyribose associated compounds, and the presence of special protein conformational structures. The very sensitive FTIR technique allowed us to highlight important changes that occur along ageing in the metabolomes of the cells deficient in the key nutrient-sensitive Tor1-Sch9 pathway, even though slight differences on chronological lifespan were detected in our conditions.

Diverse conditions support near-zero growth in yeast: Implications for the study of cell lifespan.

Baker's yeast has a finite lifespan and ages in two ways: a mother cell can only divide so many times (its replicative lifespan), and a non-dividing cell can only live so long (its chronological lifespan). Wild and laboratory yeast strains exhibit natural variation for each type of lifespan, and the genetic basis for this variation has been generalized to other eukaryotes, including metazoans. To date, yeast chronological lifespan has chiefly been studied in relation to the rate and mode of functional decline among non-dividing cells in nutrient-depleted batch culture. However, this culture method does not accurately capture two major classes of long-lived metazoan cells: cells that are terminally differentiated and metabolically active for periods that approximate animal lifespan (e.g. cardiac myocytes), and cells that are pluripotent and metabolically quiescent (e.g. stem cells). Here, we consider alternative ways of cultivating Saccharomyces cerevisiae so that these different metabolic states can be explored in non-dividing cells: (i) yeast cultured as giant colonies on semi-solid agar, (ii) yeast cultured in retentostats and provided sufficient nutrients to meet minimal energy requirements, and (iii) yeast encapsulated in a semisolid matrix and fed ad libitum in bioreactors. We review the physiology of yeast cultured under each of these conditions, and explore their potential to provide unique insights into determinants of chronological lifespan in the cells of higher eukaryotes.

Leucine depletion extends the lifespans of leucine-auxotrophic fission yeast by inducing Ecl1 family genes via the transcription factor Fil1.

Many studies show that lifespans of various model organisms can be extended by limiting the quantities of nutrients that are necessary for proliferation. In Schizosaccharomyces pombe, the Ecl1 family genes have been associated with lifespan control and are necessary for cell responses to nutrient depletion, but their functions and mechanisms of action remain uncharacterized. Herein, we show that leucine depletion extends the chronological lifespan (CLS) of leucine-auxotrophic cells. Furthermore, depletion of leucine extended CLS and caused cell miniaturization and cell cycle arrest at the G1 phase, and all of these processes depended on Ecl1 family genes. Although depletion of leucine raises the expression of ecl1+ by about 100-fold in leucine-auxotrophic cells, these conditions did not affect ecl1+ expression in leucine-auxotrophic fil1 mutants that were isolated in deletion set screens using 79 mutants disrupting a transcription factor. Fil1 is a GATA-type zinc finger transcription factor that reportedly binds directly to the upstream regions of ecl1+ and ecl2+. Accordingly, we suggest that Ecl1 family genes are induced in response to environmental stresses, such as oxidative stress and heat stress, or by nutritional depletion of nitrogen or sulfur sources or the amino acid leucine. We also propose that these genes play important roles in the maintenance of cell survival until conditions that favor proliferation are restored.

Conserved NDR/LATS kinase controls RAS GTPase activity to regulate cell growth and chronological lifespan.

Adaptation to the nutritional environment is critical for all cells. RAS GTPase is a highly conserved GTP-binding protein with crucial functions for cell growth and differentiation in response to environmental conditions. Here, we describe a novel mechanism connecting RAS GTPase to nutrient availability in fission yeast. We report that the conserved NDR/LATS kinase Orb6 responds to nutritional cues and regulates Ras1 GTPase activity. Orb6 increases the protein levels of an Ras1 GTPase activator, the guanine nucleotide exchange factor Efc25, by phosphorylating Sts5, a protein bound to efc25 mRNA. By manipulating the extent of Orb6-mediated Sts5 assembly into RNP granules, we can modulate Efc25 protein levels, Ras1 GTPase activity, and, as a result, cell growth and cell survival. Thus, we conclude that the Orb6–Sts5–Ras1 regulatory axis plays a crucial role in promoting cell adaptation, balancing the opposing demands of promoting cell growth and extending chronological lifespan.

SKO1 deficiency extends chronological lifespan in Saccharomyces cerevisiae.

Sko1 plays a key role in the control of gene expression by osmotic and oxidative stress in yeast. We demonstrate that the decrease in chronological lifespan (CLS) of hog1Δ cells was suppressed by SKO1 deletion. sko1Δ single mutant cells were shown to have a longer CLS, thus implicating Sko1 in the regulation of their CLS.

A functional unfolded protein response is required for chronological aging in Saccharomyces cerevisiae.

Progressive impairment of proteostasis and accumulation of toxic misfolded proteins are associated with the cellular aging process. Here, we employed chronologically aged yeast cells to investigate how activation of the unfolded protein response (UPR) upon accumulation of misfolded proteins in the endoplasmic reticulum (ER) affects lifespan. We found that cells lacking a functional UPR display a significantly reduced chronological lifespan, which contrasts previous findings in models of replicative aging. We find exacerbated UPR activation in aged cells, indicating an increase in misfolded protein burden in the ER during the course of aging. We also observed that caloric restriction, which promotes longevity in various model organisms, extends lifespan of UPR-deficient strains. Similarly, aging in pH-buffered media extends lifespan, albeit independently of the UPR. Thus, our data support a role for caloric restriction and reduced acid stress in improving ER homeostasis during aging. Finally, we show that UPR-mediated upregulation of the ER chaperone Kar2 and functional ER-associated degradation (ERAD) are essential for proper aging. Our work documents the central role of secretory protein homeostasis in chronological aging in yeast and highlights that the requirement for a functional UPR can differ between post-mitotic and actively dividing eukaryotic cells.

Mitochondria Associated Degradation Pathway (MAD) Function Beyond the Outer Membrane

The mitochondria-associated degradation (MAD) pathway mediates ubiquitination and removal of mitochondrial outer membrane (MOM) proteins, and targets them for degradation by the proteasome. We find that MAD, but not other mitochondrial control pathways including macroautophagy, mitophagy, or mitochondrial chaperones and proteases, is critical for yeast cellular fitness under conditions of paraquat (PQ) induced chronic, low-level oxidative stress in mitochondria. Specifically, inhibition of MAD results in increased PQ-induced defects in growth and mitochondrial quality, and reduced chronological lifespan. In light of this, we tested whether MAD function in mitochondrial proteostasis extends beyond the known substrates in MOM. Our mass spectrometry analysis revealed possible MAD substrates in different mitochondrial compartments. We confirmed that 2 matrix proteins, Kgd1p and Pim1p, are MAD substrates. Our studies reveal a broader function for MAD in mitochondrial protein surveillance beyond the MOM, and a major role in cellular and mitochondrial fitness in response to chronic, low level oxidative stress in mitochondria.

Sulfate assimilation regulates hydrogen sulfide production independent of lifespan and reactive oxygen species under methionine restriction condition in yeast.

Endogenously produced hydrogen sulfide was proposed to be an underlying mechanism of lifespan extension via methionine restriction. However, hydrogen sulfide regulation and its beneficial effects via methionine restriction remain elusive. Here, we identified the genes required to increase hydrogen sulfide production under methionine restriction condition using genome-wide high-throughput screening in yeast strains with single-gene deletions. Sulfate assimilation-related genes, such as MET1, MET3, MET5, and MET10, were found to be particularly crucial for hydrogen sulfide production. Interestingly, methionine restriction failed to increase hydrogen sulfide production in mutant strains; however, it successfully extended chronological lifespan and reduced reactive oxygen species levels. Altogether, our observations suggested that increased hydrogen sulfide production via methionine restriction is not the mechanism underlying extended yeast lifespan, even though increased hydrogen sulfide production occurred simultaneously with yeast lifespan extension under methionine restriction condition.

Tocotrienol-Rich Fraction (TRF) Improves the Viability of Wild-Type Saccharomyces cerevisiae in the Initial Stationary Phase

Palm oil tocotrienol-rich fraction (TRF) containing majorly of αα, β, γ andδδ-tocotrienols and some αα-tocopherols, was reported to have anti-ageing effects in both human and non-human model organisms, but still remains unexplored in the yeast model. It was reported to have the ability to extend the lifespan of several organisms. Chronological lifespan is one of the means to measure ageing in yeasts. The effect of TRF on the viability of three strains of Saccharomyces cerevisiae (wild-type, CTT1ΔΔand GPx2Δ) was studied. Phenotypic growth analysis of all strains was carried out for 15 hours by measuring the absorbance at OD600nm and cell counting. The optimum dose of TRF was optimised by determining the number of colony-forming unit by the wild-type strain at the end of a 24-hour treatment with TRF (ranging from 0μg/ml to 300μg/ml). TRF at 300 μg/mL showed the best result, and selected as a working dose. Treatment of cells with 300 μg/mL of TRF improved the viability of the wild-type strain in the initial stationary phase, but not on the knockout strains. These finding suggests that TRF has a potential in prolonging the chronological lifespan of S. cerevisiae, and perhaps other organisms as well.

Characterization of survival and stress resistance in S. cerevisiae mutants affected in peroxisome inheritance and proliferation, Δinp1 and Δpex11.

Baker's yeast is a valuable model system for the study of biological aging as it can be utilized for the measurement of replicative and chronological life spans in response to interventions. Whereas replicative aging in Saccharomyces cerevisiae mirrors dividing mammalian cells, chronological aging is seen in non-dividing cells. Aging is strongly influenced by the cellular organelles, especially by mitochondria which house essential functions like oxidative phosphorylation. Additionally, peroxisomes were shown to modulate the aging process, mainly by their turnover of reactive oxygen species. There is a fundamental interest in understanding how mitochondria and peroxisomes contribute to cellular aging. This work analyzes chronological aging in yeast mutants that are affected in peroxisomal proliferation and inheritance. Deletion of INP1 (retention of peroxisomes in the mother cell) or PEX11 (division of peroxisomes) leads to clearly reduced life spans compared to the wild-type control under conditions which depend on peroxisomal metabolism. Δinp1 cells are long-lived in contrast to the wild type and Δpex11 when assayed under conditions that not necessitate peroxisome function. Neither treatment affects the index of respiratory capacity, indicating fully functional mitochondria. Evaluation of stress resistances reveals that Δinp1 has significantly higher resistance to the apoptosis elicitor acetic acid. Old Δpex11 cells from an oleate cultureare more susceptible to hydrogen peroxide treatment compared to Δinp1 and the wild type. Finally, aged cells are hyper-sensitive to heat shock treatment in contrast to young cells.

Cucurbitacin B Exerts Antiaging Effects in Yeast by Regulating Autophagy and Oxidative Stress.

The budding yeast Saccharomyces cerevisiae has been used as a model organism for the basic mechanism of aging, which provides useful assay systems for measuring both replicative and chronological lifespans. In the course of our screening program for substances that extend replicative lifespan, cucurbitacin B (CuB) was found as a hit compound from a compound library, which contains cerebrosides, phenols, sesquiterpenoid, triterpenoids, and sterols isolated from natural products by our research group. Importantly, it prolonged not only the replicative lifespan but also the chronological lifespan in yeast. CuB increased ATG32 gene expression, suggesting that CuB induces autophagy. Indeed, the GFP signal generated from the cleavage of GFP-Atg8, which is a signature of autophagy, was increased upon CuB treatment. On the other hand, CuB failed to increase the chronological lifespans when either ATG2 or ATG32, essential autophagy genes, was deleted, indicating that the lifespan extension by CuB depends on autophagy induction. Furthermore, CuB significantly increased superoxide dismutase (Sod) activity and the survival rate of yeast under oxidative stress, while it decreased the amount of reactive oxygen species (ROS) and malondialdehyde (MDA) production, indicating that CuB has activity to antagonize oxidative stress. Additionally, CuB did not affect replicative lifespans of sod1, sod2, uth1, and skn7 mutants with the K6001 background, indicating that aging-related genes including SOD1, SOD2, UTH1, and SKN7 participate in the antiaging effect of CuB. These results suggest that CuB exerts antiaging activity by regulating autophagy, ROS, antioxidative ability, and aging-related genes. Finally, we discuss the possible intracellular targets of CuB based on the phenotypic comparison between the CuB and global gene deletion databases.

Redox control of yeast Sir2 activity is involved in acetic acid resistance and longevity

Yeast Sir2 is an NAD-dependent histone deacetylase related to oxidative stress and aging. In a previous study, we showed that Sir2 is regulated by S-glutathionylation of key cysteine residues located at the catalytic domain. Mutation of these residues results in strains with increased resistance to disulfide stress. In the present study, these mutant cells were highly resistant to acetic acid and had an increased chronological life span. Mutant cells had increased acetyl-CoA synthetase activity, which converts acetic acid generated by yeast metabolism to acetyl.CoA. This could explain the acetic acid resistance and lower levels of this toxic acid in the extracellular media during aging. Increased acetyl-CoA levels would raise lipid droplets, a source of energy during aging, and fuel glyoxylate-dependent gluconeogenesis. The key enzyme of this pathway, phosphoenolpyruvate carboxykinase (Pck1), showed increased activity in these Sir2 mutant cells during aging. Sir2 activity decreased when cells shifted to the diauxic phase in the mutant strains, compared to the WT strain. Since Pck1 is inactivated through Sir2-dependent deacetylation, the decline in Sir2 activity explained the rise in Pck1 activity. As a consequence, storage of sugars such as trehalose would increase. We conclude that extended longevity observed in the mutants was a combination of increased lipid droplets and trehalose, and decreased acetic acid in the extracellular media. These results offer a deeper understanding of the redox regulation of Sir2 in acetic acid resistance, which is relevant in some food and industrial biotechnology and also in the metabolism associated to calorie restriction, aging and pathologies such as diabetes.

Structure Characterization and Action Mechanism of an Antiaging New Compound from Gastrodia elata Blume.

A new compound, bis(4-hydroxybenzyl)ether mono-β-L-galactopyranoside (1), was isolated from the rhizome of Gastrodia elata Blume. Its structure was elucidated using extensive spectroscopic analysis, including 1D and 2D NMR, HR-ESI-TOF-MS, and chemical derivatization. Compound 1 extended the replicative lifespan of K6001 and the chronological lifespan of YOM36 yeast strains. To understand the mechanism of action, oxidative stress assessment, reactive oxygen species (ROS) and malondialdehyde (MDA) levels, catalase (CAT) and total glutathione peroxidase (GPx) activity assays, and replicative lifespan assay of sod1, sod2, uth1, and skn7 yeast mutant strains were performed. Results indicated the significant increase in the survival rate of yeast under oxidative stress after treatment with 1. ROS and MDA levels were reduced significantly. Meanwhile, the activity of CAT and GPx was significantly increased. The lifespan of sod1, sod2, uth1, and skn7 mutants of K6001 was not affected by 1. Furthermore, we investigated the gene expression related to longevity after administrating 1. The significant increase of Sir2 and reduction of Uth1 gene expression in the 1-treated group were observed. These results indicated that antioxidative stress played an important role in the antiaging effect of 1; Sir2 and Uth1 genes were involved in antiaging effects of 1.

Yeast Life Span and its Impact on Food Fermentations

Yeasts are very important microorganisms for food production. The high fermentative capacity, mainly of the species of the genus Saccharomyces, is a key factor for their biotechnological use, particularly to produce alcoholic beverages. As viability and vitality are essential to ensure their correct performance in industry, this review addresses the main aspects related to the cellular aging of these fungi as their senescence impacts their proper functioning. Laboratory strains of S. cerevisiae have proven a very successful model for elucidating the molecular mechanisms that control life span. Those mechanisms are shared by all eukaryotic cells. S. cerevisiae has two models of aging, replicative and chronological. Replicative life span is measured by the number of daughter cells a mother can produce. This kind of aging is relevant when the yeast biomass is reused, as in the case of beer fermentations. Chronological life span is measured by the time cells are viable in the stationary phase, and this is relevant for batch fermentations when cells are most of the time in a non-dividing state, such as wine fermentations. The molecular causes and pathways regulating both types of aging are explained in this review.

Mutations of Cys and Ser residues in the α5-subunit of the 20S proteasome from Saccharomyces cerevisiae affects gating and chronological lifespan

In addition to autophagy, proteasomes are critical for regulating intracellular protein levels and removing misfolded proteins. The 20S proteasome (20SPT), the central catalytic unit, is sometimes flanked by regulatory units at one or both ends. Additionally, proteosomal activation has been associated with increased lifespan in many organisms. Our group previously reported that the gating (open/closed) of the free 20S proteasome is redox controlled, and that S-glutathionylation of two Cys residues (Cys76 and Cys221) in the α5 subunit promotes gate opening. The present study constructed site-directed mutants of these Cys residues, and evaluated the effects these mutations have on proteosome gate opening and yeast cell survival. Notably, the double mutation of both Cys residues (Cys76 and Cys221) rendered the cells nonviable, whereas the lifespan of the yeast carrying the single mutations (α5-C76S or α5-C221S) was attenuated when compared to the wild type counterpart. Furthermore, it was found that α5-C76S or α5-C221S 20SPT were more likely to be found with the gate in a closed conformation. In contrast, a random α5-subunit double mutation (S35P/C221S) promoted gate opening, increased chronological lifespan and provided resistance to oxidative stress. The 20SPT core particle purified from the long-lived strain degraded model proteins (e.g., α-synuclein) more efficiently than preparations obtained from the wild-type counterpart, and also displayed an increased chymotrypsin-like activity. Mass spectrometric analyses of the C76S, C221S, S35P/C221S, S35P and S35P/C76S mutants provided evidence that the highly conserved Cys76 residue of the α5-subunit is the key determinant for gate opening and cellular survival. The present study reveals a sophisticated regulatory mechanism that controls gate opening, which appears to be based on the interactions among multiple residues within the α5-subunit, and consequently impacts the lifespan of yeast.

Nuclear proteasomal degradation of Saccharomyces cerevisiae inorganic pyrophosphatase Ipp1p, a nucleocytoplasmic protein whose stability depends on its subcellular localization

Inorganic pyrophosphate (PPi) is an abundant by-product of cellular metabolism. PPi-producing reactions take place in the nucleus concurrently with reactions that use PPi as a substrate. Saccharomyces cerevisiae possesses two soluble pyrophosphatases (sPPases): Ipp1p, an essential and allegedly cytosolic protein, and Ipp2p, a mitochondrial isoenzyme. However, no sPPase has yet been unambiguously described in the nucleus. In vivo studies with fluorescent fusions together with activity and immunodetection analyses demonstrated that Ipp1p is a nucleocytoplasmic protein. Mutagenesis analysis showed that this sPPase possesses a nuclear localization signal which participates in its nuclear targeting. Enforced nucleocytoplasmic targeting by fusion to heterologous nuclear import and export signals caused changes in polypeptide abundance and activity levels, indicating that Ipp1p is less stable in the nucleus that in the cytoplasm. Low nuclear levels of this sPPase are physiologically relevant and may be related to its catalytic activity, since cells expressing a functional nuclear-targeted chimaera showed impaired growth and reduced chronological lifespan, while a nuclear-targeted catalytically inactive protein was not degraded and accumulated in the nucleus. Moreover, nuclear proteasome inhibition stabilized Ipp1p whereas nuclear targeting promoted its ubiquitination and interaction with Ubp3p, a component of the ubiquitin-proteasome system. Overall, our results indicate that Ipp1p is nucleocytoplasmic, that its stability depends on its subcellular localization and that sPPase catalytic competence drives its nuclear degradation through the ubiquitin-proteasome system. This suggests a new scenario for PPi homeostasis where both nucleocytoplasmic transport and nuclear proteasome degradation of the sPPase should contribute to control nuclear levels of this ubiquitous metabolite.

Caloric restriction controls stationary phase survival through Protein Kinase A (PKA) and cytosolic pH.

Calorie restriction is the only physiological intervention that extends lifespan throughout all kingdoms of life. In the budding yeast Saccharomyces cerevisiae, cytosolic pH (pHc) controls growth and responds to nutrient availability, decreasing upon glucose depletion. We investigated the interactions between glucose availability, pHc and the central nutrient signalling cAMP‐Protein Kinase A (PKA) pathway. Glucose abundance during the growth phase enhanced acidification upon glucose depletion, via modulation of PKA activity. This actively controlled reduction in starvation pHc correlated with reduced stationary phase survival. Whereas changes in PKA activity affected both acidification and survival, targeted manipulation of starvation pHc showed that cytosolic acidification was downstream of PKA and the causal agent of the reduced chronological lifespan. Thus, caloric restriction controls stationary phase survival through PKA and cytosolic pH.

Set1-catalyzed H3K4 trimethylation antagonizes the HIR/Asf1/Rtt106 repressor complex to promote histone gene expression and chronological life span.

Aging is the main risk factor for many prevalent diseases. However, the molecular mechanisms regulating aging at the cellular level are largely unknown. Using single cell yeast as a model organism, we found that reducing yeast histone proteins accelerates chronological aging and increasing histone supply extends chronological life span. We sought to identify pathways that regulate chronological life span by controlling intracellular histone levels. Thus, we screened the histone H3/H4 mutant library to uncover histone residues and posttranslational modifications that regulate histone gene expression. We discovered 15 substitution mutations with reduced histone proteins and 5 mutations with increased histone proteins. Among these mutations, we found Set1 complex-catalyzed H3K4me3 promotes histone gene transcription and maintains normal chronological life span. Unlike the canonical functions of H3K4me3 in gene expression, H3K4me3 facilitates histone gene transcription by acting as a boundary to restrict the spread of the repressive HIR/Asf1/Rtt106 complex from histone gene promoters. Collectively, our study identified a novel mechanism by which H3K4me3 antagonizes the HIR/Asf1/Rtt106 repressor complex to promote histone gene expression and extend chronological life span.

New drugs for pharmacological extension of replicative life span in normal and progeroid cells

A high-throughput anti-aging drug screen was developed that simultaneously measures senescence-associated β-galactosidase activity and proliferation. Applied to replicatively pre-aged fibroblasts, this screen yielded violuric acid (VA) and 1-naphthoquinone-2-monoxime (N2N1) as its top two hits. These lead compounds extended the replicative life spans of normal and progeroid human cells in a dose-dependent manner and also extended the chronological life spans of mice and C. elegans. They are further shown here to function as redox catalysts in oxidations of NAD(P)H. They thus slow age-related declines in NAD(P)+/NAD(P)H ratios. VA participates in non-enzymatic electron transfers from NAD(P)H to oxidized glutathione or peroxides. N2N1 transfers electrons from NAD(P)H to cytochrome c or CoQ10 via NAD(P)H dehydrogenase (quinone) 1 (NQO1). Our results indicate that pharmacologic manipulation of NQO1 activity via redox catalysts may reveal mechanisms of senescence and aging.