Marissa Fletcher

Molecular mechanisms underlying genotype-dependent responses to dietary restriction

Dietary restriction (DR) increases lifespan and attenuates age‐related phenotypes in many organisms; however, the effect of DR on longevity of individuals in genetically heterogeneous populations is not well characterized. Here, we describe a large‐scale effort to define molecular mechanisms that underlie genotype‐specific responses to DR. The effect of DR on lifespan was determined for 166 single gene deletion strains in Saccharomyces cerevisiae. Resulting changes in mean lifespan ranged from a reduction of 79% to an increase of 103%. Vacuolar pH homeostasis, superoxide dismutase activity, and mitochondrial proteostasis were found to be strong determinants of the response to DR. Proteomic analysis of cells deficient in prohibitins revealed induction of a mitochondrial unfolded protein response (mtUPR), which has not previously been described in yeast. Mitochondrial proteotoxic stress in prohibitin mutants was suppressed by DR via reduced cytoplasmic mRNA translation. A similar relationship between prohibitins, the mtUPR, and longevity was also observed in Caenorhabditis elegans. These observations define conserved molecular processes that underlie genotype‐dependent effects of DR that may be important modulators of DR in higher organisms.

pH neutralization protects against reduction in replicative lifespan following chronological aging in yeast

Chronological and replicative aging have been studied in yeast as alternative paradigms for post-mitotic and mitotic aging, respectively. It has been known for more than a decade that cells of the S288C background aged chronologically in rich medium have reduced replicative lifespan relative to chronologically young cells. Here we report replication of this observation in the diploid BY4743 strain background. We further show that the reduction in replicative lifespan from chronological aging is accelerated when cells are chronologically aged under standard conditions in synthetic complete medium rather than rich medium. The loss of replicative potential with chronological age is attenuated by buffering the pH of the chronological aging medium to 6.0, an intervention that we have previously shown can extend chronological lifespan. These data demonstrate that extracellular acidification of the culture medium can cause intracellular damage in the chronologically aging population that is asymmetrically segregated by the mother cell to limit subsequent replicative lifespan.

Stress profiling of longevity mutants identifies Afg3 as a mitochondrial determinant of cytoplasmic mRNA translation and aging

Although environmental stress likely plays a significant role in promoting aging, the relationship remains poorly understood. To characterize this interaction in a more comprehensive manner, we examined the stress response profiles for 46 long‐lived yeast mutant strains across four different stress conditions (oxidative, ER, DNA damage, and thermal), grouping genes based on their associated stress response profiles. Unexpectedly, cells lacking the mitochondrial AAA protease gene AFG3 clustered strongly with long‐lived strains lacking cytosolic ribosomal proteins of the large subunit. Similar to these ribosomal protein mutants, afg3Δ cells show reduced cytoplasmic mRNA translation, enhanced resistance to tunicamycin that is independent of the ER unfolded protein response, and Sir2‐independent but Gcn4‐dependent lifespan extension. These data demonstrate an unexpected link between a mitochondrial protease, cytoplasmic mRNA translation, and aging.

Cell nonautonomous activation of flavin-containing monooxygenase promotes longevity and health span

Aging: All in the head—and the gut The effects of hypoxia and caloric restriction, both of which extend life span in Caenorhabditis elegans, converge on the activation of an enzyme in cells of the intestine. Leiser et al. show that the life-extending effects of hypoxia begin in neurons with transcriptional activation by hypoxia-inducible factor–1 and increased serotonergic signaling. These effects lead to increased production of flavin-containing monooxygenase-2 (FMO-2) in the intestine, which increased longevity. Finding the relevant targets of FMO-2, which also accumulates in mammals under conditions that promote longevity, may elucidate further mechanisms that promote healthy aging. Science, this issue p. 1375 Two life-span–extending pathways in the worm converge to increase production of an enzyme in the intestine. Stabilization of the hypoxia-inducible factor 1 (HIF-1) increases life span and health span in nematodes through an unknown mechanism. We report that neuronal stabilization of HIF-1 mediates these effects in Caenorhabditis elegans through a cell nonautonomous signal to the intestine, which results in activation of the xenobiotic detoxification enzyme flavin-containing monooxygenase-2 (FMO-2). This prolongevity signal requires the serotonin biosynthetic enzyme TPH-1 in neurons and the serotonin receptor SER-7 in the intestine. Intestinal FMO-2 is also activated by dietary restriction (DR) and is necessary for DR-mediated life-span extension, which suggests that this enzyme represents a point of convergence for two distinct longevity pathways. FMOs are conserved in eukaryotes and induced by multiple life span–extending interventions in mice, which suggests that these enzymes may play a critical role in promoting health and longevity across phyla.

Sir2 deletion prevents lifespan extension in 32 long-lived mutants

Activation of Sir2 orthologs is proposed to increase lifespan downstream of dietary restriction. Here, we describe an examination of the effect of 32 different lifespan‐extending mutations and four methods of DR on replicative lifespan (RLS) in the short‐lived sir2Δ yeast strain. In every case, deletion of SIR2 prevented RLS extension; however, RLS extension was restored when both SIR2 and FOB1 were deleted in several cases, demonstrating that SIR2 is not directly required for RLS extension. These findings indicate that suppression of the sir2Δ lifespan defect is a rare phenotype among longevity interventions and suggest that sir2Δ cells senesce rapidly by a mechanism distinct from that of wild‐type cells. They also demonstrate that failure to observe lifespan extension in a short‐lived background, such as cells or animals lacking sirtuins, should be interpreted with caution.

Dietary restriction and mitochondrial function link replicative and chronological aging in Saccharomyces cerevisiae.

Chronological aging of budding yeast cells results in a reduction in subsequent replicative life span through unknown mechanisms. Here we show that dietary restriction during chronological aging delays the reduction in subsequent replicative life span up to at least 23days of chronological age. We further show that among the viable portion of the control population aged 26days, individual cells with the lowest mitochondrial membrane potential have the longest subsequent replicative lifespan. These observations demonstrate that dietary restriction modulates a common molecular mechanism linking chronological and replicative aging in yeast and indicate a critical role for mitochondrial function in this process.

Life-Span Extension From Hypoxia in Caenorhabditis elegans Requires Both HIF-1 and DAF-16 and Is Antagonized by SKN-1

Stabilization of the hypoxia-inducible factor (HIF-1) protein extends longevity in Caenorhabditis elegans. However, stabilization of mammalian HIF-1α has been implicated in tumor growth and cancer development. Consequently, for the hypoxic response to benefit mammalian health, we must determine the components of the response that contribute to longevity, and separate them from those that cause harm in mammals. Here, we subject adult worms to low oxygen environments. We find that growth in hypoxia increases longevity in wild-type worms but not in animals lacking HIF-1 or DAF-16. Conversely, hypoxia shortens life span in combination with overexpression of the antioxidant stress response protein SKN-1. When combined with mutations in other longevity pathways or dietary restriction, hypoxia extends life span but to varying extents. Collectively, our results show that hypoxia modulates longevity in a complex manner, likely involving components in addition to HIF-1.

End-of-life cell cycle arrest contributes to stochasticity of yeast replicative aging

There is growing evidence that stochastic events play an important role in determining individual longevity. Studies in model organisms have demonstrated that genetically identical populations maintained under apparently equivalent environmental conditions display individual variation in life span that can be modeled by the Gompertz-Makeham law of mortality. Here, we report that within genetically identical haploid and diploid wild-type populations, shorter-lived cells tend to arrest in a budded state, while cells that arrest in an unbudded state are significantly longer-lived. This relationship is particularly notable in diploid BY4743 cells, where mother cells that arrest in a budded state have a shorter mean life span (25.6 vs. 35.6) and larger coefficient of variance with respect to individual life span (0.42 vs. 0.32) than cells that arrest in an unbudded state. Mutations that cause genomic instability tend to shorten life span and increase the proportion of the population that arrest in a budded state. These observations suggest that randomly occurring damage may contribute to stochasticity during replicative aging by causing a subset of the population to terminally arrest prematurely in the S or G2 phase of the cell cycle.

Genetic interaction with temperature is an important determinant of nematode longevity

As in other poikilotherms, longevity in C. elegans varies inversely with temperature; worms are longer‐lived at lower temperatures. While this observation may seem intuitive based on thermodynamics, the molecular and genetic basis for this phenomenon is not well understood. Several recent reports have argued that lifespan changes across temperatures are genetically controlled by temperature‐specific gene regulation. Here, we provide data that both corroborate those studies and suggest that temperature‐specific longevity is more the rule than the exception. By measuring the lifespans of worms with single modifications reported to be important for longevity at 15, 20, or 25 °C, we find that the effect of each modification on lifespan is highly dependent on temperature. Our results suggest that genetics play a major role in temperature‐associated longevity and are consistent with the hypothesis that while aging in C. elegans is slowed by decreasing temperature, the major cause(s) of death may also be modified, leading to different genes and pathways becoming more or less important at different temperatures. These differential mechanisms of age‐related death are not unlike what is observed in humans, where environmental conditions lead to development of different diseases of aging.