Global transcriptional regulation of innate immunity by ATF-7 in C. elegans.

The nematode Caenorhabditis elegans has emerged as a genetically tractable animal host in which to study evolutionarily conserved mechanisms of innate immune signaling. We previously showed that the PMK-1 p38 mitogen-activated protein kinase (MAPK) pathway regulates innate immunity of C. elegans through phosphorylation of the CREB/ATF bZIP transcription factor, ATF-7. Here, we have undertaken a genomic analysis of the transcriptional response of C. elegans to infection by Pseudomonas aeruginosa, combining genome-wide expression analysis by RNA-seq with ATF-7 chromatin immunoprecipitation followed by sequencing (ChIP-Seq). We observe that PMK-1-ATF-7 activity regulates a majority of all genes induced by pathogen infection, and observe ATF-7 occupancy in regulatory regions of pathogen-induced genes in a PMK-1-dependent manner. Moreover, functional analysis of a subset of these ATF-7-regulated pathogen-induced target genes supports a direct role for this transcriptional response in host defense. The genome-wide regulation through PMK-1- ATF-7 signaling reveals a striking level of control over the innate immune response to infection through a single transcriptional regulator.

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.

Age-Dependent Neuroendocrine Signaling from Sensory Neurons Modulates the Effect of Dietary Restriction on Longevity of Caenorhabditis elegans.

Dietary restriction extends lifespan in evolutionarily diverse animals. A role for the sensory nervous system in dietary restriction has been established in Drosophila and Caenorhabditis elegans, but little is known about how neuroendocrine signals influence the effects of dietary restriction on longevity. Here, we show that DAF-7/TGFß, which is secreted from the C. elegans amphid, promotes lifespan extension in response to dietary restriction in C. elegans. DAF-7 produced by the ASI pair of sensory neurons acts on DAF-1/TGFß receptors expressed on interneurons to inhibit the co-SMAD DAF-3. We find that increased activity of DAF-3 in the presence of diminished or deleted DAF-7 activity abrogates lifespan extension conferred by dietary restriction. We also observe that DAF-7 expression is dynamic during the lifespan of C. elegans, with a marked decrease in DAF-7 levels as animals age during adulthood. We show that this age-dependent diminished expression contributes to the reduced sensitivity of aging animals to the effects of dietary restriction. DAF-7 signaling is a pivotal regulator of metabolism and food-dependent behavior, and our studies establish a molecular link between the neuroendocrine physiology of C. elegans and the process by which dietary restriction can extend lifespan.

Age-associated vulval integrity is an important marker of nematode healthspan.

Improving healthspan, defined as the period where organisms live without frailty and/or disease, is a major goal of biomedical research. While healthspan measures in people are relatively easy to identify, developing robust markers of healthspan in model organisms has proven challenging. Studies using the nematode Caenorhabditis elegans have provided vital information on the basic mechanisms of aging; however, worm health is difficult to define, and the impact of interventions that increase lifespan on worm healthspan has been controversial. Here, we describe a marker of population healthspan in C. elegans that we term age-associated vulval integrity defects, or Avid, frequently described elsewhere as rupture or exploding. We connect the presence of this phenotype with temperature, reproduction, diet, and longevity. Our results show that Avid occurs in post-reproductive worms under common laboratory conditions at a frequency that correlates negatively with temperature; Avid is rare in worms kept at 25 °C and more frequent in worms kept at 15 °C. We describe the kinetics of Avid, link the phenotype to oocyte production, and describe how Avid involves the ejection of worm proteins and/or internal organ(s) from the vulva. Finally, we find that Avid is preventable by removing worms from food, suggesting that Avid results from the intake, digestion, and/or absorption of food. Our results show that Avid is a significant cause of death in worm populations maintained under laboratory conditions and that its prevention often correlates with worm longevity. We propose that Avid is a powerful marker of worm healthspan whose underlying molecular mechanisms may be conserved.

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

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.

A Comprehensive Analysis of Replicative Lifespan in 4,698 Single-Gene Deletion Strains Uncovers Conserved Mechanisms of Aging.

Many genes that affect replicative lifespan (RLS) in the budding yeast Saccharomyces cerevisiae also affect aging in other organisms such as C. elegans and M. musculus. We performed a systematic analysis of yeast RLS in a set of 4,698 viable single-gene deletion strains. Multiple functional gene clusters were identified, and full genome-to-genome comparison demonstrated a significant conservation in longevity pathways between yeast and C. elegans. Among the mechanisms of aging identified, deletion of tRNA exporter LOS1 robustly extended lifespan. Dietary restriction (DR) and inhibition of mechanistic Target of Rapamycin (mTOR) exclude Los1 from the nucleus in a Rad53-dependent manner. Moreover, lifespan extension from deletion of LOS1 is nonadditive with DR or mTOR inhibition, and results in Gcn4 transcription factor activation. Thus, the DNA damage response and mTOR converge on Los1-mediated nuclear tRNA export to regulate Gcn4 activity and aging.

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.

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.

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.

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.

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.

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.