Long-lived Mutants Research Articles

Anti-aging interventions affect lifespan variability in sex, strain, diet and drug dependent fashion.

Decreased forkhead box O1 (FoxO1) activity induces hyperlipidemia and increased PPARγ, leading to hyperlipidemia in association with endoplasmic reticulum (ER) stress. In the liver, aging and comorbidities such as hyperlipidemia and diabetes significantly influence a wide variety of steatosis, but the underlying mechanisms are complex and remain elusive.To establish the modulatory role of FoxO1 and the functional consequences of its altered interaction with PPARγ in the present study, we utilized a cell culture system, aged rats and diabetic db/db mice.We found that, under ER stress, FoxO1 induces PPARγ-mediated lipid accumulation in aged rat livers. Our data showed that the FoxO1-induced hepatic lipid accumulation was negatively regulated by Akt signaling. PPARγ, a key lipogenesis transcription factor, was increased in aged liver, resulting in lipid accumulation via hepatic ER stress under hyperglycemic conditions. We further demonstrated that loss of FoxO1 causes a decline in PPARγ expression and reduces lipid accumulation. In addition, the interaction between FoxO1 and PPARγ was shown to induce hepatic steatosis in aging and db/db mice.We provide evidence that, in aged rats, FoxO1 interaction with PPARγ promotes hepatic steatosis, due to hyperglycemia-induced ER stress, which causes an impairment in Akt signaling, such in aging-related diabetes.

A universal transcriptomic signature of age reveals the temporal scaling of Caenorhabditis elegans aging trajectories

We collected 60 age-dependent transcriptomes for C. elegans strains including four exceptionally long-lived mutants (mean adult lifespan extended 2.2- to 9.4-fold) and three examples of lifespan-increasing RNAi treatments. Principal Component Analysis (PCA) reveals aging as a transcriptomic drift along a single direction, consistent across the vastly diverse biological conditions and coinciding with the first principal component, a hallmark of the criticality of the underlying gene regulatory network. We therefore expected that the organism’s aging state could be characterized by a single number closely related to vitality deficit or biological age. The “aging trajectory”, i.e. the dependence of the biological age on chronological age, is then a universal stochastic function modulated by the network stiffness; a macroscopic parameter reflecting the network topology and associated with the rate of aging. To corroborate this view, we used publicly available datasets to define a transcriptomic biomarker of age and observed that the rescaling of age by lifespan simultaneously brings together aging trajectories of transcription and survival curves. In accordance with the theoretical prediction, the limiting mortality value at the plateau agrees closely with the mortality rate doubling exponent estimated at the cross-over age near the average lifespan. Finally, we used the transcriptomic signature of age to identify possible life-extending drug compounds and successfully tested a handful of the top-ranking molecules in C. elegans survival assays and achieved up to a +30% extension of mean lifespan.

Characterization of long living yeast deletion mutants that lack mitochondrial metabolism genes DSS1, PPA2 and AFG3

Molecular mechanisms of aging and longevity are still mostly unknown. Mitochondria play central roles in cellular metabolism and aging. In this study, we identified three deletion mutants of mitochondrial metabolism genes (ppa2∆, dss1∆, and afg3∆) that live longer than wild-type cells. These long-lived cells harbored significantly decreased amount of mitochondrial DNA (mtDNA) and reactive oxygen species (ROS). Compared to the serpentine nature of wild-type mitochondria, a different dynamics and distribution pattern of mitochondria were observed in the mutants. Both young and old long-lived cells produced relatively low but adequate levels of ATP for cellular activities. The status of the retrograde signaling was checked by expression of CIT2 gene and found activated in long-lived mutants. The mutant cells were also profiled for their gene expression patterns, and genes that were differentially regulated were determined. All long-lived cells comprised similar pleiotropic phenotype regarding mitochondrial dynamics and functions. Thus, this study suggests that DSS1, PPA2, and AFG3 genes modulate the lifespan by altering the mitochondrial morphology and functions.

Age-Dependent Changes in Transcription Factor FOXO Targeting in Female Drosophila.

FOXO transcription factors have long been associated with longevity control and tissue homeostasis. Although the transcriptional regulation of FOXO have been previously characterized (especially in long-lived insulin mutants and under stress conditions), how normal aging impacts the transcriptional activity of FOXO is poorly understood. Here, we conducted a chromatin immunoprecipitation sequencing (ChIP-Seq) analysis in both young (2-week-old) and aged (5-week-old) wild-type female fruit flies, Drosophila melanogaster, to evaluate the dynamics of FOXO gene targeting during aging. Intriguingly, the number of FOXO-bound genes dramatically decreases with age (from 2617 to 224). Consistent to the reduction of FOXO binding activity, many genes targeted by FOXO in young flies are transcriptionally altered with age, either up-regulated (FOXO-repressing genes) or down-regulated (FOXO-activating genes) in adult head tissue. In addition, we show that many FOXO-bound genes in wild-type flies are unique from those in insulin receptor substrate chico mutants. Distinct from chico mutants, FOXO targets specific cellular processes (e.g., actin cytoskeleton) and signaling pathways (e.g., Hippo, MAPK) in young wild-type female flies. FOXO targeting on these pathways decreases with age. Interestingly, FOXO targets in aged flies are enriched in cellular processes like chromatin organization and nucleosome assembly. Furthermore, FOXO binding to core histone genes is well maintained at aged flies. Together, our findings provide new insights into dynamic FOXO targeting under normal aging and highlight the diverse and understudied regulatory mechanisms for FOXO transcriptional activity.

Mitochondrial DNA Damage Does Not Determine C. elegans Lifespan.

The mitochondrial free radical theory of aging (mFRTA) proposes that accumulation of oxidative damage to macromolecules in mitochondria is a causative mechanism for aging. Accumulation of mitochondrial DNA (mtDNA) damage may be of particular interest in this context. While there is evidence for age-dependent accumulation of mtDNA damage, there have been only a limited number of investigations into mtDNA damage as a determinant of longevity. This lack of quantitative data regarding mtDNA damage is predominantly due to a lack of reliable assays to measure mtDNA damage. Here, we report adaptation of a quantitative real-time polymerase chain reaction (qRT-PCR) assay for the detection of sequence-specific mtDNA damage in C. elegans and apply this method to investigate the role of mtDNA damage in the aging of nematodes. We compare damage levels in old and young animals and also between wild-type animals and long-lived mutant strains or strains with modifications in ROS detoxification or production rates. We confirm an age-dependent increase in mtDNA damage levels in C. elegans but found that there is no simple relationship between mtDNA damage and lifespan. MtDNA damage levels were high in some mutants with long lifespan (and vice versa). We next investigated mtDNA damage, lifespan and healthspan effects in nematode subjected to exogenously elevated damage (UV- or γ-radiation induced). We, again, observed a complex relationship between damage and lifespan in such animals. Despite causing a significant elevation in mtDNA damage, γ-radiation did not shorten the lifespan of nematodes at any of the doses tested. When mtDNA damage levels were elevated significantly using UV-radiation, nematodes did suffer from shorter lifespan at the higher end of exposure tested. However, surprisingly, we also found hormetic lifespan and healthspan benefits in nematodes treated with intermediate doses of UV-radiation, despite the fact that mtDNA damage in these animals was also significantly elevated. Our results suggest that within a wide physiological range, the level of mtDNA damage does not control lifespan in C. elegans.

Divergent Aging of Isogenic Yeast Cells Revealed through Single-Cell Phenotypic Dynamics

Although genetic mutations that alter organisms' average lifespans have been identified in aging research, our understanding of the dynamic changes during aging remains limited. Here, we integrate single-cell imaging, microfluidics, and computational modeling to investigate phenotypic divergence and cellular heterogeneity during replicative aging of single S.cerevisiae cells. Specifically, we find that isogenic cells diverge early in life toward one of two aging paths, which are characterized by distinct age-associated phenotypes. We captured the dynamics of single cells along the paths with a stochastic discrete-state model, which accurately predicts both the measured heterogeneity and the lifespan of cells on each path within a cell population. Our analysis suggests that genetic and environmental factors influence both a cell's choice of paths and the kinetics of paths themselves. Given that these factors are highly conserved throughout eukaryotes, divergent aging might represent a general scheme in cellular aging of other organisms.

JNK modifies neuronal metabolism to promote proteostasis and longevity.

Aging is associated with a progressive loss of tissue and metabolic homeostasis. This loss can be delayed by single‐gene perturbations, increasing lifespan. How such perturbations affect metabolic and proteostatic networks to extend lifespan remains unclear. Here, we address this question by comprehensively characterizing age‐related changes in protein turnover rates in the Drosophila brain, as well as changes in the neuronal metabolome, transcriptome, and carbon flux in long‐lived animals with elevated Jun‐N‐terminal Kinase signaling. We find that these animals exhibit a delayed age‐related decline in protein turnover rates, as well as decreased steady‐state neuronal glucose‐6‐phosphate levels and elevated carbon flux into the pentose phosphate pathway due to the induction of glucose‐6‐phosphate dehydrogenase (G6PD). Over‐expressing G6PD in neurons is sufficient to phenocopy these metabolic and proteostatic changes, as well as extend lifespan. Our study identifies a link between metabolic changes and improved proteostasis in neurons that contributes to the lifespan extension in long‐lived mutants.

Of Mice, Whales, Jellyfish and Men: In Pursuit of Increased Longevity.

The quest for increased human longevity has been a goal of mankind throughout recorded history. Recent molecular studies are now providing potentially useful insights into the aging process which may help to achieve at least some aspects of this quest. This chapter will summarize the main findings of these studies with a focus on long-lived mutant mice and worms, and the longest living natural species including Galapagos giant tortoises, bowhead whales, Greenland sharks, quahog clams and the immortal jellyfish.

Monitoring Age-Related Changes in the Lactate/Pyruvate Ratio Using a Colorimetric Assay in a C. elegans Model of Increased Life Span.

In the nematode Caenorhabditis elegans (C. elegans), monitoring the lactate/pyruvate ratio in cells helps to detect imbalances in age-related energy metabolism. Here, we describe a modified small-scale extraction in C. elegans and measurement of lactate and pyruvate concentrations using colorimetric assay kits. During sample extraction, protein precipitation is the most critical step for precise determination of intracellular metabolites in C. elegans. Moreover, improved sensitivity and accuracy of colorimetric assay kits contributed to measurements of metabolites in samples derived from small-scale extraction. Using these protocols, we recently detected a metabolic alteration that occurs during aging by the monitoring the lactate/pyruvate ratio in a long-lived mutant of the mammalian tumor suppressor p53 ortholog CEP-1 in C. elegans.

Mitochondrial unfolded protein response transcription factor ATFS-1 promotes longevity in a long-lived mitochondrial mutant through activation of stress response pathways

BackgroundThe mitochondrial unfolded protein response (mitoUPR) is a stress response pathway activated by disruption of proteostasis in the mitochondria. This pathway has been proposed to influence lifespan, with studies suggesting that mitoUPR activation has complex effects on longevity.ResultsHere, we examined the contribution of the mitoUPR to the survival and lifespan of three long-lived mitochondrial mutants in Caenorhabditis elegans by modulating the levels of ATFS-1, the central transcription factor that mediates the mitoUPR. We found that clk-1, isp-1, and nuo-6 worms all exhibit an ATFS-1-dependent activation of the mitoUPR. While loss of atfs-1 during adulthood does not affect lifespan in any of these strains, absence of atfs-1 during development prevents clk-1 and isp-1 worms from reaching adulthood and reduces the lifespan of nuo-6 mutants. Examining the mechanism by which deletion of atfs-1 reverts nuo-6 lifespan to wild-type, we find that many of the transcriptional changes present in nuo-6 worms are mediated by ATFS-1. Genes exhibiting an ATFS-1-dependent upregulation in nuo-6 worms are enriched for transcripts that function in stress response and metabolism. Consistent, with this finding, loss of atfs-1 abolishes the enhanced stress resistance observed in nuo-6 mutants and prevents upregulation of multiple stress response pathways including the HIF-1-mediated hypoxia response, SKN-1-mediated oxidative stress response and DAF-16-mediated stress response.ConclusionsOur results suggest that in the long-lived mitochondrial mutant nuo-6 activation of the mitoUPR causes atfs-1-dependent changes in the expression of genes involved in stress response and metabolism, which contributes to the extended longevity observed in this mutant. This work demonstrates that the mitoUPR can modulate multiple stress response pathways and suggests that it is crucial for the development and lifespan of long-lived mitochondrial mutants.

Identification of key pathways and metabolic fingerprints of longevity in C. elegans.

Impaired insulin/IGF-1 signaling (IIS) and caloric restriction (CR) prolong lifespan in the nematode C. elegans. However, a cross comparison of these longevity pathways using a multi-omics integration approach is lacking. In this study, we aimed to identify key pathways and metabolite fingerprints of longevity that are shared between IIS and CR worm models using multi-omics integration. We generated transcriptomics and metabolomics data from long-lived worm strains, i.e. daf-2 (impaired IIS) and eat-2 (CR model) and compared them with the wild-type strain N2. Transcriptional profiling identified shared longevity signatures, such as an upregulation of lipid storage and defense responses, and downregulation of macromolecule synthesis and developmental processes. Metabolomics profiling identified an increase in the levels of glycerol‑3P, adenine, xanthine, and AMP, and a decrease in the levels of the amino acid pool, as well as the C18:0, C17:1, C19:1, C20:0 and C22:0 fatty acids. After we integrated transcriptomics and metabolomics data based on the annotations in KEGG, our results highlighted increased amino acid metabolism and an upregulation of purine metabolism as a commonality between the two long-lived mutants. Overall, our findings point towards the existence of shared metabolic pathways that are likely important for lifespan extension and provide novel insights into potential regulators and metabolic fingerprints for longevity.

Enoxacin extends lifespan of C. elegans by inhibiting miR-34-5p and promoting mitohormesis

Alterations in microRNA (miRNA) processing have been previously linked to aging. Here we used the small molecule enoxacin to pharmacologically interfere with miRNA biogenesis and study how it affects aging in C. elegans. Enoxacin extended worm lifespan and promoted survival under normal and oxidative stress conditions. Enoxacin-induced longevity required the transcription factor SKN-1/Nrf2 and was blunted by the antioxidant N-acetyl-cysteine, suggesting a prooxidant-mediated mitohormetic response. The longevity effects of enoxacin were also dependent on the miRNA pathway, consistent with changes in miRNA expression elicited by the drug. Among these differentially expressed miRNAs, the widely conserved miR-34-5p was found to play an important role in enoxacin-mediated longevity. Enoxacin treatment down-regulated miR-34-5p and did not further extend lifespan of long-lived mir-34 mutants. Moreover, N-acetyl-cysteine abrogated mir-34(gk437)-induced longevity. Evidence also points to double-stranded RNA-specific adenosine deaminases (ADARs) as new targets of enoxacin since ADAR loss-of-function abrogates enoxacin-induced lifespan extension. Thus, enoxacin increases lifespan by reducing miR-34-5p levels, interfering with the redox balance and promoting healthspan.

The miR-58 microRNA family is regulated by insulin signaling and contributes to lifespan regulation in Caenorhabditis elegans.

microRNAs regulate diverse biological processes such as development and aging by promoting degradation or inhibiting translation of their target mRNAs. In this study, we have found that the miR-58 family microRNAs regulate lifespan in C. elegans. Intriguingly, members of the miR-58 family affect lifespan differently, sometimes in opposite directions, and have complex genetic interactions. The abundances of the miR-58 family miRNAs are up-regulated in the long-lived daf-2 mutant in a daf-16-dependent manner, indicating that these miRNAs are effectors of insulin signaling in C. elegans. We also found that miR-58 is regulated by insulin signaling and partially required for the lifespan extension mediated by reduced insulin signaling, germline ablation, dietary restriction, and mild mitochondrial dysfunction. We further identified the daf-21, ins-1, and isw-1 mRNAs as endogenous targets of miR-58. Our study shows that miRNAs function in multiple lifespan extension mechanisms, and that the seed sequence is not the dominant factor defining the role of a miRNA in lifespan regulation.

Physiological and Transcriptomic Analysis of a Chronologically Long-Lived Saccharomyces cerevisiae Strain Obtained by Evolutionary Engineering.

High-throughput aging studies with yeast as a model organism involve transposon-mutagenesis and yeast knockout collection, which have been pivotal strategies for understanding the complex cellular aging process. In this study, a chronologically long-lived Saccharomyces cerevisiae mutant was successfully obtained by using another high-throughput approach, evolutionary engineering, based on systematic selection in successive batch cultures under gradually increasing levels of caloric restriction. Detailed comparative physiological and transcriptomic analyses of the chronologically long-lived mutant and the reference strain revealed enhanced levels of respiratory metabolism, upregulation of genes related to carbohydrate metabolic processes, glycogen-trehalose pathways, stress response, and repression of protein synthesis-related genes in the long-lived mutant SRM11, already in the absence of caloric restriction. Interestingly, SRM11 had also significantly higher resistance to copper stress, and higher resistance to silver, ethanol, and 2-phenylethanol stresses than the reference strain. It also had lower ethanol production levels and an enhanced ethanol catabolism. To conclude, evolutionary engineering is another powerful high-throughput method for aging research, in addition to its widespread use in industrial strain development. Additionally, the interesting results revealed by this study about the potential relationship between longevity and various cellular properties are yet to be investigated further at molecular level.

A window into extreme longevity; the circulating metabolomic signature of the naked mole-rat, a mammal that shows negligible senescence

Mouse-sized naked mole-rats (Heterocephalus glaber), unlike other mammals, do not conform to Gompertzian laws of age-related mortality; adults show no age-related change in mortality risk. Moreover, we observe negligible hallmarks of aging with well-maintained physiological and molecular functions, commonly altered with age in other species. We questioned whether naked mole-rats, living an order of magnitude longer than laboratory mice, exhibit different plasma metabolite profiles, which could then highlight novel mechanisms or targets involved in disease and longevity. Using a comprehensive, unbiased metabolomics screen, we observe striking inter-species differences in amino acid, peptide, and lipid metabolites. Low circulating levels of specific amino acids, particularly those linked to the methionine pathway, resemble those observed during the fasting period at late torpor in hibernating ground squirrels and those seen in longer-lived methionine-restricted rats. These data also concur with metabolome reports on long-lived mutant mice, including the Ames dwarf mice and calorically restricted mice, as well as fruit flies, and even show similarities to circulating metabolite differences observed in young human adults when compared to older humans. During evolution, some of these beneficial nutrient/stress response pathways may have been positively selected in the naked mole-rat. These observations suggest that interventions that modify the aging metabolomic profile to a more youthful one may enable people to lead healthier and longer lives.

Activation of DAF-16/FOXO by reactive oxygen species contributes to longevity in long-lived mitochondrial mutants in Caenorhabditis elegans.

Mild deficits in mitochondrial function have been shown to increase lifespan in multiple species including worms, flies and mice. Here, we study three C. elegans mitochondrial mutants (clk-1, isp-1 and nuo-6) to identify overlapping genetic pathways that contribute to their longevity. We find that genes regulated by the FOXO transcription factor DAF-16 are upregulated in all three strains, and that the transcriptional changes present in these worms overlap significantly with the long-lived insulin-IGF1 signaling pathway mutant daf-2. We show that DAF-16 and multiple DAF-16 interacting proteins (MATH-33, IMB-2, CST-1/2, BAR-1) are required for the full longevity of all three mitochondrial mutants. Our results suggest that the activation of DAF-16 in these mutants results from elevated levels of reactive oxygen species. Overall, this work reveals an overlapping genetic pathway required for longevity in three mitochondrial mutants, and, combined with previous work, demonstrates that DAF-16 is a downstream mediator of lifespan extension in multiple pathways of longevity.

Visible light reduces C. elegans longevity

The transparent nematode Caenorhabditis elegans can sense UV and blue-violet light to alter behavior. Because high-dose UV and blue-violet light are not a common feature outside of the laboratory setting, we asked what role, if any, could low-intensity visible light play in C. elegans physiology and longevity. Here, we show that C. elegans lifespan is inversely correlated to the time worms were exposed to visible light. While circadian control, lite-1 and tax-2 do not contribute to the lifespan reduction, we demonstrate that visible light creates photooxidative stress along with a general unfolded-protein response that decreases the lifespan. Finally, we find that long-lived mutants are more resistant to light stress, as well as wild-type worms supplemented pharmacologically with antioxidants. This study reveals that transparent nematodes are sensitive to visible light radiation and highlights the need to standardize methods for controlling the unrecognized biased effect of light during lifespan studies in laboratory conditions.

Electrophysiological Measures of Aging Pharynx Function in C. elegans Reveal Enhanced Organ Functionality in Older, Long-lived Mutants.

The function of the pharynx, an organ in the model system Caenorhabditis elegans, has been correlated with life span and motility (another measure of health) since 1980. In this study, in order to further understand the relationship between organ function and life span, we measured the age-related decline of the pharynx using an electrophysiological approach. We measured and analyzed electropharyngeograms (EPG) of wild type animals, short-lived hsf-1 mutants, and long-lived animals with genetically decreased insulin signaling or increased heat shock pathway signaling; we recorded a total of 2,478 EPGs from 1,374 individuals. As expected, the long-lived daf-2(e1370) and hsf-1OE(uthIs235) animals maintained pharynx function relatively closer to the youthful state during aging, whereas the hsf-1(sy441) and wild type animals' pharynx function deviated significantly further from the youthful state at advanced age. Measures of the amount of variation in organ function can act as biomarkers of youthful physiology as well. Intriguingly, the long-lived animals had greater variation in the duration of pharynx contraction at older ages.

A High-Throughput Screen for Yeast Replicative Lifespan Identifies Lifespan-Extending Compounds

Progress in aging research is constrained by the timerequirement of measuring lifespans. Even the most rapid model for eukaryotic aging, the replicative lifespan of Saccharomyces cerevisiae, is technically limited to only several lifespan measurements each day. Here we report a 384-well plate-based technique to measure replicative lifespan, termed High-Life. Using the High-Life technique, a single researcher can compare lifespan for more than 1,000 conditions per day. We validated the technique with long-lived mutant strains and the lifespan-extending compound ibuprofen. We also applied this technique to screen a small compound library for lifespan extension. Two hits, terreic acid and mycophenolic acid, were validated on our single-cell replicator device and found to extend mean replicative lifespan by 15% and 20%, respectively. Together, we report a technique for high-throughput lifespan measurement, and we identify two lifespan-extending compounds. Our technique could be used to efficiently drive early-stage discovery of pro-longevity therapeutics.

D-Allulose, a stereoisomer of d-fructose, extends Caenorhabditis elegans lifespan through a dietary restriction mechanism: A new candidate dietary restriction mimetic

Dietary restriction (DR) is an effective intervention known to increase lifespan in a wide variety of organisms. DR also delays the onset of aging-associated diseases. DR mimetics, compounds that can mimic the effects of DR, have been intensively explored. d-Allulose (d-Alu), the C3-epimer of d-fructose, is a rare sugar that has various health benefits, including anti-hyperglycemia and anti-obesity effects. Here, we report that d-Alu increased the lifespan of Caenorhabditis elegans both under monoxenic and axenic culture conditions. d-Alu did not further extend the lifespan of the long-lived DR model eat-2 mutant, strongly indicating that the effect is related to DR. However, d-Alu did not reduce the food intake of wild-type C. elegans. To explore the mechanisms of the d-Alu longevity effect, we examined the lifespan of d-Alu-treated mutants deficient for nutrient sensing pathway-related genes daf-16, sir-2.1, aak-2, and skn-1. As a result, d-Alu increased the lifespan of the daf-16, sir-2.1, and skn-1 mutants, but not the aak-2 mutant, indicating that the lifespan extension was dependent on the energy sensor, AMP-activated protein kinase (AMPK). d-Alu also enhanced the mRNA expression and enzyme activities of superoxide dismutase (SOD) and catalase. From these findings, we conclude that d-Alu extends lifespan by increasing oxidative stress resistance through a DR mechanism, making it a candidate DR mimetic.