Genetic suppression of cryoprotectant toxicity.

We report here a new, unbiased forward genetic method that uses transposon-mediated mutagenesis to enable the identification of mutations that confer cryoprotectant toxicity resistance (CTR). Our method is to select for resistance to the toxic effects of M22, a much-studied whole-organ vitrification solution. We report finding and characterizing six mutants that are resistant to M22. These mutants fall into six independent biochemical pathways not previously linked to cryoprotectant toxicity (CT). The genes associated with the mutations were Gm14005, Myh9, Nrg2, Pura, Fgd2, Pim1, Opa1, Hes1, Hsbp1, and Ywhag. The mechanisms of action of the mutations remain unknown, but two of the mutants involve MYC signaling, which was previously implicated in CT. Several of the mutants may up-regulate cellular stress defense pathways. Several of the M22-resistant mutants were also resistant to dimethyl sulfoxide (Me2SO), and many of the mutants showed significantly improved survival after freezing and thawing in 10% (v/v) Me2SO. This new approach to overcoming CT has many advantages over alternative methods such as transcriptomic profiling. Our method directly identifies specific genetic loci that unequivocally affect CT. More generally, our results provide the first direct evidence that CT can be reduced in mammalian cells by specific molecular interventions. Thus, this approach introduces remarkable new opportunities for pharmacological blockade of CT.

Movement decline across lifespan of Caenorhabditis elegans mutants in the insulin/insulin-like signaling pathway.

Research in aging biology has identified several pathways that are molecularly conserved across species that extend lifespan when mutated. The insulin/insulin-like signaling (IIS) pathway is one of the most widely studied of these. It has been assumed that extending lifespan also extends healthspan (the period of life with minimal functional loss). However, data supporting this assumption conflict and recent evidence suggest that life extension may, in and of itself, extend the frail period. In this study, we use Caenorhabditis elegans to further probe the link between lifespan and healthspan. Using movement decline as a measure of health, we assessed healthspan across the entire lifespan in nine IIS pathway mutants. In one series of experiments, we studied healthspan in mass cultures, and in another series, we studied individuals longitudinally. We found that long-lived mutants display prolonged mid-life movement and do not prolong the frailty period. Lastly, we observed that early-adulthood movement was not predictive of late-life movement or survival, within identical phenotypes. Overall, these observations show that extending lifespan does not prolong the period of frailty. Both genotype and a stochastic component modulate aging, and movement late in life is more variable than early-life movement.

Dietary restriction in C. elegans: recent advances.

The nematode Caenorhabditis elegans continues to serve as a useful model of life extension caused by dietary restriction. Using this model, downstream effectors of dietary restriction-induced longevity have been elucidated, including neuropeptides and cell-surface receptors. Although it remains possible that different forms of dietary restriction may utilize both specific and overlapping mechanisms to promote longevity, the nematode model has revealed roles for autophagy, metabolic energy-sensing and the hypoxic response. The nematode has also been used to identify specific tissues required for life extension via DR, including coelomocytes, intestine, and neurons.

Predicting longevity in C. elegans: fertility, mobility and gene expression.

Expression level of an hsp-16.2::gfp transgene is a predictor of longevity in Caenorhabditis elegans. Here we examine fertility, movement and longevity, comparing high-expressing ("bright") and low-expressing ("dim") animals. There was no differential fertility between bright and dim individuals, suggesting that dim worms were not excessively frail. Worms with high hsp-16.2::gfp expression had improved mobility, consistent with improved health span. We predicted that the increased longevity of the bright worms would be associated with increased expression of protective genes such as those shown to be upregulated in Age mutants. However, few genes were differentially transcribed, although internal controls (hsp-16.2 and family members) were differentially expressed. Quite surprising was the observation that expression level of the transgenic reporter was inherited by the progeny: in seven experiments bright worms consistently produced progeny that were brighter. We tested and ruled out possible artifacts such as differential copy-number of the transgene as an explanation of this differential brightness. These results suggest that a robust physiological state does not depend heavily upon transcriptional differences for its establishment, consistent with proteostatic mechanisms underlying the differential longevity.

Expression of a single-copy hsp-16.2 reporter predicts life span.

The level of green fluorescent protein expression from an hsp-16.2-based transcriptional reporter predicts life span and thermotolerance in Caenorhabditis elegans. The initial report used a high-copy number reporter integrated into chromosome IV. There was concern that the life-span prediction power of this reporter was not attributable solely to hsp-16.2 output. Specifically, prediction power could stem from disruption of some critical piece of chromatin on chromosome IV by the gpIs1 insertion, a linked mutation from the process used to create the reporter, or from an artifact of transgene regulation (multicopy transgenes are subject to regulation by C elegans chromatin surveillance machinery). Here we determine if the ability to predict life span and thermotolerance is specific to the gpIs1 insertion or a general property of hsp-16.2-based reporters. New single-copy hsp-16.2-based reporters predict life span and thermotolerance. We conclude that prediction power of hsp-16.2-based transcriptional reporters is not an artifact of any specific transgene configuration or chromatin surveillance mechanism.

Genetic dissection of late-life fertility in Caenorhabditis elegans.

The large post-reproductive life span reported for the free-living hermaphroditic nematode, Caenorhabditis elegans, which lives for about 10 days after its 5-day period of self-reproduction, seems at odds with evolutionary theory. Species with long post-reproductive life spans such as mammals are sometimes explained by a need for parental care or transfer of information. This does not seem a suitable explanation for C elegans. Previous reports have shown that C elegans can regain fertility when mated after the self-fertile period but did not report the functional limits. Here, we report the functional life span of the C elegans germ line when mating with males. We show that C elegans can regain fertility late in life (significantly later than in previous reports) and that the end of this period corresponds quite well to its 3-week total life span. Genetic analysis reveals that late-life fertility is controlled by conserved pathways involved with aging and dietary restriction.

Mortality shifts in Caenorhabditis elegans: remembrance of conditions past.

The analysis of age-specific mortality can yield insights into how anti-aging interventions operate that cannot be matched by simple assessment of longevity. Mortality, as opposed to longevity, can be used to assess the effects of an anti-aging intervention on a daily basis, rather than only after most animals have died. Various gerontogene mutations in Caenorhabditis elegans have been shown to increase longevity as much as tenfold and to decrease mortality at some ages even more. Environmental alterations, such as reduced food intake (dietary restriction) and lower temperature also result in reduced mortality soon after the intervention. Here, we ask how soon anti-aging interventions, applied during adult life, affect age-specific mortality in nematodes. Using maximum likelihood analysis, we estimated the Gompertz parameters after shifts of temperature, and of food concentration and maintenance conditions. In separate experiments, we altered expression of age-1 and daf-16, using RNAi. Using about 44 000 nematodes in total, to examine daily mortality, we find that for both types of environmental shift, mortality responded immediately in the first assessment, while RNAi-induced changes resulted in a slower response, perhaps due to delayed mechanics of RNAi action. However, under all conditions there is a permanent 'memory' of past states, such that the initial mortality component [a] of the Gompertz equation [mu(x) = ae(bx)] bears a permanent 'imprint' of that earlier state. However, 'b' (the rate of mortality increase with age) is always specified by the current conditions.

Multiple mild heat-shocks decrease the Gompertz component of mortality in Caenorhabditis elegans.

Exposure to mild heat-stress (heat-shock) can significantly increase the life expectancy of the nematode Caenorhabditis elegans. A single heat-shock early in life extends longevity by 20% or more and affects life-long mortality by decreasing initial mortality only; the rate of increase in subsequent mortality (Gompertz component) is unchanged. Repeated mild heat-shocks throughout life have a larger effect on life span than does a single heat-shock early in life. Here, we ask how multiple heat-shocks affect the mortality trajectory in nematodes and find increases of life expectancy of close to 50% and of maximum longevity as well. We examined mortality using large numbers of animals and found that multiple heat-shocks not only decrease initial mortality, but also slow the Gompertz rate of increase in mortality. Thus, multiple heat-shocks have anti-aging hormetic effects and represent an effective approach for modulating aging.

Coenzyme Q supports distinct developmental processes in Caenorhabditis elegans.

Coenzyme Q (Q) regulates aging in Caenorhabditis elegans, and its deficiency leads to a variety of pathologies in humans. We used a coq-8 deleted strain to study the role of Q in C. elegans development and how it influences life span. Endogenous Q(9) content of coq-8(ok840) knockouts was demonstrated to be about 7% of that found in the wild-type, indicating the basal biosynthesis rate is reduced in this strain. Knockouts abnormally developed both gonads and hypodermis, showed reduced fertility and shortened life span, and this was partially recovered by ingestion of exogenous Q. Knockouts produced embryos that showed arrested development at the time of initial expression of coq-8 in embryo. Uridine, whose biosynthesis depends on mitochondrial Q, improved both egg production and progeny under Q-rich dietary conditions. COQ-8 is a candidate protein for post-translational regulation of Q biosynthesis rate and its expression correlates with Q content during the life cycle in C. elegans. We show for the first time that a critical level of Q is necessary to support embryo development and fertility in C. elegans. These results suggest that extra-mitochondrial function of Q is a key factor linking development and bioenergetics in C. elegans.

The U-shaped response of initial mortality in Caenorhabditis elegans to mild heat shock: does it explain recent trends in human mortality?

U-shaped dose-response relationships (hormesis) have been documented in numerous biological, toxicological, and pharmacological investigations. For example, in response to a mild 35 degrees C heat shock, the longevity of Caenorhabditis elegans exhibits an inverted U-shaped dose-response. By applying the demographic concept of heterogeneity, we find that this U-shaped curve for longevity response is driven by a U-shaped dose-response of initial mortality. When worms are subjected to mild heat shock, the initial mortality decreases compared to the control. This initial mortality benefit increases with moderate increases in the length of heat shock, peaking at a point that coincides with the induction of damage to the worms. The dose of heat shock that coincided with this benefit in initial mortality did not affect the rate of increase in mortality.

Hormesis and aging in Caenorhabditis elegans.

Hormesis has emerged as an important manipulation for the study of aging. Although hormesis is manifested in manifold combinations of stress and model organism, the mechanisms of hormesis are only partly understood. The increased stress resistance and extended survival caused by hormesis can be manipulated to further our understanding of the roles of intrinsic and induced stress resistance in aging. Genes of the dauer/insulin/insulin-like signaling (IIS) pathway have well-established roles in aging in Caenorhabditis elegans. Here, we discuss the role of some of those genes in the induced stress resistance and induced life extension attributable to hormesis. Mutations in three genes (daf-16, daf-18, and daf-12) block hormetically induced life extension. However, of these three, only daf-18 appears to be required for a full induction of thermotolerance induced by hormesis, illustrating possible separation of the genetic requirements for stress resistance and life extension. Mutations in three other genes of this pathway (daf-3, daf-5, and age-1) do not block induced life extension or induced thermotolerance; daf-5 mutants may be unusually sensitive to hormetic conditions.

A stress-sensitive reporter predicts longevity in isogenic populations of Caenorhabditis elegans.

When both genotype and environment are held constant, 'chance' variation in the lifespan of individuals in a population is still quite large. Using isogenic populations of the nematode Caenorhabditis elegans, we show that, on the first day of adult life, chance variation in the level of induction of a green fluorescent protein (GFP) reporter coupled to a promoter from the gene hsp-16.2 predicts as much as a fourfold variation in subsequent survival. The same reporter is also a predictor of ability to withstand a subsequent lethal thermal stress. The level of induction of GFP is not heritable, and GFP expression levels in other reporter constructs are not associated with differences in longevity. HSP-16.2 itself is probably not responsible for the observed differences in survival but instead probably reflects a hidden, heterogeneous, but now quantifiable, physiological state that dictates the ability of an organism to deal with the rigors of living.

Insulin regulation of heart function in aging fruit flies.

Insulin-IGF receptor (InR) signaling has a conserved role in regulating lifespan, but little is known about the genetic control of declining organ function. Here, we describe progressive changes of heart function in aging fruit flies: from one to seven weeks of a fly's age, the resting heart rate decreases and the rate of stress-induced heart failure increases. These age-related changes are minimized or absent in long-lived flies when systemic levels of insulin-like peptides are reduced and by mutations of the only receptor, InR, or its substrate, chico. Moreover, interfering with InR signaling exclusively in the heart, by overexpressing the phosphatase dPTEN or the forkhead transcription factor dFOXO, prevents the decline in cardiac performance with age. Thus, insulin-IGF signaling influences age-dependent organ physiology and senescence directly and autonomously, in addition to its systemic effect on lifespan. The aging fly heart is a model for studying the genetics of age-sensitive organ-specific pathology.

Hormesis in Caenorhabditis elegans dauer-defective mutants.

We have shown that increased longevity and stress resistance can be induced by sub-lethal exposure to stressors (hormesis). Here we ask whether genes of the dauer formation pathway that are known to modulate life span in Caenorhabditis elegans are required for this hormesis. We find that loss-of-function mutations in any of three genes (daf-16, daf-18, or daf-12) not only reduce or abolish the ability to form dauers but also block the hormetic response increasing life span following sub-lethal heat stress. Indeed, the life expectancy of these dauer-defective mutants is decreased by the same pretreatments that increase the life expectancy of wild-type animals. Additionally, we find that daf-16 and daf-12 are not required for the induction of thermotolerance, but daf-18 is required for its full induction. Our results underscore the importance of the dauer-formation pathway in specifying life span by demonstrating a similar, but not identical, role in life extension attributed to hormesis.

Ginkgo biloba extract EGb 761 increases stress resistance and extends life span of Caenorhabditis elegans.

EGb 761, a standardized extract of Ginkgo biloba leaves, has been used in clinical trials for its beneficial effects on brain functions. In mammals, EGb 761 has been shown to enhance cognition, stress resistance, and longevity, but its molecular and cellular mechanisms are not known. In the present investigation, we used the model organism Caenorhabditis elegans to evaluate pharmacological effects of EGb 761 on aging. We tested the theory that EGb 761 augments the natural antioxidant system of C elegans, and thus increases stress resistance and longevity. We found that treatment of the wild-type worms with EGb 761 extended their median life span by 8%. Amongst several purified components of EGb 761, the flavonoid tamarixetin showed the most dramatic effect: it extended the median life span by 25%. Furthermore, EGb 761 increased the wild type's resistance to acute oxidative and thermal stress by 33% and 25%, respectively. Treatment of the prematurely aging mutant worms mev-1 with EGb 761 increased their resistance to acute oxidative and thermal stress by 33% and 11%, respectively. It appears that oxidative stress, a major determinant of life span, as well as other types of stress, can be successfully counteracted by the Ginlkgo biloba extract EGb 761.

Multiple stressors in Caenorhabditis elegans induce stress hormesis and extended longevity.

We demonstrate here that the nematode Caenorhabditis elegans displays broad hormetic abilities. Hormesis is the induction of beneficial effects by exposure to low doses of otherwise harmful chemical or physical agents. Heat as well as pretreatment with hyperbaric oxygen or juglone (a chemical that generates reactive oxygen species) significantly increased subsequent resistance to the same challenge. Cross-tolerance between juglone and oxygen was also observed. The same heat or oxygen pretreatment regimens that induced subsequent stress resistance also increased life expectancy and maximum life span of populations undergoing normal aging. Pretreatment with ultraviolet or ionizing radiation did not promote subsequent resistance or increased longevity. In dose-response studies, induced thermotolerance paralleled the induced increase in life expectancy, which is consistent with a common origin.