The transcription factor NHR-8: A new target to increase ivermectin efficacy in nematodes.

Resistance to the anthelmintic macrocyclic lactone ivermectin (IVM) has a great impact on the control of parasitic nematodes. The mechanisms by which nematodes adapt to IVM remain to be deciphered. We have identified NHR-8, a nuclear hormone receptor involved in the xenobiotic response in Caenorhabditis elegans, as a new regulator of tolerance to IVM. Loss-of-function nhr-8(ok186) C. elegans mutants subjected to larval development assays and electropharyngeogram measurements, displayed hypersensitivity to IVM, and silencing of nhr-8 in IVM-resistant worms increased IVM efficacy. In addition, compared to wild-type worms, nhr-8 mutants under IVM selection pressure failed to acquire tolerance to the drug. In addition, IVM-hypersensitive nhr-8(ok186) worms displayed low transcript levels of several genes from the xenobiotic detoxification network and a concomitant low Pgp-mediated drug efflux activity. Interestingly, some pgp and cyp genes known to impact IVM tolerance in many nematode species, were down regulated in nhr-8 mutants and inversely upregulated in IVM-resistant worms. Moreover, pgp-6 overexpression in nhr-8(ok186) C. elegans increased tolerance to IVM. Importantly, NHR-8 function was rescued in nhr-8(ok186) C. elegans with the homolog of the parasitic nematode Haemonchus contortus, and silencing of Hco-nhr-8 by RNAi on L2 H. contortus larvae increased IVM susceptibility in both susceptible and resistant H. contortus isolates. Thus, our data show that NHR-8 controls the tolerance and development of resistance to IVM in C. elegans and the molecular basis for this relates to the NHR-8-mediated upregulation of IVM detoxification genes. Since our results show that Hco-nhr-8 functions similarly to Cel-nhr-8, this study helps to better understand mechanisms underlying failure in drug efficacy and open perspectives in finding new compounds with NHR-8 antagonist activity to potentiate IVM efficacy.

Comparison of lipidome profiles of Caenorhabditis elegans-results from an inter-laboratory ring trial.

INTRODUCTION: Lipidomic profiling allows 100s if not 1000s of lipids in a sample to be detected and quantified. Modern lipidomics techniques are ultra-sensitive assays that enable the discovery of novel biomarkers in a variety of fields and provide new insight in mechanistic investigations. Despite much progress in lipidomics, there remains, as for all high throughput "omics" strategies, the need to develop strategies to standardize and integrate quality control into studies in order to enhance robustness, reproducibility, and usability of studies within specific fields and beyond. OBJECTIVES: We aimed to understand how much results from lipid profiling in the model organism Caenorhabditis elegans are influenced by different culture conditions in different laboratories. METHODS: In this work we have undertaken an inter-laboratory study, comparing the lipid profiles of N2 wild type C. elegans and daf-2(e1370) mutants lacking a functional insulin receptor. Sample were collected from worms grown in four separate laboratories under standardized growth conditions. We used an UPLC-UHR-ToF-MS system allowing chromatographic separation before MS analysis. RESULTS: We found common qualitative changes in several marker lipids in samples from the individual laboratories. On the other hand, even in this controlled experimental system, the exact fold-changes for each marker varied between laboratories. CONCLUSION: Our results thus reveal a serious limitation to the reproducibility of current lipid profiling experiments and reveal challenges to the integration of such data from different laboratories.

Lifespan-extending interventions enhance lipid-supported mitochondrial respiration in Caenorhabditis elegans.

Dietary restriction and reduced reproduction have been linked to long lifespans in the vast majority of species tested. Although decreased mitochondrial mass and/or function are hallmarks of aging, little is known about the mechanisms by which these organelles contribute to physiological aging or to the effects of lifespan-extending interventions, particularly with respect to oxidative phosphorylation and energy production. Here, we employed the nematode Caenorhabditis elegans to examine the effects of inhibition of germline proliferation and dietary restriction, both of which extend the lifespan of C. elegans, on mitochondrial respiratory activity in whole animals and isolated organelles. We found that oxygen consumption rates and mitochondrial mass were reduced in wild-type (WT) C. elegans subjected to bacterial deprivation (BD) compared with animals fed ad libitum (AL). In contrast, BD decreased the rate of oxygen uptake but not mitochondrial mass in germline-less glp-1(e2144ts) mutants. Interestingly, mitochondria isolated from animals subjected to BD and/or inhibition of germline proliferation showed no differences in complex I-mediated respiratory activity compared to control mitochondria, whereas both interventions enhanced the efficiency with which mitochondria utilized lipids as respiratory substrates. Notably, the combination of BD and inhibition of germline proliferation further increased mitochondrial lipid oxidation compared to either intervention alone. We also detected a striking correlation between lifespan extension in response to BD and/or inhibition of germline proliferation and the capacity of C. elegans to generate ATP from lipids. Our results thus suggest that the ability to oxidize lipids may be determinant in enhanced longevity.

Fatty acid desaturation links germ cell loss to longevity through NHR-80/HNF4 in C. elegans.

BACKGROUND: Preventing germline stem cell proliferation extends lifespan in nematodes and flies. So far, studies on germline-longevity signaling have focused on daf-16/FOXO and daf-12/VDR. Here, we report on NHR-80/HNF4, a nuclear receptor that specifically mediates longevity induced by depletion of the germ line through a mechanism that implicates fatty acid monodesaturation. METHODS AND FINDINGS: nhr-80/HNF4 is induced in animals lacking a germ line and is specifically required for their extended longevity. Overexpressing nhr-80/HNF4 increases the lifespan of germline-less animals. This lifespan extension can occur in the absence of daf-16/FOXO but requires the presence of the nuclear receptor DAF-12/VDR. We show that the fatty acid desaturase, FAT-6/SCD1, is a key target of NHR-80/HNF4 and promotes germline-longevity by desaturating stearic acid to oleic acid (OA). We find that NHR-80/HNF4 and OA must work in concert to promote longevity. CONCLUSIONS: Taken together, our data indicate that the NHR-80 pathway participates in the mechanism of longevity extension through depletion of the germ line. We identify fat-6 and OA as essential downstream elements although other targets must also be present. Thus, NHR-80 links fatty acid desaturation to lifespan extension through germline ablation in a daf-16/FOXO independent manner.

PHA-4/Foxa mediates diet-restriction-induced longevity of C. elegans.

Reduced food intake as a result of dietary restriction increases the lifespan of a wide variety of metazoans and delays the onset of multiple age-related pathologies. Dietary restriction elicits a genetically programmed response to nutrient availability that cannot be explained by a simple reduction in metabolism or slower growth of the organism. In the nematode worm Caenorhabditis elegans, the transcription factor PHA-4 has an essential role in the embryonic development of the foregut and is orthologous to genes encoding the mammalian family of Foxa transcription factors, Foxa1, Foxa2 and Foxa3. Foxa family members have important roles during development, but also act later in life to regulate glucagon production and glucose homeostasis, particularly in response to fasting. Here we describe a newly discovered, adult-specific function for PHA-4 in the regulation of diet-restriction-mediated longevity in C. elegans. The role of PHA-4 in lifespan determination is specific for dietary restriction, because it is not required for the increased longevity caused by other genetic pathways that regulate ageing.

Parental age effect on the longevity and healthspan in Drosophila melanogaster and Caenorhabditis elegans.

Several studies have investigated the effect of parental age on biological parameters such as reproduction, lifespan, and health; however, the results have been inconclusive, largely due to inter-species variation and/or modest effect sizes. Here, we examined the effect of parental age on the lifespan, reproductive capacity, and locomotor activity of genetic isogenic lines of the nematode Caenorhabditis elegans and the fruit fly Drosophila melanogaster. We found that the progeny of successive generations of old parents had significantly shorter lifespans than the progeny of young parents in both species. Moreover, we investigated the fertility, fecundity, and locomotor activity of C. elegans. Interestingly, both the shorter lifespan and deteriorated healthspan of the progeny were significantly improved by switching to only one generation of younger parents. Collectively, these data demonstrate that the detrimental effect of older parental age on the longevity of the progeny can be reversed, suggesting the existence of a beneficial non-genetic mechanism.

Lipase-like 5 enzyme controls mitochondrial activity in response to starvation in Caenorhabditis elegans.

The C. elegans lipase-like 5 (lipl-5) gene is predicted to code for a lipase homologous to the human gastric acid lipase. Its expression was previously shown to be modulated by nutritional or immune cues, but nothing is known about its impact on the lipid landscape and ensuing functional consequences. In the present work, we used mutants lacking LIPL-5 protein and found that lipl-5 is important for normal lipidome composition as well as its remodeling in response to food deprivation. Particularly, lipids with signaling functions such as ceramides and mitochondrial lipids were affected by lipl-5 silencing. In comparison with wild type worms, animals lacking LIPL-5 were enriched in cardiolipins linked to polyunsaturated C20 fatty acids and coenzyme Q-9. Differences in mitochondrial lipid composition were accompanied by differences in mitochondrial activity as mitochondria from well-fed lipl-5 mutants were significantly more able to oxidize respiratory substrates when compared with mitochondria from well-fed wild type worms. Strikingly, starvation elicited important changes in mitochondrial activity in wild type worms, but not in lipl-5 worms. This indicates that this lipase is a determinant of mitochondrial functional remodeling in response to food withdrawal.

Coelomocytes Regulate Starvation-Induced Fat Catabolism and Lifespan Extension through the Lipase LIPL-5 in Caenorhabditis elegans.

Dietary restriction is known to extend the lifespan and reduce fat stores in most species tested to date, but the molecular mechanisms linking these events remain unclear. Here, we found that bacterial deprivation of Caenorhabditis elegans leads to lifespan extension with concomitant mobilization of fat stores. We find that LIPL-5 expression is induced by starvation and that the LIPL-5 lipase is present in coelomocyte cells and regulates fat catabolism and longevity during the bacterial deprivation response. Either LIPL-5 or coelomocyte deficiency prevents the rapid mobilization of intestinal triacylglycerol and enhanced lifespan extension in response to bacterial deprivation, whereas the combination of both defects has no additional or synergistic effect. Thus, the capacity to mobilize fat via LIPL-5 is directly linked to an animal's capacity to withstand long-term nutrient deprivation. Our data establish a role for LIPL-5 and coelomocytes in regulating fat consumption and lifespan extension upon DR.

A salicylic acid derivative extends the lifespan of Caenorhabditis elegans by activating autophagy and the mitochondrial unfolded protein response.

Plant extracts containing salicylates are probably the most ancient remedies to reduce fever and ease aches of all kind. Recently, it has been shown that salicylates activate adenosine monophosphate-activated kinase (AMPK), which is now considered as a promising target to slow down aging and prevent age-related diseases in humans. Beneficial effects of AMPK activation on lifespan have been discovered in the model organism Caenorhabditis elegans (C. elegans). Indeed, salicylic acid and acetylsalicylic acid extend lifespan in worms by activating AMPK and the forkhead transcription factor DAF-16/FOXO. Here, we investigated whether another salicylic acid derivative 5-octanoyl salicylic acid (C8-SA), developed as a controlled skin exfoliating ingredient, had similar properties using C. elegans as a model. We show that C8-SA increases lifespan of C. elegans and that a variety of pathways and genes are required for C8-SA-mediated lifespan extension. C8-SA activates AMPK and inhibits TOR both in nematodes and in primary human keratinocytes. We also show that C8-SA can induce both autophagy and the mitochondrial unfolded protein response (UPRmit ) in nematodes. This induction of both processes is fully required for lifespan extension in the worm. In addition, we found that the activation of autophagy by C8-SA fails to occur in worms with compromised UPRmit , suggesting a mechanistic link between these two processes. Mutants that are defective in the mitochondrial unfolded protein response exhibit constitutive high autophagy levels. Taken together, these data therefore suggest that C8-SA positively impacts longevity in worms through induction of autophagy and the UPRmit .

HIF-1-dependent regulation of lifespan in Caenorhabditis elegans by the acyl-CoA-binding protein MAA-1.

In yeast, the broadly conserved acyl-CoA-binding protein (ACBP) is a negative regulator of stress resistance and longevity. Here, we have turned to the nematode C. elegans as a model organism in which to determine whether ACBPs play similar roles in multicellular organisms. We systematically inactivated each of the seven C. elegans ACBP paralogs and found that one of them, maa-1 (which encodes membrane-associated ACBP 1), is indeed involved in the regulation of longevity. In fact, loss of maa-1 promotes lifespan extension and resistance to different types of stress. Through genetic and gene expression studies we have demonstrated that HIF-1, a master transcriptional regulator of adaptation to hypoxia, plays a central role in orchestrating the anti-aging response induced by MAA-1 deficiency. This response relies on the activation of molecular chaperones known to contribute to maintenance of the proteome. Our work extends to C. elegans the role of ACBP in aging, implicates HIF-1 in the increase of lifespan of maa-1-deficient worms, and sheds light on the anti-aging function of HIF-1. Given that both ACBP and HIF-1 are highly conserved, our results suggest the possible involvement of these proteins in the age-associated decline in proteostasis in mammals.

The Role of Dafachronic Acid Signaling in Development and Longevity in Caenorhabditis elegans: Digging Deeper Using Cutting-Edge Analytical Chemistry.

Steroid hormones regulate physiological processes in species ranging from plants to humans. A wide range of steroid hormones exist, and their contributions to processes, such as growth, reproduction, development, and aging, is almost always complex. Understanding the biosynthetic pathways that generate steroid hormones and the signaling pathways that mediate their effects is thus of fundamental importance. In this work, we review recent advances in (i) the biological role of steroid hormones in the roundworm Caenorhabditis elegans and (ii) the development of novel methods to facilitate the detection and identification of these molecules. Our current understanding of steroid signaling in this simple organism serves to illustrate the challenges we face moving forward. First, it seems clear that we have not yet identified all of the enzymes responsible for steroid biosynthesis and/or degradation. Second, perturbation of steroid signaling affects a wide range of phenotypes, and subtly different steroid molecules can have distinct effects. Finally, steroid hormone levels are critically important, and minute variations in quantity can profoundly impact a phenotype. Thus, it is imperative that we develop innovative analytical tools and combine them with cutting-edge approaches including comprehensive and highly selective liquid chromatography coupled to mass spectrometry based on new methods such as supercritical fluid chromatography coupled to mass spectrometry (SFC-MS) if we are to obtain a better understanding of the biological functions of steroid signaling.

The mysterious relationship between reproduction and longevity.

A negative correlation between fertility and longevity has been documented in many species under a variety of conditions, but the association is not always observed,(1) leading to heated discussion about the nature of the reproduction-longevity relationship.(2) This debate is further fueled by the fact that no genes or molecules have been clearly shown to link the 2 traits. A recent study by Thondamal et al., in the nematode C. elegans has identified one potential link. The authors showed that the steroid signaling pathway, which regulates reproduction, is activated in response to dietary restriction (DR) and is in fact required for DR-induced lifespan extension.(3) Steroid signaling mutants subjected to DR not only failed to undergo lifespan extension but also exhibited altered germline plasticity. Interestingly, the requirement for steroid signaling was bypassed when germline plasticity was restored, suggesting that the DR response is mediated, at least in part, by signals from the germline. In this commentary, I discuss the implications of these findings. Several theories of aging have proposed the existence of an energetic trade-off between reproduction and lifespan,(4,5) but mechanistic details are lacking. I propose that revisiting and dissecting at the molecular level the link between reproduction, nutrition, and lifespan, will lead to a better understanding of the aging process and its connection to reproduction.

Fast separation and quantification of steroid hormones Δ4- and Δ7-dafachronic acid in Caenorhabditis elegans.

Separation of isomeric molecular species, e.g. double bond position isomers, is a challenging task for liquid chromatography. The two steroid hormones Δ4- and Δ7-dafachronic acid (DA) represent such an isomeric pair. DAs are 3-ketosteroids found in the nematode Caenorhabditis elegans and generated from cholesterol. Δ4- and Δ7-DA have important biological activities and are produced by two different biological pathways in C. elegans. Here we have described a fast separation method for these two isomers using a 1.3 µm core-shell particle in less than 10 min together with a simple MeOH extraction. Using this method we were able to independently quantify Δ4- and Δ7-DA in C. elegans independently from each other and limits of detection of about 5 ng/ml for each isomer were achieved with a good day-to-day reproducibility. As proof-of-principle the method has been applied to the quantification of DAs in worms fed ad libitum or under bacterial deprivation.

Steroid hormone signalling links reproduction to lifespan in dietary-restricted Caenorhabditis elegans.

Dietary restriction (DR) increases healthspan and longevity in many species, including primates, but it is often accompanied by impaired reproductive function. Whether signals associated with the reproductive system contribute to or are required for DR effects on lifespan has not been established. Here we show that expression of the cytochrome P450 DAF-9/CYP450 and production of the steroid hormone Δ(7)-dafachronic acid (DA) are increased in C. elegans subjected to DR. DA signalling through the non-canonical nuclear hormone receptor NHR-8/NHR and the nutrient-responsive kinase let-363/mTOR is essential for DR-mediated longevity. Steroid signalling also affects germline plasticity in response to nutrient deprivation and this is required to achieve lifespan extension. These data demonstrate that steroid signalling links germline physiology to lifespan when nutrients are limited, and establish a central role for let-363/mTOR in integrating signals derived from nutrients and steroid hormones.

Reproduction, fat metabolism, and life span: what is the connection?

Reduced reproduction is associated with increased fat storage and prolonged life span in multiple organisms, but the underlying regulatory mechanisms remain poorly understood. Recent studies in several species provide evidence that reproduction, fat metabolism, and longevity are directly coupled. For instance, germline removal in the nematode Caenorhabditis elegans promotes longevity in part by modulating lipid metabolism through effects on fatty acid desaturation, lipolysis, and autophagy. Here, we review these recent studies and discuss the mechanisms by which reproduction modulates fat metabolism and life span. Elucidating the relationship between these processes could contribute to our understanding of age-related diseases including metabolic disorders.

Aging yeast cells undergo a sharp entry into senescence unrelated to the loss of mitochondrial membrane potential.

In budding yeast, a mother cell can produce a finite number of daughter cells before it stops dividing and dies. Such entry into senescence is thought to result from a progressive decline in physiological function, including a loss of mitochondrial membrane potential (ΔΨ). Here, we developed a microfluidic device to monitor the dynamics of cell division and ΔΨ in real time at single-cell resolution. We show that cells do not enter senescence gradually but rather undergo an abrupt transition to a slowly dividing state. Moreover, we demonstrate that the decline in ΔΨ, which is observed only in a fraction of cells, is not responsible for entry into senescence. Rather, the loss of ΔΨ is an age-independent and heritable process that leads to clonal senescence and is therefore incompatible with daughter cell rejuvenation. These results emphasize the importance of quantitative single-cell measurements to decipher the causes of cellular aging.

Carbonylated proteins are eliminated during reproduction in C. elegans.

Oxidatively damaged proteins accumulate with age in many species (Stadtman (1992) Science257, 1220-1224). This means that damage must be reset at the time of reproduction. To visualize this resetting in the roundworm Caenorhabditis elegans, a novel immunofluorescence technique that allows the detection of carbonylated proteins in situ was developed. The application of this technique revealed that carbonylated proteins are eliminated during C. elegans reproduction. This purging occurs abruptly within the germline at the time of oocyte maturation. Surprisingly, the germline was markedly more oxidized than the surrounding somatic tissues. Because distinct mechanisms have been proposed to explain damage elimination in yeast and mice (Aguilaniu et al. (2003) Science299, 1751-1753; Hernebring et al. (2006) Proc Natl Acad Sci USA103, 7700-7705), possible common mechanisms between worms and one of these systems were tested. The results show that, unlike in yeast (Aguilaniu et al. (2003) Science299, 1751-1753; Erjavec et al. (2008) Proc Natl Acad Sci USA105, 18764-18769), the elimination of carbonylated proteins in worms does not require the presence of the longevity-ensuring gene, SIR-2.1. However, similar to findings in mice (Hernebring et al. (2006) Proc Natl Acad Sci USA103, 7700-7705), proteasome activity in the germline is required for the resetting of carbonylated proteins during reproduction in C. elegans. Thus, oxidatively damaged proteins are eliminated during reproduction in worms through the proteasome. This finding suggests that the resetting of damaged proteins during reproduction is conserved, therefore validating the use of C. elegans as a model to study the molecular basis of damage elimination.

Elimination of damaged proteins during differentiation of embryonic stem cells.

During mammalian aging, cellular proteins become increasingly damaged: for example, by carbonylation and formation of advanced glycation end products (AGEs). The means to ensure that offspring are born without such damage are unknown. Unexpectedly, we found that undifferentiated mouse ES cells contain high levels of both carbonyls and AGEs. The damaged proteins, identified as chaperones and proteins of the cytoskeleton, are the main targets for protein oxidation in aged tissues. However, the mouse ES cells rid themselves of such damage upon differentiation in vitro. This elimination of damaged proteins coincides with a considerably elevated activity of the 20S proteasome. Moreover, damaged proteins were primarily observed in the inner cell mass of blastocysts, whereas the cells that had embarked on differentiation into the trophectoderm displayed drastically reduced levels of protein damage. Thus, the elimination of protein damage occurs also during normal embryonic development in vivo. This clear-out of damaged proteins may be a part of a previously unknown rejuvenation process at the protein level that occurs at a distinct stage during early embryonic development.

Asymmetric inheritance of oxidatively damaged proteins during cytokinesis.

Carbonylated proteins were visualized in single cells of the budding yeast Saccharomyces cerevisiae, revealing that they accumulate with replicative age. Furthermore, carbonylated proteins were not inherited by daughter cells during cytokinesis. Mother cells of a yeast strain lacking the sir2 gene, a life-span determinant, failed to retain oxidatively damaged proteins during cytokinesis. These findings suggest that a genetically determined, Sir2p-dependent asymmetric inheritance of oxidatively damaged proteins may contribute to free-radical defense and the fitness of newborn cells.