The C. elegans truncated insulin receptor DAF-2B regulates survival of L1 arrested larvae.

We have previously characterized a truncated isoform of the C. elegans insulin-like receptor, DAF-2B, which retains the ligand binding domain but cannot transduce a signal due to the absence of the intracellular signaling domain. DAF-2B modifies insulin / insulin-like growth factor signaling-dependent processes, such as dauer formation and lifespan, by sequestering insulin-like peptides (ILP) and preventing signaling through full length DAF-2 receptors. Here we show that DAF-2B is also important for starvation resistance, as genetic loss of daf-2b reduces survival in arrested first stage larvae (L1). Under fed conditions, we observe daf-2b splicing capacity in both the intestine and the hypodermis, but in starved L1s this becomes predominantly hypodermal. Using a novel splicing reporter system, we observe an increase in the ratio of truncated to full length insulin receptor splicing capacity in starved L1 larvae compared with fed, that may indicate a decrease in whole body insulin responsiveness. Consistent with this, overexpression of DAF-2B from the hypodermis, but not the intestine, confers increased survival to L1 animals under starvation conditions. Our findings demonstrate that the truncated insulin receptor DAF-2B is involved in the response to L1 starvation and promotes survival when expressed from the hypodermis.

The SR protein RSP-2 influences expression of the truncated insulin receptor DAF-2B in Caenorhabditis elegans.

The alternatively spliced daf-2b transcript in Caenorhabditis elegans encodes a truncated isoform of the nematode insulin receptor that retains the extracellular ligand binding domain but lacks the intracellular signaling domain and is therefore unable to transduce a signal. To identify factors that influence expression of daf-2b, we performed a targeted RNA interference screen of rsp genes, which encode splicing factors from the serine/arginine protein family. Loss of rsp-2 significantly increased the expression of a fluorescent daf-2b splicing reporter, as well as increasing expression of endogenous daf-2b transcripts. Correspondingly, rsp-2 mutants exhibited similar phenotypes to those previously observed with DAF-2B overexpression, namely suppression of pheromone-induced dauer formation, enhancement of dauer entry in insulin signaling mutants, inhibition of dauer recovery, and increased lifespan. However, the epistatic relationship between rsp-2 and daf-2b varied according to the experimental context. Increased dauer entry and delayed dauer exit of rsp-2 mutants in an insulin signaling mutant background were partially dependent on daf-2b. Conversely, suppression of pheromone-induced dauer formation and increased lifespan in rsp-2 mutants were independent of daf-2b. These data demonstrate that C. elegans RSP-2, an ortholog of human splicing factor protein SRSF5/SRp40, is involved in regulating the expression of the truncated DAF-2B isoform. However, we also find that RSP-2 can influence dauer formation and lifespan independently of DAF-2B.

An alternatively spliced, non-signaling insulin receptor modulates insulin sensitivity via insulin peptide sequestration in C. elegans.

In the nematode C. elegans, insulin signaling regulates development and aging in response to the secretion of numerous insulin peptides. Here, we describe a novel, non-signaling isoform of the nematode insulin receptor (IR), DAF-2B, that modulates insulin signaling by sequestration of insulin peptides. DAF-2B arises via alternative splicing and retains the extracellular ligand binding domain but lacks the intracellular signaling domain. A daf-2b splicing reporter revealed active regulation of this transcript through development, particularly in the dauer larva, a diapause stage associated with longevity. CRISPR knock-in of mScarlet into the daf-2b genomic locus confirmed that DAF-2B is expressed in vivo and is likely secreted. Genetic studies indicate that DAF-2B influences dauer entry, dauer recovery and adult lifespan by altering insulin sensitivity according to the prevailing insulin milieu. Thus, in C. elegans alternative splicing at the daf-2 locus generates a truncated IR that fine-tunes insulin signaling in response to the environment.

Genetic behavioral screen identifies an orphan anti-opioid system.

Opioids target the µ-opioid receptor (MOR) to produce unrivaled pain management, but their addictive properties can lead to severe abuse. We developed a whole-animal behavioral platform for unbiased discovery of genes influencing opioid responsiveness. Using forward genetics in Caenorhabditis elegans, we identified a conserved orphan receptor, GPR139, with anti-opioid activity. GPR139 is coexpressed with MOR in opioid-sensitive brain circuits, binds to MOR, and inhibits signaling to heterotrimeric guanine nucleotide-binding proteins (G proteins). Deletion of GPR139 in mice enhanced opioid-induced inhibition of neuronal firing to modulate morphine-induced analgesia, reward, and withdrawal. Thus, GPR139 could be a useful target for increasing opioid safety. These results also demonstrate the potential of C. elegans as a scalable platform for genetic discovery of G protein-coupled receptor signaling principles.

Dysregulation of DAF-16/FOXO3A-mediated stress responses accelerates oxidative DNA damage induced aging.

DNA damage is presumed to be one type of stochastic macromolecular damage that contributes to aging, yet little is known about the precise mechanism by which DNA damage drives aging. Here, we attempt to address this gap in knowledge using DNA repair-deficient C. elegans and mice. ERCC1-XPF is a nuclear endonuclease required for genomic stability and loss of ERCC1 in humans and mice accelerates the incidence of age-related pathologies. Like mice, ercc-1 worms are UV sensitive, shorter lived, display premature functional decline and they accumulate spontaneous oxidative DNA lesions (cyclopurines) more rapidly than wild-type worms. We found that ercc-1 worms displayed early activation of DAF-16 relative to wild-type worms, which conferred resistance to multiple stressors and was important for maximal longevity of the mutant worms. However, DAF-16 activity was not maintained over the lifespan of ercc-1 animals and this decline in DAF-16 activation corresponded with a loss of stress resistance, a rise in oxidant levels and increased morbidity, all of which were cep-1/ p53 dependent. A similar early activation of FOXO3A (the mammalian homolog of DAF-16), with increased resistance to oxidative stress, followed by a decline in FOXO3A activity and an increase in oxidant abundance was observed in Ercc1-/- primary mouse embryonic fibroblasts. Likewise, in vivo, ERCC1-deficient mice had transient activation of FOXO3A in early adulthood as did middle-aged wild-type mice, followed by a late life decline. The healthspan and mean lifespan of ERCC1 deficient mice was rescued by inactivation of p53. These data indicate that activation of DAF-16/FOXO3A is a highly conserved response to genotoxic stress that is important for suppressing consequent oxidative stress. Correspondingly, dysregulation of DAF-16/FOXO3A appears to underpin shortened healthspan and lifespan, rather than the increased DNA damage burden itself.

A Simple Method for High Throughput Chemical Screening in Caenorhabditis Elegans.

Caenorhabditis elegans is a useful organism for testing chemical effects on physiology. Whole organism small molecule screens offer significant advantages for identifying biologically active chemical structures that can modify complex phenotypes such as lifespan. Described here is a simple protocol for producing hundreds of 96-well culture plates with fairly consistent numbers of C. elegans in each well. Next, we specified how to use these cultures to screen thousands of chemicals for effects on the lifespan of the nematode C. elegans. This protocol makes use of temperature sensitive sterile strains, agar plate conditions, and simple animal handling to facilitate the rapid and high throughput production of synchronized animal cultures for screening.

Barriers to the Preclinical Development of Therapeutics that Target Aging Mechanisms.

Through the progress of basic science research, fundamental mechanisms that contribute to age-related decline are being described with increasing depth and detail. Although these efforts have identified new drug targets and compounds that extend life span in model organisms, clinical trials of therapeutics that target aging processes remain scarce. Progress in aging research is hindered by barriers associated with the translation of basic science discoveries into the clinic. This report summarizes discussions held at a 2014 Geroscience Network retreat focused on identifying hurdles that currently impede the preclinical development of drugs targeting fundamental aging processes. From these discussions, it was evident that aging researchers have varied perceptions of the ideal preclinical pipeline. To forge a clear and cohesive path forward, several areas of controversy must first be resolved and new tools developed. Here, we focus on five key issues in preclinical drug development (drug discovery, lead compound development, translational preclinical biomarkers, funding, and integration between researchers and clinicians), expanding upon discussions held at the Geroscience Retreat and suggesting areas for further research. By bringing these findings to the attention of the aging research community, we hope to lay the foundation for a concerted preclinical drug development pipeline.

Synthetic Ligands of Cannabinoid Receptors Affect Dauer Formation in the Nematode Caenorhabditis elegans.

Under adverse environmental conditions the nematode Caenorhabditis elegans can enter an alternate developmental stage called the dauer larva. To identify lipophilic signaling molecules that influence this process, we screened a library of bioactive lipids and found that AM251, an antagonist of the human cannabinoid (CB) receptor, suppresses dauer entry in daf-2 insulin receptor mutants. AM251 acted synergistically with glucose supplementation indicating that the metabolic status of the animal influenced the activity of this compound. Similarly, loss of function mutations in the energy-sensing AMP-activated kinase subunit, aak-2, enhanced the dauer-suppressing effects of AM251, while constitutive activation of aak-2 in neurons was sufficient to inhibit AM251 activity. Chemical epistasis experiments indicated that AM251 acts via G-protein signaling and requires the TGF-ß ligand DAF-7, the insulin peptides DAF-28 and INS-6, and a functional ASI neuron to promote reproductive growth. AM251 also required the presence of the SER-5 serotonin receptor, but in vitro experiments suggest that this may not be via a direct interaction. Interestingly, we found that other antagonists of mammalian CB receptors also suppress dauer entry, while the nonselective CB receptor agonist, O-2545, not only inhibited the activity of AM251, but also was able to promote dauer entry when administered alone. Since worms do not have obvious orthologs of CB receptors, the effects of synthetic CBs on neuroendocrine signaling in C. elegans are likely to be mediated via another, as yet unknown, receptor mechanism. However, we cannot exclude the existence of a noncanonical CB receptor in C. elegans.

Chemical activation of a food deprivation signal extends lifespan.

Model organisms subject to dietary restriction (DR) generally live longer. Accompanying this lifespan extension are improvements in overall health, based on multiple metrics. This indicates that pharmacological treatments that mimic the effects of DR could improve health in humans. To find new chemical structures that extend lifespan, we screened 30 000 synthetic, diverse drug-like chemicals in Caenorhabditis elegans and identified several structurally related compounds that acted through DR mechanisms. The most potent of these NP1 impinges upon a food perception pathway by promoting glutamate signaling in the pharynx. This results in the overriding of a GPCR pathway involved in the perception of food and which normally acts to decrease glutamate signals. Our results describe the activation of a dietary restriction response through the pharmacological masking of a novel sensory pathway that signals the presence of food. This suggests that primary sensory pathways may represent novel targets for human pharmacology.

Characterization of N-acyl phosphatidylethanolamine-specific phospholipase-D isoforms in the nematode Caenorhabditis elegans.

N-acylethanolamines are an important class of lipid signaling molecules found in many species, including the nematode Caenorhabditis elegans (C. elegans) where they are involved in development and adult lifespan. In mammals, the relative activity of the biosynthetic enzyme N-acyl phosphatidylethanolamine-specific phospholipase-D and the hydrolytic enzyme fatty acid amide hydrolase determine N-acylethanolamine levels. C. elegans has two N-acyl phosphatidylethanolamine-specific phospholipase-D orthologs, nape-1 and nape-2, that are likely to have arisen from a gene duplication event. Here, we find that recombinant C. elegans NAPE-1 and NAPE-2 are capable of generating N-acylethanolamines in vitro, confirming their functional conservation. In vivo, they exhibit overlapping expression in the pharynx and the nervous system, but are also expressed discretely in these and other tissues, suggesting divergent roles. Indeed, nape-1 over-expression results in delayed growth and shortened lifespan only at 25°C, while nape-2 over-expression results in significant larval arrest and increased adult lifespan at 15°C. Interestingly, deletion of the N-acylethanolamine degradation enzyme faah-1 exacerbates nape-1 over-expression phenotypes, but suppresses the larval arrest phenotype of nape-2 over-expression, suggesting that faah-1 is coupled to nape-2, but not nape-1, in a negative feedback loop. We also find that over-expression of either nape-1 or nape-2 significantly enhances recovery from the dauer larval stage in the insulin signaling mutant daf-2(e1368), but only nape-1 over-expression reduces daf-2 adult lifespan, consistent with increased levels of the N-acylethanolamine eicosapentaenoyl ethanolamine. These results provide evidence that N-acylethanolamine biosynthetic enzymes in C. elegans have conserved function and suggest a temperature-dependent, functional divergence between the two isoforms.

Bacterial fatty acids enhance recovery from the dauer larva in Caenorhabditis elegans.

The dauer larva is a specialized dispersal stage in the nematode Caenorhabditis elegans that allows the animal to survive starvation for an extended period of time. The dauer does not feed, but uses chemosensation to identify new food sources and to determine whether to resume reproductive growth. Bacteria produce food signals that promote recovery of the dauer larva, but the chemical identities of these signals remain poorly defined. We find that bacterial fatty acids in the environment augment recovery from the dauer stage under permissive conditions. The effect of increased fatty acids on different dauer constitutive mutants indicates a role for insulin peptide secretion in coordinating recovery from the dauer stage in response to fatty acids. These data suggest that worms can sense the presence of fatty acids in the environment and that elevated levels can promote recovery from dauer arrest. This may be important in the natural environment where the dauer larva needs to determine whether the environment is appropriate to support reproductive growth following dauer exit.

Intramyocellular fatty-acid metabolism plays a critical role in mediating responses to dietary restriction in Drosophila melanogaster.

Changes in fat content have been associated with dietary restriction (DR), but whether they play a causal role in mediating various responses to DR remains unknown. We demonstrate that upon DR, Drosophila melanogaster shift their metabolism toward increasing fatty-acid synthesis and breakdown, which is required for various responses to DR. Inhibition of fatty-acid synthesis or oxidation genes specifically in the muscle tissue inhibited life-span extension upon DR. Furthermore, DR enhances spontaneous activity of flies, which was found to be dependent on the enhanced fatty-acid metabolism. This increase in activity was found to be at least partially required for the life-span extension upon DR. Overexpression of adipokinetic hormone (dAKH), the functional ortholog of glucagon, enhances fat metabolism, spontaneous activity, and life span. Together, these results suggest that enhanced fat metabolism in the muscle and physical activity play a key role in the protective effects of DR.

N-acylethanolamine signalling mediates the effect of diet on lifespan in Caenorhabditis elegans.

Dietary restriction is a robust means of extending adult lifespan and postponing age-related disease in many species, including yeast, nematode worms, flies and rodents. Studies of the genetic requirements for lifespan extension by dietary restriction in the nematode Caenorhabditis elegans have implicated a number of key molecules in this process, including the nutrient-sensing target of rapamycin (TOR) pathway and the Foxa transcription factor PHA-4 (ref. 7). However, little is known about the metabolic signals that coordinate the organismal response to dietary restriction and maintain homeostasis when nutrients are limited. The endocannabinoid system is an excellent candidate for such a role given its involvement in regulating nutrient intake and energy balance. Despite this, a direct role for endocannabinoid signalling in dietary restriction or lifespan determination has yet to be demonstrated, in part due to the apparent absence of endocannabinoid signalling pathways in model organisms that are amenable to lifespan analysis. N-acylethanolamines (NAEs) are lipid-derived signalling molecules, which include the mammalian endocannabinoid arachidonoyl ethanolamide. Here we identify NAEs in C. elegans, show that NAE abundance is reduced under dietary restriction and that NAE deficiency is sufficient to extend lifespan through a dietary restriction mechanism requiring PHA-4. Conversely, dietary supplementation with the nematode NAE eicosapentaenoyl ethanolamide not only inhibits dietary-restriction-induced lifespan extension in wild-type worms, but also suppresses lifespan extension in a TOR pathway mutant. This demonstrates a role for NAE signalling in ageing and indicates that NAEs represent a signal that coordinates nutrient status with metabolic changes that ultimately determine lifespan.

Compounds that confer thermal stress resistance and extended lifespan.

The observation that long-lived and relatively healthy animals can be obtained by simple genetic manipulation prompts the search for chemical compounds that have similar effects. Since aging is the most important risk factor for many socially and economically important diseases, the discovery of a wide range of chemical modulators of aging in model organisms could prompt new strategies for attacking age-related disease such as diabetes, cancer and neurodegenerative disorders [Collins, J.J., Evason, K., Kornfeld, K., 2006. Pharmacology of delayed aging and extended lifespan of Caenorhabditis elegans. Exp. Gerontol.; Floyd, R.A., 2006. Nitrones as therapeutics in age-related diseases. Aging Cell 5, 51-57; Gill, M.S., 2006. Endocrine targets for pharmacological intervention in aging in Caenorhabditis elegans. Aging Cell 5, 23-30; Hefti, F.F., Bales, R., 2006. Regulatory issues in aging pharmacology. Aging Cell 5, 3-8]. Resistance to multiple types of stress is a common trait in long-lived genetic variants of a number of species; therefore, we have tested compounds that act as stress response mimetics. We have focused on compounds with antioxidant properties and identified those that confer thermal stress resistance in the nematode Caenorhabditis elegans. Some of these compounds (lipoic acid, propyl gallate, trolox and taxifolin) also extend the normal lifespan of this simple invertebrate, consistent with the general model that enhanced stress resistance slows aging.

DAF-12-dependent rescue of dauer formation in Caenorhabditis elegans by (25S)-cholestenoic acid.

Population density, temperature and food availability all regulate the formation of the Caenorhabditis elegans dauer larva by modulating endocrine signaling pathways. The orphan nuclear receptor DAF-12 is pivotal for the decision to form a dauer or to undergo normal reproductive development. The DAF-12 ligand has been predicted to be a sterol that is metabolized by DAF-9, a cytochrome P450. Here we chemically characterize purified lipophilic nematode extracts and show that the ligand for DAF-12 contains a carboxyl moiety and is likely to be derived from a sterol. Using a candidate ligand approach we find that the C27 bile acid cholestenoic acid (5-cholesten-3beta-ol-(25S)-carboxylic acid) promotes reproductive growth in dauer-constitutive mutants in a daf-9- and daf-12-dependent manner. Furthermore, we find that cholestenoic acid can act as a DAF-12 ligand by activating DAF-12 in a cell-based transcription assay. Analysis of dauer-rescuing lipophilic extracts from nematodes by gas chromatography-mass spectrometry indicates the presence of several regioisomers of cholestenoic acid that are distinct from Delta(5)-cholestenoic acid and are not present in extracts from daf-9 mutants. These data suggest that carboxylated sterols may be key determinants of life history.

Endocrine targets for pharmacological intervention in aging in Caenorhabditis elegans.

Studies in the nematode Caenorhabditis elegans have been instrumental in defining genetic pathways that are involved in modulating lifespan. Multiple processes such as endocrine signaling, nutritional sensing and mitochondrial function play a role in determining lifespan in the worm and these mechanisms appear to be conserved across species. These discoveries have identified a range of novel targets for pharmacological manipulation of lifespan and it is likely that the nematode model will now prove useful in the discovery of compounds that slow aging. This review will focus on the endocrine targets for intervention in aging and the use of C. elegans as a system for high throughput screens of compounds for their effects on aging.

Lipophilic regulator of a developmental switch in Caenorhabditis elegans.

Abstract In Caenorhabditis elegans, the decision to develop into a reproductive adult or arrest as a dauer larva is influenced by multiple pathways including insulin-like and transforming growth factor beta (TGFbeta)-like signalling pathways. It has been proposed that lipophilic hormones act downstream of these pathways to regulate dauer formation. One likely target for such a hormone is DAF-12, an orphan nuclear hormone receptor that mediates these developmental decisions and also influences adult lifespan. In order to find lipophilic hormones we have generated lipophilic extracts from mass cultures of C. elegans and shown that they rescue the dauer constitutive phenotype of class 1 daf-2 insulin signalling mutants and the TGFbeta signalling mutant daf-7. These extracts are also able to rescue the lethal dauer phenotype of daf-9 mutants, which lack a P450 steroid hydroxylase thought to be involved in the synthesis of the DAF-12 ligand; extracts, however, have no effect on a DAF-12 ligand binding domain mutant that is predicted to be ligand insensitive. The production of this hormone appears to be DAF-9 dependent as extracts from a daf-9;daf-12 double mutant do not exhibit this activity. Preliminary fractionation of the lipophilic extracts shows that the activity is hydrophobic with some polar properties, consistent with a small lipophilic hormone. We propose that the dauer rescuing activity is a hormone synthesized by DAF-9 that acts through DAF-12.

Hormônio do crescimento ou somatotrófico: novas perspectivas na deficiência isolada de GH a partir da descrição da mutação no gene do receptor do GHRH nos indivíduos da cidade de Itabaianinha, Brasil / Growth or somatotrophic hormone: new perspectives in isolated GH deficiency after description of the mutation in the GHRH receptor gene in individuals of Itabaianinha county, Brazil

Além de influenciar o crescimento corpóreo, o hormônio do crescimento, ou somatotrófico, desempenha importante papel no metabolismo, composição corporal, perfil lipídico, estado cardiovascular e longevidade. Seu controle é multi-regulado por hormônios, metabólitos e peptídeos hipotalâmicos. Dados sobre a Deficiência Isolada de GH (DIGH) obtidos a partir da descrição da mutação IVS1+1G®A no gene do receptor do hormônio liberador do GH (GHRH-R) em indivíduos da cidade de Itabaianinha, SE, são revisados. São abordadas novas perspectivas sobre o modelo de resistência ao GHRH, a importância do GHRH no controle da secreção de GH, a freqüência das mutações do gene do GHRH-R, a relevância diagnóstica do IGF-I e os achados metabólicos, cardiovasculares e de qualidade de vida nestes indivíduos.

[Growth or somatotrophic hormone: new perspectives in isolated GH deficiency after description of the mutation in the GHRH receptor gene in individuals of Itabaianinha County, Brazil]. / Hormônio do crescimento ou somatotrófico: novas perspectivas na deficiência isolada de GH a partir da descrição da mutação no gene do receptor do GHRH nos indivíduos da cidade de Itabaianinha, Brasil.

In addition to stimulating body growth, growth or somatotrophic hormone plays an important role in metabolism, body composition, lipid profile, cardiovascular status and longevity. Its control is multiregulated by hormones, metabolites and hypothalamic peptides. Obtained data of the isolated growth hormone deficiency (IGHD) after the description of the IVS1+1G-->A GHRH receptor gene mutation in individuals of Itabaianinha County are reviewed. New perspectives about the growth hormone resistance model, the importance of GHRH in the control of GH secretion, the frequency of GHRH-R gene mutations, the diagnostic relevance of IGF-I and the metabolic, cardiovascular and quality of life findings are approached.