Publications from researchers at the Buck Institute for Research on Aging http://www.buckinstitute.org/lithgowLab © 2011 Buck Institute, All Rights Reserved Over the last ten years, various screens of small molecules have been conducted to find longsought interventions in aging. Most of these studies were performed in invertebrates but the demonstration of pharmacological lifespan extension in the mouse has created considerable excitement. Since aging is a common risk factor for several chronic diseases, there is a reasonable expectation that some compounds capable of extending lifespan will be useful for preventing a range of agerelated diseases. One of the potential targets is protein aggregation which is associated with several agerelated diseases. Genetic studies have long indicated that protein homeostasis is a critical component of longevity but recently a series of chemicals have been identified in the nematode Caenorhabditis elegans that lead to the maintenance of the homeostatic network and extend lifespan. Here we review these interventions in C. elegans and consider the potential of improving health by enhancing protein homeostasis. 2012 The Authors Aging Cell 2012 Blackwell Publishing Ltd/Anatomical Society of Great Britain and Ireland. http://www.ncbi.nlm.nih.gov/pubmed/22226190 Sat, 31 Dec 2011 00:00:00 -0800 Microglial activation and subsequent release of toxic proinflammatory factors are believed to play an important role in neuronal cell death associated with Parkinson's disease (PD). Compounds that inhibit microglia activation and suppress proinflammatory factor release have been reported to have neuroprotective effects in animal models of PD. In this study, we tested whether diadzein, a natural isoflavone found in soybean, attenuated lipopolysaccharide (LPS)induced release of inflammatory mediators in BV2, a murine microglial cell line. Diadzein pretreatment was found to significantly suppress the production of the proinflammatory factors nitric oxide and IL6 as well as their mRNA expression in conjunction with reductions in ROS production, p38 MAPK phosphorylation, and NFB activation. Furthermore, transfer of conditioned media (CM) from BV2 cells pretreated with diadzein resulted in a significantly reduction in dopaminergic neurotoxicity compared with CM from microglia stimulated with LPS alone. Together, our results suggest that diadzein's neuroprotective properties may be due to its ability to dampen induction of microglial activation and the subsequent release of soluble proinflammatory factors. This appears to be via inhibition of oxidative induction of the p38 MAP kinaseNFB pathway, resulting in reduced expression of proinflammatory genes and release of their corresponding gene products. http://www.ncbi.nlm.nih.gov/pubmed/22573480 Sat, 31 Dec 2011 00:00:00 -0800 Aluminium (Al) is highly abundant in the environment and can elicit a variety of toxic responses in biological systems. Here we characterize the effects of Al on Caenorhabditis elegans by identifying phenotypic abnormalities and disruption in wholebody metal homeostasis (metallostasis) following Al exposure in food. Widespread changes to the elemental content of adult nematodes were observed when chronically exposed to Al from the first larval stage (L1). Specifically, we saw increased barium, chromium, copper and iron content, and a reduction in calcium levels. Lifespan was decreased in worms exposed to low levels of Al, but unexpectedly increased when the Al concentration reached higher levels (4.8 mM). This biphasic phenotype was only observed when Al exposure occurred during development, as lifespan was unaffected by Al exposure during adulthood. Lower levels of Al slowed C. elegans developmental progression, and reduced hermaphrodite selffertility and adult body size. Significant developmental delay was observed even when Al exposure was restricted to embryogenesis. Similar changes in Al have been noted in association with Al toxicity in humans and other mammals, suggesting that C. elegans may be of use as a model for understanding the mechanisms of Al toxicity in mammalian systems. http://www.ncbi.nlm.nih.gov/pubmed/22534883 Sat, 31 Dec 2011 00:00:00 -0800 Genetic studies indicate that protein homeostasis is a major contributor to metazoan longevity. Collapse of protein homeostasis results in protein misfolding cascades and the accumulation of insoluble protein fibrils and aggregates, such as amyloids. A group of small molecules, traditionally used in histopathology to stain amyloid in tissues, bind protein fibrils and slow aggregation in vitro and in cell culture. We proposed that treating animals with such compounds would promote protein homeostasis in vivo and increase longevity. Here we show that exposure of adult Caenorhabditis elegans to the amyloidbinding dye Thioflavin T (ThT) resulted in a profoundly extended lifespan and slowed ageing. ThT also suppressed pathological features of mutant metastable proteins and human amyloidassociated toxicity. These beneficial effects of ThT depend on the protein homeostasis network regulator heat shock factor 1 (HSF1), the stress resistance and longevity transcription factor SKN1, molecular chaperones, autophagy and proteosomal functions. Our results demonstrate that pharmacological maintenance of the protein homeostatic network has a profound impact on ageing rates, prompting the development of novel therapeutic interventions against ageing and agerelated diseases. http://www.ncbi.nlm.nih.gov/pubmed/21451522 Fri, 31 Dec 2010 00:00:00 -0800 http://www.ncbi.nlm.nih.gov/pubmed/21512207 Fri, 31 Dec 2010 00:00:00 -0800 Dietary restriction is a robust means of extending adult lifespan and postponing agerelated 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 nutrientsensing target of rapamycin (TOR) pathway and the Foxa transcription factor PHA4 (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. Nacylethanolamines (NAEs) are lipidderived 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 PHA4. Conversely, dietary supplementation with the nematode NAE eicosapentaenoyl ethanolamide not only inhibits dietaryrestrictioninduced lifespan extension in wildtype 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. http://www.ncbi.nlm.nih.gov/pubmed/21562563 Fri, 31 Dec 2010 00:00:00 -0800 Reducing protein synthesis slows growth and development but can increase adult life span. We demonstrate that knockdown of eukaryotic translation initiation factor 4G (eIF4G), which is downregulated during starvation and dauer state, results in differential translation of genes important for growth and longevity in C.elegans. Genomewide mRNA translation state analysis showed that inhibition of IFG1, the C.elegans ortholog of eIF4G, results in a relative increase in ribosomal loading and translation of stress response genes. Some of these genes are required for life span extension when IFG1 is inhibited. Furthermore, enhanced ribosomal loading of certain mRNAs upon IFG1 inhibition was correlated with increased mRNA length. This association was supported by changes in the proteome assayed via quantitative mass spectrometry. Our results suggest that IFG1 mediates the antagonistic effects on growth and somatic maintenance by regulating mRNA translation of particular mRNAs based, in part, on transcript length. http://www.ncbi.nlm.nih.gov/pubmed/21723504 Fri, 31 Dec 2010 00:00:00 -0800 While it is generally recognized that misfolding of specific proteins can cause lateonset disease, the contribution of protein aggregation to the normal aging process is less well understood. To address this issue, a mass spectrometrybased proteomic analysis was performed to identify proteins that adopt sodium dodecyl sulfate (SDS)insoluble conformations during aging in C. elegans. SDS Insoluble proteins extracted from young and aged C. elegans were chemically labelled by isobaric tagging for relative and absolute quantification (iTRAQ) and identified by liquid chromatography and mass spectrometry. Two hundred and three proteins were identified as being significantly enriched in an SDSinsoluble fraction in aged nematodes and were largely absent from a similar protein fraction in young nematodes. The SDSinsoluble fraction in aged animals contains a diverse range of proteins including a large number of ribosomal proteins. Gene Ontology analysis revealed highly significant enrichments for energy production and translation functions. Expression of genes encoding insoluble proteins observed in aged nematodes was knockeddown using RNAi and effects on lifespan were measured. 41 of genes tested were shown to extend lifespan after RNAi treatment, compared to 18 in a control group of genes. These data indicate that genes encoding proteins that become insoluble with age are enriched for modifiers of lifespan. This demonstrates that proteomic approaches can be used to identify genes that modify lifespan. Finally, these observations indicate that accumulation of insoluble proteins with diverse functions may be a general feature of aging. http://www.ncbi.nlm.nih.gov/pubmed/22103665 Fri, 31 Dec 2010 00:00:00 -0800 The insulinlike signaling (ILS) pathway regulates metabolism and is known to modulate adult life span in C. elegans. Altered stress responses and resistance to a wide range of stressors are also associated with changes in ILS and contribute to enhanced longevity. The transcription factors DAF16 and HSF1 are key effectors of the longevity phenotype. We demonstrate that increased intrinsic thermotolerance, due to lower ILS, is not dependent on stressinduced transcriptional responses but instead requires active protein translation. Translation profiling experiments reveal genes that are posttranscriptionally regulated in response to altered ILS during heat shock in a DAF16dependent manner. Furthermore, several novel proteins are specifically required for ILS effects on thermotolerance. We propose that lowered ILS results in metabolic and physiological changes. These DAF16induced changes precondition a translational response under acute stress to modulate survival. http://www.ncbi.nlm.nih.gov/pubmed/20816092 Tue, 31 Aug 2010 00:00:00 -0700 Senescence is a cellular program that irreversibly arrests the proliferation of damaged cells and induces the secretion of the inflammatory mediators IL 6 and IL8 which are part of a larger senescence associated secretory phenotype (SASP). We screened quiescent and senescent human fibroblasts for differentially expressed microRNAS (miRNAs) and found that miRNAs 146a and 146b (miR146a/b) were significantly elevated during senescence. We suggest that delayed miR146a/b induction might be a compensatory response to restrain inflammation. Indeed, ectopic expression of miR146a/b in primary human fibroblasts suppressed IL6 and IL8 secretion and downregulated IRAK1, a crucial component of the IL1 receptor signal transduction pathway. Cells undergoing senescence without induction of a robust SASP did not express miR146a/b. Further, IL1alpha neutralizing antibodies abolished both miR146a/b expression and IL6 secretion. Our findings expand the biological contexts in which miRNA146a/b modulates inflammatory responses. They suggest that IL1 receptor signaling initiates both miR146a/b upregulation and cytokine secretion, and that miR146a/b is expressed in response to rising inflammatory cytokine levels as part of a negative feedback loop that restrains excessive SASP activity. http://www.ncbi.nlm.nih.gov/pubmed/20148189 Sun, 28 Feb 2010 00:00:00 -0800 The observation that longlived 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 agerelated 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 agerelated diseases. Aging Cell 5, 5157 Gill, M.S., 2006. Endocrine targets for pharmacological intervention in aging in Caenorhabditis elegans. Aging Cell 5, 2330 Hefti, F.F., Bales, R., 2006. Regulatory issues in aging pharmacology. Aging Cell 5, 38. Resistance to multiple types of stress is a common trait in longlived 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. http://www.ncbi.nlm.nih.gov/pubmed/18755260 Tue, 30 Sep 2008 00:00:00 -0700 In eukaryotes and many bacteria, tyrosine is degraded to produce energy via a fivestep tyrosine degradation pathway. Mutations affecting the tyrosine degradation pathway are also of medical importance as mutations affecting enzymes in the pathway are responsible for type I, type II, and type III tyrosinemia. The most severe of these is type I tyrosinemia, which is caused by mutations affecting the last enzyme in the pathway, fumarylacetoacetate hydrolase (FAH). So far, tyrosine degradation in the nematode Caenorhabditis elegans has not been studied however, genes predicted to encode enzymes in this pathway have been identified in several microarray, proteomic, and RNA interference (RNAi) screens as perhaps being involved in aging and the control of protein folding. We sought to identify and characterize the genes in the worm tyrosine degradation pathway as an initial step in understanding these findings. Here we describe the characterization of the K10C2.4, which encodes a homolog of FAH. RNAi directed against K10C2.4 produces a lethal phenotype consisting of death in young adulthood, extensive damage to the intestine, impaired fertility, and activation of oxidative stress and endoplasmic stress response pathways. This phenotype is due to alterations in tyrosine metabolism as increases in dietary tyrosine enhance it, and inhibition of upstream enzymes in tyrosine degradation with RNAi or genetic mutations reduces the phenotype. We also use our model to identify genes that suppress the damage produced by K10C2.4 RNAi in a pilot genetic screen. Our results establish worms as a model for the study of type I tyrosinemia. http://www.ncbi.nlm.nih.gov/pubmed/18227072 Fri, 29 Feb 2008 00:00:00 -0800 Lithium (Li()) has been used to treat mood affect disorders, including bipolar, for decades. This drug is neuroprotective and has several identified molecular targets. However, it has a narrow therapeutic range and the one or more underlying mechanisms of its therapeutic action are not understood. Here we describe a pharmacogenetic study of Li() in the nematode Caenorhabditis elegans. Exposure to Li() at clinically relevant concentrations throughout adulthood increases survival during normal aging (up to 46 median increase). Longevity is extended via a novel mechanism with altered expression of genes encoding nucleosomeassociated functions. Li() treatment results in reduced expression of the worm ortholog of LSD1 (T08D10.2), a histone demethylase knockdown by RNA interference of T08D10.2 is sufficient to extend longevity ( approximately 25 median increase), suggesting Li() regulates survival by modulating histone methylation and chromatin structure. http://www.ncbi.nlm.nih.gov/pubmed/17959600 Fri, 30 Nov 2007 00:00:00 -0800 Protein synthesis is a regulated cellular process that links nutrients in the environment to organismal growth and development. Here we examine the role of genes that regulate mRNA translation in determining growth, reproduction, stress resistance and lifespan. Translational control of protein synthesis by regulators such as the capbinding complex and S6 kinase play an important role during growth. We observe that inhibition of various genes in the translation initiation complex including ifg1, the worm homologue of eIF4G, which is a scaffold protein in the capbinding complex and rsks1, the worm homologue of S6 kinase, results in lifespan extension in Caenorhabditis elegans. Inhibition of ifg1 or rsks1 also slows development, reduces fecundity and increases resistance to starvation. A reduction in ifg1 expression in dauers was also observed, suggesting an inhibition of protein translation during the dauer state. Thus, mRNA translation exerts pleiotropic effects on growth, reproduction, stress resistance and lifespan in C. elegans. http://www.ncbi.nlm.nih.gov/pubmed/17266680 Wed, 31 Jan 2007 00:00:00 -0800 Aging research has a problem. The field is expanding rapidly but our understanding of aging is clouded because of the language we use when describing genes that influence aging. As researchers from different disciplines gather to solve aging problems, different terms are being used to describe genetic affects. Genetic alterations can bring about enormous changes in lifespan but the significance of these experiments is debated either the results are hailed as spectacular breakthroughs or alternatively considered laboratory artifacts unlikely to reveal useful knowledge about aging. How could this have come about and what do we need to do about it I suggest we need to listen to exactly what each other is saying and also think carefully about how we are using key terms when describing our results. Rather than a scholarly review of this important issue, this article is intended to prompt debate. Consequently, "the gloves are off". http://www.ncbi.nlm.nih.gov/pubmed/16959457 Thu, 30 Nov 2006 00:00:00 -0800 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 DAF12 is pivotal for the decision to form a dauer or to undergo normal reproductive development. The DAF12 ligand has been predicted to be a sterol that is metabolized by DAF9, a cytochrome P450. Here we chemically characterize purified lipophilic nematode extracts and show that the ligand for DAF12 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 (5cholesten3betaol(25S)carboxylic acid) promotes reproductive growth in dauerconstitutive mutants in a daf9 and daf12dependent manner. Furthermore, we find that cholestenoic acid can act as a DAF12 ligand by activating DAF12 in a cellbased transcription assay. Analysis of dauerrescuing lipophilic extracts from nematodes by gas chromatographymass 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 daf9 mutants. These data suggest that carboxylated sterols may be key determinants of life history. http://www.ncbi.nlm.nih.gov/pubmed/16913876 Mon, 31 Jul 2006 00:00:00 -0700 Mild hormetic heat treatments early in life can significantly increase the lifespan of the nematode C. elegans. We have examined the effects of heat treatments at different ages and show that treatments early in life cause the largest increases in lifespan. We also find that repeated mild heat treatments throughout life have a larger effect on lifespan compared to a single mild heat treatment early in life. We hypothesize that the magnitude of the hormetic effect is related to the levels of heat shock protein expression. Following heat treatment young worms show a dramatic increase in the levels of the small heat shock protein HSP16 whereas old worms are a 100fold less responsive. The levels of the heat shock proteins HSP4 and HSP16 correlate well with the effects on lifespan by the hormetic treatments. http://www.ncbi.nlm.nih.gov/pubmed/16826446 Mon, 31 Jul 2006 00:00:00 -0700 During the last three decades the soil nematode C. elegans has become a prominent model organism for studying aging. Initially research in the C. elegans aging field was focused on the genetics of aging and single gene mutations that dramatically increased the life span of the worm. Undoubtedly, the existence of such mutations is one of the main reasons for the popularity of the worm as model system for studying aging. However, today many different approaches are being used in the C. elegans aging field in addition to genetic manipulations that influence life span. For example, environmental manipulations such as caloric restriction and hormetic treatments, evolutionary studies, population studies, models of agerelated diseases, and drug screening for compounds that extend life span are now being investigated using this nematode. This review will focus on the most recent developments in C. elegans aging research with the aim of illustrating the diversity of the field. http://www.ncbi.nlm.nih.gov/pubmed/16803977 Wed, 31 May 2006 00:00:00 -0700 Checkpoints are evolutionarily conserved signaling mechanisms that arrest cell division and alter cellular stress resistance in response to DNA damage or stalled replication forks. To study the consequences of loss of checkpoint functions in whole animals, checkpoint genes were inactivated in the nematode C. elegans. We show that checkpoint proteins are not only essential for normal development but also determine adult somatic maintenance. Checkpoint proteins play a role in the survival of postmitotic adult cells. http://www.ncbi.nlm.nih.gov/pubmed/16741121 Wed, 31 May 2006 00:00:00 -0700 The orphan nuclear hormone receptor gene daf12 in Caenorhabditis elegans plays a key role in the regulation of development and determination of adult longevity. To understand the effects of daf12 on aging we characterized the lifespan of lossoffunction and gainoffunction daf12 alleles that have been identified on the basis of their effects on dauer development. We find that these mutations have opposing effects on longevity and resistance to oxidative and thermal stress which makes daf12 the first gene with alleles that can extend or shorten lifespan. We find that the shortened lifespan of the lossoffunction mutation is due to accelerated aging in young adulthood rather than an adverse effect of the mutation on development. Microarray analysis of worms carrying the two alleles revealed a relatively small number of genes differentially expressed between the two genotypes. Comparison of the expression profiles with the profiles associated with dauer formation and longlived daf2 mutants revealed that while the profiles are largely different, there is significant overlap among the genes downregulated, but not upregulated, in all profiles. Several of these genes downregulated in multiple longlived worms have known effects on lifespan, and many of the genes belong to a family of poorly characterized genes that are strongly downregulated in dauers, daf2 mutants, and longlived daf12 mutants. Our results point to daf12 modulating aging and stress responses in part through the repression of specific genes, and emphasize the role that the repression of genes that curtail maximal lifespan plays in lifespan determination. http://www.ncbi.nlm.nih.gov/pubmed/16626392 Fri, 31 Mar 2006 00:00:00 -0800 Relative to iron and copper we know very little about the cellular roles of manganese. Some studies claim that manganese acts as a radical scavenger in unicellular organisms, while there have been other reports that manganese causes Parkinson's diseaselike syndrome, DNA fragmentation, and interferes with cellular energy production. The goal of this study was to uncover if manganese has any free radical scavenging properties in the complex multicellular organism, Caenorhabditis elegans. We measured internal manganese in supplemented worms using inductively coupled plasma mass spectrometry (ICPMS) and the data obtained suggest that manganese supplemented to the growth medium is taken up by the worms. We found that manganese did not appear to be toxic as supplementation did not negatively effect development or fertility. In fact, supplementation at higher levels accelerated development and increased total fertility of wild type worms by 16. Manganesesupplemented wild type worms were found to be thermotolerant and, under certain conditions, longlived. In addition, the oxidatively challenged C. elegans strain mev1's short life span was significantly increased after manganese supplementation. Although manganese appears to be beneficial to C. elegans, the mode of action remains unclear. Manganese may work directly as a free radical scavenger, as it has been postulated to do so in unicellular organisms, or may work indirectly by up regulating several protective factors. http://www.ncbi.nlm.nih.gov/pubmed/16545686 Tue, 28 Feb 2006 00:00:00 -0800 http://www.ncbi.nlm.nih.gov/pubmed/16264422 Mon, 31 Oct 2005 00:00:00 -0800 The mammalian Ku heterodimer has important roles in DNA double strand break repair, telomere maintenance, cell cycle checkpointarrest, tumor suppression, and cellular stress resistance. To investigate the evolutionarily conserved functions of Ku, we knocked down expression by RNA interference (RNAi) of Ku genes in C. elegans. We found that C. elegans Ku70 (CKU70) is required for resistance to genotoxic stress, regulates cytotoxic stress responses, and influences aging. The latter effects are dependent on an IGF1/insulinlike signaling pathway previously shown to affect life span. Reduction of CKU70 activity amplifies the aging phenotype of longlived insulin receptor daf2 mutations in a daf16dependent manner. These observations support the view that organismal stress resistance determines life span and Ku70 modulates these effects. http://www.ncbi.nlm.nih.gov/pubmed/16099946 Wed, 31 Aug 2005 00:00:00 -0700 An insulin/IGFIlike signalling pathway determines the rate of aging of the adult nematode, Caenorhabditis elegans. Mutations in genes encoding this pathway can result in a doubling of lifespan. While such mutations may appear to have little effect on development or fertility, evolutionary theory predicts that large increases in lifespan will not be optimal for fitness. We demonstrate by laboratory natural selection that partial loss of function of the insulin receptorlike protein DAF2 results in dramatically reduced fitness even under laboratory conditions. Despite longlived mutants appearing healthy, they exhibit a heavy fitness cost consistent with an evolutionary theory of aging. http://www.ncbi.nlm.nih.gov/pubmed/15590605 Tue, 30 Nov 2004 00:00:00 -0800 Abstract In Caenorhabditis elegans, the decision to develop into a reproductive adult or arrest as a dauer larva is influenced by multiple pathways including insulinlike 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 DAF12, 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 daf2 insulin signalling mutants and the TGFbeta signalling mutant daf7. These extracts are also able to rescue the lethal dauer phenotype of daf9 mutants, which lack a P450 steroid hydroxylase thought to be involved in the synthesis of the DAF12 ligand extracts, however, have no effect on a DAF12 ligand binding domain mutant that is predicted to be ligand insensitive. The production of this hormone appears to be DAF9 dependent as extracts from a daf9daf12 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 DAF9 that acts through DAF12. http://www.ncbi.nlm.nih.gov/pubmed/15569358 Sun, 31 Oct 2004 00:00:00 -0700 The accumulation of damage caused by reactive oxygen species (ROS) is held to be one of the underlying causes of agerelated decline and has been shown to be involved in a number of pathological states. Inherent defense mechanisms have evolved to limit this damage by reducing the levels of ROS, which are produced mainly by the mitochondria in aerobic organisms. One such defense is superoxide dismutase 1 (SOD1). It is well established that oxidative stress results in increased transcription and translation of the SOD1 gene, but it is now known that an additional level of posttranslational control exists. A recent paper describes the presence of an inactive pool of SOD1 whose activation is wholly reliant on the presence of superoxide or oxygen and a specific coppercontaining chaperone. This mechanism highlights the importance of rapid responses in the fight against oxidative stress. http://www.ncbi.nlm.nih.gov/pubmed/15215539 Mon, 31 May 2004 00:00:00 -0700 The lifespan of Caenorhabditis elegans can be extended by the administration of synthetic superoxide dismutase/ catalase mimetics (SCMs) without any effects on development or fertility. Here we demonstrate that the mimetics, Euk134 and Euk8, confer resistance to the oxidative stressinducing agent, paraquat and to thermal stress. The protective effects of the compounds are apparent with treatments either during development or during adulthood and are independent of an insulin/IGFIlike signalling pathway also known to affect thermal and oxidative stress resistance. Worms exposed to the compounds do not induce a cellular stress response and no detrimental effects are observed. http://www.ncbi.nlm.nih.gov/pubmed/14677634 Sun, 30 Nov 2003 00:00:00 -0800 To date, more than 40 genes have been identified in the nematode Caenorhabditis elegans, which, when mutated, lead to an increase in lifespan. Of those tested, all confer an increased resistance to oxidative stress. In addition, the lifespan of C. elegans can also be extended by the administration of synthetic superoxide dismutase/catalase mimetics. These compounds also appear to confer resistance to oxidative damage, since they protect against paraquat treatment. The protective effects of these compounds are apparent with treatment during either development or adulthood. These findings have demonstrated that pharmacological intervention in the aging process is possible and that these compounds can provide important information about the underlying mechanisms. To date, such interventions have targeted known processes rather than screening compound libraries because of the limitations of assessing lifespan in nematodes. However, we have recently developed a microplatebased assay that allows for a rapid and objective score of nematode survival at rates many times higher than previously possible. This system now provides the opportunity to perform highthroughput screens for compounds that affect nematode survival in the face of acute oxidative stress and will facilitate the identification of novel drugs that extend nematode lifespan. http://www.ncbi.nlm.nih.gov/pubmed/14641049 Sun, 30 Nov 2003 00:00:00 -0800 Many genetic or environmental manipulations that extend life span in the nematode Caenorhabditis elegans (C. elegans) also enhance survival following acute stresses such as oxidative damage and thermal stress. This coupling of stress response and aging mechanisms has proved a useful tool in identifying new genes that affect the aging process without the need for performing lengthy life span analyses. Therefore, it is likely that this approach may also be applied to the identification of pharmacological agents that extend life span through enhanced resistance to oxygen radicals or other stressors. To facilitate highthroughput drug screens in the nematode, we have developed a microtitre plate survival assay that uses uptake of the fluorescent dye SYTOX green as a marker of nematode death. An increase in throughput compared with the conventional survival assay was achieved by combining automated wormhandling technology with automated realtime fluorescence detection. We have validated this assay by examining survival during acute heat stress and protection against oxidative stress with the superoxide dismutase/catalase mimetic Euk134. We propose that this novel method of survival analysis will accelerate the discovery of new pharmacological interventions in aging and oxidative stress. http://www.ncbi.nlm.nih.gov/pubmed/12957648 Sun, 31 Aug 2003 00:00:00 -0700 The cuticle of the nematode Caenorhabditis elegans is a collagenous extracellular matrix which forms the exoskeleton and defines the shape of the worm. We have characterized the C. elegans gene M142.2, and we show that this is a developmentally regulated gene important for cuticle structure. Transgenic worms expressing M142.2 promoter fused to green fluorescent protein showed that M142.2 is expressed in late embryos and L2d predauers, in the hypodermal cells which synthesize the cuticle. The same temporal pattern was seen by RTPCR using RNA purified from specific developmental stages. A recombinant fragment of M142.2 was expressed in Escherichia coli and used to raise an antiserum. Immunohistochemistry using the antiserum localized M142.2 to the periphery of the alae of L1 and dauers, forming two longitudinal ribbons over the hypodermal cells. Lossoffunction of M142.2 by RNAi resulted in a novel phenotype: dumpy dauers which lacked alae. M142.2 therefore plays a major role in the assembly of the alae and the morphology of the dauer cuticle because of its similarity to the other cut genes of the cuticle, we have named the gene cut6. http://www.ncbi.nlm.nih.gov/pubmed/12921736 Thu, 31 Jul 2003 00:00:00 -0700 Insulinlike signalling is a key determinate of lifespan in diverse species including mammals but the mechanism by which this pathway influences the rate of aging is unknown. In the roundworm Caenorhabditis elegans, mutations in the insulinlike signalling pathway extend adult lifespan and are associated with upregulation of stress response genes including those for heat shock proteins (HSPs). We tested the hypothesis that the C. elegans insulinlike signalling pathway determines longevity through modulating HSP levels. We introduced extra copies of the gene encoding HSP16 and this conferred stress resistance and longevity both in a wildtype and a longlived mutant strain. The DAF16 transcription factor is essential for maximal hsp16 expression and for lifespan extension conferred by hsp16. This demonstrates that lifespan is determined in part by insulinlike regulation of molecular chaperones. http://www.ncbi.nlm.nih.gov/pubmed/12882326 Mon, 30 Jun 2003 00:00:00 -0700 Aging is the dominant risk factor for human disease in developed countries. Could it be that a wide variety of disease states all have their origins in a common mechanism Major signaling pathways that determine the rate of aging, such as the insulin/insulinlike growth factor 1 (IGF1) pathway, might give clues to the nature of this major disease risk factor. It has now been shown that insulin/IGF1 signaling influences Caenorhabditis elegans resistance to bacteria in such a way that longlived worms are stressresistant and slow to succumb to infection. Perhaps enhanced innate immunity is a feature of genetically determined longevity. http://www.ncbi.nlm.nih.gov/pubmed/12844540 Mon, 30 Jun 2003 00:00:00 -0700 http://www.ncbi.nlm.nih.gov/pubmed/12520289 Tue, 31 Dec 2002 00:00:00 -0800 We report here the identification of a previously unknown transcription regulatory element for heat shock (HS) genes in Caenorhabditis elegans. We monitored the expression pattern of 11,917 genes from C. elegans to determine the genes that were upregulated on HS. Twenty eight genes were observed to be consistently upregulated in several different repetitions of the experiments. We analyzed the upstream regions of these genes using computational DNA pattern recognition methods. Two potential cisregulatory motifs were identified in this way. One of these motifs (TTCTAGAA) was the DNA binding motif for the heat shock factor (HSF), whereas the other (GGGTGTC) was previously unreported in the literature. We determined the significance of these motifs for the HS genes using different statistical tests and parameters. Comparative sequence analysis of orthologous HS genes from C. elegans and Caenorhabditis briggsae indicated that the identified DNA regulatory motifs are conserved across related species. The role of the identified DNA sites in regulation of HS genes was tested by in vitro mutagenesis of a green fluorescent protein (GFP) reporter transgene driven by the C. elegans hsp162 promoter. DNA sites corresponding to both motifs are shown to play a significant role in upregulation of the hsp162 gene on HS. This is one of the rare instances in which a novel regulatory element, identified using computational methods, is shown to be biologically active. The contributions of individual sites toward induction of transcription on HS are nonadditive, which indicates interaction and crosstalk between the sites, possibly through the transcription factors (TFs) binding to these sites. http://www.ncbi.nlm.nih.gov/pubmed/11997337 Tue, 30 Apr 2002 00:00:00 -0700 It is difficult to exaggerate the progress that has been made in biogerontology over the last 15 years. As with all scientific revolutions, a few experiments in a small number of laboratories have changed the way in which we think about and design experiments. As a result of these experiments, there is much evidence to suggest that a rudimentary understanding of some of the processes that cause aging will be available in the next decade. One particular area of progress is the molecular genetics of lifespan. Although one may draw some distinctions between chronological lifespan and normal aging, extended lifespan remains one of the best indicators that an intervention in an aging process has been made. The isolation of a longlived variant of a laboratory invertebrate is now essentially a trivial project but the information obtained from this approach is proving invaluable. As with most other biological problems, the most important experimental developments are coming from studying simple organisms in a reductionist fashion. http://www.ncbi.nlm.nih.gov/pubmed/11869734 Thu, 31 Jan 2002 00:00:00 -0800 http://www.ncbi.nlm.nih.gov/pubmed/11506284 Tue, 31 Jul 2001 00:00:00 -0700 We present evidence for elevated levels of heat shock protein 16 (HSP16) in an intrinsically thermotolerant, longlived strain of Caenorhabditis elegans during and after heat stress. Mutation of the age1 gene, encoding a phosphatidylinositol 3kinase catalytic subunit, results in both extended life span (Age) and increased intrinsic thermotolerance (Itt) in adult hermaphrodites. We subjected agesynchronous cohorts of worms to lethal and nonlethal thermal stress and observed the accumulation of a small (1618 kd) heatshockspecific polypeptide detected by an antibody raised against C. elegans HSP16. Strains carrying the mutation hx546 consistently accumulated HSP16 to higher levels than a wildtype strain. Significantly, overaccumulation of HSP16 in the age1(hx546) strain following heat was observed throughout the adult life span. A chimeric transgene containing the Escherichia coli betagalactosidase gene fused to a C. elegans HSP1641 transcriptional promoter was introduced into wildtype and age1(hx546) backgrounds. Heatinducible expression of the transgene was elevated in the age1(hx546) strain compared with the wildtype strain under a wide variety of heat shock and recovery conditions. These observations are consistent with a model in which Age mutations exhibit thermotolerance and extended life span as a result of elevated levels of molecular chaperones. http://www.ncbi.nlm.nih.gov/pubmed/11445592 Sat, 30 Jun 2001 00:00:00 -0700 In the nematode Caenorhabditis elegans, dauer formation, stress resistance, and longevity are determined in part by DAF2 (insulin receptorlike protein), AGE1 (phosphatidylinositol3OH kinase catalytic subunit), and DAF16 (forkhead transcription factor). Mutations in daf2 and age1 result in increased resistance to heat, oxidants, and UV. We have discovered that daf2 and age1 mutations result in increased Cd and Cu ion resistance in a 24 h toxicity assay. Lethal concentration (LC50) values for Cd and Cu ions in daf2 and age1 mutants were significantly (P0.001) higher than in wildtype nematodes. However, LC50 values in daf16age1 mutants were not significantly different, implying that metal resistance is influenced by a DAF16related function. As metallothionein (MT) proteins play a major role in metal detoxification, we examined the expression of MT genes both under noninducing conditions and after exposure to sublethal and acute heavy metal stress. MT1 mRNA levels were significantly (P0.05) higher in daf2 mutants compared to age1 mutants and wildtype C. elegans under basal conditions. After 10 mM Cd treatment, induction of MT1 and MT2 mRNA was three and twofold higher, respectively, in daf2 mutant worms than in wildtype. However, a sublethal concentration of Cd (0.1 mM) resulted in even higher (three to sevenfold) levels of both MT mRNAs in all strains. Cu did not induce MT1 or MT2 mRNAs. These results are consistent with a model in which the insulinsignaling pathway determines life span through regulation of stress protein genes http://www.ncbi.nlm.nih.gov/pubmed/11259381 Wed, 28 Feb 2001 00:00:00 -0800 We tested the theory that reactive oxygen species cause aging. We augmented the natural antioxidant systems of Caenorhabditis elegans with small synthetic superoxide dismutase/catalase mimetics. Treatment of wildtype worms increased their mean lifespan by a mean of 44 percent, and treatment of prematurely aging worms resulted in normalization of their lifespan (a 67 percent increase). It appears that oxidative stress is a major determinant of lifespan and that it can be counteracted by pharmacological intervention. http://www.ncbi.nlm.nih.gov/pubmed/10968795 Thu, 31 Aug 2000 00:00:00 -0700 http://www.ncbi.nlm.nih.gov/pubmed/10838698 Thu, 31 Aug 2000 00:00:00 -0700 http://www.ncbi.nlm.nih.gov/pubmed/10911977 Mon, 31 Jul 2000 00:00:00 -0700 http://www.ncbi.nlm.nih.gov/pubmed/10911975 Mon, 31 Jul 2000 00:00:00 -0700 http://www.ncbi.nlm.nih.gov/pubmed/10830948 Wed, 31 May 2000 00:00:00 -0700 Genetic variants with greatly extended lifespan are proving invaluable in uncovering signal transduction pathways that influence the rates of normal ageing. These studies have so far been confined to invertebrate models such as Caenorhabditis elegans and Drosophila, but there has been much speculation as to whether a similar approach could be applied to mammals. The recent publication of results on a mouse strain, mutant in a gene encoding the signaling molecule p66(shc), gives cause for optimism. The mutation renders the mouse resistant to the action of oxygen radical generators and appears to increase mean lifespan by 30. This approach may provide a boost for the modeling of human agerelated diseases. http://www.ncbi.nlm.nih.gov/pubmed/10797480 Sun, 30 Apr 2000 00:00:00 -0700 The effects of 2(4Morpholinyl)8phenyl4H1benzopyran4one (LY294002), an inhibitor of mammalian phosphatidylinositol 3OH kinase, was tested on an insulin signalinglike pathway in the nematode Caenorhabditis elegans. Populations of C. elegans were treated with LY294002 at different stages of the life cycle, and its effects on development, thermotolerance and longevity were assessed. At concentrations of 160 microM and above, LY294002 significantly induced both dauer formation and thermotolerance. Treatment of adult worms also resulted in a small, but significant, increase in life span. The results presented are consistent with the view that a neuroendocrine signaling pathway functions in adult worms to determine stress resistance and longevity. http://www.ncbi.nlm.nih.gov/pubmed/10638524 Mon, 31 Jan 2000 00:00:00 -0800 Studies of aging in the nematode Caenorhabditis elegans have revealed a relationship between stress resistance and the rate of aging: Mutations which extend mean and maximum lifespan also confer resistance to thermal stress. We review the molecular genetics of aging in C. elegans and introduce methods for obtaining novel mutants which display altered aging rates. We present the use of the "surrogate" phenotype of thermotolerance to develop a selection for novel mutations which slow aging. http://www.ncbi.nlm.nih.gov/pubmed/9732049 Sat, 31 Oct 1998 00:00:00 -0800 http://www.ncbi.nlm.nih.gov/pubmed/9646294 Mon, 31 Aug 1998 00:00:00 -0700 http://www.ncbi.nlm.nih.gov/pubmed/9668638 Fri, 31 Jul 1998 00:00:00 -0700 In the last decade it has become evident that many laboratory manipulations, both genetic and environmental, can lead to significant life extension. All or almost all of the observed lifeextension phenotypes are associated with increased resistance and/or ability to respond to environmental stress. These observations show dramatically that life span is not maximized. We suggest that latent within many speciesperhaps even humansis the ability for large increases of life expectancy. The striking correlation between the increased stress resistance of all longlived mutants in C. elegans and other species and the increased resistance of dietary restricted rodents to environmental toxins is consistent with an evolutionary conservation of a lifespan maintenance/environmental stress resistance program. We suggest that it may be possible to develop methods for life extension in mammals, including humans, using relatively straightforward manipulations, such as drug treatments. It should be obvious that these findings have tremendous implications for human society at large, and we suggest that the implications of these findings should be explored. http://www.ncbi.nlm.nih.gov/pubmed/8914487 Sat, 30 Nov 1996 00:00:00 -0800 Ageing is a complex phenomenon which remains a major challenge to modern biology. Although the evolutionary biology of ageing is well understood, the mechanisms that limit lifespan are unknown. The isolation and analysis of singlegene mutations which extend lifespan (Age mutations) is likely to reveal processes which influence ageing. Caenorhabditis elegans is the only metazoan in which Age mutations have been identified. The Age mutations not only prolong life, but also confer a complex array of other phenotypes. Some of these phenotypes provide clues to the evolutionary origins of these genes while others allude to mechanisms of lifespanextension. Many of the Age genes interact and share a second common phenotype, that of stress resistance. Rather than invertebrate ageing being determined by a 'clock mechanism', a picture is emerging of ageing as a nonadaptive process determined, in part, by resistance to intrinsic stress mediated by stressresponse genes. http://www.ncbi.nlm.nih.gov/pubmed/8885718 Sat, 30 Nov 1996 00:00:00 -0800 http://www.ncbi.nlm.nih.gov/pubmed/8658201 Wed, 31 Jul 1996 00:00:00 -0700 We have discovered that three longevity mutants of the nematode Caenorhabditis elegans also exhibit increased intrinsic thermotolerance (Itt) as young adults. Mutation of the age1 gene causes not only 65 longer life expectancy but also Itt. The Itt phenotype cosegregates with age1. Longlived spe26 and daf2 mutants also exhibit Itt. We investigated the relationship between increased thermotolerance and increased lifespan by developing conditions for environmental induction of thermotolerance. Such pretreatments at sublethal temperatures induce significant increases in thermotolerance and small but statistically highly significant increases in life expectancy, consistent with a causal connection between these two traits. Thus, when an animal's resistance to stress is increased, by either genetic or environmental manipulation, we also observe an increase in life expectancy. These results suggest that ability to respond to stress limits the life expectancy of C. elegans and might do so in other metazoa as well. http://www.ncbi.nlm.nih.gov/pubmed/7638227 Thu, 31 Aug 1995 00:00:00 -0700 We have developed a longextensionPCR strategy which amplifies approximately half of the mitochondrial genome (6.3 kb) of Caenorhabditis elegans using an individual worm as target. We analyzed three strains over their life span to assess the number of detectable deletions in the mitochondrial genome. Two of these strains are wildtype for life span while the third is mutant in the age1 gene, approximately doubling its maximum life span. At the mean life span in wildtype strains, there was a significant difference between the frequency of deletions detected in the mitochondrial genome compared with the mean number of deletions in young animals. In addition, deletions in the mitochondrial genome occur at a significantly lower rate in age1 mutants as compared with wild type. We cloned and identified the breakpoints of two deletions and found that one of the deletions had a direct repeat of 8 bp at the breakpoint. This is the largest single study (over 900 individual animals) characterizing the frequency of deletions in the mitochondrial genome as a function of age yet carried out. http://www.ncbi.nlm.nih.gov/pubmed/7753635 Wed, 31 May 1995 00:00:00 -0700 http://www.ncbi.nlm.nih.gov/pubmed/7973641 Wed, 30 Nov 1994 00:00:00 -0800 Agesynchronous cohorts of Caenorhabditis elegans were grown at 20 degrees C, then stressed at 30 degrees C or 35 degrees C. Intrinsic thermotolerance of wild type and age1 mutant strains was assessed by measuring either progeny production or survival. In addition to increased life span (Age), mutation of age1 results in a highly significant increased intrinsic thermotolerance (Itt) as measured by survival at 35 degrees C. Mean survival of Age strains is approximately 45 longer than that of nonAge strains for both sterile and nonsterile worms. Thermotolerance declines across the life span of both Age and nonAge strains, but Itt was observed at almost all ages. Unstressed age1 animals showed a consistent and significant fertility deficit. Short thermal stresses can cause a dramatic reduction in progeny production for both Age and nonAge genotypes. Mutants of age1 showed a small but consistent increased thermotolerance as measured by fertility. We propose that the enhanced ability of Age strains to cope with environmental stress may be mechanistically related to their lower agespecific mortality rates. http://www.ncbi.nlm.nih.gov/pubmed/7963273 Wed, 30 Nov 1994 00:00:00 -0800 The genetic analysis of aging processes has matured in the last ten years with reports that longlived strains of both fruit flies and nematodes have been developed. Several attempts to identify mutants in the fruit fly with increased longevity have failed and the reasons for these failures are analyzed. A major problem in obligate sexual species, such as the fruit fly, is the presence of inbreeding depression that makes the analysis of lifehistory traits in homozygotes very difficult. Nevertheless, several successful genetic analyses of aging in Drosophila suggest that with careful design, fruitful analysis of induced mutants affecting life span is possible. In the nematode Caenorhabditis elegans, mutations in the age1 gene result in a life extension of some 70 thus age1 clearly specifies a process involved in organismic senescence. This gene maps to chromosome II, well separated from a locus (fer15) which is responsible for a large fertility deficit in the original stocks. There is no tradeoff between either rate of development or fertility versus life span associated with the age1 mutation. Transgenic analyses confirm that the fertility deficit can be corrected by a wildtype fer15 transformant (transgene) however, the life span of these transformed stocks is affected by the transgenic array in an unpredictable fashion. The molecular nature of the age1 gene remains unknown and we continue in our efforts to clone the gene. http://www.ncbi.nlm.nih.gov/pubmed/8125279 Thu, 31 Mar 1994 00:00:00 -0800 Agespecific mortality rates in isogenic populations of the nematode Caenorhabditis elegans increase exponentially throughout life. In genetically heterogeneous populations, agespecific mortality increases exponentially until about 17 days and then remains constant until the last death occurs at about 60 days. This period of constant agespecific mortality results from genetic heterogeneity. Subpopulations differ in mean lifespan, but they all exhibit near exponential, albeit different, rates of increase in agespecific mortality. Thus, much of the observed heterogeneity in mortality rates later in life could result from genetic heterogeneity and not from an inherent effect of aging. http://www.ncbi.nlm.nih.gov/pubmed/8303273 Mon, 28 Feb 1994 00:00:00 -0800 Study of C. elegans has provided much information for gerontologists. The influence of the genome on life span is clearly observable, and at least one gerontogene, age1, has been defined. Data relating to important evolutionary questions has emerged and will continue to be used in testing current hypotheses. We are using an approach unbiased by theoretical constraints to delineate aging processes simultaneously at the molecular and organismal levels. Much remains to be discovered before fundamental questions posed in this article are answered to a satisfactory degree. The immediate agenda is the identification and isolation of gerontogenes which influence life span in invertebrate models. This work is well in hand and will lead to the unraveling of specific lifespandetermining processes. At this point we may be able to predict whether analogous processes also limit life in mammals. If we are fortunate and aging processes exhibit evolutionary conservation, many exciting possibilities await. Molecular tools provided by the invertebrate system can then be used to isolate homologous mammalian gerontogenes that could be subsequently utilized in highly targeted attempts to intervene in mammalian aging. This offers the most direct strategy for identifying lifespan prolongation genes in humans. http://www.ncbi.nlm.nih.gov/pubmed/1355097 Mon, 31 Aug 1992 00:00:00 -0700