genetic control of longevity in c. elegans

9
Genetic control of longevity in C. elegans Bart P. Braeckman * , Jacques R. Vanfleteren Biology Department, Ghent University, K.L.Ledeganckstraat 35, B-9000 Ghent, Belgium Received 26 April 2006; accepted 28 April 2006 Available online 10 July 2006 Abstract The nematode Caenorhabditis elegans has proven to be a very useful tool for studying the genetics of longevity. Over 70 genes have been found to influence lifespan in this worm. Those related to the Ins/IGF signaling pathway are among the best studied and will be focused on in this review. The master regulator of this pathway, the forkhead transcription factor DAF-16, can activate an enhanced life maintenance program in response to environmental and gonadal inputs. DAF-16 up- and downregulates expression of many genes lead- ing to metabolic alterations and increased stress and microbial resistance. This is generally confirmed by biochemical and physiological data. Longevity mutants are not hypometabolic and probably produce more reactive oxygen species than wild type. However, their high antioxidant capacity may result in lower oxidative damage. Enhanced molecular turnover rates may also play a role in their longevity phenotype. Ó 2006 Elsevier Inc. All rights reserved. Keywords: Caenorhabditis elegans; Ins/IGF signaling; daf-16; Aging physiology 1. Introduction Nematodes have been used in biogerontological studies from the early 1970s onwards. Initial research was conduct- ed on the vinegar eel Turbatrix aceti and was mainly focused on age-dependent alteration of enzyme activity (Gershon and Gershon, 1970; reviewed by Rothstein, 1980). The first classical study on the aging process in Cae- norhabditis elegans was published a few years later and described the non-aging phenotype of the dauer stage (Klass and Hirsh, 1976). Klass introduced C. elegans as ‘‘an excellent experimental system for the study of aging’’ for which he ‘‘identified some of the major biological and environmental factors influencing lifespan as a prelude to more detailed genetic and biochemical analyses’’ (Klass, 1977). The tremendous progress in biogerontology due to C. elegans studies carried out during the last three decades confirmed his prediction. Caenorhabditis elegans has proven to be a powerful model system for aging studies due to its simple biology. It is a terrestrial free-living nematode that feeds primarily on bacteria. Hermaphrodites are typically 1.2–1.5 mm long and 50–70 lm wide and can produce about 300 self-fertil- ized eggs under laboratory conditions. Males originate due to meiotic non-disjuction of the sex chromosome X and occur rarely in a normal population. They can fertilize hermaphrodites, increasing their fecundity to about 1000 eggs (sperm cells are a limiting factor in hermaphrodites). The C. elegans life cycle is very simple – it consists of four larval stages separated by molts – and takes only 2.5 days under optimal conditions. When young larvae are exposed to unfavorable conditions (high temperature, overcrowd- ing, food scarcity), they molt into an alternative third stage larva – the dauer. Dauers show typical diapause features: they are highly stress resistant, hypometabolic, extremely long-lived and show an altered morphology. In the labora- tory, C. elegans is commonly kept on agar plates seeded with Escherichia coli as a food source, however for long- term storage of worm strains liquid nitrogen can be used. The worm is transparent during all life stages allowing the organism to be studied in detail by simple light micros- copy (a C. elegans biology review can be found in Riddle et al., 1997 and Wood, 1988). 0531-5565/$ - see front matter Ó 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.exger.2006.04.010 * Corresponding author. Tel.: +32 9 264 87 44; fax: +32 9 264 87 93. E-mail address: [email protected] (B.P. Braeckman). www.elsevier.com/locate/expgero Experimental Gerontology 42 (2007) 90–98

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Page 1: Genetic control of longevity in C. elegans

www.elsevier.com/locate/expgero

Experimental Gerontology 42 (2007) 90–98

Genetic control of longevity in C. elegans

Bart P. Braeckman *, Jacques R. Vanfleteren

Biology Department, Ghent University, K.L.Ledeganckstraat 35, B-9000 Ghent, Belgium

Received 26 April 2006; accepted 28 April 2006Available online 10 July 2006

Abstract

The nematode Caenorhabditis elegans has proven to be a very useful tool for studying the genetics of longevity. Over 70 genes havebeen found to influence lifespan in this worm. Those related to the Ins/IGF signaling pathway are among the best studied and will befocused on in this review. The master regulator of this pathway, the forkhead transcription factor DAF-16, can activate an enhanced lifemaintenance program in response to environmental and gonadal inputs. DAF-16 up- and downregulates expression of many genes lead-ing to metabolic alterations and increased stress and microbial resistance. This is generally confirmed by biochemical and physiologicaldata. Longevity mutants are not hypometabolic and probably produce more reactive oxygen species than wild type. However, their highantioxidant capacity may result in lower oxidative damage. Enhanced molecular turnover rates may also play a role in their longevityphenotype.

� 2006 Elsevier Inc. All rights reserved.

Keywords: Caenorhabditis elegans; Ins/IGF signaling; daf-16; Aging physiology

1. Introduction

Nematodes have been used in biogerontological studiesfrom the early 1970s onwards. Initial research was conduct-ed on the vinegar eel Turbatrix aceti and was mainlyfocused on age-dependent alteration of enzyme activity(Gershon and Gershon, 1970; reviewed by Rothstein,1980). The first classical study on the aging process in Cae-

norhabditis elegans was published a few years later anddescribed the non-aging phenotype of the dauer stage(Klass and Hirsh, 1976). Klass introduced C. elegans as‘‘an excellent experimental system for the study of aging’’for which he ‘‘identified some of the major biological andenvironmental factors influencing lifespan as a prelude tomore detailed genetic and biochemical analyses’’ (Klass,1977). The tremendous progress in biogerontology due toC. elegans studies carried out during the last three decadesconfirmed his prediction.

Caenorhabditis elegans has proven to be a powerfulmodel system for aging studies due to its simple biology.

0531-5565/$ - see front matter � 2006 Elsevier Inc. All rights reserved.

doi:10.1016/j.exger.2006.04.010

* Corresponding author. Tel.: +32 9 264 87 44; fax: +32 9 264 87 93.E-mail address: [email protected] (B.P. Braeckman).

It is a terrestrial free-living nematode that feeds primarilyon bacteria. Hermaphrodites are typically 1.2–1.5 mm longand 50–70 lm wide and can produce about 300 self-fertil-ized eggs under laboratory conditions. Males originatedue to meiotic non-disjuction of the sex chromosome Xand occur rarely in a normal population. They can fertilizehermaphrodites, increasing their fecundity to about 1000eggs (sperm cells are a limiting factor in hermaphrodites).The C. elegans life cycle is very simple – it consists of fourlarval stages separated by molts – and takes only 2.5 daysunder optimal conditions. When young larvae are exposedto unfavorable conditions (high temperature, overcrowd-ing, food scarcity), they molt into an alternative third stagelarva – the dauer. Dauers show typical diapause features:they are highly stress resistant, hypometabolic, extremelylong-lived and show an altered morphology. In the labora-tory, C. elegans is commonly kept on agar plates seededwith Escherichia coli as a food source, however for long-term storage of worm strains liquid nitrogen can be used.The worm is transparent during all life stages allowingthe organism to be studied in detail by simple light micros-copy (a C. elegans biology review can be found in Riddleet al., 1997 and Wood, 1988).

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B.P. Braeckman, J.R. Vanfleteren / Experimental Gerontology 42 (2007) 90–98 91

Despite some discouraging predictions and warningsbased on evolutionary arguments, Friedman and Johnson(1988) successfully isolated the first healthy long-lived C.

elegans mutant from a series of strains that were createdby Klass a few years earlier (Klass, 1983). The mutantwas designated age-1 and lived 65% longer than wild typeat 25 �C. This report was a turning point for the geneticresearch on aging and during the next decade several newlongevity mutants were described.

2. The Ins/IGF signaling pathway and lifespan in C. elegans

Many of the genes that confer lifespan extension whentheir function is reduced were identified in the late 1990s(Braeckman et al., 2003). The majority of them act in aninsulin/IGF signaling pathway (Fig. 1) which determinesdauer formation, stress resistance and longevity.

Environmental cues such as food availability generate anervous signal that results in the release of one or moreinsulin/IGF-like peptides. In the C. elegans genome asmany as 38 insulin-like genes are predicted and some ofthem have been studied in detail (Kawano et al., 2000; Liet al., 2003; Murphy et al., 2003; Pierce et al., 2001). Thesepeptides can bind on the unique Ins/IGF-like receptorDAF-2 (Kenyon et al., 1993; Kimura et al., 1997) therebygenerating an intracellular signaling cascade. The DAF-2receptor activates a phosphatidyl-3-OH kinase consistingof a p55-like regulatory subunit (AAP-1, Wolkow et al.,

Fig. 1. Gene interactions that influence lifespan in C. elegans through DAF-literature cited in the text.

2002) and a p110 catalytic subunit (AGE-1, Morris et al.,1996). This kinase is antagonized by DAF-18, a humanPTEN homolog with IP3-phosphatase activity (Ogg andRuvkun, 1998). The PI-P3 signal activates the Akt/PKBkinase homolog PDK-1 (Paradis et al., 1999) which in turnphosphorylates AKT-1, AKT-2 (Paradis and Ruvkun,1998) and SGK-1 (Hertweck et al., 2004). Both AKT pro-teins are redundant and are primarily involved in dauerformation while SGK-1 – a serum and glucocorticoidinducible kinase homolog – controls development, stressresponse and longevity (although akt-1(RNAi) has beenreported to extend lifespan as well; Hamilton et al.,2005). These kinases inactivate DAF-16, a FOXO familytranscription factor homologous to FKHRL1, by phos-phorylation and keep it out of the nucleus (Lin et al.,1997; Ogg et al., 1997). As a consequence, it is the reduc-tion of insulin/IGF-1 signaling, due to environmental fac-tors or mutation, that activates the DAF-16 transcriptionfactor. In the nucleus, DAF-16 can activate an enhancedlife maintenance program to increase lifespan (Hendersonand Johnson, 2001; Lin et al., 2001).

Unexpectedly, nuclear localization of DAF-16 per se isnot sufficient for increasing the lifespan as a response tolow Ins/IGF signaling (Lin et al., 2001). Recently, a newcomponent of the Ins/IGF pathway, SMK-1 was identifiedas an essential regulator for DAF-16-mediated longevity(Wolff et al., 2006). SMK-1 colocalizes with DAF-16 andis involved in the innate immune, UV and oxidative stress

16 activation or inactivation. This hypothetical diagram is based on the

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functions of this transcription factor. DAF-16-mediatedheat resistance, however, is independent of SMK-1 activity.This is consistent with earlier studies on the role of the heatshock transcription factor HSF-1 in the Ins/IGF signalingpathway. HSF-1, a master regulator of stress-induciblegenes and protein folding homeostasis, is a clue factor forIns/IGF-dependent lifespan extension. This factor acts inmultiple tissues of C. elegans and many of its downstreamtargets such as small heat-shock proteins partially contrib-ute to the longevity phenotype (Garigan et al., 2002; Hsuet al., 2003; Morley and Morimoto, 2004). Another compo-nent of the Ins/IGF-signaling pathway that acts in parallelto DAF-16 to extend lifespan is the AMP-activated proteinkinase AAK-2 (Apfeld et al., 2004).

Ins/IGF signaling does not occur randomly in theworm. This was first demonstrated by Apfeld and Kenyon(1998) using genetic mosaic analysis. They found that thepresence of a limited number of daf-2(�) cells in a wild typeworm can elicit lifespan extension in a worm. Tissue-specif-ic rescue experiments confirmed this finding and suggestedthat major sites contributing to Ins/IGF-dependent life-span extension were the neurons (Wolkow et al., 2000). Afew other studies confirm that neurons are important forDAF-16-dependent longevity. Mutation in genes codingfor two synaptic proteins, homologs of syntaxin and aCa2+-dependent activator protein for secretion, extendedworm lifespan in a DAF-16-dependent fashion (Ailionet al., 1999). In the same year it was reported that sensoryneurons can link environmental cues to lifespan by meansof DAF-16 activity (Apfeld and Kenyon, 1999). Transduc-tion of this neuronal signal may require cholinergic synaps-es and muscarinic receptors (Tissenbaum et al., 2000).More recently, mutants which are chemotaxis defectivebecause they lack active TUB-1 in their ciliary neurons,were reported to show a DAF-16-dependent lifespan exten-sion (Mukhopadhyay et al., 2005). Neurons, however, donot seem to be the only cell types in which DAF-16 activitypromotes organismal longevity.

Besides environmental input, gonadal signals also regu-late lifespan in C. elegans. Laser ablation of germline pre-cursor cells resulted in a 60% lifespan increase which,again, was dependent on DAF-16 function (Arantes-Oli-veira et al., 2002; Hsin and Kenyon, 1999). However, ablat-ing the complete gonads (including the somatic cells) hadno effect on lifespan which led the authors to conclude thatthe somatic gonad provides a life-extending signal thatcounterbalances the life-shortening signal from the germcells.

The C. elegans intestine, which, besides its digestivefunction also has a storage function and produces yolk,was shown to be an important site for life-extendingDAF-16 activity (Libina et al., 2003). It was suggested thatDAF-16 activity in signaling cells is able to activate DAF-16 in specific responsive tissues, possibly via an Ins/IGF-like signal. These results appear inconsistent with the find-ings of Wolkow et al. (2000) reporting that Ins/IGF signal-ing in neurons is sufficient to rescue the longevity

phenotype of daf-2 and age-1 worms. This paradox stillneeds to be resolved.

Recently, Berman and Kenyon (2006) found that thereproductive system communicates with the intestine toincrease lifespan in a DAF-16-dependent fashion. Themodel suggests that, in the somatic gonad, the cytochromeP-450 homolog DAF-9 synthesizes a lipophilic hormone.In response to this signal, KRI-1, an intestinal ankyrinrepeat protein, promotes nuclear localization of DAF-16,which in turn regulates lifespan.

Besides spatial patterns, temporal patterns of DAF-16activity may also play an important role in its life extendingproperties. Ins/IGF signaling is involved in dauer forma-tion, stress resistance and lifespan. The dauer decision ismade during the first (sometimes the beginning of the sec-ond) larval stage implicating that Ins/IGF signaling is nec-essary in these early stages of life. Can this early activityinfluence adult lifespan or is Ins/IGF signaling necessaryduring adulthood to lengthen lifespan considerably? Usingtemporal daf-2 and daf-16 RNAi experiments, Dillin et al.(2002a) found that early DAF-16 activity was irrelevant toinfluence adult lifespan. Lifespan was extended by DAF-16activity during adulthood.

Lifespan regulation by Ins/IGF signaling appeared to beevolutionary conserved over a wide range of organisms.Homologs of some of the aforementioned pathway compo-nents were shown to regulate lifespan in yeast, Drosophila

as well as mice (for a review, see Kenyon, 2005).In C. elegans, many more mutations were reported to

extend lifespan, part of them being independent of DAF-16. They include the Clk-family, several mitochondrialelectron transport genes, mTOR pathway homologs, Junkinase and sir-2 deacetylase among others (for a recentmini review, see Schaffitzel and Hertweck, 2006). SeveralRNAi screens yielded additional genes that influence wormlifespan (Hamilton et al., 2005; Hansen et al., 2005; Leeet al., 2003a). Most of these genes are involved in signaltransduction, stress response, protein turnover, geneexpression or metabolism (which encompasses pretty muchthe majority of all cellular processes). Here, we will notelaborate on this topic.

3. Transcriptional targets of DAF-16

Because DAF-16 obviously plays a key role in theimpressive lifespan extension mediated by reduced Ins/IGF-signaling, major effort has been put into the searchfor its downstream effectors. Some of these genes were indi-vidually identified in early experiments but three recentgenome-wide studies that were published almost simulta-neously yielded a gigantic set of DAF-16-related tran-scripts (Lee et al., 2003b; McElwee et al., 2003; Murphyet al., 2003). Stress response genes, antimicrobial and met-abolic genes, and a group of genes with unknown functionwere found to act downstream of DAF-16.

Microarray data on the upregulation of stress responsegenes was generally consistent. The mitochondrial superox-

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ide dismutase sod-3 and metallothionein mtl-1 were recov-ered, confirming the earlier reports by Honda and Honda(1999) and Barsyte et al. (2001), respectively. Besides these,Murphy et al. (2003) found that the cytosolic and peroxi-somal catalases ctl-1 and ctl-2, the glutathione-S-transfer-ase gst-4 and several small heat shock proteins appearedto be upregulated by DAF-16 as well. McElwee et al.(2003) reported DAF-16-dependent upregulation of heatshock proteins (HSP-16, HSP-70, HSP-90) together withan increase of cytosolic SOD-5 expression. RNAi experi-ments carried out by Murphy et al. (2003) showed thatmany of these genes are partially responsible for the life-span extending properties of active DAF-16. The fact thatlongevity is associated with an enhanced stress response isin keeping with the oxidative stress theory of aging. How-ever, two independent studies reported that sod-3 (RNAi)did not shorten lifespan in long-lived daf-2 mutants, sug-gesting that the synthesis of mitochondrial SOD-3 per sedoes not increase lifespan (Hsu et al., 2003; McElweeet al., 2003).

Murphy et al. (2003) showed that some antimicrobialgenes such as the lysozymes lys-7 and lys-8, and the sapo-sin-like gene spp-1 were upregulated in the daf-2 Ins/IGFsignaling mutant and that they contributed to its extendedlifespan. Earlier it was found that, under laboratory condi-tions, wild type C. elegans shows bacterial packing in thegut which ultimately kills the worm (Garigan et al.,2002). Long-lived daf-2 mutants exhibit reduced packing,probably due to the activation of their innate immunesystem. Moreover, the extreme longevity of C. elegans insterile axenic medium may be (partially) due to the absenceof bacteria that otherwise may infect weak old worms(Braeckman et al., 2001).

Finally, several metabolic genes were found of whichactivity is necessary to increase lifespan in daf-2 mutants(Murphy et al., 2003). This group consisted of several genesinvolved in amino acid metabolism, ubiquitin-mediatedprotein degradation and the glyoxylate cycle enzyme isoci-trate lyase/malate synthase. These findings suggest thatprotein and energy metabolism is shifted in longevitymutants. The microarray study of McElwee et al. (2003)showed no specific differences for genes involved in proteinsynthesis between animals with active and inactive DAF-16, but, in accordance to Murphy et al. (2003), non-protea-somal proteases may play a role in the Ins/IGF longevityphenotype. In the same study, no systematic differenceswere detected for mitochondrial genes as well but therewas a tendency for reduced expression of metabolism-relat-ed genes in worms with active DAF-16.

The transmembrane tyrosine kinase OLD-1 is a tran-scriptional target of DAF-16 and a positive regulator oflongevity and stress resistance. Activity of this gene is aprerequisite to Ins/IGF-dependent longevity and it isexpressed in the whole body. Its precise function and tar-gets however, remain to be elucidated (Murakami andJohnson, 2001). The protein SCL-1 has almost identical lifeextending properties compared to OLD-1, except for the

fact that it is a putative cysteine-rich secretory proteinand thus might have extracellular signaling properties(Ookuma et al., 2003; Patterson, 2003). This protein wasalso recovered in the microarray study of Murphy et al.(2003).

Besides microarray studies on the transcriptional outputof DAF-16, the transcriptional and translational profilewas followed during the aging process of C. elegans.Two-dimensional gel electrophoresis experiments revealedthat protein synthesis decreases with increasing age, butthe general translational pattern of about 700 proteins doesnot change dramatically with age (Johnson and McCaffrey,1985; Vanfleteren and De Vreese, 1994). This was con-firmed in an early expression study (Fabian and Johnson,1995). A similar study was carried out several years laterwhen whole-genome microarray chips for C. elegans

became available (Lund et al., 2002). Here also, transcrip-tional profiles did not vary considerably over the aging pro-cess: less than 1% of the genome (164 genes) showedstatistically significant changes over time during adulthood.

Among these genes, the insulins ins-2 and ins-7 exhibitedan age-dependent decrease while ins-17 and ins-18 showedan age-dependent upregulation. The other Ins/IGF signal-ing pathway genes did not alter significantly. Interestingly,Murphy et al. (2003) found that ins-7 is a DAF-2 agonistwhich is repressed by DAF-16 activity, suggesting that thisinsulin is part of a positive feedback loop supporting theorganismic response to Ins/IGF signaling. The Lundet al. (2002) study shows that ins-7 expression decreaseswith age, suggesting gradual deterioration of this positivefeedback loop.

Many heat shock proteins were upregulated during earlyadulthood followed by a decrease later in life. Low HSPlevels at advanced age may lead to increased levels ofimproperly folded proteins and consequent loss of homeo-stasis and organismic senescence (Lund et al., 2002).

4. Physiology and biochemistry of C. elegans longevity

During the last 20 years, genetic and molecular studieshave gathered an enormous amount of invaluable informa-tion on the regulation of the aging process in C. elegans.The most important signaling pathways together with theirdownstream targets that influence aging are identified andcharacterized. Also their spatial and temporal patterns arewell studied. But is this enough to understand the molecu-lar basis of the aging process?

Transcriptomics and proteomics provide details aboutthe available protein machinery in the animals and suggestthe relative importance of certain cellular processes. How-ever, they fail to distinguish the precise balances of metab-olites, actual turnover rates of small and large biomoleculesand redox balances in their cellular context. Besides, fluxesthrough metabolic pathways are not only regulated bysheer abundance of participating enzymes but rather byphosphorylations and complex feedback mechanisms.Biochemical and physiological analyses can provide this

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information and may elucidate important molecular eventsthat take place during aging or pinpoint biochemical char-acteristics that correlate with longevity. Many of thesetechniques allow direct testing of several molecular aginghypotheses. Moreover, the existence of extreme longevitystrains in C. elegans provides an excellent tool to studythe biochemistry and physiology of longevity.

The oxidative stress theory of aging, based on the freeradical theory proposed by Harman (1956), is still the mostwidely accepted theory explaining the molecular basis ofaging. The main idea of this theory is that during normalmetabolism, oxidative byproducts are inevitably generatedand damage molecules such as nucleic acids, proteins andlipids thereby impairing their biological function. Accumu-lation of these damaged molecules compromises cellularhomeostasis and leads to senescence. According to thisview, C. elegans longevity mutants should suffer lower lev-els of oxidative stress, a prediction that can be verifiedusing several biochemical approaches. There are manyways by which oxidative stress can be lowered in an animal.The production rate of reactive oxygen species (ROS) canbe lowered, the oxidative stress response can be elevated,or the turnover rates of damaged molecules can be raised.During the last decade, we spent major effort testing thefirst two assumptions.

5. Energy metabolism in C. elegans longevity mutants

It has been widely assumed that lowering metabolicrate decreases ROS production and therefore many geron-tologists expected longevity mutants to be hypometabolic.This speculation seemed to be partially confirmed by thefact that extremely long-lived dauers exhibit low energymetabolism (Houthoofd et al., 2002; O’Riordan and Bur-nell, 1989, 1990; Vanfleteren and De Vreese, 1996). Wehave monitored the metabolic rate of several Ins/IGF sig-naling pathway mutants during adulthood and found nogeneral reduction in long-lived strains compared to wildtype (Braeckman et al., 2002; Houthoofd et al.,2005a,b). Calorimetric/respirometric ratios were generallylower in these mutants suggesting a shift towardsincreased mitochondrial efficiency. This was supportedby the high ATP levels that were found in these mutants.However, high standing ATP concentrations are not aprerequisite for longevity; elevated ATP levels were alsoreported in a short-lived ucp-4 strain (Iser et al., 2005)and RNAi of several genes involved in mitochondrialelectron transport and ATP synthase result in a pheno-type combining low ATP content with extended lifespan(Dillin et al., 2002b; Lee et al., 2003a). It was also notedthat the life extension in daf-2 mutants is independent ofthe AMP:ATP ratio (Apfeld et al., 2004).

A remarkable phenotype of daf-2 mutants is that manyalleles are hypoxia resistant (Scott et al., 2002). Although itmight be tempting to assume that this phenotype is linkedto lowered oxygen consumption and reduced ROS produc-tion and hence extended lifespan, comparison of a variety

of daf-2 alleles showed that there is no significant correla-tion between hypoxia resistance and lifespan. Moreover,oxygen consumption in daf-2 mutants is not lower com-pared to wild type (Braeckman et al., 2002; Houthoofdet al., 2005a,b).

The low calorimetric/respirometric ratio found in Ins/IGF mutants indicates that they maintain tight mitochon-drial coupling compared to wild type animals. High cou-pling efficiency results in a high membrane potential,which in turn promotes ROS generation (Brand, 2000).In other words, long-lived Ins/IGF mutants are expectedto generate more ROS than wild type. At present, thereare no reports on in vivo ROS production in these mutantsto support this prediction. This scenario seems to runcounter to the oxidative stress theory of aging but thisapparent conflict can be resolved if these mutants exhibita strongly increased ROS degrading response.

6. Stress response in C. elegans longevity mutants

Larsen (1993) and Vanfleteren (1993) were the first toreport that the Ins/IGF signaling mutant age-1 is resistantto oxidative stress by upregulating SOD and catalase activ-ity. Over the next few years it was shown that this mutantwas also resistant to other stressors such as UV and elevat-ed temperature (Lithgow et al., 1994; Murakami and John-son, 1996). Resistance to some kind of stress and longevityseem to be tightly correlated in all known C. elegans lon-gevity mutants (Johnson et al., 2001). Accordingly, overex-pression of the small heat shock protein HSP-16Aconferred lifespan extension in C. elegans (Walker andLithgow, 2003). GFP fusion to HSP-16.2 allowed research-ers to predict individual longevity in an isogenic C. elegans

cohort (Rea et al., 2005).Over the last few years we have collected a detailed data-

set on the antioxidant activity present in C. elegans longev-ity mutants. Expectedly, all Ins/IGF longevity mutantstested, exhibited a strong antioxidant response. Theyshowed higher levels of SOD and catalase throughoutadulthood (Houthoofd et al., 2005b) and a higher contentof reduced GSH (unpublished results). These increasedactivity levels may hypercompensate the expected ROSoverproduction resulting from their high and tightly cou-pled energy metabolism which in turn might lead toincreased lifespan. It is tempting to hypothesize that simpleupregulation of antioxidants or overexpression of antioxi-dant enzymes may extend lifespan. These kinds of experi-ments have not been carried out on C. elegans yet, butdata on Drosophila is ambiguous (Orr et al., 2003; Sohalet al., 2002). As mentioned above, the 10-fold upregulationof the mitochondrial SOD-3 in daf-2 mutants is not a deter-minant for their longevity phenotype (Hsu et al., 2003;McElwee et al., 2003). Moreover, mitochondrial SODactivity (KCN-insensitive) is only a fraction of the cytosol-ic, KCN-sensitive Cu/Zn SOD activity in C. elegans wildtype and Ins/IGF mutants (Vanfleteren, 1992, 1993 andunpublished observations). The high SOD activities we

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detected in Ins/IGF mutants therefore likely representupregulation of Cu/Zn SOD’s.

7. Pharmacological interventions

The fact that long-lived Ins/IGF mutants exhibitincreased oxidative stress tolerance supports the oxidativestress theory of aging. Researchers have tried to increaseoxidative stress resistance by adding compounds with anti-oxidant activity to the C. elegans diet hoping that this inter-vention would induce a considerable lifespan increase.Vitamin E is a collective name for tocotrienols and toc-opherols, small antioxidant molecules that prevent freeradical damage in the membranes (Ingold et al., 1987).Tocotrienol administration reduces protein carbonylationand extends mean lifespan (but not maximum lifespan)by a modest 20% in C. elegans (Adachi and Ishii, 2000).a-Tocoferol also extends lifespan albeit at higher concen-trations (Harrington and Harley, 1988).

More recently, a more spectacular lifespan increase(44%) was obtained by administration of the SOD mimet-ics EUK-8 and EUK-134 (Melov et al., 2000; Sampayoet al., 2003). Unfortunately, these results could not be rep-licated by other independent groups who found thesemimetics to be toxic instead (Bayne and Sohal, 2002; Kea-ney and Gems, 2003) in spite of the fact that they showedSOD-like catalytic activity and accumulated in the mito-chondria (Keaney et al., 2004). Recent evidence suggeststhat EUK components cause increased protein carbonyl-ation in C. elegans, probably by enhancing hydrogen per-oxide and accompanying hydroxyl radical generation(Matthijssens et al. unpublished results).

Other pharmaceutical interventions that were recentlyshown to increase worm lifespan are the anticonvulsantsethosuximide, trimethadione and 3,3-diethyl-2-pyrrolidi-none (Evason et al., 2005). Trimethadione showed thestrongest effect, extending mean lifespan of wild type C.

elegans with 47%, independently of DAF-16 activity. It ishypothesized that these drugs affect worm lifespan by influ-encing neuronal function (Kornfeld and Evason, 2006).

8. Molecular turnover rates

Apart from avoiding oxidative stress by reducing ROSgeneration or enhancing ROS scavenging systems, cellscan cope with oxidative stress by efficiently replacing theirdamaged molecules i.e. by increasing their turnover orrepairing rates. The role of cellular turnover processes inaging still needs major experimental attention. During thelast few decades, several models have been proposed.

Early work of Johnson and McCaffrey (1985) showedthat 35S incorporation decreases with age in C. elegans,suggesting that protein synthesis rates slow down overtime. However, the reduced incorporation of radioactivelabel at advanced age may also have been the result ofreduced pharyngeal pumping or gut transport rather thanprotein synthesis per se. However, protein degradation

rates (that are measured more directly) were also shownto decrease several-fold over age in the nematode T. aceti

(Prasanna and Lane, 1979), suggesting that protein turn-over slows down with age in nematodes. As a result, pro-tein half life increases, making these molecules moresusceptible to damage accumulation. This may result in aprotein damage catastrophe: a positive feedback loop thatdrives the aging process (Ryazanov and Nefsky, 2002). Ifprotein turnover rates are indeed powerful longevity deter-minants, Ins/IGF mutants or dietary restricted animalsshould exhibit increased turnover rates compared to wildtype (Tavernarakis and Driscoll, 2002).

Brunk and co-workers proposed the mitochondrial-lyso-somal axis theory of aging (Brunk and Terman, 2002). Theystate that, due to oxidative damage, mitochondrial turnoverby lysosomal function (autophagy) declines with age leadingto death of postmitotic cells. Interestingly, it was found thatautophagy is a prerequisite for lifespan extension in daf-2

mutants of C. elegans (Melendez et al., 2003). It is still notclear whether this process is dependent on DAF-16 activity.

By comparing transcripts of dauers and daf-2 mutants ina genome-wide microarray study, new classes of potentiallongevity genes were detected and a broad molecular agingmodel was established (McElwee et al., 2004). In short,aging arises from molecular damage inflicted by endobiotictoxins originating from diverse metabolic processes. Manyof these toxins can be detoxified by cytochrome P450,short-chain dehydrogenase/reductase and UDP-glu-curonosyltransferases, three families of enzymes that werefound to be upregulated in dauers and daf-2 mutants. Thisway of detoxification comes with a high energy cost and themodel is therefore entitled the Green Theory of Aging(Gems and McElwee, 2005).

9. Conclusion

In summary, C. elegans has proven to be an excellentmodel system for aging research due to its rapid life historyand nearly unlimited possibilities in molecular genetics.However, this model is evolutionarily remote from humansprohibiting direct extrapolations of nematode data. Never-theless, C. elegans still remains the best tool for the geneticanalysis of longevity so far (Johnson, 2003).

The Ins/IGF signaling pathway, a major control switchfor longevity, has been thoroughly characterized inC. elegans and many of its downstream targets (includingtheir temporal and spatial expression patterns) have beenidentified. The increased resistance to internal and externalstressors seems to be a major player assuring longevity. Thenext challenge consists of elucidating how these gene prod-ucts interact on lower biochemical and physiological levelsto control longevity.

Although it is possible to attain a spectacular lifespanextension in C. elegans by combining genetic and anatom-ical interventions or environmental alterations (Arantes-Oliveira et al., 2003; Houthoofd et al., 2004), a few finalquestions remain: why does the extreme long-lived worm

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still die? Is it possible to force cellular maintenance to alevel far beyond this current limit? Probably it is the dreamof many gerontologists to catch the aging process before itcatches them.

Acknowledgements

B.P.B. and J.R.V. are supported by the Fund for Scien-tific Research-Flanders (G.0025.06) and the EuropeanCommunity (LSHM-CT-20004-512020).

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