Molecular Genetic Mechanisms of Life Span Manipulation in Caenorhabditis elegans

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Molecular Genetic Mechanisms of Life Span Manipulation in Caenorhabditis elegans

SHIN MURAKAMI,a PATRICIA M. TEDESCO, JAMES R. CYPSER, ANDTHOMAS E. JOHNSON

Institute for Behavioral Genetics, University of Colorado, Boulder, Colorado 80309, USA

ABSTRACT: Aging and a limited life span are fundamental biological realities.Recent studies have demonstrated that longevity can be manipulated and haverevealed molecular mechanisms underlying longevity control in the soil nema-tode Caenorhabditis elegans. Signals from both neurons and the gonad appearto negatively regulate longevity. One tissue-specific signal involves an insulin-like phosphatidylinositol 3-OH kinase pathway, dependent upon the DAF-16forkhead transcription factor. These signals regulate mechanisms determininglongevity that include the OLD-1 (formerly referred to as TKR-1) receptor ty-rosine kinase. Interestingly, increased resistance to environmental stress showsa strong correlation with life extension.

Life span can be manipulated by both genetic and environmental factors. Genetic ex-tension of life span has been accomplished in a variety of species.1–5 Genes whosealteration causes life extension are referred to as gerontogenes.3,4 Longevity in Cae-norhabditis elegans has been extended by (1) polygenic alterations involving crossesbetween wild-type strains, (2) single-gene mutations, and (3) overexpression of life-extension genes. Most illuminating have been studies on the single-gene mutants,which have identified several gerontogenes and revealed molecular pathways, in-cluding an insulin-like signal transduction pathway that specifies life span.5,6 Inac-tivating genes in this pathway leads to increased life expectancy. In contrast, genesthat cause life extension when overexpressed represent another class: positive mod-ulators of longevity. Thus, there are at least two ways to extend life span: inactivatingnegative regulators of life span and activating positive modulators of life span.

Interestingly, the life-extension mutants in C. elegans also have revealed a strongcorrelation between life extension and increased resistance to environmentalstress.2,7,8 Here, we will discuss the genetic manipulation of longevity, the molecu-lar mechanisms underlying regulation of life span, and potential causes of longevityextension in C. elegans.

LIFE EXTENSION VERSUS LIFE SHORTENING

Life extension has been a good indicator of delayed aging and has been used toidentify gerontogenes that regulate life span. By selecting for life extension rather

aCurrent address for correspondence: Shin Murakami, Division of Biological Sciences, Uni-versity of Missouri, 310 Tucker Hall, Columbia, MO 65211.

[email protected]

41MURAKAMI et al.: LIFE SPAN MANIPULATION

than life shortening, one can be sure that these genes play an active role in regulatingthe length of life and the aging process. There are numerous ways to shorten life spanthat are nonspecific to aging; in many cases, it is very hard to demonstrate that short-er life is significantly indicative of faster aging in C. elegans and in other species.One problem with using the life-shortening phenotype is the lack of a convincingbiological marker for aging in C. elegans. Therefore, in this review, we will mainlyfocus on studies using the life-extension phenotype and will be cautious about inter-preting results from life-shortening alterations.

LIFE EXTENSION CAUSED BY MULTIPLE GENE ALTERATIONS

The first studies used crosses between the N2 and Bergerac “wild-type” strainsfollowed by inbreeding of F2 progeny to generate recombinant-inbred strains thatshow life expectancies up to 63% longer than wild-type.9 Using the RI strains, poly-genes (called quantitative trait loci or QTLs) affecting life span and other life-historytraits have been identified.10–13

SINGLE-GENE MUTATIONS: NEGATIVE REGULATORSOF LONGEVITY

To identify gerontogenes in C. elegans, forward genetics has commonly beenused. Mutations have typically been generated using a mutagenic chemical, ethylmethanesulfate (EMS); so far, age-1 and age-2 are the only mutants originally iden-tified in screens for increased longevity.14–17 This method mainly generates reduc-tion (hypomorphic) or loss-of-function (nullomorphic) mutations.18 For example,EMS tends to generate point mutations, including stop codons, by G/C to A/T transi-tions and can also cause deletions of up to 10–20% of the mutagenized genome.18–19

age-1b

The first gerontogene mutant identified was age-1.14,15 The age-1(hx546) refer-ence allele has a life expectancy 65% longer than wild type and a maximum life spanthat is 105% longer.15,20 age-1 mutations have little effect on fertility, length of re-production, or rate of development15,16,20 but are dauer constitutive at the semi-lethal temperature of 27°C.21 (The dauer is an alternative form of larvae producedunder conditions of crowding or starvation. daf mutations affect the dauer-formationpathway22 [see below].) age-1(hx546) is resistant to H2O2,23 paraquat,24 UV,8 andheat25,26 and has reduced frequency of deletions in mitochondrial DNA.27 Threeother alleles of age-1 were independently isolated on the basis of longevity alone16

and are also stress resistant but show subtle variations among themselves. Two more

bIn C. elegans, genes are given names consisting of three italicized letters, a hyphen, and anitalicized Arabic number (e.g., age-1 and old-1). The protein product of a gene is referred to bythe relevant gene name, written in nonitalic capitals, for example, the protein encoded by old-1 iscalled OLD-1.

42 ANNALS NEW YORK ACADEMY OF SCIENCES

alleles of age-1 have been isolated by selecting for increased thermotolerance (G.Lithgow, personal communication). All age-1 alleles are also Daf-c at 27°C.

Mutations in another gene, previously called daf-23, also cause life-extension,dauer-constitutive (Daf-c), and stress-resistance phenotypes.28 daf-23 mutants areDaf-c at 25°C and age-1 mutations fail to complement daf-23 mutations.21,29 Morriset al.29 have cloned the daf-23 locus, demonstrated that it shows structural homologywith mammalian phosphatidylinositol-3-OH kinase (PI3 kinase), and suggested thatdaf-23 and age-1 are the same gene based on the failure to complement. To date,however, no mutation in the PI3 kinase structural locus has been found in any of thestrains carrying the non-25°C-Daf-c alleles of age-1 (Ref. 29; Murakami, Klimin-skaya, and Johnson, unpublished). Thus, the formal possibility exists that age-1 isnot the same gene as daf-23 and is not PI3K. Nevertheless, it is commonly assumedthat PI3 kinase is the protein coded for by the age-1 gene.

C. elegans Gerontogenes That Specify Increased Longevity asHypomorphs or Nullomorphs

Except for age-1 and age-2 mutants, all life-extension mutants were isolated ini-tially by screens for other phenotypes. These mutants can be classed according totheir associated phenotypes. In all of these cases the Age (increased life span) phe-notype results from hypomorphic or nullomorphic mutations.

Daf (Dauer Larva Formation Abnormal) Genes

age-1 is a member of this class. Mutants in daf-2 are Daf-c at 25°C and result intwofold extension of life expectancy in the adult phase.30 Additionally, daf-2 inter-acts with daf-12 to cause an almost fourfold increase in life expectancy.28 daf-2bears structural homology with the human insulin receptor.31

The genes in the Daf class regulate dauer formation22 and are the most well-char-acterized gerontogenes, but not all Daf genes cause life extension. AGE-1 PI3 kinaseand DAF-2 insulin-like receptor form a genetic pathway,8,28,30,32 dependent on theDAF-16 forkhead transcription factor.33,34 Recently, PDK-1 (phosphatidylinositoldependent kinase-1) has been cloned and shown to be involved in this pathway.35

Because the mammalian insulin receptor transduces a signal through PI3 kinase andPDKs,36 it appears likely that a similar signal is transduced from the DAF-2 receptor,AGE-1 PI3 kinase and PDK-1 to the DAF-16 transcription factor (FIG. 1A). UNC-31 and UNC-64 appear to play a role in releasing a ligand of DAF-2,36 perhaps spec-ifying a neuronal signal for the DAF-2 receptor.37 Mosaic analysis suggests that thefunction of daf-2 in neuronal and/or other tissues originating from the AB cell is nec-essary for regulation of longevity.37 Thus, the age-1/daf-2 pathway is likely to trans-duce signal(s) from a limited spectrum of tissues that negatively regulate longevity.

The age-1/daf-2 pathway also includes AKT-1 and AKT-2 (AKT: protein kinaseB),38 and DAF-18 (PTEN: PI3 phosphatase)39,40 when regulating a metabolic shift,reproduction, and larval diapause (dauer stage). daf-18 may antagonize AGE-1 PI3kinase activity; however, it is unclear whether akt-1 is also involved in regulation oflongevity, since inactivation of akt-1 suppresses the dauer-constitutive phenotype ofage-1 mutations but not the longevity phenotype.38


Spe (Sperm Formation Defective) Genes

Two of the six mutant alleles of spe-26, a gene specifying proper segregation ofcellular components affecting sperm activation, result in life extensions of about65% for the hermaphrodite and the mated male,41 although the details are con-tended.42 An allele of spe-10 also confers a modest (20%) life extension.43

Clk (Clock, Abnormal Biological Timing) Genes

Wong et al.44 reported that two of four alleles of clk-1, both of which have alteredcell cycle and developmental timing, also have increased life expectancy. The clk-1

FIGURE 1. (A) Models for regulation of longevity. The age-1/daf-2 insulin-like phos-phatidylinositol 3-OH kinase pathway described within. Upon receipt of a signal from theDAF-2 insulin-like receptor,31 the AGE-1 PI3 kinase is predicted to produce PIP3, whichactivates PDK-1 and AKT-1/AKT-2.29,35,38 The AKTs may directly phosphorylate the DAF-16 forkhead transcription factor and may antagonize its function. The OLD-1 receptortyrosine kinase appears to be downstream of these signal transduction proteins (Ref. 51;Murakami and Johnson, submitted). UNC-31 and UNC-64 are neuronal proteins and maybe involved in releasing a ligand of the DAF-2 receptor.36


gene encodes a 187 amino acid protein showing some homology with yeast CAT5p,a gene required for transcriptional activation of several genes involved in gluconeo-genesis in yeast.45 They suggest that clk-1 mutant alleles have reduced metabolicrates that are responsible for the extended longevity. Lakowski and Hekimi46 haveextended these studies to include clk-2, clk-3, and gro-1, all of which have modest(typically 20–30%) extensions of life span, but only for some alleles. Using doublemutants, they have suggested that clk-1 identifies a pathway distinct from that iden-tified by daf-2.46 Other studies using double mutants show that both clk-1 and daf-2require a function of daf-167 or ctl-1, a cytosolic catalase gene.47 However, all suchinterpretations run the risk that the life-shortening effects of daf-16 and ctl-1 mutantsare nonspecific and result from an abnormal mode of death. Interestingly, the clk-1;daf-2 double mutant extends life expectancy almost fivefold.

Eat Genes

Eat mutations cause reduced food intake, probably leading to dietary restriction.Some, but not all, of them show a life extension (eat-2, eat-6, eat-13, eat-13, eat-18,and unc-26).48 Life extension in this class results from changes in eating behavior.

There are several additional gerontogene mutants that complement all known ger-ontogenes (e.g., Ref.16; Herndon et al., in preparation), suggesting that there are asyet unidentified gerontogenes in C. elegans.

Gonadal Signals

It is likely that negative signals are transduced from multiple tissues to modulatelife span. Although the postulated age-1/daf-2 insulin-like pathway may be specificto neurons, gonadal signals have also been implicated in modulating life span.49 La-ser ablation of gonad precursor cells Z1–Z4 (Z1 and Z4 cells differentiate into thesomatic gonad, and Z2 and Z3 precursor cells differentiate into the germ line) haverevealed just such an effect. The signals of Z2 and Z3 shorten life span and are de-pendent on the DAF-16 forkhead transcription factor. It seems that those of Z1 andZ4 have little effect on life span but interact with daf-2 mutants. Thus, it appears thattissue-specific signals can negatively regulate longevity. We extend our previously

FIGURE 1. (B) Our proposed model. Negative signals from specific tissues repressmolecular mechanisms for longevity in the whole body that may involve the DAF-16 fork-head transcription factor33 and the OLD-1 receptor tyrosine kinase.51 This life-extensionmechanism also confers increased stress resistance. A life-shortening signal of neurons andother tissues (neurons etc.) involves the age-1/daf-2 pathway, whereas a signal from the go-nad interacts with the DAF-2 insulin-like receptor.


published model8 to propose a molecular mechanism in which life extension is neg-atively regulated by such tissue-specific signals (FIG. 1B).

UPREGULATION OF POSITIVE REGULATORS OF LONGEVITY

We have used overexpression to upregulate genes positively modulating life span.Using a transgenic system, we identified a gerontogene that extends life span whenoverexpressed.51 The gene encodes a novel tyrosine kinase receptor gene, old-1(overexpression longevity determinant).

Although positive modulators of longevity are poorly understood, old-1 increasesstress resistance and longevity (from 40 to 100%) when overexpressed in transgenicanimals. These effects of old-1 are comparable to those of the life-extension muta-tion age-1 and are larger than those observed in clk-1 and spe-26 mutants. Important-ly, the transgenic animals overexpressing old-1 do not show altered developmentalrates and show normal induction of dauer larvae. Nevertheless, the stress resistanceand longevity of these animals is suppressed by mutations in daf-16 (as is observedfor the genes involved in the age-1/daf-2 pathway, including age-1, daf-2, pdk-1,unc-31, and unc-64), therefore formally placing old-1 in a common pathway withother gerontogenes. These results strongly support the correlation of stress resis-tance with life extension in C. elegans and illustrate the importance of (1) establish-ing the molecular mechanisms by which old-1 overexpression induces stressresistance and life extension, and (2) identifying, in general, genes involved in reg-ulating and enacting the stress response in C. elegans.

PROPOSED MOLECULAR MECHANISM OF LIFE EXTENSION

Organisms are continuously exposed to intrinsic environmental stress from insidethe cell (e.g., oxidative stress) as well as extrinsic stress from outside environments(e.g., radiation and thermal stress). Such continuous exposure often promotes mac-romolecular damage, leading to deleterious effects that we call aging and senes-cence. Our proposed stress-resistance theory of aging hypothesizes that the abilityto resist these environmental insults either by higher initial resistance to the stres-sor(s) and/or by more effective repair of critical damage is the rate-determiningevent leading to increased life expectancy.

Stress Resistance

Resistance to a variety of both intrinsic and extrinsic environmental stressors isstrongly correlated with life extension in many species. In C. elegans, all geronto-gene mutants tested show increased resistance to oxidative stress (age-1 and daf-2),23,24,52 thermal stress (age-1, clk-1, eat-2, daf-2, spe-10, and spe-26),25,26,43 andUV stress (age-1, clk-1, daf-2, daf-28, spe-10, and spe-26).8,43 Interestingly, the pos-tulated age-1/daf-2 insulin-like pathway regulates UV and oxidative stress resis-tance.8,52 Overexpression of old-1 also causes life extension and increasedresistance to UV and heat stress (Ref. 51; Murakami and Johnson, submitted).


The increased resistance to a variety of environmental stressors and the putativeroles of age-1/daf-23, daf-2, and daf-16 in a signal-transduction pathway involvingthe phosphoinositides are consistent with a model in which life span is determinedby the induction of a number of transcripts causing altered sensitivity to the environ-ment and increased ability to resist or repair environmental damage.2,8 The facts thatthe spe-26 and clk-1 Age (life-extension) alleles (but not the non-Age alleles) arealso resistant to UV and that the UV resistance of these mutants is suppressed bydaf-16 without altering fertility8 are consistent with increased resistance playing acausal role in the increased life expectancy of these mutants.

Other Possible Causes

Although extensive evidence supports increased stress resistance as a centralmechanism generating the long-life phenotype of Age mutations, competing (if notnecessarily mutually exclusive) hypotheses have been proposed. These include theconcept of a central “life span” clock that can be genetically reset by mutation,53 aswell as the view that Age mutations convey longevity primarily by altering cellularmetabolism.33,54 However, few direct metabolic or biochemical measurements havebeen made to critically test these assumptions.

The sequence similarities of daf-2 to the human insulin receptor and age-1 to hu-man PI3 kinase have led to the interpretation that these genes regulate an insulin re-sponse pathway regulating glucose metabolism.31 Indeed, age-1 and daf-2 mutantscause a metabolic shift toward fat accumulation.33 Similar metabolic shifts have alsobeen observed in daf-4 and daf-7, Daf mutants with wild-type life spans, suggestingthat fat accumulation is associated with but not necessary for prolonged life span.

The clk-1 gene appears to play a role in respiratory function in mitochondria.clk-1 animals are longer-lived and have altered cell cycles and altered rhythmic be-haviors.45 The clk-1 gene is probably orthologous to COQ7, which regulates yeastmetabolism55 through the biosynthesis of coenzyme Q. clk-1 mutations slightly im-pair mitochondrial function56 and uncouple energy production and consumption.57

Reduced rate of metabolism has also been investigated as a candidate for causinglongevity by several labs. clk-1 mutants consistently show large (70%) reductions inmetabolism. Although controversial, long-lived mutants such as age-1 and daf-2show little57,58 or modest reduction in respiration.59 In other assays, these mutantsshow no reduction in ATP levels.57,58

SPECULATIONS

Regulation of aging and longevity remains a compelling biological problem. At-tractive, but largely untestable, evolutionary models argue against direct selectionfor an adaptive effect of aging. Recent studies of single-gene mutants have revealedtissue-specific signals that negatively regulate longevity. Such signals from neuronaltissues or gonads are sufficient for modulation of longevity. These signals are medi-ated, at least in part, by the DAF-2 insulin-like receptor, the AGE-1 PI3 kinase, andPDK-1. This pathway also appears to regulate a positive molecular mechanism forlongevity that involves old-1. Other regulators will likely be identified using themethods described here as well as new methods such as biochip analyses. The cor-


relation between longevity and stress resistance could be used to isolate additionalregulators of longevity through use of stress resistance as a surrogate phenotype.

Details of the molecular mechanisms underlying stress resistance in the geronto-gene mutants are unclear. Stress resistance can be achieved by reducing stress-induced damage or by increasing ability to repair the damage. One way to reduce thedamage would be to reduce the rate of metabolism, leading to reduction of oxidativestress from respiratory reactions. This mechanism may be responsible for increasedstress resistance in long-lived DR (dietary-restricted) rodents.60 In addition, someregulators of metabolism play an active role in stress resistance or vice versa. Forexample, the age-1/daf-2 pathway regulates both metabolism33 and stress resis-tance.2,8 The age-1/daf-2 pathway also determines a dauer-formation path that canbe induced by starvation.22 Moreover, dietary restriction (including starvation) canmanipulate stress-inducible genes such as chaperones.61 Thus, we see potential linksbetween stress resistance and metabolism that could causally determine longevity.The uncovering molecular mechanisms encoded in the pathway will contribute fur-ther to the understanding of aging.

ACKNOWLEDGMENTS

This work was supported by the American Federation for Aging Research, the Ja-pan Society for the Promotion of Science, the National Institute of Health (RO1-AG08322, P01-AG08761, RO1-AG16219, and KO1-AA00195), the Glenn Founda-tion for Medical Research, and the Ellison Medical Foundation.

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Molecular Genetic Mechanisms of Life Span Manipulation in Caenorhabditis elegans