Longevity and aging

The nematode C. elegans has developed into a classical model for the study of aging. Most of this research is motivated by a possible connection with aging in humans, but the new insights accumulated over the last few years are of equal relevance to ecology and life-history theory. It also appears that the properties of the molecular machinery regulating aging are shared with Drosophila and are even conserved across the whole animal kingdom. The main reason why C. elegans became a model for aging was due to the discovery of mutants that showed extended longevity. It turned out that the genes mutated to extend longevity were the same as those associated with the formation of dauer larvae. The dauer larva is a developmentally arrested stage, the entry of which is triggered by adverse conditions such as food shortage or crowding (see Section 3.3). Here we will consider the signalling pathways associated with dauer formation as well as extended longevity.

5.2.1 The insulin signalling pathway

The first report of single-gene mutations that affected lifespan and reproduction in C. elegans dates back to 1988. The gene involved was called age-1, and mutations in this locus increased longevity and decreased hermaphrodite fertility (Friedman and Johnson 1988). Later, Kenyon et al. (1993) discovered that two other single-gene mutations could affect lifespan. One of these longevity-regulating genes was known as daf-2 (daf from dauer formation); animals mutated at the daf-2 locus lived twice as long as the wild-type worms (Fig. 5.3). It was also shown that a second gene, daf-16, is required for the lifespan-extending effect of daf-2, because the double mutant, daf-2; daf-16, had a normal lifespan (Fig. 5.3). Interestingly, the reproductive output of the daf-2 mutant was hardly decreased (the brood size was 212 ± 36 eggs in the long-lived mutant versus 278 ± 35 in the wild type) and it was also shown that ablation of the germ-line precursor cells, effectively sterilizing the adult, did not increase lifespan. Thus the increased

Daf Longevity Mutation

Days (after hatching)

Figure 5.3 Survival curves for C. elegans mutated in the daf-2 gene (daf-2(e1370)), compared with a control group (wild type). Survival curves are also shown for nematodes mutated in the daf-16 gene (daf-16(m26)) and in both genes (daf-16;daf-2). Median survival times are 17days for m26, 17 days for m26;e1370, 19days for the wild type, and 46 days for e1370. The comparison of mutants demonstrates that daf-2 dysfunction increases longevity by more than a factor of 2, and this effect requires a functional copy of daf-16. From Kenyon etal. (1993) by permission of Nature Publishing Group.

Days (after hatching)

Figure 5.3 Survival curves for C. elegans mutated in the daf-2 gene (daf-2(e1370)), compared with a control group (wild type). Survival curves are also shown for nematodes mutated in the daf-16 gene (daf-16(m26)) and in both genes (daf-16;daf-2). Median survival times are 17days for m26, 17 days for m26;e1370, 19days for the wild type, and 46 days for e1370. The comparison of mutants demonstrates that daf-2 dysfunction increases longevity by more than a factor of 2, and this effect requires a functional copy of daf-16. From Kenyon etal. (1993) by permission of Nature Publishing Group.

lifespan of the daf-2 mutant was not a consequence of lower reproduction. Later several other genes were identified that can extend longevity when altered in C. elegans, and in total approximately 70 are known today. These genes include regulators of metabolism, genes involved in sensory perception, and reproduction genes. In addition, several genes regulating longevity were found to be associated with defence against oxidative stress. One specific group of genes receiving much attention appeared to encode proteins of the insulin signalling pathway.

Insulin is a peptide hormone that in vertebrates is secreted from groups of cells associated with diverticula of the gut, in mammals taking the form of the islets of Langerhans in the pancreas. Insulinlike peptides and insulin-like growth factors (IGFs) are also present in invertebrates, usually in their highest concentrations in the gut. Insulin in mammals has a central role in carbohydrate and lipid metabolism, the best-known effect being increased uptake of glucose from the blood by muscles and adipose tissue and the formation of glycogen by the liver. Insulin and IGF do not enter their target cells but instead react with a receptor protein in the cell membrane, the insulin/IGF receptor. This protein has an extracellular domain, binding insulin or IGF, and a cytosolic domain, which acts as a tyrosine kinase: when activated it catalyses the phosphorylation of tyrosine residues in cytosolic proteins. These proteins in turn activate others in a complicated cascade, finally leading to phosphorylation of a cytosolic protein known as DAF-16. DAF-16 is a transcription factor of the so-called forkhead family. When active, this transcription factor switches on a series of genes that form a programme of dauer-larva formation. As long as DAF-16 is phosphorylated by DAF-2 signalling, it cannot enter the nucleus and so is effectively inactivated as a transcription factor (Lin et al. 2001). The secretion of insulin-like peptides is under control of the nervous system, which receives sensory input from the mouth region. So the whole system seems to be targeted towards translating information about food availability in the environment into either normal development (DAF-16 inhibited by activated DAF-2) or a dauer programme (DAF-16 activated by relaxation of DAF-2; Braeckman et al. 2001; Olsen et al. 2003; Fig. 5.4). The pathway is referred to as insulin/ IGF-1 signalling.

The fact that mutations in the DAF-2/DAF-16 pathway regulate longevity as well as dauer formation strongly suggest that the effect of daf-2 knockout on longevity is basically a dauer programme 'mis-expressed' in the adult. Indeed, Dillin et al. (2002), using RNAi to suppress daf-2 and daf-16 at different times in the life cycle, found that the same pathway regulates aging in the adult and dauer formation in the larvae. Jones et al. (2001), using SAGE (see Section 2.3) applied to dauer and non-dauer populations of C. elegans, showed that the dauer-specific transcriptome was greatly enriched in genes that previously were known to regulate longevity. The study also showed that dauer formation is not to be considered a true resting stage from a genetic point of view, because no fewer than 18% of the genes detected were specifically upregulated in the dauer larva, compared to a mixed-stage population. Similarly, Wang and Kim (2003) in a microarray study classified 1984 genes as dauer-regulated, which is 11% of the C. elegans genome. The dauer-enriched genes include several enzymes characteristic for anaerobic metabolism (Holt and Riddle 2003).

The long-lived non-dauer daf-2 mutant illustrates that it is possible to uncouple part of the dauer programme (the part that confers extended longevity) from the main programme leading to quiescence. The mutations in daf-2 and other genes of the insulin/IGF-1 signalling pathway that affect longevity are weak mutations. Strong mutations in the same genes cause the C. elegans larvae to go into dauer dormancy regardless of environmental cues. This indicates that there may be thresholds in the levels of endocrine signalling such that a mild decrease of signalling is already sufficient to start the anti-aging programme, whereas a further decrease is necessary to enter the dauer stage. These thresholds could also be dependent on temperature; some age-1 mutants that develop into long-lived adults at normal culture temperature will go into dauer diapause at high temperature (27°C).

Figure 5.4 Scheme of the insulin/IGF-1 signalling pathway, showing how extracellular insulin-like peptides may trigger a cascade of events, starting with binding to DAF-2, a receptor protein in the cell membrane and eventually leading to phosphorylation of DAF-16, a transcription factor of the forkhead family. By decreased insulin/IGF-1 signalling DAF-16 is activated, allowed to enter the nucleus, trigger a programme of dauer formation, stress resistance, and longevity, and suppress normal reproduction. P, phosphate group; PDK-1, phosphoinositide-dependent kinase 1; PI(3,4)P2, phosphatidylinositol 3,4-bisphosphate; PI(3,4,5)P3, phosphatidylinositol 3,4,5-trisphosphate. After Olsen et al. (2003), with permission from Springer.

Figure 5.4 Scheme of the insulin/IGF-1 signalling pathway, showing how extracellular insulin-like peptides may trigger a cascade of events, starting with binding to DAF-2, a receptor protein in the cell membrane and eventually leading to phosphorylation of DAF-16, a transcription factor of the forkhead family. By decreased insulin/IGF-1 signalling DAF-16 is activated, allowed to enter the nucleus, trigger a programme of dauer formation, stress resistance, and longevity, and suppress normal reproduction. P, phosphate group; PDK-1, phosphoinositide-dependent kinase 1; PI(3,4)P2, phosphatidylinositol 3,4-bisphosphate; PI(3,4,5)P3, phosphatidylinositol 3,4,5-trisphosphate. After Olsen et al. (2003), with permission from Springer.

Increased longevity similar to decreased DAF-2 signalling is also seen when the animals are cultured under dietary restriction, also called caloric restriction. This refers to a diet in which food intake is limited to 30-40% of the intake shown by animals fed ad libitum. In C. elegans this can be achieved by diluting the bacteria in the medium or by applying an axenic medium with heat-killed E. coli cells. Because the insulin signalling pathway is associated with carbohydrate metabolism and the apparent cues for dauer induction come from food availability, it seems logical to conclude that dietary restriction promotes longevity through the same DAF-2/DAF-16 pathway. However, this appears not to be the case. Houthoofd et al. (2003) did experiments with daf-2 and daf-16 mutants both exposed to dietary restriction (Table 5.1). Their data show clearly that dietary restriction causes an increase in the median survival time by a factor of two and a half, both in the wild type and in the daf-16 mutant. In addition, a restricted diet can boost the median lifespan of the daf-2 mutant (which is already increased by a factor of 1.7 compared with the wild type), to a record value of 90.9 days (maximum, 136 days). In human terms these animals would correspond to healthy 500 years old! Interestingly, extension of lifespan is this way is not accompanied by decreased metabolic activity; actually, respiration was elevated substantially by dietary restriction as were activities of antioxidant enzymes, such as superoxide dismutase (Houthoofd et al. 2002).

Another mutation that extends lifespan in C. elegans seems to act mostly independent of the insulin/IGF-1 signalling pathway. Mutations in the so-called clk-1 gene (the name derives from clock biological timing abnormality) increase longevity in association with a slowing down of the rate of many processes, including cell division, rhythmic behaviour, rate of feeding, and mito-chondrial respiration. The correlation between longevity and metabolic rate supports the rate of living hypothesis of aging, which states that aging is

Table 5.1 Survival times of different populations of C. elegans showing that effects of dietary restriction increase longevity independent of the insulin signalling pathway

Population Medium lifespan (days; mean + S.E.)

On normal medium Under dietary restriction on axenic medium

Source: After Houthoofd et al. (2003).

a consequence of accumulating metabolic damage, in particular from endogenous oxygen radicals (Finkel and Holbrook 2000; Hekimi and Guarante 2003). Reactive oxygen species such as superoxide anion (O-T), hydroxyl radical (OH*), and hydrogen peroxide (H2O2) are amply generated by the electron-transport chain in the mitochondrion. The major source is complex III, in which ubiquinone, alias coenzyme Q, resides. This electron-transport molecule has an unstable intermediate that can easily donate electrons directly to molecular oxygen rather than to complex III. The mutated clk-1 protein cannot perform the final step in the biosynthesis of ubiquinone and as a consequence the precursor, demethoxyubiquinone, is incorporated in the electron-transport chain. Paradoxically, this alternative component appears to be less prone to the production of reactive oxygen species. Another role for clk-1 has been suggested by Branicky et al. (2000). The protein could have a regulatory role by reporting to the nucleus on the metabolic state of the mitochondria, in such a way that the rate of living may be adjusted to the generation of energy. If clk is mutated nuclear genes do not receive the right information on respiration and would set the rate of living at a default level lower than normal. This theory is interesting because it would imply that the effect of respiration on aging acts through a metabolic switch, rather than through a direct impact of damage accumulation (Guarante and Kenyon 2000).

In addition to mutations, surgical operations can be applied to C. elegans to increase longevity. An interesting effect is seen after removal of the germ line. As noted in Chapter 3, C. elegans has a completely determinate developmental pattern in which the destination of every cell is fixed as soon as it comes into existence; the adult has exactly 959 cells, not including eggs and sperm cells, which descend from the zygote in a fixed lineage. Two cells, Z2 and Z3, give rise to the germ line by continuous division during development. The germ cells differentiate into sperm during the L4 stage or oocytes during adulthood (see Fig. 3.18). Removing the germ-line precursor cells by means of a laser extends the lifespan of C. elegans by about 60% and this effect requires the presence of DAF-16. Animals that lack germ cells due to a mutation are also long-lived. In a series of elegant experiments, measuring the survival curves of various mutants, Arantes-Oliveira et al. (2002) showed that the lifespan-suppressive effect of the germ line is not dependent on the sperm or oocytes themselves, but only on proliferating, active germ-line precursor cells. So, despite the obvious negative correlation between reproduction and longevity in this system, there does not seem to be a simple trade-off in the classical sense that energy allocated to reproduction would detract from maintenance and so increase the rate of aging. Rather, a signal from the germ-line stem cells directs both aging and reproduction, maybe by altering the production of a steroid hormone or by altering the response to such a hormone (Arantes-Oliveira et al. 2002).

An important issue in the metabolic network affecting aging in C. elegans is the question of which genes act upstream and which downstream in the insulin signalling pathway. Taking DAF-16 as a reference point, upstream genes are defined as the ones that affect expression or activity of DAF-16. To this category belong the neurosecretory signals leading to secretion of insulin-like factors, the genes encoding the insulin-like peptides themselves, the insulin/IGF receptor DAF-2, and the various genes in the signalling cascade leading to phosphorylation of DAF-16, such as age-1 and pdk-1. The downstream genes include the ones that are regulated by the transcription factor DAF-16 and whose expression contributes to longevity and stress resistance. The difference between upstream and downstream genes is difficult to make when looking at gene expression as such, but can be unravelled by studying mutants that are knocked out at crucial positions in the pathway. For example, Murakami and Johnson (2001) studied a transmembrane tyrosine kinase gene called old-1 , which if overexpressed in C. elegans increases longevity by a factor of 1.5; using transgenic nematodes in which the old-1 gene was fused to green fluorescent protein the authors observed that the positive effect of old-1 on longevity was absent in a mutant in which daf-16 was knocked out. This makes it likely that old-1 is regulated by DAF-16; that is, it is downstream of DAF-16.

5.2.2 Genome-wide analysis of lifespan modulation

The various single-gene mutation studies and other manipulations have demonstrated that the effect of insulin/IGF signalling on longevity in C. elegans is linked to a large number of other processes, such as reproduction, lipid metabolism, diapause entry, and stress resistance. This has made the situation increasingly complex and it became difficult to see the bigger picture. However, recent genome-wide microarray studies (Murphy et al. 2003; Golden and Melov 2004; McElwee et al. 2004) have enforced an important breakthrough, presenting an interpretative framework for earlier data and even a new theory of aging.

Murphy et al. (2003) aimed to identify all the genes that act downstream of DAF-16 by means of a cDNA microarray survey. These authors focused on genes that showed opposite expression profiles in daf-2- versus daf-16-knockout mutants. They also treated animals with RNAi of selected genes to confirm that these genes had an effect on longevity. A division in two classes was made: class-1 (lifespan-extending) genes were upregulated in daf-2 mutants and in daf-2 (RNAi) animals, but downregulated in animals in which both daf-16 and daf-2 were inhibited by RNAi. The second class of genes (lifespan-shortening) was defined by genes that displayed the opposite profile. A relatively succinct list of genes could be identified as belonging to either class and the most prominent ones of each class are listed in Table 5.2.

Inspecting the genes in the two classes, it is obvious that the first class contains many genes of stress-defence systems (Table 5.2). Heat-shock proteins are molecular chaperones that support the folding of other proteins; many of them are highly inducible by several stress factors, including a heat shock, in which they were first described. Cytochrome P450 is an enzyme that catalyses the oxidation of aromatic lipophilic compounds, including many xenobiotics, but also endogenic substances such as steroids. Catalase is an enzyme

Table 5.2 Overview of genes in C. elegans acting downstream of DAF-16, identified by differential expression using a microarray and reduced expression of daf-2 and daf-16 by RNAi

Gene Brief description

Class 1: upregulated by DAF-16 and positive regulators of longevity

Table 5.2 Overview of genes in C. elegans acting downstream of DAF-16, identified by differential expression using a microarray and reduced expression of daf-2 and daf-16 by RNAi

Gene Brief description

Class 1: upregulated by DAF-16 and positive regulators of longevity

ctl-2

Peroxisomal catalase

dod-1

Member of cytochrome P450 family

hsp-16.1

Heat-shock protein

lys-7

Enzyme associated with response to pathogenic bacteria

dod-2

Thaumatin (sweet protein) associated with plant pathogenesis

hsp12.6

Heat-shock protein, a crystalline

mtl-1

Metallothionein-like, cadmium-binding protein

gei-7

Member of family of malate synthase/isocitrate lyase

dod-3

Protein of unknown function

dod-4

Aquaporin, member of family of transmembrane channels

Class 2: downregulated by DAF-16 and negative regulators of longevity

dod-17

Protein of unknown function

nuc-1

Endonuclease associated with apoptosis

dod-18

Member of Maf-like transcription factors

dod-19

Protein of unknown function

gcy-6

Putative guanylate cyclase, catalysing cGMP second messenger

dod-20

Protein of unknown function

dod-21

Protein of unknown function

vit-5

Vitellogenin, 170 kDa yolk protein

mtl-2

Protein of unknown function

dod-22

Protein of unknown function

Notes: Only the first 10 most prominent genes are shown for each class. Source: From Murphy et al. (2003).

Notes: Only the first 10 most prominent genes are shown for each class. Source: From Murphy et al. (2003).

that supports the catalysis of hydrogen peroxide, a very reactive oxygen species that may cause damage to membranes. It is also striking that class 1 includes several proteins that are known to be involved in antibacterial defence. Stress-defence systems will be discussed in more detail in Chapter 6. The class 2 genes listed in Table 5.2 are less well defined; in addition to a yolk protein, class 2 includes many proteins of unknown function. Several of the differential expressions reported in Table 5.2 were also found in a microarray study by Golden and Melov (2004), who compared a daf-2 mutant with the wild type.

How DAF-16 upregulates or downregulates all these genes is not yet clear. Lee et al. (2003) identified 17 genes in the genome of C. elegans that were orthologous between C. elegans and D. melanogaster and had a putative binding site for DAF-16 in their promoter (between the start site and 1kbp upstream). Expression analysis confirmed that six of them were differentially expressed between a daf-2 and a daf-2; daf-16 mutant, as expected in the case of genes with a causal relationship to the insulin/IGF-1 signalling pathway. Interestingly, and in accordance with Murphy et al. (2003), both the upregulated and the downregulated genes had the consensus binding motif. However, none of the 17 genes identified by Lee et al. (2003) is shared with the list of Murphy et al. (2003), which indicates that other binding sites and trans-acting factors other than DAF-16 may be involved.

Murphy et al. (2003) also discovered that a gene encoding an insulin-like peptide, ins-7, was present among the class 2 members. This protein is not only downstream of the DAF-16 pathway, but, being insulin-like, also acts as an activator of DAF-2. So, any positive signal on DAF-2 will amplify the pathway, via inhibition of DAF-16, relieving the negative regulation of ins-7 expression and INS-7 stimulation of DAF-2. This positivefeedback loop in the system was thought to contribute to synchrony across cells in the animal. When dauer larvae sense the presence of favourable food conditions and the DAF-2 pathway is activated in some cells by increasing insulin-like peptides, an amplified signal to other cells will prevent the animals emerging from the dauer stage with a mixture of dauer and non-dauer cells.

An overview of the aging-regulator system of C. elegans is given in Fig. 5.5 (Gems and McElwee 2003). The bigger picture integrates many aspects of previous studies. It explains why there are many genes with a small additive effect on aging: they are all regulated by the same transcription factor. The presence of yolk proteins in class 2 is consistent with the link between aging and reproduction, while the presence of antioxidant enzymes in class 1 is in accordance with the well-known relationship between oxygen radicals, cell damage, and aging. Another confirmation of the picture is found in Hsu et al. (2003) who showed that the transcription factor HSF-1, which is a regulator of the heat-shock response, also influences aging. Overexpression of hsf-1 extends lifespan, while reducing it shortens lifespan. So the two transcription factors DAF-16 and HSF-1 partly regulate the same genes with similar effects on aging.

Another genome-wide survey of insulin/IGF-1 signalling-mediated longevity in C. elegans is found in the work of McElwee et al. (2003, 2004). These authors compared the expression profiles of dauer larvae with those of daf-2 mutants and argued that genes promoting longevity would have an expression signature similar to the dauer profile. It turned out that in fact around 21% of genes upregulated in dauer larvae are also

Insulin Signalling Pathway

Figure 5.5 The insulin signalling pathway of C. elegans converges on repression of the transcription factor DAF-16. DAF-16 itself downregulates expression of proteins promoting aging and upregulates expression of proteins promoting longevity. Among the factors promoting aging is an insulin-like protein, INS-7, that acts upon DAF-2 as a positive-feedback link in the network and a signal to other cells. After Gems and McElwee (2003), by permission of Nature Publishing Group.

Figure 5.5 The insulin signalling pathway of C. elegans converges on repression of the transcription factor DAF-16. DAF-16 itself downregulates expression of proteins promoting aging and upregulates expression of proteins promoting longevity. Among the factors promoting aging is an insulin-like protein, INS-7, that acts upon DAF-2 as a positive-feedback link in the network and a signal to other cells. After Gems and McElwee (2003), by permission of Nature Publishing Group.

upregulated in the daf-2 mutants, and a similar pattern holds for the downregulated genes. Like Murphy et al. (2003), McElwee et al. (2004) noted a prominent representation of genes associated with stress-defence systems among the positive regulators of longevity. Several of these genes are known to be associated with the so-called drug metabolism or biotransformation system. This system of interacting enzymes, which is studied extensively in toxicology, involves two phases in which lipophilic, often aromatic, compounds are first activated and then conjugated to form water-soluble complexes that can be excreted. Phase I of the biotransformation pathway is conducted by enzymes of the cytochrome P450 family. These are haem proteins that can oxidize aromatic compounds to phenols, epoxides, and quinones, which can then be subjected to conjugation by enzymes of phase II, which attach endogenic compounds such as sulphate, glucose, glucuronic acid, or glutathione to the activated product of phase I. The system acts against an enormous variety of lipophilic compounds including many xenobiotics (drugs, environmental pollutants, and plant secondary metabolites) as well as endogenic aromatics such as steroid hormones. We will learn more about this defence system in Section 6.2.

The upregulation of many enzymes of the drug metabolism system in long-lived daf-2 mutants suggested to McElwee et al. (2004) that this system plays a central role in protection from the metabolic damage that accumulates with age (Fig. 5.6). Various cellular processes as well as xenobiotics produce ample reactive molecules that can cause permanent damage to cellular constituents. The biotransformation system can prevent such damage and upregulation of its enzymes is assumed to promote longevity. This new theory of aging is attractive since it assigns a central biological role to the biotransformation system which did obviously not evolve only to metabolize man-made chemicals such as drugs. On the other hand, the new theory could turn out to be a bit too simplistic, since biotransformation does not only detoxify chemicals, it can also activate chemicals to intermediates that cause more harm than the substrate itself. The detoxifying role of the biotransformation system depends on a delicate balance between phase I and phase II enzyme activity; a general upregulation of all enzymes would be very ineffective and could even be damaging. In addition, many biotransformation enzymes have a great number of isoforms, with different induction profiles, where each substrate needs another isoform to be metabolized effectively.

5.2.3 Longevity-regulating systems across species

One might think that the regulation of lifespan seen in C. elegans is tied so specifically to the dauer stage, which is absent in insects and vertebrates, that it does not have a validity outside nematodes. However, the converse is true! Fundamental aspects of the longevity programme elaborated in C. elegans appear to be conserved across organisms as widely different as yeast, Drosophila, and mouse; that is, the aging mechanisms are 'public' rather than 'private' (Gems and Partridge 2001; Partridge and Gems 2002a; Partridge and Pletcher 2003). This has stimulated hope for extrapolation to human longevity assurance, but it is also of relevance to ecology, since we may expect that several aspects of the genomic determination of longevity in model species are also valid for non-models in an ecological context.

In D. melanogaster, as in nematodes, the discovery of mutants with elongated lifespans has been the trigger for aging research. A first hint that regulation of aging was conserved between nematodes and fruit flies came from the observation that, as in C. elegans, D. melanogaster mutants disturbed in the insulin/IGF signalling pathway have a longer lifespan. The proteins involved in the insulin/IGF-1 signalling pathway of Drosophila are INR (insulin/IGF receptor; homologous with DAF-2 in C. elegans), an insulin receptor substrate designated CHICO, a phosphoinositide 3-kinase, and a protein kinase B. Most attention has been paid to mutations in the gene encoding CHICO, which confer a dwarf phenotype with reduced fecundity, named after the smallest of the Marx brothers. Clancy et al. (2001), studying heterozygous and homozygous D. melanogaster chico1

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