To search for public, IIS-regulated determinants of longevity, we used previously published microarray data from long-lived mutant worms and mice with lowered IIS, and generated new microarray data for a long-lived IIS mutant in flies (see Table 1 for array data overview). For each species, raw data were analyzed using rigorous quality control procedures and the same statistical methods to maximize data comparability (see Materials and methods) 25.

In C. elegans, the increased lifespan of daf-2 mutants requires the downstream FOXO transcription factor DAF-16 (GenBank: NM_001026423) 9. We reanalyzed mRNA profile data comparing long-lived daf-2 mutants and non-long-lived daf-16; daf-2 double mutants, effectively a comparison of DAF-16 ON and DAF-16 OFF 24. This identified 953 differentially expressed genes (558 up-regulated, 395 down-regulated in daf-2, q < 0.1, here and below). Other transcript profiles of C. elegans IIS-regulated genes are available 1516, which closely resemble the gene lists studied here 24; these lists were generated using a different microarray platform (spotted DNA arrays), and we therefore chose not to include them in our analysis.

For Drosophila, we compared wild-type (Dahomey) and long-lived chico¹/+ heterozygotes 8. This identified 1,169 differentially expressed genes (893 up-regulated, 276 down-regulated in chico¹/+). Initially, we also examined transcript profiles from homozygous chico¹ mutants, which are slightly longer lived than chico¹/+. However, the proportion of genes showing differential expression was so high as to make data analysis impracticable (data not shown). This difficulty was likely due to the fact that homozygous chico¹ flies are sterile dwarfs, with different quantities of eggs and oocytes, and altered allometry of tissues and organs and, as a result, the mRNAs that they contain. By contrast, chico¹/+ flies are fertile and normal sized. Thus, the present analysis was only possible thanks to the semi-dominant effect of chico¹ on aging but not on fertility and size.

Finally, for the mouse, we reanalyzed data comparing gene expression in the liver of long-lived Prop-1^df/df (Ames dwarf) and Ghrhr^lit/lit (Little) mutants to normal-lived controls 26. Both mutants fail to secrete growth hormone, and have little circulating IGF-1. While comprehensive array datasets from these models are currently only available for the liver, the liver in mammals is a crucial insulin-sensitive tissue. Moreover, the comparable tissues in worms (the intestine) and flies (the fat body) have both been shown to be specific mediators of the longevity of IIS mutants 2728. In our analysis, 1,416 genes were differentially expressed in the Ames dwarf (761 up-regulated, 655 down-regulated in the mutant), and 1,042 in the Little mouse (575 up-regulated, 467 down-regulated in the mutant).

If IIS controls aging via regulated public mechanisms, we would expect to see similarities between transcriptional changes in long-lived mutants in each species. We initially reasoned that such similarities could occur on either of two levels. Firstly, IIS could regulate a set of orthologous genes in all species. Secondly, IIS could regulate genes contributing to similar biological processes in different species (for example, antioxidant defence) that result in increased longevity. This might or might not involve orthologous genes in the three species.

For gene-level (as opposed to process-level) analysis, we first identified orthologous pairs of genes between each species, and orthologous sets of genes between all three species (Additional data file 4). We then screened for ortholog pairs or sets (triplets) that showed significant (q < 0.1) changes in expression in each species, and in the same direction (up- or down-regulated given reduced IIS). Surprisingly, very few orthologous genes changed expression co-ordinately in different species, and the number of such genes differed little from that expected by chance alone. For example, only nine ortholog pairs were significantly up-regulated in the worm and fly datasets (approximately 14 would be expected by chance). However, four ortholog sets were up-regulated in the worm, fly and Little mouse, significantly more (p = 0.003) than expected by chance alone (Tables 2, 3, 4).

To further test whether the nine worm-fly ortholog gene pairs might be longevity determinants, we reduced expression of each gene in C. elegans using RNA-mediated interference (RNAi) in the long-lived, RNAi-hypersensitive strain rrf-3(pk1426); daf-2(m577) (Table 4; Additional data file 5). As a positive control we performed RNAi using daf-16 which, as expected, resulted in a large decrease in lifespan (57%). Of the test genes, RNAi of only one, the pantothenate kinase pnk-1, significantly shortened lifespan. However, pnk-1 RNAi also did this in a normal-lived control strain (data not shown), and it also causes sterility, larval arrest, and embryonic lethality 29. The reduced lifespan may therefore reflect a requirement for pnk-1 for overall viability rather than prevention of aging. Pantothenic acid is a component of coenzyme A, the acetylated form of which plays a key role in the citric acid cycle. Pantothenate kinase catalyzes the first step in coenzyme A synthesis. In conclusion, the transcriptional response to reduced IIS shows very little evolutionary conservation at the level of gene orthology.

The lack of conservation seen at the level of gene orthology was unexpected. It led us to wonder whether perhaps, in some cases, IIS-regulated functions might be performed in different species by paralogous genes rather than orthologous ones. To this end, we looked at expression of paralogous genes in long-lived worms, flies and mice in two ways. Firstly, we examined all sets of paralogs where there was either n ≤ 2 or n ≤ 3 paralogous genes present in the gene list for each individual species (see Materials and methods). We counted the number of paralog sets (pairs, triplets or quadruplets) where at least one gene was differentially expressed in each species, and in the same direction. Secondly, we examined all paralog sets, whatever their size, and counted the number of paralog sets where a substantial number of genes showed differential expression in the same direction (we used the arbitrary cut-off of >50%). In addition, we counted again the number of orthologs with altered expression in more than one species, using the same statistics (see Materials and methods). For each of these four levels of conservation (ortholog set, paralog sets of size n ≤ 2, n ≤ 3 or any size), we asked whether the number of ortholog or paralog sets identified were more than expected by chance alone. To this end we performed bootstrap analysis on paralogous groups, comparing the observed number of differentially expressed paralogous groups with the numbers obtained by drawing the lists of differentially expressed groups at random (see Materials and methods).

The results of this analysis are shown in additional Table 1 in Additional data file 3. As before, at the level of orthology, there was no conservation of IIS regulation. When this analysis was loosened to include small and then large paralog groups, for most comparisons, there was still no significant conservation of IIS regulation. However, one triplet comparison showed an over-representation of IIS-regulated genes in all paralog comparisons: there were up-regulated genes in worms, flies and Little mice in four paralog sets (p = 0.01) (additional Table 1 in Additional data file 3). Data for the individual four genes in each of the four models examined are shown in additional Table 2 in Additional data file 3. The four paralog sets identified two proteins that we previously identified as IIS regulated in worms and flies: pantothenate kinase and glycerol-3-phosphate dehydrogenase. The two other paralog sets were, firstly, fructose-biphosphate aldolase and, secondly, beta-glucosidase, lactase phlorizinhydrolase and related proteins. Thus three-quarters of IIS-regulated paralog sets are linked to sugar metabolism. In summary, our analysis of paralog sets supports the unexpected conclusion that there is little evolutionary conservation between C. elegans, Drosophila or mouse of IIS regulation at the gene level.

Next we asked whether similar biochemical and cellular processes show conserved regulation at the transcriptional level by IIS. To this end we screened each dataset for biologically related genes or structurally related gene families showing co-ordinately increased or decreased expression in response to reduced IIS. Using biological annotation available through GO and Interpro, each dataset was analyzed using Catmap 30. This software program assigns significance to gene categories based on their relative statistical ranking or representation within the dataset. This generated a list of gene categories showing significantly altered expression in each species; of these, a subset showed similar and significant changes in all three species (Figure 1; Table 5; Additional data file 6).

Next we tested whether the number of shared gene categories enriched for differentially regulated genes was more than predicted by chance alone. To do this, we performed bootstrap analysis of gene categories, drawing categories at random and computing p values from the number of common categories between the various combinations of gene lists (see Materials and methods). According to this analysis, for most comparisons the number of shared categories is more than predicted by chance alone, particularly where genes are up-regulated in the long-lived mutants (Additional data file 7). However, it should be borne in mind that the statistical test used assumes that the various categories are independent of one another, and in some cases this may not be the case. For example, cytochrome P450 (CYP) enzymes and GSTs can be subject to coordinate regulation 31; moreover, given that the GO annotation is not a strict hierarchy, different GO categories may be non-independent. Thus, while the conclusion that no more gene classes are seen than expected by chance alone may be relied upon, the opposite conclusion cannot be. Nonetheless, the categories represented in Table 5 do potentially correspond to conserved IIS-regulated processes. These may include public determinants of aging that are not dependent on parallel transcriptional changes in orthologous genes.

An expected outcome of this analysis was that the two microarray datasets from the mouse would share more over-represented gene categories with one another than with the two invertebrate datasets. In terms of the individual genes showing altered expression, there are strong overlaps between the Prop-1^df/df and Ghrhr^lit/lit datasets 26. However, the number of shared categories is surprisingly low (Figure 1). To some degree, this may reflect the fact that the Prop-1^df/df mutation is more pleiotropic, blocking production of thyroid stimulating hormone and prolactin in addition to growth hormone. It may also reflect the larger size of the lists of differentially expressed genes from the dwarf mouse studies, which can reduce the sensitivity of the test for overlapping gene categories. More positively, it suggests that comparing datasets from the two mouse strains has acted as a strong filter to exclude numerous gene categories unlinked to the increased lifespan phenotype.

The majority of the common up-regulated GO categories are involved in sugar catabolism and energy generation (Table 5), implying that these processes are activated in IIS mutant animals. This is likely to reflect insulin-like control of sugar homeostasis by IIS in the three organisms. It is also consistent with a recent study of genes linked to energy metabolism in the worm dataset, which implies increased conversion of fat to carbohydrate and conservation of ATP stocks 32. Among the shared down-regulated GO categories are many linked to protein biosynthesis and translation (Table 5), implying down-regulation of these processes in long lived milieus. Interestingly, it was recently discovered that lifespan in C. elegans is increased by loss of function of several genes promoting protein translation, including translation initiation factors and ribosomal proteins 202122. Thus, our results suggest that reduced protein translation may be a public mechanism of longevity assurance regulated by IIS (Figure 2).

Most of the Interpro domain gene families showing conserved up-regulation in IIS mutants are linked to cellular detoxification (that is, drug or xenobiotic metabolism) (Table 5; Figure 3). These correspond mainly to CYP, short-chain dehydrogenase/reductase (SDR; note that glucose-ribitol dehydrogenases are a type of SDR), and GST enzymes. Our analysis suggests the possibility that this detoxification system is a public mechanism of longevity assurance, protecting against the stochastic molecular damage that underlies the aging process.

The association of increased expression of gene classes linked to cellular detoxification with longevity in three species, coupled with the lack of gene-level orthology, prompted us to examine the evolutionary relationships of these gene families in more detail. To do this, we constructed phylogenetic trees for each of three families in worms, flies, and mice, and then examined the distribution of IIS-regulated gene expression. Figure 4 shows a phylogenetic tree of worm, fly and mouse GSTs, marked to show differentially expressed genes (see also Additional data file 2). We also examined the phylogenetic tree of UDP-glucuronosyltransferases (UGTs), a major class of phase 2 enzymes, which are over-represented in genes up-regulated in C. elegans daf-2 mutants and long-lived dauer larvae 24. In each case, the phylogenetic distribution of IIS-regulated genes is apparently random (Additional data file 2). Significantly, comparing worms, flies and mice, there are no orthologs for most genes in these families. In each of these large gene families, individual genes are, in most cases, the products of lineage-specific expansions 33. This is typical of proteins whose function entails recognizing diverse chemical moieties in a changing chemical environment. Such proteins include chemoreceptors and antigen recognition proteins of the innate and acquired immune systems, as well as those involved in cellular detoxification 3334.

Finally, we explored whether IIS transcriptional responses are regulated by conserved DNA binding factors. Using the program Clover (Cis-eLement OvEr-Representation) 35, we examined the upstream regions of the differentially expressed genes in each species for over-representation of known DNA-binding motifs (Additional data file 8). Many motifs were identified when examining each individual dataset. Of these, none was over-represented among genes regulated in the same direction in all three species. The FOXO1-binding site was over-represented among genes up-regulated in long-lived worms and mice; by contrast, this motif was over-represented among genes down-regulated in long-lived flies (Additional data file 8). Overexpression of FOXO increases lifespan in both worms and flies 27. These findings could imply that down-regulation of FOXO-regulated genes influences lifespan in flies (perhaps lowering damage-generating processes), while up-regulation is more important in worms and mice (perhaps increasing damage-protective processes). Furthermore, an analysis using the EASE program of gene classes over-represented in genes with putative FOXO-binding sites in worms and mice revealed little similarity between these genes at this level (data not shown). Thus, while the role of FOXO in mediating transcriptional regulation by IIS shows some evolutionary conservation, the IIS-regulated target genes of FOXO may be conserved only at the level of the gene families and the biological processes that they control - not at the level of orthology.

The role of IIS as a regulator of aging shows evolutionary conservation. The effects of IIS on lifespan reflect the action of IIS-regulated genes and biochemistries of aging and longevity. In this study, we have asked the question: are these genes and processes public (evolutionarily conserved) or private (lineage specific)? We have done this by means of a cross-species comparison of transcript changes seen in long-lived nematodes, insects and mammals with lowered IIS when compared to normal-lived controls. To be able to do this we developed a novel, multi-level cross-species comparative method, comparing gene expression at the levels of genetic orthology, paralogy (in small and large paralog sets), and gene classes. We detected little evolutionary conservation of IIS regulation at the orthologous or paralogous gene levels. However, at the genes class or process level some evolutionary conservation was observed, including several processes previously associated with aging.

The absence of detectable regulation by IIS of orthologous genes in the three animal models tested was unexpected, for several reasons. Firstly, even if the same IIS-regulated genes did not regulate aging in worms, flies and mice, one would expect that some of the genes mediating the effects of IIS on growth and sugar metabolism would be conserved at the level of orthology. Secondly, an earlier study examined putative direct transcriptional targets of FOXO in C. elegans and Drosophila, focusing on 17 C. elegans-Drosophila ortholog gene pairs with predicted DAF-16 binding sites in their promoter regions 36. There, a third of C. elegans orthologs showed IIS regulation, suggesting possible evolutionary conservation of IIS-regulated genes at the level of orthology. However, no data on IIS regulation of Drosophila orthologs were reported in that study. Our findings point to the opposite conclusion: that the set of genes regulated by IIS is largely lineage specific.

If significant numbers of orthologous genes were robustly IIS regulated in similar ways in multiple tissues, then it is likely that the analytical approaches that we have employed would have detected this. However, it remains possible that orthologous genes regulated similarly by IIS eluded our analysis, for several reasons. Firstly, microarray analysis may have failed to detect small but functionally significant changes in transcript levels, for example, genes showing IIS-regulated expression in only a small proportion of cells in C. elegans or Drosophila. Secondly, if the direction of IIS regulation is different in different tissues in the invertebrate models, this could prevent detection of IIS regulation. Thirdly, it may be that in extra-hepatic tissues, transcript profile changes resulting from Prop-1^df/df and Ghrhr^lit/lit are more similar to those in C. elegans and Drosophila IIS mutants. The liver consists mainly of dividing cells whereas, in the invertebrate models, adult somatic tissues consist largely of post-mitotic cells. Recent mouse studies suggest that age-related changes in gene expression may differ between mitotic and post-mitotic tissues 37. Fourthly, gene regulation by IIS might differ between sexes (we compared data from hermaphrodite worms, females flies and male mice). Finally, although young adults of each organism were used, it is possible that the slight differences in their relative age constituted a confounding variable. More generally, the value of transcript profile studies is limited by the fact that changes in mRNA levels may not correspond to changes in levels of protein products of mRNA translation. Further studies are warranted to establish with greater certainty the extent of evolutionary conservation of regulation of genes by IIS. For example, there may be differences in the degree of evolutionary conservation of IIS regulation by direct targets of FOXO versus genes further downstream in a FOXO-regulated cascade. It would be useful to identify direct targets of FOXO, for example, using chromatin immunoprecipitation 38 and to perform cross-species comparisons of their IIS regulation.

In contrast to our studies of orthologous or paralogous genes, our comparative analysis at the gene class level identified a number of candidate gene classes and processes showing an evolutionarily conserved pattern of regulation in long-lived mutants with reduced IIS (Table 5). We performed this analysis with the aim of identifying candidate evolutionarily conserved processes that mediate the effects of IIS on aging. However, IIS is also a major regulator of growth and metabolism (including sugar homeostasis), so the presence of any of the gene categories in Table 5 may reflect a role in these other processes, rather than in aging. For example and as expected, many categories associated with sugar catabolism are up-regulated in the long-lived mutants in all three species, consistent with lowered insulin signaling. This demonstrates that methods used here are sensitive enough to identify known insulin-regulated gene categories.

Clearly, the presence of any of the gene categories in Table 5 may reflect a role in aging or in processes not linked to aging. However, a number of the gene categories present are linked to one or the other of two biological processes recently implicated in the control of aging. These are protein biosynthesis (for example, GO:0006412 protein biosynthesis, GO:0043037 translation, and GO:0045182 translation regulator activity) and GST activity (IPR004045 Glutathione-S-transferase N-terminal and IPR004046 Glutathione-S-transferase C-terminal). Data in Table 5 imply that protein biosynthesis and GST activity are down-regulated and up-regulated, respectively, in long-lived mutant worms, flies and mice. Potentially, this contributes to longevity (Figure 2).

Several recent studies imply that increased protein biosynthesis accelerates aging. Lowered expression of a number of genes involved in mRNA translation, ribosomal proteins, translation initiation factors and ribosomal protein S6 kinase results in reduced rates of protein biosynthesis and increased lifespan in C. elegans 202122. Similarly, deletion of ribosomal protein genes can increase replicative lifespan in the budding yeast S. cerevisiae 19. Over-representation of genes associated with protein biosynthesis among those down-regulated in long-lived C. elegans, Drosophila and mice implicates this process as a public, IIS-regulated mechanism controlling aging. However, it should be noted that the individual genes involved in protein biosynthesis whose expression was shown to affect C. elegans aging were not themselves IIS regulated 21. How lowered protein synthesis might increase lifespan is unknown, although in C. elegans these perturbations increase heat stress resistance, suggesting that lowered protein synthesis leads to induction of somatic maintenance functions 21.

GSTs detoxify a wide range of electrophilic (that is, oxidizing) and often toxic compounds by conjugation with glutathione (GSH) 39. Such electrophiles can otherwise react with nucleophilic centers, for example, in proteins, causing molecular damage. Within biogerontology, there is a growing consensus that the primary cause of biological aging is accumulation of damage at the molecular level. Studies to date broadly support the view that longevity-assurance processes prevent accumulation of damage by promoting somatic maintenance processes 404142. The mechanisms involved include reduction or removal of the causes of molecular damage, and repair or turnover of damaged molecules. Thus, a role of GSTs in protection against aging is easy to rationalize.

More importantly, there is some direct experimental evidence for a role of GSTs in longevity assurance. The C. elegans genes gst-5 and gst-10 encode GSTs that detoxify 4-hydroxy-2-nonenal (HNE), which is a major product of peroxidation of membrane lipids and a mediator of the pathophysiological effects of oxidative stress 43. RNAi knockdown of either of these genes reduces both HNE-conjugating activity and lifespan 2344. Overexpression of GST-10 or of murine mGSTA4-4 (also active against HNE) increases HNE-conjugating activity and, significantly, lifespan 23. The over-representation of GST genes among genes up-regulated in long-lived mutant C. elegans, Drosophila and mice with reduced IIS suggests that GST activity may represent a public, IIS-regulated mechanism of longevity assurance.

The possible broader implications of the observed association between GST gene expression and extended lifespan (Table 5) may be considered in three overlapping biochemical contexts: defence against reactive oxygen species (ROS), the biology of GSH, and broad spectrum detoxification (that is, drug metabolism). GSTs play a major role in detoxifying a broad range of oxidized breakdown products of macromolecules that form during periods of oxidative stress 39. These pro-oxidant products include α,β-unsaturated carbonyls such as HNE, hydroperoxides and epoxides. ROS such as superoxide and hydrogen peroxide have long been viewed as potential major contributors to the molecular damage that underlies aging 45. Thus, elevated GST levels could reflect a broader up-regulation of antioxidant defenses in these three long-lived models. However, looking at transcript levels for genes encoding superoxide dismutase (SOD), which scavenges superoxide, we see that while several sod genes are up-regulated in C. elegans, this is not the case in Drosophila or the mouse (Table 6). Consistent with this, increased SOD has been observed in daf-2 C. elegans 46, but not chico¹/+ Drosophila 8. In terms of hydrogen peroxide scavengers, there is some evidence of increased catalase mRNA levels in long-lived C. elegans and Drosophila, but not in the mouse. In C. elegans, there is a tandem array of three very similar genes encoding catalase, ctl-1, ctl-2 and ctl-3 47. Our microarray analysis shows strongly increased expression of ctl-3 in daf-2 animals (q < 0.003); however, for the purposes of analysis in this study, ctl-3 data were excluded due to predicted promiscuity in probe binding between clt-3 and ctl-1. In Drosophila there is a possible increase in catalase mRNA levels (log2 fold change 0.3, q = 0.045). The absence of increased transcript levels of catalase and Mn SOD genes in Prop-1^df/df mouse liver was unexpected, since increased catalase levels have been reported in this tissue 48. Overall, our transcript profile comparison provides little support for the view that direct defense against superoxide and hydrogen peroxide is a regulated public mechanism of longevity assurance.

A second perspective on possible GST function in aging is within the context of a broader, GSH-associated biochemistry. Besides its role in detoxification by GSTs, GSH itself acts as an antioxidant 39, and the ratio of reduced to oxidized GSH is a determinant of cellular redox status. GSH-mediated processes can clearly influence aging. For example, in Drosophila overexpression of glutamate cysteine ligase (γ-glutamylcysteine synthetase), the major rate-limiting enzyme in GSH biosynthesis, extends lifespan 49. Moreover, overexpression of methionine sulfoxide reductase, an enzyme that uses GSH to restore oxidized methionine in proteins by reducing methionine sulfoxide, also increases Drosophila lifespan 50.

Hepatic metabolism in Prop-1^df/df (Ames dwarf) mice appears to be geared up for increased GSH production and usage 5152535455. Both GSH levels and GSH/GSSG ratios are increased 53, and there is increased activity of the trans-sulfuration pathway, implying increased flux of thiols from methionine to cysteine and GSH 5155. Possibly increased GSH production retards aging by supporting a range of mechanisms that protect against an age-related increase in levels of toxic electrophiles.

Beyond the biology of GSH, GSTs may be viewed as part of a wider system of cellular detoxification involving two phases: phase 1 (functionalization reactions), and phase 2 (conjugative reactions) 31 (Figure 3). CYPs and short-chain dehydrogenase reductases (SDRs) are major effectors of phase 1 metabolism, which through oxidative (CYP) or reductive (SDR) chemistry can bioactivate toxic molecules. Activated metabolites from phase 1 are substrates for effectors of phase 2 metabolism, such as the GSTs, UDP-glucuronosyl/UDP-glucosyltransferases (UGTs) and sulfotransferases. Phase 2 reactions can both detoxify and increase solubility of toxic moieties, aiding excretion. In mammals, this system acts in a coordinate fashion to dispose of a very broad range of xenobiotic and endobiotic compounds, including toxins, drugs, carcinogens and damaged cellular constituents 31.

Interestingly, CYPs and SDRs are also over-represented among genes up-regulated in long-lived C. elegans, Drosophila and mice (Table 5) (though UGTs and sulfotransferases are not). This suggests that the cellular detoxification more broadly might play a role in longevity assurance. Genes encoding CYPs, SDRs and UGTs are also over-represented among genes whose expression is increased in long-lived C. elegans dauer larvae relative to larvae that have exited the dauer stage 2456. In mice, caloric restriction and Prop1^df/df have additive effects on longevity. Phase 1 and phase 2 detoxification genes are up-regulated in both contexts and, in some cases, show additive increases in expression in Prop1^df/df mice subjected to caloric restriction 57. In summary, a growing number of studies show correlations between cellular (phase 1, 2) detoxification and longevity.

Studies in C. elegans imply that IIS exerts its effects most strongly during the reproductive period in the first few days of adulthood 58. This could imply that damaging aspects of protein synthesis and generation of toxins that drug-metabolizing enzymes (DMEs) protect against are elevated during this period, perhaps due to reproduction.

Our results suggest that protein translation, GST activity and possibly the broader cellular detoxification system may represent 'semi-public' mechanisms of longevity determination: the processes show evolutionary conservation while the individual genes do not. In the case of GSTs, this could imply that different toxins are being cleared in different evolutionary lineages, that is, that the cause of aging, the diverse harmful molecular species that this system targets, may differ between species. Thus, although damage-causing toxins appear implicated as a cause of aging-related damage in all three species, the specific toxins involved may include some that are evolutionarily conserved and others that are lineage-specific.

The lack of gene orthology between DMEs might seem to suggest that damage-causing toxins are private. However, in at least one case this is not the case. Up-regulation of GSTs that detoxify HNE occurs both in C. elegans daf-2 mutants (gst-10 17) and liver of Prop1^df/df and Ghrhr^lit/lit mice (GSTA4 in both cases 26), although these genes are not orthologous (Figure 1b in Additional data file 2). Moreover, expression of murine GSTA4 in C. elegans lowers HNE levels and increases lifespan 23. This demonstrates that convergent evolution can lead to similar substrate specificities in non-orthologous DMEs. Significantly, a major source of HNE is oxidative damage to lipid, consistent with reactive oxygen species acting as a public mechanism of aging 6.

In principle, toxins contributing to aging that are lineage-specific could contribute to the lineage specificity of aging-related pathologies. According to this view, aging involves stochastic mechanisms that are partially public and partially private. A summary overview of this interpretation is shown in Figure 5. Here, public regulators of lifespan (for example, IIS) regulate semi-public mechanisms of longevity assurance (for example, cellular detoxification), which act on both private and public types of damage generation (for example, toxins). In the specific example discussed above, IIS regulates a semi-public mechanism of longevity assurance (GSTs with HNE-conjugating activity) acting against a public mechanism of aging (HNE toxicity).