No evolutionary conservation of regulation by IIS at the level of gene 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
, focusing on 17 C. elegans
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
. 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-1df/df
are more similar to those in C. elegans
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 ). 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 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 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 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 ).
Decreased protein biosynthesis: a candidate longevity assurance process in multiple animal species
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
]. Similarly, deletion of ribosomal protein genes can increase replicative lifespan in the budding yeast S. cerevisiae
]. Over-representation of genes associated with protein biosynthesis among those down-regulated in long-lived C. elegans
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
GST activity: a candidate longevity assurance process in multiple animal species
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 [40
]. 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
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 [23
]. 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
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 ) 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 ). Consistent with this, increased SOD has been observed in daf-2 C. elegans
], but not chico1
]. In terms of hydrogen peroxide scavengers, there is some evidence of increased catalase mRNA levels in long-lived C. elegans
, but not in the mouse. In C. elegans
, there is a tandem array of three very similar genes encoding catalase, ctl-1
]. Our microarray analysis shows strongly increased expression of ctl-3
< 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
. 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-1df/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.
Expression of SOD genes in mutant worms, flies, mice with reduced insulin/IGF-1 signaling
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
Hepatic metabolism in Prop-1df/df
(Ames dwarf) mice appears to be geared up for increased GSH production and usage [51
]. 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 [51
]. 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 ). 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
and mice (Table ) (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 [24
]. In mice, caloric restriction and Prop1df/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 Prop1df/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.
An overview of evolutionary conservation of biological mechanisms controlling aging
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
]) and liver of Prop1df/df
in both cases [26
]), although these genes are not orthologous (Figure 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 . 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).
Figure 5 Different determinants of longevity may be public, semi-public or private. Our results suggest that public regulators of lifespan regulate semi-public mechanisms of longevity assurance, which may in turn act on a combination of private and public mechanisms (more ...)