The Tick-Tock of Aging?

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Science  23 Dec 2005:
Vol. 310, Issue 5756, pp. 1911-1913
DOI: 10.1126/science.1122816

The relationship between organismal development and aging has long been a matter of intense debate. It seems natural to posit that developmental timing mechanisms that culminate in reproductive maturity continue to affect post-reproductive biology, with consequences for total organism life span. On the other hand, evolutionary theories of aging discount regulated aging per se, because the force of selection declines with age and drops precipitously after reproductive potential ends. In other words, a program that actively ages the organism is unlikely to be selected for in evolution. Instead, aging is thought to entail the passive stochastic accumulation of damage to molecules, cells, and organs, leading to loss of fertility and organismal demise. Therefore, the notion that regulated intrinsic biological timers control aging seems superficially untenable.

Just this possibility, however, has been raised by Boehm and Slack on page 1954 of this issue (1). The have found that components of a nematode's (Caenorhabditis elegans) heterochronic circuit—namely the lin-4 microRNA and its target, the nuclear protein encoded by lin-14—not only perturb developmental timing but also influence organismal life span. They do so by regulating insulin/IGF-1 (insulin-like growth factor-1) signaling, a cellular regulatory pathway whose modest decrease in activity leads to increased longevity across taxa (2).

Just as each cell in a developing organism has a positional identity that is determined by gradients of morphogens and hierarchies of transcription factor activity, cells also have a temporal identity dictated by regulatory signaling cascades. Pioneering work in C. elegans led to the discovery of the heterochronic loci (3), which constitute a regulatory circuit that confers temporal identity to the various tissues. These genes determine cellular programs of division, migration, and differentiation that are appropriate for a specific developmental stage. In addition, the interactions among these heterochronic loci ensure the proper succession of larval temporal fates. Normally, C. elegans develops to adulthood through four larval stages, L1 to L4. Worms with mutations in the heterochronic loci inappropriately express cellular programs at the wrong stage that could result, for example, in the expression of adult features in the juvenile or conversely, juvenile features in the adult. A molecular analysis has revealed that most heterochronic loci are evolutionarily conserved. Perhaps most striking are the examples of lin-4 and let-7 microRNAs, short 21-to 24-nucleotide RNAs that post-transcriptionally regulate gene expression (4-6). First discovered in the worm, orthologs are found conserved across species, including human (7). This spawned the discovery of large families of similar molecules whose diverse functions are only just beginning to be explored. As part of an early larval timer, a rise in lin-4 microRNA expression triggers larval stage L2 and later developmental programs by decreasing the expression of a molecular target, lin-14. It does so by binding with imperfect complementarity to sequences in the 3′-untranslated region of the messenger RNA (mRNA) that encodes lin-14, inhibiting translation and mRNA stability (4, 5). A decrease in the expression of lin-14 mRNA and protein is seen in the developing worm, but expression persists in some tissues in the adult.

Regulation of adult life span by the lin-4 microRNA.

(Left) When lin-4microRNA activity is high, expression of lin-14 mRNA and protein are low. Hence, the DAF-16 transcription factor is active and promotes long life. (Right) When lin-4 activity is low, lin-14 activity is high, and DAF-16 is inhibited, resulting in short life. lin-4 and lin-14gene products may work downstream of, or in parallel to, DAF-2 (the insulin-like receptor) to modulate DAF-16. Proteins are depicted as oval shapes.

However, the role of lin-14 and other heterochronic loci in the adult worm has been little explored, largely because cells of the adult are postmitotic (except for germline cells) and therefore do not display any overt stage-specific cellular programs. Clearly, though, adult animals undergo germline maturation, growth, homeostasis, and metabolic changes, as well as seemingly coordinated shifts in gene expression (8). Conceivably, such changes could arise from intrinsic timing mechanisms at work in the adult. Hence, Boehm and Slack sought to test the hypothesis that the heterochronic loci influence adult longevity.

Remarkably, they found that overexpression of lin-4 microRNA results in a modest increase in adult life span (by 15%), and conversely, that loss of a functional lin-4 gene shortens life (by 53%). These adult phenotypes, as in larvae, depend on lin-14. Worms lacking a functional lin-14 gene (loss-of-function mutants) were long-lived (by 28%), whereas those that overexpress lin-14 (gain-of-function mutants) were short-lived (by 63%). In worms lacking both lin-4 and lin-14, the long-lived lin-14 phenotype largely prevailed (a life span 14% longer than wild type), suggesting that lin-4 works at least in part through lin-14. Similarly, the developmental phenotypes of lin-14 mutants (precocious) prevail over lin-4 (delayed) in the lin-4 lin-14 double mutant worms. The fact that the same epistatic relations hold for lin-4 and lin-14 in both the developing larva and aging adult supports the idea that a similar regulatory pathway is at work.

The question then arises as to whether longevity is determined by developmental events or adult events. To address this, Boehm and Slack took advantage of conditional manipulation of lin-14 expression. By using a temperature-sensitive allele, they bypassed developmental defects and performed shifts to the nonpermissive temperature that caused a decrease in lin-14 expression in the adult. Interestingly, post-developmental temperature shifts still caused extended life span. Accordingly, decreasing lin-14 expression by RNA interference in adults also extends life, which suggests a function independent of development and specific for adults. As correlates of longevity, lin-14 loss-of-function mutants were found to be more resistant to heat stress and slower to accumulate lipofuscin, a marker of aged tissues.

Molecular genetic studies first identified insulin/IGF-1 signaling as a key modulator of nematode life span (9, 10). Remarkably, the same was later shown to be true for flies and mice (2). Specifically, decreased insulin/IGF-1 signaling results in the nuclear translocation of a transcription factor called DAF-16/FOXO. This transcription factor turns on genes for stress resistance, DNA repair, innate immunity, and heat shock, and, as a consequence, the worm's life span is doubled.

Given the central role of insulin/IGF-1 signaling in aging, Boehm and Slack asked whether lin-4 and lin-14 impinged on this pathway. Indeed, they found that worms with a mutation in daf-16 as well as in hsf-1, a longevity gene encoding a transcription factor for turning on heat shock proteins, abolished longevity mediated by lin-14. Moreover, longevity of a worm with a mutation in daf-2, the gene encoding the insulin-like receptor, was not further increased by a loss-of-function mutation in lin-14. Finally, a short-lived lin-4 mutation largely suppressed the longevity of the daf-2/insulin-like receptor mutant, placing lin-4 activity downstream or parallel to that of the receptor. These genetic studies argue that lin-4 and lin-14 could somehow regulate daf-16 via insulin/IGF-1 signal transduction or a parallel signaling pathway (see the figure).

The union of signaling pathways that control developmental timing and life span is not without precedent, because the worm nuclear hormone receptor DAF-12 operates in both (11). However, if a conserved microRNA and its target converge on DAF-16/FOXO to influence adult longevity, this raises the intriguing notion that intrinsic developmental clocks can modulate aging. Alternatively, lin-4 and lin-14 may have undescribed metabolic outputs somewhat independent of a timer. Notably, the heterochronic circuit is initialized by food cues, with nutrient inputs at distinct points of L1 and L3 diapause, periods of arrested development entered under conditions of starvation. In particular, both lin-4 and daf-16 are required for entry into the L3 dauer diapause, which is a long-lived stress-resistant stage.

In either scenario, the results of Boehm and Slack raise a myriad of questions. How do lin-4 and lin-14 converge on daf-16? What tissues are involved? Is this a mechanism of regulation observed in the wild-type worms under some conditions? What other signaling pathway components are involved? Do microRNAs regulate longevity in higher animals? If so, what are they and what are their targets?

Finally, how might the paradox of intrinsic timers and “regulated” aging be reconciled with the evolutionary theories of aging? One possibility is that the tempo of reproductive development needs to be coordinated between the tissues, as well as with nutrient availability and the environment. Under conditions of adversity, regulatory signaling pathways that delay reproduction and increase somatic endurance could adaptively retard organismal decline. When exercised in the adult, such mechanisms could secondarily extend life. Another notion is that developmental timing mechanisms that determine a species' life plan may somehow influence the life span. Perhaps the great natural variation in animal life spans is determined by such global temporal regulators.


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