Sci. Aging Knowl. Environ., 12 October 2005
Vol. 2005, Issue 41, p. pe31
[DOI: 10.1126/sageke.2005.41.pe31]

PERSPECTIVES

C. elegans Gives the Dirt on Aging

Maren Hertweck

Laboratory of Bioinformatics and Molecular Genetics, Institute of Biology 3, Albert-Ludwigs University of Freiburg, Schänzlestrasse 1, D-79104 Freiburg, Germany. E-mail: maren.hertweck{at}biologie.uni-freiburg.de

http://sageke.sciencemag.org/cgi/content/full/2005/41/pe31

Key Words: C. elegans • insulin/IGF-1 • DAF-2; SKN-1 • Tubby • HSP-16.2 • anticonvulsants

Introduction

There is steadily growing interest in understanding the genetic mechanisms that underlie aging--in part, a result of the remarkable increase in human life expectancy in the Western world and the exponential growth of the aging population (including researchers). Plenty of evidence gathered over the past 10 years indicates that Caenorhabditis elegans is an ideal model organism to study cellular and organismal aging (see Johnson Subfield History), including the relevant signaling pathways that regulate nutrition, metabolism, stress response, and life span. More recently, the success of whole-genome sequencing and the implementation of tools for genome-wide monitoring of gene expression and analyses offer an opportunity to identify on a large scale new genes and pathways associated with aging.

The results of such studies were discussed at the 15th Biennial C. elegans Conference held in June at the University of California, Los Angeles campus, a wonderful and stimulating location for discussing science. The biannual conference is the only worldwide international meeting focusing exclusively on C. elegans. This year, a separate session of the meeting was devoted entirely to aging-related research. This Perspective reviews some of the highlights from that session.

Going Genome-Wide

The aging-related research session opened with a presentation by Malene Hansen (University of California, San Francisco) and Siu Sylvia Lee (Cornell University, Ithaca, New York), who independently reported data from the first genome-wide, functional screens for longevity genes in C. elegans (1, 2).

C. elegans life span is known to be influenced by several genetic pathways and processes including the DAF-2/insulin/IGF-1 pathway, the reproductive system, food intake, and mitochondrial activity (Fig. 1). The forkhead transcription factor DAF-16/FOXO appears to be a key regulator of life-span extension in animals defective in insulin signaling and mutants lacking germline signaling (3). In addition, overexpression of the SIR-2.1 histone deacetylase and the JNK-1 Jun kinase can increase life span in a DAF-16-dependent manner (see "Feeling Spunky With JNK"). In contrast, DAF-16 is not required for the extension of life span induced by dietary restriction or by perturbations of mitochondrial activities.



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Fig. 1. Signaling pathways and processes that contribute to the complex regulation of life span in C. elegans. Inactivation of DAF-2/insulin, TOR, and germline signaling, and decreased mitochondrial respiration and food intake, as well as overexpression of SIR-2 histone deacetylase and JNK-1 Jun N-terminal kinase, increase life span in a DAF-16/FOXO-dependent manner. Representative genes involved in these processes are marked in blue.

 
To identify new genes acting in these pathways and determine whether additional pathways might also influence aging, Hansen and Lee screened for animals exhibiting enhanced longevity after the function of different genes was down-regulated using RNA interference (RNAi). The researchers used a genomic RNAi bacterial feeding library containing 16,757 C. elegans open reading frames (about 87% of the total number). Hansen identified 23 new candidate longevity genes and assigned these genes to specific pathways. Most of the genes identified fell into one of four classes: genes that influence life span through DAF-16/FOXO (e.g., ttr-1, maoc-1, sinh-1, gpi-1, and ddl-1, -2, and -3), genes that influence respiration (e.g., nuo-3, -4, and -5, cco-2, atp-4 and -5, and asb-2), genes that affect the response to dietary restriction (e.g., sams-1, rab-10, drr-1, and drr-2), and genes that affect integrin signaling (pat-4 and pat-6). One of the most important findings of this study was that dietary restriction extends life span by reducing RNA concentrations of several key genes, including sams-1, which encodes S-adenosyl methionine synthetase, an enzyme required for the methylation of many macromolecules. This finding is consistent with other reports indicating that aging is associated with posttranscriptional modifications of proteins.

Lee reported that knocking down the expression of 89 different genes by RNAi extended C. elegans life span. Genetic epistasis analyses indicated that some of the genes identified in this screen act upstream of the DAF-16/FOXO transcription factor and the SIR-2.1 deacetylase, whereas others function independently of these two signaling molecules and probably define new pathways that control life span, including ones involved in metabolism, signal transduction, protein turnover, and gene expression. The large number of longevity regulators reported by Lee may provide new entry points for further investigations that will likely reveal important insights into the molecular control of organismal longevity.

Making Microarrays

Two presentations discussed the use of microarray analysis to address different questions about aging. Josh McElwee, from University College London, asked the question: What processes regulated by insulin/IGF-1 signaling (IIS) produce potent effects on aging? In particular, his work focused on whether the processes regulated by IIS in nematodes and fruit flies are evolutionarily conserved (or "public"). First, he sought orthologous gene pairs showing similar IIS regulation, but did not find any. However, when IIS-regulated genes were compared at a process level, McElwee found that several gene classes involved in phase 1 and phase 2 drug metabolism (which dispose of toxic lipophilic and electrophilic metabolites in cells) were up-regulated in both long-lived worms and flies. Interestingly, the analysis of genes showing altered expression in long-lived dwarf mice gave similar results. Based on C. elegans studies, Gems and McElwee proposed that phase 1 and phase 2 drug detoxifications are public mechanisms the cell uses to ensure longevity--this "publicness" is detectable only at the level of process rather than orthology (4).

Pamela L. Larsen of the University of Texas Health Science Center at San Antonio, Texas, discussed the relation between environmental factors and longevity. Cooler temperatures show a direct, conserved relation with longevity, as well as the DAF-2 insulin/IGF-1 signaling pathway, in different species. In C. elegans, Larsen has shown that the relation between phenotype (longevity) and environment (temperature) depends on the status of the DAF-2 insulin receptor. To uncover early changes in gene expression caused by either cultivation temperature or mutation of the daf-2 gene, Larsen employed microarray analysis followed by real-time reverse transcription polymerase chain reaction analysis. She concluded that some of the transcriptional changes identified might contribute to differences in life expectancy, metabolic rate, body size, or fat storage. Genetic analyses to test this hypothesis are now under way.

Stressed Out

The oxidative damage theory of aging argues that senescence--a type of cellular death (see "More Than a Sum of Our Cells") --occurs as a result of cumulative cellular and systemic damage from oxidative stress (see "The Two Faces of Oxygen"). Several stress defense mechanisms exist in C. elegans. Jae Hyung An of the Joslin Diabetes Center, Boston, Massachusetts, looked at one such mechanism involving the conserved SKN-1 oxidative stress response protein. The activity of SKN-1--a functional homolog of the human stress-defense proteins Nrf1 and Nrf2, which is required for oxidative stress response and normal life span in C. elegans (5)--is controlled by the glycogen synthase kinase-3 (GSK-3) signaling pathway. SKN-1 is a basic region transcription factor that localizes to intestinal nuclei and induces the expression of several genes in response to oxidative stress. In the absence of stress, GSK-3 phosphorylates SKN-1, preventing it from localizing to the intestinal nuclei and activating transcription. One of the targets of SKN-1 is the phase 2 oxidative stress response gene gcs-1, which encodes glutamate cysteine ligase, an enzyme that is rate limiting for glutathione synthesis.

Shane L. Rea from the University of Colorado, Boulder, discussed oxidation from a different angle. C. elegans Mit mutants have a defect in the mitochondrial electron transport chain, which leads to endogenous oxidative stress (6). Mit mutants normally experience an elevated level of oxidative stress that, in turn, acts to initiate a life-prolonging, protective response. As a result, these mutants tend to live longer than the wild-type counterparts. Glutathione-S-transferase (GST-4) is a robust marker of oxidative stress response in C. elegans and is transcriptionally activated by SKN-1. However, Rea found that different Mit mutants use different survival strategies, some of which are dependent on SKN-1, whereas others are not. The work suggests that several survival pathways exist in C. elegans.

The glyoxalase pathway is also responsible for removing oxidative damage. Michael Morcos of the University of Heidelberg, Germany, presented work on the role of the C. elegans glyoxalase-I (CeGly1) gene in determining life span. In aged animals, the expression of CeGly1 is substantially reduced. Overexpression of CeGly1 in aged animals reduces formation of methylglyoxal-derived advanced glycation end products and reactive oxygen species (ROS) and increases life span. These data suggest that posttranscriptional protein modifications are involved in determining life span and that the aging process is associated with a down-regulation of enzymatic defense systems.

Anders Olsen from the Buck Institute, Novato, California, described a novel function of an evolutionary conserved checkpoint pathway in determining organismal resistance to stress and, thereby, life span. In a genetic screen for increased stress resistance, he identified a mutation in the cid-1 gene, the homolog of the S. pombe checkpoint gene Cid1, which controls progression through the cell cycle. The mutant was highly resistant to cytotoxic and genotoxic stress and lived longer than wild-type animals. Down-regulation of two other DNA-damage checkpoint genes, chk-1 and cdc-25, with RNAi resulted in similar phenotypes, demonstrating that these checkpoint proteins that act during DNA synthesis (S phase) and mitosis (M phase) represent a novel class of enzymes that determine stress response and life span in nondividing cells.

Metabolism, Reproduction, and Other Factors

One of the few single-gene mutations known to cause obesity in mammals occurs in the tubby gene (7). Mutations in tubby result in adult-onset obesity, insulin resistance, and infertility. In C. elegans, deletion of the tubby ortholog, tub-1, leads to an accumulation of triglycerides--in other words, increased fat storage. Arnab Mukhopadhyay of the University of Massachusetts Medical School, Worcester, discussed a novel role for tub-1 in life-span regulation through the IIS pathway (8) (see "Fat-Free Longevity"). A yeast two-hybrid screen identified a RabGTPase-activating protein called RBG-3 that interacts with TUB-1. The rbg-3 gene is expressed in the same set of neurons as tub-1 and functions in tub-1-dependent fat storage, but has no effect on life span. Thus, Mukhopadhyay concluded that tub-1 controls life span and fat storage through two distinct downstream mechanisms, thereby decoupling DAF-16 and RGB-3 from one another.

A couple of presentations described new markers of physiological aging in C. elegans. Beate Gerstbrein of Rutgers University, Piscataway, New Jersey, used in vivo spectrofluorimetry to reveal biomarkers indicative of healthspan in C. elegans (9). In many species, autofluorescent lipofuscin and advanced glycation end products (also called age pigments) accumulate inside cells of aging organisms, yet little is known about their formation under physiological conditions or their specific contribution to the complex aging process (see Gray Review). Gerstbrein explained that in wild-type worms, age pigments increase during adulthood, accumulating slowly during the reproductive phase and more rapidly during the postreproductive period. In long-lived insulin-signaling mutants and dietary-restricted mutants, age pigments accumulate much more slowly. In contrast, elimination of the transcription factor DAF-16/FOXO results in shorter life and increased accumulation of age pigments, supporting the notion that daf-16 is progeric. Surprisingly, mutations resulting in increased mitochondrial ROS production do not affect age-pigment accumulation, indicating that oxidative stress does not play a role in generating these species in vivo. Regardless of the actual mechanisms involved, Gerstbrein concluded that age pigment accumulation provides a quantitative measurement of physiological aging in C. elegans.

Thomas E. Johnson from the University of Colorado, Boulder, discussed stochastic effects that make a big difference in how long worms live (10) (see "How Long Do I Have, Doc?"). According to his analysis, neither the genetic background nor environmental cues can account for the huge variation in life span typically reported by almost every longevity study performed in C. elegans over the past 25 years. Using isogenic populations of the nematode, in which genetic variation is zero, Johnson found that on the first day of adulthood, "chance" variations in the concentrations of a green fluorescent protein (GFP) reporter fused to the promoter from the heat shock protein gene hsp-16.2 remarkably predict up to fourfold variations in life span. The same reporter also predicts the subsequent thermal-stress resistance in these organisms. Therefore, his research group selected worms displaying the brightest and dimmest GFP signals from an entire population and found that the two groups are distinct with regard to life expectancy and heat stress resistance. Johnson argued that HSP-16.2 itself is unlikely to be responsible for the observed differences, but rather reflects a hidden, heterogeneous, but now quantifiable, physiological state that dictates the ability of an organism to deal with the rigors of living.

Fountain of Youth

Genetic studies have elucidated several mechanisms that regulate aging, as illustrated by the presentations at the C. elegans meeting, but the development of "treatments" for aging remains a challenge. Kimberley Evason of Washington University, St. Louis, Missouri, presented data on anticonvulsant drugs that extend C. elegans life span (11). Among 19 drugs tested, three anticonvulsants--ethosuximide (ETH), trimethadione, and 3,3-diethyl-2-pyrolidinone--increased life span and delayed age-related declines in physiological processes, such as fast body movement and pharyngeal pumping. ETH showed the most prominent effect on life span by extending the mean age of survival by 17%. To identify molecular targets of ETH and trimethadione, Evanson conducted a forward genetic screen under concentrations of ETH and trimethadione that cause lethality. She identified several lethality-resistant mutants. The molecular characterization of the genes involved may shed light on the mechanisms by which these drugs extend life span and, in turn, how normal aging is regulated.

Conclusion

Several signaling pathways are either up- or down-regulated during organismal aging in C. elegans. Whereas some of the key players in these pathways are known, genetic screens have identified an unprecedented number of new candidates, some of established functions and others needing to be elucidated. Furthermore, current work suggests that additional survival pathways exist in the worm and that posttranscriptional modifications of proteins constitute an important mechanism in aging. The presentations highlighted in this Perspective show a glimpse into the rapid progress being made in understanding the complex mechanism of aging in C. elegans, which will, in turn, provide important insights into human aging and possible therapies.


October 12, 2005
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Citation: M. Hertweck, C. elegans Gives the Dirt on Aging. Sci. Aging Knowl. Environ. 2005 (41), pe31 (2005).








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