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Corresponding author: Cynthia Kenyon, Box 0448, Rm. HSE 1521A, San Francisco, CA 94143-0448., ckenyon{at}biochem.ucsf.edu (E-mail)
Communicating editor: P. ANDERSON
 | ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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The genetic analysis of life span has revealed many interesting genes and pathways; however, our understanding of aging has been limited by the lack of a way to assay the aging process itself. Here we show that the tissues of aging worms have a characteristic appearance that is easy to recognize and quantify using Nomarski optics. We have used this assay to determine whether life-span mutations affect the rate of aging, to identify animals that age more rapidly than normal, and to infer the cause of death in C. elegans. Mutations that reduce insulin/IGF-1 signaling double the life span of C. elegans, and we find that tissue decline is slowed in these mutants. Thus this endocrine system appears to influence the rate at which tissues age. This effect extends even to the germline, which is the only mitotically active tissue in the adult. We find that Nomarski microscopy also allows a ready distinction between short-lived mutants that age more rapidly than normal and those that are simply sick, and we have identified an RNAi clone that confers a dramatic rapid-aging phenotype. This clone encodes the C. elegans heat-shock factor (HSF), a transcription factor that regulates the response to heat and oxidative stress. This suggests that heat-shock proteins, many of which act as chaperones, may function in normal animals to slow the rate of aging. Finally, we have identified a cause of death of C. elegans: namely, proliferating bacteria. This suggests that increased susceptibility to bacterial infections contributes to mortality in these animals, just as it does in humans.
IN our daily lives, we unconsciously quantify aging by evaluating the physical characteristics of an individual. Signs of aging are easy to recognize in humans: people in their twenties look different from those in their fifties or eighties. Likewise, old and young Caenorhabditis elegans look different when examined using a low-power dissecting microscope. Old animals move more slowly and become progressively more flaccid with age. These changes are retarded in long-lived insulin/IGF-1 signaling mutants, which remain active much longer than normal (
Having a rapid way to monitor additional aspects of the aging process would be useful in several ways. First, it might help to elucidate the means by which mutations affecting life span affect the process of aging itself. In addition, it might provide a way to identify mutants that age more rapidly than normal. Finally, it might provide clues about the causes of death in this animal. In this study, we have found that Nomarski differential interference contrast microscopy provides an effective, rapid, and convenient means of visualizing many features of tissue aging. We find that extensive tissue deterioration takes place during aging, not only in the postmitotic somatic tissues of the animal, but also in a mitotic lineage, the germline. As with old humans, this pattern of deterioration gives the animals a characteristic appearance that becomes easy to recognize.
The life span of C. elegans is regulated hormonally by an insulin/IGF-1-like signaling pathway. Wild-type animals live just a few weeks; however, mutants with reduced activity of DAF-2, an insulin/IGF-1-like receptor (
At the cellular level, one can imagine several ways in which insulin/IGF-1 signaling mutations could extend life span. If nematodes die from a single lethal event (analogous to a heart attack), then mutations in the insulin/IGF-1 signaling pathway could extend life span by decreasing the probability of this event. Alternatively, if nematodes, like humans, age in a progressive fashion that involves a decline in tissue integrity, a mutation could extend life span by slowing the entire aging process. The final possibility is a trivial one, namely, that the commonly used N2 laboratory strain of C. elegans has suffered detrimental mutations that shorten life span and that mutations in the daf-2 pathway correct this defect. This is unlikely, however, because insulin/IGF-1 pathway mutants live much longer than any of four wild C. elegans strains (
In this study, we have developed a protocol for evaluating and quantifying age-related changes and used this protocol to compare the tissues of wild-type animals to those of long-lived and short-lived insulin/IGF-1 signaling mutants over the course of their lives. Our findings indicate that insulin/IGF-1 signaling influences life span by changing the rate at which the tissues age and that this pathway governs the aging not only of the postmitotic somatic cells, but of mitotic lineages as well.
The pathways that govern many biological processes, such as the cell cycle, or the timing of developmental events, include genes that function to advance the process, as well as genes that function to retard the process. Because of the difficulty of distinguishing mutations that cause progeria (accelerated aging) from mutations that kill the animal for reasons unrelated to aging, the analysis of life span relies mainly on a single class of regulatory genes: genes whose wild-type function is to shorten life span. Loss-of-function mutations in these genes will extend life span. Thus, having a way to quickly identify rapidly aging mutants (many of which would define genes whose wild-type function extends life span) would be very powerful. Several short-lived mutants that accumulate lipofuscin granules more rapidly than normal have been postulated to age rapidly, on the basis of this one age-related phenotype (
The changes we observed in old animals may simply correlate with age, as wrinkles do in humans, or they may be a pathology that contributes directly to death. We observed that bacteria, C. elegans' food source, often accumulated in the pharynx and gut of worms near death, and we hypothesized that the presence of proliferating bacteria in the body of an aged worm might have a deleterious effect on its health. Accordingly, we grew the animals on live bacteria that were unable to proliferate and observed a sharp decrease in the frequency of constipation and a 30–40% increase in mean life span. We conclude that some feature of proliferating bacteria, possibly their ability to cause infections in older animals, shortens the life span of this organism.
| MATERIALS AND METHODS |
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| TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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C. elegans strains used:
The following strains were used: N2, daf-16(mu86)I, daf-2(e1370)III, daf-2(mu150)III, ced-3(n1286)IV, DH1033 bIs1[vit-2::gfp; rol-6]; sqt-1(sc103), daf-2(e1370)III; him-5(e1490)V, CF512 fer-15(b26)II; fem-1(hc17)IV. daf-2(mu150) was recovered from an EMS mutagenesis screen of >11,000 haploid genomes in a temperature-sensitive sterile background [CF512 fer-15(b26)II; fem-1(hc17)IV; J. APFELD, H. HSIN, B. ALBINDER, B. TSUNG, J. DORMAN and C. KENYON, unpublished data]. Dominance, complementation, and linkage tests using the dauer phenotype were conducted by N. Oliveira, and complementation tests using the life-span phenotype were conducted by N. Libina (Kenyon laboratory). C. Murphy outcrossed the mutant and conducted preliminary life-span analysis.
Life-span analysis:
Wild-type, daf-2, and daf-16 animals raised at 20° were shifted to 25° at the L4 molt and transferred to new plates every other day thereafter until progeny production ceased. Animals were judged to be dead when they no longer responded to gentle prodding. Animals that crawled off the plate, became desiccated on the sides of the plate, displayed extruded internal organs, or died from internally hatched progeny were censored. Censored animals were incorporated into the data set until the day of their disqualification, as described previously (
Photography of autofluorescence:
Endogenous gut fluorescence was photographed using a 525-nm bandpass filter. Images were collected without automatic gain control in order to preserve the relative intensity of different animals' fluorescence. Two-, 5-, and 10-day-old adults were photographed on the same day to avoid effects of light source variation on apparent fluorescence intensity.
Visualization of yolk:
The vit-2::GFP fusion strain (see above) was a kind gift of David Hirsch and Barth Grant. Animals were allowed to age and were photographed using both Nomarski optics and epifluorescence (525 nm).
Nomarski analysis:
Animals that had been cultured at 25° were placed on a 2% agarose pad in M9 buffer containing 2 mM sodium azide and covered with a coverslip. Control experiments indicated that sodium azide did not affect the age-related phenotypes we observed. Delicate older animals of all genotypes occasionally ruptured and were lost during this process (
10%). Images were captured using a CCD camera coupled to Universal Imaging's MetaMorph Imaging System (version 3.6). Image files were contrast balanced and rotated when necessary using PhotoShop 5.0.
Quantification of tissue damage:
C. elegans heads were photographed as described above. In a blind experiment, photographs of heads were given a score of 1–5, with 1 representing a youthful, unsullied appearance and 2–4 denoting low, medium, and high levels of overall deterioration. A rare score of 5 was assigned to animals so deteriorated as to be nearly unrecognizable. Please note that there is no strict quantitative relationship between the extent of damage in these different classes: an animal receiving a score of 2 does not necessarily have twice as much damage as an animal receiving a score of 1. Instead, this scoring system is more analogous to grading of student exams, in which a grade of A–F is assigned on the basis of relative performance. This system makes it possible to carry out statistical analysis by nonparametric methods that simply ask whether members of certain subgroups are more likely to receive low (or high) ranks than are members of other subgroups. Fig 3 shows representative photographs of heads earning these scores.
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Photographs of germ cells in the distal gonad also were rated on a scale of 1–5 on the basis of these criteria. In addition, these photographs were also assigned a cumulative value that represented the presence and extent of several of the correlates of aging we often observed: graininess; large, well-separated nuclei; cavities; and fewer nuclei. Each correlate of aging was scored separately and then summed to give the final score.
Scores were assigned without knowledge of the age or genotype of the worm in the photograph. Overall scores were reevaluated at least once, and a naive observer was asked to score a selection of photographs in a double-blind experiment.
Statistical analysis of tissue damage:
Nonparametric analysis of head scores was conducted using the Kruskal-Wallis test to determine if there were significant differences between multiple groups, followed by a pairwise comparison, the Mann-Whitney test. All statistical analysis was conducted using Statview 5.0.1 (SAS) software.
RNA interference:
Bacterial RNAi clones that shortened adult life span were identified by a procedure described elsewhere (A. DILLIN, D. GARIGAN, D. CRAWFORD, J. RAMOND and C. KENYON, unpublished results). We then used Nomarski optics to observe the tissue phenotypes of C. elegans cultured on these bacteria.
Life spans on nonproliferating bacteria: Antibiotic treatment:
The OP50 strain typically used as a C. elegans food source was transformed with a vector containing an ampicillin resistance gene, which also confers resistance to carbenicillin. Conventional or drug-resistant OP50 was seeded onto NG plates and allowed to grow for 2 days before the addition of 80 µl of 0.5 M carbenicillin. C. elegans eggs were transferred to these plates, and worms were transferred as necessary to similar plates. Carbenicillin-sensitive bacteria were periodically streaked onto drug-containing and drug-free culture plates to ensure that they were not proliferating but were capable of recovery on drug-free media. An identical procedure was followed for life-span assays on kanamycin-sensitive and -resistant bacteria. After treatment with 80 µl of 10 mM kanamycin, which is a bactericide, drug-sensitive bacteria were streaked onto drug-free plates to confirm that they had been killed. In these experiments, we examined fer-15(b26); fem-1(hc17) mutants at 25°. These animals do not produce progeny at this temperature, thus simplifying the procedure.
UV treatment:
To kill bacteria, NG plates seeded with OP50 bacteria were exposed to 302 nm ultraviolet light for 30 min. As a control, unseeded plates were also treated in this way and seeded subsequently. Bleached wild-type C. elegans eggs were transferred to these plates and incubated at 25°. Worms were transferred as necessary to similar plates.
| RESULTS |
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| TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Decline of tissue integrity in aging wild-type animals:
Nomarski microscopy is commonly used to observe the development of C. elegans. In young animals, the nuclei and nucleoli of all cells are easy to see using this method. It is also possible to see the boundaries of many cells and tissues, such as the muscles, gonad, epidermal seam cells, and certain neurons. We observed that in young adults the cells and tissues appeared similar to those of late juvenile stages, except that the nuclear boundaries were less distinct. This lack of definition became more pronounced as the animals grew older. For example, by day 10, it was very difficult to see the nuclei of the epidermal cells (Fig 1A). (Throughout the article, the ages given refer to days of adulthood; for example, "day 10" refers to the tenth day of adulthood.) Neuronal nuclei, which have a wrinkled appearance, remained visible throughout the life of the animal, although they, too, grew less distinct with time. In young animals, the cytoplasm and nucleoplasm of most cells was smooth and uniform (Fig 1B). However, as the animals grew older, both began to show signs of deterioration (Fig 1C and Fig D). Necrotic cavities of various sizes appeared, often containing vibrating particles that appeared to display Brownian motion. Tissues often acquired a curdled texture (Fig 1D).
Older animals also accumulated shiny, mobile patches of a substance that appeared to be yolk. In young animals, yolk is transported from its site of synthesis in the intestine into the gonad, where it is incorporated into embryos. It is possible that yolk accumulates in old animals when the production of embryos ceases. We examined animals expressing a green fluorescent protein (GFP)-tagged yolk protein and confirmed that this substance was in fact yolk (Fig 1F and Fig G). We also observed shiny but less mobile nonfluorescent material in the bodies of GFP-expressing worms; similar, less mobile material (presumably something other than yolk) was also present in non-GFP-expressing worms. Finally, as reported previously (
The head is a particularly informative and compact area composed of multiple tissue types. It contains the pharynx, a neuromuscular pump that ingests and grinds bacteria, as well as nervous tissue, muscle, and epidermis. Using Nomarski optics, we were able to evaluate the general character of this body region, but because cellular boundaries can be indistinct, we were often not able to resolve individual tissue types with certainty. Fig 3 shows representative photographs of heads of animals of different ages, illustrating the changes that occur in tissue integrity during aging.
To quantify the changes we observed, we analyzed photographs of 83 individual wild-type worm heads. At every age, some animals exhibited more extensive tissue deterioration than did others. This was not surprising, since some animals of the same genotype live longer than others. Each animal's head received a grade on the basis of the extent of deterioration it exhibited, as described in MATERIALS AND METHODS. As shown in the scatterplot in Fig 4, we observed a steady trend toward increasing damage with age. To evaluate these findings quantitatively, we used the Kruskal-Wallis test, a nonparametric statistical test, to ask whether there was a statistically significant relationship between an animal's age and its rank within the overall population. We found that, indeed, the average level of tissue deterioration in the overall population increased with age in a statisically significant manner (P < 0.0001; Fig 4 and Fig 5 and legends). These findings imply that the tissues of C. elegans deteriorate in a progressive fashion as the animals grow older. This tissue decline correlates with the decreased mobility and flaccid appearance of old worms that are visible with a low-power dissecting microscope.
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Tissue deterioration occurs in the germline of aging wild-type animals:
The only cells that are able to divide in C. elegans adults are the germline stem cells located near the distal tip of the gonad (
Mutations in the insulin/IGF-1 pathway change the rate at which both mitotic and postmitotic tissues age:
Next, we asked whether mutations in the insulin/IGF-1 signaling system influence tissue aging. To do this, we examined two long-lived but otherwise quite dissimilar daf-2 mutants, daf-2(e1370) and daf-2(mu150), as well as the short-lived daf-16(mu86) null mutant, at different ages using Nomarski optics. The daf-2(e1370) allele has been characterized previously (
We found that the quality of tissue deterioration that took place in both daf-2 and daf-16 mutants resembled that of wild type, suggesting that both types of mutants age in a normal way. All of the mutants displayed increased lipofuscin-like intestinal fluorescence at relatively old ages (Fig 2, and data not shown). We found that both the somatic tissues and the germlines of daf-2(mu150) and daf-16(mu86) mutants exhibited age-related damage that appeared identical to that seen in wild type (Fig 3 and data not shown). daf-2(e1370) mutants tended to look less damaged even very late in life; however, because they were prone to die from internally hatching progeny, their germlines were difficult to evaluate because they were often obscured by quiescent L1 progeny. Their bodies were somewhat opaque, and they had less yolk in the body cavity than did wild type, even at advanced ages (perhaps because yolk was still exported into progeny). Also, the frequency of constipation and bacterial packing in the pharynx was markedly reduced in daf-2(e1370) mutants (see Fig 1E and Fig 3), although it was present in some infirm individuals. Nevertheless, overall, the bodies of aging e1370 mutants resembled those of wild type. Interestingly, the tissues of aging daf-2(mu150) animals were indistinguishable from those of wild type.
Although the character of the tissue deterioration we observed in daf-2 mutants was similar to that of wild type, we found that the rate of this deterioration was dramatically slowed (Fig 3 Fig 4 Fig 5). For example, in daf-2(e1370) mutants, the outlines of epidermal nuclei (shown in Fig 1A) were clearly visible until at least 20 days of adulthood, compared to 5 days in wild type. In addition, it was not until daf-2 animals were 20 days old that we began to see cavities and "curdled" tissues.
We quantified the extent of tissue damage by scoring photographs of individual heads and germ cells in the distal gonad (Fig 4). Nonparametric analysis of scores assigned to daf-2 and wild-type animals of the same age confirmed our impression that daf-2 animals exhibited significantly less age-related damage in both body regions. For example, in 5-day-old adults, comparison of scores by the Mann-Whitney statistical test showed daf-2 and wild-type tissue to be significantly different. (N2 vs. e1370 heads, P < 0.0001; N2 vs. mu150 heads, P = 0.0003; N2 vs. e1370 germ cells, P = 0.001; N2 vs. mu150 germ cells, P = 0.001; Fig 5). We also examined the tissues of daf-16 mutants, whose mean life spans were 10–20% shorter than normal at 25°. daf-16 animals did not look dramatically older than age-matched wild-type animals until the tenth day of adulthood. These differences were apparent for both heads (Mann-Whitney, P = 0.01) and germ cells (Mann-Whitney, P = 0.02). daf-16 animals at this age were predicted to be dead within 2 days, whereas wild-type animals have a longer life expectancy (maximum daf-16 life span, 12 days; maximum wild-type life span, 19 days). In contrast, daf-2 mutants, which significantly outlive both wild-type and daf-16 animals, began to look younger than daf-16 mutants as early as 2 days of adulthood (Mann-Whitney, P = 0.04).
Nomarski optics can be used to assay for progeria:
A major goal in the study of C. elegans' aging is to be able to identify mutations that accelerate the aging process. From a screen of 2445 individual clones of a chromosome I bacterial RNAi library (80% of control animals were still alive. To ask which of these RNAi clones might accelerate aging, we examined 5-day-old RNAi-treated adults by Nomarski optics. Four of the five clones clearly did not cause 5-day-old worms to look older than normal. For example, the gld-1(RNAi) clone, which causes ectopic germline proliferation, caused germ cells to accumulate in the body cavity of day-5 adults but did not affect tissue morphology. Another clone, Y53C10A.1, caused a tissue degradation phenotype that did not resemble that of normal aging (Fig 6). However, one clone, Y53C10A.12, produced a truly striking progeric phenotype. When viewed by Nomarski optics, 5-day-old animals grown on Y53C10A.12 dsRNA at 25° looked like normal animals at a much older age; they resembled wild-type animals that were 10–15 days old. They contained cavities and deteriorated tissue that had the curdled texture characteristic of old animals. In addition, their pharynx and gastrointestinal tracts were packed with bacteria (Fig 6 and Fig 7). These animals were also short lived and progeric at 20°, where they had a mean life span that was 38% shorter than that of control animals (10.7 vs. 17.2 days; Fig 8). We quantified the phenotypes of day-7 animals grown at 20° by photographing them and scoring them blind, as described above. Of 13 control animals examined, 1 received a score of 1, 11 received a score of 2, and 1 received a score of 3. In contrast, 5/14 animals cultured on hsf-1 dsRNA received a score of 3 and 9/14 received a score of 4 (P < 0.0001; Mann-Whitney test). Thus, unlike the other four genes whose RNAi phenotypes we examined, this gene, which encodes the C. elegans heat-shock factor, is a candidate for a gene that functions in normal animals to prolong youthfulness and retard the aging process.
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Apoptosis is unlikely to influence aging in C. elegans:
One of the goals of this study was to identify possible causes of death in C. elegans. In C. elegans, 131 cells undergo programmed cell death, or apoptosis, during development, and additional apoptosis occurs among the meiotic precursor cells in the germline (
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C. elegans is killed by its food:
C. elegans is normally cultured on a lawn of OP50 bacteria (
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Previously, 16%. We also determined the life spans of animals grown on UV-irradiated bacteria and found that, under our conditions, mean life span was increased 30–40% (Fig 10). Why do UV-irradiated bacteria extend life span? UV irradiation damages cellular components and may also induce the bacterial SOS response, which is associated with many physiological changes that could conceivably trigger a life-span-extension response. To ask whether the effect was specific to UV, we also determined the life spans of animals grown on bacteria killed with kanamycin. These, too, lived longer than normal (Fig 10). Finally, to ask whether life-span extension required the death of the bacteria, we determined the life spans of animals grown on live bacteria whose growth was arrested with carbenicillin. We found that this, too, increased life span (Fig 10). Thus we conclude that live bacteria per se are not harmful to C. elegans; instead, something associated with bacterial growth and proliferation kills them.
| DISCUSSION |
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| TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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We have found that Nomarski microscopy provides a powerful way to monitor and quantify the aging process in C. elegans. This technique does not permit a detailed study of individual tissue types, because many of the boundaries between cells and tissues cannot be resolved. It does, however, permit a general assessment of the quality of cells and tissues that make up most of the body mass. Using this method, we find that tissues of wild-type animals deteriorate progressively during aging. Like old humans, old worms have a very characteristic appearance, which is easily recognized by the experienced eye.
One objective of this study was to learn how mutations in the insulin/IGF-1 signaling pathway influence the aging of the body. The relevance of this pathway to aging has been questioned by some who have asserted that mutations in this pathway do not affect the rate at which the aging process takes place (
Some have argued that the genetic analysis of aging in C. elegans will not reveal universal mechanisms of aging control, because, unlike many higher organisms, cells in the adult are postmitotic. This criticism has been tempered by the finding that SIR2 protein, which regulates the mitotic life span of yeast, also functions in the C. elegans insulin/IGF-1 system (
Another goal of this study was to learn whether Nomarski optics would allow us to distinguish short-lived mutants that were simply sick from those that might be aging prematurely. This type of analysis seemed likely to be informative: in the case of humans, a young person with a terminal illness, who has a short expected life span, would never be confused with old person. We were pleased to find that a cursory examination allowed us to discard, immediately, four of five short-lived RNAi-treated strains from further consideration as progeric animals. Interestingly, one clone did cause animals that were young to resemble normal, old animals. These animals' tissues displayed the curdled texture and cavity-ridden appearance so characteristic of old animals. Furthermore, they were constipated, suggesting that their physiology, too, was similar to that of older animals. Thus we conclude that this RNAi clone is an excellent candidate for a gene that prevents progeria.
Nomarski analysis will never provide definitive proof that a short-lived mutant is aging precociously. Specifically, it is possible that these animals are in a physiological state that is not the same as that of normal old animals but nevertheless causes them to resemble old animals when viewed with Nomarski optics. The case for progeria would be strengthened by examining gene expression profiles to confirm that animals that appear older than normal also display gene expression patterns characteristic of old animals. In addition, if a gene encodes a protein that carries out a rate-limiting step in the aging process, then overexpression should lengthen life span. In spite of these caveats, we would like to emphasize that the morphology of these animals was quite striking. As shown in Fig 1, the Nomarski aging phenotype is quite complex, involving many different morphological features (tissue texture, constipation, etc.), and these RNAi-treated young animals closely resembled normal old animals. They were analogous to 45-year-old people who looked 70.
The identity of this progeric RNAi clone was very interesting: it encodes a homolog of HSF, which is the transcription factor that activates expression of heat-shock genes in response to heat shock and oxidative stress. Thus the finding that hsf-1(RNAi) shortens life span and produces a progeric Nomarski phenotype suggests the hypothesis that this transcription factor, and thus presumably the stress response it regulates, acts in normal animals to prolong youthfulness and extend life span by retarding the aging process. One possibility is that improperly folded proteins that accumulate during aging activate heat-shock factor, which in turn would be expected to activate the expression of heat-shock genes (
Although this is the first demonstration that the heat-shock system acts in normal animals to prevent a syndrome that resembles precocious aging, a link between the heat-shock response and aging has been demonstrated previously. The expression of heat-shock proteins has been shown to increase during aging in wild-type C. elegans (
In addition to providing a way of examining the aging process in long- and short-lived mutants, Nomarski examination also suggested possible causes of death. One of the most striking phenotypes exhibited by older animals was bacterial packing in the pharynx and the anterior and posterior regions of the intestine. This packing was never observed in young animals, which must therefore be resistant to it. We found that bacterial packing was greatly reduced if the bacteria were incapable of proliferation. This suggests that, as the animal ages, it loses the capacity to prevent bacterial proliferation in its gastrointestinal tract. Bacterial packing in the pharynx could be due to the decreased rate of pumping observed in older animals (
In addition to reducing bacterial packing, feeding worms bacteria that were incapable of proliferation extended life span substantially—by 30–40% (Fig 10). A trivial explanation for this is that the animals cannot use the nonproliferating bacteria as a food source and become calorically restricted as a result. Caloric restriction is known to extend life span (
Interestingly, when we examined the tissues of five long-lived animals shown at the top of Fig 10 at 15 days of adulthood, we found that they looked very old, just as old as age-matched controls (data not shown). This suggests that bacteria may not influence the rate of tissue aging of the animals, but instead that they may cause a catastrophic death once the animals' health has declined sufficiently. One possibility is that the bacterial packing we saw in the gastrointestinal tract kills the animals. Since this packing is markedly reduced in the absence of bacterial proliferation, this is a plausible explanation. A related explanation, also suggested by
Because daf-2 mutants live 100% longer than normal, much longer than the 30–40% extension conferred by nonproliferating bacteria, it is unlikely that they are long-lived simply because they are able to resist bacterial growth or infections. However, many life-span mutants in C. elegans do live 30% longer than normal. It should be possible to determine whether any of these mutants are long-lived because they are resistant to bacteria, since their life spans should not be increased further by growth on nonproliferating bacteria.
| ACKNOWLEDGMENTS |
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We thank Paige Nittler, Douglas Crawford, Andrew Dillin, Lisa Williams, Natasha Libina, Joy Alcedo, and Coleen Murphy for helpful conversations. We also thank Peter Bacchetti of the UCSF Biostatistics Consulting Service for statistical expertise. The mu150 mutant was isolated in a mutagenesis screen conducted in the Kenyon lab by J. Apfeld, H. Hsin, B. Albinder, B. Tsung, and J. Dorman; outcross and complementation tests were performed by N. Oliveira, N. Libina, and C. Murphy. J. Alcedo initially analyzed the life span of cell death mutants. A. Dillin directed, and D. Garigan, D. Crawford, and J. Ramond assisted with, the RNAi library screen in the Kenyon lab that yielded the five clones that decreased life span. We thank Barth Grant and David Hirsch, the Caenorhabditis Genetics Center, and Cori Bargmann for strains. This work was supported by an American Federation for Aging Research Scholarship to D.G., a Canadian Institutes of Health Research fellowship to A-L. H., and a grant from the Ellison Foundation to C.K., who is the Herbert Boyer Professor of Biochemistry and Biophysics. A.G.F. was supported by a U.S. Army Breast Cancer Research Fellowship, R.S.K. by a Howard Hughes Medical Institute Predoctoral Fellowship, and J.A. by a Wellcome Trust Senior Research Fellowship (054523).
Manuscript received December 3, 2001; Accepted for publication April 11, 2002.
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