Old worms, new aging genes: biologists look into DNA for the secrets of long life.

For more than a decade, Cynthia Kenyon has watched microscopic worms of the species Caenorhabditis elegans live far longer than they should. She has seen mutant strains of this worm, which is normally dead and gone after a mere 2 or 3 weeks, last well into their second month. It's as if a person lived to be 200 years old. Kenyon's long-lived worms are a result of mutations in individual genes. That's a radical notion to many scientists who have long thought of aging as an uncontrollable process of deterioration that isn't regulated by single genes.

"There have to be genes that affect life span," counters Kenyon of the University of California, San Francisco. Noting the dramatic differences in life span among various animals--a mouse may last for 2 years while a bat can live for half a century--Kenyon has become convinced that longevity has evolved in animals many times. She argues that her long-lived nematodes can reveal some of the fundamental molecular biology that controls longevity in more-complex organisms, even people.

In 1993, Kenyon and her colleagues jump-started the field of aging genetics when they reported on a mutant strain of C. elegans that lives twice as long as normal. It showed the largest proportional lifespan extension of any animal known at the time. Researchers eventually determined that this long-lived nematode strain arose from a defect in a hormone-triggered cascade of molecular signals that resembles one in people that is prompted by the hormone insulin. Mutations affecting a similar hormone-driven cascade in fruit flies can lengthen the lives of these insects as well.

Over the past few months, Kenyon's team and several other groups of worm researchers have documented an unexpectedly large number of genes controlled by this hormonal system, including genes involved in stress responses and antimicrobial actions. This aging pathway appears to be at work in mammals, also. Two research teams have shown that altering how mice respond to insulin or a related hormone can extend the animals' lives, raising the prospect that manipulating these hormones in people could slow aging or enable them to age with better health.

"There's a possibility in humans that a similar aging pathway is at work," says Catherine Wolkow of the National Institute of Aging in Bethesda, Md.

WRINKLED WORMS Some scientists challenge Kenyon's work by claiming that her long-lived nematodes aren't actually aging slowly. Perhaps, these critics say, the genetically altered worms become old and frail at the normal pace but simply have had a major cause of death eliminated. Settling that controversy requires a routine way of measuring the aging process.

In her initial work with C. elegans, Kenyon gauged the increasing age of a worm by its decreasing mobility. More recently, she and her colleagues trained high-powered microscopes on aging nematodes and documented many changes in various tissues. Among other signs of deterioration, cell boundaries become less distinct, and the insides of cells go from smooth to curdled and become filled with cavities. In complementary work, Monica Driscoll of Rutgers University in Piscataway, N.J., and her coworkers found that worm muscle fibers lose their organized appearance as worms age (SN: 10/26/02, p. 260).

Like elderly people, who have wrinkles and other signs of age, "old worms have a particular look to them," Kenyon says.

Now that biologists have an idea of what happens to a worm as it grows old, they may be able to make better sense of all the genes they've identified over the past decade that affect aging. Indeed, the number of these longevity genes continues to grow. At a recent annual international meeting of C. elegans researchers, Kenyon's group reported unearthing more than 30 previously unrecognized genes that, when mutated, extend the nematode life span.

At the moment, the best-characterized genetic pathway of worm aging is the one Kenyon's group described in 1993. In their original report, the scientists showed that mutations in two genes, dubbed daf-2 and daf-16, had major effects on nematode longevity. Worms with a certain mutation in daf-2 had a doubled life span, but worms with mutations in both genes had a normal life span.

Kenyon's group was able to deduce from these observations that a working daf-16 gene tends to trigger other worm genes that would promote longevity, while a functioning daf-2 gene normally suppresses the activity of daf-16 or its protein.

Why would the worm, or any animal, have a gene such as daf-2 whose apparent purpose is to limit the organism's life span?

The gene appears to be part of a genetic system that allows worms to regulate their development--and, thereby, their lifespan--depending on whether environmental conditions are suitable for reproduction. When nutrients are scarce, growing worms don't develop fully, but instead take on a thinner, sexually immature form known as dauer. In this state, the organism can hang on for months and increase its chances of encountering richer living conditions. Worm biologists consider these long-lived dauers akin to the spores that bacteria form to ride out tough conditions.

Long before Kenyon's work, other researchers linked daf-2 and daf-16 to this arrested form of development. The genes' names derive from "dauer formation." Completely knocking out the activity of daf-2 sends a developing worm right into the dauer state, whether or not nutrients are scarce. Kenyon found something more intriguing: Certain subtle mutations in the gene enabled a developing worm to bypass the dauer state but still have an abnormally long life span.

In 1997, a research group led by Gary Ruvkun of Massachusetts General Hospital in Boston finally identified the DNA sequence of daf-2. To everyone's surprise, the scientists found that its protein, DAF-2, resembles human cell-surface proteins, or receptors, that respond to insulin and another hormone known as insulin-like growth factor-1 (IGF-1). The worm's receptor is a primitive version of these human receptors, says Ronald Kahn, director of the Joslin Diabetes Center in Boston.

As for daf-16, it turned out to encode a DNA-binding protein that turns on other genes. Known as a transcription factor, this protein, DAF-16, is apparently suppressed when a hormone triggers DAF-2. The daf-2 mutations, therefore, extend a worm's lifespan because they unleash DAF-16, enabling it to trigger genes that tend to promote longevity.

PULLING OUT ALL THE STOPS In their 1993 report, Kenyon and her colleagues speculated that the identification of genes under the sway of daf-16" could lead to a general understanding of how life span can be extended." In a flurry of recent publications, some of those genes have finally come to light.

In the April Aging Cell, a recently launched journal devoted to the molecular biology of aging, James H. Thomas of the University of Washington in Seattle and his colleagues identified several dozen C. elegans genes under the control of DAF-16. To do this, the scientists sought genes active in worms with mutations in daf-2 and compared these to genes active in worms with mutations in both daf-2 and daf-16.

The investigators also pinpointed genes containing a particular DNA sequence that DAF-16 binds to, implying that the protein controls the activity of those genes. Many of the genes governed by this protein are known to have roles in the metabolism and stress responses of worms.

In a paper published in the April 25 Science, Ruvkun's group unveiled its own list of genes regulated by the protein DAF-16. He and his colleagues searched the complete DNA sequences of C. elegans and the fruit fly Drosophila melanogaster for locations where DAF-16 might bind. They identified 17 cases in which the two animals have a similar gene with that characteristic sequence. They also found that the activity of six of those genes in the worms is affected by mutations of daf-2 and daf-16. The data indicate that DAF-16 turns on some of the six genes and suppresses others.

Ruvkun's team used a technique called RNA interference to turn off these six genes, one at a time, in normal worms. Several of the inactivations extended the life span of C. elegans, but not as much as the doubling normally produced by daf-2 mutations.

Through her own comparison of gene activity in worms with daf-2 and daf-16 mutations, Kenyon has also weighed in on the issue of DAF-16 targets. She and her colleagues found that DAF-16 turns on a variety of genes that make antimicrobial proteins. A DAF-16-boosted immune response may explain why long-lived mutant strains of C. elegans tend to be more resistant to death from bacterial infestation than normal worms are; Kenyon has found that aging worms typically die when the bacteria they eat overrun their bodies.

Kenyon's team also found that DAF-16 controls the production of many proteins that cells use to thwart damage to DNA and other molecules in response to factors such as heat or highly reactive molecules known as free radicals. A popular theory holds that aging is the result of a slow accumulation of free-radical damage.

Long-lived worms with a mutant daf-2 make extra amounts of enzymes that defuse free radicals, experiments by Kenyon and others have shown. The worms also make more so-called heat shock proteins, which prevent other proteins from folding abnormally or aggregating into clumps. In the April Aging Cell, Gordon J. Lithgow of the Buck Institute for Age Research in Novato, Calif., and his colleagues demonstrated the importance of these protective proteins. The researchers introduced extra copies of the gene for one heat shock protein called hsp-16 into worms, and that alone increased the animal's average life span by more than 10 percent.

Last year, Kenyon and her colleagues added another character to the unfolding stow. In the July 2002 Genetics, they revealed a key partner for DAF-16 in coordinating the worm's antiaging stress response: a transcription factor called heat shock factor (HSF). When the researchers used RNA interference to deactivate the gene for HSF, worms died earlier than normal. By monitoring the worms' tissues under microscopes, Kenyon's group showed that this premature death resulted from accelerated aging.

In the May 16 Science, the researchers flesh out the stow yet more by showing that daf-2 mutants don't have a doubled life span if the gene for HSF is also mutated. That raises HSF to DAF-16's level of importance. Indeed, Kenyon's group found a group of worm genes, including some that encode heat shock proteins, whose activities are influenced by both proteins working in concert.

Wolkow notes that some investigators had hoped that DAF-16 controlled just a few genes. If that were the case, it would in theory make it easier to extend people's life span by manipulating those genes. "It looks like it's much more complicated," Wolkow says. "I don't think we knew that at the outset."

"The worm seems to be pulling out all the stops. It's doing all sorts of things to increase life span," adds Kenyon. "It's a lot of little contributions from lots of genes."

THE BIG QUESTION Evidence is building that investigations into worm aging could have a payoff in mammals. Whereas worms have a single receptor matched to insulinlike signals, mammals have developed distinct hormonal pathways for insulin and IGF-1, each characterized by its own dedicated receptor.

In one series of experiments, Martin Holzenberger of Saint Antoine Hospital in Paris and his colleagues created strains of mice in which one or both copies of the rodent gene for the IGF-1 receptor had mutations. Mice lacking any normal copies died as embryos. However, mice with one working copy developed normally and lived, on average, 26 percent longer than did animals with two normal copies of the IGF-1-receptor gene. Holzenberger's group reported these results in the Jan. 9 Nature.

Similar results have emerged from the study of mice lacking some insulin receptors. A research team led by Kahn has created mouse strains that lack insulin receptors in specific tissues such as liver, brain, and fat. In the Jan. 24 Science, Kahn and his colleagues reported that mice missing the receptors in their fat tissue live 18 percent longer on average than typical mice.

The long-lived mice were also leaner, despite eating normal amounts of food, Kahn's group found. That's not surprising, because one of insulin's roles is to signal cells to store fat, but the researchers' findings could help uncover why severely calorie-restricted diets extend the life spans of many animals (SN: 3/15/97,p.162). It may not be the reduction in calories that's crucial for boosting longevity, but rather the animals' leanness, says Kahn. His group is now putting the mutant mice on diets to see whether there's any additional life span extension.

Kahn also plans to study the longevity of other mutant mouse strains to deter mine how insulin signaling in each tissue contributes to aging. He's particularly interested in mice that don't have insulin receptors in their brains, since studies in flies and worms have indicated that the nervous system has a key role in mediating insulin's impact on aging.

When she looks at the progress researchers have made on aging over the past decade, Kenyon admits she's stunned. "There wasn't a field when we started," she says. "Now, there are a lot of people working on aging. We've learned a huge amount."

For all their recent success, however, worm researchers have plenty of questions about the biology of aging that should keep them going for generations. Lithgow says he still can't answer a fundamental question: "Why do worms live 20 days and not 20 years?"