Sci. Aging Knowl. Environ., 20 March 2002
Stay Mellow, Stay Young
A cell is a worm is a mouse is a boy. At least that's what researchers hope when they delve into the genetics of aging. Mutations in single genes can extend the life-span of the roundworm Caenorhabditis elegans, but the picture of how those genes contribute to longevity in mammals--if they do at all--remains muddy. New work links proteins involved in worm and mammalian aging and also ties in reactive oxygen species (ROS), destructive molecules that are thought to contribute to aging in a variety of creatures (see "The Two Faces of Oxygen").
"What's cool is that now we have a connection between a C. elegans pathway that mediates response to stress and one in vertebrates that mediates a similar response to stress--and they're both involved in longevity," says molecular geneticist Cynthia Kenyon of the University of California, San Francisco. "That's really beautiful." Beautiful, but not simple. Although the growing number of similar aging themes among diverse organisms is clarifying some general ideas, the details--and variations--are turning out to be a complex business.
Previous work on C. elegans implicated daf-16, a component of an insulin-like signaling pathway, in control of life-span. (See "Growing Old Together" for an overview of the pathway.) Other genes require the protein to exert their life-extending effects, and overproduction of DAF-16 itself modestly prolongs life. The daf-16 gene resembles members of the vertebrate forkhead family of genes, whose protein products turn other genes on and off.
In C. elegans, DAF-16 is thought to increase longevity in part by spurring the manufacture of antioxidant molecules such as the enzyme catalase, which breaks down the oxidant hydrogen peroxide. This observation inspired cardiologist Shino Nemoto and molecular biologist Toren Finkel of the National Heart, Lung, and Blood Institute in Bethesda, Maryland, to probe whether a mammalian DAF-16-like protein, FKHRL1, fosters catalase production. The researchers attached the first portion of the catalase gene to the gene that encodes luciferase, an enzyme that causes cells to glow. Rat cells doctored to churn out the forkhead protein produced light, and the more FKHRL1 they made, the brighter they glowed, the team reported in the 7 March issue of Science Express. This observation suggests that FKHRL1 turns on the catalase gene. Although these results don't gauge catalase production per se, additional tests showed that FKHRL1 enhances cells' ability to neutralize hydrogen peroxide. Furthermore, the protein promotes cellular survival in the face of a hydrogen peroxide onslaught.
These results seem straightforward in light of the worm data, but they clash with a number of studies in mammals that link forkhead activity to cellular demise. For example, Michael Greenberg at Harvard Medical School in Boston and colleagues reported last year that shutting off FKHRL1 in mammalian cells promotes their survival. This finding and those from other labs imply that FKHRL1 normally induces cell death. In contrast, Nemoto and Finkel's data suggest that the same protein protects mammalian cells. Many researchers, including Greenberg and Finkel, don't seem discouraged by the apparent discrepancy between the two sets of results, attributing it to different experimental conditions. Instead, they are trying to untangle details of FKHRL1 regulation; Finkel is concentrating on how oxidants govern the protein's activity.
If the forkhead protein works the same way in mammals as DAF-16 does in worms, Nemoto and Finkel reasoned, ROS should signal FKHRL1 to crank up production of antioxidants such as catalase. Yet at least one type of ROS seems to curb FKHRL1's activity, the researchers found. When they doused cultured rat cells with hydrogen peroxide, FKHRL1 molecules acquired a chemical group--phosphate--known to send the protein into retirement. The phosphorylated protein vacates the nucleus and collects in the cytoplasm. In keeping with this observation, addition of antioxidants increased the protein's activity, as measured by expression of a test gene that's controlled by FKHRL1. Together, the results suggest that oxidants foil FKHRL1's attempts to turn on its target genes. Although FKHRL1 appears to respond to stress differently than DAF-16 does, the fact that both proteins react to oxidative assault is still significant, says molecular geneticist Tom Johnson of the University of Colorado, Boulder: "It's a nice demonstration of a correlation between nematodes and mammals. The general response to stress has been conserved, but the details have evolved considerably."
Why such a system--which apparently withdraws antioxidant defenses in response to ROS--would benefit an animal is not yet clear, but what's bad for the cell is not necessarily bad for the whole animal. Scientists already know of a gene--p66shc--that appears to illustrate this idea. Mice without p66shc live 30% longer than normal mice do, according to a 1999 report by experimental oncologist Pier Giuseppe Pelicci and his colleagues at the European Institute of Oncology in Milan, Italy. Furthermore, environmental insults such as hydrogen peroxide or ultraviolet light cause normal cells to kill themselves, whereas p66shc-deficient cells live on. Perhaps the p66shc protein renders cells sensitive to the oxidants and thereby triggers a cell's demise. Although such behavior dooms individual cells, it might enhance the organism's fitness. Genes that promote cell death but limit life-span might keep cancer at bay before and during an animal's reproductive years (see "Cancer and Aging: Yin, Yang, and p53").
If ROS control FKHRL1 activity in the cell, Nemoto and Finkel speculated, then p66shc might indirectly hold the reins of FKHRL1 by regulating the amount of oxidants in the cell. To test this idea, they measured hydrogen peroxide in mouse cells with and without p66shc. Slightly less hydrogen peroxide amassed in cells that lack the protein. The difference increased dramatically when the cells were starved for growth factors and other molecules, a treatment that induces oxidative stress, among other perturbations. Under these conditions, the amount of hydrogen peroxide shot up: Normal cells contained about four times the amount of this ROS as did those that lacked p66shc. Data from Pelicci's lab, in press in Oncogene, back up these findings. The researchers conclude that ROS buildup depends on p66shc. Therefore, p66shc's influence on life-span might hinge on its control of FKHRL1 activity through oxidant concentration.
They then further probed the relation between the forkhead protein and longevity. Because animals that lack p66shc live longer than normal, eliminating p66shc from cells allowed the researchers to find out whether FKHRL1, like DAF-16, kicks into high gear under conditions that extend life-span. Under normal culture conditions, FKHRL1 activity was similar in cells that lack p66shc and those that contain the protein. At first glance, this result seems to contradict the discovery that p66shc promotes the accumulation of hydrogen peroxide, which in turn inhibits FKHRL1 activity. However, the researchers then tested whether the absence of p66shc affected FKHRL1's ability to respond to an oxidative onslaught. Hydrogen peroxide treatment increased phosphorylation of FKHRL1--and thus inactivated the protein--only in cells with p66shc. This result suggests that ROS require p66shc to squelch the activity of the forkhead protein.
To further explore the interplay between oxidative assault, p66shc, and FKHRL1, the researchers deprived cells with and without p66shc of growth factors and other molecules. Under these conditions, the team observed a different scenario than the one they documented in unstarved cells. FKHRL1 activity spiked in cells without p66shc, whereas it remained constant in those that harbored the protein. These results from p66shc-deficient cells match the researchers' prediction, they say: The rise in ROS no longer thwarts FKHRL1 activity, because p66shc is not present. In contrast, because they knew that hydrogen peroxide puts the kibosh on FKHRL1 in cells that carry intact p66shc, they expected the protein's activity to fall in these cells. The cells' response to starvation involves more than a simple increase in hydrogen peroxide concentration, Finkel says, and other changes apparently override the suppressive effect of ROS on FKHRL1: "The most likely interpretation of our data is that [FKHRL1] activity is positively and negatively regulated by multiple inputs including ROS."
Together, Finkel says, the findings flesh out Pelicci's discovery that p66shc sensitizes cells to oxidants; in response to ROS, the protein turns off the production of antioxidants such as catalase through the action of FKHRL1. Furthermore, the new results suggest a model that explains how the absence of p66shc extends life-span by orchestrating the response to various signals, including oxidative stress. Eliminating p66shc from the cell unhooks FKHRL1 from control by oxidants. As a result, conditions that spur ROS production and would normally foil FKHRL1 fail to check the forkhead protein. FKHRL1 instead remains active in the nucleus, where it can turn on production of catalase and perhaps other defensive enzymes. By breaking down destructive molecules, these enzymes help keep cells--and potentially entire organisms--spry.
Although the paper is the first to tie oxidative stress to a forkhead protein and p66shc, the effect of FKHRL1 on longevity remains speculative. "The right word is 'implication,' " says Pelicci. "There are no genetic data in this paper to show that FKHRL1 in mammals is a life-span determinant." However, he adds, "this paper clearly shows that FKHRL1 activity is controlled by p66[shc], which is a known longevity gene in mammals." As scientists unravel the molecular details of aging, they're encouraged by the growing number of similarities between worms and mammals. The new results add to that list and could help illuminate the complicated relation between stress, cell death, and longevity.
March 20, 2002
Caroline Seydel is a science writer in Los Angeles, California, where she's learning more about stress than she ever wanted to know.
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