Experimental 'gene switch' increases lifespan with no ill effects

October 24, 2002

By experimentally switching genes off or on at specific stages in an animal's lifecycle, UCSF scientists have discovered that vigor and lifespan can be significantly extended with no side effects. Many researchers believe that increasing lifespan will dampen reproduction. But the new study of the tiny roundworm commonly known as C. elegans shows that silencing a key gene only in adulthood increases longevity with no effect on reproduction.

The focus of the research is a set of genes that control a hormonal pathway. Since the same or a similar pathway is found in many organisms including humans, the results offer the tantalizing prospect of safely and effectively extending the human lifespan as well.

The research is published in the October 25 issue of the journal SCIENCE.

Many experiments in different organisms have supported the evolutionary principle that everything comes at a price - that tweaking hormone levels to boost lifespan will inevitably cause abnormal reproduction. But in the new study, UCSF scientists report for the first time that the hormonal pathways controlling reproduction and longevity act independently and can be decoupled.

Typically, to study a gene's effect, researchers knock out the gene's activity permanently. But the scientists were able to use a new technique to turn genes off at different times and thereby learn that the genes had different effects at different stages in the life of the worm. They were also able to turn the "off" genes "on" again to further probe the timing of their influence.

The UCSF scientists studied a now well-known gene called daf-2. In previously research they showed that partially disabling the gene doubles the worm's lifespan. The gene encodes a receptor for insulin as well as for a hormone called insulin-like growth factor. The same or related hormone pathways have since been shown to affect lifespan in fruit flies and mice, and therefore are likely to control lifespan in humans as well. The gene also affects reproduction, but the new research shows that the gene acts at different stages in the lifecycle to control reproduction and lifespan, so the two effects can be decoupled, said Cynthia Kenyon, PhD, the Herbert Boyer Professor of Biochemistry and Biophysics at UCSF and senior author on the SCIENCE paper.

"A lot of evolutionary biologists predicted that you couldn't lengthen lifespan with out inhibiting reproduction," Kenyon said. "But that's not true. These worms live much longer than normal and they reproduce perfectly normally. They look great, they're vigorous. These animals are having their cake and eating it too."

"As we uncover more about how these and related genes function we hope to learn how youthfulness and longevity can be extended in humans without any side effects as well."

Kenyon made international news in 1993 when she discovered that blocking daf-2 activity in C. elegans -- more formally, Caenorhadtis elegans -- doubled the normal two-week life of the worms. Their added weeks were not spent as doddering old worms, either.

"You could look at them under the microscope and see that they were lively and youthful," Kenyon said. In the new experiments, Kenyon and her colleagues used a fairly new technique that allowed them to silence daf-2 or a second gene, daf-16, either just after the worms had emerged from eggs, or as young adults. All genes are made of DNA, but protein-making instructions are sent out of the nucleus in the form of a related molecule, RNA. The technique can partially disable, or "knock down" any given RNA sequence - and hence any given gene - simply by introducing double stranded RNA that matches it. The two strands separate when cleaved by an enzyme called Dicer, and one of the strands hybridizes with the animal's own RNA, leading to its degradation. Perhaps evolved as a viral strategy to attack and disable hosts, RNA interference, or RNAi as it is called, can be used to knock down the activity of any gene at any time in the life of an organism.

Kenyon's team engineered bacteria to produce double-stranded RNA corresponding to one of the worm's genes, and then allowed the worms to feed on the bacteria. In some experiments, they used RNAi to partially disable daf-2 activity; in others, they inhibited a gene called daf-16, which Kenyon's group had previously determined acts "downstream" of daf-2 in the pathway to extend lifespan.

They discovered that if daf-2 activity is knocked down just after the worms have hatched, the worms live about twice as long as normal and are more resistant to stress, but their reproduction is delayed. However, if daf-2 is allowed to function normally until young adulthood, and then is knocked down, the worms are long-lived, stress-resistant and reproduce normally - the best of all possible worlds.

Previous research by Kenyon and others had shown that inhibiting daf-2 activity in C. elegans, or related genes in insulin-like pathways in other organisms, was a two-edged sword. The animals gained longer lives but their reproduction was suppressed. It now appears that these findings - consistent as they were with evolutionary theory - are the result of using too crude a tool -- turning the gene off throughout the life of the organisms. By refining the experimental switch, Kenyon's group discovered that daf-2 is involved in both the lifespan pathway and the reproduction pathway, but that the two act independently.

"It is very exciting to find the two pathways are not inextricably connected," Kenyon said. "This means that by understanding the timing of their activity, we may be able to gain the benefits - longevity - without the drawbacks."

A decade of research on aging and related processes has shown that daf-2 gene activity shuts down when the worm's food source is scarce or environmental conditions are otherwise not ripe for reproduction. Under these conditions, juvenile worms go into a state of suspended animation, known as the dauer, and reproduction is delayed until food is restored and they come out of this phase. But the new study shows that if the gene is not inhibited until the organism already is a young adult, the worms gain increased longevity without experiencing a dauer stage or compromised reproduction.

The researchers were able to refine their scrutiny of the gene's effect even more by exploiting the Dicer enzyme required in the RNAi process. They were able to turn off the "off" switch of RNAi - using it against itself to turn genes back on. Dicer normally acts to cut up the double-stranded RNAi into small pieces that bind to the host's RNA. But Kenyon's group realized they could introduce double stranded RNA corresponding to the Dicer gene as well and inhibit RNAi itself. When they did this, the effectiveness of RNAi was reduced, and the worm's RNAi-silenced genes were turned back on. By knocking down daf-2 during early life stages, but restoring it in adults, they were able to show that the worms gained no increased lifespan protection, and that therefore the gene's life extension powers act only in the adult.

Kenyon and UCSF have applied for a patent for the use of the Dicer gene to turn off RNAi, which may be therapeutically useful. Genes could be turned off when their activity causes disease, but turned back on when the suppression is no longer needed.

The team found that daf-2-deficient worms are both longer-lived and resistant to oxidative stress - damage to cells caused by charged, oxygen-bearing molecules. This supports the view that vulnerability to oxidative stress is a key -- if not the key -- cause of aging, the researchers state. Oxidative damage disrupts normal cellular function and leads to cell death. Many people take antioxidant supplements in a strategy -- still unproven -- to stave off the aging effects of oxidative damage.
Lead author on the paper is Andrew Dillin, PhD, now an assistant professor in molecular and cell biology at the Salk Institute, who participated on the research while a postdoctoral scientist in Kenyon's UCSF laboratory. Co-author is Douglas K. Crawford, a graduate student in Kenyon's lab.

The research is funded by the National Institutes of Health.

University of California - San Francisco

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