Team Identifies a New Mechanism That Extends Lifespan of a Nematode

By Eric Sauter

Caenorhabditis elegans is a nematode or roundworm, approximately 1 mm in length, the first multi-cellular organism to have its entire genome sequenced. This unassuming (not to mention transparent) creature has been used extensively as a model organism, one that has provided a number of biological insights into our own species over the years. Even in comparison to the ubiquitous fruit fly and mouse, C. elegans may, in fact, be the most widely studied organism in the universe.

Now, scientists at The Scripps Research Institute have used this semi-famous nematode to uncover a new mechanism that helps determine the worm's lifespan—and that, if inhibited, actually extends that lifespan, albeit by a relatively small amount. The findings, which were published in the August 3 (Vol. 317; No. 5838) issue of the journal Science, should help significantly in the study of diabetes and aging in humans.

In the new study, Scripps Research scientists used a new automated approach to tandem mass spectrometry to identify signaling targets of the insulin receptor-like protein DAF-2. The DAF-2 signaling pathway in C. elegans is similar to the insulin-like growth factor (IGF-1) pathway in humans; it controls the expression of a large number of protein targets that regulate metabolism, development, and aging.

"There are two important aspects to our study," said Meng-Qiu Dong, the lead author of the paper and research associate in the Yates lab at Scripps Research. "First, there is the technical aspect of using advanced mass spectrometry to compare protein abundance between wild type nematodes and mutant types. Secondly, we discovered a new compensatory mechanism involved in insulin signaling and lifespan. Previously, it was widely assumed all changes in long-lived daf-2 mutants should extend lifespan. Our results show that it's not that simple."

In the new quantitative proteomic study, Scripps Research scientists were able to identify 86 proteins that were more (or less) abundant in long-lived DAF-2 mutant roundworms than in wild type worms. Genetic studies of a subset of these proteins indicated that they act in one or more processes that are regulated by DAF-2, including entry into the dauer developmental stage and aging. The dauer phase is an alternative stage of the worm's development process. Dauer animals live much longer in comparison to worms that develop normally; lifespan increases of as much as four to eight times have been reported.

The Scripps Research scientists were able to identify these new proteins through a combination of improvements in mass spectrometry.

"Our new tandem mass spectrometry techniques are to proteins what the microarray is to genomics and messenger RNA profiling," Dong said. "The new method lets us look at a complex mix of proteins and find meaningful quantitative information of individual proteins. We are able to quantify the abundance of these proteins—in both wild and mutant type worms—far better than what we could with traditional techniques."

Instead of measuring messenger RNA, which is not a final gene product, the scientists made direct measurements of downstream proteins, a novel approach that allowed for the identification of protein targets not regulated at the transcription level. Mass spectrometry converts individual molecules into ions, allowing them to be manipulated by electric and magnetic fields and, consequently, measured and analyzed; tandem mass spectrometry involves multiple analyzers and is used for structural and sequencing studies.

These new techniques, which were first described by Scripps Research scientists in a 2004 article published in the journal Nature Methods, can easily be used to determine changes in protein abundance that result from either genetic or pharmacological changes, the study noted.

Diabetes Link

What the scientists discovered with this new technology was a compensatory mechanism that was activated in response to reduced DAF-2 signaling—and that helped maintain normal DAF-2 signaling levels.

The protein, TAX-6 (or CNA-1, the C. elegans calcineurin A protein) turned out to be part of a mechanism that helps maintain DAF-2 signaling at normal levels through a complex feedback loop—although TAX-6 facilitates DAF-2 signaling, TAX-6 itself is regulated by DAF-2 because reduced DAF-2 signaling results in an increased abundance of TAX-6.

As a result, the study concluded that the lifespan of daf-2 mutant worms was the result of two types of changes. One type of changes extends lifespan. The other, such as the protein abundance changes of TAX-6 and several proteins that function in carbohydrate metabolism or translation, actually shortens it; inhibition of the TAX-6 compensatory mechanism extends lifespan, at least in wild type worms.

"TAX-6, which is a calcineurin protein, is important because it shows that there is another layer of regulation," Dong said. "Admittedly, inactivation of tax-6 results in only a small lifespan extension, but this is a single gene. There may be others that function in parallel to tax-6. We don't know what might happen if you inhibit all the compensatory regulation, but a big increase of lifespan is a possibility."

Because calcineurin is conserved in both humans and nematodes, she said, there may be significant links between the inhibition of this protein and the onset of diabetes in humans. Studies have shown that a significant number of transplant patients who receive calcineurin inhibitors as immunosupressors were stricken with new onset diabetes mellitus. One such study, completed in 2006 and published in the journal Clinical Transplantation, reported that new diabetes cases occurred in approximately 15 to 20 percent of kidney transplant patients and 15 percent of those with liver transplants.

"Our new study is something of a primer," she said. "We hope more people will follow up on the diabetes aspect of this because calcineurin inhibitors are a risk factor. We also hope that scientists will become aware of our advancements in mass spectrometry. It's a great new tool and we want more people take advantage of it."

In addition to Meng-Qiu Dong, other authors of the study,Quantitative Mass Spectrometry Identifies Insulin Signaling Targets in C. Elegans, include John D. Venable, Tao Xu, Sung Kyu Park, Daniel Cociorva, Jeffrey R. Johnson, and John R. Yates III of The Scripps Research Institute; and Andrew Dillin of the Salk Institute for Biological Studies; and Nora Au of the University of California, San Diego. See Science at http://www.sciencemag.org/cgi/content/abstract/sci;317/5838/660.

The study was supported by the National Institutes of Health.

Send comments to: mikaono[at]scripps.edu

"Previously, it was widely assumed all changes in long-lived daf-2 mutants should extend lifespan. Our results show that it's not that simple."

—Meng-Qiu Dong

"Our new tandem mass spectrometry techniques are to proteins what the microarray is to genomics and messenger RNA profiling."

—Meng-Qiu Dong