~ Gene Expression and the Search for Fountain of Youth

Genomics and Proteomics


Whether Spanish conquistador Juan Ponce de Leon actually set out to find the Fountain of Youth in 1513 is an interesting and unsolvable debate. Some accounts, including elementary school textbooks, state this pursuit as fact. Others, including the online working resource Wikipedia, state flatly that "the popular story that Ponce de Leon was searching for the Fountain of Youth is misconceived."

Some 500 years later, Ponce de Leon's fabled quest continues to fascinate humankind and, in its own way, drive scientific research. This is not the search for a mythological body of water, but rather, a figurative fountain in the form of gene expression research. It's too late to help Ponce de Leon, but it could offer his ancestors the chance to live longer and healthier lives.

But when, and at what cost? The answer may be sooner than many people realize. Researchers have already succeeded in increasing the life spans of simple organisms such as Drosophila melanogaster and Caenorhabditis elegans through genetic manipulation. For those who assume that it is a large leap from successes in model organisms to major changes in the life span of humans, Stuart Kim, professor of developmental biology and genetics, Stanford University, Stanford, Calif., gives a word of caution. "It's easy to change the life span of an animal," Kim says. "We know how to change the life span in worms, flies, and mice. [I think we are not that] far away from doing some sort of therapy that would change human life span. This year, next year, who knows?"

Making senescense of gene expression

Genetic manipulation and the concept of immortality have been popular comic book and science fiction topics for decades. Immortality in particular is a tantalizing subject, in part because it violates a well-understood principle: we're all going to die. If a car accident or heroin overdose doesn't get us in an instant, Huntington's disease or cancer or Alzheimer's disease will get us a piece at a time. No one gets the drop on Father Time.

In principle, genetic manipulations could change all that. The more researchers learn about senescence, or the state or process of aging, the more they are finding ways to slow it, and the goal for some is to stop it altogether.

"Aging is complex," Kim says. "Almost everything changes as animals get older. You need to look at all the changes together to try to figure out what is different between the young adult and the older adult. We want so-called 'biomarkers for aging.' We've been working hard to do that for one model organism, the nematode, and now we're looking at another ," he says, "the human."

To oversimplify a bit, the number of times a cell can divide is limited; thus, older organisms become more susceptible to age-related cellular meltdowns. One of those malfunctions, Parkinson's disease, has been the research focus of Mark Cookson, PhD, chief of cell biology and gene expression at the National Institute on Aging (NIA), Bethesda, Md. Cookson's work focused on understanding mechanisms leading to neural cell damage in Parkinson's disease, using expression arrays to find underlying pathways that lead to cell death. In one project, Cookson's team is examining altered patterns of gene expression for genes that produce similar phenotypes, in an effort to clarify the contribution of multiple gene products to neurodegeneration.

"Like all degenerative diseases, Parkinson's [disease] has a bunch of neurons that die in a progressive manner," says Cookson. "By the end of the disease, there are big populations missing. One of the questions we asked about Parkinson's is if genes that are changed share a common promoter element. The problem was that, because there are multiple regulatory elements, that data set was almost impossible to sort through."

Cookson and his group found about 10 genes on which they are amassing data for some kind of supervised clustering approach to figure out if they're similar or different in terms of gene expression. "You expect them all eventually to look the same, because they're all neurons that are going to die," Cookson says. "But there is some real argument in the field about whether these are similar disease processes or not."

Cookson is aware that his research foothold is planted somewhere along the slippery slope between improvements in human health and gene manipulation, but he doesn't seem conflicted about it. "There is a person who's unwell, and you can try to help their quality of life," he says. "There will always be a push that people want to live longer and live healthier. Fortunately, what's happening in the scientific community and in the society in general is that people are aware that healthy living is as important as long life. Hopefully, what we're doing is minimizing disease processes, and we're looking at successful aging rather than just increasing life span. But all of those things are related scientifically and logically."

The debate over that relationship is just beginning. In an interview with online magazine Salon , geneticist and author Gregory Stock framed the debate nicely. "People talk about . . . genetic enhancement and anti-aging interventions as if they're independent from medical technologies, but in fact what we're doing is unraveling biology. There are huge numbers of people who would like solutions for Alzheimer's disease, heart disease, and cancer. A natural spin-off is the kind of knowledge that allows us to do some things that many people are far more conflicted about. In order to stop the one, you are basically taking real people with real diseases and saying they'll just have to be sacrificed."

Here come the mutants

The origins of anti-aging research began with the sequencing of the human genome. "That gave us the power to start looking at biological processes in completeness, by scanning all of the genes using full genome DNA chips," says Stanford's Kim. "You can see in parallel all the genes that changed in whatever developmental process you're interested in." Gene chips from Affymetrix, Santa Clara, Calif., and others are used extensively in cancer research, and now are being employed to make sense of gene expression differences in what Kim calls "big-picture analyses used to study development and response to stress."

The tiny, transparent nematode C. elegans was the first target, primarily because its entire nervous system had been mapped. "You can't just change every gene in the mouse one by one and see how it affects the life span of the mouse," says Cynthia Kenyon, PhD, professor of biochemistry and biophysics at the University of California, San Francisco. "It's a humongous undertaking. And you can't do clinical trials for anti-aging medications. You can't make human mutants."

Kim echoes the worm's utility in longevity research. "C. elegans grows in two weeks and is a terrific genetic model organism," he says. "The work done by Cynthia Kenyon and others has made the worm the best model system for aging."

Kenyon is determined to find out how much a 959-cell worm can really tell us about cell death, and, by extension, aging in humans. "People said to me, 'Why study the worm? You won't learn anything about people from studying the worm.' They were all totally wrong. Turns out there is a whole set of genes whose only job in the world is to determine the pattern of an animal. The reason a dog looks different than a cat is that some of those genes are turned up or down, but they're the same genes."

Another common misconception, says Kenyon, is that people just get old, and nothing can be done about it. "I thought that there must be some kind of machinery, like a clock, that controls the rate at which the animal ages," she says. "The clock can be set to go fast in a mouse, which has a two-year life span, or it can be set to go slowly in a human, which has an 80-year life span. We began to change genes at random to see which ones influence life span, and we found [the] daf-2 [gene]."

By adding copies of daf-2 to C. elegans , Kenyon and others were able to expand the worm's life span sixfold. "Everybody who sees our long-lived worms thinks they're magic," she says. "What we find in these model organisms is [when we] change gene activitives, we don't just increase life span, we also postpone the time of onset of a lot of different age-related diseases. The goal is to do both. There are worms that are long-lived and don't appear to be healthy. That, to me, is the nightmare."

More recently, Kenyon has been studying the direct connections between aging and disease, particularly in Huntington's disease. In particular, she is interested in how the changing hormone genes, also known as chaperones, can affect the activity states of a lot of different genes. "Hundreds of chaperones become more active or less active when they age," says Kenyon. "Some of them are used to fight infections, and others prevent the function of antioxidants. In the long-lived mutants, chaperones are turned up, and you can change the time of onset of Huntington's disease. The chaperones are preventing their own genes from clumping together."

Trading in for a different model

"If I had to characterize aging research right now, I almost feel like it's too easy to alter the life span of flies, worms, and mice," says Stanford's Kim. "There are currently something like 200 different genes that affect the life span of worms, and these 200 genes are in all sorts of different processes, such as affecting insulin pathways, or chromatin, or mitochondria. There are lots of ways to succeed. Smaller experiments have been done on flies and mice, but if you look at how many genes have been tried, and how many affect life span in flies and mice, it's about the same number. [In] about 2% of genes, if you change them in the right way, you can make the animal age faster or age slower."

Kenyon says there is a clear path from gene expression in model organisms to expressing analogous genes in humans. "We think it's likely that the pathways we've identified that control aging in these animals, because they work in mice, are reasonably likely to have a similar effect in humans," she says. "But we don't know that."

Kim has also done a lot of work with C. elegans . "Aging is so different between a worm and a human. [The worm] ages 5,000 times faster than a human. For any mechanisms you discover, they have to have a huge plasticity in mechanism in order to be relevant for an 80-year-old human and a two-week-old worm. You very well could figure out exactly what causes a worm to age, but that might have nothing to do with what causes a human to age, even at the most fundamental level."

A basic tenet of gene therapy is that significant gene interactions should be conserved through evolution. That is, similar pairs of genes from different organisms should show similar gene expression. "I'm not very confident that aging is conserved," Kim says. "A three-week-old worm doesn't exist in the wild. A two-year-old mouse doesn't exist in the wild, and an 80-year-old person doesn't exist until very recently. In natural populations in the wild, old animals tend not to be there. That means that normal Darwinian selection doesn't apply for old animals."

Because of that, Kim started to seriously doubt whether the events occurring in old animals could be evolutionarily conserved in the same way that a cell's biological and developmental processes are conserved. If old animals aren't present, their fitness can't be selected for. Thus, Kim began to shift his work from model organisms to tissue taken from human kidneys. The kidney is a good organ to study because its performance is easily measured and is known to decline with age. Beginning at age 40, Kim says, the filtration function of the kidney starts to decline.

Kidney genes in young and old

What Kim found so far is that a young kidney and an old kidney look surprisingly similar at the gene expression level. But he found some biomarkers that track physiological age, and he plans to publish those results shortly. "We just now have molecular markers for aging in the kidney," he says. "If I did the same thing, we could have molecular markers for aging in the muscle, liver, or skin. These markers don't tell how many years you've lived; they tell your relative health. You could turn these into terrific clinical markers, and you would know whether someone is 40 and looks like they're on a bad trajectory, or if they're 80 and you could treat them as a 40-year-old."

Such results could have signficant implications in kidney transplantation, for example. Older kidneys are categorically excluded from transplantation, because, says Kim, young kidneys are marginally more efficient. But if markers can tell researchers the relative physiological fitness of a kidney, they could better predict how fit the organ is for transplantation. They could also expand the available pool of kidneys to include many that are currently excluded. "The 60-year-old kidney isn't that much worse off than the 40-year-old kidney," Kim says. "It's not 10-fold worse or twofold worse, it's 50% worse. But I could find a small subclass of 60-year-old kidneys that look like they're 40."

Ecology, ethics, and evolution

Such real-world results from genetic studies provide a framework for discussion about the implications of longer-lived humans, but do not answer larger questions about the unintended consequences of genetic manipulations. What happens to the planet's finite resources if birth rates continue unabated, but the death rate slows significantly? What are the potential social implications of children having not only parents, grandparents, and (for some) great-grandparents, but also great-great-grandparents and so on?

More germane to pure science are the potential unpredictable effects of genetic manipulation, which are thus far unknown. Some scientists believe cells have a finite life span as a way to prevent unchecked cell divisions, also known as tumors. Would genetic manipulation prevent cell death, while at the same time promoting unchecked growth of cancerous cells?

With an eye toward such sticky issues, microarray manufacturer Affymetrix Inc., Santa Clara, Calif., maintains an Ethics Advisory Committee to consider how to ensure the beneficial uses of their technology and to minimize the risk for misuse. "We believe that any manufacturer of a technology that has multiple uses has an obligation to evaluate potential ethical issues," says Thane Kreiner, PhD, senior vice president of corporate affairs for Affymetrix.

Kenyon, Kim, and Cookson all agree that such issues need to be discussed at more length than they have been to this point. "I haven't thought of this very much at all," says Kim. "I'm perfectly happy with everyone curing cancer and heart disease, which will extend human life span by 15 years or 20 years. Let's say we extended human life span not by just curing one disease, but by making people relatively younger so that they were less susceptible to disease, that's almost the same thing. If that worked really well, it might be not just 20 to 30 years, it might be a lot longer."

"I think we're fairly safe in wanting people to age well," says NIA's Cookson. "I don't know if we want people to last longer, but certainly successful aging is something that most people would be really happy with."

The idea of changing genes makes Kenyon uncomfortable, because it is irreversible. "I'm not thinking about changing genes; I'm thinking about making drugs that deal with the activities of proteins that are encoded by these genes. Once you start fooling around with genes, I feel less comfortable with it. What's happening now is that we're extending life span because we're curing diseases.

In principle, if everyone aged twice as slowly, you'd still have the same proportion of old and young. [People] wouldn't get social security at age 65 if they still seemed like they were 40. If there is life span adjustment, it will be incremental. Society will have some time to adapt."

Microarrays Enable Whole-Genome Approach Bill Schu Improvements in microarray technology have enabled researchers to look at the entire genome. Mark Cookson, PhD, chief of cell biology and gene expression at the National Institute on Aging, Bethesda, Md., has worked extensively with the Affymetrix GeneChip arrays. "For us, we wanted to know what the cells would tell us if we looked at as much as we possibly could," Cookson says. "We changed how we approached an experiment, which is to look at the full compliment of things, rather than defining a pathway, having a hypothesis, testing it, and then going back and refining it. We started taking a more whole genome approach."

"Researchers are already using GeneChip tiling arrays to study transcription across the complete genome, not just the 2% historically considered to be important or [that are] 'encoding genes'," says Lianne McLean, senior director of gene expression marketing. "Early studies using these arrays found that 10 times more of the genome is transcribed into RNA than previously thought."

Cookson is trying to use the data that comes off the chips rather than spending a long time validating it and following it up and trying to find out what the pathways are. "We are now looking at and comparing different types of stresses," he says. "Let's say you think protein inhibition is important in Parkinson's disease. One way to use arrays that I think is quite powerful, is to do that experiment, find out everything that's regulated, and then compare it to the thing that you know causes the disease?the mutant protein."

The power of microarrays is just beginning to emerge, says Cookson. "If we have a relatively unbiased snapshot of everything that's going on, we've got a much better chance of actually parsing them out into the right compartments. The platforms that we're using are getting better and better."

Next up for Affymetrix is the development of all-exon microarrays that enable scientists to study splice variants (see related article article on page 25). "Exon arrays will enable researchers to study the different variants of transcripts at a genome-wide scale for the first time," says McLean. "Splice variation can result in one gene being transcribed into multiple RNAs, each resulting in a different protein, with potentially vast differences in biological function. Meaningful measures of gene expression require probes that can measure gene expression at the exon level, [that is,] probes that measure more than just a generic gene. Our goal is to make it possible for both individual scientists and high-throughput laboratories to ask whatever questions they desire about genome variability or genome function in any experiment." Please visit http://genpromag.com for more information.

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