Partnership in The Fight Against Aging

SECOND OF 2 PARTS

Companies and academic researchers are racing to find the control mechanism for aging, in order to block aging itself. In this second installment, covering an important conference on the topic, Dr. Gregory M. Fahy updates us on longevity genes, calorie restriction and telomeres.

Powerful forces are being unleashed in the war against aging. The genes that control aging are rapidly being identified, drugs have been found that appear to be effective in reversing age-related changes without toxicity, and links to aging phenomena observed in widely divergent species, such as worms, yeast and humans, are becoming apparent.

Significantly, the economic implications of successful aging intervention are becoming real, meaning that more interest from Wall Street and other investors is forthcoming to bring products and techniques to market. Anti-aging research is no longer solely in the realm of "pure science," but is nearing a practical stage to be of benefit to most people living today.

Perhaps the most spectacular presentation at the Conference on Age-Related Diseases, held in Las Vegas in December, was given by Cynthia Kenyon, the Herbert Boyer Distinguished Professor of Biochemistry and Biophysics at the University of California, San Francisco. Describing the elements of a general scientific tour de force, she revealed that a special longevity gene in a nematode worm called Caenorhabditis elegans is normally triggered by calorie restriction, but can also be unleashed to double the life span of the normal adult. Further, the gene acts through a central signaling mechanism, and may function in a manner analogous to similar established mechanisms in human beings.

As detailed in previous issues of Life Extension, the basic way C. elegans usually balloons its life span under natural conditions is by blocking its own development. The worm usually goes through four larval stages prior to becoming an adult, but if its calorie intake is reduced or if overcrowding takes place, the worms go from the second larval stage into a state of arrested development, called the dauer stage. Worms in the dauer state live far longer than normal worms, allowing them to survive until food becomes more available or until crowding subsides, whereupon they complete development, become adults, and live out their 15-day life spans. (Aging postponement through developmental arrest has also been reported in insects, molluscs and mammals.)

Cynthia Kenyon of the University of California, San Francisco, described the elements of a general scientific tour de force: That a special longevity gene in the worm Caenorhabditis elegans, normally triggered by calorie restriction, can also be unleashed to double the life span of the normal adult. Further, the gene acts through a central signaling mechanism, and may function in a manner analogous to similar established mechanisms in human beings.

Kenyon explained that the genes called daf-2 and daf-16 (daf refers to "dauer formation") jointly govern life span in C. elegans. Daf-2 action normally causes maturation to adulthood and a normal life span, while daf-16 action tends to produce the dauer state and a long life span (causing Kenyon and her colleagues to affectionately call it "sweet 16"). Unless calorie restriction or crowding is imposed, daf-16 is silent and daf-2 imparts a normal life span. However, if mutations knock out daf-2, the result is a greatly extended life span or a permanent dauer state.

If the technology can ever be applied to humans, it conjures up images of people being six years old forever. The really interesting observation was that merely weakening daf-2 activity, rather than entirely knocking it out, allows daf-2 to facilitate the maturation of the worm, while also permitting daf-16 to produce long life spans-without arresting the worms in a pre-adult state.

Daf-2 activity is weakened by using a temperature-sensitive daf-2 mutant that permits maturation at low temperature, but then becomes inactive when the worms are warmed. When daf-2 becomes inactive, daf-16 is allowed to act in the adults, a very unusual situation. These adult worms appear normal, and do not exhibit most of the characteristics of dauer larvae.

Also of great importance is the fact that the metabolic rate of these super long-lived adults is the same as that of non calorie-restricted, normal adults, so the observed life span extension is not due to "living slower" or producing fewer free radicals. Something much more intriguing is involved. Daf-2 is a member of the insulin receptor and insulin-like growth factor-I receptor family. Therefore, the "death signal"-that is, the signal to mature and age at a normal rate-presumably is induced by the binding of the worm version of insulin to the daf-2 protein in response to increasing sugar levels following feeding. (Worm insulin-like molecules are known to exist.)

But daf-2 is no ordinary insulin receptor. Kenyon believes that daf-2, rather than acting locally, serves a signaling role mediated by neurons and endocrine cells. Using a special technique in which chromosome fragments that contain daf-2 are distributed at random throughout the cells of a daf-2-deficient worm as it develops, Kenyon tried to find out whether daf-2 acts only locally, or at a distance. What she found is that daf-2 in neurons and endocrine cells blocked dauer formation in cells elsewhere in the body, even though those cells lacked daf-2. Conversely, when daf-2 was missing in neurons and endocrine cells, the other cells went into a dauer state even when they had daf-2.

This means there must be a signal created by the daf-2 in neurons or endocrine cells that travels throughout the body and controls whether the animal as a whole goes into a dauer state or not. This signal is a kind of master anti-youth hormone, and it no doubt will be found soon.

But daf-16 is the more interesting gene. Kenyon's search for other genes that act like daf-16 failed, indicating that this is a unique master gene for life-span extension. In fact, when daf-16 was essentially deleted from otherwise normal worms, they matured normally and had a normal life span. This means that the only thing daf-16 is naturally used for is increasing life span, in association with producing the dauer state. Kenyon is now investigating whether the effect of calorie restriction on life span requires daf-16.

Daf-16 is a member of the HNF3, or forkhead, family of transcription factor proteins. Therefore, the main function of daf-16 is to turn on other genes, something that is required for life span extension in the C. elegans worm. Finding which youth-preserving genes are turned on by daf-16 will obviously be of great interest.

The daf-16 gene resembles analogous mammalian HNF3 genes, including genes that affect the risk of cancer. Particularly tantalizing is the fact that there are examples in mammals in which insulin completely blocks the function of HNF3 forkhead proteins.

The obvious implication: The worm mechanisms also may apply to humans, and might directly lead to human life span-extension therapies, probably without the need for calorie restriction.

The thrust of almost all calorie-restriction research is to find the mechanisms by which calorie restriction works to extend life span, and then find ways for humans to do the same thing without having to undergo the discomfort of excessively reducing their food intake. Kenyon's worms seem especially close to showing us how this might be accomplished.

Underscoring such possibilities, Kenyon notes that human cells can use C. elegans genes and vice versa, including genes that govern many vital functions such as cell division, programmed cell death, cell migration, cell differentiation, and tissue pattern formation, with the "transplanted" genes working just fine in the radically alien host cells. This implies that a human analogue of daf-16 could have an active role in humans.

It is hard not to conclude from Kenyon's talk that gerontology is converging rapidly on some of the core mechanisms of both human aging and human life-span extension.

This is, of course, provided that calorie restriction actually does work in humans and other primates. This was addressed by Mark Lane, a senior staff fellow at the National Institute on Aging's Gerontology Research Center in Baltimore, Md. The NIA study on primate calorie restriction was started about 11 years ago and involves 200 rhesus monkeys (see "Calorie Restriction in Monkeys," July 1998, of which Dr. Lane was a co-author). Calories are restricted by 30 percent, a process phased in by reducing the calorie intake 10 percent per month over three months.

It is still too soon to know what is happening to aging per se, but the following observations have been made: First, the restricted animals are not emaciated, but are simply smaller. Secondly, restricted animals have lower cholesterol, triglycerides, blood pressure and fasting insulin (enhanced insulin sensitivity). Further, serum DHEA sulfate has fallen noticeably in the control monkeys, but not in the restricted ones.

In summary, 16 changes that have been induced by calorie restriction in rodents also have been replicated in these primates. Similar studies underway at the University of Wisconsin and at the University of Maryland are showing results consistent with those found in the NIA study. Lane concluded that calorie restriction appears to be reducing risk factors for aging and disease, and that obese animals respond in a manner similar to non-obese animals.

Previous studies have indicated that a common denominator is improvement in insulin sensitivity (glucose control). Lane further linked glucose metabolism to the calorie restriction effect by citing the link between insulin signaling and aging in models such as C. elegans. He also mentioned that the glucose analogue 2-deoxyglucose, which can't give rise to useful energy for the cell, mimics calorie restriction in that it reduces tumor growth, body temperature (even producing torpor) and cell cycling, and facilitates necessary programmed cell death (apoptosis).

Presentations were given by Mike West, previously of Geron Corp., left, Jan Vijg of the Harvard Medical School, and Minori Sugawara, of Agene Research Institute Co., bottom

In a pilot study, the diets of rodents were spiked with three doses of 2-deoxyglucose (0.2, 0.4, and 0.6 percent of food weight). These animals gained almost as much weight, and ate almost as much food, as did control animals, yet their insulin sensitivity was improved after three and six months of treatment. Other candidate glucose analogues also are under study.

At the end of the first morning of the IBC meeting, there was a panel discussion focused on central questions, including: If a gene is identified in a model system of aging, will it tell us anything about human disease? Are there universal mechanisms of aging? Is there a correlation between "in vitro aging" and other models of aging? Jack Egan, the senior director of pre-clinical research for Alteon Inc., a Ramsey, N.J.-based publically traded company, felt genes governing the ATP/ADP ratio and insulin resistance would be relevant to human disease, and he believes in the usefulness of in vitro models. Egan also said aging is a disease.

Kenyon said she felt her worms are not unique, that universal aging mechanisms do exist, and that random damage might be an effect of aging rather than a cause. Dr. Jan Vijg of the Harvard Medical School felt that aging was nothing more than pathology, and that in-vitro aging is not the cause of organismal aging.

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Dr. Minori Sugawara, of the Agene Research Institute Company, in Kamakura, Japan, held out that the "wrn" mutation-which causes Werner's syndrome, a disease characterized by rapid premature human aging-is a single-gene mutation that overlaps aging.

Michael Rose, a University of California, Irvine, professor, who also consults with private industry, submitted that energy metabolism is a common theme and an example of what could be universal. Kenyon countered that mice live for only two years, but that animals with much higher rates of energy metabolism can live much longer (for example, bats can live 15 years and canaries 13 years).

Huber Werner, with the National Institute on Aging, suggested that mitochondrial aging could be important, and that birds make fewer superoxide radicals per oxygen used than rodents. Werner's comments were rebutted by Vijg, who noted that there are so many mitochondria that the loss of a few mutated mitochondria may not be important, and that the mitochondrial mutation rate is not different from the nuclear DNA mutation rate in several cases. Werner drew attention to an NIA request for proposals on genes and gene regions that are candidates for aging modulators.

Vijg explained his system for screening rodent DNA for background mutations that might underlie the aging process. His results showed that the number of mutations present in the DNA at any given time was constant between the time of sexual maturation and the time rodents die, about 35 months.

Therefore, mutations do not accumulate during the time that aging takes place, making mutations an unlikely cause of aging. The hypothesis that the exponentially rising incidence of cancer might be due to an exponentially rising number of mutations was also contradicted by these results. Clearly, something else is controlling cancer incidence besides mutations or even DNA repair. In any case, Vijg is now shifting his attention to changes in gene expression that lead to aging.

Another compelling avenue of aging research is the telomere theory of aging. As detailed extensively in Life Extension, telomeres are regions of DNA at the end of each chromosome that are thought to shorten each time a cell divides. Michael West, at the time of the meeting vice president for new technologies for Geron Corp., of Menlo Park, Calif., explained Geron's hypothesis: That telomere shortening is the clock of cell aging, which leads to several types of product possibilities. One of these is the use of naturally immortal primordial stem cells to rejuvenate other cells. Another possibility is to find a way to inhibit telomerase-the enzyme that maintains telomere length in cancer cells-as a universal anti-cancer target, to prevent cancer cells from being immortal. In support of this concept, the death or cure of neuroblastoma patients is predictable based on their cancers' telomerase activity, and 85 to 90 percent of malignancies studied have turned-on telomerase activity. Collected urine can be checked for telomerase activity to detect bladder cancer.

A therapeutic use of telomere biology could entail removing cells from a patient, recharging them with telomerase, and putting them back to replace old, dividing cell types.

West noted that prolonging the lives of cells used for making biotechnological pharmaceuticals would be another therapeutic use of telomere biology. Further, cells could be removed from a patient, recharged with telomerase, and put back in the body to replace old, dividing cell types. This would be easy to do with bone marrow cells. Similarly, reversing senescent gene expression by restoring telomere length should reduce wrinkling, fragility, and other age-related changes in skin.

West also spoke of using known, naturally immortal primordial stem cells that, when injected into muscle, spontaneously give rise to blood cells, neurons, various kinds of muscle cells, hair follicles, intestinal cells, teeth, and other complex tissues. He said there is no other known technology for making complex tissues, and that this could be an inexhaustible resource for making cells and tissues for transplantation. West's new company, Origen, will develop primordial stem cells to make therapeutics.

West said the dream is to manipulate the human genome as we wish. This stem cell technology opens the door to that because if we can put genes into specified areas of the genome and overcome cell senescence, we can do gene targeting without limit and make any kind of cells we want. Therefore, this is a technology platform for very open-ended gene therapy and gene augmentation. West also contemplated transgenic cell-based screening, using a product he called Lifescreen. In this assay, cells of various kinds are put onto a biochip.

Despite the exciting discoveries made by Geron and research scientists associated with the company's efforts, not all scientists agree with the telomere theory of aging. Cynthia Kenyon, for one, challenged West's statement that telomere shortening is the clock of cell aging. She noted that while her worms age, all of the cells in the worm are non-dividing cells. Since telomeres shorten only when cells divide, Kenyon argued that telomere shortening doesn't appear to be a factor in aging C. elegans worms.

Similarly, she noted, mice grow old and die while still having long telomeres, and when telomerase is removed from mice, they live just fine for six generations despite the resulting telomere shortening. She also said that low levels of telomerase are, indeed, found in human tissues and that you need very little to maintain telomere length. She was concerned that lengthening telomeres to help aging cells would interfere with anti-cancer therapy.

West agreed that not all aging is cell aging (replicative senescence). But immunosenescence, age-related macular degeneration, and other human aging processes appear to be related to cell senescence. Telomere shortening is probably not germane to Kenyon's worms, but is germane to aging in yeast. As for mice, these animals do not get heart disease and other conditions that are characteristic of human aging and thus may not die or age for the same reasons that humans do. Further, mice have very long telomeres compared with humans, and so can get by better without telomerase. Mouse cells are easy to immortalize, whereas human and bird cells are classically hard to immortalize.

Walter Funk, also from Geron, noted that many changes in gene expression have been documented as cells approach the "Hayflick limit"-the replicative senescence point during reproduction in tissue culture dishes in the lab-but the relevance of these findings to aging in the whole body needs to be better established.

Funk reported it is possible to restore telomerase activity to human cells, and implied that he would be using such rejuvenated cells in his future experiments to show that cell rejuvenation can allow old cells to give rise to young (and, by implication, natural) human skin.

Funk concurred with Francis Bacon, who noted several hundred years ago, "The lengthening of the thread of life itself" has not received enough attention.