W O R K S H O P R E P O R T

The Promise of Basic Research on Aging

I N T E R N A T I O N A L

L O N G E V I T Y

CENTER - USA, L T D.

The Promise of Basic Research on Aging

A N I N T E R D I S C I P L I N A R Y W O R K S H O P O F T H E

I N T E R N AT I O N A L L O N G E V I T Y C E N T E R - U S A , LT D .

S P O N S O R E D B Y T H E B R O O K D A L E F O U N D AT I O N G R O U P

W I T H A D D I T I O N A L S U P P O R T F R O M T H E I N S T I T U T E F O R T H E S T U D Y O F A G I N G

A N D A N E D U C AT I O N A L G R A N T F R O M P F I Z E R I N C

N e w Y o r k

F e b r u a r y 10-11, 1999 STEVEN N. AUSTAD, PH.D. GEORGE M. MARTIN, M.D. University of Idaho University of Washington

ACOB RODY J A. B , M.D. RICHARD A. MILLER, M.D., PH.D. University of Illinois - Chicago University of Michigan

ROBERT N. BUTLER, M.D. S. JAY OLSHANSKY, PH.D. International Longevity Center-USA, Ltd. University of Chicago

JUDITH CAMPISI, PH.D. Lawrence Berkeley National Laboratory OLIVIA M. PEREIRA-SMITH, PH.D. Baylor College of Medicine ANTHONY CERAMI, PH.D. The Kenneth S. Warren Laboratories, Inc. JAMES R. SMITH, PH.D. 2 Baylor College of Medicine GENE COHEN, M.D., PH.D. George Washington University RICHARD L. SPROTT, PH.D. The Ellison Medical Foundation VINCENT J. CRISTOFALO, PH.D. Lankenau Medical Research Center Thomas Jefferson University HUBER R. WARNER, PH.D. National Institute on Aging

DAVID A. DRACHMAN, M.D. University of Massachusetts Medical Center MICHAEL D. WEST, PH.D. Advanced Technology CALEB E. FINCH, PH.D.

University of Southern California T. FRANKLIN WILLIAMS, M.D. University of Rochester IRWIN FRIDOVICH, PH.D. Duke University Medical Center JOHN R. WILMOTH, PH.D. University of California - Berkeley CALVIN B. HARLEY, PH.D. WOODRING E. WRIGHT, M.D., PH.D. RICHARD J. HAVLIK, M.D., M.P.H. University of Texas Southwestern National Institute on Aging Medical Center MICHAEL AHLIJANIAN NINA M. HILL, PH.D. Pfizer Inc Pfizer Inc

FELIX J. BAKER, PH.D. SHIRO HORIUCHI, PH.D. Tisch Family Interests Rockefeller University

COMMANDER JOHN DUGENE Naval War College MARK HORN, M.D. Pfizer Inc JOE FECZKO, M.D. Pfizer Inc LAURA JOHANNES Wall Street Journal HOWARD FILLIT, M.D. Institute for the Study of Aging KAZUO KOSHIYA, PH.D. 3 Yamanouchi USA, Inc. TILMAN FRIEDRICH, M.D. Pfizer Inc GREGORY STOCK, PH.D.

RAYMOND HANDLAN University of California, Los Angeles Atlantic Philanthropic Service School of Medicine P R E A M B L E

T H E W O R K S H O P R E P O R T

C O M M E N TA R Y

L I T E R AT U R E C I T E D

G L O S S A R Y in gene expression, but also an investigation of these processes on a tissue by tissue basis. It is also important to deduce the actual mecha- There are many unanswered questions about nism(s) by which genetic interventions extend what causes the adverse effects associated with the maximum life span of model organisms, as aging, and even what aging actually is, includ- there are already several examples of interven- ing the potential for continuing creativity and tions of this kind in various model systems contributions to society. Answers to these (Table 1). questions will give us powerful insights into the mechanisms of aging and the causes and Wo rds used to describe the processes of treatment of age-related diseases. Currently, the aging tend to suffer from imprecision. For losses of function and costs of medical care are the purpose of this discussion, we are using concentrated in the last years of life; thus, it is Caleb Finch’s suggestions (Finch, 1990). clear that maintaining healthful functioning and Aging will refer to changes that occur during reducing the morbidity associated with aging the life span, not all of which need to be could have a significant effect on overall life sat- adverse. , on the other hand, isfaction and cost of medical care in the United refers to “age-related changes in an org a n i s m States. With these issues in mind, a multidisci- that adversely affect its vitality and func- plinary workshop was held in tions,” and is due to the inevitable passage of on February 10-11, 1999 sponsored by the biological time. Thus, senescence clearly International Longevity Center-USA to discuss implies a degenerative process. Georg e 7 “The Aging Factor in Health and Disease.” M a rtin suggests the utility of defining a peri- The goal of this workshop was to identify od of the life course called “sageing,” as the promising areas of research which could lead to i n t e rval between decline in re p roductive fit- biological interventions to prevent, delay, or ness and the onset of senescence. During this reverse the adverse effects associated with period a series of adaptive physiological and aging, in particular the burden of disease. behavioral changes may occur in response to both intrinsic and extrinsic challenges. Assuming that there are both genetic and envi- Maximum life span will refer to the empirical ronmental risk factors for aging, the important value observed for the longest surviving indi- general questions become: do these risk factors vidual in a population; it is not a fixed value, affect aging and disease independently of each and will depend upon environmental condi- other; do age-related changes make humans tions. If an organism lives longer in re s p o n s e more vulnerable to disease; or does disease to some intervention, it may either be senesc- occur independently, which then hastens aging? ing more slowly overall, or the interv e n t i o n Viewed from the perspective of individual tis- may have uniquely prevented some critical sues, or specific processes, these options may degenerative pro c e s s . not be mutually exclusive, as what might obtain for one tissue, may not for another. The participants at this workshop discussed U n f o rt u n a t e l y, this means that a complete what general questions need to be answered for understanding of aging will require not only an a thorough understanding of both aging and investigation of general processes such as senescence, what approaches appear to be use- oxidative damage and repair, glycation, changes ful for answering these questions, and the need T a b l e 1

Genes with Significant Effects on Life Spans in Model Systems

MODEL SYSTEM GENE BIOCHEMICAL COMMENTS REFERENCE FUNCTION

Yeast V-Ha-ras Viral oncogene controlled expres- Chen et al., sion of ras extends 1990 life span 100%

Fruit fly methuselah Homologous to mth mutation Lin et al., (mth) membrane GTP- extends life span 1998 binding protein by 35%

Fruit fly sod-1 Cu/Zn-superoxide expression of sod-1 Parkes et al., dismutase in motorneurons 1998 extends life span 8 up to 40%

Nematode age 1/daf23 phosphatidyl- age 1 mutation Morris et al., inositol-3-kirase extends life span by 1996 up to 100%

Nematode daf2 homologous to daf2 mutation Kimura et al., human gene for extends life span by 1997 insulin receptor at least 100%

Mice thioredoxin reduce oxidized transgenic overex- Yodoi et al., groups in protein pression extends 1999 life span by 30%

Human cells TERT catalytic subunit of maintains Bodnar et al., telomerase length; extends 1998 proliferative Jiang et al., potential of cells 1999 in culture (indefinitely?) to support the infrastructure, in terms of people The purpose of this document is to highlight and equipment, re q u i red for adequate and call broad attention to the challenges we p ro g ress in basic gerontological re s e a rc h .1 face and the urgency of achieving advances in T h e re is much new technology being devel- our knowledge, through summarizing some of oped which has the potential to help unlock the promising new opportunities to understand the secrets of aging. However, this will not aging. Taking advantage of these opportunities be an easy task, and it is important to distin- will promote the development of effective inter- guish between hyperbole and genuine scien- ventions to prevent or retard critical adverse tific pro g ress which underlies eff o rts to mod- aspects of the aging experience, and in so doing ify the rate of senescence. improve the quality of life of older Americans.

F i g u r e 1

Period life table estimated survivorship for U.S. population

1.0 9

0.8

0.6

0.4

0.2

0.0

0 20 40 60 80 100

1990

1960

1930

1900 of , and the discovery of antibiotics (see F i g u re 1). Substantial future pro g ress will depend upon obtaining a much better under- standing of what aging is, particularly the adverse components, and how to intervene to I n t ro d u c t i o n either prevent, reverse or retard these adverse age-related changes. In 1825, an English actuary named Benjamin Gompertz observed that death rates in humans As a result of the striking increase in the life rise exponentially after sexual maturity, and he expectancy of Americans in the 20th century, the proposed a simple formula to define the rela- of America is now anticipating a tionship between the force of mortality and age doubling of Americans aged 65 or older, from 35 within a population. This is often used to char- million in 2000 to at least 69 million in 2020. acterize the age-related changes in mortality of This doubling in the number of individuals aged a genetically heterogeneous population, 65 or more could bring with it large increases in whether human or animal. The Gompert z di s a b i l i t y , loss of function, and the cost of health curve implies no finite maximum age for a ca r e for this segment of the population. Thus, it species, strain or population, but does predict is imperative that the biomedical community d e c reasing probabilities of survival with make substantial advances in reducing the major increasing age. The crucial question now is: causes of disability in the elderly population. Can the slope of the Gompertz curve be altered This can only occur with appropriate levels of 10 by biological interventions, i.e., can basic investment in aging res e a r ch. Edward Schneider re s e a rch on aging provide interventions to estimates that the United States currently spends improve quality of life by decelerating the rate only 0.3% as much on aging res e a r ch as it does of senescence. on health care services for older people (S c h n e i d e r , 1999). Money spent on res e a r ch to A different, but simpler way to present survival better understand the mechanisms of aging and data is to plot the fraction of individuals the causes of age-related disease may lead to remaining in a population as a function of the in t e r ventions to both postpone the need for age of the population, i.e., a survival curve. ag i n g - r elated health care, and partially pay for From such a plot it is possible to: 1) detect itself in reduced health care costs. Whether early deaths which presumably are not due to these practical benefits will start to accrue in the usual senescence - related changes; 2) deter- years or in decades will depend on the vigor of mine the median age of death of the individuals the investment. within the population; and 3) estimate the max- imum life span of the species, i.e., an age at which the chance of survival of any individual is Aging vs. Age-related Pathology: negligibly low. The past century has been char- The Role of Age-Related acterized by large decreases in early deaths Changes caused by infectious diseases, and a dramatic i n c rease in the average life expectancy of A continuing debate has been whether a pattern of humans due to improvements in sanitation, aging free of disease can be dissected away from health care, housing, nutrition, the development the overlapping development of age-related dis- eases such as cancer, cardiovascular disease, osteo- 1) pathologically neutral age-related changes, po r osis, osteoarthritis, diabetes, and a large variety e.g., graying of hair; of neurodegenerative diseases, including 2) changes which may contribute to the devel- Alzheimer disease. The most substantial attempt opment of one or more age-related pathologies, to do this has been the 40 year old Baltimore e.g., accumulation of oxidative damage; and Longitudinal Study on Aging (BLSA), which has 3) changes which may cause or indicate overt attempted to describe age-related changes by mak- pa t h o l o g y , e.g., the development of plaques and ing successive measurements over a period of time. tangles in the brain as risk factors for Alzheimer Only apparently healthy individuals are accepted disease. Distinguishing among these possibili- into the study. Whereas many facts have emerge d ties is necessary to guide and prioritize the fr om this study, the foremost is that the effects of development of anti-senescence interve n t i o n s . aging are extremely variable from person to per- son, so it is almost impossible to evaluate the The longer term challenge is to understand the “aging status” of an individual based on one, or molecular bases for the rate at which species even a few, biochemical, physiological or physical senesce, and individuals within a species me a s u r ements. The implication of this is that it senesce, and ultimately to develop strategies to has not yet been possible to develop a panel of d e c rease that rate. This has already been human biomarkers which can reliably estimate the accomplished to some extent in simple animal biological age of an individual, as opposed to the models, using death as the endpoint. In some ch r onological age. A res e a r ch initiative to develop cases life span has been extended several fold, a panel of biomarkers in rodents (mice and rats) although it is not clear whether this can be con- 11 has produced a few promising leads, but has not strued to mean that the rate of senescence in yet produced a panel of validated, reliable tests. these model systems has been modified. Success in extending the maximum life span of Another observation from the BLSA is that mammalian models has been much more mod- most measurements show gradual changes with est, and has been achieved mainly through age, rather than precipitous changes. It is thus caloric restriction. So far with humans, longevi- assumed that precipitous changes are more ty has been increased th r ough the prev e n t i o n likely to be associated with the development of and treatment of life-threatening disease. specific age-related pathology. Jacob Brody and Edward Schneider have previously identi- By now it is clear that some age-related changes fied the occurrence of at least two distinct types are risk factors for disease, and that these of associations; aging-dependent and age- changes can be both intrinsic and extrinsic, and dependent (Brody and Schneider, 1986). In either random or programmed (genetic). Some particular, there are certain genetically deter- of what we already know about these risk fac- mined changes leading to deficits which are tors is summarized in the next two sections. age-dependent, i.e., they express themselves at predictable ages, which are apparently not nor- mal aging, e.g., Huntington disease. Ho w e v e r , Intrinsic Stochastic Factors age-related changes that are associated with in Aging no r mal aging also contribute to the development of pathology. Thus, the immediate challenge is It is now well established that aging is very to distinguish among: “plastic” in the sense that individual humans, and other animals, display individually different Other structural changes occur during aging patterns of change during aging. This implies with possible negative implications. For exam- that environmental factors, both negative and ple, the conformation of individual proteins positive, have an impact. Research during the may change, leading to loss of function, and past 25 years has identified a number of nega- sometimes aggregation. Changes in the aggre- tive factors such as oxidative damage to pro- gation state of specific proteins have long been teins, DNA and lipids; glycation; changes in associated with Alzheimer disease and related protein conformation; and induction of muta- n e u rodegenerative disorders (Hardy and tions. Whereas cells have robust repair systems Gwinn-Hardy, 1998); whether there is a causal to reverse many of these processes, repair is sel- relationship is still a matter for debate. dom complete or instantaneous. F i n a l l y, the importance of apoptosis during Oxidative damage is an intrinsic process in aging is now recognized (Wa rner et al., or ganisms which req u i r e oxygen for life, as dam- 1997). Apoptosis is a genetically re g u l a t e d , aging oxygen radicals are continuously pro- inducible cell death program. It is known duced during cellular respiration. Although it is that apoptosis is induced by oxidative stre s s reasonable to assume that some persisting and DNA damage, but the exact induction oxidative damage is acceptable, particularly if it pathway is not known. When apoptosis occurs in non-critical molecules or molecules occurs in post-mitotic tissue such as the cen- pr esent in great excess, unrep a i r ed damage to tral nervous system, the result may be cata- molecules involved in critical rate-limiting s t rophic, as in the case of neuro d e g e n e r a t i v e 12 pr ocesses are likely to have negative effects dur- diseases such as Alzheimer disease, ing aging. Thus, it is not surprising that there Parkinson disease, Huntington disease, etc. ar e several examples of extension of life span by On the other hand, apoptosis appears to play ov e r- e x p r ession of genes coding for anti-oxidant an important role in reducing cancer. enzymes (Table 1).

Glycation is another intrinsic process which is Genetic Factors in Aging unavoidable in most animals. Glycation involves a non-enzymatic reaction between the Recent technological advances can now provide carbonyl group of a reducing sugar, usually glu- unprecedented opportunities to understand the cose, and the primary amino groups of proteins. genetic basis of aging, and with that the devel- This initial reaction to form a Schiff’s base is opment of interventions to improve the quality usually followed by a complex series of reac- of life in older persons. These advances include tions, most of which are poorly characterized, the ability to: 1) isolate and obtain the sequence often leading ultimately to protein cross-link- of individual genes; 2) generate genetically ing. This overall process not only alters the altered mice, e.g., transgenic mice, to character- structure and function of the proteins them- ize the functional role of such genes; 3) develop selves, but also leads to more global effects such sensitive and rapid techniques to measure the as blood vessel wall and connective tissue stiff- expression of thousands of genes in a single ening. Several compounds are currently in assay; 4) detect small differences (polymor- development to either prevent or reverse this phisms) in the DNA sequence of any gene kind of damage. among individuals in a population; and 5) determine the entire sequence of the genome of life span at the cellular level, by inducing the humans, as well as that of useful animal models. e x p ression of telomerase, will have a similar It is anticipated that the sequence of the human e ffect at the organismal level. Te l o m e r a s e genome may be accomplished by 2003, or per- deficiency in mice does lead to some pathol- haps sooner. The complete sequences of popu- ogy due to reduced proliferative potential lar model organisms such as Caenorhabditis ele - (Rudolph et al., 1999), as the grad- gans (a nematode), Saccharomyces cerevisiae(a ually shorten in successive generations. In yeast), and Escherichia coli (a bacterium) have p a rt i c u l a r, these mice suffer from slow already been determined (Goffeau et al., 1996; wound healing, ulcerative skin lesions, Blattner et al., 1997; the C. elegans sequencing reduced ability to respond to immune system c o n s o rtium, 1998). These analytical tech- s t ress, intestinal pathology, and re d u c e d niques, and the knowledge which can be l o n g e v i t y. Although some of these changes obtained with their appropriate use, will allow a re characteristic of human aging, the full the biomedical community not only to evaluate s p e c t rum of age-related diseases is not seen. the effectiveness of a wide variety of biological These mice are cancer- p rone, a trait appar- interventions, but also to determine the genetic ently linked to genetic instability caused by basis for the wide diversity in the individual critical telomere loss. It remains to be deter- patterns of aging. mined how a lack of telomerase will aff e c t p ro g ression of rapidly growing tumors. The observation that a strain of long-lived f ruit flies can be derived from an outbre d 13 population of flies (Rose, 1984), indicates The Need for Animal Models that genetic factors do play a role in aging. The case has been made best in short - l i v e d Whereas the ultimate goal is to understand model organisms, such as yeast, nematodes, human aging, many experiments are too inva- f ruit flies and mice. A list of genes (Table 1) sive to be performed on humans. Furthermore, implicated in regulating longevity and aging the long life span, genetic heterogeneity, and includes genes for proteins with anti-oxidant diversity of environmental exposure of humans activities, e.g., superoxide dismutase, cata- make them unsuitable for many kinds of exper- lase, and thioredoxin; and signal transduc- imentation. Thus, animal models have been tion proteins, e.g., insulin re c e p t o r, phos- used extensively to study normal aging (Sprott phatidyl inositol-3-kinase, ras and GTP- and Austad, 1996). Mice have been a particu- binding protein (the methuselah gene pro d- lar favorite because of their short life span, well- uct). All of these genes have human characterized , easy husbandry and low homologs. A case has also been made for a cost. Also, they can be easily raised in a genetic role in human longevity (Herskind et p a t h o g e n - f ree facility. For similar re a s o n s , al., 1996); based on a study of Danish twin except for the relative paucity of genetic infor- pairs, these authors estimated that the mation, the rat is also a favored model, espe- longevity in humans is about 25% due to cially because of its larger size. Neither the genetic factors. mouse nor the rat genome has yet been sequenced, but emphasis is now being placed It is not yet known whether the ability to on obtaining the mouse sequence (Battey et al., p revent replicative senescence and extend 1999). It is clear that much of the pathology occurring in mice and rats is similar to that Because of this, there is a real need to devel- found in humans, although Alzheimer disease op a non-human primate model to use as a and stroke are not found in old mice and rats. better surrogate for human aging. Although However, human pathology can often be repro- rhesus monkeys are currently being used in duced in genetically altered mice, and the pop- experiments to determine whether the exten- ularity of mice has increased dramatically sion of life span and re t a rdation of age-re l a t- because of this ability to create transgenically ed disease by caloric restriction might work altered mice which mimic human diseases. in humans (Couzin, 1998), these monkeys a re fairly long-lived (up to 40 years). Thus, Almost all studies done on mice and rats so small, relatively short-lived prosimians, e.g., far have been done using genetically lemurs, or new world monkeys, e.g., mar- homogenous strains. This made sense in mosets, with life spans in the range of 12 to early gerontological studies focused mainly 20 years have been suggested as a more suit- on characterizing species-specific aging phe- able model of human aging, but no colonies nomena. However, the recent emphasis on a re currently available for such use. identification of genetic factors which influ- ence life span and the rate of aging can ben- Another underutilized potential animal efit from the use of genetically hetero g e- model for studying aging is the avian model neous models (McClearn, 1997). No com- (Holmes and Austad, 1995). Birds are high- m e rcial colony of such a model currently is ly aerobic and have high circulating glucose available, but pre l i m i n a ry experiments with levels, but despite these characteristics, they 14 several institution-based colonies suggest f requently live longer than similar sized that they may indeed be useful for identifica- mammals. It would be useful to know how tion of genetic loci with significant effects on various bird species overcome the appare n t life span (Harrison and Broderick, 1997; disadvantages of high glucose concentration Miller et al., 1998). One unique value of and high rate of oxygen metabolism. Thus, such a colony is that it more closely re f l e c t s despite the relative paucity of genetic infor- the genetic diversity of human populations, mation about bird species, there is a need for and thus provides an opportunity to ask s t a n d a rdized, accessible bird colonies for questions about genetic associations with those wishing to tackle these gero n t o l o g i c a l- a g e - related phenotypes. ly important questions.

However, the above mammalian models have F i n a l l y, it can be asked what human popula- several limitations. Although these rodents do tions provide opportunities for studying have fairly short life spans (two to four years, human aging which are not yet being fully depending on strain), these animals are too utilized. There appear to be several. These long-lived to permit routine screening for long- include groups with small founder popula- lived mutants, as has been done with nema- tions, sib pairs and twins, large family gro u p s todes and fruit flies. for which it is possible to obtain substantial clinical data, and individuals who display Also, these rodents rarely develop athero- successful aging phenotypes. These latter s c l e rosis or neurodegenerative disease, and include very old individuals, e.g., centenari- mice have very high incidence of cancer. ans, who are relatively free of disease and dis- a b i l i t y, and older individuals who are part i c u- m i c ro a rrays to characterize gene expre s s i o n larly fit and healthy, e.g., master athletes. p a t t e rns in any tissue in any species of inter- DNA samples and relevant clinical data could est, as a function of age, and under a variety p rove extremely useful for establishing asso- of conditions, especially in response to possi- ciation between genes and aging-related phe- ble anti-senescence interventions; and 3) notypes in these human populations. advances in technology for high-thro u g h p u t DNA sequencing (Lander, 1999; C h a k r a v a rti, 1999). Unfort u n a t e l y, these New Tools and Resources for techniques re q u i re expensive equipment and Aging Research related materials which are not yet available in most laboratories. Thus, there is a need to The application of molecular biological tech- p rovide the equipment and infrastru c t u re niques to studies in experimental gero n t o l o g y needed to use these technologies, and/or has led to remarkable advances in under- p rovide support for these technologies using standing the genetic basis of aging in invert e- m i c ro a rray chips and filters produced com- brate animal model systems. These have m e rc i a l l y. This includes the need for an eff i- included the identification of several genes cient data management system to perm i t which alter the rate of aging in nematodes ready access by interested laboratories to ( M o rris et al., 1996; Kimura et al., 1997), and p e rmit data sharing, and thus avoid expen- f ruit flies (Lin et al., 1998; Parkes et al., 1998). sive duplication of eff o rt . The production and characterization of genet- 15 ically altered mice (transgenic and gene The ability to characterize patterns of gene knockout mice) are also providing useful ani- e x p ression in animal model systems is only mal models for the study of age-re l a t e d the first step in understanding the signifi- pathology (Williams et al., 1998; Morgan et cance of age-related changes in gene expre s- al., 1999), and age-related diseases such as sion in humans. Information about such pat- Alzheimer disease (Hsiao et al., 1996). These t e rns might provide useful biomarkers of kinds of approaches are generating import a n t aging. However, in order to obtain baseline i n f o rmation about the genetic basis of aging data in humans, a bank of human surg i c a l in these particular animal models, and it is and autopsy material will be re q u i red. Such hoped, relevant insights about human aging. material must be very carefully collected and Examples of these genes are shown in Table 1. maintained to be of any use for study of gene e x p ression patterns, because RNA is very Other new technologies, which will pro v i d e unstable in such tissues after death. Such a even greater insights into both species-spe- bank should contain samples of all major cific aging and the genetic basis of aging, are human tissues, collected over a wide range of now becoming available. These include: 1) ages, from healthy individuals with well-char- the polymorphisms, including single acterized clinical histories. Samples should nucleotide polymorphisms, in any gene of also be annotated with a thorough pathologi- known sequence, and the possibility of cal analysis obtained at the time the autopsy demonstrating an association between any samples are obtained. Similar tissue banks p a rticular polymorphism and an aging-re l a t- will be needed for the model systems chosen ed phenotype; 2) use of gene expre s s i o n for animal studies. Avenues and Specific Goals of . IDENTIFICATION OF LINKAGE BETWEEN Basic Research on Aging DNA POLYMORPHISMS AND LONGEVITY AND/OR AGE-RELATED PATHOLOGY. The conviction that basic research on aging is underfunded comes from the recognition that Longevity differs dramatically among individu- there are many unanswered questions about the als of the same species. These differences are basic mechanisms of aging, and that the assumed to be due to differences among indi- answers to these questions may give us power- viduals due both to their environment and their ful insights about aging and the treatment of genomes, but are also related to chance events age-related diseases. The following goals are during development and aging. Any association examples of research questions which need to study linking genomic diff e rences to either be answered in our desire to understand the longevity or age-related pathology would pro- mechanistic basis of age-related changes. vide insight about the genetic basis of senes- cence either in humans or the specific animal . model being studied. IDENTIFICATION OF THE GENETIC . DIFFERENCES WHICH ARE CAUSALLY THE ROLE OF OXIDATIVE DAMAGE ASSOCIATED WITH LIFE SPAN IN SENESCENCE. DIFFERENCES BETWEEN SPECIES. It is widely assumed that some forms of age- 16 The genes responsible for the dramatic differ- related pathology result, at least in part, from ences in longevity among animal species are oxidative damage. The unrealized challenge is currently unknown, even between species that to tack real numbers onto this vague generaliza- are closely related. The identification of these tion; i.e., what proportion of dysfunction, in genes and demonstration of their function in which cells and tissues, is really due to oxidative damage? Are there critical proteins which are vivo, would be a significant accomplishment for oxidized, thereby compromising overall cellular understanding the genetic basis of senescence and tissue function? Another challenge is to in general. develop interventions that either reduce the rate of production of oxygen free radicals or . increase the activity of anti-oxidant defense sys- GENERATION OF ANIMAL MODELS WITH tems. Interventions that consistently slow the SIGNIFICANTLY POSTPONED SENESCENCE, rate of senescence in model systems would sug- AND IDENTIFICATION OF THE gest promising clinical trial opportunities in GENES RESPONSIBLE FOR THE human populations, especially in delaying LIFE SPAN EXTENSION. degenerative changes in post-mitotic tissues. The generation of mice with a significantly . extended life span, and identification of the CALORIC RESTRICTION. genes responsible, will continue to be an i m p o rtant goal, because it is likely that such Although it has been known for more than 60 i n f o rmation will provide insights into the years that caloric restriction extends the max- genetic basis of senescence in humans. imum life span of most animal species in which it has been adequately tested, the F i b roblasts from WS patients, which have mechanism by which it does so is not known reduced proliferative capacity, also lose telomer- for any species. Research to elucidate the ic DNA significantly faster than normal fibrob - causal mechanism(s) could suggest surro g a t e lasts, implicating the WRN pr otein in telomere i n t e rventions to re t a rd senescence without dynamics as well. Elucidation of the actual rol e actually restricting calories. of the WRN pr otein in vivo could provide sig- nificant insight into the causes of several age- . related diseases such as cancer, cardi o v a s c u l a r BIOMARKERS OF AGING. disease and cataract. For example, a common DNA polymorphism in WRN is a risk factor for The lack of a panel of biomarkers of aging my o c a r dial infarction (Ye et al., 1997). means that there are few, if any, indicators of the rate of aging in any particular individual. Such a panel is needed to test promising inter- . IN VIVO. ventions, first in animal models, and eventually in the human population, because measuring Since the finding by Leonard Hayflick that survival is a very inefficient and crude alterna- human cells do not divide indefinitely when tive. The particular challenge is to demonstrate g rown in culture, cultured human cells, prin- an empirical link between putative biomarkers cipally fibroblasts, have been a much-used, of aging, which can be measured continuously but controversial model for human aging. It over the life span of an individual, and the tim- is now known that when human cells lose 17 ing of adverse events, including death or the their ability to divide, they also lose func- onset of disease or disability. Reversing these tional capacity, in the sense of acquiring new events is the ultimate goal of interventions. and deleterious activities, although they do not die. This has been termed the cellular . senescent phenotype. This loss of pro l i f e r a- WERNER SYNDROME: A HUMAN MODEL tive and functional capacity can be induced OF RAPID SENESCENCE. by telomere shortening, certain types of DNA damage, and some oncogenic stimuli. Patients with Wer ner syndrome (WS), develop P re l i m i n a ry data showing that senescent cells many of the adverse phenotypes of aging, but at do exist in vivo (Dimri et al., 1995), suggest an accelerated rate. WRN, the gene defective in that accumulation of such cells could have WS, has now been cloned and shown to code for negative implications for the integrity of the a protein with both helicase and exonuclease extracellular matrix in the vicinity of such ac t i v i t y . However, there are at least five other cells (Campisi, 1997). helicase genes with related functions in the human genome. The role of the WRN ge n e Replicative senescence in human fibroblasts can pr oduct and related proteins in ret a r ding senes- be prevented by transgenic expression of the cence in humans is not yet known, but they catalytic subunit of telomerase (Bodnar et al., could be involved in any or all of the following: 1998). Such cells become capable of continued DNA replication, DNA rep a i r , transcription, pr oliferation well beyond their usual limit. This recombination, or chromosome segre g a t i o n . suggests the potential to reverse any in vivo ph e - notype which might be due to loss of prol i f e r a - in this area to determine which horm o n e tive capacity, e.g., wound healing, immune replacement therapies are both effective and senescence, provided telomerase activity can be safe (Lamberts et al., 1997). For example, selectively res t o r ed in the cells of interest. p re l i m i n a ry studies with growth horm o n e . (GH) indicated that muscle mass and a gen- CELL REPLACEMENT AND STEM CELLS. eral feeling of well-being are increased by GH (Rudman et al., 1990), but safety re m a i n s Cells in which the telomerase gene has been an important concern as serious side eff e c t s reactivated are possible candidates for use in may occur. replacing cells lost through apoptosis due to excessive damage, wear, or trauma. The above examples give credence to the idea F o rt u n a t e l y, it is now apparent that such that research on aging has tremendous poten- cells are not necessarily carcinogenic even tial to raise our level of understanding enough though they express telomerase (Jiang et al., to lead to successful interventions in both 1999). An alternative approach is the use of aging, and specific age-related diseases. Any stem cells which retain pluripotent capabili- delay or reduction in age-related disability and t y. Such cells have recently been isolated, disease will increase the number of years of and have tremendous potential to treat a healthy life and is a worthy goal. variety of age-related degenerative diseases ( G e a rh a rt, 1998). However, there re m a i n To facilitate such prog r ess in understanding many technical and conceptual hurdles to aging there is a critical need not only for 18 c o n t rol the diff e rentiation of such cells once in c r eased funding for aging res e a r ch, but also to they have been transplanted. If, and when, pr ovide funding for res e a r ch infrastruc t u r e in these hurdles can be overcome thro u g h the form of equipment and trained personnel. re s e a rch, applications of this technology to reverse the adverse effects of aging appear to. have significant potential. HORMONE REPLACEMENT. 1 The research goals articulated in this docu- While it is seductive to believe that re s t o r a- ment are a product of both the discussions in tion of hormone levels in older individuals to the ILC workshop held in New York City, the levels found in young individuals will February 10-11, and a workshop recently spon- reverse aging, this may or may not be tru e . sored by the UCLA Program on Medicine, Thus, while re s e a rch to determine the eff e c t s Technology and Society, and convened by Dr. of manipulating hormone levels has enor- Gregory Stock. mous potential, e.g., estrogen re p l a c e m e n t t h e r a p y, the general question is complex This latter meeting held in Los Angeles, which because hormones have both positive and extended the discussions begun in New York negative effects, and it may be difficult to involved several attendees at the ILC confer- adequately mimic the natural daily variations ence, as well as Drs. Michael Rose, Cynthia of each hormone. Much re s e a rch is needed Kenyon, Jan Vijg and James Nelson. on Aging for his comprehensive and insightful narrative. We are grateful to the outstanding contributors to American , in fields BY ROBERT N. BUTLER , M.D. ranging from cell and molecular biology to AND T. FRANKLIN WILLIAMS, M.D. chemistry to demography and epidemiology, who despite other pressing demands made time Together we had the responsibility (and the plea- to participate. su r e) of helping establish and direct the National Institute on Aging over nearly two decades, dur- We believe this state-of-the-art scholarly rep o r t ing its formative period between 1975 and 1991. should become a campaign document to encour- We witnessed the remarkable advances in the age further support of basic biological studies of understanding of the basic biology of aging and aging by government, foundations, individual age-linked diseases as well as of the social and ph i l a n t h r opists and the for-p r ofit sector. These behavioral aspects of aging. Although funding potential supporters should appreciate the grow - for the Institute has grown considerably from an ing importance of the demographic revolution as original $12 million to nearly $500 million annu- we come to the close of the century and the mil- al l y , there remain many challenging res e a rc h lennium. The United States now enjoys the op p o r tunities, as well as needs of the Institute's highest life expectancy in its history - 76.5 years, various programs. We personally favor increa s - while at the same time benefiting from dramatic ing support for the NIA in general, but in this dr ops in disability rates. There have been excit- 19 document our focus is upon the opportu n i t i e s ing developments such as the cultivation of posed by the intellectual vigor of modern biolo- pluripotent cells, identification of molecular time gy . We believe insufficient funds are available to clocks in aging (telomeres), nuclear transfer or study the molecular and cellular processes that cloning, and the introduction of a variety of sig- define the biology of aging, for example, the fact nificant theories, all of which should be caref u l l y that the "force of mortality" rises with the pas- evaluated. There is the promise of importa n t sage of time, as delineated by Benjamin steps in the prevention and treatment of age- Go m p e r tz 175 years ago. related disorders. The prospects for "reg e n e r a - tive medicine" and genomics lie just before us. It is time to dramatically increase the National For this reason, we sought financial support for Institutes of Health's investment in aging a workshop to explore the impact of aging fac- res e a r ch, now only about $100 million out of the tors in health and disease, to evaluate the state $15 billion budget of the NIH. of knowledge and to consider issues of science policy and funding. The Aging Factor in Health and Disease: The Promise of Basic Research on Arguably, if it were possible to compress mor- Aging is the result of a two-day workshop sup- bidity - both in length and degree - biomedical p o rted by the The Brookdale Foundation research in general, and aging research in par- Group, the Institute for the Study of Aging, ticular might be credited with the resulting and Pfizer, Inc. We are exceptionally indebted reduced health costs. Added research funding to Dr. Huber Warner of the National Institute might even pay for itself in health cost savings. A recent examination of disability rates shows a Campisi J. 1997. Aging and cancer: The significant 2.1% decline since 1982. double-edge sword of replicative senescence. Consequently, there are some 1.2 million fewer J. Am. Ger. Soc. 45: 482-488. disabled persons than were anticipated. This reduces Medicare expenses and, of course, Chakravarti A. 1999. Population genetics - more importantly, advances quality of life. making sense out of sequence. Nature Genetics 21: 56-60. Additional funding for investigator- i n i t i a t e d research, programs and centers should be sup- Chen JB, Sun J, Jazwinski SM. 1990. Prolongation of the yeast life span by V-Ha-ras plemented with new investments in animal oncogene. Molec. Microbiol. 4: 2081-2086. models, laboratory resources, research training and banking of human tissues. The search for Couzin K. 1998. Low-calorie diets may slow an appropriate panel of biomarkers is essential. monkey’s aging. Science 282: 1018. At present there is no single set of biomarkers to evaluate the many interventions sold in the Dimri GP, Lee X, Basile G, et al. 1995. A marketplace under the misleading term “anti- biomarker that identifies senescent human aging medicine.” Contemporary enthusiasm for cells in culture and in aging skin in vivo. healthful, satisfying longer life must be matched Proc. Natl. Acad. Sci. USA 92: 9363-9367. by new funding and critical science - it will not

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Goffeau A, Barrell BG, Bussey H, et al. 1996. Life with 6000 genes. Science 274: 563-567. Battey J, Jordan E, Cox D, Dove W. 1999. An action plan for mouse genomics. Gearhart J. 1998. New potential for human Nature Genetics 21: 73-75. embryonic stem cells. Science 282: 1061-1062.

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Bodnar AG, Ouellete M, Frolkis M, et al. Harrison DE, Broderick TH. 1997. 1998. Extension of life-span by introduction Selection for maximum longevity in mice. of telomerase into normal human cells. Exp. Gerontol. 32: 65-78. Science 279: 349-352. Hershkind AM, McGue M, Holm NV, et al. Brody JA, Schneider EL. 1986. Diseases and 1996. The heritability of human longevity: disorders of aging: An hypothesis. J. Chron. A population-based study of 2872 Danish twin Dis. 39: 871-876. pairs born 1870-1900. Hum. Genet. 97: 319-323. Holmes D, Austad S. 1995. Birds as animal regulatable promoter systems to transgenic models for comparative biology of aging: models for the study of aging. A prospectus. J. Gerontology 50A: B59-B66. J. Gerontology 54A: B30-B40.

Hsiao K, Chapman P, Nilsen S, et al. 1996. Morris J, Tissenbaum HA, Ruvkun G. 1996. Correlative memory deficits, AB elevation, and A phosphatidylinositol-3-OH kinase family amyloid plagues in transgenic mice. member regulating longevity and diapause in Science 274: 99-102. Caenorhabditis elegans. Nature 382: 536-539.

Jiang XR, Jimenez G, Chang E, et al. 1999. Parkes TL, Elia AJ, Dickinson D, et al. 1998. Telomerase expression in human somatic cells Extension of Drosophila lifespan by overex- does not induce changes associated with a pression of human SOD1 in motorneurons. transformed phenotype. Nature Genetics 19: 171-174. Nature Genetics 21: 111-114. Rose MR. 1984. Laboratory evolution of Kimura KD, Tissenbaum HA, Liu Y, Ruvkun postponed senescence in Drosophila G. 1997. daf-2 an insulin receptor like gene melanogaster. Evolution 38: 1004-1010. that regulates longevity and diapause in Caenorhabditis elegans. Science 277: 942-945. Rudman D, Feller AG, Nagraj HS, et al. 1990. Effects of human growth hormone in Lamberts SWJ, van den Beld AW, van der Lely men over 60 years old. 21 A-J. 1997. The endocrinology of aging. N. Engl. J. Med. 323: 1-6. Science 278: 419-424. Rudolph KL, Chang L, Lee H-W, et al. 1999. Lander ES. 1999. Array of hope. Nature Longevity, stress response, and cancer in aging Genetics 21: 3-4. telomerase-deficient mice. Cell 96: 701-712.

Lin Y-J, Seroude L, Benzer S. 1998. Schneider EL. 1999. Aging in the third Extended life-span and stress resistance in the millenium. Science 283: 796-797. Drosophila mutant methuselah. Science 282: 943-946. Sprott RL, Austad SN. 1996. Handbook of the Biology of Aging, 4th Edition. McClearn GE. 1997 Heterogeneous Academic Press, Inc. pp. 3-23. reference populations in animal research on aging. ILAR J. 38: 119-123. The C. elegans sequencing consortium. 1998. Genome sequence of the nematode C. elegans: Miller RA, Chrisp C, Jackson AU, Burke D. A platform for investigating biology. 1998. Marker loci associated with lifespan in Science 282: 2012-2018. genetically heterogeneous mice. J. Gerontology 53A: M257-M263. Vlassara H, Fuh H, Makita Z, et al. 1992. Exogenous advanced glycosylation products Morgan WM, Richardson A, Sharp ZD, induce complex vascular dysfunction in Walter CA. 1999. Application of exogenously normal animals, a model for diabetic and aging complications. APOPTOSIS - a genetically regulated program Proc. Natl. Acad. Sci. USA 89: 12043-12047. leading to cell suicide.

Warner HR, Hodes RJ, Pocinki K. 1997. BIOMARKER (OF AGING) - an age-related change What does cell death have to do with aging? which reflects the physiological age of an indi- J. Am. Ger. Soc. 45: 1140-1146. vidual, in contrast to its chronological age.

Williams MD, Van Remmen H, Conrad CC, et CARBONYL GROUP - the reactive portion of al. 1998. Increased oxidative damage is sugar molecules such as glucose; composed of a correlated to altered mitochondrial function in carbon atom and an oxygen atom; combines heterozygous manganese superoxide dismutase with an amino group to form a Schiff’s base knockout mice. during glycation. J. Biol. Chem. 273: 28510-28515.

Ye L, Miki T, Nakura J, et al. 1997. CATALASE - an enzyme which destroys hydrogen Association of a polymorphic variant of the peroxide, converting it to water and oxygen. Werner helicase gene with myocardial infarction in a Japanese population. DNA REPLICATION - the process of copying Am. J. Med. Genet. 68: 494-499. DNA to make two identical copies before cell division occurs. Yodoi J, Hirota K, Oono T, et al. 1999. Redox regulation of signal transduction DNA POLYMORPHISM - a difference in DNA 22 pathways by thioredoxin superfamily. structure found in a small, but significant seg- 1999 World Congress on Oxidants and ment of a given population (in contrast to a Antioxidants in Biology, p. 45 (Abstr.). mutation which is rare).

FIBROBLAST - one of the major cell types found in human skin; fibroblasts have been developed as a model system for studying cellular aging.

GLY C AT I O N - the non-enzymatic re a c t i o n AGING - changes that occur during the life between a reducing sugar and the amino group span, not all of which need to be adverse. of proteins; often leads to protein crosslinking.

AL Z H E I M E R D I S E A S E - an aging-dependent HIGHTHROUGHPUT (TECHNOLOGY) - technolo- disease characterized by loss of memory. Risk gy permitting the assay of thousands of samples factors include both genetic and environmental simultaneously without individual handling. factors. Age of onset varies from the late 40s for patients with early-onset genetic risk factors, to HUN T I N G T O ND I S E A S E - an age-dependent genet- 65 and older for most other patients. ic disease due primarily to the loss of neurons in the striatum; age of onset is about 40 years. AMI N O GR O U P - the reactive group found in amines, composed of a nitrogen atom and two LIFE EXPECTANCY - the average number of hy d r ogen atoms (i.e., NH2); reacts with carbonyl remaining years an individual can expect to live gr oups to form a Schiff’s base during glycation. at any given age. LIPID - a class of molecules which are not water- RECOMBINATION - the process whereby two dis- soluble, and form the major component of bio- tinct pieces of DNA exchange genetic material. logical membranes. SC H I F F’S B A S E - the product of an interaction LONGEVITY - the length of life of an individual, between an amino group and a carbonyl or the average length of life of a population of g ro u p . individuals. SEN E S C E N C E - ag e - r elated changes in an orga n - MU TAT I O N - base substitution in DNA which ism that adversely affect its vitality and functions. occurs infrequently within a population of n o rmal individuals (in contrast to a polymor- SUPEROXIDE DISMUTASE (SOD) - an anti-oxi- phism which may occur more frequently in a dant enzyme which converts the superoxide p o p u l a t i o n ) . anion to hydrogen peroxide.

NEM AT O D E - a small worm, usually soil- TE L O M E R A S E - an enzyme that synthesizes dwelling, which has been developed for bio- telomeric DNA. medical research because of its well character- ized developmental program; it is a useful TELOMERE - the non-coding DNA at the ends model system for studying aging because of its of chromosomes consisting of long stretches of short life span. short repetitive DNA sequences. 23 NON-ENZYMATIC - not catalysed by enzymes. THIOREDOXIN - a protein capable of reversing oxidative damage to other proteins. ONCOGENIC - causing cancer. TRANSCRIPTION - the process of transferring OXIDATIVE DAMAGE - changes in structure of genetic information from DNA to RNA by biological molecules due to their interaction copying the DNA sequence. with oxygen radicals. TRANSGENE - a gene from one organism insert- OXYGENRADICAL - a very reactive form of oxy- ed into the genome of another organism (usual- gen, produced continuously in mitochondria ly refers to mice). during normal metabolism. TR A N S L AT I O N - the process of joining animo PHENOTYPE - the visible properties of an organ- acids together in specific sequence to form ism resulting from the interaction between its p ro t e i n s . genes and the environment. WE R N E R S Y N D R O M E - a genetic disease char- RECEPTOR - a multimolecular protein complex, acterized by pre m a t u re development of usually found on the surface of cells, which adverse age-related changes such as cataracts, binds specific extracellular signalling c a rdiovascular disease, cancer; cataracts may molecules, thereby transducing a signal to the develop as early as the 20s, with average age inside of the cell. of death at 45-50 years. The International Longevity Center - USA, Ltd. (ILC-USA) is a not-for- p rofit, non-par- tisan re s e a rch and education org a n i z a t i o n whose mission is to help societies addre s s longevity and population aging in positive and productive ways and highlight older p e o p l e ’s productivity and contributions to their families and society as a whole.

24 The organization is part of a multinational re s e a rch and education consortium, which includes centers in the U.S., , Gre a t Britain, France, and the Dominican Republic. These centers work both autonomously and collaboratively to study how greater life expectancy and incre a s e d p ro p o rtions of older people impact nations a round the world.

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