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Developmental Genomics and Its Relation to Aging
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PERSPECTIVE
Developmental Genomics and Its Relation to Aging David Schlessinger1 and Minoru S. H. Ko Laboratory of Genetics, Gerontology Research Center, National Institute on Aging, Baltimore, Maryland 21224
Negative and Positive Views of Aging Negative views of aging are deeply embedded in Western culture. As is often the case, some current ideas can be traced back to Hellenic antecedents. Aristotle laid the foundations for modern genetics (Grene, 1968), with an insistence on careful observation and an emphasis on heredity, reproduction, adaptation in response to the struggle for survival, and relevant effects of diet and locomotion. In his thinking about the course of biological life, Aristotle contrasted the phase of “becoming” (that is, development) with that of “passing away” (that is, degeneration). Aristotle’s idea that each species tends to the fulfillment of its ideal form was in keeping with the Hellenic formulation of the division of life into temporal compartments, with development culminating in transient “acme.” In classic thought, human acme, as exemplified in Attic sculpture, occurred at about age 18, and it was all downhill from there. Had Aristotle been acquainted with the Analects as well as Plato’s Symposium, he might have pondered Confucius’ account of his progressive, slow process of education until he reached an important realization when he was 70. Even in Aristotle’s restricted surroundings, venerable Athenian judges were considered the wisest and most circumspect, and he himself was writing the Lyceum lessons (that we still read!) in his sixth decade. Nevertheless, the impressive verve and beauty of youthful heroes like his tutee Alexander dominated his thinking, and the notion that human life can be divided into growth and decay has lasted to the present day. For example, Martin and Mian (1997) quote the (facetious?) formulation that “first we ripen and then we rot.” Current versions of this up-and-down view of human life are often based on the “evolutionary theory of aging.” Discussions (reviewed, for example, by Finch, 1990; Austad, 1997) comment that once individuals are essentially beyond reproductive age, negative consequences of genes and mutations are not subject to selection and accumulate to cause the infirmities of the aged (Medawar, 1957; Williams, 1957). Such formulations, however, have difficulty rationalizing the 1 To whom correspondence should be addressed at Gerontology Research Center, Box 31, 5600 Nathan Shock Drive, National Institute on Aging, Baltimore, MD 21224-6825. Telephone: (410) 5588337. Fax: (410) 558-8331. E-mail: [email protected].
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substantial hereditary component in human longevity (Martin, 1978; McGue et al., 1993; Finch and Tanzi, 1997)— or the substantial differences in longevity between species and between variant individuals in a particular species (cf. Kirkwood, 1985; Lithgow, 1996; Tower, 1996; Rogina et al., 1997; Tissenbaum and Ruvkun, 1998). Modern evolutionary theory can take a more positive view of potential selective advantages of longevity. Developments based on the work of Hamilton (1964) consider the unit of selection as the tribe/family/group rather than the individual and discuss the evolution of phenomena such as cooperation and altruism (e.g., Dugatkin, 1990). Selection based on kinship in several species has been commented on increasingly, for example, by Carey and Gruenfelder (1997). Such treatments, however, have not generally followed up the ways in which “cultural evolution” (Medawar, 1957) could provide information with selectable value. As a putative example, consider the climatological evidence that miniperiods of severe weather can be intercalated with relatively mild weather. [According to recent studies, the variation can be within several decades, even in the arctic; Overpeck et al. (1997).] One can imagine the plight of a preliterate society suddenly faced with the recurrence of a mini-ice age. Members of a group whose experience spanned the interval back to the last mini-ice age, and who could transmit information about how such a period had been weathered, could provide considerable selective advantage to the survival of the group. Presumably there would thus be some selective pressure for longevity, though a few such members of a tribe could be sufficient. The positive features of aging are not limited to sheer staying power. In addition to the maturity and perspective traditionally associated with the moderation of hormone levels in adult life, creativity seems to show no obligatory agerelated decline. For example, if one considers artists, performers remain vibrant throughout their lives, and the last works of composers are often their best. Verdi wrote Aida, Otello, and Falstaff in his seventies, premiering Falstaff when he was 80. Beethoven produced nothing like the Missa Solemnis or Opus 131 quartet until his last years; Haydn wrote his greatest “London” symphonies and The Creation in his seventh decade. Similar observations hold for every art: consider Monet at Giverny in his final years or Titian painting brilliantly in his nineties. Aging as a Part of Metazoan Maturation
Turnover and Its Limitations Positive selective features are one indication that aging is not simply a “decay” phase after development, but rather an integral part of metazoan maturation. D’Arcy Thompson (1984) analyzed the inescapable transience of exponential growth: the size of complex organisms and their constituent parts is limited by functional constraints, so that net growth must cease. As growth slows and stops, homeostasis depends increasingly on the balance of synthesis and turnover of cells
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SPECIAL FEATURE and tissues. Some authors have noted that some organisms can develop with essentially fully replaceable parts. This alternative, which would avoid the dependence on intracellular turnover, is precluded for human aging for two reasons. First, no mechanism is available for the replacement of some structural members—such as collagen. [One might imagine more aggressive turnover pathways that might alleviate this, but we are currently limited to those evolution has provided us.] Second, and more intractable, is the fact that one would not want to replace some portions of the body. In particular, memory and learning would be totally disrupted by the replacement of all brain cells by new ones. If an individual adult is to continue to exist, ultimate decay of brain cells that no longer divide is inevitable. Tissues differ in the degree of their dependence on turnover. Renewing tissues that retain stem cells can be more resistant to the effects of aging. This is because errors in DNA can be corrected by recombination and selection, and errors in protein can be diluted by net growth. In contrast, static cells and components cannot rescue mistakes or mutational events by dilution or recombination. They are in effect placed in a predicament similar to that of the Y chromosome in evolution, whose gene content atrophies progressively. An analogue of Muller’s ratchet (Muller, 1964; Rice, 1987) comes into play in aging tissues, and function is increasingly limited by irreplaceable loss. Thus, in primates, aging has evolved as a balance of opposing forces: progressive deterioration of components of the body (and elimination of entire individuals) and the benefit to fitness of having a number of individuals of considerable age who continue as part of a population cohort. The balancing mechanism starts with events in embryonic and fetal life that determine features of the course of aging.
Dependence of Aging on Embryonic Events Aging can be visualized as starting in the embryo. Embryonic events can be seen as critical for the understanding and possible alleviation of aging-related pathology. As an example, many theories of aging are predicated on the difference between indefinitely growing, totipotential embryonic stem cells (and germ cell progeny) and mortal differentiating cells that are already committed to cellular senescence (Hayflick, 1992). There are many indications that the process and its rate are genetically determined. Among different mouse strains, for example, a trend toward longer life span is correlated with a larger pool of hematopoietic stem cells (de Haan et al., 1997). Standard systems show that the loss of replicative capacity, and presumably of the relative numbers of types of cells, is correlated with telomere shortening (Blasco et al., 1997) and the activation or repression of a variety of genes (e.g., Linskens et al., 1995; Dimri et al., 1996; Smith et al., 1996; Garkavtsev et al., 1997; Haber, 1997; Yeager et al., 1998). The prevention or reversal of the process is also complex: it is not simply an imitation of “viral transformation” (Gotoh et al., 1979; Rubelj et al., 1997), but involves the regulation of genes such as telomerase, which can
extend life span (Bodnar et al., 1998), and others that can provoke senescence (Vojta et al., 1996). In part because of the complexity of the systems, experts dispute whether there is an “aging clock” in cellular senescence, but everyone agrees that a switch of some type initiates differentiation and loss of immortality. For example, Suda et al. (1987) showed that embryonic stem cells from mouse grow for an indefinite number of generations. This is especially noteworthy since mouse cells, including embryonic fibroblasts that have begun their mortal existence, show extraordinarily high rates of autotransformation and development of chromosomal aberrations and changes in epistatic methylation mechanisms (Holliday, 1996). Notably, theories of aging that are predicated on the accumulation of insults all take this initial transition for granted. For example (see Tower, 1996), consider the notion that oxidative stress provokes damage that is unremedied and contributes to age-related pathology and ultimate cell death. A number of correlated phenomena make this an attractive possibility, but mouse embryonic stem cells are exposed to the same insults as other cells and presumably should accumulate damage at the same rate. Yet they do not apparently age. Special status is also accorded germ cells, which are clearly “spared” progressive deterioration or loss of growth potential over generations. Sparing is another way of saying that the cells do not begin the aging process. [One implication of these findings is that repair processes may be more effective in “immortal” cells, and the findings that Werner and Cockayne syndromes are caused by lesions in repair-related genes (Yu et al., 1996; Mallery et al., 1998) are consistent with that view.] The underlying specification of the physiological process must be based on gene programs, and such theories place a premium on genes involved in the “immortalization” or “mortalization” process. In a second link between embryonic and aging events, every system of the body shows age-related loss—including osteoporosis, depletion of neurons and glomeruli, etc. No one thinks that the formation of tissues or organs involves a detailed mechanism that is the reverse of turnover and depletion. But if we understood how differentiated cells and specialized tissues form, conditions or regimens might be developed to prevent their loss or even to regenerate or replace them. Also, development often involves selective apoptosis or “programmed cell death”; are there comparable mechanisms involved in a programmed cell death in later life, based on a putative “programmed cell aging”? In cogent instances, critical events in embryonic and fetal life have a direct bearing on the subsequent etiology of agerelated conditions. Inherited conditions provide especially clear instances. We consider three that we have had experience with in recent years: (1) Premature ovarian failure. In about 1% of women, gonadal failure occurs before the age of 40. In all instances, the termination of a woman’s reproductive life occurs when her supply of mature follicles is exhausted (Davis, 1996). Of course this is by definition an aging phenomenon, but the net supply of follicles is already set in fetal life. Thus, to understand menopause— or premature ovarian failure, in which follicular atresia occurs at an increased rate in the embryo—
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SPECIAL FEATURE one must understand ovarian and follicular embryonic development. (2) X-linked ectodermal dysplasia. Individuals with lesions in ectodysplasin A, the product of the EDA gene (Kere et al., 1996) [and mice with lesions in the cognate Tabby gene (Srivastava et al., 1997)], show a defect in the development of skin appendages, including hair, teeth, and eccrine sweat glands. This provides an entry point for the analysis of the developmental process that leads to these specialized tissues, but the analysis could be highly relevant to the preservation or regeneration, for example, of hair in aging individuals. Comparable lines of investigation could provide information relevant to osteoporosis, end-stage kidney failure, and other baneful features of age-related pathophysiology. (3) Overgrowth syndromes. In these conditions, exemplified by Beckwith-Wiedemann syndrome (Li et al., 1997) and the X-linked Simpson-Golabi-Behmel Syndrome (Pilia et al., 1996), individuals grow to considerable height, with the overgrowth of a number of primarily mesoderm-derived internal tissues and organs. Biological questions of importance here relate to the mechanism by which the set point for organ size is determined. The individuals with this condition are susceptible to an increased incidence of embryonal tumors and are also subject to life-threatening problems based on heart overgrowth. Problems arise in childhood and in aging adults, but again, the critical events occur in embryonic life. Investigation of mechanistic details relating early and late phenomena are just beginning. As an example, growth and development—and the overgrowth syndromes (Eggenschwiler et al., 1997; Morison et al., 1996)—involve the action of insulin-like growth factors in promoting cellular expansion and proliferation. Recent studies hint that the insulin growth factor (IGF) protein system is also involved in the model aging process in nematodes (e.g., Tissenbaum and Ruvkun, 1998). Genetics of Aging and Developmental Genomics
Analysis of Aging and Longevity Genetic analysis of aging-related conditions is very much hampered precisely by the fact that they affect old people. Although the conditions depend on developmental events earlier in life, the parents of affected individuals are often dead by the time the disease is scored, and the children of those affected often have a number of decades to go before they can be scored. Genetic analysis has traditionally been made even more complex by the nagging problems of heterogeneity and variable penetrance. To overcome some of these difficulties, one can turn to the analysis of early-onset forms of aging-related disorders, preferably coupled to core phenotypes that are easily scoreable. The successes of this approach have been notable. Our knowledge of the genetics of Alzheimer disease has been extended extraordinarily by the discovery of presenilins through the analysis of early-onset pedigrees (Haass, 1996). As another outstanding example, the rate of aging has be-
come a field of biochemical study with the discovery of helicase mutants in early-onset aging in Werner syndrome (Yu et al., 1996) and other progeroid (Martin, 1978; Mallery et al., 1998) syndromes. The analysis of the basis for menopause starting from early-onset premature ovarian failure (see above) is another example, and other phenotypes, such as early-onset recurrent unipolar depression, are likely to be susceptible to such an approach. In this regard, there is a considerable body of longitudinal information on the course of system-specific normal and abnormal aging, particularly from the Baltimore Longitudinal Study of Aging (Shock et al., 1984) that can provide parameters and trajectories to aid in scoring conditions at a relatively early age. Early-onset conditions can be studied by any of the standard techniques, including linkage analysis (or detections of translocations or microdeletions) followed by refined mapping; linkage disequilibrium mapping through haplotype analysis; and association analysis, looking for increased prevalence of an allele in individuals affected by a condition or disease. All of these approaches are obviously facilitated, however, by the use of specialized populations, in which heterogeneity is demonstrably lower than in outbred populations, and have been used successfully in many reported studies, for example, those of the “Finnish diseases” (Norio, 1994). Such specialized population resources are especially important for the study of the extreme phenotype of aging, “longevity.” Longevity can be scored unequivocally, since, from the analysis of Gavrilov and collaborators (1997, 1998), an individual who is 90 years or older can be safely classified in a special group with those who are 100 years or older. But this criterion of course eliminates the possibility of finding an early-onset form of longevity. In fact, one must wait till advanced ages to score individuals, simply to be able to discount confounding effects such as accidental death. The unavailability of parental and offspring DNA from scored individuals for such a cohort is further complicated by the likelihood that alleles for a number of condition-specific risk factors will have been depleted in this population (distorting associations). However, methods based on haplotype studies in interrelated, delimited populations retain their power. A likely experimental design would be to collect sib pairs and multiple sibs over 90 years old and apply the potent modern haplotypic analyses.
Developmental Genomic Analysis For aging-related phenomena, like all other simple and complex traits, initial studies are designed to identify candidate genes by positional cloning. Especially for conditions with a strong developmental component, it would be useful to know what genes are involved and when they are expressed. Developmental genomic analysis aims to determine expression patterns on a systematic basis for genes expressed at specific times and places during the life of a species. It can help to complete and annotate the catalogue of genes that is required for the analysis of embryonic
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SPECIAL FEATURE phenomena that relate to aging, for developmental biology, and for the Genome Initiative. As a specific example, the sequencing of collections of cDNAs made from specific stages in embryonic life are producing 30 –50% new genes [that is, genes not present in the current databases; (Ko et al., submitted for publication; and work in progress)]. Many of those genes are represented for only brief periods and in specific subregions of the embryo. Thus, the fundamental achievement of a complete catalogue of genes is both required for and requires the analysis of embryonic and fetal gene expression. [We note that the methods for determining expression pattern may vary, with the use of SAGE (Velculescu et al., 1995), microarrays (Schena et al., 1995), etc., but those methods all become increasingly powerful as the available catalogue of genes becomes more complete.] The generation of the catalogue would be in line with current discussions of “functional genomics,” but the analysis of embryonic events requires further information that can again be obtained more efficiently by systematic genomic approaches. In pilot studies, it has been demonstrated that a large number of genes can be placed on the mouse genome by the use of interspecific backcross panels (Ko et al., 1994; and work in progress), and a large number of cDNA species can be examined by in situ hybridization to whole mount and sections of embryos (Komiya et al., 1997). The result is an increasingly complete account not only of the list of genes, but of their map location and of the time and place of their expression. Genome approaches should be adaptable to the systematic exploration of the repertoire of genes that are turned on or off as differentiation and mortalization take hold. For example, ES cells cultured in vitro and induced to differentiate could be studied for differential gene expression. Summary Thinking about aging has begun to change. Only in recent decades, in effective arguments for the establishment of the National Institute on Aging (see Longevity, pp.76 –77, June 1991), Florence Mahoney put forward the idea that human performance should be optimized throughout life and that this goal could be abetted by systematic research on aging and the aging process. The notion of “optimization” can be formulated as the alleviation of the ills of aging to promote creative, long life. Genomic approaches are now providing the basis for much of medical research, and aging research is no exception. One no longer needs to recall that all the genetic approaches referred to here benefit from genomic approaches. For example, the discovery of the Werner syndrome gene (Yu et al., 1996) employed genome maps and long-range sequencing techniques, and one powerful approach to the cloning of genes that control growth started from the use of monochromosomal hybrids to score function (Smith et al., 1996) and continued with positional cloning methods. In this Perspective, we have focused on the expectation that all such analyses, including those of genes that affect longevity in model organisms or humans,
can be accelerated by “developmental genomic” catalogues. The determination of gene cohorts and their expression patterns both achieves a logical completion of the Human Genome Project and provides a starting point for human biology research throughout the life span. From the perspective of individual existence, the proposed approach has a particular appeal. Each of us, in a singular way, reenacts the myth of Theseus, facing the minotaur as we age, but we could now be guided through the labyrinth of human development by the jeweled thread of Ariadne—in double-helical form. ACKNOWLEDGMENTS We thank Dr. Victor McKusick for encouraging us to write this Perspective. The work of the authors’ laboratories was largely performed during their tenure as faculty members at Washington University School of Medicine (D.S.) and Wayne State University (M.S.H.K.). Part of the discussion is adapted from the Mahoney Lecture given by D.S. on September 22, 1997, and benefited from comments by Drs. Joel Kupperman, Lucio Luzzatto, and Giuseppe Pilia.
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PERSPECTIVE
Developmental Genomics and Its Relation to Aging David Schlessinger1 and Minoru S. H. Ko Laboratory of Genetics, Gerontology Research Center, National Institute on Aging, Baltimore, Maryland 21224
Negative and Positive Views of Aging Negative views of aging are deeply embedded in Western culture. As is often the case, some current ideas can be traced back to Hellenic antecedents. Aristotle laid the foundations for modern genetics (Grene, 1968), with an insistence on careful observation and an emphasis on heredity, reproduction, adaptation in response to the struggle for survival, and relevant effects of diet and locomotion. In his thinking about the course of biological life, Aristotle contrasted the phase of “becoming” (that is, development) with that of “passing away” (that is, degeneration). Aristotle’s idea that each species tends to the fulfillment of its ideal form was in keeping with the Hellenic formulation of the division of life into temporal compartments, with development culminating in transient “acme.” In classic thought, human acme, as exemplified in Attic sculpture, occurred at about age 18, and it was all downhill from there. Had Aristotle been acquainted with the Analects as well as Plato’s Symposium, he might have pondered Confucius’ account of his progressive, slow process of education until he reached an important realization when he was 70. Even in Aristotle’s restricted surroundings, venerable Athenian judges were considered the wisest and most circumspect, and he himself was writing the Lyceum lessons (that we still read!) in his sixth decade. Nevertheless, the impressive verve and beauty of youthful heroes like his tutee Alexander dominated his thinking, and the notion that human life can be divided into growth and decay has lasted to the present day. For example, Martin and Mian (1997) quote the (facetious?) formulation that “first we ripen and then we rot.” Current versions of this up-and-down view of human life are often based on the “evolutionary theory of aging.” Discussions (reviewed, for example, by Finch, 1990; Austad, 1997) comment that once individuals are essentially beyond reproductive age, negative consequences of genes and mutations are not subject to selection and accumulate to cause the infirmities of the aged (Medawar, 1957; Williams, 1957). Such formulations, however, have difficulty rationalizing the 1 To whom correspondence should be addressed at Gerontology Research Center, Box 31, 5600 Nathan Shock Drive, National Institute on Aging, Baltimore, MD 21224-6825. Telephone: (410) 5588337. Fax: (410) 558-8331. E-mail: [email protected].
0888-7543/98 $25.00 Copyright © 1998 by Academic Press All rights of reproduction in any form reserved.
substantial hereditary component in human longevity (Martin, 1978; McGue et al., 1993; Finch and Tanzi, 1997)— or the substantial differences in longevity between species and between variant individuals in a particular species (cf. Kirkwood, 1985; Lithgow, 1996; Tower, 1996; Rogina et al., 1997; Tissenbaum and Ruvkun, 1998). Modern evolutionary theory can take a more positive view of potential selective advantages of longevity. Developments based on the work of Hamilton (1964) consider the unit of selection as the tribe/family/group rather than the individual and discuss the evolution of phenomena such as cooperation and altruism (e.g., Dugatkin, 1990). Selection based on kinship in several species has been commented on increasingly, for example, by Carey and Gruenfelder (1997). Such treatments, however, have not generally followed up the ways in which “cultural evolution” (Medawar, 1957) could provide information with selectable value. As a putative example, consider the climatological evidence that miniperiods of severe weather can be intercalated with relatively mild weather. [According to recent studies, the variation can be within several decades, even in the arctic; Overpeck et al. (1997).] One can imagine the plight of a preliterate society suddenly faced with the recurrence of a mini-ice age. Members of a group whose experience spanned the interval back to the last mini-ice age, and who could transmit information about how such a period had been weathered, could provide considerable selective advantage to the survival of the group. Presumably there would thus be some selective pressure for longevity, though a few such members of a tribe could be sufficient. The positive features of aging are not limited to sheer staying power. In addition to the maturity and perspective traditionally associated with the moderation of hormone levels in adult life, creativity seems to show no obligatory agerelated decline. For example, if one considers artists, performers remain vibrant throughout their lives, and the last works of composers are often their best. Verdi wrote Aida, Otello, and Falstaff in his seventies, premiering Falstaff when he was 80. Beethoven produced nothing like the Missa Solemnis or Opus 131 quartet until his last years; Haydn wrote his greatest “London” symphonies and The Creation in his seventh decade. Similar observations hold for every art: consider Monet at Giverny in his final years or Titian painting brilliantly in his nineties. Aging as a Part of Metazoan Maturation
Turnover and Its Limitations Positive selective features are one indication that aging is not simply a “decay” phase after development, but rather an integral part of metazoan maturation. D’Arcy Thompson (1984) analyzed the inescapable transience of exponential growth: the size of complex organisms and their constituent parts is limited by functional constraints, so that net growth must cease. As growth slows and stops, homeostasis depends increasingly on the balance of synthesis and turnover of cells
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SPECIAL FEATURE and tissues. Some authors have noted that some organisms can develop with essentially fully replaceable parts. This alternative, which would avoid the dependence on intracellular turnover, is precluded for human aging for two reasons. First, no mechanism is available for the replacement of some structural members—such as collagen. [One might imagine more aggressive turnover pathways that might alleviate this, but we are currently limited to those evolution has provided us.] Second, and more intractable, is the fact that one would not want to replace some portions of the body. In particular, memory and learning would be totally disrupted by the replacement of all brain cells by new ones. If an individual adult is to continue to exist, ultimate decay of brain cells that no longer divide is inevitable. Tissues differ in the degree of their dependence on turnover. Renewing tissues that retain stem cells can be more resistant to the effects of aging. This is because errors in DNA can be corrected by recombination and selection, and errors in protein can be diluted by net growth. In contrast, static cells and components cannot rescue mistakes or mutational events by dilution or recombination. They are in effect placed in a predicament similar to that of the Y chromosome in evolution, whose gene content atrophies progressively. An analogue of Muller’s ratchet (Muller, 1964; Rice, 1987) comes into play in aging tissues, and function is increasingly limited by irreplaceable loss. Thus, in primates, aging has evolved as a balance of opposing forces: progressive deterioration of components of the body (and elimination of entire individuals) and the benefit to fitness of having a number of individuals of considerable age who continue as part of a population cohort. The balancing mechanism starts with events in embryonic and fetal life that determine features of the course of aging.
Dependence of Aging on Embryonic Events Aging can be visualized as starting in the embryo. Embryonic events can be seen as critical for the understanding and possible alleviation of aging-related pathology. As an example, many theories of aging are predicated on the difference between indefinitely growing, totipotential embryonic stem cells (and germ cell progeny) and mortal differentiating cells that are already committed to cellular senescence (Hayflick, 1992). There are many indications that the process and its rate are genetically determined. Among different mouse strains, for example, a trend toward longer life span is correlated with a larger pool of hematopoietic stem cells (de Haan et al., 1997). Standard systems show that the loss of replicative capacity, and presumably of the relative numbers of types of cells, is correlated with telomere shortening (Blasco et al., 1997) and the activation or repression of a variety of genes (e.g., Linskens et al., 1995; Dimri et al., 1996; Smith et al., 1996; Garkavtsev et al., 1997; Haber, 1997; Yeager et al., 1998). The prevention or reversal of the process is also complex: it is not simply an imitation of “viral transformation” (Gotoh et al., 1979; Rubelj et al., 1997), but involves the regulation of genes such as telomerase, which can
extend life span (Bodnar et al., 1998), and others that can provoke senescence (Vojta et al., 1996). In part because of the complexity of the systems, experts dispute whether there is an “aging clock” in cellular senescence, but everyone agrees that a switch of some type initiates differentiation and loss of immortality. For example, Suda et al. (1987) showed that embryonic stem cells from mouse grow for an indefinite number of generations. This is especially noteworthy since mouse cells, including embryonic fibroblasts that have begun their mortal existence, show extraordinarily high rates of autotransformation and development of chromosomal aberrations and changes in epistatic methylation mechanisms (Holliday, 1996). Notably, theories of aging that are predicated on the accumulation of insults all take this initial transition for granted. For example (see Tower, 1996), consider the notion that oxidative stress provokes damage that is unremedied and contributes to age-related pathology and ultimate cell death. A number of correlated phenomena make this an attractive possibility, but mouse embryonic stem cells are exposed to the same insults as other cells and presumably should accumulate damage at the same rate. Yet they do not apparently age. Special status is also accorded germ cells, which are clearly “spared” progressive deterioration or loss of growth potential over generations. Sparing is another way of saying that the cells do not begin the aging process. [One implication of these findings is that repair processes may be more effective in “immortal” cells, and the findings that Werner and Cockayne syndromes are caused by lesions in repair-related genes (Yu et al., 1996; Mallery et al., 1998) are consistent with that view.] The underlying specification of the physiological process must be based on gene programs, and such theories place a premium on genes involved in the “immortalization” or “mortalization” process. In a second link between embryonic and aging events, every system of the body shows age-related loss—including osteoporosis, depletion of neurons and glomeruli, etc. No one thinks that the formation of tissues or organs involves a detailed mechanism that is the reverse of turnover and depletion. But if we understood how differentiated cells and specialized tissues form, conditions or regimens might be developed to prevent their loss or even to regenerate or replace them. Also, development often involves selective apoptosis or “programmed cell death”; are there comparable mechanisms involved in a programmed cell death in later life, based on a putative “programmed cell aging”? In cogent instances, critical events in embryonic and fetal life have a direct bearing on the subsequent etiology of agerelated conditions. Inherited conditions provide especially clear instances. We consider three that we have had experience with in recent years: (1) Premature ovarian failure. In about 1% of women, gonadal failure occurs before the age of 40. In all instances, the termination of a woman’s reproductive life occurs when her supply of mature follicles is exhausted (Davis, 1996). Of course this is by definition an aging phenomenon, but the net supply of follicles is already set in fetal life. Thus, to understand menopause— or premature ovarian failure, in which follicular atresia occurs at an increased rate in the embryo—
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SPECIAL FEATURE one must understand ovarian and follicular embryonic development. (2) X-linked ectodermal dysplasia. Individuals with lesions in ectodysplasin A, the product of the EDA gene (Kere et al., 1996) [and mice with lesions in the cognate Tabby gene (Srivastava et al., 1997)], show a defect in the development of skin appendages, including hair, teeth, and eccrine sweat glands. This provides an entry point for the analysis of the developmental process that leads to these specialized tissues, but the analysis could be highly relevant to the preservation or regeneration, for example, of hair in aging individuals. Comparable lines of investigation could provide information relevant to osteoporosis, end-stage kidney failure, and other baneful features of age-related pathophysiology. (3) Overgrowth syndromes. In these conditions, exemplified by Beckwith-Wiedemann syndrome (Li et al., 1997) and the X-linked Simpson-Golabi-Behmel Syndrome (Pilia et al., 1996), individuals grow to considerable height, with the overgrowth of a number of primarily mesoderm-derived internal tissues and organs. Biological questions of importance here relate to the mechanism by which the set point for organ size is determined. The individuals with this condition are susceptible to an increased incidence of embryonal tumors and are also subject to life-threatening problems based on heart overgrowth. Problems arise in childhood and in aging adults, but again, the critical events occur in embryonic life. Investigation of mechanistic details relating early and late phenomena are just beginning. As an example, growth and development—and the overgrowth syndromes (Eggenschwiler et al., 1997; Morison et al., 1996)—involve the action of insulin-like growth factors in promoting cellular expansion and proliferation. Recent studies hint that the insulin growth factor (IGF) protein system is also involved in the model aging process in nematodes (e.g., Tissenbaum and Ruvkun, 1998). Genetics of Aging and Developmental Genomics
Analysis of Aging and Longevity Genetic analysis of aging-related conditions is very much hampered precisely by the fact that they affect old people. Although the conditions depend on developmental events earlier in life, the parents of affected individuals are often dead by the time the disease is scored, and the children of those affected often have a number of decades to go before they can be scored. Genetic analysis has traditionally been made even more complex by the nagging problems of heterogeneity and variable penetrance. To overcome some of these difficulties, one can turn to the analysis of early-onset forms of aging-related disorders, preferably coupled to core phenotypes that are easily scoreable. The successes of this approach have been notable. Our knowledge of the genetics of Alzheimer disease has been extended extraordinarily by the discovery of presenilins through the analysis of early-onset pedigrees (Haass, 1996). As another outstanding example, the rate of aging has be-
come a field of biochemical study with the discovery of helicase mutants in early-onset aging in Werner syndrome (Yu et al., 1996) and other progeroid (Martin, 1978; Mallery et al., 1998) syndromes. The analysis of the basis for menopause starting from early-onset premature ovarian failure (see above) is another example, and other phenotypes, such as early-onset recurrent unipolar depression, are likely to be susceptible to such an approach. In this regard, there is a considerable body of longitudinal information on the course of system-specific normal and abnormal aging, particularly from the Baltimore Longitudinal Study of Aging (Shock et al., 1984) that can provide parameters and trajectories to aid in scoring conditions at a relatively early age. Early-onset conditions can be studied by any of the standard techniques, including linkage analysis (or detections of translocations or microdeletions) followed by refined mapping; linkage disequilibrium mapping through haplotype analysis; and association analysis, looking for increased prevalence of an allele in individuals affected by a condition or disease. All of these approaches are obviously facilitated, however, by the use of specialized populations, in which heterogeneity is demonstrably lower than in outbred populations, and have been used successfully in many reported studies, for example, those of the “Finnish diseases” (Norio, 1994). Such specialized population resources are especially important for the study of the extreme phenotype of aging, “longevity.” Longevity can be scored unequivocally, since, from the analysis of Gavrilov and collaborators (1997, 1998), an individual who is 90 years or older can be safely classified in a special group with those who are 100 years or older. But this criterion of course eliminates the possibility of finding an early-onset form of longevity. In fact, one must wait till advanced ages to score individuals, simply to be able to discount confounding effects such as accidental death. The unavailability of parental and offspring DNA from scored individuals for such a cohort is further complicated by the likelihood that alleles for a number of condition-specific risk factors will have been depleted in this population (distorting associations). However, methods based on haplotype studies in interrelated, delimited populations retain their power. A likely experimental design would be to collect sib pairs and multiple sibs over 90 years old and apply the potent modern haplotypic analyses.
Developmental Genomic Analysis For aging-related phenomena, like all other simple and complex traits, initial studies are designed to identify candidate genes by positional cloning. Especially for conditions with a strong developmental component, it would be useful to know what genes are involved and when they are expressed. Developmental genomic analysis aims to determine expression patterns on a systematic basis for genes expressed at specific times and places during the life of a species. It can help to complete and annotate the catalogue of genes that is required for the analysis of embryonic
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SPECIAL FEATURE phenomena that relate to aging, for developmental biology, and for the Genome Initiative. As a specific example, the sequencing of collections of cDNAs made from specific stages in embryonic life are producing 30 –50% new genes [that is, genes not present in the current databases; (Ko et al., submitted for publication; and work in progress)]. Many of those genes are represented for only brief periods and in specific subregions of the embryo. Thus, the fundamental achievement of a complete catalogue of genes is both required for and requires the analysis of embryonic and fetal gene expression. [We note that the methods for determining expression pattern may vary, with the use of SAGE (Velculescu et al., 1995), microarrays (Schena et al., 1995), etc., but those methods all become increasingly powerful as the available catalogue of genes becomes more complete.] The generation of the catalogue would be in line with current discussions of “functional genomics,” but the analysis of embryonic events requires further information that can again be obtained more efficiently by systematic genomic approaches. In pilot studies, it has been demonstrated that a large number of genes can be placed on the mouse genome by the use of interspecific backcross panels (Ko et al., 1994; and work in progress), and a large number of cDNA species can be examined by in situ hybridization to whole mount and sections of embryos (Komiya et al., 1997). The result is an increasingly complete account not only of the list of genes, but of their map location and of the time and place of their expression. Genome approaches should be adaptable to the systematic exploration of the repertoire of genes that are turned on or off as differentiation and mortalization take hold. For example, ES cells cultured in vitro and induced to differentiate could be studied for differential gene expression. Summary Thinking about aging has begun to change. Only in recent decades, in effective arguments for the establishment of the National Institute on Aging (see Longevity, pp.76 –77, June 1991), Florence Mahoney put forward the idea that human performance should be optimized throughout life and that this goal could be abetted by systematic research on aging and the aging process. The notion of “optimization” can be formulated as the alleviation of the ills of aging to promote creative, long life. Genomic approaches are now providing the basis for much of medical research, and aging research is no exception. One no longer needs to recall that all the genetic approaches referred to here benefit from genomic approaches. For example, the discovery of the Werner syndrome gene (Yu et al., 1996) employed genome maps and long-range sequencing techniques, and one powerful approach to the cloning of genes that control growth started from the use of monochromosomal hybrids to score function (Smith et al., 1996) and continued with positional cloning methods. In this Perspective, we have focused on the expectation that all such analyses, including those of genes that affect longevity in model organisms or humans,
can be accelerated by “developmental genomic” catalogues. The determination of gene cohorts and their expression patterns both achieves a logical completion of the Human Genome Project and provides a starting point for human biology research throughout the life span. From the perspective of individual existence, the proposed approach has a particular appeal. Each of us, in a singular way, reenacts the myth of Theseus, facing the minotaur as we age, but we could now be guided through the labyrinth of human development by the jeweled thread of Ariadne—in double-helical form. ACKNOWLEDGMENTS We thank Dr. Victor McKusick for encouraging us to write this Perspective. The work of the authors’ laboratories was largely performed during their tenure as faculty members at Washington University School of Medicine (D.S.) and Wayne State University (M.S.H.K.). Part of the discussion is adapted from the Mahoney Lecture given by D.S. on September 22, 1997, and benefited from comments by Drs. Joel Kupperman, Lucio Luzzatto, and Giuseppe Pilia.
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