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or age-related pathology
Mechanisms of Ageing and Development 124 (2003) 581 /587 www.elsevier.com/locate/mechagedev
Progress Paper
Models of accelerated ageing can be informative about the molecular mechanisms of ageing and/or age-related pathology Huber R. Warner *, Felipe Sierra Biology of Ageing Program, National Institute on Ageing, Bethesda, MD 20892, USA Received 20 December 2002; received in revised form 24 January 2003; accepted 27 January 2003
Abstract During the past ten years considerable progress has been made in discovering genes that regulate longevity by identifying single gene mutations that lead to increased longevity. The initial success in nematodes was quickly followed by comparable success in fruit flies and mice. In contrast, mutations that cause a decrease in longevity have been largely discounted as unlikely to be informative about aging mechanisms. However, the recent creation of several mutant mouse models that develop a variety of aging-like phenotypes and die prematurely, suggests that such models may be useful in understanding aging mechanisms, particularly as they relate to progressive tissue and organ dysfunction. A possible common feature of these models may be an imbalance between loss of cells by apoptosis and subsequent cell replacement, leading gradually to a net loss of cells in multiple tissues. # 2003 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Accelerated ageing; Molecular mechanisms; Age-related pathology
1. Introduction Ageing is difficult to define unambiguously, and the complexity of the ageing process diminishes the probability that any one theory can adequately explain all or even most of its root causes. Early research on ageing was focused on obtaining data to either support or disprove one or more of the many theories that had been proposed to explain ageing. These theories include both random damage theories (e.g. free radical and DNA damage) and programmed ageing theories (e.g. immunological or endocrine decline), and the concept that some age-related changes may be programmed, whereas, others are stochastic and unpredictable, is now generally accepted. However, research on these various theories has so far failed to divulge the critical mechanisms causing ageing, suggesting the need for new thinking about how best to study and elucidate the biological basis of ageing.
* Corresponding author. Tel.: /1-301-496-4996; fax: /1-301-4020010. E-mail addresses: [email protected] (H.R. Warner), [email protected] (F. Sierra).
1.1. Generalized functional decline leading to homeostatic failure as a unifying theory of ageing Most gerontologists agree that ageing leads to a failure to maintain and/or return to homeostasis after exposure to a stress, no matter what system they happen to be studying. In discussing a recent paper by De Boer et al. (2002) on the role of DNA damage and repair in ageing, Hasty and Vijg (2002) suggest the ‘broad-based hypothesis . . . that generalized homeostatic failure leads to age-related decline’. The ability to successfully resist and survive various kinds of biochemical stresses must be a major factor in the ability of a living organism to maintain homeostasis. Lithgow and Walker (2002) recently reviewed the evidence indicating that stress resistance contributes to longevity regulation in the nematode, Caenorhabditis elegans . Stress resistance and longevity also appear to be associated in the fruit fly, Drosophila melanogaster (Lin et al., 1998). Because both of these organisms are comprised of mostly postmitotic cells, this raises the important question of whether invertebrate and mostly post-mitotic organisms can model the events occurring in mammals, where most organs are regularly undergoing cell death and replacement. Indeed, homeostatic failure in these models might
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imply the inability to maintain an adequate number of functional cells in each tissue during ageing. In turn, this can be regulated at two different levels: the number of cells, and the functional quality of the available cells. Thus, the study of mutant and transgenic mice may provide a more potentially fruitful line of investigation of ageing mechanisms in mammals. In this discussion we will focus on the number of cells existing in the tissue, rather than their functional quality. 1.2. Appropriate animal models for studying ageing Long-lived nematode, fruit fly and mouse mutants have been described and their relevance to ageing mechanisms is currently being assessed. In contrast, because there are so many pathological conditions that appear to have no relevance to ageing but nevertheless shorten life span, many gerontologists hold the view that animal models with reduced life expectancy are of little or even no relevance in studying ageing compared with models with extended life expectancy. This may be particularly true in the case of the invertebrate models of ageing such as yeast, fruit flies and nematodes, because we know almost nothing about the pathology of these organisms and why they die. In contrast to fruit flies and nematodes, we know a good deal about mouse pathology and why mice die (Lipman et al., 1999). Thus, it seems likely that mice prematurely displaying a variety of phenotypes associated with ageing, as well as mice displaying delayed ageing, i.e. extended life expectancy, could well be informative about risk factors for various aspects of human ageing. 1.3. Mouse models of delayed ageing At least seven long-lived transgenic and mutant mice have been described in the literature in the past few years. These include four mouse mutants that either fail to produce growth hormone or to respond to it (Bartke et al., 2001; Flurkey et al., 2001). These mice are all small, and at least three of these mutants have low levels of circulating IGF-I and insulin, suggesting that the signal produced by their insulin signaling pathway might be attenuated. Their longevity is increased by 30/60% in agreement with long-lived nematode (Morris et al., 1996; Kimura et al., 1997) and fruit fly (Tatar et al., 2001; Clancy et al., 2001) mutants having reduced insulin signaling. The most recent addition to this collection of IGF-I level/insulin-signaling pathway mutants is a heterozygous knockout mouse lacking one copy of the gene for the IGF-I receptor (Holzenberger et al., 2003). Females with this Igf -Ir / genotype have an average life expectancy about 33% longer than the wild type, but are not dwarf. They are resistant to paraquat and also have reduced levels of the phosphorylated p66shc protein. Male Igf -Ir / mice are only mildly
long-lived, whereas Igf -Ir / mice show multiple defects, including severe neurological dysfunction. A sixth long-lived mouse contains a mutation in the p66shc gene that increases the ability of mouse cells to resist apoptosis induced by either hydrogen peroxide or ultraviolet light (Migliaccio et al., 1999). The p66shc gene product is part of a signal transduction pathway that is activated by reactive oxygen species, leading to apoptosis (Nemoto and Finkel, 2002). The p66shc gene negatively regulates the transcription factor, FKHRL1, that in turn appears to regulate transcription of the gene coding for catalase. Thus, when the p66shc protein is absent, FKHRL1 remains active and overproduces catalase and possibly other anti-oxidant enzymes, thereby increasing resistance to oxidative stress. The phenotype of this mutant partially overlaps the phenotype of the long-lived C. elegans age-1 mutant that overproduces catalase and superoxide dismutase, but also displays reduced insulin signaling (Larsen, 1993; Morris et al., 1996). Overall, the results with the Igf -Ir / and p66shc mice suggest a causal linkage between insulin signaling, response to oxidative stress and longevity regulation. The seventh long-lived mouse mutant apparently genetically recapitulates the caloric restriction paradigm (Miskin and Masos, 1997). While the above examples demonstrate some concordance between invertebrate and mammalian organisms with regard to longevity regulation, thereby providing support for the concept that invertebrate models may be informative about mammalian ageing, there is no direct evidence available yet to suggest that either the insulin-signaling pathway or resistance to oxidative stress regulates longevity in humans. 1.4. Transgenic mouse models of accelerated ageing On the opposite side of the coin are at least four mouse models that are not only short-lived, but also prematurely display one or more ageing-associated phenotypes. These include mice defective in XPD function (De Boer et al., 2002), mice with abnormal p53 function (Tyner et al., 2002), mice defective in joining DNA double-strand breaks (Vogel et al., 1999), and the Klotho mouse (Kuro-o et al., 1997). The properties of these four mouse models are summarized in Table 1, and the possible relevance of these accelerated ageing mouse models is discussed below with particular reference to putative human premature ageing syndromes. 1.4.1. XPD De Boer et al. (2002) recently created transgenic mice carrying a mutation in the XPD gene that codes for a DNA helicase involved in both repair of DNA damage and transcription. The mutation does not knock out the
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Table 1 Phenotypes of mouse mutants of accelerated ageing Phenotype
Mouse mutant Klotho
Ku80
XPD
p53 /m
Biochemical defect
Unknown 8 /9
Lacks a DNA helicase DNA repair defective B/50
Hyperactive p53
Mean life expectancy (weeks) Control (%) Onset of growth failure Osteoporosis Subcutaneous fat loss Hair Wound healing Cancer risk
Defective repair of DNA double strand breaks 35
96
B/10 3 /4 weeks
35 From birth?
B/50 12 weeks
70 /18 month
Premature onset Premature onset Reduced number of follicles NR NR
Premature onset Premature onset NR NR Occurs earlier, but lower incidence Immune deficiency
Premature onset Premature onset Premature graying NR NR
Premature onset Premature onset Minimal graying Reduced Reduced
Other phenotypes
Arteriosclerosis; premature thymus atropy; abnormal walking behavior
General organ atrophy
NR, not reported.
helicase activity, but significantly reduces it. As a result, these mice have substantially impaired transcription and mildly impaired DNA repair. The phenotype of these transgenic mice includes not only osteoporosis, loss of female fertility, and premature graying of hair, but also reduced life span (median life expectancy B/12 months vs. /24 months for the controls). These mice appear to be normal at birth and remain normal for about 4 months, although they weigh 10 /20% less as early as 2 weeks of age. However, by 6 months of age a variety of defects begin to appear as indicated above. The molecular basis for the age-related onset of the ageing-like symptoms is not known, but De Boer et al. suggest that the failure to repair DNA damage and allow transcription to proceed may trigger programmed cell death, ‘leading to functional decline and depletion of cell renewal capacity’. 1.4.2. p53 A comparable mutant mouse model was produced by Tyner et al. (2002). They reported the unexpected result that deletion of a portion of the p53 gene that codes for the carboxy terminus of the p53 protein can lead to a gain of p53 tumor suppressor function. The p53 protein plays a critical role in regulating cell division, response to environmental stress, and programmed cell death, and more than 50% of human cancers are associated with one or more mutations in the p53 gene. Tyner et al. (2002) reported that mice containing one good copy of the p53 gene, and one of the new mutant copies are very resistant to cancer, but also show premature signs of ageing such as osteoporosis, slow wound healing, muscle and general organ atrophy, and loss of hair and subcutaneous adipose tissue. The life expectancy of these mice is reduced by 20/25%. The authors suggest
that cells in the mutant mice are being lost in multiple tissues faster than they can be replaced. As in the case of the xpd -defective mice, the authors suggest that these mice may be experiencing excessive cell death, thereby prematurely exhausting their stem cell reserves so that ‘sufficient numbers of mature cells cannot be provided to maintain organ homeostasis’. 1.4.3. Ku-80 Another DNA repair deficient model is the Ku-80deficient mouse (Vogel et al., 1999; Lim et al., 2000). The average longevity of this mouse is only 35 weeks, compared with 100 weeks for the control. Besides reduced longevity, this mouse also prematurely develops ageing-associated phenotypes such as osteoporosis, skin atrophy and follicular loss, beginning by the age of about 37 weeks. Cancer also develops prematurely, although at a much lower incidence than in Ku -80/ mice. The Ku-80-deficient mice are also markedly smaller than normal mice, a phenotype observable as early as 2.5 weeks after birth. Hence, the Ku-80 / mice share some of the same phenotypes observed in the p53/m and xpd / mice, and may also be experiencing non-replaceable cell loss. 1.4.4. Klotho The klotho mouse produced by Kuro-o et al. (1997) also displays several ageing-associated phenotypes, including skin atrophy, lipodystrophy, osteoporosis and arteriosclerosis, besides shortened longevity. The mean life span of kl / mice is about 7 weeks, but growth failure begins at about 3/4 weeks. The kl insertion mutation occurs in an unidentified gene and is autosomal recessive. The gene has been cloned and sequenced; it has a transmembrane domain, and shares
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homology with known b-glucosidases. A human homolog has also been identified and has 86% identify with the mouse gene. More recent evidence suggests that the wild-type klotho protein suppresses insulin signaling (Utsugi et al., 2000). Thus, the kl / mouse may have a hyperactive insulin-signaling pathway and its reduced longevity may be due to some of the same factors that reduce longevity in mice in response to over-production of growth hormone (Wolf et al., 1993). The report of a fifth such mouse model is currently ‘in press’ (Wong et al., 2003). According to the authors, their Atm-telomerase-mouse model has a ‘generalized proliferation defect . . . evident in all cell types and tissues examined, and this defect extended to tissue stem/ progenitor cell compartments, thereby providing a basis for progressive multi-organ system compromise, accelerated ageing and premature death’. There are other short-lived mice that could be included in this discussion, including mice deficient in anti-oxidant defense enzymes such as methionine sulfoxide reductase (Moskovitz et al., 2001), and the SAMP mouse strains (Takeda, 1999). The above examples were chosen for discussion here because their development of age-associated phenotypes is well described. The central question about the above studies is whether the shortened longevity observed in these mutant mice has any relevance to ageing in non-mutant mice. Other important questions include: (1) What do these mice die of? (2) What causes the early onset of growth failure in these mouse models? (3) Are the ageing phenotypes observed relevant to development of agerelated pathology in non-mutant mice? and (4) What accounts for the timing of onset of premature ageing phenotypes in these mutant mice? Many gerontologists assume, although it has not been fully unequivocally proven, that cells are constantly being lost at a slow rate due to continual intrinsic damage caused by the generation of reactive oxygen species that accompanies normal mitochondrial metabolism, especially in post-mitotic tissues and cells (Beckman and Ames, 1998). This loss of damaged cells must be constantly balanced by cell replacement either through proliferation of neighboring ‘like’ cells in proliferative tissues such as liver, or by proliferation of stem cells (Fig. 1). When cells reach a state known as cellular or replicative senescence (Hayflick, 1965), they would no longer be able to proliferate to replace lost cells, but only limited evidence is currently available to suggest that such senescent cells actually exist in vivo (Dimri et al., 1995; Hornsby, 2002), and would cause problems if they do (Krtolica et al., 2001). If cell senescence does prevent proliferative repair, then this could lead to loss of tissue homeostasis if an adequate stem cell population is not available to replace cells lost from the tissue. Unfortunately, very little is currently known about the roles of stem cell populations in
Fig. 1. Maintaining tissue homeostasis by repair and/or cell replacement.
maintaining homeostasis, or how these change with ageing. If stem cell populations do become rapidly depleted by excessive cell death as suggested by Tyner et al. (2002) and De Boer et al. (2002), would the phenotypes that result be informative about normal ageing? Although we currently know little about age-related changes in stem cell populations, it might be instructive to consider whether these mice provide opportunities to study the development of age-related pathology on an accelerated scale. The most obvious pathology to consider is osteoporosis, which shows early onset in XPD , p53/m and klotho mutants, as well as in Werner’s syndrome in humans (Martin and Oshima, 2000). Although there may be many factors contributing to osteoporosis, the general model is that bone resorption by osteoclasts exceeds bone deposition by osteoblasts, leading to net bone loss with increasing age. Thus, it would be important to learn whether either the XPD , p53 /m or the klotho genotype affects cell loss and cell replacement rates within the osteoblast and osteoclast populations in mice, and how the ratio of these populations changes with increasing age in these mice. This might be possible if these rates are accelerated in these two mutant strains, and if so, the results might well provide an explanation for the early onset of osteoporosis in these mutant mice. Whether such a result would be informative about osteoporosis in humans would have to be tested directly. A similar opportunity exists for studying tissue/organ atrophy in the p53 mutant where general organ atrophy is observed by 24 months of age (Tyner et al., 2002). Exaggerated rates of cell turnover, with an imbalance between cell loss and replacement, might be measurable in this mutant, and if so would provide at least one explanation for phenotypes occurring during normal ageing, e.g. sarcopenia, slow wound healing, etc. 1.5. Human accelerated ageing syndromes as models for studying ageing The most important question is whether any of these accelerated mouse strains can be informative about
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human ageing. In 1978 and again in 1990, pathologist George Martin analyzed the known genetic syndromes in man that might have potential relevance to the pathobiology of ageing. Although none of these was or should be considered to be a ‘pure’ example of premature ageing, he chose syndromes that display one or more ageing-like phenotypes, such as premature graying of hair, cataracts, cardiovascular disease, diabetes, osteoporosis, neoplasms characteristic of the elderly population and dementias. He called these segmental progeroid syndromes. As a result of this analysis, Dr Martin estimated that up to about 7% of the human genome ‘could play a role in modulating specific aspects of the senescent phenotype in man’ (Martin, 1990). He also suggested that these syndromes would be useful for investigating the molecular, cellular and genetic basis of specific aspects of human ageing, even though the syndrome itself may not be an exact representation of premature ageing. Using these same criteria, the mouse models discussed above might represent bona fide models of murine segmental progeroid syndromes. Three segmental human progeroid syndromes may provide an opportunity for comparative research on various aspects of cell turnover in humans. These are Bloom (BS), Hutchinson /Gilford (HGS), and Werner (WS) syndromes. Table 2 briefly summarizes some of the phenotypes of these three progeroid syndromes. BS and WS are caused by defects in genes for two different DNA helicase activities (Ellis et al., 1995; Gray et al., 1997). These helicase deficiencies may cause the build-up of unresolved replication, repair and/or transcription complexes in cells, leading ultimately to programmed cell death. In contrast, HGS is caused by a mutation at an unique site in the gene for lamin A, a major component of the nuclear envelope (Eriksson et al., in press). Mutations at other sites in this gene are responsible for a variety of pathologies including Emery /Dreifuss muscular dystrophy, skeletal and cardiomyopathy, lipodystrophy, Charcot /Marie /Tooth disorder and mandibuloacral dyslasia (Burke and Stew-
art, 2002). Why lamin A mutations cause these pathologies is not known, but excessive cell death due to aberrant nuclear structure and function appears to be one possibility. In fact, caspases disrupt the nuclear membrane during apoptosis (Faleiro and Lazebnik, 2000) and lamin A is one of the substrates for these caspases (Takahashi et al., 1996). Fibroblasts taken from patients with any of these syndromes have very limited proliferative capacity when cultured, suggesting that many of these cells may have already reached replicative senescence (Campisi, 1996). Although none of these syndromes is associated with growth hormone deficiency, patients with BS are born small, remain smaller than normal through development, and live only into their 20s, while patients with HGS and WS both appear normal at birth, but begin to show growth defects between 1 and 2, and 15/20 years, respectively. It would be interesting to know how the age-related onset of growth defects in these three syndromes is related to the amount of programmed cell death occurring, and the ability to replace these lost cells, in the various tissues in these patients. Such knowledge should also be relevant to understanding the slow loss of tissue mass and function in apparently normal human subjects during ageing, both in general, and as a direct cause of age-related pathology.
2. Discussion Martin and Oshima (2000) argued that studies of human genes in which mutations can lead to accelerated emergence of senescent phenotypes ‘may lead to a clearer understanding of the nature of senescence, and could provide clues for ways in which ageing might be retarded’. Recent papers on the development of some accelerated ageing-like phenotypes in several transgenic mouse strains, as well as the molecular characterization of some of the human segmental progeroid syndromes, suggest a need to include tissue dynamics involving programmed cell death, cell proliferation, and stem cell
Table 2 Comparison of several human progeroid syndromes Phenotype
Biochemical defect Mean life expectancy (years) Normal (%) Onset of growth failure Osteoporosis Subcutaneous fat loss Slow wound healing Cancer risk
585
Human syndrome HGS
BS
WS
Lamin A 13 17 Year 2 No? Yes ? Normal?
Lacks a DNA helicase 25 33 In utero No? ? ? Elevated
Lacks a DNA helicase 50 67 Year 15 /18 Premature onset Yes Yes Elevated
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replacement capacity in this context (Fig. 1). The experimental evidence obtained thus far suggests that a better understanding of the factors directly affecting the ability to maintain tissue cell number and function will provide a better conceptual framework for future research aimed at understanding the root causes of ageing, and this could lead to interventions to reduce frailty in the elderly. The suggestion that cells from these mouse and human mutant models are especially vulnerable to apoptosis can be directly tested as apoptosis can be studied in in vitro systems (Lazebnik et al., 1993). When multiple phenotypes of ageing are delayed by a given mutation, such as the dwarf dw /dw mutants studied by Flurkey et al. (2001), we may be most comfortable that we are truly studying ageing per se. However, limiting our study only to animal models that delay the development of ageing phenotypes seems too restrictive. We believe it is unwise to automatically discard as irrelevant animal models that accelerate the appearance of one or more ageing-like phenotypes, and in so doing may provide a unique opportunity to study the molecular causes of at least that particular phenotype. The above discussion is admittedly speculative with regard to whether these mouse and human mutants developing ageing-like phenotypes can shed light on the underlying molecular and cellular mechanisms of ageing in non-mutant mice or humans. Obviously not every mouse model that dies prematurely should be assumed to be informative about ageing, or even informative about some segmental aspect of ageing. To decide whether it may be or probably is not, and whether it is worth studying, requires some educated guesses about the role of ageing in the usual development of the phenotype, and whether the mutation is likely to mimic and/or enhance that role or to follow an entirely different pathway in inducing the ageing-like phenotype. It is incumbent on the investigator to make the case for studying any model of ageing, particularly those where ageing or some phenotype of ageing is accelerated. Such an opportunity should not be overlooked simply because the model represents accelerated ageing (reduced longevity) rather than delayed ageing (extended longevity).
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Progress Paper
Models of accelerated ageing can be informative about the molecular mechanisms of ageing and/or age-related pathology Huber R. Warner *, Felipe Sierra Biology of Ageing Program, National Institute on Ageing, Bethesda, MD 20892, USA Received 20 December 2002; received in revised form 24 January 2003; accepted 27 January 2003
Abstract During the past ten years considerable progress has been made in discovering genes that regulate longevity by identifying single gene mutations that lead to increased longevity. The initial success in nematodes was quickly followed by comparable success in fruit flies and mice. In contrast, mutations that cause a decrease in longevity have been largely discounted as unlikely to be informative about aging mechanisms. However, the recent creation of several mutant mouse models that develop a variety of aging-like phenotypes and die prematurely, suggests that such models may be useful in understanding aging mechanisms, particularly as they relate to progressive tissue and organ dysfunction. A possible common feature of these models may be an imbalance between loss of cells by apoptosis and subsequent cell replacement, leading gradually to a net loss of cells in multiple tissues. # 2003 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Accelerated ageing; Molecular mechanisms; Age-related pathology
1. Introduction Ageing is difficult to define unambiguously, and the complexity of the ageing process diminishes the probability that any one theory can adequately explain all or even most of its root causes. Early research on ageing was focused on obtaining data to either support or disprove one or more of the many theories that had been proposed to explain ageing. These theories include both random damage theories (e.g. free radical and DNA damage) and programmed ageing theories (e.g. immunological or endocrine decline), and the concept that some age-related changes may be programmed, whereas, others are stochastic and unpredictable, is now generally accepted. However, research on these various theories has so far failed to divulge the critical mechanisms causing ageing, suggesting the need for new thinking about how best to study and elucidate the biological basis of ageing.
* Corresponding author. Tel.: /1-301-496-4996; fax: /1-301-4020010. E-mail addresses: [email protected] (H.R. Warner), [email protected] (F. Sierra).
1.1. Generalized functional decline leading to homeostatic failure as a unifying theory of ageing Most gerontologists agree that ageing leads to a failure to maintain and/or return to homeostasis after exposure to a stress, no matter what system they happen to be studying. In discussing a recent paper by De Boer et al. (2002) on the role of DNA damage and repair in ageing, Hasty and Vijg (2002) suggest the ‘broad-based hypothesis . . . that generalized homeostatic failure leads to age-related decline’. The ability to successfully resist and survive various kinds of biochemical stresses must be a major factor in the ability of a living organism to maintain homeostasis. Lithgow and Walker (2002) recently reviewed the evidence indicating that stress resistance contributes to longevity regulation in the nematode, Caenorhabditis elegans . Stress resistance and longevity also appear to be associated in the fruit fly, Drosophila melanogaster (Lin et al., 1998). Because both of these organisms are comprised of mostly postmitotic cells, this raises the important question of whether invertebrate and mostly post-mitotic organisms can model the events occurring in mammals, where most organs are regularly undergoing cell death and replacement. Indeed, homeostatic failure in these models might
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imply the inability to maintain an adequate number of functional cells in each tissue during ageing. In turn, this can be regulated at two different levels: the number of cells, and the functional quality of the available cells. Thus, the study of mutant and transgenic mice may provide a more potentially fruitful line of investigation of ageing mechanisms in mammals. In this discussion we will focus on the number of cells existing in the tissue, rather than their functional quality. 1.2. Appropriate animal models for studying ageing Long-lived nematode, fruit fly and mouse mutants have been described and their relevance to ageing mechanisms is currently being assessed. In contrast, because there are so many pathological conditions that appear to have no relevance to ageing but nevertheless shorten life span, many gerontologists hold the view that animal models with reduced life expectancy are of little or even no relevance in studying ageing compared with models with extended life expectancy. This may be particularly true in the case of the invertebrate models of ageing such as yeast, fruit flies and nematodes, because we know almost nothing about the pathology of these organisms and why they die. In contrast to fruit flies and nematodes, we know a good deal about mouse pathology and why mice die (Lipman et al., 1999). Thus, it seems likely that mice prematurely displaying a variety of phenotypes associated with ageing, as well as mice displaying delayed ageing, i.e. extended life expectancy, could well be informative about risk factors for various aspects of human ageing. 1.3. Mouse models of delayed ageing At least seven long-lived transgenic and mutant mice have been described in the literature in the past few years. These include four mouse mutants that either fail to produce growth hormone or to respond to it (Bartke et al., 2001; Flurkey et al., 2001). These mice are all small, and at least three of these mutants have low levels of circulating IGF-I and insulin, suggesting that the signal produced by their insulin signaling pathway might be attenuated. Their longevity is increased by 30/60% in agreement with long-lived nematode (Morris et al., 1996; Kimura et al., 1997) and fruit fly (Tatar et al., 2001; Clancy et al., 2001) mutants having reduced insulin signaling. The most recent addition to this collection of IGF-I level/insulin-signaling pathway mutants is a heterozygous knockout mouse lacking one copy of the gene for the IGF-I receptor (Holzenberger et al., 2003). Females with this Igf -Ir / genotype have an average life expectancy about 33% longer than the wild type, but are not dwarf. They are resistant to paraquat and also have reduced levels of the phosphorylated p66shc protein. Male Igf -Ir / mice are only mildly
long-lived, whereas Igf -Ir / mice show multiple defects, including severe neurological dysfunction. A sixth long-lived mouse contains a mutation in the p66shc gene that increases the ability of mouse cells to resist apoptosis induced by either hydrogen peroxide or ultraviolet light (Migliaccio et al., 1999). The p66shc gene product is part of a signal transduction pathway that is activated by reactive oxygen species, leading to apoptosis (Nemoto and Finkel, 2002). The p66shc gene negatively regulates the transcription factor, FKHRL1, that in turn appears to regulate transcription of the gene coding for catalase. Thus, when the p66shc protein is absent, FKHRL1 remains active and overproduces catalase and possibly other anti-oxidant enzymes, thereby increasing resistance to oxidative stress. The phenotype of this mutant partially overlaps the phenotype of the long-lived C. elegans age-1 mutant that overproduces catalase and superoxide dismutase, but also displays reduced insulin signaling (Larsen, 1993; Morris et al., 1996). Overall, the results with the Igf -Ir / and p66shc mice suggest a causal linkage between insulin signaling, response to oxidative stress and longevity regulation. The seventh long-lived mouse mutant apparently genetically recapitulates the caloric restriction paradigm (Miskin and Masos, 1997). While the above examples demonstrate some concordance between invertebrate and mammalian organisms with regard to longevity regulation, thereby providing support for the concept that invertebrate models may be informative about mammalian ageing, there is no direct evidence available yet to suggest that either the insulin-signaling pathway or resistance to oxidative stress regulates longevity in humans. 1.4. Transgenic mouse models of accelerated ageing On the opposite side of the coin are at least four mouse models that are not only short-lived, but also prematurely display one or more ageing-associated phenotypes. These include mice defective in XPD function (De Boer et al., 2002), mice with abnormal p53 function (Tyner et al., 2002), mice defective in joining DNA double-strand breaks (Vogel et al., 1999), and the Klotho mouse (Kuro-o et al., 1997). The properties of these four mouse models are summarized in Table 1, and the possible relevance of these accelerated ageing mouse models is discussed below with particular reference to putative human premature ageing syndromes. 1.4.1. XPD De Boer et al. (2002) recently created transgenic mice carrying a mutation in the XPD gene that codes for a DNA helicase involved in both repair of DNA damage and transcription. The mutation does not knock out the
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Table 1 Phenotypes of mouse mutants of accelerated ageing Phenotype
Mouse mutant Klotho
Ku80
XPD
p53 /m
Biochemical defect
Unknown 8 /9
Lacks a DNA helicase DNA repair defective B/50
Hyperactive p53
Mean life expectancy (weeks) Control (%) Onset of growth failure Osteoporosis Subcutaneous fat loss Hair Wound healing Cancer risk
Defective repair of DNA double strand breaks 35
96
B/10 3 /4 weeks
35 From birth?
B/50 12 weeks
70 /18 month
Premature onset Premature onset Reduced number of follicles NR NR
Premature onset Premature onset NR NR Occurs earlier, but lower incidence Immune deficiency
Premature onset Premature onset Premature graying NR NR
Premature onset Premature onset Minimal graying Reduced Reduced
Other phenotypes
Arteriosclerosis; premature thymus atropy; abnormal walking behavior
General organ atrophy
NR, not reported.
helicase activity, but significantly reduces it. As a result, these mice have substantially impaired transcription and mildly impaired DNA repair. The phenotype of these transgenic mice includes not only osteoporosis, loss of female fertility, and premature graying of hair, but also reduced life span (median life expectancy B/12 months vs. /24 months for the controls). These mice appear to be normal at birth and remain normal for about 4 months, although they weigh 10 /20% less as early as 2 weeks of age. However, by 6 months of age a variety of defects begin to appear as indicated above. The molecular basis for the age-related onset of the ageing-like symptoms is not known, but De Boer et al. suggest that the failure to repair DNA damage and allow transcription to proceed may trigger programmed cell death, ‘leading to functional decline and depletion of cell renewal capacity’. 1.4.2. p53 A comparable mutant mouse model was produced by Tyner et al. (2002). They reported the unexpected result that deletion of a portion of the p53 gene that codes for the carboxy terminus of the p53 protein can lead to a gain of p53 tumor suppressor function. The p53 protein plays a critical role in regulating cell division, response to environmental stress, and programmed cell death, and more than 50% of human cancers are associated with one or more mutations in the p53 gene. Tyner et al. (2002) reported that mice containing one good copy of the p53 gene, and one of the new mutant copies are very resistant to cancer, but also show premature signs of ageing such as osteoporosis, slow wound healing, muscle and general organ atrophy, and loss of hair and subcutaneous adipose tissue. The life expectancy of these mice is reduced by 20/25%. The authors suggest
that cells in the mutant mice are being lost in multiple tissues faster than they can be replaced. As in the case of the xpd -defective mice, the authors suggest that these mice may be experiencing excessive cell death, thereby prematurely exhausting their stem cell reserves so that ‘sufficient numbers of mature cells cannot be provided to maintain organ homeostasis’. 1.4.3. Ku-80 Another DNA repair deficient model is the Ku-80deficient mouse (Vogel et al., 1999; Lim et al., 2000). The average longevity of this mouse is only 35 weeks, compared with 100 weeks for the control. Besides reduced longevity, this mouse also prematurely develops ageing-associated phenotypes such as osteoporosis, skin atrophy and follicular loss, beginning by the age of about 37 weeks. Cancer also develops prematurely, although at a much lower incidence than in Ku -80/ mice. The Ku-80-deficient mice are also markedly smaller than normal mice, a phenotype observable as early as 2.5 weeks after birth. Hence, the Ku-80 / mice share some of the same phenotypes observed in the p53/m and xpd / mice, and may also be experiencing non-replaceable cell loss. 1.4.4. Klotho The klotho mouse produced by Kuro-o et al. (1997) also displays several ageing-associated phenotypes, including skin atrophy, lipodystrophy, osteoporosis and arteriosclerosis, besides shortened longevity. The mean life span of kl / mice is about 7 weeks, but growth failure begins at about 3/4 weeks. The kl insertion mutation occurs in an unidentified gene and is autosomal recessive. The gene has been cloned and sequenced; it has a transmembrane domain, and shares
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homology with known b-glucosidases. A human homolog has also been identified and has 86% identify with the mouse gene. More recent evidence suggests that the wild-type klotho protein suppresses insulin signaling (Utsugi et al., 2000). Thus, the kl / mouse may have a hyperactive insulin-signaling pathway and its reduced longevity may be due to some of the same factors that reduce longevity in mice in response to over-production of growth hormone (Wolf et al., 1993). The report of a fifth such mouse model is currently ‘in press’ (Wong et al., 2003). According to the authors, their Atm-telomerase-mouse model has a ‘generalized proliferation defect . . . evident in all cell types and tissues examined, and this defect extended to tissue stem/ progenitor cell compartments, thereby providing a basis for progressive multi-organ system compromise, accelerated ageing and premature death’. There are other short-lived mice that could be included in this discussion, including mice deficient in anti-oxidant defense enzymes such as methionine sulfoxide reductase (Moskovitz et al., 2001), and the SAMP mouse strains (Takeda, 1999). The above examples were chosen for discussion here because their development of age-associated phenotypes is well described. The central question about the above studies is whether the shortened longevity observed in these mutant mice has any relevance to ageing in non-mutant mice. Other important questions include: (1) What do these mice die of? (2) What causes the early onset of growth failure in these mouse models? (3) Are the ageing phenotypes observed relevant to development of agerelated pathology in non-mutant mice? and (4) What accounts for the timing of onset of premature ageing phenotypes in these mutant mice? Many gerontologists assume, although it has not been fully unequivocally proven, that cells are constantly being lost at a slow rate due to continual intrinsic damage caused by the generation of reactive oxygen species that accompanies normal mitochondrial metabolism, especially in post-mitotic tissues and cells (Beckman and Ames, 1998). This loss of damaged cells must be constantly balanced by cell replacement either through proliferation of neighboring ‘like’ cells in proliferative tissues such as liver, or by proliferation of stem cells (Fig. 1). When cells reach a state known as cellular or replicative senescence (Hayflick, 1965), they would no longer be able to proliferate to replace lost cells, but only limited evidence is currently available to suggest that such senescent cells actually exist in vivo (Dimri et al., 1995; Hornsby, 2002), and would cause problems if they do (Krtolica et al., 2001). If cell senescence does prevent proliferative repair, then this could lead to loss of tissue homeostasis if an adequate stem cell population is not available to replace cells lost from the tissue. Unfortunately, very little is currently known about the roles of stem cell populations in
Fig. 1. Maintaining tissue homeostasis by repair and/or cell replacement.
maintaining homeostasis, or how these change with ageing. If stem cell populations do become rapidly depleted by excessive cell death as suggested by Tyner et al. (2002) and De Boer et al. (2002), would the phenotypes that result be informative about normal ageing? Although we currently know little about age-related changes in stem cell populations, it might be instructive to consider whether these mice provide opportunities to study the development of age-related pathology on an accelerated scale. The most obvious pathology to consider is osteoporosis, which shows early onset in XPD , p53/m and klotho mutants, as well as in Werner’s syndrome in humans (Martin and Oshima, 2000). Although there may be many factors contributing to osteoporosis, the general model is that bone resorption by osteoclasts exceeds bone deposition by osteoblasts, leading to net bone loss with increasing age. Thus, it would be important to learn whether either the XPD , p53 /m or the klotho genotype affects cell loss and cell replacement rates within the osteoblast and osteoclast populations in mice, and how the ratio of these populations changes with increasing age in these mice. This might be possible if these rates are accelerated in these two mutant strains, and if so, the results might well provide an explanation for the early onset of osteoporosis in these mutant mice. Whether such a result would be informative about osteoporosis in humans would have to be tested directly. A similar opportunity exists for studying tissue/organ atrophy in the p53 mutant where general organ atrophy is observed by 24 months of age (Tyner et al., 2002). Exaggerated rates of cell turnover, with an imbalance between cell loss and replacement, might be measurable in this mutant, and if so would provide at least one explanation for phenotypes occurring during normal ageing, e.g. sarcopenia, slow wound healing, etc. 1.5. Human accelerated ageing syndromes as models for studying ageing The most important question is whether any of these accelerated mouse strains can be informative about
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human ageing. In 1978 and again in 1990, pathologist George Martin analyzed the known genetic syndromes in man that might have potential relevance to the pathobiology of ageing. Although none of these was or should be considered to be a ‘pure’ example of premature ageing, he chose syndromes that display one or more ageing-like phenotypes, such as premature graying of hair, cataracts, cardiovascular disease, diabetes, osteoporosis, neoplasms characteristic of the elderly population and dementias. He called these segmental progeroid syndromes. As a result of this analysis, Dr Martin estimated that up to about 7% of the human genome ‘could play a role in modulating specific aspects of the senescent phenotype in man’ (Martin, 1990). He also suggested that these syndromes would be useful for investigating the molecular, cellular and genetic basis of specific aspects of human ageing, even though the syndrome itself may not be an exact representation of premature ageing. Using these same criteria, the mouse models discussed above might represent bona fide models of murine segmental progeroid syndromes. Three segmental human progeroid syndromes may provide an opportunity for comparative research on various aspects of cell turnover in humans. These are Bloom (BS), Hutchinson /Gilford (HGS), and Werner (WS) syndromes. Table 2 briefly summarizes some of the phenotypes of these three progeroid syndromes. BS and WS are caused by defects in genes for two different DNA helicase activities (Ellis et al., 1995; Gray et al., 1997). These helicase deficiencies may cause the build-up of unresolved replication, repair and/or transcription complexes in cells, leading ultimately to programmed cell death. In contrast, HGS is caused by a mutation at an unique site in the gene for lamin A, a major component of the nuclear envelope (Eriksson et al., in press). Mutations at other sites in this gene are responsible for a variety of pathologies including Emery /Dreifuss muscular dystrophy, skeletal and cardiomyopathy, lipodystrophy, Charcot /Marie /Tooth disorder and mandibuloacral dyslasia (Burke and Stew-
art, 2002). Why lamin A mutations cause these pathologies is not known, but excessive cell death due to aberrant nuclear structure and function appears to be one possibility. In fact, caspases disrupt the nuclear membrane during apoptosis (Faleiro and Lazebnik, 2000) and lamin A is one of the substrates for these caspases (Takahashi et al., 1996). Fibroblasts taken from patients with any of these syndromes have very limited proliferative capacity when cultured, suggesting that many of these cells may have already reached replicative senescence (Campisi, 1996). Although none of these syndromes is associated with growth hormone deficiency, patients with BS are born small, remain smaller than normal through development, and live only into their 20s, while patients with HGS and WS both appear normal at birth, but begin to show growth defects between 1 and 2, and 15/20 years, respectively. It would be interesting to know how the age-related onset of growth defects in these three syndromes is related to the amount of programmed cell death occurring, and the ability to replace these lost cells, in the various tissues in these patients. Such knowledge should also be relevant to understanding the slow loss of tissue mass and function in apparently normal human subjects during ageing, both in general, and as a direct cause of age-related pathology.
2. Discussion Martin and Oshima (2000) argued that studies of human genes in which mutations can lead to accelerated emergence of senescent phenotypes ‘may lead to a clearer understanding of the nature of senescence, and could provide clues for ways in which ageing might be retarded’. Recent papers on the development of some accelerated ageing-like phenotypes in several transgenic mouse strains, as well as the molecular characterization of some of the human segmental progeroid syndromes, suggest a need to include tissue dynamics involving programmed cell death, cell proliferation, and stem cell
Table 2 Comparison of several human progeroid syndromes Phenotype
Biochemical defect Mean life expectancy (years) Normal (%) Onset of growth failure Osteoporosis Subcutaneous fat loss Slow wound healing Cancer risk
585
Human syndrome HGS
BS
WS
Lamin A 13 17 Year 2 No? Yes ? Normal?
Lacks a DNA helicase 25 33 In utero No? ? ? Elevated
Lacks a DNA helicase 50 67 Year 15 /18 Premature onset Yes Yes Elevated
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replacement capacity in this context (Fig. 1). The experimental evidence obtained thus far suggests that a better understanding of the factors directly affecting the ability to maintain tissue cell number and function will provide a better conceptual framework for future research aimed at understanding the root causes of ageing, and this could lead to interventions to reduce frailty in the elderly. The suggestion that cells from these mouse and human mutant models are especially vulnerable to apoptosis can be directly tested as apoptosis can be studied in in vitro systems (Lazebnik et al., 1993). When multiple phenotypes of ageing are delayed by a given mutation, such as the dwarf dw /dw mutants studied by Flurkey et al. (2001), we may be most comfortable that we are truly studying ageing per se. However, limiting our study only to animal models that delay the development of ageing phenotypes seems too restrictive. We believe it is unwise to automatically discard as irrelevant animal models that accelerate the appearance of one or more ageing-like phenotypes, and in so doing may provide a unique opportunity to study the molecular causes of at least that particular phenotype. The above discussion is admittedly speculative with regard to whether these mouse and human mutants developing ageing-like phenotypes can shed light on the underlying molecular and cellular mechanisms of ageing in non-mutant mice or humans. Obviously not every mouse model that dies prematurely should be assumed to be informative about ageing, or even informative about some segmental aspect of ageing. To decide whether it may be or probably is not, and whether it is worth studying, requires some educated guesses about the role of ageing in the usual development of the phenotype, and whether the mutation is likely to mimic and/or enhance that role or to follow an entirely different pathway in inducing the ageing-like phenotype. It is incumbent on the investigator to make the case for studying any model of ageing, particularly those where ageing or some phenotype of ageing is accelerated. Such an opportunity should not be overlooked simply because the model represents accelerated ageing (reduced longevity) rather than delayed ageing (extended longevity).
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