Genetics of longevity

Experimental Gerontology, Vol. 33, Nos. 7/8, pp. 773–783, 1998 Copyright © 1998 Elsevier Science Inc. Printed in the USA. All rights reserved 0531-5565/98 $19.00 1 .00

PII S0531-5565(98)00027-8

GENETICS OF LONGEVITY

S. MICHAL JAZWINSKI Department of Biochemistry and Molecular Biology, Louisiana State University Medical Center, 1901 Perdido St., Box P7-2, New Orleans, Louisiana 70112

Abstract—Recent studies on the genetics of aging in the yeast Saccharomyces cerevisiae, the roundworm Caenorhabditis elegans, and the fruit fly Drosophila melanogaster have converged revealing the central role of metabolic capacity and resistance to stress in determining life span. Signal transduction has emerged from these studies as an important molecular mechanism underlying longevity. In their broad features, the results obtained in these genetic models are applicable to the dietary restriction paradigm in mammals, suggesting a general significance. It will be of interest to determine whether many of the molecular details will also pertain. The examination of centenarian populations for the frequency of certain alleles of pertinent genes may provide insights into the relevance of the conclusions of studies in invertebrates to human aging. These population genetic studies can be augmented by mechanistic studies in transgenic mice. © 1998 Elsevier Science Inc. Key words: Saccharomyces cerevisiae, Caenorhabditis elegans, Drosophila melanogaster, dietary restriction, longevity genes

INTRODUCTION THE GENETIC ANALYSIS of aging has seen unprecedented progress in the past few years. This development has been fueled to a great extent by studies in invertebrate genetic model systems. This is not surprising, given the facility of genetic analysis in lower eukaryotes. However, there are more subtle reasons underlying this situation. Despite much effort, there are no reliable biomarkers of aging in hand in any organism. Thus, the best predictor of mortality remains life span itself. As a consequence, the genetics of aging becomes the genetics of longevity. The end-point assay is life span. This in itself has additional virtues. The question that is addressed is the biological basis of life maintenance, rather than hereditary factors contributing to disease. Another major reason for the special utility of invertebrates is seen in two of the premier genetic models, Saccharomyces cerevisiae (yeast) and Caenorhabditis elegans (roundworm). Yeasts are clonal, and roundworms are obligate inbreeders (hermaphrodites). This renders them naturally inbred. Perhaps not coincidentally, major, single-gene effects on longevity have been easily

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found in these species (reviewed in Jazwinski, 1996). The third major invertebrate model, Drosophila melanogaster (fruit fly), has not been exploited as yet in this fashion; however, there are no fundamental reasons why this cannot happen (Pletcher et al., 1998). Thus far, the achievements in the fruit fly are the result of artificial selection studies (reviewed in Jazwinski, 1996). The results of genetic studies on aging in invertebrates have been exciting indeed, but it is not clear a priori that any of the information obtained is applicable to mammalian aging, especially human aging. It is necessary to verify the results obtained in mammalian systems. Obviously, advances in the genetics of aging in mice will be instrumental. However, these are only in their early stages. In the interim, there are four other available verification schemes. First, a comparison of the data and conclusions of studies across the invertebrate models can be illuminating. Given the large phylogenetic distances separating the yeast, the worm, and the fruit fly, it is hard to imagine that broad biological principles will not be gleaned from studies using these models (Jazwinski, 1996). Second, the homologues of the genes identified as determinants of longevity in each of the three species can be cloned from the other two species. This can serve as a tool to generate transgenics either missing the homologue or overexpressing it in the species from which it was cloned. This would serve as a direct test for analogous function and bolster the conclusions from the comparative studies mentioned above. Third, the same principles can be applied to generate transgenic mice. Obviously, a positive result in such a study would constitute direct proof of conservation of longevity gene function in mammals. Fourth, population genetic studies can be carried out in humans, using human homologues of the invertebrate longevity genes as probes. To date, only limited associative genetic studies on aging have been carried out in humans, and these have focused on potential disease risk factors (reviewed in Jazwinski, 1996). It is clear that the third and fourth schemes listed above, though most definitive, are also the most arduous. There is, however, one more verification alternative. This involves comparison of the invertebrate findings with those emerging from dietary restriction studies in rodents (Richardson and Pahlavani, 1994; Masoro, 1995). One human experimental model that will not be discussed in any detail here is the cellular senescence paradigm (reviewed in Smith and Pereira-Smith, 1996). There is still much discussion as to the applicability of this model to aging in vivo, although recently it has become more muted. Recent studies have revealed the major factor involved in cessation of cell proliferation in normal human cells. This is the absence of telomerase activity and the attendant erosion of chromosomal telomeres (Bodnar et al., 1998). Senescent cells remain alive for prolonged periods of time in culture, and they appear to be phenotypically different from “young” cells that have not exhausted their proliferative capacity. These differences could constitute contributory factors to aging in vivo, as such cells can be detected, albeit infrequently, in tissues. A major outstanding issue is the lack of aging phenotypes in transgenic mice lacking telomerase activity (Blasco et al., 1997), which has been rationalized to be due to the larger size of murine telomeres. Certainly, the transfer of findings in the cellular senescence model to aging in vivo has as much distance to span as the transfer of discoveries from invertebrate models. The studies in the invertebrate models at least identify factors limiting for longevity in an organism. In studies with isolated cells, this stricture is no longer applicable. Unless an intrinsic aging program is postulated, for which evidence and evolutionary rationale is lacking, the cells in culture are free to respond to the selective forces in culture. Relevance to the topic of this symposium requires an additional consideration. Nervous tissue is postmitotic in adult life. The experimental models described above involve postmitotic in some cases and mitotic cells in other cases. This does not mean that some of the determinants

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of aging are not identical in both cases. However, some differences are to be expected. Clearly, telomere shortening will not be an issue in nervous tissue, because it is dependent on the DNA replication that is necessary for cell division. Just as dependent on replication is the accumulation of extrachromosomal ribosomal DNA circles in yeast (Sinclair and Guarente, 1997). The fruit fly and the roundworm consist of postmitotic cells if one ignores the gonads. Yeast life span is measured by the number of divisions individual cells undergo. Certainly, the first two organisms may model postmitotic nervous tissue. Surprisingly, as will be seen, the same is true of yeasts. The genetic analysis of aging has revealed four processes that are important for determining life span. These are metabolic capacity and efficiency, stress responses, integrity of gene regulation, and genetic stability (Jazwinski, 1996). The significance of the first two of these for aging of nervous tissue is particularly acute. This discussion will, therefore, focus on these two parameters. One other question of general importance that may be asked at this point concerns the relative contributions of genes and environment to longevity. This is a difficult question, and it depends not only on the organism concerned but also on how the question is posed. The answer in invertebrate models can be as high as just under 50%. In humans, it has been recently estimated to be about 35% (Finch and Tanzi, 1997). Metabolic capacity The genetics of aging began in earnest when fruit flies displaying postponed senescence were artificially selected in the laboratory from wild outbred lines (Luckinbill et al., 1984; Rose, 1984). These experiments proved that genes determine life span, and subsequent analysis showed that longevity is a polygenic trait. Quantitative genetic analysis is now being applied to determine the genes involved. However, most of what we currently know is derived from physiological analysis of the long-lived lines that were obtained. The artificial selection resulted, as it were, in the gathering together in the long-lived lines of alleles extant in the starting population that were conducive to long life. These alleles together rendered the long-lived flies physiologically distinct from the randomly bred control lines. The long-lived flies showed marked metabolic differences compared to the controls. These included higher glycogen and lipid stores (Service et al., 1985; Service, 1987; Graves et al., 1992; Arking et al., 1993; Dudas and Arking, 1995). Underlying these changes in energy reserves, there were marked enzymatic and metabolic differences. Flies displayed changes in glucose-6-phosphate dehydrogenase isoforms, which are associated with increased flux through the pentose phosphate pathway and could result in the accumulation of glycogen and lipid (Luckinbill et al., 1989). Furthermore, they showed more efficient nutrient use (Riha and Luckinbill, 1996). These changes were associated with greater flight frequency and flight duration (Graves et al., 1988). The long-lived flies had an expanded metabolic capacity at any temperature, as measured by lifetime oxygen consumption and egg production (Arking et al., 1988). Recombinant-inbred strains (RIs) have been generated in C. elegans that show a variety of life spans, both shorter and longer than the parent strains (Johnson, 1987). These strains lose motor activity at a rate inversely proportional to their longevity; thus, they display a metabolic capacity that increases with their longevity. In both the fruit fly and the worm long-lived lines and strains, extension of life span is associated with an expanded metabolic capacity. An exciting avenue of investigation into the mechanisms of aging began with the discovery that the daf-2 gene of C. elegans determines adult longevity (Kenyon et al., 1993; Larsen et al.,

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1995). This gene and other daf genes are involved in dauer larva development. The dauer is an alternate larval state that arises under stressful environmental conditions, such as starvation, heat, or crowding. It is a dispersal form that allows the worm to survive through a profound reduction in motility and total food abstinence. When conditions become auspicious, the worm resumes development and becomes an adult. Various daf mutants either prevent dauer formation or make it constitutive. The daf-2 mutants form dauers at elevated temperature. When raised at normal temperature, they have a normal life span. If the temperature is elevated after they become adults, they exhibit a twofold increase in longevity with no overt signs of dauer formation. (It still remains to be determined what features of the dauer may be preserved.) The fact that the dauer developmental pathway is involved comes from the fact that the daf-16 dauer gene, which lies downstream of daf-2, must be present and active for the life extension to be observed. This was the first demonstration of a pathway involved in determining longevity. Historically, the first gene to be implicated in longevity was the worm gene age-1, identified by mutation (Klass, 1983; Friedman and Johnson, 1988). This gene was shown to reside in the daf-2/daf-16 pathway for longevity, and the mutant was found to be a weak dauer mutant (Larsen et al., 1995; Dorman et al., 1995). The gene was finally cloned as the daf-23 gene (Morris et al., 1996), which had previously been shown to be part of the daf-2/daf-16 pathway for longevity (Larsen et al., 1995). The daf-23/age-1 gene encodes a phosphatidylinositol-3-OH kinase (Morris et al., 1996), which is clearly a signal transduction protein. This story has recently become more complete with the cloning of daf-2 (Kimura et al., 1997) and daf-16 (Lin et al., 1997; Ogg et al., 1997). The former encodes an insulin receptor/IGF-1 receptor homologue, while the latter codes for a protein that belongs to the forkhead family of transcription factors that includes such proteins as hepatic nuclear factor-3 (HNF-3). The involvement of an insulin receptor-related protein immediately suggests important metabolic consequences of daf-2 signaling. HNF-3 has been implicated in insulin regulation of metabolic gene transcription (Lai et al., 1990). In mammals, insulin regulates the phosphoenolpyruvate carboxykinase and Glut4 glucose transporter genes transcriptionally through HNF-3 (O’Brien et al., 1995). Many of the effects of insulin are the result of its antagonistic effect on HNF-3. This is analogous to the effect of daf-2 on daf-16. Among daf-16–mediated responses is glycogen and fat storage (Ogg et al., 1997). In addition to these effects at the transcriptional level, the daf-2 pathway is likely to exert its effects on metabolism at the protein level as well. Phosphatidylinositol-3-OH kinase is involved in the regulation of metabolic enzyme activities and glucose transport, acting through AKT/PKB kinase (Tanti et al., 1996; Toker and Cantley, 1997; Hemmings, 1997). Activation of the daf-2 pathway for life extension induces a broad array of enzymatic changes that signal a shift from utilization of the Krebs cycle to the glyoxylate cycle (Vanfleteren and DeVreese, 1995). This may signal changes in glycogen and lipid metabolism and the mobilization of acetate. There remain many details that must yet be fleshed out. However, it is already obvious that signal transduction from the cell surface to the nucleus plays an essential role in the determination of life span (Fig. 1). The genetic analysis of aging in yeast leads to similar conclusions to those that have emerged from the worm studies. Yeasts display a finite replicative capacity; that is, individual cells can only divide a limited number of times before they die (Mortimer and Johnston, 1959; Muller et al., 1980). However, the daughter cells that they produce have the potential for a full life span. Thus, individual yeasts are mortal, but the entire population or culture is immortal. As they proceed through their life spans, yeasts show a variety of changes (Jazwinski, 1993), and they exhibit an exponential increase in mortality rate with age (Pohley, 1987, Jazwinski et al., 1989).

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FIG. 1. The C. elegans daf-2 pathway for longevity. Binding of the putative DAF-2 ligand to its receptor DAF-2, an insulin receptor homologue, is proposed to result in the autophosphorylation of the receptor. This attracts the phosphatidylinositol-3-OH kinase AGE-1 to bind resulting in the production of PIP3 , which leads to activation of the kinases AKT and GSK-3. These enzymes are proposed to regulate metabolic enzymes and glucose transport and to inhibit the forkhead transcription factor DAF-16, which regulates the expression of metabolic genes. (Consult further Kimura et al., 1997; Lin et al., 1997; Ogg et al., 1997).

Close to a dozen genes that determine yeast longevity have been identified (reviewed in Jazwinski, 1996). One of the yeast longevity genes is RAS2 (Chen et al., 1990; Sun et al., 1994), which encodes a homologue of the mammalian signal transduction protein c-H-ras (Barbacid, 1987). Overexpression of RAS2 extends life span, while deletion curtails it. This gene impinges upon many

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FIG. 2. The role of the Ras2 pathway in yeast cellular homeostasis. Ras2 senses the nutritional status of the cell, and it processes internal metabolic signals. It also modulates the response to environmental and internally generated stress, such as oxidative stress. Ira1 and Ira2 are negative regulators of Ras2, and Cdc25 is a positive regulator. Ras2 signals downstream events either through adenyl cyclase or a MAP kinase pathway that includes Cdc42, Ste20, Ste11, Ste7, and Kss1. There may be other signal transduction pathways in which it participates. The outputs of these Ras2 pathways regulate metabolic activity, cell proliferation, and response to stress.

physiological processes (Tatchell, 1993). One of its main roles is as part of a nutrient sensor. Overexpression of RAS2 not only extends life span, but it also postpones senescence, as measured by the effect on the age-dependent increase in cell generation time (Sun et al., 1994) and loss of cell polarity (Jazwinski et al., 1998). RAS2 functions in at least two separate signal transduction pathways (Fig. 2). One of these involves stimulation of adenylate cyclase (Tatchell, 1993), and the other is a classical MAP kinase pathway (Roberts et al., 1997). These pathways regulate metabolic activity and cell proliferation; they also modulate response to stress. It is obvious, by definition, that extension of life span involves expansion of metabolic capacity in yeasts. This is because the measure of life span is the number of cell divisions or daughter cells produced. Yeast life span can also be prolonged by a petite mutation (P.A. Kirchman, S. Kim, and S.M. Jazwinski, unpublished). Petites are yeasts that lack fully functional mitochondria. The life extension is not due to elimination of respiratory activity or oxidative stress. Instead, it is caused by the activation of a signal transduction pathway involving interorganelle communication

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between the mitochondrion, the nucleus, and the peroxisome. The downstream targets of this pathway include transcription factors belonging to the bHLHZip family, which have numerous homologues in mammals. These transcription factors regulate the levels of numerous metabolic enzymes. The net effect is to shift from the use of a calorically rich carbon source (glucose) to ones that have a lower caloric content. The worm longevity gene clk-1 is a homologue of the yeast COQ7 gene, a regulator of mitochondrial activity (Ewbank et al., 1997), further merging the conclusions from studies in the two organisms. There are many similarities between the mechanisms of life extension described above for worms and yeasts and the dietary restriction paradigm. Dietary restriction not only extends life span, but it also postpones many of the changes associated with aging in mammals (Richardson and Pahlavani, 1994; Masoro, 1995). An important feature of dietary restriction is the lowering of blood glucose and insulin levels, among a variety of hormonal changes. Restricted animals use as much glucose and oxygen on a per-weight basis as ad libitum-fed animals. Thus, they have adapted to efficient functioning on a diet of lower caloric content. This resembles the situation in petite yeasts. The analogies with the daf pathway are obvious, considering the life extension obtained by attenuation of daf-2 signaling. Stress resistance The expansion of metabolic capacity associated with life extension takes in tow the hazards of enhanced oxidative stress due to increased oxidative metabolism. Thus, the response to oxidative stress can be considered a primary stress response. The relationship between resistance to various stressors is bolstered by the fact that a variety of stress responses in yeast are controlled at the transcriptional level through the stress response regulatory element or STRE (Marchler et al., 1993). This includes the response to oxidative stress to which the inflammatory response is related in mammals through p38 kinase (Han et al., 1994), a homologue of the Hog1 kinase in yeasts. The p38 and Hog1 kinase belong to MAP kinase pathways that are responsible for stress resistance in mammals and yeast, respectively. As mentioned earlier, RAS2 controls stress responses in yeast. One way in which this is effected is by the modulation by Ras2 of the response from the STRE (Marchler et al., 1993). Further evidence for the significance of resistance to stress in determining life span comes from the isolation of yeast mutants with extended longevity by selection for resistance to starvation and cold stress (Kennedy et al., 1995). The significance of the involvement of RAS2 in stress responses for determination of yeast longevity can be seen in the response to ultraviolet radiation (UV). RAS2 is required for resistance to UV (Engelberg et al., 1994). Resistance to this stressor displays a biphasic profile as a function of age that parallels the expression of RAS2 during the yeast life span (Kale and Jazwinski, 1996). Mobilization of stress responses can have a salutory effect on yeast longevity. Induction of thermal tolerance by subjecting yeasts to a sublethal temperature reduces the mortality rate approximately 10-fold (S. Shama, C.-Y. Lai, and S.M. Jazwinski, unpublished). This effect is transient, and the mortality rate returns ultimately to that found in the control. The heat-shock protein Hsp104 is required for this life extension, as it is for induction of thermal tolerance. The age-1/daf-23 mutant is resistant to oxidative stress, notably in old age (Larsen, 1993; Vanfleteren, 1993). This is associated with increased levels of catalase and Cu, Zn-superoxide dismutase. Thus, increased longevity is correlated with enhanced resistance to oxidative stress. This mutant and the daf-2 mutant also display enhanced thermal tolerance (Lithgow et al., 1994;

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1995). Furthermore, induction of thermal tolerance extends life span in C. elegans (Lithgow et al., 1995). It has been shown that the daf-2/daf-16 pathway is not only responsible for adult longevity, but it also regulates resistance to thermal stress and UV (Murakami and Johnson, 1996). All of these studies provide ample evidence that in the worm, extended longevity is associated with a commensurate increase in stress resistance. The situation in the fruit fly is very similar with regard to stress and longevity. The flies selected for postponed senescence show enhanced responses to starvation, dessication, heat, and ethanol (Service et al., 1985; Service, 1987; Graves et al., 1992) in the case of one set of lines. Further selection for resistance to starvation and dessication resulted in additional gains in longevity (Rose et al., 1992). Another set of lines shows enhanced resistance to oxidative stress associated with elevated levels of antioxidant enzymes (Arking et al., 1993; Dudas and Arking, 1995). The mortality rate of fruit flies can be transiently lowered, much as in yeast, by induction of thermal tolerance (Khazaeli et al., 1996). This enhancement of life span is proportional to the dosage of the Hsp70 gene (Tatar et al., 1997). The life span of the fruit fly has been prolonged by the overexpression of Cu, Zn-superoxide dismutase and catalase, and this extension is correlated with a reduction in oxidative damage to proteins (Orr and Sohal, 1994). Together, all of these studies clearly show that resistance to stress goes hand in hand with longevity. Given the results in the invertebrate model systems, it is not surprising that a correlation between enhanced resistance to stress and longevity is found in dietarily restricted animals. Dietary restriction has been found to maintain the levels of antioxidant enzyme activities late in life (Xia et al., 1995). It has also been found to enhance the resistance of older animals to heat stress (Heydari et al., 1993). Furthermore, dietary restriction has been found to increase glucocorticoid levels (Sabatino et al., 1991). One hypothesis that might explain the significance of this fact is that the elevated glucocorticoid is associated with a sublethal stress response that has a life span-enhancing effect similar to that found in yeast, worms, and fruit flies. Human genetics of aging A variety of studies has been carried out on the association of certain alleles of various genes with longevity, using centenarian populations. All of these studies suffer from a lack of replication in other populations, save for the analysis of the distribution of the APOE gene with longevity. As mentioned earlier, the choice of genes for analysis has resided in the assumption of various risk factors for disease, particularly age-related disease. The study that has gained the most attention was the French centenarian study (Schacter et al., 1994). This study associated the apoE4 isoform with shortened life span and the apoE2 isoform with longevity. This association is perhaps not unexpected, because the apoE4 isoform has been implicated in both heart disease and Alzheimer’s disease (Davignon et al., 1988; Strittmatter et al., 1993). Various scenarios can be sketched out for the significance of the apoE4 isoform in these disorders. Both diseases possess certain features characteristic of inflammation, which is related to the response to oxidative stress by virtue of at least one common signal transduction pathway, as noted earlier. The APOE study has been replicated in a Finnish population (Louhija et al., 1994). Postmitotic tissues such as the brain are highly dependent on oxidative metabolism. This means that mitochondrial function must remain intact throughout life. The danger of oxidative stress is first localized to the mitochondrion, whose genome can suffer mutations and deletions during aging (Cortopassi and Arnheim, 1990; Wallace, 1992). It is not really clear how much impact on tissue function these deletions carry. In the filamentous fungus, Podospora anserina, the degeneration of the mitochondrial genome can be devastating, and it is the cause of aging

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(Osiewacz and Hermanns, 1992). One might expect that certain human mitochondrial genomes might be more prone to the types of loss described here, and that this would have an impact on longevity. In fact, certain mitochondrial haplogroups have been associated with longevity in a Japanese population (Tanaka et al., 1998). The human genetics of aging field is wide open. There is much to be done. Disease risk factors will undoubtedly continue to be of great interest. For example, one allele of the Werner’s gene, which is a gene involved in a premature aging syndrome in humans, is associated with an increased risk of cardiac disease (Ye et al., 1997). However, it will be of particular interest to test candidate genes developed in invertebrate model systems for their role in human aging. Acknowledgments—The work in the author’s laboratory was supported by grants from the National Institute on Aging of the National Institutes of Health (U.S.P.H.S.) and by the Glenn Foundation for Medical Research.

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