Aging, life span, genetics and the fruit fly

Clinical Neuroscience Research 2 (2003) 270–278 www.elsevier.com/locate/clires

Aging, life span, genetics and the fruit fly Stephen L. Helfanda,*, Sharon K. Inouyeb a

Department of Genetics and Developmental Biology, MC 3301, University of Connecticut Health Center, 263 Farmington Avenue, Farmington, CT 06030, USA b Department of Internal Medicine, Yale University School of Medicine, Yale New Haven Hospital, 20 York Street, Tompkins 15, New Haven, CT 06504, USA (203) 688-7302

Abstract Although the process of aging has been the subject of intense study for many years we still know very little about it. Recent studies from diverse fields including molecular genetics, clinical epidemiology and demography have begun to challenge the validity of some of the most basic assumptions about the aging process. These challenges have lead to a reexamination and a revitalization of the field of aging research. The pace of biomedical science has greatly accelerated, in large part due to the use of model organisms such as Drosophila. The conservation of molecular and physiological systems shared between humans, mice, flies and nematodes has allowed for the rapid testing of aging theories, translation of information between these model organisms and the development of general principles for understanding the process of aging. q 2003 Elsevier Science B.V. All rights reserved. Keywords: Aging; Life span; Senescence; Drosophila melanogaster; Genes; Molecular genetics

individual has to make, and although, we have many psychological expedients to blunt its impact, the fact of this effective fixity of life-span, and of the decline in activity and health which often determine it, is always in the background of the human mind.

1. Introduction 1.1. Are aging myths obstacles to research? The knowledge that every one of us will someday die is a central part of our existence as human beings and perhaps the most powerful force shaping our actions and behaviors throughout our lives. If we kept throughout life the same resistance to stress, injury and disease, which we had at the age of 10, about one-half of us here today might expect to survive in 700 years’ time (10 –15% live past 2000 years). The reason that we cannot is that in man, and in many, but probably not all, other animals, the power of self-adjustment and self-maintenance declines with the passage of time, and the probability of disease and death increases. The increase in man becomes eventually so steep that while exceptional individuals may outlast a century, there is an effective limit, depending upon our present age, upon the number of years for which any of us can reasonably expect to go on living. The uniformity of this process is one of the earliest unpleasant discoveries, which every * Corresponding author. Tel.: þ1-860-679-4200; fax: þ 1-860-679-8345. E-mail addresses: [email protected] (S.L. Helfand), [email protected] (S.K. Inouye).

Alex Comfort ‘Ageing: The Biology of Senescence’ (1979) [1]. Why is it that we do not keep the same power of resistance that we had at age 10? How is it that disabilities accumulate and general frailty increases with time? And what can we do about it? These are some of the questions that have haunted people from the beginning of human understanding of our mortality. Answers have been sought from such disparate areas of human thought as religion and science with widely different outcomes and successes. Scientific explanations of the process of aging have acquired an almost commonsensical aura. Results from recent studies in disciplines as diverse as molecular genetics and biodemography are beginning to challenge many of these commonly-held assumptions on aging that underlie and direct research on these important issues. Many of these assumptions, which have served for years as guides for aging research, may also serve as obstacles to other areas of research that could prove to be equally important and productive.

1566-2772/03/$ - see front matter q 2003 Elsevier Science B.V. All rights reserved. doi:10.1016/S1566-2772(03)00003-3

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2. Reported findings and analysis by topics 2.1. Is a decline with age an inevitable part of life? A longstanding tenet is that a decline in abilities is an unavoidable effect of the aging process [2,3]. It is assumed that the benefits of multicellular life and sexual reproduction come at a cost that takes the form of a decline in physiological function with age. If the decline in our abilities with age, senescence, is an inevitable consequence of all multicellular life and a fundamental law of biology then what hope can we have that research will find a way of halting or delaying its onslaught? Finding organisms that do not show a decline in activity and vitality with age might refute this commonly held assumption, dramatically change the way we look at age-related decline, and provide a means of understanding, through comparative analysis, what is it that leads to age-related declines and what we can do about it. In his comprehensive review of the field, Caleb Finch, lists a number of species from different phyla that show no obvious loss in function with age [4] and defines a class that he refers to as gradual senescence [5]. These include tortoises and turtles, rockfish, lobster, clams, and a number of plant species. Some of these organisms such as the Rockfish, Sebastes aleutianus, have been found to live exceptionally long, over 200 years [6]; and the oldest threetoed box turtle, Terrapene Carolina triunguis, which have been found in the wild are still fertile at 70 years of age [7]. These examples of lack of functional decline with age, or at least gradual senescence, suggest that senescence is not an inevitable law of biology. In humans as well it has been suggested that age by itself does not cause functional disability [2,8,9]. Studies in older humans have shown that if factors associated with disease and other impairments are removed from the analysis, then the age of the individual no longer contributes independently to poor outcomes. Studies of functional disability have furthermore shown large variability across agematched populations and more recently a general decrease in the rate of functional decline. From this it may be inferred that although a decline with age or senescence appears to be a common part of our lives, both organisms with exceptionally slow rates of decline, and improvement in rates of decline in humans, suggest that it is not an inevitable consequence of life, and a re-examination of its causes may be justified and potentially rewarding. 2.2. Is adult life a period of passive decline or dynamic well-regulated change? Among the many different assumptions held concerning aging and senescence is that adult life is a period of passive decline. From evolutionary principles comes the conclusion that after the period of peak reproduction, the force of selection will decline, and things will deteriorate, and do so in a random passive manner [4]. Biochemical and molecular

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studies document a general decline in the synthesis of many different macromolecules with age, most notably RNA and protein [4]. The conclusion drawn from these studies is that as life progresses the ability to make macromolecules is continuously lost and this ever-increasing erosion in the reliability of macromolecular synthesis leads to the phenomena we see as age-related decline or senescence. This idea that the adult portion of life is one of passive decline has recently come under review as more sophisticated techniques for examining changes in the expression of macromolecules have emerged. These studies have demonstrated that the molecular life of aging mammals and insects may be better characterized as one of dynamic wellregulated change. In mammals many different species of mRNA and protein show dramatic increases as well as decreases with age in vivo and in vitro [10]. The work of Roy and his colleagues have demonstrated that the androgen receptor in the rat liver shows a complex triphasic pattern of regulation of expression over the life of the adult rat. The mRNA for the androgen receptor increases, plateaus and then shows a late period of decline in the liver. A combination of cis and trans-acting factors has been identified to conspire to cause this stereotypic temporal pattern of change with age [11,12]. In Drosophila melanogaster the use of reporter genes and enhancer-trap lines has allowed the visualization of changes in the expression of single genes at the level of individual or small subsets of cells [13 –16]. The precision afforded by these techniques has shown that the expression of many different genes changes in stereotypic patterns with age. Some genes increase their level of expression with age while others decrease expression. These temporal patterns of expression can be quite complex. In addition to the dynamic nature of change, what may be even more exciting is that these changes appear stereotypic. Alterations in the level of gene expression for a particular gene show a signature pattern that is reproducible from animal to animal [13]. Gene expression during adult life is not only dynamic but it is carefully regulated, similar to what is seen during development. Recent studies using microarrays have also shown that there are a number of genes in both Drosophila, mice, and monkeys that are changing in a dynamic and wellregulated manner during aging [17 –21]. The well-regulated age-dependent pattern of expression seen with a number of genes may also be useful as biomarkers of aging. Other studies have shown that the ability to synthesize macromolecules appears to be preserved all throughout life in Drosophila melanogaster [22]. In light of these findings, the decrease in bulk synthesis of RNA and protein previously demonstrated probably reflects an active selective regulation of gene expression and not a passive loss of macromolecular expression resulting from a debilitation of the machinery necessary for synthesis. Since the functional decline seen with aging is not due to an unremitting accelerating loss of the macromolecular machinery of life, it opens up the possibility that specific changes are responsible

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for senescence and aging. The goal of aging research may begin to emphasize the identification of changes that are causally related to the decline in activity and vitality of the organism with age. Taken together, results from many different species are beginning to suggest that adult life, far from being a condition of biological stasis, is a dynamic ever-changing part of the overall life history of an animal. While the dramatic stereotypic changes that occur during embryogenesis are obvious, subtler, but no less characteristic, changes occur all throughout life. In humans, milestones such as puberty, menopause, and ‘metabolic’ changes appear at particular times in life and mark age-related events. This is graphically illustrated by the term ‘premature graying’ of the hair. The use of the term premature implies that there is a normal time during adult life at which the transition to gray hair should occur. Sequential characteristic age-related changes have been observed in most species. The demonstration of well-regulated change, however, does not mean that the process of aging need be a directed one. Few would believe that aging is a process directed at creating functionally impaired old organisms. It does suggest though that rather than an exceedingly complex and incomprehensible process, the finding of characteristic well-ordered change provides us with the opportunity of exploring the mechanisms behind these specific changes, and the possibility of intervening by delaying the onset, timing and pace, of these changes.

But is it true that everything falls apart together? An alternative, that only certain things are declining, would dramatically change how we look at the aging process and the emphasis of aging research. Physicians have known for years, as many of us have observed, that in humans not all organ systems fail at the same time. The report of a dramatic extension in the life span of fruitflies with the selective expression of SOD in motorneurons [23] shows that the maintenance of some physiological systems is more critical to life span extension than others. These observations suggest that a global loss of homeostasis may not be a major feature of the aging process. Further data against the idea of a global loss of homeostasis with aging was found by assessing the regulation of gene expression with age. Examination of the level of expression of six different genes, throughout adult life in Drosophila melanogaster, demonstrated no evidence of a loss of gene regulation with age [24]. Taken together, the findings of life span extension with selective SOD overexpression, and preservation of gene regulation with age, cast doubt on the notion that all physiological functions decline with age. Aging may be characterized by the loss of some kinds of homeostasis but not by a pervasive loss of all homeostasis. Thus, rather than having to give up at the enormity of the problem, that all things are falling apart together, the identification of specific elements that may be involved in controlling the overall process of aging, would surely lead to fruitful information.

2.3. Do all things fall apart together—at the same time?

2.4. Is aging associated with an ever-increasing rate of mortality?

Whether adult life is one of active regulated change or not it is nonetheless still true that, for most organisms, such as ourselves and fruit flies, we can expect a finite number of years of life and a decline in our facilities as we age. What are the mechanisms behind this decline and what if anything can we do about it? A rational approach to this question might involve a description of the functional decline. In the field of aging research it appears that how we describe the functional decline has profound influences upon the types of possible mechanisms entertained, and in turn, the direction of investigations to be considered. It is commonly believed for example, that aging is associated with a global decline in homeostasis and loss of all physiological functions. This has been assumed for years and is portrayed in the Oliver Wendell Holmes poem ‘The Deacon’s Masterpiece’ about the description of a one-horse, horse-drawn carriage that is perfectly built and falls apart all together and at once. “How it went to pieces all at once, All at once, and nothing first, Just as bubbles do when they burst.” The Deacon’s Masterpiece Oliver Wendell Holmes

One of the central tenets of aging research is the demographic demonstration that there is a steady increase in mortality with increasing age. First demonstrated by Benjamin Gompertz, an actuary, in 1825, as an exponential increase in mortality with age, and referred by him as ‘the law of mortality’ it has largely defined the field of aging research since that time [25]. The so-called Gompertzian curve has for example, provided the scientific underpinning for the common-sensical notion of species specific life spans, which indicate that there is an inherent limit to the life span of each species. More simply, dogs live 15– 20 years and not 80 years, mice live 2– 3 years and not 50 years. The demographic support for the species-specific life span comes from the exponential nature of Gompertzian curve. If mortality rates increase exponentially then at some point, determined by the slope of the mortality curve, a time will be reached where no further members of that species would statistically be expected to survive. In a series of studies, Vaupel and associates have demonstrated that the mortality curve is not exponential in nature but shows a late life plateau in mortality [26 – 28]. In fact, in several species, including medflies, fruitflies, nematodes, and humans, a decline in mortality rate has been seen [29]. The decline in mortality rates in humans is not seen until after 80 years of

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age, explaining why this had not been previously noticed. These studies have forced a reconsideration of the evolutionary biology theories of senescence [30] as well as many other aspects of how we view aging. 2.5. Are single gene alterations that extend life span useful? As theoretical obstacles are diminishing, other approaches such as molecular genetic ones have begun to be used to examine the mechanisms of aging with increasing frequency. The use of single gene alterations for dissecting out the mechanisms underlying complex biological phenomena, which has been so successful for studying development, recently has begun to make inroads into aging research. Single gene mutations that dramatically extend life span have been identified in yeast, nematode worm, fruitfly, and mice [31 – 45]. These mutations have revealed the importance of physiological systems associated with hormone signal transduction, mitochondrial function, chromosomal stability, food intake, intermediary metabolism, and the growth hormone-prolactin-thyroid stimulating hormone system [34,37,45– 48]. 2.6. Why use model organisms for studying aging? There are a number of model organisms used for studying aging. Among the most prominent are the mouse (Mus musculus), fruitfly (Drosophila melanogaster), nematode (Caenohrabditis elegans), and yeast (Saccharomyces cerevisiae). The choice of these particular organisms is based on several features. In comparison to humans, each of these model organisms has a relatively short life span. The life span of mice is 2 –3 years, for flies 2 – 3 months, for nematodes 2– 3 weeks, and yeast 7 –14 days. Each of these organisms has been the subject of years of intensive molecular and genetic studies and a great deal of information, as well as techniques, are available for exploitation in aging research. Manipulations for altering life span are known for each of these organisms, allowing the experimenter to alter life span, and examine in detail, how this changes specific molecular and physiological elements. 2.7. The uniformity of life Perhaps the most important discovery of the past halfcentury is the recognition of how much we share with other organisms—the ‘uniformity of life’. While it was reasonable to expect that molecules making up the structural features of cells would be conserved, such as tubulin, actin and myosin, the conservation among regulatory molecules that has emerged as a general theme in biomedicine was not anticipated. Molecules responsible for the regulation of complex hierarchical decisions, such as the determination of the body plan or the generation of specific organs are highly conserved across distant species (see the Nobel Prize in

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Medicine 1995: http://www.nobel.se/medicine/educational/ poster/1995/index.html [49]. An example is the relationship between the human Pax-6 gene and the fly eyes absent and eyeless genes. The sequence of the protein from the Pax-6 gene in humans, associated with hereditary Aniridia is over 50% identical to the fruit fly eyes absent and eyeless genes, responsible for the development of the compound insect eye. Amazingly, the human Pax-6 gene can be genetically engineered into the fly and substitute for the eyes absent and eyeless genes in the fly. When the human Pax-6 gene is expressed in ectopic positions in the fly it results in the development of an entirely normal appearing compound eye in these ectopic positions [50]. This and several other experiments using human genes in flies has demonstrated that there are apparently more similarities among organisms than we could have ever imagined [49]. The importance of the recognition of how many molecular genetic features humans share with other organisms is that many of the physiological systems conserved among these model organisms will almost certainly be conserved in humans. This also appears to be true for aging where, for example, alterations in the insulin-signaling pathway in nematodes, flies and mice have lead to life span extension in each of these diverse organisms [37,46,51,52]. 2.8. Why use Drosophila as a model system for studying aging? The fruit fly, Drosophila melanogaster, has a variety of features making it a particularly useful model organism for studying aging [53]. Included in this list are the fly’s relatively short life span, ease of maintenance, genetic and environmental manipulations that alter life span, previous information on aging, storehouse of stocks available with altered genes, molecular genetic techniques, full Drosophila genomic sequence, and its proven success in pioneering the understanding of complex biological phenomena such as development and behavior. The life history and biology of the fly provides two further advantages for aging research. First is the fact that as opposed to other species, in which it can sometimes be difficult to be certain when adult life begins, different aspects of life history in the fly are neatly portioned into readily identifiable morphologically distinct stages; growth and development is associated with the worm-like larval stages and, after metamorphosis, the sexually mature adult fly emerges from the pupal case. The process we usually think of as aging is associated with the adult fly. The second feature of the adult fly that is important for aging research is the fact that, except for cells associated with the gonads and some cells in the gut, the adult fly is entirely made up of post-mitotic cells [54,55]. Similar to the organs in humans, such as the central nervous system and heart, made up of rarely dividing neurons and cardiac cells, that are present throughout life, and thus age along with the individual, most cells of the fly are also aging with the fly. It has been suggested by Arking that with

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regard to aging, the adult fly be considered a set of synchronously aging cells [56]. Therefore, if one of the purposes of aging research is to make sense of how post mitotic cells born with the organism change with age, then the adult fly is an excellent model system. 2.9. The lowly fly’s contribution to aging research Use of Drosophila melanogaster and closely related species have been responsible for demonstrating a number of important fundamental aspects of the aging process. In the early part of the 20th century, Northrop and Loeb used changes in ambient temperature to alter life span in flies in order to demonstrate that life span obeyed the properties of normal chemical and physical laws [57,58]. Drosophila was used by Pearl in the 1920s to show that life span is an inherited trait and in the development of the rate-of-living hypothesis [59 – 63]. In the 1980s, a number of laboratories made use of laboratory selection approaches in Drosophila to show that the inheritance of life span was a plastic property and could be dramatically extended [64 – 69]. Some of the more recent contributions, as noted above, have included the use of Drosophila for demographic studies, identifying the late life plateau in mortality and casting doubt on the concept of a species-specific life span—the principle that there are biological limits to life span in each species [27]. Studies of gene expression have shown the dynamic well-regulated nature of adult life, dispelled the idea that adult life and aging is associated with global loss of homeostasis, and have suggested a possible system for biomarkers of aging in mammalian model systems and humans [13 –17,53]. 2.10. Genetic manipulations that extend life span in flies (and other organisms) The real strength of Drosophila as a model system for studying aging are the powerful molecular genetic approaches and techniques that can be used to directly examine aging and life span extension. At present, techniques are available in Drosophila that allow the experimenter to increase or decrease the level of expression of any single gene or groups of genes, in any subset of cells, at any time throughout the life of the fly [70]. This unprecedented control has opened the way to use molecular genetic approaches to test and confirm theories of aging and search for new genes and physiological systems important in the aging process. These two complementary approaches are usually referred to as the candidate gene approach and the random single gene alteration approach. 2.11. Candidate gene approach By virtue of the conservation of molecular genetic and physiological features among different organisms, the powerful molecular genetic approaches in Drosophila can

be used to rapidly test theories of aging as well as specific molecular pathways for their effects on life span and aging. 2.11.1. Antioxidant enzymes The most prominent theory of aging is the oxidative stress hypothesis [71 – 74]. One of the results of normal metabolism is the formation of a variety of reactive oxygen species (ROS). The oxidative stress hypothesis states that there is an imbalance between the formation and detoxification of ROS. This imbalance results in a progressive accumulation in oxidative damage to DNA, proteins, and lipids. The theory states that the accumulation of oxidative damage causes the loss of important cellular functions resulting in the senescent changes of aging and ultimately death. Drosophila has played a major role in attempting to test this theory by using molecular genetic techniques to decrease or increase the levels of antioxidant enzymes important in detoxifying ROS and examining the effects on life span. Decreasing the function of either of the two major antioxidant enzymes in Drosophila, catalase or SOD, results in a decrease in life span, supporting the oxidative stress hypothesis of aging, reviewed by Helfand and Rogina [53]. However, the fact that in each of these cases enzymatic activity has also been decreased during the critical phases of development make it difficult to be certain that damage acquired during development might not also contribute to the shortened adult life span. Increasing antioxidant enzyme activities using transgenic approaches have resulted in mixed, mostly disappointing results on adult life span. Independently increasing SOD or catalase throughout life does not increase life span to any significant extent. Positive effects on life span have been reported by either a combined SOD and catalase overexpression or increasing SOD activity only during adult life. Overall the effects on life span are small and do not provide much support for the oxidative stress hypothesis, reviewed by Helfand and Rogina [53]. However, in only one study was the effect of antioxidant overexpression on the accumulation of oxidative damage measured. It may be that the level, temporal patterns or tissue distribution of antioxidants is critical for life span extension. 2.11.2. Stress related proteins Many theories of aging including the oxidative stress hypothesis postulate an accumulation of damage to a variety of proteins as a causative agent in the process of aging. The ability to protect, repair or remove damaged proteins may play a role in aging. In support of this idea are the observations that mild stresses such as low levels of radiation or heat shock can extend life span, a term called hormesis [75]. An example of such a protective response may be the observation that Parkinson’s disease has a much lower incidence in smokers [76,77]. Attempts to create the beneficial effects of stress without the stress itself, through transgenic increases in stress related proteins such as hsp70

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have shown limited success. However, chaperones and heat shock related proteins have been shown to dramatically improve a number of Drosophila neurodegenerative diseases reviewed in the literature [76,78]. All of these neurodegenerative diseases in the fly were induced by expression of human genes associated with Parkinson’s disease (alpha synuclein), Huntinton’s disease (Huntingtin), and other trinucleotide associated neurodegenerative disorders (SCA1, SCA3) [78]. The possibility that the induction or amplification of stress response related proteins may extend healthy life span is actively being evaluated using Drosophila.

accessibility to the machinery involved in gene expression. These include histone deacetylases such as rpd 3 and Sir2. A reduction in activity of rpd 3 in yeast increases life span [82], while an increase in Sir2 increases life span in both yeast and C. elegans [83,84]. It has recently been demonstrated that in Drosophila a reduction in rpd 3 activity increases Sir2 expression and life span in both male and female flies [85]. It has been proposed that this may be through a pathway related to caloric restriction [85]. Interestingly, drugs that affect histone deacetylases, such as phenylbuturate (PBA) have also been shown to increase life span in Drosophila [86].

2.11.3. Repair of damaged proteins Other systems for the repair or removal of damaged proteins have also shown promise for extending life span in Drosophila. Protein carboxy methyltransferase (PCMT), which is involved in repairing damaged isoaspartyl residues in proteins formed during aging and methionine sulfoxide reductase, which repairs oxidized methionines in proteins have both been reported to extend life span when overexpressed in transgenic flies [79,80].

2.12. Unbiased random single gene approach

2.11.4. Insulin-like signaling and life span extension The effect of insulin-like signaling on longevity is a good example of how Drosophila can contribute to understanding the process of aging through a directed candidate gene approach. Much of the recent excitement in the field of aging research, particularly among molecular geneticists, was initiated by the finding in 1993 that a decrease in the activity of the daf-2 gene doubled the life span of the nematode, C. elegans [32]. Isolation of additional life extending genes in the same genetic pathway and the identification of this being an insulin-like signaling pathway has generated great interest in the aging field. The use of Drosophila molecular genetics has shown that elements of the insulin-like signaling pathway in Drosophila are also involved in longevity determination [46]. Loss-of-function mutations in both the Insulin-like receptor (InR) gene and insulin-like receptor substrate gene, chico, increased life span in female, but not male flies [41,42]. The confirmation of the importance of the insulin-like signaling pathway in Drosophila led to the examination of similar systems in mice that has been richly rewarding. Reducing the level of expression of the IGF-1 gene all throughout the body in mice or eliminating all IGF-1 activity specifically in only fat cells of mice each result in life span extension [51,52]. Interestingly, it has been reported that mutations in chico may increase life span by affecting the caloric restriction pathway [81]. 2.11.5. Chromatin silencing and life span A number of genes are known to be important for the integrity of chromosome structure and gene expression in Drosophila and other species. Among these are proteins involved in controlling the packing of DNA and its

While the molecular genetic tools and approaches in Drosophila make it an excellent model organism for testing hypotheses and candidate genes, the real strength of Drosophila in aging research is the use of genetics for performing unbiased screens in search of genes that affect life span. The identification of life-extending genes demonstrates a direct causal relationship between the physiological system affected by the genetic alteration and life span. This is an exceedingly powerful approach that has been successfully employed to understand the mysteries of development and is ideally suited for other complex biological phenomena, such as aging. In the single gene alteration approach, individual genes are altered, either by mutation or by over/ectopic expression, and the life span of the resultant genetically altered animal is measured. This method seeks to alter each and every gene, one at a time, and measure the effect on life span. It does not require any a priori knowledge of the aging process and has the additional advantage of having no bias. Since this approach can cause genetic alterations that sometimes significantly alter the gene’s protein product or distribution of expression, it can create new genetic changes that might never have been engineered in a candidate gene approach, and may never be seen in nature. It is an excellent method for exploring a complex physiological or biological system where little is known, such as aging. 2.13. Identification of genes that extend life span in Drosophila In aging studies, the usual phenotype being sought is a genetic alteration that extends life span. Screening for genetic alterations that shorten life span should also yield genes important in the normal process of aging, but sorting out which ones are involved in normal aging, and which lead to a short life as a result of some pathological process, is difficult to do [53]. It is assumed that genes that result in life span extension will usually do so by affecting systems that are important in the determination of longevity. Some of these systems may prove to be more interesting than others. For example, it is known that a reduction in

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metabolic rate, physical activity or reproduction can increase life span in Drosophila and related insects [53]. Genetic alterations that extend life span by reducing metabolic rate or physical activity (causing a slowing down or debilitation of the animal) or reducing reproduction (rendering the animal sub-fertile) are generally thought to be less interesting, as well as having unacceptable tradeoffs when being considered as interventions for agricultural or human use. Therefore, it is important to test whether a genetic alteration that extends life span in flies decreases metabolic rates, physical activity or reproduction. There are two examples of life-extending mutations in Drosophila genes that were discovered by screening single gene mutations: methuselah (mth) [38] and Indy (I’m not dead yet) [40]. A 35% increase in the life span of males and females without a loss in reproduction is found when there is a partial reduction in the activity of the mth gene [38]. The mth gene codes for a member of a subfamily of G-coupled transmembrane receptor-like proteins that has homology to the secretin family [87]. The ligand that binds to MTH is not yet known, but mth appears to play a role in excitatory neurosecretion in larval neuromuscular junctions [88]. Complete knockout of the gene is embryonic lethal. The long-lived mth mutant animals are resistant to a number of different stresses including heat, starvation, dessication, and paraquat (oxygen radical generator). Although it is not yet understood how decreases in mth leads to life-span extension the relationship between mth’s effect on life span and stress resistance provides further support for the hypothesis that stress resistance is an important element in life span determination [75]. A partial decrease in the expression of the Indy gene, a dicarboxylate transporter, found in the plasma membrane of tissues important in intermediary metabolism in flies, leads to a near doubling of the average life span of male and female flies, without a loss in physical activity or reproduction [40]. Indy long-lived animals show little to no tradeoffs in order to achieve long life. There is no measurable decrease in resting metabolic rates or in relevant physiological systems such as flight performance and reproduction [89]. Interestingly, while reproduction is normal or better under normal laboratory conditions, when food quality is decreased reproduction is dramatically decreased, suggesting that the long-lived mutants have reduced Indy activity just enough to extend life span, but not enough to affect critical physiological systems [89]. It has been argued that any life span extending intervention will have unacceptable tradeoffs in metabolic rate, growth, physical activity or early-life fecundity. The Indy mutation appears to be an example of a mutation that challenges these rigid notions where under optimal environmental conditions there is long life with no observable tradeoff. Mutations such as Indy are particularly interesting to aging researchers since it is likely to be altering metabolism in a manner that

extends life span, but without any significant tradeoffs, as long as optimal environmental conditions are preserved. 2.14. Long-lived fly mutants and caloric restriction—a point of convergence A number of important principles have begun to emerge from the molecular genetic studies on Drosophila. There are already at least two examples of a conservation in physiological systems involved in determining life span between flies and other species including yeast, nematodes and mammals. The insulin-signaling pathway involved in longevity determination in nematodes and mammals [37,51,52] and the rpd3/Sir2 systems important in yeast and nematodes [37,84] have both been shown to extend life span in flies [46,85]. Caloric restriction, the only known way of extending life span in mammals [90], is also known to extend life span in flies, yeast, and nematodes [39,91 –95]. Interestingly, three out of the five single gene mutations that significantly increase life span (Indy, chico, rpd 3) have all been linked to caloric restriction or caloric restriction-like metabolic changes [81,85,96]. These early findings provide a strong justification for optimism among molecular geneticists, and suggest that the application of a combination of candidate and random gene alteration approaches will be successful in dissecting apart the process of aging, and identifying key physiological systems involved in determining longevity.

3. Discussion 3.1. What will the future bring? What recent studies on the biology of senescence and aging tell us is that despite the intimate nature of this process we know very little about it. Many of the assumptions that we make concerning the nature and progression of the aging process are not supported by empirical data. Rather than viewing this as a depressing state of affairs, it may be taken as a grand opportunity. The use of molecular genetic approaches, so successful in understanding the complex biological phenomena of development are already beginning to yield results in our quest to understand aging. There is great conservation between different organisms suggesting that what is learned in one model system will be true for others. Model systems can be used to rapidly confirm hypotheses and identify new physiological systems important in the process of aging and determination of longevity. If adult life is really a period of dynamic change, homeostasis is not lost in a global manner, and the rate of mortality does plateau then we have much to consider and try to understand in order to achieve the goal of ancient and modern alchemists and philosophers—a much longer and healthier life.

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Acknowledgements This work is dedicated to Benjamin and Jordan Helfand, and to the memory of Joshua Helfand. We would like to thank Blanka Rogina, Rob Reenan, Marvin Tanzer, and Marc Lalande for encouragement and intellectual and technical support. This work was funded in part by grants (RO1AG16667 to SLH and RO1AG12551 and K24AG00949 to SKI) from the National Institute on Aging, the Patrick and Catherine Weldon Donaghue Medical Research Foundation (DF99-134 to SLH and DF98-105 to SKI), and the Ellison Medical Foundation (to SLH). SLH is a member of the Scientific Advisory Board of Elixir, Pharmaceuticals, Inc., Cambridge, MA.

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