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Guest editorial: mechanisms of development and aging
Mechanisms of Ageing and Development, 12 (1980) 213-219
213
© Elsevier Sequoia S.A., Lausanne - Printed in the Netherlands
GUEST EDITORIAL: MECHANISMS OF DEVELOPMENT AND AGING
B. E. WRIGHT and P. F. DAVISON Departments o f Developmental Biology and Fine Structure, Boston Biomedical Research Institute, Boston, Massachusetts 02114 (U.S.A.J
(Received September 5, 1979; in revised form October 15, 1979)
SUMMARY The view is expressed that an inappropriate emphasis and research effort is currently directed towards simplistic approaches to the problem of aging; it is concluded that an understanding of the biochemical mechanisms underlying the aging process must be sought using a SYSTEMS analysis, at a level of complexity and integration much greater than that encompassed by any theory thus far considered.
AGING AS A MULTICELLULARPROCESS It is perhaps time to reconsider and re-evaluate the popular tendency to search for mechanisms of development and aging at the cellular and subceUular level. The reductionist philosophies and approaches have clearly been justified by the remarkable understanding that molecular biology has brought to many specific aspects of biological regulation and disease. However, we consider the developmental process to be a series of parallel, interdependent changes dictated by complex reactions between organs, cells and cellular components which, therefore, cannot be understood in terms of simplistic mechanisms such as sequential gene activation. This point of view has been elaborated upon in the past [1, 2]. Given the complexity of differentiation, how can we justifiably search for an understanding of the end product of differentiation, senescence, in terms of single mechanisms? Senescent degeneration, in general, is not a clearly definable step from normal to ineffective performance, but a progressive diminution in functional efficacy. It is likely that the failing capacity of a differentiated, aging organism reflects the deteriorating performance and interactions of a great many organs, organelles and biochemical activities [3, 4]. For example, fortuitous antigenic determinants or infecting organisms may engender the accumulation of autoimmune products which could diminish renal function; the latter impairment may slow the excretion of toxic materials and metabolites, and so on. Many structures and biochemical functions become impaired in parallel during the aging process: the integrity of tissues and organelles, membrane
214 permeability, transport, insufficient mobilization of energy sources, respiration, flux through the citric acid cycle, the rates of critical biosynthetic reactions, ad infinitum. Interdependent organs and metabolic networks which gradually become less efficient through many developmental mechanisms and environmental insults eventually interact with mutually destructive effects that result in the death of the organism. There is no evidence that one kind of defect is always more critical to aging than another, nor that all the cells within an organism become defective in unison. For example, fibroblasts from old people can initiate wound healing, and can become established in cell culture even when taken after the death of the individual. It therefore seems reasonable to seek mechanisms and rate-limiting events controlling the aging process from the analysis, as a whole, of the many complex physiological and metabolic interactions within the organism. The necessity of a SYSTEMS analysis in understanding organ function, as well as hormonal, neuroendocrine and homeostatic control mechanisms, has been recognized for some time by physiologists [5-12], who are now recommending this approach to the analysis of aging [3, 4]. The view that senescence is essentially the accumulative failure of many components within a multicellular organism encompasses but cannot be explained by simple models of aging, such as the damage induced by free radicals, or the hypothesis of error accumulation (which may well explain the death of individual cells) [13, 14], or theories based on Hayflick's observations of the limited in vitro lifespan of diploid fibroblasts [15, 16]. Clearly, single-cell models cannot be used to analyze the interaction of different cell types. Moreover, post-replicative cells in culture and those in the intact organism are not comparable: fibroblasts cultured to a post-replicative state [15, 16] cannot be a model for neurons which have persisted in that state for many decades.
ROLE OF DEVELOPMENTAND ENVIRONMENTIN AGING In assessing which kinds of events contribute most critically to the aging process, it appears to be very difficult if not impossible to distinguish experimentally between developmental and environmental effects [17], because of considerations such as the following. Normal development of a multicellular organism could not occur in isolation; for example, in a sterile, radiation-free, zero-gravity environment. Development results in part from the interaction of the organism with those same environmental factors contributing to terminal differentiation or senescence; for example, exercise is necessary for normal muscular development and maintenance, yet such exercise leads to progressive wear of the cartilage of the weight-beating joints [ 18]. On the other hand, at zero gravity, where such wear might be minimized, bones become decalcified as the experiences of the astronauts have shown. The post-mitotic state (for example, neurons, cardiac muscle) is a concomitant of differentiation and is presumably essential to the survival of the organism. It is widely appreciated (see ref. 19, for example) that terminal differentiation is an inevitable prelude to senescence because many such cell populations apparently cannot be replaced; an adult organism whose tissues cannot persist indefinitely must have
215 a limited lifespan. We would define senescence as the manifestation of accrued functional deficits which reflect the internal and external insults inflicted on the individual by development and by the environment during the strenuous process of living.
INFLUENCINGTHE AGING PROCESS The question is often asked, whether it might be possible to slow the process of aging by providing an ideal environment and diet. Aside from the obvious benefits of reducing exposure to toxic substances, radiation and infection, what sort of environment could prevent neuronal loss, damaged cartilage, autoimmune or vascular accidents? Suspended in an abiotic, radiation-shielded environment at zero gravity and fed by a balanced, detoxified diet, the resultant organism would surely be abnormal in its vascular, muscular, dental and bone development - but would it live longer? We surmise that such an organism would be unlikely to exceed the maximum lifespan already found for the species living under normal conditions, because the interactions between cell populations which use the same food sources and excrete similar waste products cannot all be mutually supportive; furthermore, biosynthetic and metabolic error are mathematical certainties. Therefore, unless repair mechanisms are perfect (and what biological processes are?) some diminished cellular functions must gradually appear, and injuries must accumulate in the course of time. Even in a hypothetical, ideal environment, changes such as altered hormonal secretions acting on a post-replicative cell population must ultimately result in progressive cell loss and organ malfunction. These factors alone are sufficient to ensure that post-replicative cell populations cannot endure indefinitely. We believe a critical component of the aging process is the inability of an organism to maintain homeostasis, and to recover completely from internal or external change or injury. Repair mechanisms must have been selected by evolutionary pressures to preserve an organism until reproduction, but there appears to be no compelling pressure to preserve organisms much beyond. From an evolutionary point of view, once an organism is post-replicative it is not essential for the propagation of a species. The importance of repair mechanisms to slowing the aging process is emphasized by the observation that, even at the DNA level, there is a positive correlation between a species' lifespan and the efficacy of healing damage by radiation or other deleterious agents [20-22].
ROLE OF GENES IN AGING Many investigators speculating on the developmental (as opposed to environmental) mechanisms of aging have noted that genetic factors "determine" a species' lifespan, and therefore conclude that "aging" genes must be involved; mutants of such genes have also been sought. We believe that such a conclusion is a logical non-sequitur. Within a species, segregation of populations showing varying lifespans are, of course, well known. However, inherited, selected characteristics do not result from gene function alone. They are
216 determined by a unique interaction of genes, nuclear milieu, cytoplasm, cellular structures and the external environment, which may include the whims of man. Variation in the lifespan of the domestic dog is an excellent example, in that the very large breeds have significantly shorter lifespans than breeds closer to the progenitor's size [23]. Such lifespans have been imposed by mankind through phenotypic selection; however, it is quite unnecessary to invoke the action of (structural or regulatory) DNA as being primarily responsible for controlling these variations in lifespan [24, 25]. In the case of the giant domestic dog, shorter lifespans are very likely the consequence of their increase in size, and are dictated by such factors as the physiological demand placed upon heart muscles, and circulatory, excretory and other organ systems. If processes such as physiological imbalance limit the lifespans of individuals within a species, these factors undoubtedly also play a role in limiting the spans of different species. Size, of course, is not always correlated with lifespan; witness the tortoise, the owl and the elephant [23] ! Such comparisons clearly illustrate the complexity of factors limiting the length of a life. While organs and tissues have been engineered by the process of natural or artificial selection to be adequate to bring the organism to fruitful replication, the irreversible consequences of development, the rigors of living, and the progressive accumulation of defects are certain to bring the organism to death. Genes don't die - organisms do. Biochemically, DNA is probably the most stable of the cellular components, and the least likely to be affected during aging. In the sense that genes are essential to the creation and differentiation of an organism, they are responsible for its aging and death; but this applies to many other cellular components as well. Why is it frequently assumed that alterations in gene expression constitute the major type of cellular activity controlling (limiting) the rate of the aging process? What is the evidence for this? Indeed, how can we interpret or use information at the genetic level until we can relate specific gene products to known biochemical events which are rate-limiting at particular points in time during the aging process? No gene, metabolite, cellular structure or function can be uniquely important to the aging process, if they are all essential, involved, affected and interdependent. Under these circumstances, we are not going to fred the "secret" of aging by analyzing the genetics of death; that is, by comparing and combining long- and short-lived mice, mortal and immortal cells, and so on. H o w lifespans are inherited is n o t the p r o b l e m - the problem is h o w and w h y a life c o m e s to an end. A genetic analysis of lifespans is comparable to conducting an elaborate study of a group of watches to determine the rate at which each one ticks, and the lifespan of each watch. It would be more productive and mechanistically meaningful first to describe how and why any one of them stops ticking; only then will it be possible to track down the responsible events. In summary, it seems to us that an explanation of the aging process must realistically be sought at a level of complexity and integration that is much greater than the activation of lethal genes or other simple processes. The theory of cataclysmic error, proposed 16 years ago, has stimulated a great deal of experimental and theoretical work. However, preoccupation with unrealistic theories may impede our recognition of and confrontation with the interdependent, multiple mechanisms which probably result in aging of the differentiated organism. To search for the "ultimate cause" of senescence is
217 as futile as searching for the "ultimate cause" of differentiation, as one of us has previously discussed in detail [ 1 , 2 ] .
DEFINING THE AGING PROCESS IN SPECIFIC SYSTEMS Aging (as opposed to death) must be defined both verbally and experimentally in a particular SYSTEM as a prerequisite to the analysis of underlying mechanisms. Such a definition m!.ght include a parallel pattern of deterioration of functions common to all living cells (perhaps energy metabolism), as well as specific metabolic functions unique to the particular organism or tissue, such as the synthesis of collagen in skin, of glycogen in liver, and so on. In the field of biochemical differentiation, the specialized kind of metabolism under analysis is usually clearly defined; for example, the accumulation of silk protein (fibroin) by larvae spinning a cocoon, the synthesis of an eye pigment in Drosophila, or the accumulation of cellulose in the cellular slime mold. In aging systems, what specific substances or kinds of metabolism characterize terminal differentiation? In models for aging, such as human fibroblasts, what are the biochemical changes we seek to analyze and understand? How can we def'me the rate-limiting events and study the mechanisms underlying these alterations in metabolism until they are described? Does there exist an "essence of aging" in nature for us to discover? Such a viewpoint does not appear to be very productive. There are, however, a great many aging SYSTEMS in which we can describe specific metabolic processes clearly related to the progressive series of events eventually resulting in death. Only then can the biochemical mechanisms and rate-limiting factors" controlling these metabolic processes be deffmed and analyzed.
NECESSITY OF A SYSTEMS ANALYSIS A process as complicated as aging appears to demand an integrative SYSTEMS analysis; the organism ages as a whole and must therefore be analyzed and understood as a SYSTEM in dynamic, in vivo terms. If, as seems likely, aging is a multi-rate-limitingevents-process, it must be described in all its complexity before we can hope to identify the most critical of the rate-limiting events at successive periods, and hence be able to intervene specifically and perhaps slow the aging process. At the level of physiological mechanisms, SYSTEMS analyses of aging are already under consideration [3, 4] ; at a biochemical level, microbial [26, 27] or other simple experimental material is presently amenable to analysis. The most rapid progress would probably be achieved if many investigators contributed to an in-depth SYSTEMS analysis of one simple, multicellular, normal organism that ages and dies. As Lederberg said in 1966 [28], " I f any one system in developmental biology received a fraction of the convergent attention give the T phages, we might be more optimistic about the pace of future research." How can we study the mechanism of a process before we have def'med an.d described it in realistic
218 terms, under the dynamic conditions of the living organism? We must have a framework - a basis for deciding which events are consequences of, and which are rate-limiting for, the aging process at successive specified intervals of time. Once the complexity of the aging process is acknowledged, the necessity of a SYSTEMS analysis will be inescapable. Only then will the essential tools and experimental approaches for such an analysis be developed and used. Although it is difficult, it is not impossible to gather the variety o f requisite data for a SYSTEMS analysis, in terms of the dynamic metabolic conditions of a living, aging organism. Moreover, it is essential, in order to understand the mechanisms involved in such a complicated process. A necessary beginning is a detailed description of biochemical events which might be expected to change during aging in particular organs or organisms; for example, metabolite and endproduct levels, macromolecular turnover, flux through critical biosynthetic pathways, and so on. This information, together with our extensive and sophisticated understanding of physiological and metabolic regulation, computer technology and steady-state kinetics, should allow the construction o f realistic models with predictive value. Thus, steady-state models of metabolism in young, mature and senescent organs or organisms can be constructed and compared, in order to define those variables most critical (rate-limiting) in proceeding from the young to the old model. Such studies could then lead to the formulation of transition models, simulating the transformation of metabolism in young to old organisms as a function of time [ 2 6 , 2 7 ] . Such simulation analysis should eventually indicate those aspects of the aging SYSTEM primarily responsible for the impaired performance and eventual death of the organism.
ACKNOWLEDGMENTS The viewpoints presented here developed as a result of work supported by the National Institute of Health grants AGO0260 (B.E.W.), AG00433 (B.E.W.) and AG00584 (P.F.D.).
REFERENCES 1 2 3 4 5
B. E. Wright, Critical Variables in Differentiation, Prentice Hall, Englewood Cliffs, N.J., 1973. B. E. Wright, Causality in biological systems. Trends Biochem. Sci., 4 (1979) N110-N111. N. W. Shock, Systems physiology and aging. Fed. Am. Soc. Exp. Biol., 38 (1979) 161-162. C. E. Finch, Neuroendocrine mechanisms and aging, ibid., 38 (1979) 178-183. D. F. Heath, The redistribution of carbon label by the reactions involved in glycolyis, gluconeogenesis and the tricarboxylic acid cycle in rat liver in vivo. Biochera. J., 110 (1968) 313-334. 6 D. F. Heath and C. J. ThreffaU, The interaction of glycolysis, gluconeogenesis and the tricarboxylic acid cycle in rat liver in vivo. Biochem. J., 110 (1968) 337-362. 7 M. J. Achs and D. Garfinkel, Computer simulation of energy metabolism in anoxic perfused rat heart. Am. J. Physiol., 232 (1977) R164-R174. 8 M. J. Achs and D. Garfinkel, Computer simulation of rat heart metabolism after adding glucose to the perfusate, ibid., 232 (1977) R175-R184.
219 9 E. R. Carson and E. A. Jones, Use of kinetic analysis and mathematical modelling in the study of metabolic pathways in vivo. Applications to hepatic organic anion metabolism, iV. Engl. J. Med., 300 (1979) 1016-1027 and 1078-1086. 10 M. Berman, The formulation and testing of models. Ann. N. Y. Acad. ScL, 108 (1963) 182-194. 11 E. Ackerman, L. C. Gatewood and J. W. Rosevear, Blood glucose regulation and diabetes, concepts and models of biomathematies. In F. Heinmets (ed.), Simulation Techniques and Methods, Vol. 1, Marcel Dekker, New York, 1969, pp. 131-156. 12 S. M. Skinner, R. E. Clark and N. Baker, Complete solution of the three-compartment model in steady-state after single injection of radioactive tracer. Am. J. Physiol., 196 (1959) 238-244. 13 L. E. Orgel, The maintenance of the accuracy of protein synthesis and its relevance to aging. Proc. Natl. Acad. Sci. U.S.A., 49 (1963) 517-521. 14 L. E. Or~',el, A correction. Proc. Natl. Acad. Sci. U.S.A., 67 (1970) 1476. 15 L. Hayflick, The limited in vitro lifetime of human diploid cell strains. Exp. CellRes., 37 (1965) 614-636. 16 E. Bell, L. F. Mark, D. S. Levinstone, C. Merrill, S. Sher, I. T. Young and M. Eden, Loss of division potential in vitro: aging or differentiation. Science, 202 (1978) 1158-1163. 17 W. D. Denckla, Systems analysis of possible mechanisms of mammalian aging. Mech. Ageing Dev., 6 (1977) 143-152. 18 P. BuUough, J. Goodfellow and J. O'Connor, The relationship between degenerative changes a n d load-bearing in the h u m a n h i p . Z Bone Jr. Surg. Am. Vol., 55 (1973) 746-758. 19 B. L. Strehler, Time, Cells and Aging, Academic Press, New York, 1977, Chapter IX, Part III. 20 J. Epstein, J. R. Williams and J. B. Little, Deficient DNA repair in human progeroid ceils. Proc. Natl. Acad. Sci. U.S.A., 70 (1973) 977-981. 21 R. W. Hart and R. B. Setlow, Correlation between deoxyribonucleic acid excision-repair and life-span in a number of mammalian species. Proc. Natl. Acad. Sci. U.S.A., 71 (1974)2169-2173. 22 R. B. Setlow, Repair deficient human disorders and cancer. Nature, 271 (1978) 713-717. 23 A. Comfort, Aging, the Biology o f Senescence, Holt, Reinhart and Winston, New York, 1964, p. 61. 24 G. M. Martin, Genetic and evolutionary aspects of aging. Fed. Proc., 38 (1979) 1962-1967. 25 R. G. Cutler, Evolution 6f human longevity: a critical overview. Mech. Ageing Dev., 9 (1979) 337. 26 B. E. Wright, A. Tai, K. A. Killick and D. A. Thomas, The effects of exogenous glucose, uracil and inorganic phosphate on differentiation in Dictyostelium discoideum. Arch. Biochem. Biophys., 192 (1979) 489--499. 27 B. E. Wright, A. Tai and K. A. Killick, Fourth expansion and glucose perturbation of the Dictyostelium kinetic model. Eur. J. Biochem., 74 (1977) 217-225. 28 J. Lederberg, Remarks. I n A . Moscona and A. Momoy (eds.), Current Topics in Developmental Biology, Vol. 1, Academic Press, New York, 1966, p. xii.
213
© Elsevier Sequoia S.A., Lausanne - Printed in the Netherlands
GUEST EDITORIAL: MECHANISMS OF DEVELOPMENT AND AGING
B. E. WRIGHT and P. F. DAVISON Departments o f Developmental Biology and Fine Structure, Boston Biomedical Research Institute, Boston, Massachusetts 02114 (U.S.A.J
(Received September 5, 1979; in revised form October 15, 1979)
SUMMARY The view is expressed that an inappropriate emphasis and research effort is currently directed towards simplistic approaches to the problem of aging; it is concluded that an understanding of the biochemical mechanisms underlying the aging process must be sought using a SYSTEMS analysis, at a level of complexity and integration much greater than that encompassed by any theory thus far considered.
AGING AS A MULTICELLULARPROCESS It is perhaps time to reconsider and re-evaluate the popular tendency to search for mechanisms of development and aging at the cellular and subceUular level. The reductionist philosophies and approaches have clearly been justified by the remarkable understanding that molecular biology has brought to many specific aspects of biological regulation and disease. However, we consider the developmental process to be a series of parallel, interdependent changes dictated by complex reactions between organs, cells and cellular components which, therefore, cannot be understood in terms of simplistic mechanisms such as sequential gene activation. This point of view has been elaborated upon in the past [1, 2]. Given the complexity of differentiation, how can we justifiably search for an understanding of the end product of differentiation, senescence, in terms of single mechanisms? Senescent degeneration, in general, is not a clearly definable step from normal to ineffective performance, but a progressive diminution in functional efficacy. It is likely that the failing capacity of a differentiated, aging organism reflects the deteriorating performance and interactions of a great many organs, organelles and biochemical activities [3, 4]. For example, fortuitous antigenic determinants or infecting organisms may engender the accumulation of autoimmune products which could diminish renal function; the latter impairment may slow the excretion of toxic materials and metabolites, and so on. Many structures and biochemical functions become impaired in parallel during the aging process: the integrity of tissues and organelles, membrane
214 permeability, transport, insufficient mobilization of energy sources, respiration, flux through the citric acid cycle, the rates of critical biosynthetic reactions, ad infinitum. Interdependent organs and metabolic networks which gradually become less efficient through many developmental mechanisms and environmental insults eventually interact with mutually destructive effects that result in the death of the organism. There is no evidence that one kind of defect is always more critical to aging than another, nor that all the cells within an organism become defective in unison. For example, fibroblasts from old people can initiate wound healing, and can become established in cell culture even when taken after the death of the individual. It therefore seems reasonable to seek mechanisms and rate-limiting events controlling the aging process from the analysis, as a whole, of the many complex physiological and metabolic interactions within the organism. The necessity of a SYSTEMS analysis in understanding organ function, as well as hormonal, neuroendocrine and homeostatic control mechanisms, has been recognized for some time by physiologists [5-12], who are now recommending this approach to the analysis of aging [3, 4]. The view that senescence is essentially the accumulative failure of many components within a multicellular organism encompasses but cannot be explained by simple models of aging, such as the damage induced by free radicals, or the hypothesis of error accumulation (which may well explain the death of individual cells) [13, 14], or theories based on Hayflick's observations of the limited in vitro lifespan of diploid fibroblasts [15, 16]. Clearly, single-cell models cannot be used to analyze the interaction of different cell types. Moreover, post-replicative cells in culture and those in the intact organism are not comparable: fibroblasts cultured to a post-replicative state [15, 16] cannot be a model for neurons which have persisted in that state for many decades.
ROLE OF DEVELOPMENTAND ENVIRONMENTIN AGING In assessing which kinds of events contribute most critically to the aging process, it appears to be very difficult if not impossible to distinguish experimentally between developmental and environmental effects [17], because of considerations such as the following. Normal development of a multicellular organism could not occur in isolation; for example, in a sterile, radiation-free, zero-gravity environment. Development results in part from the interaction of the organism with those same environmental factors contributing to terminal differentiation or senescence; for example, exercise is necessary for normal muscular development and maintenance, yet such exercise leads to progressive wear of the cartilage of the weight-beating joints [ 18]. On the other hand, at zero gravity, where such wear might be minimized, bones become decalcified as the experiences of the astronauts have shown. The post-mitotic state (for example, neurons, cardiac muscle) is a concomitant of differentiation and is presumably essential to the survival of the organism. It is widely appreciated (see ref. 19, for example) that terminal differentiation is an inevitable prelude to senescence because many such cell populations apparently cannot be replaced; an adult organism whose tissues cannot persist indefinitely must have
215 a limited lifespan. We would define senescence as the manifestation of accrued functional deficits which reflect the internal and external insults inflicted on the individual by development and by the environment during the strenuous process of living.
INFLUENCINGTHE AGING PROCESS The question is often asked, whether it might be possible to slow the process of aging by providing an ideal environment and diet. Aside from the obvious benefits of reducing exposure to toxic substances, radiation and infection, what sort of environment could prevent neuronal loss, damaged cartilage, autoimmune or vascular accidents? Suspended in an abiotic, radiation-shielded environment at zero gravity and fed by a balanced, detoxified diet, the resultant organism would surely be abnormal in its vascular, muscular, dental and bone development - but would it live longer? We surmise that such an organism would be unlikely to exceed the maximum lifespan already found for the species living under normal conditions, because the interactions between cell populations which use the same food sources and excrete similar waste products cannot all be mutually supportive; furthermore, biosynthetic and metabolic error are mathematical certainties. Therefore, unless repair mechanisms are perfect (and what biological processes are?) some diminished cellular functions must gradually appear, and injuries must accumulate in the course of time. Even in a hypothetical, ideal environment, changes such as altered hormonal secretions acting on a post-replicative cell population must ultimately result in progressive cell loss and organ malfunction. These factors alone are sufficient to ensure that post-replicative cell populations cannot endure indefinitely. We believe a critical component of the aging process is the inability of an organism to maintain homeostasis, and to recover completely from internal or external change or injury. Repair mechanisms must have been selected by evolutionary pressures to preserve an organism until reproduction, but there appears to be no compelling pressure to preserve organisms much beyond. From an evolutionary point of view, once an organism is post-replicative it is not essential for the propagation of a species. The importance of repair mechanisms to slowing the aging process is emphasized by the observation that, even at the DNA level, there is a positive correlation between a species' lifespan and the efficacy of healing damage by radiation or other deleterious agents [20-22].
ROLE OF GENES IN AGING Many investigators speculating on the developmental (as opposed to environmental) mechanisms of aging have noted that genetic factors "determine" a species' lifespan, and therefore conclude that "aging" genes must be involved; mutants of such genes have also been sought. We believe that such a conclusion is a logical non-sequitur. Within a species, segregation of populations showing varying lifespans are, of course, well known. However, inherited, selected characteristics do not result from gene function alone. They are
216 determined by a unique interaction of genes, nuclear milieu, cytoplasm, cellular structures and the external environment, which may include the whims of man. Variation in the lifespan of the domestic dog is an excellent example, in that the very large breeds have significantly shorter lifespans than breeds closer to the progenitor's size [23]. Such lifespans have been imposed by mankind through phenotypic selection; however, it is quite unnecessary to invoke the action of (structural or regulatory) DNA as being primarily responsible for controlling these variations in lifespan [24, 25]. In the case of the giant domestic dog, shorter lifespans are very likely the consequence of their increase in size, and are dictated by such factors as the physiological demand placed upon heart muscles, and circulatory, excretory and other organ systems. If processes such as physiological imbalance limit the lifespans of individuals within a species, these factors undoubtedly also play a role in limiting the spans of different species. Size, of course, is not always correlated with lifespan; witness the tortoise, the owl and the elephant [23] ! Such comparisons clearly illustrate the complexity of factors limiting the length of a life. While organs and tissues have been engineered by the process of natural or artificial selection to be adequate to bring the organism to fruitful replication, the irreversible consequences of development, the rigors of living, and the progressive accumulation of defects are certain to bring the organism to death. Genes don't die - organisms do. Biochemically, DNA is probably the most stable of the cellular components, and the least likely to be affected during aging. In the sense that genes are essential to the creation and differentiation of an organism, they are responsible for its aging and death; but this applies to many other cellular components as well. Why is it frequently assumed that alterations in gene expression constitute the major type of cellular activity controlling (limiting) the rate of the aging process? What is the evidence for this? Indeed, how can we interpret or use information at the genetic level until we can relate specific gene products to known biochemical events which are rate-limiting at particular points in time during the aging process? No gene, metabolite, cellular structure or function can be uniquely important to the aging process, if they are all essential, involved, affected and interdependent. Under these circumstances, we are not going to fred the "secret" of aging by analyzing the genetics of death; that is, by comparing and combining long- and short-lived mice, mortal and immortal cells, and so on. H o w lifespans are inherited is n o t the p r o b l e m - the problem is h o w and w h y a life c o m e s to an end. A genetic analysis of lifespans is comparable to conducting an elaborate study of a group of watches to determine the rate at which each one ticks, and the lifespan of each watch. It would be more productive and mechanistically meaningful first to describe how and why any one of them stops ticking; only then will it be possible to track down the responsible events. In summary, it seems to us that an explanation of the aging process must realistically be sought at a level of complexity and integration that is much greater than the activation of lethal genes or other simple processes. The theory of cataclysmic error, proposed 16 years ago, has stimulated a great deal of experimental and theoretical work. However, preoccupation with unrealistic theories may impede our recognition of and confrontation with the interdependent, multiple mechanisms which probably result in aging of the differentiated organism. To search for the "ultimate cause" of senescence is
217 as futile as searching for the "ultimate cause" of differentiation, as one of us has previously discussed in detail [ 1 , 2 ] .
DEFINING THE AGING PROCESS IN SPECIFIC SYSTEMS Aging (as opposed to death) must be defined both verbally and experimentally in a particular SYSTEM as a prerequisite to the analysis of underlying mechanisms. Such a definition m!.ght include a parallel pattern of deterioration of functions common to all living cells (perhaps energy metabolism), as well as specific metabolic functions unique to the particular organism or tissue, such as the synthesis of collagen in skin, of glycogen in liver, and so on. In the field of biochemical differentiation, the specialized kind of metabolism under analysis is usually clearly defined; for example, the accumulation of silk protein (fibroin) by larvae spinning a cocoon, the synthesis of an eye pigment in Drosophila, or the accumulation of cellulose in the cellular slime mold. In aging systems, what specific substances or kinds of metabolism characterize terminal differentiation? In models for aging, such as human fibroblasts, what are the biochemical changes we seek to analyze and understand? How can we def'me the rate-limiting events and study the mechanisms underlying these alterations in metabolism until they are described? Does there exist an "essence of aging" in nature for us to discover? Such a viewpoint does not appear to be very productive. There are, however, a great many aging SYSTEMS in which we can describe specific metabolic processes clearly related to the progressive series of events eventually resulting in death. Only then can the biochemical mechanisms and rate-limiting factors" controlling these metabolic processes be deffmed and analyzed.
NECESSITY OF A SYSTEMS ANALYSIS A process as complicated as aging appears to demand an integrative SYSTEMS analysis; the organism ages as a whole and must therefore be analyzed and understood as a SYSTEM in dynamic, in vivo terms. If, as seems likely, aging is a multi-rate-limitingevents-process, it must be described in all its complexity before we can hope to identify the most critical of the rate-limiting events at successive periods, and hence be able to intervene specifically and perhaps slow the aging process. At the level of physiological mechanisms, SYSTEMS analyses of aging are already under consideration [3, 4] ; at a biochemical level, microbial [26, 27] or other simple experimental material is presently amenable to analysis. The most rapid progress would probably be achieved if many investigators contributed to an in-depth SYSTEMS analysis of one simple, multicellular, normal organism that ages and dies. As Lederberg said in 1966 [28], " I f any one system in developmental biology received a fraction of the convergent attention give the T phages, we might be more optimistic about the pace of future research." How can we study the mechanism of a process before we have def'med an.d described it in realistic
218 terms, under the dynamic conditions of the living organism? We must have a framework - a basis for deciding which events are consequences of, and which are rate-limiting for, the aging process at successive specified intervals of time. Once the complexity of the aging process is acknowledged, the necessity of a SYSTEMS analysis will be inescapable. Only then will the essential tools and experimental approaches for such an analysis be developed and used. Although it is difficult, it is not impossible to gather the variety o f requisite data for a SYSTEMS analysis, in terms of the dynamic metabolic conditions of a living, aging organism. Moreover, it is essential, in order to understand the mechanisms involved in such a complicated process. A necessary beginning is a detailed description of biochemical events which might be expected to change during aging in particular organs or organisms; for example, metabolite and endproduct levels, macromolecular turnover, flux through critical biosynthetic pathways, and so on. This information, together with our extensive and sophisticated understanding of physiological and metabolic regulation, computer technology and steady-state kinetics, should allow the construction o f realistic models with predictive value. Thus, steady-state models of metabolism in young, mature and senescent organs or organisms can be constructed and compared, in order to define those variables most critical (rate-limiting) in proceeding from the young to the old model. Such studies could then lead to the formulation of transition models, simulating the transformation of metabolism in young to old organisms as a function of time [ 2 6 , 2 7 ] . Such simulation analysis should eventually indicate those aspects of the aging SYSTEM primarily responsible for the impaired performance and eventual death of the organism.
ACKNOWLEDGMENTS The viewpoints presented here developed as a result of work supported by the National Institute of Health grants AGO0260 (B.E.W.), AG00433 (B.E.W.) and AG00584 (P.F.D.).
REFERENCES 1 2 3 4 5
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