Robert Arking

Selection for delayed senescence in Drosophila melanogaster

Understanding the mechanism whereby the aging process is controlled has proven to be a uniquely difficult biological problem. Many theories have been put forth offering explanations for the phenomenon of senescence on a variety of different levels ranging from cellular, biochemical, and physiological to genetic and evolutionary. Many of these explanations are nonexclusive, which adds redundancy to confusion in considering the whole body of theory. Many of the cellular and/or biochemical mechanisms proposed amount to little more than detailed discussions of various possible gene end-products, which are themselves the subject of genetic and evolutionary theories. And even among these, no single theory predominates. J. B. S. Haldane (1941) and P. B. Medawar (1952) advanced the first theory of senescence incorporating a modem genetic and evolutionary perspective on the aging process. Their theory postulates the existence of specialized age-of-onset modifier genes which repress the action of other genes that are deleterious until an advanced age has been reached. Little harm results from the expression of the mutations then, however, and senescence gradually ensues with their derepression. In this theory, selection modifies life span by simply increasing or decreasing the period over which such modifiers are effective. Williams (19 57) later expanded on this, introducing the notion that the genes influencing senescence might themselves act pleiotropically with reciprocal effects at early and late ages. In this theory, the beneficial effects of genes early in life are weighed in evolution against their late life effects; youthful vigor must be accompanied by an early senescence and short life, while a delayed senescence and long life occur at the cost of youthful vitality. Apart from further extension of these ideas by Hamilton (1966) and Emlen (1 970), no new major theories of the evolution of senescence have arisen since Williams (1957). One reason for this may be that until recently, the few experimental tests performed contributed comparatively little substantiating information toward these theories. Early attempts at modifying life span through artificial selection include that of Glass (1960), who withheld mating in Drosophila to enforce an early versus late age-specific pattern of reproduction. This produced a slight increase in the longevity of late-reproducing lines. Wattiaux (1968) also found an increase in longevity in Drosophila under selection for an agespecific pattern of reproduction. This was followed by Sokals (1970) study showing that continuous reproduction at an early age reduced median life span in Tribolium. Mertz (1975) found similar trends in an even later study. Taylor and Condra (1980) and Barclay and Gregory (1982) report changes in the longevity of Drosophila populations under rand K-selection or when exposed to predation. Concurrently with these, Lints and Hoste (1974, 1977) published the results of a well designed and extensive experiment that also selected for increased longevity in D. melanogaster through an early or late age-specific schedule of reproduction. But life span fluctuated wildly throughout the 13 generations of selection here, declining by 70% in the first few generations and then recovering. Further experiments (Lints et al., 1979)

Forward and reverse selection for longevity in Drosophila is characterized by alteration of antioxidant gene expression and oxidative damage patterns

Patterns of antioxidant gene expression and of oxidative damage were measured throughout the adult life span of a selected long-lived strain (La) of Drosophila melanogaster and compared to that of their normal-lived progenitor strain (Ra). Extended longevity in the La strain is correlated with enhanced antioxidant defense system gene expression, accumulation of CuZnSOD protein, and an increase in ADS enzyme activities. Extended longevity is strongly associated with a significantly increased resistance to oxidative stress. Reverse-selecting this long-lived strain for shortened longevity (RevLa strain) yields a significant decrease in longevity accompanied by reversion to normal levels of its antioxidant defense system gene expression patterns and antioxidant enzyme patterns. The significant effects of forward and reverse selection in these strains seem limited to the ADS enzymes; 11 other enzymes with primarily metabolic functions show no obvious effect of selection on their activity levels whereas six other enzymes postulated to play a role in flux control may actually be involved in NADPH reoxidation and thus support the enhanced activities of the ADS enzymes. Thus, alterations in the longevity of these Drosophila strains are directly correlated with corresponding alterations in; 1) the mRNA levels of certain antioxidant defense system genes; 2) the protein level of at least one antioxidant defense system gene; 3) the activity levels of the corresponding antioxidant defense system enzymes, and 4) the ability of the organism to resist the biological damage arising from oxidative stress.

Successful selection for increased longevity in Drosophila: Analysis of the survival data and presentation of a hypothesis on the genetic regulation of longevity

Long lived strains of Drosophila melanogaster have been generated via 25 generations of artificial selection. The mean and the maximum lifespans have been increased both absolutely as well as relative to the controls. The mean lifespan of the selected line now exceeds the maximum lifespan of the controls. The data shows that this increase is entirely accounted for by a genetically based delay in the onset of senescence. Identification and analysis of biomarker data involving reproductive functions supports this interpretation and leads to a suggestion of the processes involved in the lifespan extension. This increase in the duration of the pre-senescent period is under both genetic and environmental control. Senescence itself is not under genetic control and appears to occur stochastically. Selection for decreased longevity was unsuccessful, supporting the concept of a minimum species specific lifespan. A testable hypothesis regarding the biphasic mode of gene regulation of senescence is presented in which a gene-environment interaction takes place in larval life that results in a temporal reprogramming of other, presumably structural, genes which act in adult life at a time prior to the onset of senescence.

Exercise-Training in Young Drosophila melanogaster Reduces Age-Related Decline in Mobility and Cardiac Performance

Declining mobility is a major concern, as well as a major source of health care costs, among the elderly population. Lack of mobility is a primary cause of entry into managed care facilities, and a contributing factor to the frequency of damaging falls. Exercise-based therapies have shown great promise in sustaining mobility in elderly patients, as well as in rodent models. However, the genetic basis of the changing physiological responses to exercise during aging is not well understood. Here, we describe the first exercise-training paradigm in an invertebrate genetic model system. Flies are exercised by a mechanized platform, known as the Power Tower, that rapidly, repeatedly, induces their innate instinct for negative geotaxis. When young flies are subjected to a carefully controlled, ramped paradigm of exercise-training, they display significant reduction in age-related decline in mobility and cardiac performance. Fly lines with improved mitochondrial efficiency display some of the phenotypes observed in wild-type exercised flies. The exercise response in flies is influenced by the amount of protein and lipid, but not carbohydrate, in the diet. The development of an exercise-training model in Drosophila melanogaster opens the way to direct testing of single-gene based genetic therapies for improved mobility in aged animals, as well as unbiased genetic screens for loci involved in the changing response to exercise during aging.

Identical longevity phenotypes are characterized by different patterns of gene expression and oxidative damage.

Some years ago we applied simultaneously an identical regime of selection for late-life reproduction to several normal-lived sister lines (Ra and Rb) so as to produce several selected long-lived sister lines (La and Lb). The long-lived La and Lb sister lines had statistically identical longevity phenotypes and paraquat resistance phenotypes; however, we noticed some statistically different responses of the two strains at the biochemical level. Extensive work with the La strain showed that transcriptional alterations in antioxidant gene expression are robustly associated with its extended longevity. We decided to critically test the assumption of phenotypic equivalence by subjecting the Lb strain to the same series of molecular assays as was the La strain. The two sister strains are characterized by significantly different mechanisms and patterns of antioxidant gene expression, antioxidant enzyme activity, and oxidative damage. We find that the Lb strain appears to depend on the transcriptional activation of different genes than does the La strain, and on a post-translational up-regulation of at least one other antioxidant defense gene. The phenotypic equivalence observed at the organism level need not hold at the molecular genetic level. This finding suggests that there is more than one molecular mechanism by which antioxidant defense genes can bring about an increased resistance to oxidative stress. The theoretical and empirical implications of these findings are discussed.

Genomic plasticity, energy allocations, and the extended longevity phenotypes of Drosophila

The antagonistic pleiotropy theory of the evolution of aging is shown to be too simple to fully apply to the situation in which Drosophila are selected directly for delayed female fecundity and indirectly for extended longevity. We re-evaluated our own previously reported selection experiments using previously unreported data, as well as new data from the literature. The facts that led to this re-evaluation were: (1) the recognition that there are at least three different extended longevity phenotypes; (2) the existence of metabolic and mitochondrial differences between normal- and long-lived organisms; and most importantly; (3) the observation that animals selected for extended longevity are both more fecund and longer-lived than their progenitor control animals. This latter observation appears to contradict the theory. A revised interpretation of the events underlying the selection process indicates that there is a two-step change in energy allocations leading to a complex phenotype. Initial selection first allows the up-regulation of the antioxidant defense system genes and a shift to the use of the pentose shunt. This is later followed by alterations in mitochondrial fatty acid composition and other changes necessary to reduce the leakage of H(2)O(2) from the mitochondria into the cytosol. The recaptured energy available from the latter step is diverted from somatic maintenance back into reproduction, resulting in animals that are both long-lived and fecund. Literature review suggests the involvement of mitochondrial and antioxidant changes are likely universal in the Type 1 extended longevity phenotype.

Larval regulation of adult longevity in a genetically-selected long-lived strain of Drosophila

Our previous work has shown that the major genes involved in the expression of the extended-longevity phenotype are located on the third chromosome. Furthermore, their expression is negatively and positively influenced by chromosomes 2 and 1, respectively. In this report we show that the expression of the extended-longevity phenotype is dependent on the larval environment. A controlled chromosome substitution experiment was carried out using a strain selected for long life (L) and its parent (R) strain. Twenty different combinations of the three major chromosomes were conducted and their longevities were determined under both high (HD) and low (LD) larval density conditions. The extended-longevity phenotype was only expressed under HD conditions. The chromosome interactions were not apparent under LD conditions. Density-shift experiments delineate a critical period for expression of the extended-longevity phenotype, extending from 60 h after egg laying (AEL) to 96 h AEL, during which the developing animal must be exposed to HD conditions if the extended-longevity phenotype is to be expressed. The change from HD to LD conditions is accompanied by statistically significant increases in body weight. The possible role of a dietary restriction phenomenon is examined and the implications of these findings discussed. It is now apparent, however, that the extended-longevity phenotype in Drosophila is a developmental genetic process.

Metabolic Alterations and Shifts in Energy Allocations Are Corequisites for the Expression of Extended Longevity Genes in Drosophila

Evolutionary theories suggest that the expression of extended longevity depends on the organisms ability to shift energy from reproduction to somatic maintenance. New data led us to reexamine our older data and integrate the two into a larger picture of the genetic and metabolic alterations required if the animal is to live long. Our Ra normal‐lived control strain can express any one of three different extended longevity phenotypes, only one of which involves significant and proportional increases in both mean and maximum longevity and thus a delayed onset of senescence. This phenotype is dependent on the up‐regulation of the antioxidant defense system (ADS) genes and enzymes. Animals that express this phenotype typically have a pattern of altered specific activities in metabolically important enzymes, suggesting they are necessary to support the NAD+/NADP+ reducing system required for the continued high ADS enzyme activities. Fecundity data suggests that the energy required for this higher level of somatic maintenance initially came from a reduced egg production. This was only transient, however, for the females significantly increased their fecundity in later generations while still maintaining their longevity. The energy required for this enhanced fecundity was probably obtained from an increased metabolic efficiency, for the mitochondria of the La long‐lived strain are metabolically more efficient and have a lower leakage of reactive oxygen species (ROS) to the cytosol. Selection pressures that do not lead to these shifts in energy allocations result in extended longevity phenotypes characterized by increased early survival or increased late survival but not by a delayed onset of senescence.

Immunological Confirmation of Elevated Levels of CuZn Superoxide Dismutase Protein in an Artificially Selected Long-Lived Strain of Drosophila melanogaster

Oxidative stress-induced damage is a major causal factor leading to the loss of function characteristic of the aging process. Various antioxidant defenses are marshalled by the organism so as to combat this oxidative damage and delay the onset of senscence. CuZnSOD is one of the major antioxidant enzymes and has been shown to play an important role in the extended longevity of Drosophila melanogaster. Although assays exist with which to measure the CuZnSOD RNA prevalence and enzyme activity, there existed no antibodies that permitted the measurement of the actual amount of Drosophila enzyme protein present. Development of such a tool would enhance our ability to understand mechanisms of antioxidant gene expression in this organism. We have developed a polyclonal antibody against synthetic SOD peptides that is specific for Drosophila CuZnSOD as shown by Western blots. It is very sensitive when tested against native Drosophila CuZnSOD protein. Its use in our experimental system confirms the prior RNA and enzyme activity measurements that indicate that our genetically selected long-lived strain has significantly higher levels of CuZnSOD protein than does the appropriate control strain.

Chromosomal localization and regulation of the longevity determinant genes in a selected strain of Drosophila melanogaster.

A controlled chromosome substitution experiment was performed on a strain (NDC-L) selected for long life to determine if the genes responsible for the extended-longevity phenotype could be localized to any particular chromosome(s). All 27 different possible combinations of the three major chromosomes of Drosophila melanogaster were constructed and longevities were determined on 3875 individual animals of both sexes and analysed. The results are statistically significant and demonstrate that mean longevity is specified primarily by recessive genes on the third chromosome (c3). The extended longevity phenotype (ELP) is only expressed in those lines which are homozygous for the NDC-L type c3. Loci on the first (c1) and second (c2) chromosomes interact, both positively (c1) and negatively (c2), respectively, such that c1 represses c2 which in turn represses c3. The ELP is fully expressed in the mutual presence and mutual absence of c1 and c2. The significance of these results is discussed in the context of broader categories of molecular genetic mechanisms suggested previously to be involved in the modulation of longevity in Drosophila.