Summary of genetic interventions testing the relationship between resistance to oxidative stress and altered longevity
Manipulation Genes involved Effect on life span Effect on stress resistance Reference
Selection for long life
CuZnSOD, MnSOD Increased
Catalase reduced Cat
Catalase increased SOD1 reduced SOD1 increased
SOD1 & cat increased
SOD2 reduced SOD2 increased
Increased (some strains) Increased
No increase over SOD alone
Increased ox stress, also increased starvation & dessication resistance No data
Increased (some strains) Increased
Decreased No data No effect
Arking et al. (2000a)
Mackay & Bewley (1989)
Phillips etal. (1989)
Parkes et al. (1998)
Kirby et al. (2002) Sun et al. (2002) Mockett et al. (1999)
gene, and of several heat shock protein (hsp) genes. Both of these gene sets are involved in stress resistance. The other QTLs seem to be involved in maintaining female fertility. (One of these other QTLs appears to involve the ecdysone receptor (EcR) gene, which is involved in reproductive processes; see the section on Reproductive Effects.) The several genes involved exert their effects over the first six weeks of life but not thereafter. There are other minor contributors to the extended longevity phenotype (Curtsinger et al., 1998), but the available data strongly indicates that the La strains live long primarily because of a specific up-regulation of anti-oxidant defense genes.
This finding supported our candidate gene approach in which we assayed the quantitative changes in the mRNA levels and the antioxidant enzyme activity levels of several loci during the development and early adult life of the normal-lived Ra and long-lived La strains (Dudas and Arking, 1995). In addition, we used antibodies to measure the actual amount of CuZnSOD protein present in these strains (Hari et al., 1997). The mRNA data demonstrate that, at day 5 in the L strain, there appears to be a coordinately regulated significant increase in the mRNA levels of CuZnSOD, CAT, and xanthine dehydrogenase (XDH). There is a nonsignificant increase in glutathione-S-transferase (GST) mRNA during the same time period. These increases in mRNA levels are accompanied by significant increases in the enzyme activity of CuZnSOD, CAT, and GST. Other experiments showed that the amount of SOD-specific protein is proportionately increased in the La strain during the same time period (Hari et al., 1998). Thus it seems reasonable to conclude that these alterations in gene expression in the long-lived strain are the result of a transcription-level change that alters the enzymatic arsenal available to the organisms. But these changes in gene expression have biological meaning only if they reduce the amount of oxidative damage in the long-lived animals.
The higher level of antioxidant gene expression and enzyme activities do in fact bring about a life-long and significant reduction in the levels of the most common oxidative damages in proteins or lipids in the long-lived L strain. There exists an inverse correlation between the levels of antioxidant enzyme activity (Dudas and Arking, 19956) and the levels of oxidative damage (Arking et al., 2000a). It seems reasonable to conclude that these animals developed the ability, as a consequence of artificial selection, to turn on a regulatory process that coordi-nately activates the antioxidant defense genes early in life, thereby protecting the animals against the oxidative damage to vital molecules and thus delaying the onset of senescence until their antioxidant defenses fall to normal levels. Reverse-selecting these long-lived strains for shortened longevity (Arking et al., 2000a,b) reverts their antioxidant gene expression patterns to control levels. Only the antioxidant genes (and certain other enzymes operationally connected to them) show these correlated and coordinate changes in gene expression. Other metabolically important enzymes which do not affect the antioxidant proteins do not show any significant changes as a result of selection (Arking et al., 2000a). The reversal in life span was accompanied by a specific reversal in the expression of only the antioxidant genes and genes necessary to their function. Incidentally, an independent replicate strain (Lb) has the same longevity patterns as does the La line but uses different specific patterns of antioxidant gene expression (Arking et al., 2002b). One interpretation of these data is that the overall oxidative stress level of the organism may be more important than which particular antioxidant gene is over-expressed. Another is that different signaling pathways may be involved.
Taken all together, this series of experiments reveals the existence of a causal relationship between antioxidant gene expression, oxidative stress resistance, levels of oxidative damage, and longevity in these selected strains of Drosophila.
In a parallel, simultaneous, and independent experiment, Michael Rose (1984) used a different wild-type progenitor stock but the same indirect selection protocols as used for the Wayne State University (WSU) lines to create a set of long-lived strains termed the University of California-Irvine (UCI) long-lived lines. Interestingly enough, the physiological traits associated with the UCI selected lines overlap those associated with the WSU lines, for both sets of strains of strains are significantly more resistant to environmental stresses than their respective controls. The two are resistant to a different spectrum of stressors, which may well be the result of the different genetic backgrounds used in their different progenitor stocks. The UCI lines are resistant to starvation, dessication, and oxidative stress; and selection for increased starvation or dessication resistance was later shown to lead to increased longevity (Rose et al., 1992; Harshman et al., 1999). The WSU lines are mildly resistant to dessication but mostly resistant to oxidative stress (Force et al., 1995). Although several kinds of stress resistance are associated with extended longevity, it may not be a coincidence that the only common stress resistance in all these strains is that of oxidative stress.
Genetic data generally support these selection experiments. Phillips et al. (1989) created a CuZnSOD-null mutant of Drosophila and showed that the absence of this enzyme activity significantly decreased viability and longevity. Subsequent analysis by the same group (Parkes et al., 1998) showed that the absence of the CuZnSOD gene has a number of important pleiotropic effects such as (1) adult sensitivity to paraquat, (2) male sterility, (3) female semisterility, (4) adult hyperoxia sensitivity, (5) larval radiation sensitivity, (6) developmental sensitivity to glutathione depletion (an important antioxidant molecule), and (7) adult life-span reduction. Before one could confidently interpret these results, it was necessary to determine whether these alterations stemmed from an increase in the rate of aging or from some abnormal pathology. An experiment was done showing that these CuZnSOD-null mutants showed an acceleration, relative to the wild-type control, of the normal age-related temporal changes in the expression of certain other genes (Rogina et al. 2000). Since the acceleration in the temporal expression of these other genes was proportional to the shortened life span, then this was interpreted as showing that the shortened life span of the CuZnSOD-null mutants is due, not to an abnormal pathological process, but to an increase in the rate of aging. This interpretation suggests that the aging rate is directly proportional to the animals' level of antioxidant capacity and resistance to oxidative stress, a finding in keeping with the selection results discussed above.
In contrast to the SOD data, acatalesemic mutants of Drosophila are essentially normal when reared under standard conditions as long as they have at least 3% of the normal catalase expression level (Mackay and Bewley, 1989), a finding consistent with the transgene work done on this gene (Orr and Sohal, 1992), which suggests that catalase is not normally a rate-limiting factor in longevity.
Transgenic technology, which allowed the insertion of an extra gene(s) into an otherwise normal organism, was early used to test the effects of increasing the level of CuZnSOD gene expression on the longevity and aging of the altered animals. The early experiments inserted single copies of either CuZnSOD or catalase into the test organisms, but their results were inconclusive for a variety of reasons. However, the tandem over-expression of both CuZnSOD and catalase in the same animal did extend median and maximum longevity by up to 34% in some lines while simultaneously retarding oxidative damage and increasing oxidative resistance (Orr and Sohal, 1994; Sohal et al., 1995). Sun and Tower (1999) used a controllable transgenic system that allowed them to control when CuZnSOD over-expression would take place in adult flies. This system allowed increases in mean life span of up to 48%. These two transgenic alterations of gene expression ostensibly affected all tissues of the organism at all stages. But there is much information showing that most genes have characteristic tissue and stage-specific expression patterns. Thus it was important when Parkes et al. (1998, 1999) showed that a GAL4-UAS transgene expression system which selectively targeted CuZnSOD expression to the adult motor neuron was capable both of (a) restoring the normal adult life span of CuZnSOD-null mutants and (b) extending by 40% the adult life span of an otherwise wild-type fly. Over-expression of CuZnSOD in the adult central nervous system, adult muscle or larval body has no effect on adult longevity. It turns out that adult Drosophila have a surprising lack of CuZnSOD activity in their central nervous system relative to the rest of the body (Klichko et al., 1999). It may be that the flies' motor neurons may have the lowest age-related failure threshold of the whole body and would normally be the first critical tissue to fail. Thus using transgenes to increase their resistance to oxidative stress may have the effect of postponing the age at failure of this critical tissue and thus result in lengthening the life span.
Scientific progress does not occur in a straight line. The validity of the three transgene experiments described above have been challenged by two of the researchers involved on the basis that an increased longevity was observed only in those cases where the control flies had a comparatively short life span (Orr and Sohal, 2003). In their rethinking of the matter, shorter lived control lines are helped by CuZnSOD over-expression, but genetically robust controls showed little or no effect. This reassessment casts doubt only on the efficacy of increasing life span by increasing only CuZnSOD expression; it does not affect experiments showing that suites of antioxidant genes are over-expressed in long-lived strains (e.g., Table 25.3) or that different antioxidant genes act together in a cooperative manner in the fly (Missirlis et al., 2001) or experiments involving robust control flies. It is a likely possibility that the reason for the failure of some transgenes to significantly affect the life span of some flies is that an organism with an inefficient metabolism and low levels of available ATP is simply not in a position to effectively reallocate the energy saved due to lowered oxidative damage levels to increased somatic maintenance. In the La strain, the effective use of the increased antioxidative defense enzymes requires the simultaneous alterations of metabolism (e.g., shifting from glycolysis to the pentose shunt) so as to support the enzyme functions, as well as mitochondrial changes that yield an increased efficiency and thus increase the levels of available ATP (Arking et al., 2002). Pathways—both genetic and metabolic—need to be changed if an animal is to live long. Altering the expression of one gene without altering the necessary pathways may bring about only a weak effect.
MnSOD is the mitochondrial version of superoxide dismutase. Given the crucial role of mitochondria in energy metabolism and ROS generation, it seemed logical that this gene product would likely play an important role in modulating the life span. This assumption is borne out by our selection data (Arking et al., 2000a) and by the transgenic data of Sun et al. (2000). These researchers used their controllable transgene system to induce the over-expression of MnSOD only in adult flies but not in the developmental stages, thus avoiding complications in the analysis. They reported that MnSOD showed increases in expression of up to 75%. This yielded a 33% increase in mean life span and a 37% increase in maximum life span. The simultaneous over-expression of both CuZnSOD and MnSOD led to a complicated situation wherein each transgene partially inhibited the over-expression of the other, but nonetheless still resulted in the two genes having partially additive effects on life span (Sun et al., 2004). Phillips and his colleagues (2000) have also used transgenes to over-express the MnSOD
gene in wild-type animals and find that they obtain life span extensions of about 30%. They also note that the over-expressed MnSOD gives an incomplete rescue of the CuZnSOD-null mutant, thus establishing that the two enzymes operate in functionally different compartments. Conversely, destroying the MnSOD mRNA and thus silencing this one gene in a normal animal results in a disruption of mitochondrial function, an increased sensitivity to oxidative stress, and a striking ~80% reduction in mean and maximum life span (Kirby et al., 2002). In contrast, Mockett et al. (1999) reported that their transgenic MnSOD lines did over-express MnSOD mRNA, protein and enzyme activity but did not show an increased life span relative to the controls. This finding may well have to do with the genetic background of their control strain; if so, it represents a limitation, but not a refutation, of the ability of any one specific antioxidant gene to increase life span for the reasons presented above.
Given the complexity of the stress resistance process, it is inevitable that other genes are involved. Glutathione is perhaps the most abundant low molecular weight antioxidant present in the animal and represents a potential clue to other candidate genes. Mockett et al. (1999) over-expressed the glutathione reductase gene in transgenic Drosophila and obtained up to 100% overexpression of the enzyme. Longevity was significantly enhanced under hyperoxic conditions but not under normoxic condtions, suggesting that glutathione reduc-tase may not be a rate-limiting factor in antiaging defenses under normal conditions but may well be one when the level of oxidative stress is elevated. This is very similar to the effects of glutathione-S-transferase on nematode longevity (Leiers et al., 2003).
Another set of candidate genes are those which regulate the expression of the antioxidant structural genes. Four different mutant searches have identified such genes. In the first, Lin et al. (1998) did P-element mutagenesis of the 3rd chromosome and screened for long life (relative to the white control strain) at 29oC. One homozygous mutant, named methuselah (mth), lived up to 35% longer and was more resistant to paraquat, starvation and high temperature. The mth gene appears to code for a transmembrane G protein-coupled receptor presumably involved in the regulation of stress response genes. Other data suggest it may be a negative regulator of these genes. Recent data suggest that the ligand for this mth receptor is the stunted (sun) protein, but the functional pathways involved are not yet known. The second search also used P-element mutagenesis but focused on the second chromosome and identified two groups of transacting mutants, one of which acted as if they were normally positive regulators of CuZnSOD and catalase in wild-type animals and the other of which acted as if they were normally negative regulators. The third approach showed that up-regulation of the JNK signaling pathway made the animals much more resistant to oxidative stress while increasing both their mean and maximun life spans (Wang et al., 2003). Finally, the fourth approach used microarrays to conduct a genome-wide search for genes which responded to chronic exposure to oxidative and other stresses (Giradot et al., 2004). The data show the existence of both general and specific responses to these different stressors, and indicate the existence of a complex interlocking network of stress response genes. The point is that four independent experiments have demonstrated that single genes seem to extend longevity by up-regulating the animal's ability to withstand various types of stress, and that these individual genes may well be components of a larger network of stress response genes.
The heat-shock protein (HSP) genes were initially found in Drosophila. Much work has demonstrated that these proteins have significant effects on stress resistance and longevity in all organisms. There are two complementary findings: first, the expression of the HSP genes is affected by aging; and second, the up-regulation of some (but not all) of these genes can significantly affect longevity. Aging animals usually express abnormal patterns of HSP expression relative to young animals (Niedzwiecki and Fleming, 1990). Two reports (Wheeler et al., 1995; King and Tower, 2000) show that hsp22 mRNA is up-regulated during aging, particularly in the head. In addition, an earlier onset of hsp22 and hsp23 mRNA accumulation in Drosophila flies selected for increased longevity was also reported (Kurapati et al., 2000), suggesting a possible correlation between hsp22 levels and longevity. Using genetic techniques to overexpress hsp22 in motorneurons led to a 30% increase in longevity and stress resistance (Morrow et al., 2004a). Conversely, knocking out the hsp22 gene so that flies could not express the HSP22 protein led to a 40% decrease in their longevity, coupled with an increased sensitivity to stress (Morrow et al., 2004b). Finally, a ubiquitous over-expression of hsp22 throughout the entire body also led to a reduction in life span as well as an increased sensitivity to heat and oxidative stress (Bhole et al., 2004). Looking at the data as a whole, we can see that increased longevity and stress resistance depend not just on the over-expression of the hsp22 gene, but on its being expressed in the appropriate tissues at the appropriate time and in a balanced manner relative to the expression of stress resistance genes in other tissues. Failure to achieve this balanced expression harms, rather than helps, the organism. This may come about because an unbalanced expression of one stress response gene may inappropriately down-regulate other stress response genes. This hypothesis assumes that there exists a stress response gene network of some sort, as has been noted in the nematode (Morley and Miramoto, 2004). Empirical evidence to support this assumption is provided by a DNA hybridization screen which identified at least 13 genes which were activated by exposure to heat, oxidants, and starvation (Wang et al., 2004). The involvement of the HSP in extended longevity is shown by the fact that a beneficial effect of HSPs on aging and stress resistance is observed when organisms are preconditioned by exposure to a mild stress before being exposed to a subsequent damaging stress (hormesis: see LeBourg et al., 2001; Hercus et al., 2003).
Taken all together, the different types of experiments discussed above show that (1) there are a variety of different but interacting stress resistance pathways and (2) almost all tested long-lived animals are also stress-resistant. Both Parsons (2003) and Rattan (2004) have long argued the existence of a tight relationship between stress resistance and longevity. The reciprocal experiments in which one directly up-regulates a known stress response gene, such as hsp26 or hsp27, have been done and results in flies having a significant increase in both stress resistance and longevity (Wang et al., 2004). This experiment shows that at least some stress resistant animals are also long-lived. Taken together, the several sets of experiments verify the fundamentals of Parsons's (2003) argument.
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