by Nick Lane
There are good grounds for thinking that stepping up resistance to oxidative stress is a common means of slowing ageing in all animals, although the effects may be less pronounced than in worms. I shall outline three pieces of interlinked evidence that I find convincing: first, the effects of SOD and catalase; second, the effect of DNA-repair enzymes; and third, the apparent mechanism of calorie restriction.
In 1994, William Orr and Rajinder Sohal, at the Southern Methodist University in Dallas, reported in Science the first direct evidence that increased levels of antioxidants could slow down the rate of ageing. Orr and Sohal genetically engineered Drosophila so that they produced extra cytosolic SOD (see Chapter 10) and catalase. The genetically engineered (transgenic) flies lived up to a third longer than normal ones. Of particular importance, neither enzyme by itself had any effect on lifespan — the two work together and their production needs to be coordinated. When produced together, the extension in both mean and maximum lifespan
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was linked with a greater resistance to ionizing radiation, and less oxidative damage to DNA and proteins. The transgenic flies were also more active in old age, equating to a 30 per cent increase in their lifetime energy potential (equivalent to a greater number of heartbeats). The effect of increasing SOD and catalase levels is therefore not simply a matter of decreasing the rate of living: the transgenic flies lived at the same rate as normal flies, but for longer. More recent studies have achieved lifespan extensions of 50 per cent, using improved genetic engineering techniques.3
The requirement for SOD and catalase to work together hints at the importance of antioxidant networking; and of course SOD and catalase do not work in isolation. Stress-resistance is the product of many factors, including efficient protein turnover and DNA repair — our second example.
The importance of DNA repair in people is highlighted by a distress-ing example where it is defective — Werner’s syndrome. This rare genetic disorder causes people to age at an accelerated rate. Their hair goes white and they suffer from various symptoms of premature ageing, including cataracts, muscle atrophy, bone loss, diabetes, atherosclerosis and cancer.
Those afflicted usually die of age-related diseases such as heart disease and cancer by their early 40s. In the struggle to understand this intractable syndrome, scientists had hoped to learn something about the ageing process in general, and so help everyone; but, in fact, the spectrum of ailments is not really representative of normal ageing, and in the end frustrated researchers dismissed Werner’s syndrome as a ‘caricature of ageing’. Then came a big step forward: in 1997, the gene responsible for the syndrome was isolated. It encodes an unusual dual-function enzyme: one enzymatic function unwinds the DNA double helix (a helicase action), while the other excises and replaces any erroneous letters (an exonuclease action). The protein thus repairs errors in DNA caused by replication, recombination or spontaneous mutations — many of which are produced by oxygen free radicals.
Most people suffering from Werner’s syndrome appear to have a 3 In all these studies the cytosolic form of SOD was overproduced, rather than the mitochondrial form. This might be an important distinction. Mitochondrial SOD can protect both the inside of the mitochondria and the surounding cytosol from superoxide radicals, as most superoxide radicals escape from the mitochondria. In contrast, cytosolic SOD can only provide partial protection to the cytoplasm. To the best of my knowledge, transgenic Drosophila, engineered to overproduce mitochondrial SOD and catalase, have not yet been produced. I would be surprised if they did not have even longer life extensions.
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mutation in the helicase part of the enzyme and cannot repair damaged DNA properly. Among other things, this mutation increases vulnerability to ultraviolet radiation, which damages DNA. Such vulnerability is the opposite of stress-resistant organisms, which are able to withstand high levels of ultraviolet radiation. We might predict, then, that DNA-repair enzymes should be among those whose manufacture is increased in long-lived mutant organisms, such as daf-2 mutant nematodes. Although this has not been formally demonstrated, we do know that stress-resistance and longevity are associated with better DNA repair. If cells from animals with varying maximal lifespans are cultured, and then exposed to ultraviolet rays or other types of stress (such as hydrogen peroxide), we can measure the amount of DNA repair taking place. Such studies have generally shown a positive correlation between the maximum lifespan of animals and their ability to repair DNA.
These two examples suggest that lifespan is modulated by stress-resistance. Stress-resistance, in turn, is mediated (at least in part) by changes in the levels of stress proteins such as SOD, catalase, metallothionein and DNA-repair enzymes. The master-switch role of daf-16
shows that the expression of these genes is coordinated in simple animals.
My third example, calorie restriction, suggests that, even in complex animals, the stress response can be coordinated by a relatively simple switch. The profile of the response itself, however, is not always analogous to that in simple organisms.
We are only just beginning to unravel the mechanisms through which calorie restriction (see Chapter 11, page 229) extends lifespan. The mechanism certainly involves the number of calories, rather than a reduction in any particular source of calories, such as fats or carbohydrates. In general, calorie-restricted diets cut the overall intake of calories by about 30–40 per cent, while maintaining a balanced diet in other respects. It is therefore not the same as malnutrition or starvation (which for most people would involve a calorie restriction of between 50 and 60 per cent).
Ever since the effects of calorie restriction were first noticed in the 1930s, researchers have interpreted them in terms of the rate-of-living theory — eating less lowers the metabolic rate and oxygen consumption.
In this light, calorie restriction has always seemed inherently futile, even though lifespan can be extended by 30 to 50 per cent in virtually all animals: who wants 50 per cent more life if the price we must pay is not just serious dieting, but also 50 per cent less energy? Even a couch potato
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might prefer to live fast and die young. Yet calorie restriction is turning out to be far more interesting than anyone had imagined. For a start, it does not necessarily reduce metabolic rate at all. When measured in terms of the metabolic rate per kilogram of lean weight, oxygen consumption may actually increase. Calorie restriction can therefore increase the lifetime energy potential — we get more heartbeats. For male rats, this increase is in the order of 50 per cent. The effects of calorie restriction are mediated by concerted changes in gene expression. The benefits are well worth queuing for. In all animals studied so far, calorie restriction delays ageing, not just the timing of death. This is true for more than 80 per cent of the 300 indices of ageing tested in rodents, including physical activity, behaviour, learning, immune responsiveness, enzyme activity, gene expression, hormonal action, protein synthesis and glucose tolerance.
The net effect of calorie restriction is to increase stress-resistance.
Blood glucose levels fall, and this in turn lowers insulin levels. Metabolism is switched away from sex and towards bodily maintenance. Resistance to oxidative stress increases, especially in tissues where damage is normally highest, such as the brain, heart and skeletal muscle. Exactly how this effect is achieved is, curiously, an open question. Consistent changes in antioxidant enzymes are yet to be reported. We might predict that a spectrum of stress-related genes would be activated, including those for SOD, catalase, metallothionein and the DNA-repair enzymes, but this is not consistently the case.4 One is tempted to say ‘who cares’, if the benefits apply to us; but again, we are not certain if they do. Any direct trial would take decades to complete.
In 1987, the National Institute of Ageing in Baltimore, Maryland and the Wisconsin Regional Primate Research Center in Madison, began two trials in primates — 200 rhesus and squi
rrel monkeys. In April 2001, the Wisconsin team, headed by Richard Weindruch, published an interim report on the effects of calorie restriction on gene expression, and thus the types and levels of proteins synthesized, in rhesus monkeys. To measure the effects, they determined which out of a selected sample of 7000
genes were switched on and which were switched off in the calorie-4 Although calorie restriction does not produce an ongoing stress response in rats, calorie-restricted rats are better able to mount a stress response to heat shock (where the animal is subjected briefly to a higher temperature than normal) than are normal ageing rats. In other words, in normal ageing, the ongoing stress response to mitochondrial leakage blunts the acute response to sudden stresses (such as heat shock), whereas calorie restriction reins back the chronic stress response and so sharpens the response to sudden stresses.
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restricted monkeys and in a parallel group of fully fed monkeys of the same age. Their findings were surprising and intriguing. Although stress-resistance was increased in the calorie-restricted group, as expected, there was hardly any difference in the level of production of stress proteins in fully fed ageing monkeys, compared with middle-aged monkeys subjected to calorie restriction. Instead, calorie restriction had three big effects. First, it strengthened the internal structure of cells, more than doubling the rate of synthesis of almost all structural proteins. Second, it lowered the synthesis of proteins that promote inflammation, such as tumour necrosis factor (TNF-␣) and the enzyme nitric oxide synthase.
Third, it lowered the expression of genes responsible for oxygen respiration, in particular cytochrome c (to 1/23 of normal!). This last effect is consistent with a reduction in the metabolic rate — live slow, die old.5
Far from gearing up to combat stress, the shifts in gene expression in calorie-restricted monkeys almost seem to skirt the issue. If anything, the expression of stress proteins goes down rather than up. A possible explanation is as follows. We already live twice as long as the chimpanzees, which in turn live longer than rhesus monkeys. The mechanism underpinning our extra years is likely to be better stress-resistance. As we shall see in the next chapter, age-related diseases also produce a stress response, which draws on many of the same gene products. For example, ageing rhesus monkeys increase the synthesis of at least 18 stress proteins, including metallothionein and various DNA-repair enzymes. Unless calorie restriction is started very early in life, it is hard to see how the imposition of one stress response over another could extend our lives much further. Instead, in rhesus monkeys at least, the changes in gene expression brought about by calorie restriction in middle-age seem designed to lower the level of metabolic stress imposed on the system. In other words the important parameter is not stress-resistance per se, but the degree of stress on the system. This can be lowered by improving stress-resistance, or by lowering the level of stress imposed.
5 There is another intriguing possibility here. Release of cytochrome c from mitochondria initiates apoptosis, or programmed cell death. It is conceivable that lower levels of cytochrome c in cells might cut the number of cells that undergo apoptosis in ageing organs.
Function would then be maintained for longer. This is pure speculation, but I daresay Richard Weindruch will be looking into the possibility.
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It is time to pause and take stock. The disposable soma theory argues that longevity is a trade-off between the resources committed to reproduction and those committed to bodily maintenance. We maintain our bodies in two ways — by preventing damage from happening in the first place, and by repairing any damage actually done. The amount of damage prevented depends, in large part, on the intrinsic rates of production and elimination of free radicals. The amount of damage repaired depends on the rate of turnover of DNA, membranes and proteins, in which damaged molecules are replaced new for old. The repair work can only be performed efficiently if the machinery is not itself damaged. Free radicals are undiscriminating, and damage the repair machinery, and the DNA encoding it, as easily as anything else. In the end, then, poor prevention leads to poor repair.
The rate of ageing is determined by the level of resources committed to prevention and repair. These resources are programmed genetically, but their deployment is influenced by environmental factors, such as the availability of food or the likelihood of sex. The switch between sex and longevity is conserved in nematodes, Drosophila, rats and humans, but the genetic response to the switch varies. In nematodes, longevity is apparently achieved by increasing production of stress proteins; in rhesus monkeys, by suppressing oxygen metabolism, and so metabolic rate.
Given the parsimony of natural selection, the response elicited is always likely to be the most cost-effective, and so will depend on the level of stress-resistance already built into the system. Nematodes have low levels of a small number of different stress proteins, and so can easily up their levels. Rhesus monkeys, on the other hand, have much higher levels of numerous different stress proteins. Rather than having to make more of all of these, it is less costly to suppress metabolism instead. Regardless of the actual mechanism, the outcome in every case is to reduce the stress on the system, which enables animals to weather out the hard times and breed again when conditions improve. Thus, stress can be avoided either through the countering action of stress proteins, or by lowering the rate of respiration and the intensity of inflammation. Either way, the secret of a long life is low metabolic stress.
Similar mechanisms appear to have underpinned the evolution of longevity in species freed from predation and starvation. Lifespan reflects the rate of accumulation of damage, which varies with the metabolic rate, the production and elimination of free radicals, and the capacity for repair. Seen in this light, metabolic rate might even have been a factor in
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the evolution of gigantism, discussed in Chapter 5. Larger size permits a lower metabolic rate, hence a longer lifespan. In modern species, high oxygen levels reduce lifespan; in Carboniferous times, then, it is conceivable that greater size might have been a means of dealing with high atmospheric oxygen by lowering the metabolic rate. Be that as it may, the consistent factors underpinning lifespan extension in all cases are efficient prevention and repair of damage caused by free radicals. We can reasonably conclude that oxygen free radicals are a primary cause of ageing, and that ageing can be slowed down, in principle, by altering the expression of genes responsible for bodily maintenance.
If ageing really is caused by free radicals, we need to answer two difficult questions. First, how is ageing apparently deferred until after sexual maturation, a period, for us, of three decades or more? Second, how do some cells, such as bacteria, cancer cells and sex cells, avoid ageing?
Indeed, it is not just single cells that avoid ageing: some animals, such as Hydra (a small, tentacled, freshwater relative of the sea anemone) appear to escape ageing altogether. They live in shallow, oxygenated waters and show no signs of senescence. How do they contrive to avoid the damaging effects of free radicals?
The first question — how is ageing deferred — can be answered by thinking about the peculiar nature of the mitochondria, and the way in which they operate in the cell. Recall that mitochondria were once free-living bacteria, which eventually evolved into the organelles responsible for oxygen metabolism in plants and animals alike. We saw in Chapter 8
that mitochondria have retained vestiges of their independent past, in particular their own DNA and their ancestral way of dividing, simply splitting in two by binary fission: an asexual process. Mitochondria are therefore asexual genetic systems that replicate themselves within a sexually reproducing organism. Their own DNA is critical to their function. If their DNA is damaged, mitochondria cannot work: it is impossible to build a mitochondrion from nuclear genes alone. Thus, all oxygen-breathing animals are totally dependent on the integrity of mitochondrial DNA. If damaged mitoch
ondria are passed on to the next generation, the offspring will be compromised or die.
The mitochondrial theory of ageing was first proposed by Denham Harman, author of the free-radical theory, as a refinement of his original
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theory. His ideas were later developed by Jaime Miquel, at the Institute of Neurosciences in Alicante, Spain, and others. Essentially, the idea is as follows. Free radicals are formed continuously in the immediate vicinity of mitochondrial DNA. Mitochondrial DNA is naked — it is not coated in proteins — and so is exposed to attack. Even worse, repair of mitochondrial DNA is said to be rudimentary. As a result, errors accumulate quickly. Because mitochondria very rarely indulge in ‘sex’ by fusing together, these errors cannot be cleansed by recombination and so they persist. The persistence of mutations is confirmed by the rapid mutation rate of mitochondrial DNA, compared with nuclear DNA, over evolutionary time.6 We are left in an odd situation, in which the most toxic compartment of the cell shelters the most vulnerable DNA. A vicious circle develops. Mutated mitochondrial genes direct the production of faulty respiratory proteins, which leak more free radicals, causing more DNA damage. The spiralling descent seems to lead inexorably to ageing and death. Indeed, it is astonishing that we survive as long as we do.
In 1988, Christoph Richter, Jeen-Woo Park and Bruce Ames, at Berkeley, measured the amount of damage to mitochondrial DNA compared with nuclear DNA (which is, of course, cordoned off behind its own membranes and wrapped in proteins, at a safe distance from the mitochondria). Their findings seemed to provide good support for the mitochondrial theory of ageing: the apparent load of oxidative damage to mitochondrial DNA was nearly 20 times that of nuclear DNA. During the 1990s, several research teams attempted to replicate these early results.
Extrapolated from some 20 papers, the scatter of results almost defies belief. According to Bruce Ames and Kenneth Beckman, in a refreshingly honest reappraisal published in 1999 (it is always good to see scientists rising above their own theories) the range of estimates of oxidative damage spans more than 60 000-fold! There is no suggestion that anyone is fabricating data — it is simply that even the most sophisticated modern 6 Mitochondrial DNA evolves over thousands of years, but is not recombined through sex.