Uncertainty has not stopped many neo-Cornarists from committing themselves to lives of rigorous dieting. Caloric restriction has become a health fad like any other, with its own books and gurus. The diet usually consists of about a thousand calories per day, which is necessarily supplemented with a battery of vitamins and minerals. A thousand calories is about the minimum number needed to sustain the life of an average-sized man, though not enough to sustain his sex drive (or, to judge by pictures, his sex appeal). Whether these ultra-puritans will reap their reward is an open question. The severe caloric restriction experienced by the Dutch population during the Hongerwinter of 1944–45 certainly had no detectable beneficial effect on the long-term mortality rates of the survivors, but it could be argued that it takes decades of near-starvation for its virtues to become apparent.
Caloric restriction works in rats, mice, fruit flies and nematode worms. Why it does so remains mysterious. One explanation goes back to the deleterious effects of reproduction. Caloric-restricted animals have fewer offspring than those allowed to eat all they want; perhaps the energy savings that come with not reproducing are enough to ensure longevity. But there is probably more to it than this. In caloric-restricted fruit flies not only are the genes involved in reproduction largely switched off, but those involved in resistance to infection (the fly’s immune system) are turned on, so that immunity proteins are produced at higher levels than they would be normally. This result suggests at least two reasons for the longevity of caloric-restricted animals. There may be many others besides. About two thousand of the fifteen thousand genes in the fly’s genome show a response to caloric restriction. It is quite possible that caloric restriction works its magic by the cumulative benefits of dozens of different molecular pathways.
This should hardly come as a surprise. Evolutionary theory predicts that ageing is caused by the independent destruction of many different systems; if caloric restriction has such pervasive effects on health, then it too must work by maintaining the body in many different ways. Even so, many gerontologists still seek a single explanation for all the diverse manifestations of ageing and the way in which caloric restriction delays them. One idea is that ageing is caused by a kind of insidious poison that is a consequence of the very condition of being alive.
THE BREATH OF DEATH
‘We term sleep a death and yet it is waking that kills us,’ observed Thomas Browne in his Religio medici. That living itself is the cause of our decline – either by exhausting some vital substance or else by gradual self-poisoning – is one of the oldest ideas in the history of ageing science. In its most recent version, ageing is caused by small, pernicious molecules capable of oxidising DNA, proteins, lipids, indeed almost anything they come into contact with. In the course of normal respiration, oxygen is reduced to water. But this is an imperfect process, and several other molecular species called ‘free radicals’ are produced as by-products. These molecules, which have chemical formulas such as •OH (the • signifying an unpaired electron), are especially abundant in mitochondria, the sub-cellular structures in which respiration takes place. From there they leak into the rest of the cell, attacking other structures as they go.
The free radical theory postulates that ageing is caused by the accumulated damage that these molecules inflict upon cells over the course of years. An abundance of correlative evidence supports this. Free radicals certainly damage cells, and the kind of damage they do becomes more common in old age. Most disturbingly, they cause mutations. The DNA of each human cell receives ten thousand oxidative hits per day. While many of these are repaired, many are not. Old rats have about two million mutations per cell, about twice as many as young rats do. Most of these mutations will have no effect on the health of a given cell. But should the radical hit a gene vital for the survival of a cell it might well kill it. Should it hit a proliferation-control gene in a stem cell it might initiate a cancer. Should it hit a gene in the cells that give rise to rise to sperm and eggs, it may be transmitted to future generations.
Free radicals are clearly pernicious. But do they cause some or all of ageing? Perhaps. Long-lived animals – be they innately so or else calorie-restricted – seem to be exceptionally resistant to toxins such as paraquat, a weed-killer that works by inducing the production of free radicals. More direct evidence comes from genetic manipulations in a variety of animals. Animal cells contain a battery of defences against free radicals, among them a group of enzymes devoted to scavenging free radicals, the superoxide dismutases. Several different kinds of evidence suggest that they protect against some of ageing’s effects.
An especially active form of superoxide dismutase seems to contribute to the longevities of the fruit flies, alluded to previously, that were the result of generations of gerontocratic reproduction. The founding population of these flies was polymorphic for two varieties of superoxide dismutase. Selection changed the frequencies of these variants so that the more active form became much more common in the populations of long-lived flies than in the short-lived ones. This wasn’t just a matter of chance: the experiment was replicated five times, and the same result was found each time. In an even more direct demonstration of the benefits of this enzyme, flies were engineered to express human superoxide dismutase – apparently more potent than the fly’s own – in their motor neurons. They lived 40 per cent longer than un-engineered controls, a particularly interesting result for it implies that superoxide dismutase can protect the nervous system. Finally, in the last few years many mutants have been found in nematode worms and fruit flies that seem to confer extraordinary longevity (one of them has even been named Methuselah after the patriarch who, Genesis assures us, lived to the age of 969). These mutants do not alter the sequences of superoxide dismutase genes themselves but rather affect genes that control when and how superoxide dismutase is activated. It is, it seems, hard to make a long-lived fly or worm without boosting superoxide dismutase by one means or another.
All these results suggest the following chain of argument: extra superoxide dismutase postpones ageing (at least in worms and flies); superoxide dismutase protects against free radicals; hence free radicals cause ageing. Does this imply that the means for postponing ageing in humans are at hand? Might we not simply engineer ourselves with a more effective superoxide dismutase and so gain years of life? The short answer seems to be no. Moreover, the reason that this won’t work casts some doubt upon one of the premises of the foregoing argument.
Our genomes contain three genes that encode superoxide dismutases. Mutations in one of these, SOD1, have been known for years. These mutations are gain-of-function and dominant: they give a hyperactive protein. It may be thought that this is precisely the sort of mutation that, by analogy with fruit flies and worms, might give a human lifespan of 120 years. In fact, they kill by the age of fifty or so. SOD1 mutations cause amytrophic lateral sclerosis (ALS), a particularly ferocious neurological disease in which the motor neurons of the spinal cord, brain stem and motor cortex are progressively destroyed, leading to paralysis and death. In America the disorder is known as Lou Gehrig disease after the baseball player who suffered and died from it. Nowhere is the issue of physician-assisted suicide as pressing as it is in ALS.
These mutations pose a paradox. They suggest that superoxide dismutase kills motor neurons in humans, even as it protects them in flies. Why? For the last ten years this paradox has been resolved along the following lines. Superoxide dismutase is only the first step in an enzymatic pathway that neutralises free radicals. It converts the free radical oxygen anion, O•2, to another molecule, H2O2, more commonly known as hydrogen peroxide, whose destructive effects upon biological tissue can be gauged by its fame as the active ingredient in chemical drain-cleaners and the classic suicide blonde. It takes another enzyme, catalase, to neutralise hydrogen peroxide by converting it to water. Perhaps an imbalance in the activity of these two enzymes in humans, but not flies, leads to a build-up of hydrogen peroxide in neurons and kills them.
It is a reasonable explanation, but it appears to be quite wrong. The reason that SOD1 mutations kill motor neurons has nothing to do with free radicals or hydrogen peroxide poisoning. Rather, their deleterious effects seem to be related to some other, slightly mysterious, role that superoxide dismutase has in the brain. Neurons are strange cells. They are large, have long protrusions called axons, and a whole special cellular architecture that goes with this. Besides scavenging free radicals, superoxide dismutase appears to have some role in this architecture. Biologists have adopted a lovely phrase to describe such multitasking proteins – they call them ‘moonlighters’. Moonlighting SOD1 may also contribute to another neurological disorder, Down’s syndrome. Children with Down’s syndrome have three copies of chromosome 21 – the chromosome on which the SOD1 gene resides – instead of the usual two. Hundreds of different genes reside on this chromosome, and any or all of them might contribute to the distinctive features of Down’s (mental retardation, the facial abnormalities, heart problems to name but a few), but the extra copy of SOD1 has long been fingered as one of the more destructive.
If superoxide dismutase moonlights, then the argument proposed above is predicated on a false premise. And with it goes one of the few good reasons for believing the whole free radical theory of ageing. The proponents of this theory (and among scientists they surely number in the thousands) may well feel that this is a harsh assessment of the only mechanistic account of the origin of ageing that has any pretensions to generality. It is certainly still possible that superoxide dismutase’s seemingly beneficial effects on ageing are mostly due to free radical scavenging, but this remains to be shown. For the time being, however, few would disagree that superoxide dismutase can be struck from the list of elixirs that might one day stave off the decline of our later years.
A WRINKLE
Even if free radicals are not the sole, or even major, source of mutations, mutations may still cause at least some aspects of ageing. Mutations may be especially destructive in those tissues, such as skin, whose cells divide continually throughout life. Some of us keep relatively youthful complexions well into old age, while others wrinkle when young. This variety partly depends on the exposure to the elements, sun most obviously, that each of us has received; ultraviolet light is a powerful mutagen. But even sheltered skin ages. And for all the parasols, veils and sun-block in the world, no thirty-five-year-old’s skin has ever glowed as it glowed when she was fifteen.
Wrinkling is a manifestation of a deeper inability of epidermal cells to replace themselves and maintain the integrity of the connective tissue of our skins. It is a problem that pervades our bodies. This is evident from people whose skins and connective tissues age with unusual, indeed catastrophic, rapidity. An inherited disorder called Werner’s syndrome causes its victims to go grey and bald when still in their teens. In their twenties, the testicles atrophy in men as the ovarian follicles do in women – a kind of premature menopause. In their thirties sufferers need lens transplants to cure cataracts, and their arteries stiffen and become covered in fat deposits. In their forties they die, usually from heart attacks.
Werner’s syndrome is one of a group of inherited rapid-ageing disorders called ‘progerias’. The disorder is caused by mutations that disable a protein that maintains the integrity of DNA during replication. Cells that lack the protein have very high mutation rates. This barrage of mutations causes the cells to die instead of proliferating, or else to produce abnormal proteins. Tissues, such as skin, which rely on large numbers of dividing cells in order to maintain their integrity, fall apart. Perhaps something similar happens to us all, only at a much slower rate.
As we age, vitality slips away from our cells. This can be seen in the laboratory. It has long been possible to grow human cells in petri-dishes by means of elaborate and delicate protocols. No matter how salubrious their environment, however, freshly harvested cells will divide only a certain number of times and then divide no more. Their decline is gradual, and is caused by some intrinsic limit. Many have suggested that this cellular senescence is not merely a consequence of the ageing body but its direct cause.
Supporting this idea, cells taken from human foetuses can divide for about twice as many generations as can those from ninety-year-olds before sinking into decline. Perhaps, then, elderly people have many cells that are closer to the end of their replicative lifespans and which are, therefore, unable to contribute to repairing the wear and tear of everyday life as well as they might. When, therefore, in 1998, the molecular cause of the limit to cell division was discovered, and then broken, the thrill was tangible. If cellular senescence could be cured, perhaps so could ageing.
Each time a cell divides, its chromosomes must be replicated as well. But the enzymes that replicate chromosomal DNA are unable to replicate the ends of the chromosomes. These ends are, therefore, protected by sequences, thousands of base-pairs long, called telomeres that are gradually whittled away over the course of many cell divisions at a rate of about a hundred base-pairs per cell division. When the telomeres are gone, the cell can no longer divide and it dies. It is the rate of whittling that sets the fundamental clock of ageing. Or so the argument goes.
What is needed, then, is a way to prevent the attrition of telomeres. Not all cells lose their telomeres. The germ cells that give rise to eggs and sperm possess a complex enzyme called telomerase that maintains their telomeres and so confers upon them the immortality that they must necessarily have. The loss of telomeres that occurs in the rest of the body’s cells is precisely due to the fact that they do not contain this enzyme. If telomerase is engineered into cells that normally lack the enzyme, their telomeres are preserved division after division. The cells also became immortal.
If the route to cellular immortality is so easy, why have we not taken it? The reason is quite simple: immortality is a property of cancers. Nearly all tumor cells have, somewhere in their history, undergone mutations that cause them to have telomerase where other cells do not. The absence of telomerase in our cells is probably one of the first defences we have against the multiplication of rogue cells. Besides, there is still little to show that short telomeres do, in fact, cause ageing. Only one experiment has addressed the problem directly: an experiment in which telomerase-defective mice were engineered and then bred for six generations.
Mice, it seems, can get by without telomerase for at least a while. The first generation of telomerase-defective mice that was ever produced showed no signs of premature ageing. In a way this is not surprising. These mice had telomeres as long as those of any other mice, for mice, like us, inherit their telomeres from their parents, and their parents were normal. For want of telomerase in their germ cells, however, each successive generation of these mutant mice started life with ever shorter telomeres. The effects became apparent by the fourth generation when the male mice proved to have few viable sperm. By the sixth generation they had none at all. Females were not sterile, but they produced fewer eggs than normal, and those they did produce often gave rise to defective embryos. By the sixth generation, too, male and female mice alike began to age prematurely. Like humans, mice go bald and grey with age, and the sixth-generation mice did so while still young.
These results provide at best mixed support for the idea that a want of telomeres causes ageing. Sufficiently short telomeres can clearly cause premature ageing; but since this happens only after six generations of attrition, they cannot be the cause of normal ageing in mice. While it is tempting to dismiss the whittling away of telomeres as an explanation of ageing in humans, it is probably too soon to do so. Laboratory mice have extraordinarily long telomeres – far longer than ours. If our telomeres are rather short at the start of our lives and must, by virtue of our greater size and longevity, undergo far more attrition than a mouse’s, it remains quite possible that they matter to us.
One way to prove the point would be to clone a human. Clones should start life with abnormally short telomeres, for they are produced without t
he aid of germ cells and so their telomeres are never renewed. Successive generations of clones should have shorter and shorter telomeres and age with increasing rapidity – all the more so if the clone-donors are elderly. What with the global ban on human cloning this experiment is not likely to be carried out soon – unless by UFO cultists or renegade Italian obstetricians. But, of course, it has been done in animals. Sheep 6LL3, a.k.a ‘Dolly’, got her chromosomes from the udder-cells of a six-year-old Finn Dorset. She therefore began life with substantially worn-down telomeres. Many thought that she would age fast. Some arthritis aside, however, she was quite healthy; there was nothing exotic about the viral disease that prompted her euthanasia at the age of six. Clones of other animals such as cattle and mice often suffer from a variety of health problems such as obesity, but none have been reported to be progeric. Still, these are early days.
Telomerase-mutant humans would be informative too. There is another progeria, rarer than Werner’s but even more severe, in which catastrophic ageing begins in childhood. The victims of this disorder usually die by the age of twelve or so, again from heart attacks, by which time they are to all appearances very small octogenarians. Their symptoms suggest defective telomeres. Even if this grim disease can be explained by too-rapid cellular senescence, we will have penetrated only a small way into ageing’s mysteries. For while the progerias hasten some aspects of physical decline, they leave the minds of their victims untouched.
MAKING A CENTURY
In the last ten years there has been a revolution in the study of ageing. Much of it has come from the study of the nematode worm Caenorhabditis elegans. This worm is only about 1 millimetre long, and it is possible to grow thousands of them in petri-dishes. They are perfectly transparent. Under a powerful microscope it is possible to see every single one of the 959 cells in their living bodies. For whatever reason, it has been especially easy to identify worm mutants that are extraordinarily long-lived. Some of these mutant worms live twice as long as normal worms do: forty-two days – in human terms, about 150 years.
Mutants Page 24