by Nick Lane
Thus, there is no ‘mixing’ of mitochondrial genes. If a Turkish woman marries a native American, all children born to them will have pure Turkish mitochondrial DNA. Because of the rate of evolution, races that diverged from each other thousands of years ago can be distinguished from each other through their mitochondrial DNA, whereas those within a group, all of whom inherited their mitochondrial DNA down the maternal line, share similar mitochondrial DNA. This is the basis of ‘mitochondrial Eve’, the mythical mother of mankind who passed her mitochondrial DNA to all those living on the planet today.
According to Bryan Sykes, a specialist in mitochondrial DNA at Oxford, and author of The Seven Daughters of Eve, all modern Europeans are descended from seven mythical women, representatives of seven different tribes who migrated into Europe at different times.
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techniques generate copious artefactual errors. Ames and Beckman concluded:
In summary, despite considerable popularity and intuitive merit, the theory that mitochondrial DNA is more heavily damaged by oxidative damage than nuclear DNA does not stand on firm ground. Due to the variation between competing methods of analyzing oxidative damage, it must be concluded that the background level of oxidative damage of mitochondrial DNA is not yet known with certainty; nor, for that matter, does there exist a firm estimate of oxidative damage in nuclear DNA with which to compare it.
Do these messy experimental shenanigans signal the demise of the mitochondrial theory of ageing? In its original form, probably. There are a number of biological objections too. For example, even though there are fewer, larger, less-efficient mitochondria in ageing tissues, they are in some sort of working order: there are few signs of the catastrophic damage predicted by the mitochondrial theory of ageing. In relation to this, seriously damaged mitochondria ought to destabilize cells and set off the cell-suicide programme — apoptosis. But examination of ageing organs suggests that apoptosis does not take place on the scale predicted by the mitochondrial theory. So how do the leaky mitochondria maintain their integrity? Well, they have multiple copies of their genes, which are kept in functional clusters to ensure they have at least one working copy of each gene. Then, it seems, mitochondria are better at repairing their DNA than was once thought: an enzyme responsible for correcting oxidative damage to mitochondrial DNA was isolated in 1997. Mitochondria can also tolerate a large number of mutations — they apparently have a mechanism for editing erroneous RNA to make workable proteins. Finally, an evolutionary thought: if mitochondrial DNA is really so vulnerable, why did it persist there — why was it not all transferred to the nucleus?
Genetic studies suggest that there is no physical reason why it should not have done, so there must be some benefit to DNA remaining in the mitochondria.7 In sum, these considerations suggest that the mitochondrial theory, as originally stated, is biologically naive.
7 One possibility put forward by John Allen (who we shall meet later in the chapter) is that mitochondrial genes allow a rapid response to sudden changes in oxygen level, nutrient supply or the presence of respiratory poisons. The energy status of the cell is so critical that cells need to respond swiftly and appropriately to sudden change. Having to rely on bureau-cratic nuclear genes to do this is like waiting on the government to make decisions about the disposition of troops on the ground in a war. Mitochondrial DNA is thus a kind of front-
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And yet . . . there is too strong a link between metabolism and ageing, across all species, to dismiss the mitochondrial theory out of hand. Mitochondrial DNA sequences certainly change relatively rapidly (over generations), which implies that their DNA does suffer more mutations than nuclear DNA. In addition, mitochondria from ageing tissues are unquestionably damaged to some extent, even if not catastrophically so. A more subtle version of the mitochondrial theory must be true. I favour a model put forward by Tom Kirkwood, working this time with the German biochemist Axel Kowald, and known as the MARS model (Mitochondria, Aberrant proteins, Radicals, Scavengers). The idea typifies Kirkwood’s contribution to ageing research. Trained originally as a mathematician, he took a step back from the details of competing theories, and considered the broader network of interactions within cells. In particular, Kirkwood and Kowald asked what would happen to protein turnover if mitochondrial function declined only slightly. They made three assumptions: first that free radicals would escape from the mitochondria to damage other cellular components, such as the protein-synthesis apparatus; second, that prevention and repair is never 100 per cent efficient; and third, that mildly damaged, albeit functional, mitochondria produce less energy than their undamaged cousins, ultimately causing a cellular energy deficit (in other words, the cell cannot produce as much energy as it needs).
Kirkwood and Kowald built these three assumptions into a computer model, to see how well they could simulate the pace of the changes that occur during ageing. The detailed equations presented in their 1996 paper are enough to make most biochemists tear their hair, but their conclusions make good intuitive sense. A very slight mismatch between the rate of free-radical production and the ability of the cell to repair that damage, coupled with a growing energy deficit, leads to an insidious decline in mitochondrial function. The decline unfolds over many decades, until finally a threshold is crossed. At this point, the mitochondria probably resemble those isolated from old tissues. The emphasis now shifts from the mitochondria to the protein-synthesizing machinery. In a relatively line rapid reaction unit, enabling the sensitive local control of critical gene expression and respiratory function. Mitochondrial DNA might be called ‘altruistic’, in that it serves the greater good; but in reality cells and organisms have a selective advantage if they retain DNA in their mitochondria, and so will survive better than cells that eliminate their ‘altruistic’ DNA. This reminds us that the ‘selective unit’ is always the organism (even if the organism is a single cell) and not individual genes.
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short space of time, compared with the overall process, the cell’s ability to maintain its biochemical equilibrium collapses. Once the equilibrium is lost, it is only a matter of time before the cell dies. This process matches both the timescale and the acceleration of ageing observed in real life.
Critically, at no time does the model system actively lower the performance of its maintenance systems. The cell’s resources were, in fact, insufficient from the beginning, and this allowed the gradual undermining of its integrity.
Although simultaneous equations inevitably simplify the real cell, I agree with Kirkwood and Kowald’s conclusion that the model provides a plausible framework for understanding the process of ageing. It distinguishes between what is theoretically possible and what is improbable. In the absence of convincing experimental data, they seem to be barking up the right tree. If they are, then there is a big implication. Mitochondrial respiration will eventually undermine the integrity of cells. The speed at which this happens depends on the ability of cells to protect themselves, but no cell is 100 per cent efficient, so all creatures containing mitochondria should die. This returns us to the second difficult question: how do some cells, and even some simple animals, avoid ageing?
When August Weismann first distinguished between the mortal body and the immortal germ line at the end of the nineteenth century, he made a remarkable prediction: that all somatic (body) cells would have a finite lifespan. For much of the twentieth century, Weismann’s prediction remained controversial. The debate was put on a more empirical footing in 1965 by the American biologist Leonard Hayflick, who finally proved that human fibroblasts (connective tissue cells involved in wound healing, and easily grown in culture) can divide no more than 50 to 70 times before succumbing to ‘replicative senescence’ and dying. Thus, unlike bacteria, fibroblasts cannot be cultured indefinitely: in the end, the entire population dies out, apparently of old age. The potential number of divisions
that a single cell can make before dying (or more precisely, the number of population doublings) became known as the Hayflick limit.
Different cell types have different Hayflick limits, but we now know that essentially all somatic cells senesce and finally die.
There are intriguing variations on this theme. Fibroblasts taken from short-lived species, such as mice, have a lower Hayflick limit than fibro-
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blasts from long-lived species, such as humans (about 15 cell divisions compared with 70). This relationship is robust across all species tested.
The Hayflick limit also varies with the age of the donor. If fibroblasts are cultured from an old donor, they divide fewer times before senescing and dying than those taken from a young individual. Presumably, they had already used up some part of their limit while dividing in the body, and so had fewer divisions left to them. Cells taken from people with Werner’s syndrome, which causes accelerated ageing, also quickly curl up and die.
The implication is startling: cells can count. When they have counted up to their limit, they die. The limit is encoded in the genes. Genetic diseases in which ageing is accelerated have a lower limit.
Cancer cells are an exception. They behave more like bacteria. Cancer cells somehow get around the Hayflick limit and continue to multiply indefinitely. The most famous example is the tumour of the unfortunate black American Henrietta Lacks, who died of cervical cancer in Baltimore in 1951. Doctors took a sample of her tumour in the 1940s, and cultured the cells to see what kind of a tumour it was. The cells, known as HeLa cells, were so vigorous that they are still being grown in research centres across the world 60 years later. They show no signs of senescence.
In total, they now weigh more than 400 times Henrietta’s own body weight.
The story of the Hayflick limit came to a head in 1990, when Cal Harley, founder of the Californian biotechnology company Geron Corporation, made a connection between the ability of cells to count and the length of their telomeres — the ‘tips’ at the ends of individual chromosomes. Telomeres are often said to resemble the ends of a shoelace — their purpose is to prevent ‘fraying’; in other words, to preserve the integrity of the chromosome. They are also said to be the secret of eternal life. They are not, as we shall see.
Telomeres are a characteristic biological fudge: they are needed because our DNA replication machinery was inherited from bacterial ancestors with circular chromosomes, whereas those of all eukaryotes are linear. Because of the way the biochemical machinery for replicating DNA works, it is impossible to replicate the extreme ends of a linear DNA molecule. As a result, the chromosome gets shorter each time it is copied.
The solution? A fudge. Evolution cannot whip new DNA-replicating machinery out of a hat, just like that, but it is easy enough to add a bit of extra non-coding DNA to each end of the chromosome, to which the enzymes can bind at the beginning and end of replication. The loss of this
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extra DNA does not matter, at least until its loss is complete: then the chromosomes start to fray and the cell can divide no longer.
These extra caps of non-coding DNA, then, are the telomeres. What Cal Harley showed was that they get steadily shorter in human fibroblasts growing in culture. Each time a cell divides it must replicate its DNA, so a little bit of telomere is lost with every cell division. Human fibroblasts lose all their telomeres after a maximum of about 70 cell divisions. Thus, the shortening telomeres function as a biological clock, which sets a limit on the number of times a cell can divide. This limit is determined by the original length of the telomere and the rate at which it is used up — but in general, the longer it was at the beginning, the more cell divisions are possible.
How do cancer cells escape? It seems they make use of an enzyme, called telomerase, which regenerates the telomeres, so that their length is not perpetually truncated. Telomerase is thought to be present in all cancer cells. These cells do not magic the enzyme out of thin air. The gene is present in all our cells, but is normally switched off. In our bodies, it is usually used only by stem cells, unspecialized cells that can divide and differentiate to produce new tissues, and by sex cells, whose raison d’être is reproduction. In 1997, scientists at Geron succeeded in cloning part of the gene for telomerase. When they introduced it into cultured human somatic cells, along with a promoter gene to make sure that the transfected telomerase was active, the new gene made the cells essentially immortal. The cell population was able to continue dividing indefinitely, but did not behave like cancer cells, which tend to form clumps similar to tumours, even in a petri dish. The findings were published in Science in 1998 and generated huge excitement — here was the secret of eternal youth! The product of a single gene could overcome ageing, or at least replicative senescence, in somatic cells.
The excitement over telomerase echoes the long human quest for immortality. Gilgamesh would have been enraptured. The gene-centric molecular biologists who advocate programmed ageing were triumphant. If our lifespan is set by the length of a piece of DNA, then the specific length must have been ‘programmed’ in some way to ensure that our lives are the right length, presumably for the good of the species. Evolutionary biologists took a different view. As we saw in the last chapter, if selection pressure falls with age, then there can be no unfolding programme for ageing. If this is the case, the telomeres must have a different significance.
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Their apparent control of senescence in cell culture must be an artefact that is irrelevant to their role in the body.
These diametrically opposed interpretations of a central fact, which is not itself in dispute — that telomerase confers immortality on cells grown in culture — shows the importance of theory in science. Facts mean little in isolation unless they can be interpreted within the wider framework of a theory; and it is usually those needling little facts, which resist interpretation by any theory, that bring dogmas crashing down. In the case of telomerase, however, there is no need for a radical reinterpretation. Telomerase is necessary, but not in itself sufficient, for cells with straight chromosomes to go on replicating themselves indefinitely. It is almost irrelevant to the ageing of our bodies.
Many cells in the adult body do not divide, and therefore do not lose telomere length. They do not need telomerase because they do not have a problem with shrinking telomeres. The brain, heart, major arteries and the ‘skeletal’ muscles that enable us to move are composed largely of specialized cells that have a job to do, that do not divide and are not easily replaced. A centenarian’s brain has nerve cells (neurons) that are 100
years old. Even though we do not understand the workings of the conscious mind, it clearly resides, in one sense or another, in the great network of connections formed between neurons throughout our lives. With the 100 billion neurons we start out with, we make some 200 million million connections. It is hard to imagine how this fantastic web of connections could be replicated by replacing old neurons with new ones, which would have to reproduce the exact spatial connections of their defunct predecessors. If they failed, our minds would change, our memor-ies would be wiped or transfigured. Some songbirds that sing a new song each year are thought to replace certain neurons: something similar would surely be true for us. We might live forever, but unless we wrote it down we’d never know. The problem, then, is that the evolved structure of the human body is simply not compatible with eternal life, unless we can find a way of replacing worn-out neurons — and here we enter the realms of science fiction.
Cells that do divide regularly, such as stem cells and the cells that give rise to sperm, have active telomerase. They have no problem with shrinking telomeres either. Even circulating immune cells, which do not express telomerase when quiescent, reactivate it when stimulated to proliferate by bacteria. In other words, if our immune cells need to undergo many rounds of cell division, they have all the telomeres they need. All
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that remains are certain epithelial cell types, such as kidney cells and liver cells, and fibroblasts, which do divide in the body, but only when needed.
These cells do not produce telomerase and potentially face the Hayflick limit, but it is questionable whether they ever reach it. Fibroblasts taken from elderly donors can usually still divide between 20 and 50 times before becoming senescent and dying: clearly they never reached their limit in the body. If they have no telomerase, it is because they do not need it.
A few other miscellaneous facts confirm the same story. There is a poor correlation between telomere length and the maximum lifespan of different species. Mice have far longer telomeres than humans, even though we live 25 times longer. Different species of mice, all with the same maximum lifespan, have very different telomere lengths. Remarkably, ‘knock-out’ mice lacking the gene for telomerase have normal lifespans until the third generation, when they do show signs of accelerated ageing, the significance of which is uncertain. Finally, the number of cell doublings needed to make a body does not relate to the subsequent rate of ageing. The cells of an elephant must divide many more times to produce an elephant than must those of a mouse to produce a mouse; yet elephants live far longer. In short, it seems fair to say that, for all the hullabaloo, telomerase does not hold the secret of eternal life. Without the enzyme, eternal cell replication is not possible in eukaryotes, due to a glitch in the DNA-replication machinery passed down by evolution.
Telomerase thus facilitates cell division in the same way that a light switch facilitates lighting a room: it is technically helpful, but just as the light switch is not the source of the light, telomerase is not the spring of everlasting life. So why is telomerase switched off in epithelial cells? Some argue that a limit on the possible number of cell replications might protect against cancer, but this seems unlikely. The Hayflick limit is too high to be relevant: it is equivalent to the Chinese government decreeing that, to safeguard against population growth, it will impose a limit of 70