Power, Sex, Suicide: Mitochondria and the Meaning of Life (52 page)

So how fast do mitochondrial mutations really accumulate? It’s difficult to say for sure because the rate of change over generations is restrained by natural selection. For most mitochondrial genes, the evolution rate is about 10- to 20-fold the rate found in nuclear genes, but in the control region it can be as much as fifty times faster. Because mutations are only ‘fixed’ in the genome if they don’t cause catastrophic damage (otherwise they are eliminated by selection) the actual rate of change must be faster than this. To get an idea of how fast the change might really be, Anthony Linnane, at Monash University in Australia,
and his collaborators at the University of Nagoya in Japan, considered yeast in their classic
Lancet
paper published in 1989. Yeast is revealing because, as any brewer or vintner knows, it doesn’t depend on oxygen: yeast can also ferment to produce alcohol and carbon dioxide. Fermentation takes place outside the mitochondria, so yeast can tolerate serious damage to their mitochondria and still survive. Such damage was first noted in the 1940s with the discovery of ‘petite’ strains of yeast, whose growth is stunted. It turned out that the petite mutation involves the deletion of a large section of mitochondrial DNA, rendering the dispossessed mutant unable to respire. Critically, the petite mutation crops up spontaneously in cell cultures at a rate of about 1 in 10 to 1 in 1000 cells, depending on the yeast strain. In contrast, nuclear mutations occur at an almost infinitesimally slow rate, which is similar in both yeast and higher eukaryotes like animals—about 1 in every 100 million cells. In other words, if yeast is anything to go by, mitochondrial mutations accumulate at least 100 000 times faster than nuclear mutations. If such a fast mutation rate is true of animals too, then it could certainly account for ageing; indeed it would be hard to explain why we don’t drop dead almost immediately.

The search was on: how quickly do mitochondrial mutations accumulate in the tissues of animals and people? It has to be said that this area is controversial and a consensus is only now emerging. Part of the problem is the technology used to measure the mutations: the techniques used to sequence DNA letters sometimes amplify mutated sequences at the expense of ‘normal’ sequences, making it very difficult to quantify the extent of damage. In consequence, results from different laboratories can be extremely variable, sometimes ranging by as much as 10 000-fold. As so often in all walks of life, those people who hope to find mitochondrial mutations tend to find them, whereas the doubters inevitably find but a few. This is almost certainly not because researchers are deliberately fabricating their data, but rather where and how they look: it’s possible for both sides to be right.

Against this background, it’s perhaps rash for me to attempt a categorical statement, but attempt I shall. The picture that is beginning to emerge does indeed suggest that both sides are right. It seems there is a difference in the fate of mutant mitochondria, depending on the location of the mutation—whether this lies in the control region of the mitochondrial genome, or in a coding region.

Mutations in the control region (which binds the factors responsible for copying mitochondrial DNA) can prosper, and even stage a clonal take-over of entire tissues. These mutations don’t necessarily cause much functional deficit. A ground-breaking study, published in
Science
in 1999 by Giuseppe Attardi (one of the pioneers of mitochondrial DNA sequencing) and his colleagues at Caltech, showed that individual mutations in the control region can
accumulate at over 50 per cent of the total mitochondrial DNA in the tissues of older people, but are largely absent in young people. We can take it that certain types of mutation
do
build up with age to high levels, but we can’t say whether these mutations are harmful, as they don’t affect the protein-coding genes. Certainly not all of them are harmful. Another important study published by Attardi’s group in 2003 showed that one mutation in the control region is actually associated with
greater
longevity in an Italian population. In this case the mutation, a single letter change, cropped up five times more often in centenarians than in the general population, implying that it might have offered some kind of survival advantage.

In contrast, mutations in the functional protein- or RNA-coding regions very rarely accumulate at levels above about 1 per cent, which is far too low to cause a significant energy deficit. Interestingly, functional mitochondrial mutations, such as cytochrome oxidase deficiency,
are
amplified clonally within particular
cells
, so that the mutants come to predominate in those cells. This happens, for example, in some neurones, heart-muscle cells, and indeed the ragged red fibres of ageing muscles. However, the total proportion of such mutants in the tissue as a whole rarely rises above 1 per cent. There are two possible explanations for this. One is that different cells accumulate different mutations, so that any particular mutation is just the tip of an enormous iceberg of diverse mutations. The other explanation is that most mitochondrial mutations simply don’t accumulate at very high levels in ageing tissues. Perhaps surprisingly, it’s this latter explanation that seems closest to the truth. Several studies have shown that most mitochondria in ageing tissues have basically normal DNA, except perhaps in the control region, and moreover, are capable of virtually normal respiration. Given that the proportion of mutant mitochondria needed to undermine a cell’s performance in mitochondrial diseases is as high as 60 per cent, a total mutation load of just a few per cent seems insufficient to account for ageing, at least by the tenets of the original mitochondrial theory.

So what’s going on here? I find myself asking the obtuse question, ‘Are we really so different to yeast?’ I doubt that question will cost many readers much sleep. But it ought to! Yeasts accumulate mitochondrial mutations rapidly, yet for the most part, we don’t. From an energetic point of view, we function in a similar way to yeast; the only difference is that we depend on our mitochondria, whereas yeasts don’t. Perhaps this difference gives the game away—necessity. Let’s say we accumulate mutations in the control region simply because they don’t matter a great deal: they have little impact on function (as indeed is implicit in the studies of human inheritance discussed in
Part 6
), whereas most functional mutations do
not
accumulate because they
do
matter. That sounds fair enough, but it implies that selection for the best mitochondria is taking place in tissues (even in tissues composed of long-lived cells like heart and
brain). So we are faced with two possibilities. Either the mitochondrial theory of ageing is completely wrong, or mitochondrial mutations do occur at a similar rate to yeasts, but the mutants are eliminated by selection within a tissue for the best mitochondria. If so, then mitochondrial function must be far more dynamic than had been envisaged in the original mitochondrial theory of ageing. Which is it?

After the previous chapter, you might be forgiven for supposing that the mitochondrial theory of ageing is claptrap. After all, most of its predictions seem utterly false. One prediction is that antioxidants should prolong maximal lifespan, and this does not seem to be true. Another is that mitochondrial DNA mutations should accumulate with ageing, but only the least important ones actually do. Another is that the proportion of free radicals escaping the respiratory chains is constant, so lifespan should vary with metabolic rate; but this is only true in general, and fails to explain exceptions like bats, birds, humans, and the exercise paradox (the fact that athletes, who consume more oxygen over a lifetime, don’t age faster than couch potatoes). In fact, the only prediction of the original theory that seems to be true is that mitochondria are the main source of free radicals in the cell. Hardly the lineaments of a vigorous, healthy theory.

It’s time to return to an idea that we parked in the previous chapter: the proportion of free radicals escaping from the respiratory chains is
not
constant and unavoidable, but is subject to natural selection. Over evolutionary time, the rate of free-radical leakage is set at the optimal level for each species. In this way, long-lived animals have a fast metabolic rate while leaking relatively few free radicals, whereas short-lived animals typically combine a fast metabolic rate with leaky mitochondria and plenty of antioxidants. We posed the question: What is the cost of well-sealed mitochondria? Why would a rat
not
benefit from cutting back its investment in antioxidants by sealing its mitochondria better? What does it have to lose?

Let’s think all the way back to
Part 3
, and in particular to John Allen’s explanation for the very existence of mitochondrial DNA (see
pages 141
–
144
). You might recall that he argued it was no fluke that a hard-core of genes survives
in every species
that relies on oxygen for respiration. The reason, he suggested, was to balance the requirements of respiration, as an imbalance in the components of the respiratory chain can lead to inefficient respiration and free-radical leakage. We saw that it is necessary to retain a local contingent of genes to pinpoint the need for reinforcements in particular mitochondria, rather than all
mitochondria at once, regardless of need, which is what would happen if control were retained by the bureaucratic confederation of genes in the nucleus. Allen’s essential point is that the mitochondrial genes survive because the benefits of keeping them on site outweigh the disadvantages.

How might one particular mitochondrion signal its need to produce more respiratory chain components? We’re now entering into the realms of twenty-first-century science, and it’s best to admit that at present little is known. As we saw in
Part 3
, Allen suggests that they do so by modulating the rate of free-radical production from the respiratory chains: the free radicals themselves act as the signal to start building more respiratory complexes. This suggests an immediate reason why a rat might lose out by restricting free-radical leakage—it would muffle the strength of the signal, and so require a more refined detection system. We’ll see later how birds might have got around this problem, and why it wasn’t worth it for rats.

What might happen if there is not enough cytochrome oxidase in a particular mitochondrion? This is the scenario that we considered in
Chapter 8
. Respiration becomes partially blocked, and electrons back up in the respiratory chains, making them more reactive. Oxygen levels rise, as less is consumed by respiration. The combination of high oxygen with slow electron flow means that free-radical production increases. According to Allen, this is exactly the signal required to produce more complexes, to correct the deficit. How the mitochondria detect the rise in free-radical leakage is unknown, but various plausible possibilities exist. For example, mitochondrial transcription factors (which initiate protein synthesis) might be activated by free radicals; or the stability of the RNA might depend on free-radical attack. Examples of both are known, but neither has been proved to take place in the mitochondria. Either way, the rise in free-radical leakage should lead to more core respiratory proteins being made from mitochondrial DNA. These insert themselves into the inner membrane, and once implanted they behave as beacons and assembly points for the additional proteins encoded by the nuclear genes. When the full complex is assembled, the blockage of respiration is corrected. Free-radical leakage falls again, and so the system is switched off. Overall, then, this system behaves like a thermostat, in which a fall in room temperature is itself the signal used to switch on the boiler. The rising temperature then switches off the boiler, so regulating the room temperature between two fixed limits. But of course, if the room temperature didn’t fluctuate up and down, the system couldn’t work at all. Similarly, if the rate of free-radical leakage from respiratory chains didn’t fluctuate, there could be no self-correction to an appropriate number of respiratory complexes.

What happens if the free-radical signalling fails? If the synthesis of new respiratory proteins from the mitochondrial genes fails to stem the leak of free radicals, then the lipids of the inner membrane, such as cardiolipin, are
oxidized. In
Part 5
, we noted that cardiolipin binds cytochrome oxidase, so if cardiolipin is oxidized, cytochrome oxidase is released from its shackles. This in turn blocks the passage of electrons down the respiratory chain altogether, so respiration grinds to a halt. Without the constant flow of electrons needed to maintain it, the membrane potential collapses, and apoptotic proteins are spilled out into the cell. If this turn of events happens in a single mitochondrion, the cell does not necessarily commit apoptosis—there appears to be a threshold. When only a few mitochondria expire at one moment, then the signal for apoptosis is not strong enough to cause the cell to die, and instead the mitochondria themselves are broken down. In contrast, if a large number of mitochondria simultaneously spill out their contents, then the cell as a whole does get the point, and goes on to commit apoptosis.

This flexible signalling system is a far cry from the spirit of the original mitochondrial theory of ageing. The original theory supposed that free radicals are purely detrimental; that the continued existence of mitochondrial DNA was a diabolical fluke of evolution; and that free-radical damage spiralled out of control, leading to the degeneration and misery of ageing. We now appreciate that free radicals are
not
purely detrimental—they carry out an essential signalling role—and that the bizarre survival of mitochondrial DNA is not a fluke, but is actually necessary for cellular and bodily health. Furthermore, mitochondria are better protected against free-radical damage than had once been assumed. Not only is mitochondrial DNA present in multiple copies (usually five to ten copies in every mitochondrion), but recent work shows that mitochondria are reasonably efficient at repairing damage to their genes, and (as we saw in
Part 6
) are capable of recombination to fix genetic damage.