Power, Sex, Suicide

by Nick Lane

  Paradoxically, in the long term, a cell can only correct against energetic deficiency by producing more mitochondria. If the mitochondria are defective, the cell attempts to correct the problem by producing more mitochondria—hence the tendency of defective mitochondria to ‘take over’ cells. For many years, cells can preferentially amplify the least-damaged mitochondria. The overall mitochondrial population is in continuous flux, with a turnover time of perhaps several weeks. Mitochondria either divide, if their energy deficit is fairly mild, or they die. The mitochondria that die are broken down, and their constituents recycled by the cell. This means that the most damaged mitochondria are continuously eliminated from the population. In this way, cells can spin out their lives almost indefinitely by constantly correcting the deficit. Our neurones, for example, are usually as old as we are ourselves: they are rarely, if ever, replaced, yet their function doesn’t spiral out of control in an error catastrophe, but rather declines imperceptibly. What isn’t possible though, is any return to the fountain of youth. While the most devastating mitochondrial mutations can be eliminated from cells, there is no way of restoring their pristine function, short of not using the mitochondria at all (which is how egg cells, and to a degree adult stem cells, do reset their clocks).

  The more a cell relies on defective mitochondria, the more oxidizing the intra-cellular conditions become (oxidizing means a tendency to steal electrons). When I say ‘oxidizing’, however, I don’t mean the cell loses control of its internal environment. It retains control by adapting its behaviour, establishing a new status quo. Most proteins, lipids, carbohydrates, and DNA are not affected by the change—again, in disagreement with the predictions of the original mitochondrial theory, which anticipated evidence of accumulating oxidation. Most studies searching for such evidence have failed to find any serious difference between young and old tissues. What is affected is the spectrum of operative genes, and there is plenty of evidence to support this change. The shift in operative genes hinges on the activity of transcription factors—and the activity of some of the most important of these depends on their redox state (which is to say, whether they’re oxidized or reduced, having lost or gained electrons). Many transcription factors are oxidized by free radicals, and reduced again by dedicated enzymes; the dynamic balance between the two states determines their activity.

  The principle here is similar to lowering a canary down a mine-shaft, to test for poisonous gases. If the canary is dead on raising it from the shaft, then the miner can take an appropriate precaution, such as only venturing down if wearing a gas mask. Redox-sensitive transcription factors behave like the canary, warning the cell of impending danger, and enabling it to take evasive action. Rather than the fabric of the cell as a whole being oxidized, which is as much as to say dead, the ‘canary’ transcription factors are oxidized first. Their oxidation sets in motion the changes necessary to prevent any further oxidation. For example, NRF-1 and NRF-2 (the ‘nuclear respiratory factors’) are transcription factors that coordinate the expression of genes needed for generating new mitochondria. Both factors are sensitive to redox state, which dictates the strength of their binding to DNA. If the conditions in the cell become more oxidizing, then NRF-1 stimulates the genesis of new mitochondria to restore the balance, and for good measure also induces the expression of a battery of other genes, which protect against stress in the interim. NRF-2 appears to do the opposite, becoming more active when the conditions are ‘reducing’, and falling inactive when oxidized.

  When the cell drifts to a more oxidizing internal state, then, a small posse of redox-sensitive transcription factors shifts the spectrum of active nuclear genes. The shift is away from the normal ‘house-keeping’ genes and towards those genes that protect the cell against stress, including some mediators that summon the help of immune and inflammatory cells. I argued in Oxygen that their activation helps to account for the chronic low-grade inflammation that underpins the diseases of advancing age, such as arthritis and atherosclerosis. While the exact spectrum of active genes varies from tissue to tissue, and with the degree of stress, in general the tissues establish a new ‘steady-state’ equilibrium, in which more resources are directed towards self-maintenance, and so fewer can be dedicated to their original tasks. This situation is liable to be stable for decades. We may notice we have less energy, or take longer to recover from minor ailments, and so on, but we’re hardly in a state of terminal decline.

  So, overall, what happens is this. If conditions become oxidizing within a particular mitochondrion, then the mitochondrial genes are actively transcribed to form more respiratory complexes. If this resolves the situation, then all is well and good. However, if this fails to resolve the situation, then conditions in the cell as a whole become more oxidizing, and this activates transcription factors like NRF-1. Their activation shifts the spectrum of nuclear genes in operation, which in turn stimulates the genesis of more mitochondria and protects the cell against stress. The new arrangement stabilizes the cell again, albeit in a new status quo that can influence vulnerability to inflammatory conditions. But there is little oxidation of the fabric of cells and tissues, and because only the least damaged mitochondria tend to proliferate there is little overt sign of mitochondrial mutations and damage. In other words, the use of free radicals to signal danger explains why we don’t see the spiralling, catastrophic damage predicted by the original mitochondrial theory. And this in turn explains why the cell doesn’t accumulate too many antioxidants—it needs just the right amount, so that it’s sensitive to changes in the redox state of transcription factors. That is why I said earlier that biology is more dynamic than ‘mere’ free-radical chemistry: there is little that’s accidental going on here; rather, a continuing adaptation to the metabolic undercurrents of the cell.

  So how do mitochondria kill us in the end? In time, some cells run out of normal mitochondria. When the next call comes to generate more, these cells have little option but to amplify their defective mitochondria clonally, and this is why particular cells are ultimately taken over by defective clones. But why do we only see a few cells with defective mitochondria at any one time, even in tissues of elderly people? Because now another level of signalling imposes itself. When cells finally work themselves into this state they are eliminated, along with their faulty mitochondria, by apoptosis, and that’s why we don’t detect high levels of mitochondrial mutations across ageing tissues. But there is a high cost for such purification—the gradual loss of tissue function, and with it ageing and death.

  Disease and death

  The ultimate fate of the cell depends on its ability to cope with its normal energetic demands, which vary with the metabolic requirements of the tissue. As in mitochondrial diseases, if the cell is normally highly active, then any significant mitochondrial deficiency will lead to a swift execution by apoptosis. What exactly constitutes the signal for apoptosis is uncertain, and again depends on the tissue, but two mitochondrial factors are probably involved—the proportion of damaged mitochondria, and the ATP levels in the cell as a whole. Of course, these two are interlinked. Clonal expansion of dysfunctional mitochondria inevitably leads to a more general failure to match ATP production to demand. In most cells, once the ATP levels fall below a particular threshold, the cell inexorably commits itself to apoptosis. Because cells with dysfunctional mitochondria eliminate themselves, it’s rare to observe heavy loads of mitochondrial mutations, even in the tissues of elderly people.

  The fate of the tissue, and the function of whole organs, depends on the types of cell from which they’re composed. If the cells are replaceable, by way of the division of stem cells that preserve an unsullied mitochondrial population, the loss of cells by apoptosis doesn’t necessarily perturb the status quo, so long as a dynamic balance is maintained in the cell population. But if the cells forced to die are more or less irreplaceable, like neurones or heart-muscle cells, then the tissue becomes depleted of functional cells, and the survivors are placed under greater
strain, pushing them closer to their limits—their own particular metabolic threshold. Any other factors that force cells closer to their limits could precipitate a specific disease. In other words, as cells draw closer to their limits, with advancing age, various random factors are more likely to push them over the abyss to apoptosis. Such factors may include environmental assaults such as smoking and infections, and physiological traumas such as heart attacks, but also any genes associated with disease.

  This link between metabolic threshold and disease is critical. It explains how mitochondria can be responsible for a whole gamut of diseases, even if they appear to be completely irrelevant. This simple insight explains why rats succumb to the diseases of old age within a few years, whereas it takes decades for humans. What’s more, it helps explain why birds don’t age in a particularly ‘pathological’ way, and how we can cure many of our own diseases at a single stroke. It explains in short how we can be more like elves.

  I’ve been enumerating the failings of the original mitochondrial theory of ageing. Here’s another: it’s very difficult to link the underlying process of ageing with the occurrence of age-related diseases. To be sure, there was a hypothetical relationship between free-radical production and the onset of disease, but if this is taken at face value then the theory is forced to predict that all the diseases of old age are caused by free radicals. Obviously, this is not true. Medical research has shown that most diseases of old age are an appallingly complex amalgam of genetic and environmental factors—and most of them have little to do with free radicals and mitochondria, at least not directly. Proponents of the mitochondrial theory have spent years trying to identify specific links between genes and free-radical production, but to little avail. Mutations in some genes are associated with free-radical production, but this is not the rule. Of more than a hundred different genetic defects known to cause degeneration of the retina, for example, only a few affect free-radical production at all.

  The solution was put forward in a beautiful paper by Alan Wright and his colleagues in Edinburgh, and published in Nature Genetics in 2004. I personally think this paper is one of the most important to emerge for a long time, for it gives a new, unifying framework for considering the diseases of old age, which ought to replace the current paradigm—both fallacious and counterproductive, in my view.

  The paradigm underlying most medical research today is gene-centric. The approach is first to pinpoint the gene, then to find out what it does and how it works, then dream up some pharmacological way of correcting the problem, and finally to apply the pharmacological solution. I think this paradigm is fallacious, as it is based on a view of ageing that now seems incorrect—the idea that ageing is little more than a dustbin of late-acting genetic mutations, which have broadly independent effects and so must be targeted individually. This is the hypothesis, you might recall, of Haldane and Medawar, which I criticized earlier on the grounds that recent genetic research shows that ageing is far more flexible. Extend the lifespan, and all diseases of old age are postponed by a commensurate period, if not indefinitely. More than forty different mutations extend lifespan in nematodes, fruit flies, and mice, and all of them postpone the onset of degenerative diseases in general. In other words, the diseases of old age are tied to the primary process of ageing, which is somewhat flexible. The best way of targeting the diseases of old age is therefore to tackle the underlying process of ageing itself.

  Wright and colleagues considered specific mutations in genes known to increase the risk of particular neurodegenerative diseases. Rather than asking what these genes do, they wondered what happens when the same mutation is found in different animals with differing lifespans. Of course, the same mutations are often found, and not only by chance. Animal models are essential for medical research, and genetic models of diseases figure at the centre of research today. So all that Wright and colleagues needed to do was to track down data on animal models in which the same genetic mutations cause equivalent neurodegenerative diseases. Nothing else was different. They came up with ten mutations that they could flesh out with data from five species with a wide range of lifespans—mouse, rat, dog, pig, and human. The ten mutations caused different diseases, but the same mutations produced the same disease in each species. The main difference was the timing. In the case of mice, the mutations produced disease within a year or two; for people, it might take a hundred times as long to cause exactly the same disease.

  It’s important to appreciate that the ten mutations are all inherited genetic mutations of nuclear DNA. None of them have anything to do with mitochondria or free-radical production directly. Wright and colleagues considered mutations in the HD gene in Huntington’s disease, the SNCA gene in familial Parkinson’s disease, and the APP gene in familial Alzheimer’s disease, plus a number of genes causing degenerative diseases of the retina, leading to blindness. In each case, the pharma industry is ploughing billions of dollars into research, as any effective treatment would recoup billions of dollars every year. More human ingenuity goes into this research than into rocket science these days. In no case has a really serious clinical breakthrough been made—the kind of breakthrough that leads to a genuine cure, or a delay in the onset of symptoms beyond months, or at best a few years. As Wright and colleagues put it, with nice understatement: ‘There are few situations in which neurodegeneration rates can be altered as substantially as the differences between species shown here.’ In other words, we can’t begin to slow down the progression of diseases, through medical interventions, by anything like as much as happens naturally in different species.

  Wright and colleagues plotted out the time of onset and the progression of disease from mild symptoms through to severe illness in the different animals. What they found was a very tight correlation between disease progression and the underlying rate of production of free radicals from mitochondria. In other words, in the species that produced free radicals quickly, the disease set in early and was quick to progress, despite no direct link with free-radical production. Conversely, in animals that leak free radicals slowly, the onset of diseases was delayed many-fold, and they progressed more slowly. This relationship could hardly be chance, for the correlation was too tight; clearly the onset of disease is tied, in some way, to the physiological factors that regulate longevity. Nor could the relationship be ascribed to differences in the genes themselves, for in each case the defects were exactly equivalent, and the biochemical pathways were conserved. It could not be ascribed to free radicals in general, as most of the genes didn’t change free-radical production directly. And it couldn’t be linked to other aspects of metabolic rate, as the metabolic rate doesn’t correlate with lifespan in many cases, including birds and bats—and, critically in this case, humans.

  The most likely reason for the correlation, said Wright, is that in all these degenerative diseases, the cells are lost by apoptosis—and free-radical production influences the threshold for apoptosis. Each of the genetic defects creates cellular stress, culminating in the loss of cells by apoptosis. The probability of apoptosis depends on the overall degree of stress, and the ability of the cell to keep meeting its metabolic demands. If it fails to meet its demands, it commits apoptosis. And the likelihood that it will fail depends on the overall metabolic status of the cell, which is calibrated by mitochondrial free-radical leakage as we have seen. The speed at which cells activate the retrograde response and amplify defective populations of mitochondria, leading to an ATP deficit, depends on the underlying rate of free-radical leakage. Species that leak free radicals rapidly are closer to the threshold, and so more likely to lose cells by apoptosis.

  Of course, all this is correlative, and it is hard to prove that a relationship is causal. But one study published in Nature in 2004 suggests that there is indeed a causal relationship. The study won several of its senior authors, among them Howard Jacobs and Nils-Göran Larsson, of the Karolinska Institute in Stockholm, the prestigious EU Descartes Prize for research in the life sciences. Th
e team introduced a mutant form of a gene into mice, termed knockin mice, as they have a gene knocked in (which is to say that a functional gene is added to the genome, rather than knocked out, the more common approach). The knockin gene in this case encoded an enzyme known as a proof-reading enzyme. Like an editor, a proof-reading enzyme corrects any errors introduced during DNA replication. In their study, however, the researchers introduced a gene that encoded a faulty version of this enzyme, which, ironically, was prone to errors. Like a bad editor, an error-prone version of the proof-reading enzyme leaves behind more errors than usual. The gene introduced in this study encoded a proof-reading enzyme specialized to work in the mitochondria, so that the errors it left behind were in mitochondrial, rather than nuclear, DNA. Having succeeded in setting the slapdash editor to work, the investigators were duly rewarded with a several-fold rise in the usual levels of mitochondrial errors, or mutations. There were two intriguing findings. The finding that captured the headlines was that the affected mice had foreshortened lives, coupled with an early onset of several age-related conditions, including weight-loss, hair loss, osteoporosis and kyphosis (curvature of the spine), reduced fertility, and heart failure. But perhaps the most intriguing aspect of the study was that the number of mutations did not rise with the age of the mice. As the mice got older, the number of mitochondrial mutations in body tissues remained relatively constant, as happens in humans—there was no big increase in mutational load during ageing.

  Although the reason was not ascertained, I imagine that any cells that acquired an unworkable load of mutations were simply eliminated by apoptosis, giving an impression that mitochondrial mutations did not accumulate with age. Overall, the study confirms the importance of mitochondrial mutations in ageing, but does not conform to the expectations of the original mitochondrial theory of ageing, which predicts a large accumulation of mitochondrial mutations leading into an ‘error catastrophe’. But the findings do support the more subtle version of the mitochondrial theory, in which free-radical signals and apoptosis continually relive the burden of mutation.