AGEING is as inevitable as death and taxes. And when your skin starts to sag, your hair turns grey and your muscles slowly lose their strength, you can’t help dreaming of forcing the sands of time back up into the hourglass.
Yet before we can hope to stem the flow, let alone reverse it, we need to know just what this sand consists of. Pinning down the molecular changes that underlie the ageing process is not easy. But it has long been suspected that mitochondria, the energy-generating structures within almost every cell of the body, play a key role. And in the past couple of years, researchers have produced strong evidence that this is indeed the case, that the decline of mitochondria determines when our bodies begin to crumble.
And some don’t stop there. The techniques they are developing to cure mitochondrial diseases, they say, might someday allow us to rejuvenate our mitochondria – and thus delay the onset of old age.
Advertisement
Essentially, what mitochondria do is burn sugars to produce energy. It’s a slow and controlled form of burning, but it can still be dangerous. Any errors or interruptions result in the production of highly reactive free radicals that can damage DNA.
The vast majority of a cell’s DNA is tucked away in the nucleus, far from the danger. But mitochondria have their own genome, a small circular bit of DNA containing just 13 protein-coding genes. Not only is this DNA at ground zero of the free-radical barrage, it also lacks the sophisticated machinery for repairing DNA damage found in the nucleus of cells – making it especially vulnerable to mutations.
What’s more, a single cell can have hundreds of mitochondria, each containing as many as 10 copies of this genome, and this DNA is replaced much more often than nuclear DNA. Each time it replicates, there is a risk of mutations – especially since the enzyme that copies mtDNA is more error-prone than the enzymes that copy DNA in the cell’s nucleus.
Not surprisingly then, mtDNA tends to accumulate mutations. The theory is that this might be the key to the ageing process: with each mutation, a grain of sand falls through the hourglass.
The evidence, however, has been circumstantial. For instance, a wide variety of mutations have been found in the mitochondria of older people. And the mitochondria of elderly people have far more mutations than mitochondria taken from the same people 15 or more years earlier, Giuseppe Attardi’s team at the California Institute of Technology in Pasadena showed in 1999. “In the aged human, essentially every mitochondrial genome has a mutation,” says Rafal Smigrodzki, a mitochondrial researcher at biotech company Gencia, based in Charlottesville, Virginia.
Other pointers come from rare genetic diseases caused by inherited mutations in mtDNA. Many resemble aspects of ageing. In particular, mitochondrial diseases tend to develop gradually, sometimes relatively late in life, and tend to affect the same tissues that fail as we age – the brain, heart and muscle.
But to prove that mitochondrial DNA damage is the sand in the hourglass, researchers need more direct evidence – and this is now beginning to emerge. A team led by Nils-Göran Larsson of the Karolinska Institute in Stockholm, Sweden, recently disabled the proof-reading ability of the enzyme for copying mtDNA in mice. The modified mice accumulate mtDNA mutations three to five times as fast as normal mice. These “mutator” mice appear normal at first, but at about six months of age, when normal mice are in their prime, they begin to lose weight and fertility, their hair starts to fall out, their posture becomes stooped and they develop enlarged hearts, osteoporosis, anaemia and hearing loss.
“The mice lived 20 per cent longer, and signs of ageing arose later”
The mice live for barely a year, less than half the normal lifespan, Larsson reported in 2004 (Nature, vol 429, p 417). Another team that developed its own strain of mutator mice reported a similar result a year later (Science, vol 309, p 481).
Many researchers see mutator mice as the first direct evidence of a link between mitochondrial mutations and ageing. But not everyone agrees. “This is another instance in which somebody makes a very sick mouse and points out a number of ways the sickness they see reminds them of some aspect of ageing, so they call it premature ageing,” says Richard Miller, a gerontologist at the University of Michigan in Ann Arbor.
His point is that it is easy to make mice die young. To convince sceptics that mitochondrial DNA damage is involved in ageing you need to demonstrate that reducing this damage makes mice live longer.
And last year, for the first time, researchers did exactly that. A team led by gerontologist Peter Rabinovitch at the University of Washington in Seattle genetically modified mice to produce more of an enzyme called catalase, which mops up free radicals. If the damage caused by these free radicals is important in ageing, they reasoned, mice that mop them up quicker should live longer.
Simply increasing the production of catalase made little difference. Next, the team altered the catalase so it would be delivered to the nucleus, reasoning that it might prevent free radicals causing mutations in nuclear DNA. That made even less difference. Finally, Rabinovitch’s team added a tag to the catalase that marks it for delivery to mitochondria.
“At the time these experiments began, seven or eight years ago, we weren’t betting on that working,” he says. “As it turns out, the home run was with the mitochondrial localisation.” These mice lived almost 20 per cent longer, and signs of ageing such as heart disease and cataracts also arose later than normal (Science, vol 308, p 1909). “It provides some of the most direct evidence for the importance of free radicals, and mitochondrially generated free radicals in particular, in the ageing of mammals,” says Rabinovitch. “That’s the kind of evidence you really want,” agrees Leonard Guarante, a molecular biologist at the Massachusetts Institute of Technology. “But like any interesting result, you want to see it replicated.”
“If you cannot prevent mutations building up in mitochondria, there’s only one option: fix them”
The results strongly suggest that mtDNA damage is the sand in the hourglass, or at least a major part of it. But what exactly is going on? Douglas Wallace of the University of California at Irvine thinks he knows. We start life with a clean slate, mitochondrially speaking. To prevent us inheriting our mother’s damaged mtDNA, egg cells go through a process that usually winnows out any mitochondria with mutations. But even before we leave the womb, the damage starts to build up.
All 13 of the proteins coded for by mtDNA form part of the machinery that oxidises sugars to generate energy, so mutations disrupt this process. This does not matter too much at first, since each mitochondrion has several copies of mtDNA. But any faults raise free-radical levels, causing more mutations, which raise free-radical levels still further. So as cells start to accumulate mtDNA damage, the rate of mutation accelerates.
Cells can tolerate a high proportion of mutant mitochondrial genomes – up to 90 per cent can have a particular mutation without causing major problems. But once a certain threshold is passed, energy production in a cell falls dramatically.
After this, the situation can soon reach crisis point. Rising levels of free radicals trigger a self-destruct mechanism called the mitochondrial permeability transition: pores open in the mitochondria, releasing chemicals that make the cell commit suicide.
And when cells in a particular tissue – the brain, heart or skin – start dying faster than they can be replaced, the classic signs of old age start to appear. “That’s what ageing is all about,” says Wallace.
According to this hypothesis, the integrity of our mtDNA is the weakest link in our body’s armour, the place where the whips and scorns of time first make themselves felt. “I would say this is the central factor in our ageing,” says Wallace.
If ageing is down to our mtDNA, what can we do about it? Rabinovitch’s work shows that antioxidants can extend lifespan by mopping up radicals – but only if they get into mitochondria. So it’s not clear whether eating lots of fruit and vegetables or popping pills will make much difference. And even if we could design antioxidants specifically to protect mitochondria, the benefits would be limited, because other factors such as UV radiation and thermal effects are damaging too. “Even the very best mitochondrial system is not going to make you live past 125,” says Wallace.
If you can’t protect the DNA in the mitochondria, maybe the answer is to move as many of the 13 genes as possible into the better-protected nucleus. People could perhaps be treated with stem cells modified in this way. After all, many other genes have moved from the mitochondria to the nucleus since the bacterial ancestors of mitochondria first took up residence in cells around a billion years ago; malaria parasites have just three genes in their mitochondria. But this is beyond the abilities of genetic engineers at present.
This leaves only one option: fix the mutations. If decrepit mitochondrial genomes were replaced with shiny new mutation-free ones, it should, in effect, slow ageing.
Unfortunately, mitochondrial genomes have so far resisted the usual sorts of genetic manipulations that geneticists perform on nuclear DNA. “In the nucleus you can do all kinds of things. You can switch genes on and off at will, and you can introduce new genes. These are standard techniques that have been around for 30 years,” says Smigrodzki. “So far, nobody has published a method for easy introduction of mitochondrial genomes into living cells.”
The challenge is getting DNA through the double membrane of mitochondria, which, unlike the nucleus, does not have pores that allow DNA through. What’s more, mitochondria are negatively charged and thus repel DNA, which also has a negative charge. But Smigrodzki is part of a team at Gencia, led by Shaharyar Khan, that claims to have overcome this problem. The trick, the researchers say, is in the packaging.
Khan’s team coats the DNA to be inserted with protective proteins that bear two amino-acid sequences. The first sequence, exploited by many biologists, allows the proteins to cross cell membranes and so enter cells. The second is a positively charged “mailing label” that allows proteins to enter mitochondria.
In 2004, Khan and James Bennett reported that they had used this technique to introduce a fluorescence gene into mitochondria in cells growing in a dish – a huge achievement in itself. Yet the team claims to have gone even further. In September 2005, Smigrodzki told an anti-ageing conference in Cambridge, UK, that the team added complete mitochondrial genomes to mitochondria in the liver and brain cells of live mice, simply by injecting the mtDNA constructs into their abdomens.
Ambitious aims
Khan’s team has submitted its results to a major journal. But until the full details are revealed, other researchers are reserving judgement. “If it’s right, it’s really exciting,” says Robert Lightowlers, a leading mitochondrion specialist at the University of Newcastle upon Tyne, UK. “He’s saying he can make whole mitochondrial DNA molecules in the test tube, which in itself is quite impressive, and can then get these molecules into mitochondria. But there are no details of the actual experimentation at all. Nobody knows whether this is real or not.” For instance, Lightowlers doubts that the protective proteins, which are the mitochondrial version of the histones that bind to nuclear DNA, can cross the membrane without unfolding and dropping their DNA cargo.
But if the Gencia team really can do what it claims, the technique raises many interesting possibilities. Most immediately, it would let researchers replace ageing mitochondrial genomes in animals with pristine, mutation-free ones, to see whether this extends lifespan. And Smigrodzki has an even more ambitious goal: replacing mtDNA in people.
Gencia’s aim, he says, is to use the technique to treat people with inherited mitochondrial diseases by adding healthy genes to compensate for mutant ones. But they might eventually be able to use mitochondrial replacement as a treatment for ageing itself. “It’s a gleam in our eye,” Smigrodzki says.
Replacing mitochondria poses a unique challenge because cells have so many copies of mtDNA (see Diagram). Getting one mitochondrial genome into a cell will make no difference at all if that cell has another 5000 copies with mutations.
To make a difference, at least some of the old mtDNA will first have to be destroyed, most likely by cutting it up with enzymes. Luckily, the proteins that mtDNA codes for are long-lived, so a cell can manage without mtDNA for the few hours the new mtDNA takes to proliferate. But the mtDNA-destroying enzymes will probably have to be delivered in the form of genes coding for them, so this part of the treatment will have all the problems associated with gene therapy.
Next, you need to get enough copies of the new mtDNA into the body to ensure that at least one mitochondrion in each cell gets at least one copy. Smigrodzki told the Cambridge meeting that the Gencia team has managed all these steps in cultured cells, but once again the details have not yet been published. “You’d like to know what the level of import is. Is it one molecule per cell, or is it a thousand?” says Lightowlers. “If you’re getting 500 copies in, that’s potentially very big news indeed.”
The bottom line, however, is that until someone rejuvenates mitochondria in an animal, we won’t know how much it will slow the ageing process. Guarente points out that boosting the antioxidants within mitochondria makes mice live just 20 per cent longer, while caloric restriction extends animals’ lifespan by 50 per cent.
“My instinct is that mitochondrial damage is one of many things that are going wrong in cells,” he says. “The question is, is it the dominant thing? If it’s not, then fixing it is not going to do much for maintaining youth.” If Gencia’s mitochondrial-engineering techniques really do work, then if nothing else, that’s one question we should soon be able to answer.
