TREADING on a nail is normally a painful experience, but not for Jack. The 75-year-old鈥檚 long-standing diabetes had gradually damaged the nerves in his feet until he had little sensation left. Despite walking around all day with a nail piercing his skin through his shoe, he didn鈥檛 feel a thing.
But pain would have been a small price to pay to prevent what happened next: the wound became infected. The first Jack knew was when a bad smell prompted him to examine his foot. To his horror, a large abscess had formed, oozing blood and pus. Doctors tried various measures to help it heal, but thanks to the poor blood supply to Jack鈥檚 feet, also caused by diabetes, none was successful. Soon gangrene set in, and the only option left was amputation.
Jack is not a real person, but the story represents a surprisingly common complication of type 2 diabetes. Minor wounds, even from something as trivial as a stone in the shoe, are a leading cause of limb amputations in the west, second only to physical injuries such as those from a car crash. Why diabetes should cause such nerve damage is unclear, but one controversial theory suggests the culprit is a breakdown of the biological conveyor belts that transport substances along the long neurons that extend from the limbs to the spinal cord and the brain.
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It is not just diabetes specialists who are interested: researchers are discovering that the same thing might play a role in a surprising range of neurological disorders 鈥� from motor neuron disease to stroke, and from Alzheimer鈥檚 disease to multiple sclerosis. And the lifetime risk of acquiring a disorder in which this transport system fails approaches 50 per cent in the west. Transport failure is not always the root cause, but it can be part of a feedback loop that worsens symptoms. A growing number of scientists and doctors now believe that finding a way to protect neurons from the consequences of transport breakdown could bring enormous medical benefits.
Neurons are so vulnerable to transport problems because of their extraordinary length. Most cells are around 10 to 20 micrometres in diameter 鈥� up to a thousand would fit on a full stop. Neurons, on the other hand, can be nearly 2 metres in length in humans, with the longest running from your toe to the base of your brain.
The neuron鈥檚 nucleus and surrounding area is called the cell body (see Diagram). This is the control centre of the neuron and is located either in the brain, or in or near the spinal cord. The long stringy part of the neuron that runs from the cell body to a muscle or a sensory receptor in the skin, for example, is known as the axon. Next time you visit a zoo, take a second look at the giraffe. The axons linking its hind feet to its brain are 5 metres long. To put this in perspective, if the cell body were scaled up to the size of a swimming pool, the axon would span the Atlantic.
The first hints that axons require constant transport of substances from the cell body came in 1850, in work by the British biologist Augustus Waller. He severed the nerves to a frog鈥檚 tongue and throat and found that, while the cell bodies and the axons still attached to them seemed to survive, the axons on the other side of the cut underwent 鈥渃oagulation or curdling鈥� into 鈥減articles of various sizes鈥� within a few days 鈥� a process termed Wallerian degeneration after its discoverer. Waller concluded that the cell body somehow nourished the nerve fibres. In what now seems a remarkably prescient comment, he observed: 鈥淚t is particularly with reference to nervous diseases that it will be most desirable to extend these researches.鈥�
We now know that the axon requires a host of different chemicals to survive and function as an efficient transmitter of electrical impulses. These include structural proteins; mitochondria 鈥� a cell鈥檚 energy factories, found all along the axon; and neurotransmitter molecules 鈥� the chemical signals sent from the end of the axon to the target cell or organ. Most of these components are made in the cell body, and so demand an impressive transport system to move over huge cellular distances. Traffic in the other direction includes components for recycling.
In the early 1970s it was discovered that the transport system consists of long tracks of protein molecules called microtubules. 鈥淢otor鈥� proteins 鈥渨alk鈥� along them by alternating between two shapes, with different proteins carrying various cargoes. A family of proteins called kinesins carries substances away from the cell body, and a motor named dynein goes in the other direction. It can take anything from a couple of days to eight months for cargoes to reach the tip of the axon.
鈥淚f a giraffe neuron鈥檚 cell body were the size of a swimming pool, its axon would span the Atlantic鈥�
The first condition in which a failure of axon transport was identified as the culprit was motor neuron disease (MND, also called amyotrophic lateral sclerosis or Lou Gehrig鈥檚 disease). MND is a progressive, paralysing condition in which the neurons that enable us to contract our muscles degenerate and eventually die.
The mechanisms behind the neuron degeneration seen in MND are unclear. A tightly orchestrated process of cell death called apoptosis plays an important role in a host of diseases, and many researchers suspected MND could be one of them. So in the mid-1990s, Ann Kato, a neuroscientist at the University of Geneva, Switzerland, tried blocking apoptosis in pmn mice, a strain that undergoes its own version of MND, to see if this would slow the progression of the disease. However, she found that the animals鈥� axons continued to degenerate and the disease continued unabated. 鈥淚t was disappointing at first, but we decided it was telling us something important about axons,鈥� says Kato.
If apoptosis weren鈥檛 responsible, what was? In 2002, a team led by Jean-Louis Guenet at the Pasteur Institute in Paris, France, found that pmn mice cannot correctly make a protein called tubulin-specific chaperone, which helps microtubule building blocks take on the right shape. So the mutation disrupted microtubule assembly and thus axon transport.
Evidence that human MND could be caused by transport failure quickly followed. A team led by neurologist Ken Fischbeck at the National Institutes of 午夜福利1000集合 in Bethesda, Maryland, found that some MND patients have a faulty version of a protein that links cargoes to the molecular motor dynein. Then neurologist Jonathan Glass and colleagues at Emory University in Atlanta, Georgia, found that in another form of MND, axons degenerate well before the cell body.
Mouse survivors
The next task was to find out just how transport failure kills axons. The answer stemmed from work begun in the late 1980s. While studying Wallerian degeneration in apparently ordinary lab mice, University of Oxford neuroscientists Hugh Perry, Michael Brown and their colleagues found that cut nerves in the mice degenerated at one-tenth the normal rate, surviving weeks instead of days. They named the strain slow Wallerian degeneration (WldS) mice and traced the trait back to a spontaneous genetic mutation.
Many scientists now believe that WldS mice, along with various other observations, suggest that Wallerian degeneration is not a nourishment problem at all, but a regulated program of axon death akin to apoptosis in other types of cell. In healthy nerves, axon transport seems to deliver something that prevents this suicide. If it fails or nerves are injured, the axon self-destructs.
After a 10-year slog, the protective WldS gene was finally identified by my (Coleman鈥檚) group in Cologne, Germany, after working with a team led by Perry, now at the University of Southampton (Nature Neuroscience, vol 4, p 1199). We are still trying to work out how WldS prevents axon suicide. It might work indirectly, as the WldS prevents axon suicide. It might work indirectly, as the WldS protein expressed by the gene seems to stay in the cell body, suggesting it has accomplices that carry out its work in the axon.
In 2003, Kato鈥檚 group showed that inserting the WldS gene into pmn mice protected their axons, delayed disease symptoms, and extended the animals鈥� lifespan by 40 per cent. So WldS succeeded where anti-apoptotic genes had failed. 鈥淎t last we had a way to slow the disease down 鈥� something to build on,鈥� says Kato. Wallerian degeneration was no longer just a lab model, but an important pathway of axon death in disease.
The role of transport breakdown is clearest in MND, but it may well be implicated in several other diseases. In diabetic nerve damage, a widely held view has been that the condition is caused by poor blood supply to the limbs, as the high metabolic demands of nerves cannot be met. But in the early 1980s it was proposed that diabetics鈥� high blood glucose levels alter axon proteins, including those of the transport machinery. While this theory is hotly contested, we know that glucose can disrupt the formation of microtubules, and axon transport has been shown to be disturbed in animal models of diabetes.
Other diseases in which blocked axon transport has been implicated include stroke, HIV-associated dementia, multiple sclerosis and the rare inherited neurological conditions Charcot-Marie-Tooth disease and hereditary spastic paraplegia. And only this year research led by Larry Goldstein at the University of California, San Diego, was published linking transport failure with Alzheimer鈥檚 disease (Science, vol 307, p 1282).
The characteristic tangles and plaques seen in the brains of people with this condition were first described by the German psychiatrist Alois Alzheimer back in 1906. We now know the plaques contain a protein called beta-amyloid, as well as swollen axons 鈥� now a known sign of neuron transport breakdown. Swollen axons occur throughout the brains of Alzheimer鈥檚 patients, not just in the plaques, and they appear before any plaques are formed, suggesting they play an early role in the disease. But a key breakthrough in the early 1990s diverted attention away from axons. Researchers found that some rare inherited forms of Alzheimer鈥檚 are caused by mutations in the proteins that generate beta-amyloid or in its precursor form.
But Goldstein鈥檚 work suggests the role of axon transport should be revisited. The researchers used mice genetically engineered to develop Alzheimer鈥檚 disease young, because they had a copy of the mutated amyloid-precursor gene inserted into their DNA. They blocked axon transport in the mice by also mutating one of their genes for the molecular motor kinesin. As expected, the number of swollen axons increased. More excitingly, the amount of beta-amyloid in the brain and the number of amyloid plaques increased massively. 鈥淲hen we studied the neurons in detail, we saw a traffic jam of transport-related proteins,鈥� says Goldstein. He suggests that more beta-amyloid is produced when its precursor protein is held up in the axon traffic jam, and this worsens the disease symptoms.
We do not yet know how to correct defects in axon transport, but it may be possible in the foreseeable future to stop, or at least delay, the progression to axon suicide. This seems to be how the WldS gene works in pmn mice, and as we begin to understand its role we may find a way to mimic its effect in humans. Axon degeneration can also be delayed in animal models of multiple sclerosis, stroke and other diseases using drugs that prevent abnormal flows of sodium and calcium ions into axons. These ion imbalances may be an indirect result of blocked axonal transport.
Ironically, axon transport could itself be used to deliver certain therapies to the nervous system. Gene therapy to the spinal cord usually requires complex and risky surgery. But earlier this year researchers from the UK biotech firm Oxford Biomedica showed that axon transport could deliver a gene therapy vector from an injection site in a muscle to cell nuclei in the spinal cord (Nature Medicine, vol 11, p 429). This approach significantly delayed the development of motor neuron disease in mice.
鈥淎t last we had a way to slow the neuron disease down 鈥� something to build on鈥�
Exciting though this new field of research is, there is likely to be more to the story. With most neurological diseases several systems are likely to be involved, and addressing axon transport will not solve all the problems. Beyond doubt, though, axon transport has been a neglected area, and as researchers increasingly direct their attention to the issue, patients suffering from many common diseases are likely to reap the benefits. Waller may have been wrong about axon nourishment, but his prediction about the importance of Wallerian degeneration in neurological disease was spot on.
Motors in miniature
Living cells are home to the world鈥檚 smallest railway system. It consists of molecular motors that 鈥渨alk鈥� in steps of 8 nanometres along tracks made of filamentous proteins called microtubules. The motors carry cargoes such as molecular building blocks and chemical messengers. Nanotechnologists would like to hijack this intricate transport system for a host of other applications, such as assembling miniature electronic devices for the nanocomputers of the future.
Researchers led by Viola Vogel at the University of Washington in Seattle are developing a high-resolution imaging device by turning the transport system on its head. Instead of the motors walking over the microtubules, the motors are glued onto the surface of whatever needs to be viewed, allowing them to move fragments of microtubules over themselves.
By recording and analysing the pattern of movement of the microtubules, Vogel and her team can build up a map that provides topographical information.
Controlling the loading and unloading of cargo as well as the direction and speed of the movement is no mean feat when you are dealing with individual molecules. But the team is making progress. The speed of movement, for example, can be controlled by varying the amount of chemical fuel available to the motors.
