THE BSE epidemic in the UK was an unmitigated disaster. It devastated the beef industry, destroyed peoples’ livelihoods, unleashed a terrible disease upon the population and cost billions of pounds of public money.
But to a small group of biochemists there was a silver lining. BSE and its human equivalent, vCJD, belong to an obscure group of diseases called prion diseases, transmitted through an implausible mechanism involving shape-shifting proteins that formed clumps in the brain. Without BSE, prion science would probably have remained a backwater. Once the disease struck, however, it went mainstream.
Now, as the US ramps up its BSE testing programme and the threat of a global epidemic becomes ever more real (see “American nightmare”), prion science has come of age. At the time of the UK outbreak there were multiple unknowns and controversies – not least over the very nature of prions themselves. Today, new insights are settling these questions and could accelerate efforts to grapple with the big challenges, such as how to prevent transmission to humans and, perhaps most importantly, discover how to prevent a similar disaster from ever happening again.
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It was 22 years ago that Stanley Prusiner of the University of California at San Francisco presented the world with an unlikely and controversial proposition. Prusiner’s “prions” were pure proteins – devoid of DNA, RNA or genes – that were supposedly able to transmit infectious brain disease such as BSE. It was a highly unorthodox idea and, not surprisingly, took a while to catch on: even Prusiner’s 1997 Nobel prize for the discovery didn’t quite silence the sceptics.
This year, though, prion biologists got their proof, showing shown that a single protein can exhibit all of the prions’ mysterious abilities. For all but a few die-hard sceptics, this has established once and for all the existence of protein-only infectious agents.
As a result, the stage is set for scientists to tackle the next big question of prion biology: how can the same infectious protein exist as several different “strains”, each causing a different disease? This is more than an academic question. It holds the key to explaining why some prions spread from one species to another – as BSE does from cattle to humans – while others apparently do not. It will also tell us how great a risk humans face from emerging prion diseases, such as chronic wasting disease in elk and deer.
Even to prion proponents the existence of different strains seemed hard to reconcile with the most popular prion model, in which prions have a dual personality, a sort of Dr Jekyll and Mr Hyde. Prusiner’s hypothesis concerned the mammalian brain protein PrP – the one involved in BSE, CJD and scrapie. In its good (Jekyll) form, molecules of the protein sit innocuously on the surface of brain cells. But when the protein switches shape to become the Mr Hyde prion form, PrP clumps together and forms fibres called amyloids, which choke nerve cells to death. What’s more, each Mr Hyde can convert any Jekyll it meets into another Hyde, in a deadly chain reaction.
Nothing in this model gave any reason to think that the prions could exist in multiple forms – yet that’s exactly what they seemed to do. Researchers discovered, for instance, that when scrapie prions from sheep were passed from mouse to mouse, they separated into about 20 different strains, each with a distinct incubation period and pattern of brain damage. Yeast biologists have found something similar. Yeast prion strains are designated “weak” or “strong” based on how efficiently they convert their Jekyll counterparts to the Mr Hyde form.
There seemed no way that a single protein, made of an invariant sequence of amino acids, could produce such variety. And so sceptics maintained that prions must contain some sort of concealed genetic material that encoded instructions for strain behaviour. Prions might play a role in disease, they said, but only genetic information could display the type of diversity seen in different strains.
Now prion proponents have the evidence they say should silence the doubters. Part of the proof comes from studies in yeast by two independent research teams, Chih-Yen King and Ruben Diaz-Avalos at Florida State University in Tallahassee and a group led by Jonathan Weissman at the University of California, San Francisco.
Weissman’s group had already shown that they could synthesise prion forms of the yeast protein Sup35 in a test tube and cause them to form clumps. And when inserted into yeast cells, the prions converted normal Sup35 molecules into new prions. This remains the best direct evidence so far for the protein-only hypothesis (żěè¶ĚĘÓƵ, 5 August 2000, p 11).
However, that experiment didn’t address the issue of strains. Now the two groups have shown that Sup35 can also form different strains of prion. Both groups worked with purified Sup35 made in bacteria to eliminate the possibility that another molecule could be involved in strain formation. Previous work showed that changing the temperature at which the clumps formed led to slightly different properties. On a hunch that these different forms may also act as different strains, the researchers purified Sup35 and let it clump at different temperatures.
As hoped, this led to clumps with different properties. Structural studies revealed subtle differences in how the atoms were arranged in clumps formed at 4 °C versus 37 °C. Similarly, the clumps showed different resistance to degradation by heat and enzymes. And crucially, the different clumps acted as different strains when put back into yeast cells. Those formed at 37 °C behaved as weak prions generation after generation, and those born at 4 °C behaved as strong prions. Just as intriguingly, when later removed from yeast cells, the prions descended from 4 °C ancestors maintained their physical properties even if they replicated for generations at higher temperatures (Nature, vol 428, p 319 and p 323).
Here was clinching proof that the same protein could form different strains of prion. “There is no wiggle room around that fact,” says Weissman.
Final proof
“This is the final proof of the protein-only theory,” says Mick Tuite of the University of Kent in Canterbury, UK. “It will take a while for other people to replicate and verify these experiments. But I think these will emerge as the most important papers in this field in the last 10 years.”
Not everyone agrees. Laura Manuelidis of Yale University, one of the most outspoken critics of Prusiner’s protein-only idea, argues that experiments in yeast will never address all the issues of human prion diseases. “These results are very relevant for understanding how proteins form amyloids,” she says. “But they say very little about how infectious agents cause BSE or vCJD.” To silence all doubt, she says, mammalian biologists need to catch up to their yeast colleagues and provide better evidence of protein-only mammalian prions.
As żěè¶ĚĘÓƵ went to press, Prusiner’s team reported significant progress towards this goal. They made amyloid from PrP produced in bacteria, then injected these synthetic prions into the brains of mice. The result: a prion disease that can be passed to other animals (Science, vol 305, p 673). What is more, prions made in slightly different ways caused different types of brain damage, suggesting they behave as different strains. Prion researchers will be scrutinising the results to determine whether they are final proof of the protein-only hypothesis.
Clearly, a better understanding of strains could have big practical pay-offs, since they have emerged as an important public health issue. For example, there are hints that a second form of prion disease has recently emerged in cattle (see “Out of sight, out of mind”). Presumably this is caused by a different strain of prion, but as yet no one knows whether it is a threat to human health.
Another big issue is the so-called “species barrier”, which apparently prevents some prion diseases such as scrapie from jumping into humans but allows others, notably BSE, to do so, albeit at a low rate. The assumption has always been that there is some fundamental difference between certain species’ prion proteins that stops one from transforming the other into an infectious form.
But research into strains has shed new light on this question. Fred Cohen, a long-time collaborator of Prusiner’s at UCSF, has found prion strains that cause disease in mice so slowly the animals usually die from other causes first. But if you give the strain a whole extra lifetime in which to develop, by grinding up the brains of infected animals and injecting them into other mice, the second generation develops a prion disease. “If you only looked at one generation, you’d say there must be a powerful species barrier operating here. But the difference is much more subtle,” he says.
In other words there is no absolute species barrier – it’s just that some strains of prion are so slow to act you never see them. Back-of-the-envelope calculations suggest that even a fairly small difference in the energy required for a prion strain to transmit its shape can alter transformation rates 100-fold. This might explain the incredibly wide range of disease progression rates among prion diseases, ranging from a few months in a mouse to decades-long incubation in humans.
The implications of this are somewhat worrying. If some strains can jump the species barrier more readily than others, could we one day face a new strain of BSE, chronic wasting disease or even scrapie that easily converts human PrP to prions? For now, there is no evidence that any such strain has emerged. “However,” cautions Byron Caughey, who studies prions at the Rocky Mountain Laboratories in Hamilton, Montana, “it’s something we should be concerned about and stay vigilant for.”
