prions

prions (rhyming with aeons) is an acronym for ‘proteinaceous infectious particles’. The term was coined in 1982 by Stanley B. Prusiner, a neurologist at the University of California at San Francisco, who proposed that a new type of pathogen consisting solely of protein is responsible for a school of deadly neurodegenerative diseases called Transmissible Spongiform Encephalopathies (TSEs). These include scrapie in sheep, bovine spongiform encephalopathy (BSE or ‘mad cow disease’) in cattle and Creutzfeldt-Jakob Disease (CJD) in people.

Inheritance and infection

TSEs come in an array of strains and types, each causing a distinct pattern of brain damage and clinical signs. The ‘drowsy’ and ‘hyper’ lines of sheep scrapie first alerted researchers to such variations in the 1950s. In the late 1970s a type was discovered in North American captive elk that causes a wasting disease. In some human strains, such as CJD, symptoms progress from disturbances of balance and co-ordination to blindness and deep dementia. Others produce sleep disorders.

Some TSEs look like genetic conditions. For example, the very rare human TSE Gerstmann– Straussler–Scheinker Syndrome (GSS) appears to be strictly familial, striking distant cousins on opposite sides of the globe with eerie similarity. By contrast, other TSEs can clearly be the result of infection. Pioneering work by French veterinarians in the 1930s and 1940s illustrated that scrapie could be spread between sheep and goats by injection.

Among humans, the disease kuru, found in the south Pacific, was also shown to be transmissible. In the 1950s, it was the leading cause of death in the Fore-speaking tribe of the Eastern Highlands of Papua New Guinea, until an international team of researchers discovered that it was spread by funeral rites in which the dead were revered by eating or handling their organs. The West suffered cases of what experts came to dub ‘high tech cannibalism’: since the 1970s, corneal transplants, dural grafts and contaminated human growth hormone extracted from cadaveric pituitary glands have all been shown to be potential vectors for the spread of CJD.

Mechanism of infection

TSE infection has some very odd features. Victims mount no obvious immune response, and the agent responsible is extraordinarily resilient. The solvents used for the storage of pituitary glands for production of growth hormone should have killed all known pathogens. The infectivity of brain matter from scrapie-positive sheep survives exposure to formaldehyde and even ultraviolet radiation. The latter observation prompted a suggestion by British researchers that the scrapie agent, unlike viruses and bacteria, might not contain nucleic acid (DNA or RNA), since this would have been destroyed by ultraviolet radiation. Prusiner cited this evidence when he proposed the prion model in 1982.

Since then, Prusiner and supporters of his ideas around the world have tackled TSEs with a series of dramatic experiments using the latest molecular techniques. They have treated diseased brain tissue with detergents and centrifuges and harvested the encrusted, suspect prion. After several groups determined the genetic sequence of that protein, Prusiner realized that it was a fragment of a normal protein (prion protein or PrP), the function of which is still uncertain, which is found in healthy nerve cells. They have gone on to argue, but not to prove, that once mutated, this protein becomes an aberrant prion, e.g. PrP (Scrapie), which might then convert similar healthy protein to the diseased form through a domino-style process that Prusiner calls ‘conformation’. This conversion can be sparked, Prusiner speculates, in three different ways: a person can inherit ‘weak’ proteins genetically inclined to mutate; a person's natural prion protein can spontaneously mutate; or the mutated form can be transmitted through food, surgery, or drugs, seeding the transformation of the host animal's natural protein.

Continuing controversy

In 1997, Prusiner was awarded the Nobel Prize for Physiology or Medicine for elucidating an ‘entirely new genre of disease-causing agents’. However, for UK government scientists at the coalface of the British BSE crisis, and other TSE specialists in the United States, the award was premature.

Prions, they observed, had never been shown to cause disease. Only four days before Prusiner's Nobel Prize was announced, the prion scarcely merited a mention in an article in the journal Nature by the leading researcher Moira Bruce of the Neuropathogenesis Unit (NPU) in Edinburgh. Bruce described evidence that the same agent that had infected more than a million British cattle was responsible for the variant form of CJD (vCJD), which had started to strike young Britons. Two groups of test mice experimentally infected with either diseased cattle brain or human tissue from victims of vCJD, had very similar patterns of brain damage after very similar incubation times. In presenting her evidence, Bruce only once mentioned the word prion, and couched it in a distancing pair of quotation marks.

Bruce insists that the TSE agent ‘behaves exactly like a virus’, though her group thinks that it may be an unconventional sort, which they call a ‘virino’. The argument between the virus/virino and prion camps is built on styles of investigation that could scarcely be more different. Bruce's group is inheritor of a line of research founded on traditional biological observation. Much of what we know about the pathogenesis of these diseases comes from this group, and the Institute for Animal Health in Compton, Berkshire.

They inoculated mice with extracts of brain tissue from sheep with scrapie, then observed the emergence of infection over two years or more. They established that the strains of scrapie can be recognized by the incubation time and pattern of brain damage in such host mice. They detected the presence in the test mice of a gene that clearly affects incubation times, which they named Sinc, for ‘Scrapie Incubation’. It turns out that Sinc is the PrP gene.

They also discovered that the host animal must have a healthy immune system for infection to take hold. Infection somehow rides the organs of the immune system and eventually floods out into the central nervous system, proceeding up the spinal cord to the brain, causing holes and protein deposits (plaques).

In 1993, presenting her findings to the Royal Society in London, Bruce demonstrated that when another species (monkey, sheep, antelope, cat) has been infected with material from a cow with BSE, the infectious material from that new species still exhibits the characteristics of BSE in her strain-typing tests. The prion conformation model has yet to cope adequately with this finding. All the other species have very different natural prion proteins. The conformation and thus progression of the disease should logically vary according to the particular chemical composition of the victim's own prion protein, which is supposed to be transformed into an aberrant prion by the initial infection. To Bruce, the obvious explanation for the persistent properties of BSE in so many different species was that the BSE agent is a virus-like agent, possessing its own DNA or RNA, which, as in a viral infection, causes the production of more infectious agent just like itself.

To Prusiner, the failure of the opposition to isolate a virus or a nucleic acid is critical. He and his collaborators have shown that mice genetically engineered to stop them producing their own PrP cannot be infected with TSEs from other animals. To them, this is evidence that the protein is the agent. The virus camp sees PrP as a receptor for a foreign agent.

Prusiner and his supporters come back to the toughness of the aberrant protein and its resilience in the face of enzymes, radiation, formaldehyde, and heat. However, prion-sceptics point to work from 1991 indicating that TSE agents are probably not indestructible, just devilishly tricky to get at. And other viruses can survive formaldehyde. During rendering, autoclaving and hormone extraction, protein fragments toughen and aggregate. Whether it is this toughening, or native impenetrability, that protects the TSE agents, their inactivation remains a key challenge in agriculture and medicine.

The most important inroads paved so far by prionism have come in the field of molecular genetics. Prionists have found mutations in the PrP gene that point to genetic susceptibility to TSEs. Neurologist John Collinge, of Imperial College London, with Prusiner in the early 1990s, discovered the PrP mutation involved in the seemingly familial prion disease GSS.

It turns out that the natural prion protein usually carries two delicate tree-like carbohydrate structures, called glycoforms. Prusiner and collaborators in Oxford and Ohio have observed that glycoforms change during disease. Prionists construe the change as a destabilizing part of the protein conformation process. The viral camp sees it as a classic side-effect of a foreign agent getting inside a cell and disrupting protein glycosylation.

Whatever causes the change, pathologists around the world now use glycoform analysis to help them determine the strain of TSE they are seeing in patients. Not enough is known about the protein-sugar variation to determine whether or not it can serve as a stand-alone test to, say, distinguish scrapie from BSE.

The prionists even claim to have demonstrated the conformation process in a test tube, by mixing normal PrP with the aberrant scrapie version, although only limited amounts were converted before the process fizzled out.

Despite impressive progress for the prion model the scientific case is not proven. Bruce's group still believes that the tough protein revealed by Prusiner's research simply cloaks and protects an independent nucleic acid, making up a virino. The prionists, they argue, have not adequately accounted for strain variation in scrapie, or the persistence of a particular TSE's characteristics, whatever its host.

What began as an obscure argument over a rare class of neurological diseases, and continues as an intense scientific controversy, is now at the heart of a world-wide public health crisis. Estimates of the number of Britons likely to succumb to vCJD now swing from hundreds to hundreds of thousands. And the rest of Europe is now battling to stem BSE in its own herds. A current challenge is development of reliable tests that can quickly detect the difference between normal and diseased prions, for screening of food and blood.

Emily Green, and Colin Blakemore

Bibliography

Collinge, J. (2000) Concise Oxford Textbook of Medicine Chapter 13.17 Oxford University Press, Oxford, 855.
Farquhar, C. F.,, Somerville, R. A., & and Bruce, M. E. (1998) Straining the prion hypothesis, Nature 391, 345–346
Prusiner, S. B. (1982) Novel Proteinaceous Infectious Particles Cause Scrapie, Science, 216: 136–144.

See also dementia; infection; microorganisms; virus.