A prion is a protein composed of about 250 amino acids. The term 'prion', meaning 'proteinaceous infectious particle' was first coined by Stanley B Prusiner¹ in 1982 to refer to an infectious agent that caused a range of neurodegenerative diseases in animals, including humans. He proposed that this agent consisted of a protein and nothing more. The research he had carried out indicated that no genetic material whatsoever, was involved in the transmission of prion diseases. This was viewed as a scientific heresy at the time. Every other disease studied up to that point, employed DNA or RNA, whether the microbial agent was a bacterium, a fungus, a nematode, a protozoan, a virus - all required genes to carry instructions to replicate.

Proteins are manufactured in cells as they are required by the body. Their form and function is genetically determined. Each protein is made up of amino acids strung together in a specific sequence. The type of protein depends on the types and sequence of its amino acids. These control the way the protein is folded which, in turn, determines the function of the protein. Proteins are contortionists - folded and twisted into very complicated shapes.

It appears that when a prion comes into contact with a normal protein, it is somehow able to 'persuade' the normal protein to assume a slightly different shape - a shape identical to that of the prion. Once this process has started, it continues. Like the domino effect, it is self-propagating.

Once the scientists knew what they were looking for, they found evidence of several different prions that are implicated in a range of diseases in various animal species and, surprisingly, in the evolution of yeasts².

The Diseases

Prions are now known to cause quite a large number of diseases, such as CJD (Creutzfeld-Jacob Disease), New Variant CJD, Kuru, FFI (Fatal Familial Insomnia) and GSS (Gerstmann-Straussler-Scheinker Syndrome) in humans; BSE (Bovine Spongiform Encephalopathy) in cattle; Scrapie in sheep; CWD (Chronic Wasting Disease) in elk and Feline Spongiform Encephalopathy in cats. These diseases are collectively known as the TSEs (Transmissible Spongiform Encephalopathies). Prions are also suspected of causing a range of diseases such as Multiple Sclerosis³, Huntington's Chorea and Alpers Syndrome, where the link between disease and prion is yet to be proven. All of the prion diseases are fatal, untreatable and very difficult to diagnose currently.

The symptoms exhibited in the known prion diseases are broadly similar, usually involving a loss of co-ordination and increasing dementia. There is some variability between diseases however. The victims of FFI, for example, completely lose their ability to sleep.

The misfolded proteins have been found to collect at particular sites in the body. The main reason they cause so much damage, is that they tend to aggregate in brain cells where they form 'plaques', then holes appear and the brain becomes spongy and unable to function properly. The precise chain of events is still unknown, but it is thought that the prions build up in intracellular vesicles to the point where the cells burst. Holes are left at the sites of burst cells and masses of prions are released to propagate more abnormal proteins. They have also been found to gather in parts of the lymphatic system and because of this, sheep can be tested for Scrapie by examining their tonsils.

Scrapie was the first of the prion diseases to come under close scrutiny. This disease has been known for about 200 years. It is thought to have arisen in Spain, then spread to the rest of Europe and beyond.

Transmission

In more recent times, cattle have been suffering from an illness with similar symptoms to those exhibited by sheep with scrapie. Even more recently, some young humans have also contracted an illness very like the sheep and cattle diseases, displaying diminishing co-ordination and increasing dementia. The human disease turned out to be New Variant CJD. CJD had been recognised in the early 1920s and was thought to affect one person per million of the human population, worldwide (though this was probably an underestimate). However, it normally struck victims in their mid-fifties and the new disease was hitting adolescents and young adults. Some people began to wonder if there might be a connection between what was popularly referred to as 'mad cow disease' (BSE) and the illness increasingly showing up in young people. They were right to wonder.

People who knew that sheep offal and bone were being fed to cattle, started to suspect that this was the source of the bovine encephalopathy. To further complicate matters, cattle were also being fed on cattle remains. So, not only were herbivores being fed on animal products, but they were being turned into cannibals. Cattle are exquisitely well designed to eat and digest grass - food takes 70 to 100 hours to pass through a cow, much longer than a carnivore. Their complex, multiple stomachs and very long digestive system did not evolve to digest animal products. It seemed a serious possibility that the scrapie agent could infect cattle fed on diseased sheep and, indeed, on other infected cattle. If that could happen, then it seemed all together likely that humans might be infected by eating diseased animals in the same way. After all, the victims of kuru were known to acquire the disease from ritual cannibalism.

Studies over the last two decades have revealed that prion diseases can be inherited genetically, they can arise sporadically and they can be acquired though diet. Also, unhappily, doctors have sometimes accidentally infected patients with prion diseases by using contaminated surgical instruments and by carrying out procedures such as corneal transplantation.

Prions are extraordinarily robust. Normal proteins are easily broken down by the body's own enzymes. Prions are not broken down by the body's enzymes or by the type of acid found in the gut (which is fairly strong). Bleach and heat leave it sufficiently intact to remain infectious. It has been shown that it can remain infectious after heating to 360 degrees C for an hour. Ultra violet radiation does not damage it significantly. In one experiment, infective tissue was buried in soil for three years and was still infectious when recovered. Until quite recently, doctors were unaware of exactly how resilient and infectious prions are. No wonder some unfortunate patients were accidentally infected by inadequately sterilized instruments

Unexpected Dual Action

The next big surprise after discovering that a protein alone, could be an infectious agent, was that prion diseases could also be inherited genetically. This was unheard of - an infectious disease agent that could also be inherited genetically. It was another 'heresy' that, initially, seemed beyond belief. Scientific research has confirmed that prion diseases can be both acquired and inherited. This seemed to suggest that, even though prions were the non-genetic agents of infectious disease, nevertheless, genes probably did play some role.

A further mystery involved cases of prion diseases in people who had neither family history nor any known opportunity to become infected. For example, although slightly more than 1 in 10 cases of CJD are inherited, the rest seem to arise sporadically. New variant CJD, on the other hand, is acquired from eating beef.

The Road to Discovery

The first important thing the scientists discovered about prions was that they did not make use of nucleic acid. The main indication of this extraordinary fact was that after the infectious matter had been exposed to ultra violet light, which would destroy any genes it might contain, the material remained infectious. Stanley Prusiner then managed to isolate and purify the infectious material. He eventually concluded that it was made entirely of protein when he found that subjecting it to treatments that would unfold proteins, reduced or eliminated its infectious potential.

Soon after this discovery, he and his team started to refer to the scrapie prions as 'PrP'. However, they later found that all the mammals they examined carried a gene for making PrP and that it did not cause these animals to get ill. This confused them initially, until they found that PrP could exist in two different forms - the normally folded, cellular form and the abnormally folded, scrapie form. Furthermore, they found that the normal form they found in animal cells was easily broken down by the cells' own enzymes, called proteases, whereas the abnormal form was resistant to break down by enzymes. They distinguished between the normal and abnormal types by calling the normal prion 'cellular PrP' or 'PrPC' and the abnormal prion 'scrapie PrP' or 'PrPSc'. All disease causing prions are now referred to as 'PrPSc' - even the ones that are found in cattle, humans and other animals.

The next part of this story focuses on what Professor Prusiner and his team discovered about the genes of people with inherited Gerstmann-Straussler-Scheinker disease. Recall that the prion proteins are made up of about 250 amino acids. The instructions for making amino acids are written in 'words' of just three characters - that is to say, just three nucleotide base pairs (base pairs are the rungs of the DNA ladder seen in double helix images) each. These groups of three are called 'codons'. Three base pairs times 250 amino acids makes 750 base pairs. They found a mutation in just one out of the 750 base pairs of the gene that codes for this particular protein, in families with GSS. They found that the misfolded protein differed from the normal protein by a single amino acid.

After that, they and other teams looked at the genes of families with a variety of hereditary prion diseases and found 18 more 'point mutations', at least five of which have been shown to be implicated in the diseases, so far. These mutations often cause one amino acid to be swapped for another. It is thought that this single amino acid is sufficient to weaken and destabilise the protein at that point in the sequence of amino acids. Although normally folded PrP is made to begin with, in time, one molecule will flip to the abnormal conformation spontaneously and this will have a cascade effect on other cellular PrP in close proximity. That is the reason people with inherited prion diseases do not develop them until they are older. Alpers disease is a possible exception to the rule as it affects children.

The mutated genes suspected of causing inherited CJD were used in vivisection experiments to genetically alter mice which then went on to develop an encephalopathy. Prions from the brains of these poor mice were then used to infect other, non-transgenic mice who also developed the disease, thus showing that a heritable prion disease can lead to an infectious disease - another first! Prion diseases are clearly very different from the diseases that are familiar to us.

The scientists have good reason to believe that when PrPSc comes into contact with normal, cellular PrP, it causes it to change shape. When normal PrP is mixed in a test tube with PrPSc, it changes to PrPSc after a short interval of contact with the abnormal protein. One reason that this is a particularly interesting result, is that the change happens to the perfectly normal cellular protein that was generated by a non-mutated gene and therefore has its full compliment of correctly sequenced amino acids. They do not know why this happens yet. Research is on-going and there is still a lot to learn.