Prions, once dismissed as an impossibility, have now gained wide recognition as extraordinary agents that cause a number of infectious, Genetic and spontaneous disorders.

by: Stanley B. Prusiner

Description & History
Fifteen years ago I evoked a good deal of skepticism when I proposed that the infectious agents causing certain degenerative disorders of the central nervous system in animals and, more rarely, in humans might consist of protein and nothing else. At the time, the notion was heretical. Dogma held that the conveyers of transmissible diseases required Genetic material, composed of nucleic acid (DNA or RNA), in order to establish an infection in a host.

Even viruses, among the simplest microbes, rely on such material to direct synthesis of the proteins needed for survival and replication. Later, many scientists were similarly dubious when my colleagues and I suggested that these 'proteinaceous infectious particles'--or 'prions,' as I called the disease-causing agents--could underlie inherited, as well as communicable, diseases. Such dual behavior was then unknown to medical science. And we met resistance again when we concluded that prions (pronounced 'pree-ons') multiply in an incredible way; they convert normal protein molecules into dangerous ones simply by inducing the benign molecules to change their shape.

Today, however, a wealth of experimental and clinical data has made a convincing case that we are correct on all three counts. Prions are indeed responsible for transmissible and inherited disorders of protein conformation. They can also cause sporadic disease, in which neither transmission between individuals nor inheritance is evident. Moreover, there are hints that the prions causing the diseases explored thus far may not be the only ones. Prions made of rather different proteins may contribute to other neurodegenerative diseases that are quite prevalent in humans. They might even participate in illnesses that attack muscles.

The known prion diseases, all fatal, are sometimes referred to as Spongiform Encephalopathies. They are so named because they frequently cause the brain to become riddled with holes. These ills, which can brew for years (or even for decades in humans) are widespread in animals. The most common form is scrapie, found in sheep and goats. Afflicted animals lose coordination and eventually become so incapacitated that they cannot stand. They also become irritable and, in some cases, develop an intense itch that leads them to scrape off their wool or hair (hence the name "scrapie").

The other prion diseases of animals go by such names as transmissible mink encephalopathy, chronic wasting disease of mule deer and elk, feline spongiform encephalopathy and bovine spongiform encephalopathy. The last, often called mad cow disease, is the most worrisome. Gerald A. H. Wells and John W. Wilesmith of the Central Veterinary Laboratory in Weybridge, England, identified the condition in 1986, after it began striking cows in Great Britain, causing them to became uncoordinated and unusually apprehensive.

The source of the emerging epidemic was soon traced to a food supplement that included meat and bone meal from dead sheep. The methods for processing sheep carcasses had been changed in the late 1970s. Where once they would have eliminated the scrapie agent in the supplement, now they apparently did not. The British government banned the use of animal-derived feed supplements in 1988, and the epidemic has probably peaked. Nevertheless, many people continue to worry that they will eventually fall ill as a result of having consumed tainted meat.

The human prion diseases are more obscure. Kuru has been seen only among the Fore Highlanders of Papua New Guinea. They call it the "laughing death." Vincent Zigas of the Australian Public Health Service and D. Carleton Gajdusek of the U.S. National Institutes of Health described it in 1957, noting that many highlanders became afflicted with a strange, fatal disease marked by loss of coordination (ataxia) and often later by dementia. The affected individuals probably acquired kuru through ritual cannibalism: the Fore tribe reportedly honored the dead by eating their brains. The practice has since stopped, and kuru has virtually disappeared.

Creutzfeldt-Jakob disease, in contrast, occurs worldwide and usually becomes evident as dementia. Most of the time it appears sporadically, striking one person in a million, typically around age 60. About 10 to 15 percent of cases are inherited, and a small number are, sadly, iatrogenic--spread inadvertently by the attempt to treat some other medical problem. Iatrogenic Creutzfeldt-Jakob disease has apparently been transmitted by corneal transplantation, implantation of dura mater or electrodes in the brain, use of contaminated surgical instruments, and injection of growth hormone derived from human pituitaries (before recombinant growth hormone became available).

The two remaining human disorders are Gerstmann-Straussler-Scheinker disease (which is (2) manifest as ataxia and other signs of damage to the cerebellum) and fatal familial insomnia (in which dementia follows difficulty sleeping). Both these conditions are usually inherited and typically appear in midlife. Fatal familial insomnia was discovered only recently, by Elio Lugaresi and Rossella Medori of the Univ of Bologna and Pierluigi Gambetti of Case Western Reserve University.

In Search of the Cause

In the course of reading, I came across an astonishing report in which Tikvah Alper and her colleagues at the Hammersmith Hospital in London suggested that the scrapie agent might lack nucleic acid, which usually can be degraded by ultraviolet or ionizing radiation. When the nucleic acid in extracts of scrapie-infected brains was presumably destroyed by those treatments, the extracts retained their ability to transmit scrapie. If the organizm did lack DNA and RNA, the finding would mean that it was not a virus or any other known type of infectious agent, all of which contain Genetic material. What, then, was it?

Investigators had many ideas--including, jokingly, linoleum and kryptonite--but no hard answers. I immediately began trying to solve this mystery when I set up a laboratory at U.C.S.F. in 1974. The first step had to be a mechanical one--purifying the infectious material in scrapie-infected brains so that its composition could be analyzed. The task was daunting; many investigators had tried and failed in the past. But with the optimism of youth, I forged ahead [see "Prions," by Stanley B. Prusiner; SCIENTIFIC AMERICAN, October 1984]. By 1982 my colleagues and I had made good progress, producing extracts of hamster brains consisting almost exclusively of infectious material. We had, furthermore, subjected the extracts to a range of tests designed to reveal the composition of the disease-causing component.

Amazing Discovery

I thus introduced the term "prion" to distinguish this class of disease conveyer from viruses, bacteria, fungi and other known Pathogens. Not long afterward, we determined that scrapie prions contained a single protein that we called PrP, for "prion protein." Now the major question became; Where did the instructions specifying the sequence of amino acids in PrP reside? Were they carried by an undetected piece of DNA that traveled with PrP, or were they, perhaps, contained in a Gene housed in the chromosomes of cells?

The key to this riddle was the identification in 1984 of some 15 amino acids at one end of the PrP protein. My group identified this short amino acid sequence in collaboration with Leroy E. Hood and his co-workers at the California Institute of Technology. Knowledge of the sequence allowed us and others to construct molecular probes, or detectors, able to indicate whether mammalian cells carried the PrP gene. With probes produced by Hood's team, Bruno Oesch, working in the laboratory of Charles Weissmann at the Univ of Zurich, showed that hamster cells do contain a Gene for PrP.

At about the same time, Bruce Cheseboro of the NIH Rocky Mountain Laboratories made his own probes and established that mouse cells harbor the Gene as well. That work made it possible to isolate the Gene and to establish that it resides not in prions but in the chromosomes of hamsters, mice, humans and all other mammals that have been examined. What is more, most of the time, these animals make PrP without getting sick.

One interpretation of such findings was that we had made a terrible mistake: PrP had nothing to do with prion diseases. Another possibility was that PrP could be produced in two forms, one that generated disease and one that did not. We soon showed the latter interpretation to be correct. The critical clue was the fact that the PrP found in infected brains resisted breakdown by cellular enzymes called proteases. Most proteins in cells are degraded fairly easily. I therefore suspected that if a normal, (3) nonthreatening form of PrP existed, it too would be susceptible to degradation.

Ronald A. Barry in my laboratory then identified this hypothetical protease-sensitive form. It thus became clear that scrapie-causing PrP is a variant of a normal protein. We therefore called the normal protein "cellular PrP" and the infectious (protease-resistant) form "scrapie PrP." The latter term is now used to refer to the protein molecules that constitute the prions causing all scrapie-like diseases of animals and humans.

Prion Diseases Can Be Inherited

Fortunately, work on a different problem led us to an alternative approach for demonstrating that prions could transmit scrapie without the help of any accompanying nucleic acid. In many cases, the scrapielike illnesses of humans seemed to occur without having been spread from one host to another, and in some families they appeared to be inherited. (Today researchers know that about 10 percent of human prion diseases are familial, felling half of the members of the affected families.)

It was this last pattern that drew our attention. Could it be that prions were more unusual than we originally thought? Were they responsible for the appearance of both hereditary and transmissible illnesses? In 1988 Karen Hsiao in my laboratory and I uncovered some of the earliest data showing that human prion diseases can certainly be inherited. We acquired clones of a PrP Gene obtained from a man who had Gerstmann-Straussler-Scheinker disease in his family and was dying of it himself. Then we compared his Gene with PrP Genes obtained from a healthy population and found a tiny abnormality known as a point mutation.

To grasp the nature of this mutation, it helps to know something about the organization of Genes. Genes consist of two strands of the DNA building blocks called nucleotides, which differ from one another in the bases they carry. The bases on one strand combine with the bases on the other strand to form base pairs: the "rungs" on the familiar DNA "ladder." In addition to holding the DNA ladder together, these pairs spell out the sequence of amino acids that must be strung together to make a particular protein. Three base pairs together--a unit called a codon--specify a single amino acid.

In our dying patient, just one base pair (out of more than 750) had been exchanged for a different pair. The change, in turn, had altered the information carried by codon 102, causing the amino acid leucine to be substituted for the amino acid proline in the man's PrP protein. With the help of Tim J. Crow of Northwick Park Hospital in London and Jurg Ott of Columbia Univand their colleagues, we discovered the same mutation in Genes from a large number of patients with Gerstmann-Straussler-Scheinker disease, and we showed that the high incidence in the affected families was statistically significant.

In other words, we established Genetic linkage between the mutation and the disease--a finding that strongly implies the mutation is the cause. Over the past six years work by many investigators has uncovered 18 mutations in families with inherited prion diseases; for five of these mutations, enough cases have now been collected to demonstrate Genetic linkage. The discovery of mutations gave us a way to eliminate the possibility that a nucleic acid was traveling with prion proteins and directing their multiplication.

We could now create Genetically altered mice carrying a mutated PrP gene. If the presence of the altered gene in these "transgenic" animals led by itself to scrapie, and if the brain tissue of the transgenic animals then caused scrapie in healthy animals, we would have solid evidence that the protein encoded by the mutated gene had been solely responsible for the transfer of disease.

Studies I conducted with Hsiao, Darlene Groth in my group and Stephen J. DeArmond, head of a separate laboratory at U.C.S.F., have now shown that scrapie can be generated and transmitted in this way [see BOX at end of this article]. These results in animals resemble those obtained in 1981, when Gajdusek, Colin L. Masters and Clarence J. Gibbs, Jr., all at the National Institutes of Health, transmitted apparently inherited Gerstmann-Straussler-Scheinker disease to monkeys. They also resemble the findings of Jun Tateishi and Tetsuyuki Kitamoto of Kyushu Univin Japan, who transmitted inherited Creutzfeldt-Jakob disease to mice.

Together the collected transmission studies persuasively argue that prions do, after all, (4) represent an unprecedented class of infectious agents, composed only of a modified mammalian protein. And the conclusion is strengthened by the fact that assiduous searching for a scrapie-specific nucleic acid (especially by Detlev H. Riesner of Heinrich Heine Univin Dusseldorf) has produced no evidence that such Genetic material is attached to prions. Scientists who continue to favor the virus theory might say that we still have not proved our case.

If the PrP Gene coded for a protein that, when mutated, facilitated infection by a ubiquitous virus, the mutation would lead to viral infection of the brain. Then injection of brain extracts from the mutant animal would spread the infection to another host. Yet in the absence of any evidence of a virus, this hypothesis looks to be untenable. In addition to showing that a protein can multiply and cause disease without help from nucleic acids, we have gained insight into how scrapie PrP propagates in cells. Many details remain to be worked out, but one aspect appears quite clear: the main difference between normal PrP and scrapie PrP is conformational.

Evidently, the scrapie protein propagates itself by contacting normal PrP molecules and somehow causing them to unfold and flip from their usual conformation to the scrapie shape. This change initiates a cascade in which newly converted molecules change the shape of other normal PrP molecules, and so on. These events apparently occur on a membrane in the cell interior.

We started to think that the differences between cellular and scrapie forms of PrP must be conformational after other possibilities began to seem unlikely. For instance, it has long been known that the infectious form often has the same amino acid sequence as the normal type. Of course, molecules that start off being identical can later be chemically modified in ways that alter their activity. But intensive investigations by Neil Stahl and Michael A. Baldwin in my laboratory have turned up no differences of this kind.

One Protein, Two Shapes

His calculations imply that the protein probably folds into a compact structure having four helices in its core. Less is known about the structure, or structures, adopted by scrapie PrP. The evidence supporting the proposition that scrapie PrP can induce an alpha-helical PrP molecule to switch to a beta-sheet form comes primarily from two important studies by investigators in my group.

Maria Gasset learned that synthetic peptides (short strings of amino acids) corresponding to three of the four putative alpha-helical regions of PrP can fold into beta sheets. And Jack Nguyen has shown that in their beta-sheet conformation, such peptides can impose a beta-sheet structure on helical PrP peptides. More recently Byron W. Caughey of the Rocky Mountain Laboratories and Peter T. Lansbury of the Massachusetts Institute of Technology have reported that cellular PrP can be converted into scrapie PrP in a test tube by mixing the two proteins together.

PrP molecules arising from mutated genes probably do not adopt the scrapie conformation as soon as they are synthesized. Otherwise, people carrying mutant Genes would become sick in early childhood. We suspect that mutations in the PrP Gene render the resulting proteins susceptible to flipping from an alpha-helical to a beta-sheet shape. Presumably, it takes time until one of the molecules spontaneously flips over and still more time for scrapie PrP to accumulate and damage the brain enough to cause symptoms.

Fred Cohen and I think we might be able to explain why the various mutations that have been noted in PrP genes could facilitate folding into the beta-sheet form. Many of the human mutations give rise to the substitution of one amino acid for another within the four putative helices or at their borders. Insertion of incorrect amino acids at those positions might destabilize a helix, thus increasing the likelihood that the affected helix and its neighbors will refold into a beta-sheet conformation.

Conversely, Hermann Schatzel in my laboratory finds that the harmless differences distinguishing the PrP gene of humans from those of apes and monkeys affect amino acids lying outside of the proposed helical domains--where the divergent amino acids probably would not profoundly influence the stability of the helical regions.

Treatment Ideas Emerge(5)

We do know with certainty that cleavage of scrapie PrP is what produces PrP fragments that accumulate as plaques in the brains of some patients. Those aggregates resemble plaques seen in Alzheimer's disease, although the Alzheimer's clumps consist of a different protein. The PrP plaques are a useful sign of prion infection, but they seem not to be a major cause of impairment. In many people and animals with prion disease, the plaques do not arise at all.

Even though we do not yet know much about how PrP scrapie harms brain tissue, we can foresee that an understanding of the three-dimensional structure of the PrP protein will lead to therapies. If, for example, the four-helix-bundle model of PrP is correct, drug developers might be able to design a compound that would bind to a central pocket that could be formed by the four helices. So bound, the drug would stabilize these helices and prevent their conversion into beta sheets.

Another idea for therapy is inspired by research in which Weissmann and his colleagues applied gene-targeting technology to create mice that lacked the PrP Gene and so could not make PrP. By knocking out a Gene and noting the consequences of its loss, one can often deduce the usual functions of the gene's protein product. In this case, however, the animals missing PrP displayed no detectable abnormalities.

If it turns out that PrP is truly inessential, then physicians might one day consider delivering so-called antisense or antigene therapies to the brains of patients with prion diseases. Such therapies aim to block Genes from giving rise to unwanted proteins and could potentially shut down production of cellular PrP [see "The New Genetic Medicines," by Jack S. Cohen and Michael E. Hogan; SCIENTIFIC AMERICAN, December 1994].

They would thereby block PrP from propagating itself. It is worth noting that the knockout mice provided a welcomed opportunity to challenge the prion hypothesis. If the animals became ill after inoculation with prions, their sickness would have indicated that prions could multiply even in the absence of a pre-existing pool of PrP molecules. As I expected, inoculation with prions did not produce scrapie, and no evidence of prion replication could be detected.

The enigma of how scrapie PrP multiplies and causes disease is not the only puzzle starting to be solved. Another long-standing question--the mystery of how prions consisting of a single kind of protein can vary markedly in their effects--is beginning to be answered as well. Lain H. Pattison of the Agriculture Research Council in Compton, England, initially called attention to this phenomenon. Years ago he obtained prions from two separate sets of goats. One isolate made inoculated animals drowsy, whereas the second made them hyperactive. Similarly, it is now evident that some prions cause disease quickly, whereas others do so slowly.

The Mystery of "Strains"

Similarly, one "conformer" might be attracted to Neuronal populations in one part of the brain, whereas another might be attracted to Neurons elsewhere, thus producing different symptoms. Considering that PrP can fold in at least two ways, it would not be surprising to find it can collapse into other structures as well. Since the mid-1980s we have also sought insight into a phenomenon known as the species barrier. This concept refers to the fact that something makes it difficult for prions made by one species to cause disease in animals of another species. The cause of this difficulty is of considerable interest today because of the epidemic of mad cow disease in Britain. We and others have been trying to find out whether the species barrier is strong (6) enough to prevent the spread of prion disease from cows to humans.

Breaking the Barrier

Moreover, on the basis of this and other experiments, we realized that the barrier resides in the amino acid sequence of PrP: the more the sequence of a scrapie PrP molecule resembles the PrP sequence of its host, the more likely it is that the host will acquire prion disease. In one of those other experiments, for example, we examined transgenic mice carrying the Syrian hamster PrP Gene in addition to their own mouse gene. Those mice make normal forms of both hamster and mouse PrP. When we inoculated the animals with mouse prions, they made more mouse prions. When we inoculated them with hamster prions, they made hamster prions.

From this behavior, we learned that prions preferentially interact with cellular PrP of homologous, or like, composition. The attraction of scrapie PrP for cellular PrP having the same sequence probably explains why scrapie managed to spread to cows in England from food consisting of sheep tissue: sheep and bovine PrP differ only at seven positions. In contrast, the sequence difference between human and bovine PrP is large: the molecules diverge at more than 30 positions. Because the variance is great, the likelihood of transmission from cows to people would seem to be low.

Consistent with this assessment are epidemiological studies by W. Bryan Matthews, a professor emeritus at the Univ of Oxford. Matthews found no link between scrapie in sheep and the occurrence of Creutzfeldt-Jakob disease in sheep-farming countries. On the other hand, two farmers who had "mad cows" in their herds have recently died of Creutzfeldt-Jakob disease. Their deaths may have nothing to do with the bovine epidemic, but the situation bears watching. It may turn out that certain parts of the PrP molecule are more important than others for breaking the species barrier.

If that is the case, and if cow PrP closely resembles human PrP in the critical regions, then the likelihood of danger might turn out to be higher than a simple comparison of the complete amino acid sequences would suggest. We began to consider the possibility that some parts of the PrP molecule might be particularly important to the species barrier after a study related to this blockade took an odd turn. My colleague Glenn C. Telling had created transgenic mice carrying a hybrid PrP Gene that consisted of human codes flanked on either side by mouse codes; this Gene gave rise to a hybrid protein.

Then he introduced brain tissue from patients who had died of Creutzfeldt-Jakob disease or Gerstmann-Straussler-Scheinker disease into the transgenic animals. Oddly enough, the animals became ill much more frequently and faster than did mice carrying a full human PrP gene, which diverges from mouse PrP at 28 positions. This outcome implied that similarity in the central region of the PrP molecule may be more critical than it is in the other segments. The result also lent support to earlier indications--uncovered by Shu-Lian Yang in DeArmond's laboratory and Albert Taraboulos in my group--that molecules made by the host can influence the behavior of scrapie PrP.

We speculate that in the hybrid-gene study, a mouse protein, possibly a "chaperone" normally involved in folding nascent protein chains, recognized one of the two mouse-derived regions of the hybrid PrP protein. This chaperone bound to that region and helped to refold the hybrid molecule into the scrapie conformation. The chaperone did not provide similar help in mice making a totally human PrP protein, presumably because the human protein lacked a binding site for the mouse factor.

The List May Grow

When we pursued the cause, we found that making excessive amounts of PrP can (7) eventually lead to neurodegeneration and, surprisingly, to destruction of both muscles and peripheral nerves. These discoveries widen the spectrum of prion diseases and are prompting a search for human prion diseases that affect the peripheral nervous system and muscles. Investigations of animals that overproduce PrP have yielded another benefit as well. They offer a clue as to how the sporadic form of Creutzfeldt-Jakob disease might arise. For a time I suspected that sporadic disease might begin when the wear and tear of living led to a mutation of the PrP Gene in at least one cell in the body. Eventually, the mutated protein might switch to the scrapie form and gradually propagate itself, until the buildup of scrapie PrP crossed the threshold to overt disease.

The mouse studies suggest that at some point in the lives of the one in a million individuals who acquire sporadic Creutzfeldt-Jakob disease, cellular PrP may spontaneously convert to the scrapie form. The experiments also raise the possibility that people who become afflicted with sporadic Creutzfeldt-Jakob disease overproduce PrP, but we do not yet know if, in fact, they do.

All the known prion diseases in humans have now been modeled in mice. With our most recent work we have inadvertently developed an animal model for sporadic prion disease. Mice inoculated with brain extracts from scrapie-infected animals and from humans afflicted with Creutzfeldt-Jakob disease have long provided a model for the infectious forms of prion disorders. And the inherited prion diseases have been modeled in transgenic mice carrying mutant PrP Genes.

These murine representations of the human prion afflictions should not only extend understanding of how prions cause brain degeneration, they should also create opportunities to evaluate therapies for these devastating maladies.

Striking Similarities

If a virus were involved in these illnesses, white cells would be expected to appear. Recent findings in yeast encourage speculation that prions unrelated in amino acid sequence to the PrP protein could exist. Reed B. Wickner of the NIH reports that a protein called Ure2p might sometimes change its conformation, thereby affecting its activity in the cell. In one shape, the protein is active; in the other, it is silent. The collected studies described here argue persuasively that the prion is an entirely new class of infectious Pathogen and that prion diseases result from aberrations of protein conformation. Whether changes in protein shape are responsible for common neurodegenerative diseases, such as Alzheimer's, remains unknown, but it is a possibility that should not be ignored.

BOX: A Persuasive Experiment

Then the workers injected brain tissue from the diseased animals into Genetically altered mice making low levels of the same mutant PrP protein. (Such mice were chosen as recipients because scrapie PrP is most attracted to PrP molecules having the same composition.) Uninoculated mice did not become ill (indicating that making low levels of the aberrant protein was safe), but many of the treated ones did. Moreover, brain tissue transferred from the diseased recipients to their healthy counterparts caused illness once again. If the aberrant protein were unable to transmit infection, none of the inoculated animals would have sickened.

STANLEY B. PRUSINER is professor of neurology and biochemistry at the University of (8) California School of Medicine, San Francisco. Born: May 28, 1942; Place of birth: U.S.A.; Residence: San Francisco, U.S.A.; Affiliation: Dept of Neurology, Univ of California, School of Medicine, San Francisco, CA 94143, USA.

He is a member of the National Academy of Sciences, the Institute of Medicine and the American Academy of Arts and Sciences. He has won many awards for his research into prions, from the Albert Lasker Basic Medical Research Award and the Paul Ehrlich Award to his latest achievement:

He is the 1997 Nobel Laureate in Medicine for his discovery of Prions - a new biological principle of infection.

This is his second article for Scientific American.

Further Reading

Molecular Biology Of Prion Diseases. S. B. Prusiner in "Science," Vol. 252, pages 1515-1522; June 14, 1991.

Prion Diseases Of Humans And Animals. Edited by S. B. Prusiner, J. Collinge, J. Powell and B. Anderton. Ellis Horwood, 1992.

Fatal Familial Insomnia: Inherited Prion Diseases, Sleep, And The Thalamus. Edited by C. Guilleminault et al. Raven Press, 1994.

Molecular Biology Of Prion Diseases. Special issue of "Philosophical Transactions of the Royal Society of London, Series B," Vol. 343, No. 1306; March 29, 1994.

Structural Clues To Prion Replication. F. E. Cohen, K.-M. Pan, Z. Huang, M. Baldwin, R. J. Fletterick and S. B. Prusiner in "Science," Vol. 264, pages 530-531; April 22, 1994.

Scientific American January 1995 Volume 272 Number 1 Pages 48-57. Scientific American (ISSN 0036-8733), published monthly by Scientific American, Inc., 415 Madison Avenue, New York, N.Y. 10017-1111.

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