Prions - a new biological principle of infection

Summary
The 1997 Nobel Prize in Physiology or Medicine is awarded to the American Stanley Prusiner for his pioneering discovery of an entirely new genre of disease-causing agents and the elucidation of the underlying priciples of their mode of action. Stanley Prusiner has added prions to the list of well known infectious agents including bacteria, viruses, fungi and parasites. Prions exist normally as innocuous cellular proteins, however, prions possess an innate capacity to convert their structures into highly stabile conformations that ultimately result in the formation of harmful particles, the causative agents of several deadly brain diseases of the dementia type in humans and animals. Prion diseases may be inherited, laterally transmitted, or occur spontaneously. Regions within diseased brains have a characteristic porous and spongy appearance, evidence of extensive nerve cell death, and affected individuals exhibit neurological symptoms including impaired muscle control, loss of mental acuity, memory loss and insomnia. Stanley Prusiner's discovery provides important insights that may furnish the basis to understand the biological mechanisms underlying other types of dementia-related diseases, for example Alzheimer's disease, and establishes a foundation for drug development and new types of medical treatment strategies.
The prize winning research was initiated 25 years ago
In 1972 Stanley Prusiner began his work after one of his patients died of dementia resulting from Creutzfeldt-Jakob disease (CJD). It had previously been shown that CJD, kuru, and scrapie, a similar disease affecting sheep, could be transmitted through extracts of diseased brains. There were many theories regarding the nature of the infectious agent, including one that postulated that the infectious agent lacked nucleic acid, a sensational hypothesis since at the time all known infectious agents contained the hereditary material DNA or RNA. Prusiner took up the challenge to precisely identify the infectious agent and ten years later in 1982 he and his colleagues successfully produced a preparation derived from diseased hamster brains that contained a single infectious agent. All experimental evidence indicated that the infectious agent was comprised of a single protein, and Prusiner named this protein a prion, an acronym derived from "proteinaceous infectious particle." It should be noted that the scientific community greeted this discovery with great skepticism, however, an unwavering Prusiner continued the arduous task to define the precise nature of this novel infectious agent.
The infectious prion particle forms within the body
Where was the gene encoding the prion, the piece of DNA that determined the sequence of the amino acids comprising the prion protein? Perhaps the gene was closely associated with the protein itself as in a small virus? The answers to these questions came in 1984 when Prusiner and colleagues isolated a gene probe and subsequently showed that the prion gene was found in all animals tested, including man. This startling finding raised even more questions. Could prions really be the causative agent of several dementia-type brain diseases when the gene was endogenous to all mammals? Prusiner must have made a mistake! The solution to this problem became evident with the sensational discovery that the prion protein, designated PrP, could fold into two distinct conformations, one that resulted in disease (scrapie PrP = PrPSc) and the other normal (PrP = PrPc). It was subsequently shown that the disease-causing prion protein had infectious properties and could initiate a chain reaction so that normal PrPc protein is converted into the more stabile PrPSc form. The PrPSc prion protein is extremely stabile and is resistant to proteolysis, organic solvents and high temperatures (even greater than 100^o C). With time, non-symptomatic incubation periods vary from months to years, the disease-causing PrPSc can accumulate to levels that result in brain tissue damage. In analogy to a well known literary work, the normal PrPc can be compared to the friendly Dr. Jekyll and the disease causing PrPSc to the dangerous Mr. Hyde, the same entity but in two different manifestations.
Mutations in the prion gene cause hereditary brain diseases
The long incubation time for prion based disease hampered the initial efforts to purify the prion protein. In order to assess purification schemes Prusiner was forced to use scores of mice and in each experiment wait patiently for approximately 200 days for the appearance of disease symptoms. The purification efforts accelerated when it was demonstrated that scrapie could be transferred to hamsters, animals that exhibited markedly shortened incubation times. Together with other scientists, Prusiner cloned the prion gene and demonstrated that the normal prion protein was an ordinary component of white blood cells (lymphocytes) and was found in many other tissues as well. Normal prion proteins are particularly abundant on the surface of nerve cells in the brain. Prusiner found that the hereditary forms of prion diseases, CJD and GSS (see the last section), were due to mutations in the prion gene. Proof that these mutations caused disease was obtained when the mutant genes were introduced into the germline of mice. These transgenic mice came down with a scrapie-like disease. In 1992 prion researchers obtained conclusive evidence for the role of the prion protein in the pathogenesis of brain disease when they managed to abolish the gene encoding the prion protein in mice, creating so called prion knock-out mice. These prion knock-out mice were found to be completely resistant to infection when exposed to disease-causing prion protein preparations. Importantly, when the prion gene was reintroduced into these knock-out mice, they once again became susceptible to infection. Strangely enough, mice lacking the prion gene are apparently healthy, suggesting that the normal prion protein is not an essential protein in mice, its role in the nervous system remains a mystery.
Structural variant disease-causing prions accumulate in different regions of the brain
Specific mutations within the prion gene give rise to structurally variant disease-causing prion proteins. These structural prion variants accumulate in different regions of the brain. Dependent upon the region of the brain that becomes infected, different symptoms, typical for the particular type of disease are evident. When the cerebellum is infected the ability to coordinate body movements declines. Memory and mental acuity are affected if the cerebral cortex is infected. Thalamus specific prions disturb sleep leading to insomnia, and prions infecting the brain stem primarily affect body movement.
Other dementias may have a similar background
Prusiner's pioneering work has opened new avenues for understanding the pathogenesis of more common dementia-type illnesses. For example, there are indications that Alzheimer's disease is caused when certain, non-prion, proteins undergo a conformational change that leads to the formation of harmful deposits or plaques in the brain. Prusiner's work has also established a theoretical basis for the treatment of prion diseases. It may be possible to develop pharmacological agents that prevent the conversion of harmless normal prion proteins to the disease-causing prion conformation.
Intrinsic defense mechanisms do not exist against prions
Prions are much smaller than viruses. The immune response does not react to prions since they are present as natural proteins from birth. They are not poisonous, but rather become deleterious only by converting into a structure that enables disease causing prion proteins to interact with one another forming thread-like structures and aggregates that ultimately destroy nerve cells. The mechanistic basis underlying prion protein aggregation and their cummulative destructive mechanism is still not well understood. In contrast to other infectious agents, prion particles are proteins and lack nucleic acid. The ability to transmit a prion infection from one species to another varies considerably and is dependent upon what is known as a species barrier. This barrier reflects how structurally related the prions of different species are.
Prion diseases in animals and man
Without exception, all known prion diseases lead to the death of those affected. There are, however, great variations in pre-symptomatic incubation times and how aggressively the disease progresses.
Scrapie, a prion disease of sheep, was first documented in Iceland during the 18th century. Scrapie was transferred to Scotland in the 1940s. Similar prion diseases are known to affect other animals, e.g., mink, cats, deer and moose.
Bovine Spongiform Encephalopathy (BSE) - Mad cow disease is a prion disease that has recently received a great deal of publicity. In England BSE was transmitted to cows through feedstuff supplemented with offals from scrapie-infected sheep. The BSE epidemic first became evident in 1985. Due to the long incubation time the epidemic did not peak until 1992. In this year alone roughly 37,000 animals were affected.
Kuru among the Fore-people in New Guinea was studied by Carleton Gajdusek (recipient of the the 1976 Nobel Prize in Physiology or Medicine). Kuru was shown to be transmitted in connection with certain cannibalistic rituals and was thought to be due to an unidentified "slow virus". The infectious agent has now been identified as a prion. Duration of illness from first symptoms to death: 3 to 12 months.
Gertsmann-Sträussler-Scheinker (GSS) disease is a hereditary dementia resulting from a mutation in the gene encoding the human prion protein. Approximately 50 families with GSS mutations have been identified. Duration of illness from evidence of first symptoms to death: 2 to 6 years.
Fatal Familial Insomnia (FFI) is due to another mutation in the gene encoding the human prion protein. Nine families have been found that carry the FFI mutation. Duration of illness from evidence of first symptoms to death: roughly one year.
Creutzfeldt-Jakob Disease (CJD) affects about one in a million people. In 85-90% of the cases it has been shown that CJD occurs spontaneously. Ten to fifteen per cent of the CJD cases are caused by mutations in the prion protein gene. In rare instances CJD is the consequence of infection. Previously infections were transmitted through growth hormone preparations prepared from the pituitary gland of infected individuals, or brain membrane transplants. About 100 families are known carriers of CJD mutations. Duration of illness from evidence of first symptoms to death: roughly one year.
A new variant of CJD that may have arisen through BSE-transmission. Since 1995 about 20 patients have been identified that exhibit CJD-like symptoms. Psychological symptoms with depression have dominated, but involuntary muscle contractions and difficulties to walk are also common.

Science Volume 278, Number 5336 Issue of 10 October 1997, p 214
Gretchen Vogel
The Nobel committee often honors scientists who spent years working against strong opposition on controversial ideas, but usually the prize arrives long after the dust has settled. Not so this year for the prize in physiology or medicine. Stockholm's Karolinska Institute announced Monday that it had chosen to honor Stanley Prusiner "for his discovery of prions--a new biological principle of infection." The University of California, San Francisco, professor of neurology, virology, and biochemistry has championed the idea that infectious proteins can cause a range of degenerative brain diseases by misfolding and causing other proteins to do likewise. The committee also departed from tradition by awarding the prize to a single researcher--the first time it has done so since 1987, and only the 10th time in the last 50 years. While many of Prusiner's colleagues have come to accept the once-heretical prion theory, most say it still faces some crucial unanswered questions. Many argue, for example, that definitive proof that prions can cause disease by themselves is still lacking and that a cofactor such as a virus cannot be ruled out. Nevertheless, they say, Prusiner's work so far in making his case is worthy of the prize. "The distance he has brought [the field] is unbelievable," says Peter Lansbury, a biochemist at Brigham and Women's Hospital in Boston who studies the possible role of prion-type processes in Alzheimer's disease. In a statement, Charles Weissmann of the University of Zurich--who some have argued should have shared the prize--called Prusiner "a true pioneer and iconoclast" who "has waged a scientific battle for over 2 decades to convince his colleagues and the world that the infectious agent responsible for diseases such as scrapie, "mad cow disease," and Creutzfeldt-Jakob disease [CJD] is an abnormal form of a protein ... and has accumulated the evidence which has convinced the vast majority of scientists of the correctness of his view." This year's prize is the second awarded for work with such degenerative brain diseases. D. Carleton Gajdusek won in 1976 for his work a decade earlier demonstrating that kuru--a brain disease that affected highlanders in New Guinea who practiced ritualized cannibalism--was infectious. At the time, Gajdusek's work led many to blame the malady on a slow-acting virus, but it is now widely considered to be a prion disease. Prusiner coined the term in 1982 to describe the "proteinaceous infectious particles" he blamed for causing scrapie in sheep and hamsters. He suggested that scrapie and a collection of other wasting brain diseases, some inherited, some infectious, and some sporadic, were all due to a common process: a misfolded protein that propagates and kills brain cells. In doing so, he was picking up on an idea proposed in the 1960s, when radiation biologist Tikvah Alper, of Hammersmith Hospital in London, and physicist J. S. Griffith of Bedford College, London, suggested that an infectious agent that lacked nucleic acid could cause disease. Alper, studying scrapie in sheep, found that brain tissue remained infectious even after she subjected it to radiation that would destroy any DNA or RNA. Griffith suggested in a separate paper that perhaps a protein, which would usually prefer one folding pattern, could somehow misfold and then catalyze other proteins to do so. Such an idea seemed to threaten the very foundations of molecular biology, which held that nucleic acids were the only way to transmit information from one generation to the next. Inspired by a patient who died of the wasting brain condition CJD in 1972, Prusiner set out to determine the causative agent behind the disease, which resembles both kuru and scrapie. He and his colleagues reported in Science in 1982 that they had found an unusual protein in the brains of scrapie-infected hamsters that did not seem to be present in healthy animals. A year later, they identified the protein and called it PrP for prion protein. In the next decade, a series of experiments, many led by Prusiner, demonstrated that PrP actually is present in healthy animals, but in a different form from the one found in diseased brains. The studies also showed that mice lacking PrP are resistant to prion diseases. Taken together, the results have convinced many scientists that the protein is indeed the agent behind CJD, scrapie, mad cow disease, and others. Key questions remain, however. "The most important bit of information has yet to come forward: What triggers the normal cell protein to transform into the [disease-causing] isotype of the protein?" says Clarence Gibbs, a virologist at the National Institute of Neurological Disorders and Stroke and a longtime colleague of Gajdusek. (Prusiner addresses part of that question on page 245, where he suggests that a possible missing element, dubbed protein X, might help chaperone the PrP protein into its infectious shape.) And no one has been able to inject a prion protein synthesized in the test tube--and therefore free of any possible contaminating virus or other nucleic acid--into a healthy animal and make it sick. "I think it's speculation that the protein itself is infectious," says Laura Manuelidis, a neuropathologist at Yale University who has argued that a virus or other particle is involved. Prusiner acknowledges that there are still many uncertainties. "There are all these other experiments that should be done," he says. "I want to know more about all these details." Although Prusiner had been mentioned frequently as a Nobel candidate, many expected the award would wait for some of those uncertainties to be resolved. Byron Caughey, of the National Institutes of Health's Rocky Mountain Laboratories in Hamilton, Montana, said in a statement that the award is "somewhat surprising in view of the incomplete resolution of these questions." Ralf Pettersson, deputy chair of the Nobel Committee at the Karolinska Institute, says the panel was not bothered by the unanswered questions. The prize was awarded, he says, for the discovery of the prion and its role in the disease process. "The committee is well aware of where the field stands," he says. "The details have to be solved in the future. But no one can object to the essential role of the prion protein" in these brain diseases. Lansbury adds that Prusiner "is really a trailblazer. ... He's captured the imagination of a huge segment of the scientific population." And those imaginations should in no way be limited by this week's prize, Gibbs advises: "There's another Nobel Prize somewhere in this field."

UPn (UPI US & World) Mon, Oct 6, 1997 By SUSAN MILIUS
BETHESDA, Md., Oct. 6 (UPI) -- On the day University of California's Dr. Stanley Prusiner learned he was the 1997 Nobel Prize winner in medicine, he is sitting on an advisory panel of the Food and Drug Administration in Bethesda, Md. He is taking part in a two-day meeting that is examining public health issues linked to the novel infectious agent that are central to his Nobel Prize-winning research. He discovered a brain-killing protein that infects and replicates without any apparent use of genes. He corned the word prions ("PREE-ons") for the naturally occurring proteins that can mutate, and induce neighboring proteins to mutate.
They have been implicated as the cause of mad cow disease and a new variant of Creutsfeld-Jakob ("CROYTS-feld" YAH-cub") disease. The confirmed death toll of these diseases is at least 20 people, and hundreds of thousands of cattle, mostly in the United Kingdom and none in the United States. The award has brought drama and confusion to the suburban Washington, D.C., Holiday Inn, where the FDA often quietly holds its advisory panel meetings. FDA staff on site outside the meeting greet arriving press with a half-teasing, half exhausted, "I bet I know what you want. Reporters get a startled double take from the staff if they ask a question about the substance of the meeting itself.
But tracking Prusiner is easy. Just watch for the lightening storm of photo flashing that starts as soon as he emerges into a public hallway. Photographers jog backward in front of him and besides him, shooting every detail of a Nobel laureate punching an elevator button. Blinking in the glare he says, "You must have a lot of film." Responding to photographers' questions about when he was going to speak publicly about , he says, "No, no, I've got to get into the meeting."

Reuters Online Service Mon, Oct 6, 1997 By Alicia Ault
BETHESDA, Md. (Reuters) - American neurologist Stanley Prusiner, awarded the Nobel Prize for Medicine on Monday, said the prize added great weight to his theory that tiny proteins cause mad cow disease and its human equivalent. Prusiner discovered and named the prion, the usually benign brain protein that mutates into a new form blamed for causing the brain-wasting illness Creutzfeldt-Jakob disease (CJD) and its cousin, bovine spongiform encephalopathy (BSE or mad cow disease).

"This prize is ample support for what I'm saying," Prusiner said.

Prusiner's work transformed research into such brain diseases and provided an explanation for the epidemic of mad cow disease that decimated British herds in the 1980s and is still affecting them today. It also helped explain how BSE could lead to human CJD -- an always fatal brain illness that usually affects only one in a million people.
Prusiner, a 55-year-old professor of neurology at the University of California in San Francisco, was in Bethesda for a U.S. Food and Drug Administration committee meeting on prion disease when he learned the news. He said he got the call at 5:05 a.m. EDT (0905 GMT). "I was delighted," he said, adding that his wife received the call first in San Francisco and knew before he did. He joked that he would use the $1 million prize money to pay his tax bill, telling reporters:

"I am going to use it to pay my taxes." He later grumbled: "This must be the only country in the world that taxes prize money."

Prusiner began his work in 1972, when he was a resident or junior doctor at a San Francisco hospital. One of his patients, a 60-year-old woman, died of CJD. His work with prions took on new importance with the outbreak of BSE in Britain. There is now a global ban on British beef products but experts fear people may have developed a new strain of CJD after eating infected beef products in the 1980s.
Twenty-one people have died from the new strain in Britain, and scientists have clearly linked it to BSE. They say it is too early to tell if an epidemic in humans will mirror the BSE epidemic. Many scientists were slow to accept the idea of prions and argued that some sort of virus must be responsible for the diseases, which are extremely rare in humans and which also are sometimes linked with a genetic mutation. Prusiner said it was right that science was not quick to accept controversial new ideas.

"Most new radical ideas turn out to be incorrect," he told reporters. "I think science is a process." He said it was the data that were important. "There is an amount of data now that has been accumulating over the years that argues very persuasively that BSE and CJD are caused by these pathogens that we call prions," he said. But, he added: "There are a group of people who will never be convinced."

The next step was to work on drugs to treat CJD. "What we'd like to do is develop an effective therapy for these diseases," he said. Prusiner said the next five to 10 years would see a drug that would stop progression of CJD as soon as it was detected. Scientists are working now on a test that will show if someone has CJD -- now it can only be diagnosed with a brain biopsy, usually performed after death. Prusiner, clearly eager to get back into the FDA meeting, spoke only briefly with reporters. "This meeting's important," he said as he left.

Reuters World Report Mon, Oct 6, 1997 By Belinda Goldsmith
STOCKHOLM, Oct 6 (Reuter) - An American neurologist, whose discovery of a disease-causing agent could have helped prevent the outbreak of "mad cow" disease but for a sceptical scientific community, was on Monday awarded the 1997 Nobel Medicine Prize.
Stanley Prusiner discovered the prion, or proteinaceous infectious particle, an innocent cellular protein present in all humans and animals which can transform into a deadly particle, causing several fatal brain diseases. He found prion diseases such as Creutzfeldt-Jakob Disease (CJD) and scrapie could be inherited, transmitted or occur spontaneously -- a discovery that could have major implications for Alzheimer's Disease.
Although his find dates back to the early 1980s, it took several years for the medical world to accept his discovery -- too late to stop the outbreak of mad cow disease in Britain. The condition is caused by feeding cows offal from sheep infected with scrapie, a disease which attacks the central nervous system.

"During the whole of the 1980s, prion was very controversial," Ralf Pettersson, vice-chairman of the Medical Nobel Assembly at Sweden's Karolinska Institute, told Reuters. "Acceptance took a while. This could have delayed moves. It was more a political decision (in Britain) about when to take action, and by then it was too late."

Mad cow disease -- or Bovine Spongiform Encephalopathy (BSE) -- first surfaced in the mid-1980s but it was several years before Britain banned the use of cattle feed containing offal. Since BSE was identified, about 165,000 cattle have died and one million have been culled as a preventative measure. As of September, 1997, there had been 24 known human victims, suffering a new form of CJD that may have arisen through BSE transmission.
Prusiner, 55, a professor of biochemistry at the University of California in San Francisco (UCSF), began his work in 1972 after one of his patients died of dementia resulting from CJD. Ten years later he and his team produced a preparation derived from diseased hamsters' brains that contained a single agent he called a prion, which can be added to the list of well known infectious agents like bacteria, viruses and fungi
. A prion is much smaller than a virus and neither humans nor animals have any in-built defence mechanism, as prions are present as natural proteins from birth and not poisonous. It is converted prions that interact with one another, forming a thread-like structure, and ultimately destroy nerve cells in the brain, causing impaired muscle control, memory loss and insomnia in affected individuals. Pettersson said Prusiner's pioneering work had opened new avenues to understand more common dementia-type illnesses, such as Alzheimer's Disease.

"We now have a theoretical basis for designing new drugs to prevent the transformation to a pathological form of protein," Pettersson told a news conference. "It will help in research to understand other dementia in forms like Alzheimer's which have many features in common."

The Nobel Medicine Prize was the first award in the 1997 Nobel season and will be followed by the ... physics and chemistry prizes on October 15.

Reuters North America Mon, Oct 6, 1997
By Maggie Fox, Health and Science Correspondent
WASHINGTON (Reuter) - It is neither a bacterium nor virus and resembles nothing so far identified as infectious. But the prion is probably behind a fatal and relentless human brain disease and the killing of a million British cattle. Stanley Prusiner won the 1997 Nobel Prize for Medicine Monday for discovering the tiny prion, a normally benign brain protein that sometimes mutates into a form that causes brain disease.
The American neurologist's work was controversial from the start, with scientists questioning how something as normal and widely present as a protein could cause disease. But Prusiner showed the proteins could take on a mutated form and these mutants could cause other proteins to change. They move very slowly but over years in animals and over decades in humans the brain takes on a spongy appearance that gives the diseases, transmissable spongiform encephalopathies, their name. They include bovine spongiform encephalopathy (BSE), known more widely as "mad cow disease," scrapie, reported in sheep for centuries, and the human version -- Creutzfeldt-Jakob disease (CJD).
Cats, deer and mink, along with a range of other animals, can develop similar diseases. British scientists believe cattle first started getting BSE -- which has only been reported in British cattle and in a few European cases -- when rendering methods changed. Cattle in many countries are routinely fed the remains of other farm animals. The theory is that sheep infected with scrapie were included in this protein feed, and when chemical rendering was replaced by high-heat methods, prions survived to infect the cattle. Very high temperature cooking does not destroy prions. They have been found in the brain, the spleen, parts of the intestine and other body products including, possibly, blood.
Britain eventually outlawed the use of such feed and exports of its beef and beef products have been banned. The epidemic is expected to die out within 10 years. But a new fear arose when cases of a mysterious strain of CJD arose. Normally a one-in-a-million disease that strikes the elderly because of its long incubation period, this CJD hit people under 40. So far 21 people have been diagnosed with the new CJD and doctors say it is too early to forecast a human epidemic.
Prusiner, a 55-year-old professor of neurology and biochemistry at the University of California San Francisco, began working with CJD in 1972. He was a resident, or junior doctor, at a San Francisco hospital and watched in dismay as a 60-year-old woman died of CJD. Published work convinced him to study such diseases. No one else had been able to identify the infectious agent. But Prusiner and other experts knew there had to be one -- children given human growth hormone from human brains sometimes suffered from CJD. Then came the BSE outbreak. Mice injected with BSE-infected brain matter get a disease that looks like CJD. Molecular tests have shown that BSE and the new strain of CJD are virtually the same disease.
Many scientists were slow to accept the idea of prions and said some sort of virus must be responsible for the diseases. Prusiner said it was only right science was not quick to accept controversial new ideas.

"Most new radical ideas turn out to be incorrect," he told reporters after learning of the prize. "I think science is a process."