©2004 Nature Publishing Group

[PSI+] using amyloid fibers derived from a recombinant Sup35p fragment [Sup-NM] at 4°C, 23°C and 37°C. White and pink and/or sectored colonies are strong and weak [PSI+] variants, respectively. (Nature, 428:323–7, 2004)

In the relative quiet following the outbreak of bovine spongiform encephalopathy in the United Kingdom, BSE returned to the headlines recently with a sole case found in the United States and new strains of BSE prion protein identified in France, Italy, and Japan. And, in May, French researchers said they found scrapie prion in sheep muscle, showing for the first time that prions have a direct path to the grocery store.1

While these events made headlines, other discoveries in the prion world also were occurring. Researchers have started, and only started, to get to the core of some fundamental questions involving prions. One of these is...


The term prion describes unusual proteins widely believed to be infectious and responsible for TSEs in mammals. Besides BSE, the various strains include scrapie in sheep, chronic wasting disease in deer and elk, and Creutzfeldt-Jakob disease in humans. CJD can be inherited, infectious, or sporadic.

In 1967, John Griffith first suggested the "protein-only" model, based on his observations of the scrapie causative agent.5 Then, in 1982, Prusiner, who coined the term prion,6 discovered a large enough peptide sequence that enabled him to identify the gene encoding a protein called PrP. He showed that the protein originated from the host, and not from a foreign infectious agent.

These infectious proteins were dubbed PrPSc, after scrapie. Although still subject to vigorous debate, the vast majority of prion researchers believe that the infectious agent is not a virus or bacterium, since treatments that destroy nucleic acids do not remove infectivity. The only entity consistently isolated from infected brain matter is insoluble, protease-resistant proteina-ceous material consisting of misfolded polymers known as amy-loids. These amyloid fibers act as the template or seed, upon which PrPC gets converted into more PrPSc. The newly made amyloids in turn act as seeds, converting even more cellular protein into the misfolded prion form.

Researchers rely on mice and hamsters to study TSEs in vivo, but the only way to study the disease in humans is by tissue culture. The mouse model is used predominantly for TSE research in vivo because of mouse susceptibility. Prions discovered in yeast and fungi are used to study amyloid formation and physiological function; the phenotypic effects are evident within days.


Biophysicists Chih-Yen King and Ruben Diaz-Avalos, at the Institute of Molecular Biophysics, Florida State University, Tallahassee, purified distinctive strains of yeast amyloid fibers.2 These were used as templates to generate more fibers in vitro, which then were introduced into uninfected yeast. The strain-specific information of the original amyloid was propagated, indicating that the prion strains result from different conformations of the same prion protein. "What we demonstrated was that protein was the only agent [involved] and that strain specificity was preserved in vitro," says Diaz-Avalos.


©2004 Nature Publishing Group

In this schematic of the transformation procedure, the [PSI+] status is assessed by plating on SD-URA, which contains trace amounts of adenine. (Nature, 428:323–7, 2004)

Trying a different approach, Jonathan Weissman, a professor in the department of cellular and molecular pharmacology at the University of California, San Francisco, and his team answered the same question.3 They created different amyloid conformations by exposing recombinant yeast prion protein to various temperatures. When they infected yeast with each of these amyloids, the conformations were stably propagated in subsequent generations.

Neil Cashman, a neurologist at the Center for Research in Neurodegenerative Disease at Sunnybrook and Women's College Health Sciences Center and the University of Toronto, seemed impressed with the evidence. "These ... papers describe very elegant experiments that demonstrate strain specificity, the ability to prepare seeds ex vivo (in vitro purification) and then to put those seeds intracellularly in yeast and watch physical and chemical characteristics emerge."

Weissman extrapolated the findings to the barrier issue, and to scrapie. "It's not just the [amino acid] sequence or where a prion came from that determines whether a species barrier is going to protect you from infection. It's also what shape the protein misfolded into, and we believe now that that's what happened with scrapie."

Tuite stresses, however, that this link between a defined conformation and distinct inherited phenotype has been established only in yeast. "It's very hard to do in animals, [in which] it takes two years to establish infection. In yeast, it takes two to three days."


©2004 Nature Publishing Group

An experimental scheme demonstrates that Sup35 amyloid particles transmit the [PSI] strain phenotype. The filled black squares are endogenous, full-length Sup35 aggregates; the filled green circles are Sup35[1-61]-GFP-labeled particles; the open green circles are Sup35[1-61]-GFP aggregates derived from Escherichia coli. (Nature, 428:319–23, 2004)

To better understand prion species barriers and prion strain diversity in mammalian TSEs, Surewicz and his group created a bare-bones model. They developed an in vitro assay designed to convert a short form of a PrP protein, designated PrP*, into PrP amyloids.7 Using PrP* prion templates for hamster, mouse, and human, they showed that barriers between different species could be overcome and new ones created, by prion conformation alone, without altering the underlying nucleic acid sequence. In vitro, they showed that hamster PrP* can seed conversion of mouse PrP* but not human PrP*. Mouse and human PrP* can seed conversion of each other into amyloid PrP*, but cannot convert hamster PrP*, providing evidence of a species barrier in vitro.

In another experiment, changing a single key amino acid was enough to create new prion strains, such as "hamsterized" human PrP* and "humanized" hamster PrP*. Mouse PrP* seeded by hamster PrP* generated hamster amyloid, providing further evidence for a link between conformation, strains, and species barriers.7,8

While the mammalian findings must be interpreted with more caution than the yeast system, these initial results are first steps in a new direction. "Really, for the first time, [Surewicz] has demonstrated strain propagation in vitro in a reductionist model," says Cashman. Surewicz's data strongly suggest that TSE transmissibility is due not only to differences in the amino acid sequence of prions among species, but also to the strains' conformational properties, which supports the yeast findings. "Our work nicely corresponds with the yeast prion papers," Surewicz explains. "They provide very strong support of the protein-only hypothesis."

Some are not convinced. "They've [prion experts] had over 20 years to show that PrP is infectious, and there have been lots of experiments, and they've never been able to show that PrP, in any form, has any significant infectivity in normal animals," says Manuelidis. "Nobody knows what the molecular agent is ... I don't have the answer. I suspect a virus, but it's not known."

She also questions the results of the Diaz-Avalos and Tanaka papers.2,3"Injecting stuff into a yeast cell – obviously the cell divides and each daughter cell gets some of the cytoplasm. Proteins can be folded abnormally and processed .... Just about any protein overexpressed abnormally is going to express disease."

Diaz-Avalos disagrees. "If you have something in the cytoplasm and it can't reproduce, you would dilute it out eventually. But the fact that you keep getting infection tells you that an infectious agent is somehow reproducing whenever it finds more molecules that are similar." In their experiments, phenotypes that he attributes to infection were stable for thousands of yeast generations.


©2004 Nature Publishing Group

Along with the colony colors, graphic indicates the amount of Sup35p protein in soluble (s) lane or aggregated (p) lane form. [PSI-] yeast cells contain a soluble form of the Sup35p protein. In the strong [PSI+] strain, more than 95% of the Sup35 protein is in the nonfunctional, aggregated fraction (lane p). In the weak [PSI+] strain, a significant level of Sup35p is also seen in the soluble fraction. The unfractionated total protein sample is shown as t. (Nature, 428:265–7, 2004)

Manuelidis proposes a different origin for prion diseases. Her team's work suggests that the CJD agent is virus-like, based on immune-response profiles. Not only did the team find an immune response, but it also found that the CJD agent triggered different signaling pathways than those activated by abnormal PrP and other amyloid proteins. "If CJD is caused by a host protein, it should not elicit a response from the host," reasons Manuelidis.

In a recent report,9 Manuelidis and her group show that transcriptional changes in interferon-sensitive genes are seen in mice microglia at 30 days after inoculation. In the disease's early stages, 10 other related genes were upregulated. This activity, she says, was seen well before abnormal PrP and clinical signs of disease were detectable. The increased expression of these interferon-sensitive genes was similar to that seen by many latent or persistent viruses, which can hide from host immune recognition but still trip inflammatory pathways, providing a clue to their presence. "This tells you that the host is responding to a foreign agent."


Elucidating a clear function for the cellular PrP protein is an area that immunologist R. Anthony Williamson of The Scripps Research Institute, La Jolla, Calif., says has been largely neglected. The focus, he says, has predominately been on the infectious aspect of prions because they are "so unusual and unorthodox."

Williamson and colleagues tested their hypothesis that PrPC plays a role in neuronal survival.10 Using mouse monoclonal antibodies, they injected PrPC-specific mAbs into the hippocampus and cerebellum of mice, cross-linking PrPC to the antibodies in vivo. Over the next few days they saw rapid and extensive apoptosis among hippocampal and cerebellar neurons. However, when they injected PrPC-specific antibodies into transgenic mice that do not express PrPC, no neurodegeneration occurred. Furthermore, they showed that complement played no role in triggering the neurodegeneration observed.

Extrapolating this finding to prion disease, the researchers say they think that the scrapie prion may be interfering similarly by cross-linking with PrPC, thereby generating neurodegeneration.11 "This is a potential role for the cellular protein, for better or worse," explains Williamson. "We're hypothesizing that this protein forms part of the pathway that controls survival or death." Inappropriate triggering of this pathway may then lead to unwanted cell death. Furthermore, he adds, "our findings suggest a possible route for PrPSc to trigger or induce a signal cascade that leads to neuronal loss in prion-infected tissues."

A recent paper by neurologist John Collinge and colleagues, Medical Research Council Institute of Neurology in London, appears consistent with these latest findings.12 The researchers used double trans-genic mice in which the presence of PrPC is controlled by a recombinase enzyme, which removed PrPC from neurons and nonneuronal cells. They showed that depleting endogenous cellular neuronal PrPC in mice with established prion infection could reverse spongiform changes and prevent loss of neurons and disease progression. "This paper shows grounds for the optimal means by which these diseases may be treated," says Reed Wickner of the National Institute of Diabetes and Digestive and Kidney Diseases. Says Williamson: "Their work ties in nicely with our hypothesis that PrPC signaling is part of the neurode-generative cascade in prion-infected tissues."

In other work, two papers published by Eric Kandel and his group at Columbia University reported that the CPEB protein may be a functional form of prion.13,14 CPEB regulates local translation of neuronal proteins that maintain long-term memory in the snail, aplysia. CPEB has prion properties in that it forms amyloids and is self-perpetuating.

"Learning and memory [are] really a building, strengthening, and modulating of the processes of one neuron to another," says Susan Lindquist, director of the Whitehead Institute for Biomedical Research at Massachusetts Institute of Technology, and coauthor on one paper.14 The role for CPEB in learning and memory is evidenced by a local increase in translation at the synaptic ends of each neuron. "The protein can then undergo a conformational change like a prion and converts to an active state," explains Lindquist. "The beauty of that is that you want to keep the memory long-term and local [at neuronal processes]. The essence of [CPEB] as a prion is that once it converts it becomes a self-perpetuating [molecule] that stays local."

Says Kandel: "We're the first to show a functional form of prion that perpetuates normal physiological function. The future now is to take that purified form and put it back in aplysia and see if that self-perpetuating mechanism operates in the brain, and then extend that from aplysia to the mouse." These results, says Tuite, suggest a possible positive role for the prion form of CPEB that shows a gain rather than a loss of function.


Considering that TSEs are such comparatively rare diseases and seem to pose minimal threat to humans, it's logical to ask why they receive so much attention. They matter for many reasons, explains Surachai Supattapone of Dartmouth Medical School. For starters, they're fatal. Another reason, says Wickner, is that a TSE could become an epidemic. This is particularly problematic because its symptoms do not appear until later in life.

Among wildlife, one TSE is an epidemic. Chronic wasting disease was found among deer and elk in 1967 in Colorado. Since then, the disease has spread eastward and into Canada and now affects 20% of the deer population. "It's not a minor disease and we can't control its spread," says Supattapone. "You can't control it through mandating feed practices."


©2004 Nature Publishing Group

Based on Prp* derivative of PrP containing amino acid residues 23–144. The arrows indicate the transmission of prion (amyloid) properties. (Mol Cell, 14:147–52, 2004)

On the consumer side, the picture is equally grim, only in this case, it's for economic reasons. "The disease can be economically devastating," Supattapone adds. "It affects the ability of the cattle industry to export products." From a public safety standpoint, the concern is that unscrupulous individuals would rather rush suspect animals to market than euthanize them. While no documented cases of scrapie passing from sheep to humans have been seen, new variant CJD, which affects younger adults, is believed to have arisen from consumption of tainted beef in the United Kingdom.

Prions also serve as model proteins to study the pathology of a wide variety of neurodegenerative diseases. Only one out of every two million people develop or acquire CJD, but one percent of the population develops Parkinson disease, amyotrophic lateral sclerosis, and Alzheimer disease, explains Wickner. Besides, prions arouse scientific curiosity, adds Supattapone. "Studying prions is like studying RNA viruses: It represents studying an entire new class of infectious agents."

Nicole Johnston nicolejohnston@mcmaster.ca is a freelance writer in Hamilton, Ontario.


Forty years ago, there was but one way to treat someone with humangrowth hormone (hGH) deficiency, and that was with human pituitary hormone taken from cadavers. But in 1985, an endocrinologist made a discovery that quickly stopped the practice.

He found that one of his young patients had Creutzfeldt-Jakob disease, quite a surprise since, back then, CJD was almost exclusively a rare disease found in the elderly. When two more growth hormone recipients came down with the disease, scientists quickly made the connection: One or more of the many cadaver pituitaries used for a single treatment had contained the agent causing CJD, which is the human variant of bovine spongiform encephalopathy. Patients were switched quickly to treatment with synthetic growth hormone, which by then had become available.

Since 1985, researchers at the National Institutes of Health and elsewhere have been tracking patients treated with cadaver-generated hGH. A recent study reports that of 7,700 patients in the United States who had received hGH from cadavers, 26 died of CJD.1 According to lead author James L. Mills, chief of the Pediatric Epidemiology Section of the National Institute of Child Health and Human Development, that rate is actually lower than expected. "It's not something you could call good news," he says, "but it's relatively good that people in the United States have had the low rates that they've had."

Though not foreseen when it first began, the study had significant implications unrelated to CJD incidence. It found no link between hGH administration and incidence of colon cancer, an association suggested by a smaller, previous study done in the United Kingdom.

Further, the study found that more hGH recipients died because of poor administration of adrenocorticotropic hormone (ACTH, also missing in many hGH-deficient patients), than because of CJD. Patients with the additional ACTH deficiency are supposed to take more ACTH as hormone production from their adrenal gland drops. The study found that either many failed to do so, or they became ill and accidentally vomited the medication. "We need to educate the medical community and the patients with these problems to make sure they know how to handle these crises when they come up," says Charles Sklar, director of the long-term follow-up program at Memorial Sloan-Kettering Cancer Center.

- Eugene Russo

"Long-term mortality in the United States cohort of pituitary-derived growth hormone recipients," Mills JL, J Pediatr , 2004 Vol 144, 430-6



Earliest reports describing scrapie among sheep in Europe.

Early 1900s

A new brain disease, Kuru, attributed to ritual cannibalism, is observed among the South Fore tribe of Papua New Guinea.

Early 1920s

German neurologists Hans Gerhard Creutzfeldt and Alfons Maria Jakob first describe Creutzfeldt-Jakob disease.


French veterinarians demonstrate that scrapie is a transmissible disease after infecting two healthy sheep with brain or spinal cord tissue from diseased sheep.


Discoveries shed light on the scrapie agent, such as its notable resistance to well-established inactivation agents and its physical distribution in infected tissue.


The protein-only model is first suggested. Scientists first propose that the scrapie agent lacks nucleic acid after doses of ionizing radiation fail to prevent subsequent infection.


Stanley Prusiner coins the term "prion" to describe these infectious proteins and identifies a peptide sequence from highly purified material that corresponds to a host protein called PrP. This discovery supports the protein-only model.


Changes in how animal remains are rendered enable the scrapie agent to infect cows, leading to an epidemic of mad cow disease in the United Kingdom.


Prions are discovered in yeast and fungi and become widely used as models for the study of prion phenomenon.


A new variant of CJD (nvCJD) that only affects individuals under 40 and leads to rapid decline, is first described among 20 young people and is traced to the consumption of tainted meat.


In vitro experimental evidence provides the strongest evidence to date validating the protein-only model.

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