Published Online December 31, 2009 Science DOI: 10.1126/science.1183218
Darwinian Evolution of Prions in Cell Culture
Jiali Li, Shawn Browning, Sukhvir P. Mahal, Anja M. Oelschlegel, Charles Weissmann*
Prions are infectious proteins consisting mainly of PrPSc, a ? sheet-rich conformer of the normal host protein PrPC, and occur in different strains. Strain identity is thought to be encoded by PrPSc conformation. We found that biologically cloned prion populations gradually became heterogeneous by accumulating "mutants," and selective pressures resulted in the emergence of different mutants as major constituents of the evolving population. Thus, when transferred from brain to cultured cells, "cell-adapted" prions outcompeted their "brain-adapted" counterparts, and the opposite occurred when prions were returned from cells to brain. Similarly, the inhibitor swainsonine selected for a resistant substrain, whereas, in its absence, the susceptible substrain outgrew its resistant counterpart. Prions, albeit devoid of a nucleic acid genome, are thus subject to mutation and selective amplification.
Department of Infectology, Scripps Florida, 130 Scripps Way, Jupiter, FL 33458, USA.
* To whom correspondence should be addressed. E-mail: email@example.com
Received for publication 12 October 2009. Accepted for publication 17 December 2009.
Public release date: 31-Dec-2009
Contact: Keith McKeown firstname.lastname@example.org 858-784-8134 Scripps Research Institute
Scripps Florida scientists show 'lifeless' prions capable of evolutionary change and adaptation Research may point to more effective therapeutic targets for deadly prion diseases JUPITER, FL – Scientists from The Scripps Research Institute have determined for the first time that prions, bits of infectious protein devoid of DNA or RNA that can cause fatal neurodegenerative disease, are capable of Darwinian evolution.
The study from Scripps Florida in Jupiter shows that prions can develop large numbers of mutations at the protein level and, through natural selection, these mutations can eventually bring about such evolutionary adaptations as drug resistance, a phenomenon previously known to occur only in bacteria and viruses. These breakthrough findings also suggest that the normal prion protein – which occurs naturally in human cells – may prove to be a more effective therapeutic target than its abnormal toxic relation.
The study was published in the December 31, 2009 issue of the journal Science Express, an advance, online edition of the prestigious journal Science.
"On the face of it, you have exactly the same process of mutation and adaptive change in prions as you see in viruses," said Charles Weissmann, M.D., Ph.D., the head of Scripps Florida's Department of Infectology, who led the study. "This means that this pattern of Darwinian evolution appears to be universally active. In viruses, mutation is linked to changes in nucleic acid sequence that leads to resistance. Now, this adaptability has moved one level down – to prions and protein folding – and it's clear that you do not need nucleic acid for the process of evolution."
Infectious prions (short for proteinaceous infectious particles) are associated with some 20 different diseases in humans and animals, including mad cow disease and a rare human form, Creutzfeldt-Jakob disease. All these diseases are untreatable and eventually fatal. Prions, which are composed solely of protein, are classified by distinct strains, originally characterized by their incubation time and the disease they cause. Prions have the ability to reproduce, despite the fact that they contain no nucleic acid genome.
Mammalian cells normally produce cellular prion protein or PrPC. During infection, abnormal or misfolded protein – known as PrPSc – converts the normal host prion protein into its toxic form by changing its conformation or shape. The end-stage consists of large assemblies (polymers) of these misfolded proteins, which cause massive tissue and cell damage.
"It was generally thought that once cellular prion protein was converted into the abnormal form, there was no further change," Weissmann said. "But there have been hints that something was happening. When you transmit prions from sheep to mice, they become more virulent over time. Now we know that the abnormal prions replicate, and create variants, perhaps at a low level initially. But once they are transferred to a new host, natural selection will eventually choose the more virulent and aggressive variants."
In the first part of the study, Weissmann and his colleagues transferred prion populations from infected brain cells to culture cells. When transplanted, cell-adapted prions developed and out-competed their brain-adapted counterparts, confirming prions' ability to adapt to new surroundings, a hallmark of Darwinian evolution. When returned to brain, brain-adapted prions again took over the population.
To confirm the findings and to explore the issue of evolution of drug resistance, Weissmann and his colleagues used the drug swainsonine or swa, which is found in plants and fungi, and has been shown to inhibit certain prion strains. In cultures where the drug was present, the team found that a drug-resistant sub-strain of prion evolved to become predominant. When the drug was withdrawn, the sub-strain that was susceptible to swainsonine again grew to become the major component of the population.
Weissmann notes that the findings have implications for the development of therapeutic targets for prion disease. Instead of developing drugs to target abnormal proteins, it could be more efficient to try to limit the supply of normally produced prions – in essence, reducing the amount of fuel being fed into the fire. Weissmann and his colleagues have shown some 15 years ago that genetically engineered mice devoid of the normal prion protein develop and function quite normally (and are resistant to prion disease!).
"It will likely be very difficult to inhibit the production of a specific natural protein pharmacologically," Weissmann said, "You may end up interfering with some other critical physiological process, but nonetheless, finding a way to inhibit the production of normal prion protein is a project currently being pursued in collaboration with Scripps Florida Professor Corinne Lasmezas in our department."
Another implication of the findings, according to the study, is that drug-resistant variants either exist in the prion population at a low level prior to exposure or are generated during exposure to the drug. Indeed, the researchers found some prions secreted by infected cells were resistant to the drug before exposure, but only at levels less than one percent.
The scientists show that prion variants constantly arise in a particular population. These variants, or "mutants", are believed to differ in the way the prion protein is folded. As a consequence, prion populations are, in fact, comprised of multiple sub-strains.
This, Weissmann noted, is reminiscent of something he helped define some 30 years ago – the evolutionary concept of quasi-species. The idea was first conceived by Manfred Eigen, a German biophysicist who won the Nobel Prize in Chemistry in 1967. Basically stated, a quasi-species is a complex, self-perpetuating population of diverse and related entities that act as a whole. It was Weissmann, however, who provided the first confirmation of the theory through the study of a particular bacteriophage – a virus that infects bacteria – while he was director of the Institut für Molekularbiologie in Zürich, Switzerland.
"The proof of the quasi-species concept is a discovery we made over 30 years ago," he said. "We found that an RNA virus population, which was thought to have only one sequence, was constantly creating mutations and eliminating the unfavorable ones. In these quasi-populations, much like we have now found in prions, you begin with a single particle, but it becomes very heterogeneous as it grows into a larger population."
There are some unknown dynamics at work in the prion population that leads to this increased heterogeneity, Weissmann added, that still need to be explored.
"It's amusing that something we did 30 years has come back to us," he said. "But we know that mutation and natural selection occur in living organisms and now we know that they also occur in a non-living organism. I suppose anything that can't do that wouldn't stand much of a chance of survival."
The joint first authors of the Science study, "Darwinian Evolution of Prions in Cell Culture," are Jiali Li and Shawn Browning of The Scripps Research Institute. Other authors include Sukhvir P. Mahal and Anja M. Oelschlegel also of The Scripps Research Institute. Weissmann notes that after the manuscript was accepted by Science, an article by Ghaemmanghami et al. appeared in PLoS Pathogens that described emergence of prions resistant to a completely different drug, quinacrine, providing additional support to the Scripps Research team's conclusions.
The Scripps Research study was supported by a grant from the National Institutes of Health and by a generous donation to the Weissmann laboratory from the Alafi Family Foundation.
About The Scripps Research Institute
The Scripps Research Institute is one of the world's largest independent, non-profit biomedical research organizations, at the forefront of basic biomedical science that seeks to comprehend the most fundamental processes of life. Scripps Research is internationally recognized for its discoveries in immunology, molecular and cellular biology, chemistry, neurosciences, autoimmune, cardiovascular, and infectious diseases, and synthetic vaccine development. Established in its current configuration in 1961, it employs approximately 3,000 scientists, postdoctoral fellows, scientific and other technicians, doctoral degree graduate students, and administrative and technical support personnel. Scripps Research is headquartered in La Jolla, California. It also includes Scripps Florida, whose researchers focus on basic biomedical science, drug discovery, and technology development. Scripps Florida is located in Jupiter, Florida.
Page last updated at 00:06 GMT, Friday, 1 January 2010 'Lifeless' prion proteins are 'capable of evolution' (pic) Scrapie prion protein from hamster brain (pic) Abnormal prion proteins cause at least 20 fatal diseases
Scientists have shown for the first time that "lifeless" prion proteins, devoid of all genetic material, can evolve just like higher forms of life.
The Scripps Research Institute in the US says the prions can change to suit their environment and go on to develop drug resistance.
Prions are associated with 20 different brain diseases in humans and animals.
The scientists say their work suggests new approaches might be necessary to develop therapies for these diseases.
In the study, published in the journal Science, the scientists transferred prion populations from brain cells to other cells in culture and observed the prions that adapted to the new cellular environment out-competed their brain-adapted counterparts.
When returned to the brain cells, the brain-adapted prions again took over the population.
Charles Weissmann, head of Scripps Florida's department of infectology who led the study, said: "On the face of it, you have exactly the same process of mutation and adaptive change in prions as you see in viruses.
This is a timely reminder that prion concerns are not going away and that controls to stop abnormal prions being transmitted to humans through the food system or through blood transfusions must be vigorously maintained Professor John Collinge, Medical Research Council Prion Unit
"This means that this pattern of Darwinian evolution appears to be universally active.
"In viruses, mutation is linked to changes in nucleic acid sequence that leads to resistance.
"Now, this adaptability has moved one level down- to prions and protein folding - and it's clear that you do not need nucleic acid (DNA or RNA) for the process of evolution."
Mammalian cells normally produce cellular prion protein or PrPC.
During infections, such as the human form of mad cow disease known as vCJD, abnormal or misfolded proteins convert the normal host prion protein into its toxic form by changing its conformation or shape.
"It was generally thought that once cellular prion protein was converted into the abnormal form, there was no further change", Mr Weissmann said.
"But there have been hints that something was happening.
"When you transmit prions from sheep to mice, they become more virulent over time.
PRION DISEASES Human prion diseases such as Creutzfeldt Jakob disease (CJD) can arise sporadically, be acquired by infection or be inherited because of a mutant gene coding for the prion protein They are relatively rare but have occurred in epidemic form in Papua New Guinea as a result of brain cannibalism Animal prion diseases include scrapie in sheep and goats, chronic wasting disease in deer and elk and transmissible mink encephalopathy Bovine spongiform encephalopathy (BSE) first appeared in UK in mid-1980s It is estimated that more than two million UK cattle were infected Variant CJD (vCJD) caused by the same prion strain as BSE was first recognised in the mid-1990s
"Now we know that the abnormal prions replicate, and create variants, perhaps at a low level initially.
"But once they are transferred to a new host, natural selection will eventually choose the more virulent and aggressive variants."
Professor John Collinge, of the Medical Research Council's (MRC) Prion Unit, described the research as exciting confirmation of a hypothesis that he had proposed two years ago, that there could be a "cloud" or whole array of prion proteins in the body.
He called it the cloud hypothesis.
He said: "The prion protein is not a clone, it is a quasi-species that can create different protein strains even in the same animal.
"The abnormal prion proteins multiply by converting normal prion proteins.
"The implication of Charles Weissmann's work is that it would be better to cut off that supply of normal prion proteins rather than risk the abnormal prion adapting to a drug and evolving into a new more virulent form.
"You would do this by trying to block the sites on the normal prion protein that the abnormal form locks on to to do its conversion.
"We know there is an antibody that can do this in mice and the Medical Research Council's Prion Unit have managed to engineer a human antibody to do this.
"It is currently undergoing safety tests and we hope to move to clinical trials by the end of 2011"
Professor Collinge said the MRC was also trying to find more conventional chemical compounds to do this and has been collaborating with the chemical company GlaxoSmithKline (GSK).
He said: "They have given us access to their chemical libraries, which contain millions of compounds, and we have already identified some that may work well.
"This is a timely reminder that prion concerns are not going away and that controls to stop abnormal prions being transmitted to humans through the food system or through blood transfusions must be vigorously maintained."
PLEASE SEE BELOW ;
"So far, there is no evidence for spontaneous PrPSc formation in any animal or human TSE."
Prions: Protein Aggregation and Infectious Diseases
ADRIANO AGUZZI AND ANNA MARIA CALELLA
Institute of Neuropathology, University Hospital of Zurich, Zurich, Switzerland
3. Sporadic Creutzfeldt-Jakob disease Approximately 85% of all human prion diseases are sporadic forms of CJD. For sCJD, there is no association with a mutant PRNP allele, nor is there any epidemiological evidence for exposure to a TSE agent through contact with people or animals infected with TSEs. sCJD cases are currently subclassified according to the methionine/valine polymorphism at codon 129 of the PRNP gene and the size and glycoform ratio of proteaseresistant prion protein identified on western blot (type 1 or type 2) (174). Heterozygosity (Met/Val) at PrP codon 129 appears to be associated with a lower risk (378) and/or prolonged incubation time (119, 387). The lack of routine laboratory testing for preclinical diagnosis makes the search for agent sources and other risk factors extremely difficult. At present, the means of acquisition of a TSE agent in these patients remains a mystery. So far, there is no evidence for spontaneous PrPSc formation in any animal or human TSE. In humans, the peak age incidence of sporadic CJD is 55-60 years. However, if spontaneous misfolding were the primary event, one might expect a continuously increasing incidence with age because more time would allow more opportunity for rare misfolding events.
Physiol Rev . VOL 89 . OCTOBER 2009 . www.prv.org
Friday, January 01, 2010
Human Prion Diseases in the United States
Monday, December 14, 2009
Similarities between Forms of Sheep Scrapie and Creutzfeldt-Jakob Disease
Are Encoded by Distinct Prion Types
Wednesday, December 30, 2009
Is there evidence of vertical transmission of variant CJD ?