How prions make you sick

dendritic spineTransmissible spongiform encephalopathies (TSEs) are rare, but always fatal, neurodegenerative disorders of humans and other mammals. They are characterized by long incubation periods, spongiform changes in the brain associated with loss of neurons, and the absence of host responses. TSEs are caused by infectious proteins called prions. Insight into how prions cause TSEs comes from the observation that exposure of neurons to prions causes retraction of dendritic spines (link to paper).

Early alterations in the nervous system caused by prions include changes in the synapse such as retraction of dendritic spines, the projections where synaptic contacts occur (illustrated; image credit). Understanding these pathologies has been difficult due to a lack of an appropriate neuronal culture system.

To determine if prions are toxic for neurons, primary neuronal cultures were prepared from mice and grown on layers of astrocytes. Addition of an infected brain homogenate from mice that had been inoculated with the scrapie prion, PrPsc, led within 24 hours to retraction of dendritic spines and a reduction in their number and area. Similar effects on dendritic spines were also observed when purified PrPsc was used.

No effects of brain homogenates were observed using neurons prepared from mice lacking the prion gene prnp. This observation might have been predicted because prion diseases do not occur in mice lacking the prnp gene. However only an N-terminal domain of PrPc (amino acids 23-31) is required for the loss of dendritic spines. It seems likely that this part of PrPc on neurons binds the pathogenic PrPsc form, leading to neuronal loss.

Normal prions (PrPc) are completely digested with the enzyme proteinase K, while the pathogenic prion PrPsc is relatively resistant. Proteinase K treated PrPsc retained the ability to cause retraction of dendritic spines, showing that amino acids 23-90 of the protein are not needed for synaptotoxicity.

Dendritic spines are responsible for excitatory postsynaptic transmission and have roles in learning and memory. Their retraction by pathogenic prions are likely early changes leading to the pathogenic consequences of TSEs. How prions cause spine retractions can now be determined using cultured neurons. It will also be possible to determine if similar mechanisms are involved in dendritic spine loss associated with other neurodegenerative diseases, such as Alzheimer’s, Huntington’s, and Parkinson’s diseases.

 

Prions in bacteria

prion conversionBacteria do not develop transmissible spongiform encephalopathies, but they have been found to produce prions – proteins that can adopt alternative conformations with different functions.

Prion diseases, a frequent topic on this blog, are caused by misfolding of a normal cellular prion protein (illustrated; image copyright ASM Press). Prion proteins are found in other organisms, where the alternative conformation confers a new, non-pathogenic function to the protein. At least 12 different prion proteins have been found in yeast, and they confer the ability to grow more efficiently under certain conditions. Now prions have been discovered in bacteria (link to article).

A search of 60,000 bacterial genomes for proteins with prion-forming domains revealed one in the transcription termination protein Rho from Clostridium botulinum (Cb-Rho). When produced in E. coli, the protein forms amyloid – protein aggregates in the form of fibrils – that are characteristic of prions. A 68 amino acid stretch of Cb-Rho can functionally substitute for the prion-forming domain of a yeast prion-forming protein. This protein, called Sup35, can read stop codons in the prion state, and this phenotype was recapitulated in yeast by the Clostridium prion.

The Cb-Rho prion can convert between prion and non-prion conformations in E. coli. This property was demonstrated by placing a Rho-dependent terminator between a promoter and the lacZ gene, the product of which produces a blue color. In the prion state, Rho has decreased activity, leading to blue cells. In the non-prion state, normal termination leads to pale blue colonies. A mixture of blue and pale blue colonies was observed, showing that Rho exists in the prion and non-prion states.

The prion conformation was also shown to be heritable. Blue colonies always gave rise to blue colonies, while pale blue colonies formed pale blue colonies. The blue colony color lasted for over 120 generations.

The finding of a prion in bacteria indicates that this form of protein-based heredity arose before eukaryotes emerged on Earth. Similar prion-like protein domains have also been found in other phyla of bacteria, suggesting the existence of an important source of epigenetic diversity that can allow bacterial growth under diverse conditions. Exactly how bacterial prions confer new functions will be exciting to discover.

Last time we learned that eukaryotes probably didn’t invent the nucleus. Now we find that prions likely emerged first in bacteria. Did eukaryotes invent anything?

TWiV 426: I’m Axl, and I’ll be your cervid today

The sages of TWiV explain how chronic wasting disease of cervids could be caused by spontaneous misfolding of prion protein, and the role of the membrane protein Axl in Zika virus entry into cells.

You can find TWiV #426 at microbe.tv/twiv, or listen below.

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TWiV 424: FLERVergnügen

Trudy joins the the TWiVlords to discuss new tests for detecting prions in the blood, and evidence showing that foamy retroviruses originated in the seas with their jawed vertebrate hosts at least 450 million years ago.

You can find TWiV #424 at microbe.tv/twiv, or listen below.

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A blood test for prion disease

PCMA for prionsA sensitive and specific blood test has been developed that could be used to limit the risk of transmission of prion disease through the blood supply (link to papers one and two).

Prion diseases, also known as spongiform encephalopathies, are uniformly fatal, chronic degenerative neurological diseases caused by misfolding of a cellular protein, PrPC. Transmissible encephalopathies may be acquired by organ transplant, receiving contaminated blood, or the ingestion of contaminated food.

In the 1990s a new spongiform encephalopathy, variant Creutzfeld-Jakob disease or vCJD, began to appear in Great Britain. Variant Creutzfeld-Jakob disease is caused by prions acquired by the consumption of cattle with bovine spongiform encephalopathy, also a prion disease affectionately known as mad cow disease. To date 231 cases of vCJD have been reported, mainly in the UK and France.

Although the spread of BSE has been controlled by surveillance and feeding restrictions, it is estimated that millions of people were exposed to BSE prions. The concern is that some of these individuals might be infected but show no symptoms of disease. If they donate blood, they may transmit infection to others. It is known that several cases of vCJD have been transmitted from infected blood donors, so further transmission is a major concern. So far prion diseases have only been diagnosed after death, by detection of conformationally altered prion proteins in the brain.

Two sensitive and specific assays for vCJD prions have now been developed that show promise for non-invasive pre-symptomatic diagnosis of the disease. They are both based on a technology called protein misfolding cyclic amplification (PMCA, illustrated; image copyright ASM Press, 2015). A small amount of the normal human prion protein, PrPC (produced in transgenic mice) is mixed with plasma. The samples are incubated to allow formation of prion oligomers, followed by disruption by a pulse of sonication to disrupt the oligomers. The cycle is repeated multiple times, much like polymerase chain reaction (PCR) which is used to amplify small amounts of DNA. Prions are detected by western blot analysis after treatment with proteinase K. The misfolded, pathogenic prions, PrPSC , are not completely digested with this enzyme.

In one study, PMCA was used to analyze blood samples from 14 cases of vCJD and 153 controls, which included healthy individuals and those with other neurological diseases, including sporadic CJD (sCJD – not caused by ingestion of contaminated beef). All 14 samples from cases of vCJD were positive in the PMCA assay, but not any of the other samples.

In a second study, the PMCA assay was positive in samples from all 18 patients with vCJD. Of 134 control samples, just one was positive for vCDJ, from a patient with sCJD. Furthermore, the assay detected vCJD prions in archived blood samples from donors who gave blood before developing symptoms of the disease.

These findings suggest that the new assays can detect vCJD prions in the blood before the appearance of the neurological symptoms of spongiform encephalopathy. While additional samples must be analyzed to validate the results, they are nonetheless promising as a way to prevent spread of the disease via the blood supply. Unfortunately, if you are diagnosed with vCJD by one of these assays, that is the only positive outcome – there are as yet no treatments for any spongiform encephalopathy.

Structure of an infectious prion

prion conversionPrions are not viruses – they are infectious proteins that lack nucleic acids. Nevertheless, virologists have always been fascinated by prions – they appear in virology textbooks (where else would you put them?) and are taught in virology classes. I’ve written about prions on this blog (five articles, to be exact – look under P in the Table of Contents) and I’m fascinated by their biology and transmission. That’s why the newly solved structure of an infectious prion protein is the topic of the sixth prion article at virology blog.

Spongiform encephalopathies are neurodegenerative diseases caused by misfolding of normal cellular prion proteins. Human spongiform encephalopathies are placed into three groups: infectious, familial or genetic, and sporadic, distinguished by how the disease is acquired initially. In all cases, the pathogenic protein is the host-encoded PrPC protein with an altered conformation, called PrPsc. In the simplest case, PrPSc converts normal PrPC protein into more copies of the pathogenic form (illustrated).

The structure of the normal PrPprotein, solved some time ago, revealed that it is largely alpha-helical with little beta-strand content. The structure of PrPSc protein has been elusive, because it forms aggregates and amyloid fibrils. It has been suggested that the PrPSc protein has more beta-strand content than the normal protein, but how this property would lead to prion replication was unknown. Clearly solving the structure of prion protein was needed to fully understand the biology of this unusual pathogen.

The structure of PrPSc protein has now been solved by cryo-electron microscopy and image reconstruction (link to paper). The protein was purified from transgenic mice programmed to produce a form of  PrPSc protein that is not anchored to the cell membrane, and which is also underglycosylated. The protein causes disease in mice but is more homogeneous and forms fibrillar plaques, allowing gentler purification methods.

prion structureThe structure of this form of the PrPSc protein reveals that it consists of two intertwined fibrils (red in the image) which most likely consist of a series of repeated beta-strands, or rungs, called a beta-solenoid. The structure provides clues about how a pathogenic prion protein converts a normal PrPC into PrPSc . The upper and lower rungs of beta-solenoids are likely the initiation points for hydrogen-bonding with new PrPC molecules – in many proteins with beta-solenoids, they are blocked to prevent propagation of beta-sheets. Once added to the fibrils, the ends would serve to recruit additional proteins, and the chain lengthens.

The authors note that the molecular interactions that control prion templating, including hydrogen-bonding, charge and hydrophobic interactions, aromatic stacking, and steric constraints, also play roles in DNA replication.

The structure of PrPSc protein provides a mechanism for prion replication by incorporation of additional molecules into a growing beta-solenoid. I wonder if incorporation into fibrils is the sole driving force for converting PrPCprotein into PrPSc, or if PrPC is conformationally altered before it ever encounters a growing fibril.

 

Prion contamination in the emergency room

prion conversionHere is a follow-up to last week’s article that described a case of variant Creutzfeldt-Jacob disease in a Texas resident caused by ingestion of BSE-contaminated beef 14 years ago.

A 59 year old male patient was admitted to the trauma unit in Lancaster, PA with a self-inflicted gunshot wound to the head. There was substantial bleeding and brain tissue extrusion from the bullet exit wound. While the patient was intubated, examination of his electronic health records revealed a previous diagnosis of Creutzfeldt-Jacob disease (CJD). After discussion with his family, the breathing tube was removed and the patient expired.

After discovering that the patient had CJD, TSE (transmissible spongiform encephalopathy) decontamination protocols were initiated. Equipment and surfaces that had been exposed to highly infectious brain tissues were identified. Because prions are extremely difficult to destroy, it was decided to incinerate many pieces of equipment costing tens of thousands of dollars. This decision was taken to protect workers in the trauma unit and future hospital patients from hospital-acquired CJD.

The usual sterilization conditions (121 degrees Celsius for 20 minutes under high pressure) do not destroy prion protein infectivity. Consequently the World Health Organization recommends incineration of potentially contaminated materials. While environmental transmission of prion diseases has not been reported, WHO suggests rinsing surfaces with sodium hydroxide or sodium hypochlorite for 1 hour, followed by flooding with water, to remove prions.

This case illustrates the problems associated with an unusual infectious agent, the prion, that is difficult to inactivate. It also shows the value of electronic health records. Without such readily accessible information, the discovery that the patient had CJD would have been substantially delayed, leading to further contamination.

Creutzfeldt-Jacob associated deaths have increased slowly but steadily in the US since 1979. The number of cases will likely continue to increase until early diagnosis tests become routinely available, and drugs are developed that can cure the disease.

A case of prion disease acquired from contaminated beef

prion conversionSpongiform encephalopathies are neurodegenerative diseases caused by misfolding of normal cellular prion proteins. A 2014 case of variant Creutzfeldt-Jacob prion disease in the United States was probably caused by eating beef from animals with bovine spongiform encephalopathy (BSE), or mad cow disease.

Human spongiform encephalopathies are placed into three groups: infectious, familial or genetic, and sporadic, distinguished by how the disease is acquired initially. In the mid 1980s, a prion disease called bovine spongiform encephalopathy appeared in cows in the United Kingdom. It is believed to have been transmitted to cows by feeding them meat and bone meal, a high protein supplement prepared from the offal of sheep, cattle, pigs, and chicken. Some of the animals prepared for feed likely had a prion disease. Cases of variant Creutzfeld-Jakob disease, a new spongiform encephalopathy of humans, began to appear in 1994 in Great Britain. They were characterized by a lower mean age of the patients (26 years), longer duration of illness, and differences in other clinical and pathological characteristics. Variant Creutzfeldt-Jakob disease (vCJD) is caused by prions transmitted by the consumption of cattle with bovine spongiform encephalopathy.

In late 2012 a male Texas resident began showing symptoms of depression and anxiety, followed by delusions, hallucinations, and other changes in behavior. Over the next 18 months the patient’s condition deteriorated, leading to inability to ambulate or speak, and after several episodes of aspiration pneumonia and sepsis the patient died. During the illness prion disease was suspected, but tests for this condition were negative. After death, examination of brain biopsies revealed typical prion plaques, and misfolded prion proteins were found in urine, confirming the diagnosis of variant Creutzfeldt-Jacob disease.

The source of the patient’s prion disease was likely consumption of contaminated beef from cows with bovine spongiform encephalopathy. The patient probably acquired the infection in Russia, Lebanon, or Kuwait, three countries that had received BSE-contaminated beef from the UK, and and where he had previously lived. He resided in the US for 14 years before developing symptoms, an incubation period consistent with models of the vCJD epidemic.

This Texas patient is the fourth worldwide since 2012 to be diagnosed with vCJD; the others were from the UK and France. There are likely to be additional cases of vCJD in the future: surveys of archived appendix tissues in the UK show that 1 in 2,000 persons born during 1941-1985 have asymptomatic vCJD infection. These individuals could transmit the misfolded prion proteins to others via transplantation, blood transfusion, or surgical instruments (prion infectivity is not destroyed by autoclaving).

The good news is that vCJD is rare: there have been 230 reported cases of vCJD worldwide caused by consumption of BSE beef. The bad news is that vCJD will probably continue to appear in humans for many years, not only from the aftermath of the BSE epidemic. Rare cows spontaneously develop BSE, and because cattle are slaughtered before disease symptoms are evident, contaminated meat could enter the food supply.

TWiV 343: The silence of the turnips

On episode #343 of the science show This Week in Virology, the TWiVerinoes discuss the potential for prion spread by plants, global circulation patterns of influenza virus, and the roles of Argonautes and a viral protein in RNA silencing in plants.

You can find TWiV #343 at www.microbe.tv/twiv.

Prions in plants

prions in plants

Chronic wasting disease is a prion disease of cervids (deer, elk, moose) that is potentially a threat to human health. A role for environmental prion contamination in transmission is supported by the finding that plants can take up prions from the soil and transmit them to animals.

A concern is that prions of chronic wasting disease could be transmitted to cows grazing in pastures contaminated by cervids. Consumption of infected cows would then pass the disease on to humans. When deer are fed prions they excrete them in the feces before developing clinical signs of infection, and prions can also be detected in deer saliva. In the laboratory, brain homogenates from infected deer can transmit the disease to cows.

To determine whether prions can enter plants, wheat grass roots and leaves were exposed to brain homogenates from hamsters that had died of prion disease. The plant materials were then washed and amounts of prions were determined by protein misfolding cyclic amplification. Prions readily bound these plant tissues, at low concentrations and after as little as 2 minutes of incubation. Mouse, cervid, and human prions also bound to plant roots and leaves. When living wheat grass leaves were sprayed with a 1% hamster brain homogenate, prions could attach to the leaves and be detected for 49 days.

To determine if prions in plants could infect animals, plants were exposed to brain homogenates, washed thoroughly, and then fed to hamsters. The positive control for this experiment was to feed hamsters the brain homogenates. All animals fed infected plants or brain homogenates succumbed to prion disease.

Plants can also take up prions from animal waste. This conclusion was reached by incubating leaves and roots for 1 hour with urine or feces obtained from prion-infected hamsters or cervids. Prions were readily detected in these samples, even after extensive washing.

Experiments were also done to examine whether plants could take up prions from the soil. Barley grass plants were grown on soil that had been mixed with hamster brain homogenate, and then 1-3 weeks later, stem and leaves were assayed for the presence of prions. Small amounts of prions were detected in stems from all plants, while 1 in 4 plants contained prions in leaves, at levels that should be able to infect an animal.

These results show that prions can bind to plants and be taken into the roots, where they may travel to the stem and leaves. Therefore it is possible that prions excreted by deer could pass on to other animals, such as grazing cows, or even humans consuming contaminated plants (illustrated – image credit). Cooking plants will not eliminate infectivity, just as cooking contaminated beef did not halt the spread of bovine spongiform encephalopathy. Keeping cervids out of grazing or growing fields should be considered as a way to manage the risk of prions entering the human food chain.