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Making headway on prion diseases

The search for answers about how BSE and other TSE prions work is still ongoing at the Alberta Prion Research Institute

Dr. David Westaway

As the Alberta Prion Research Institute (APRI) enters its 10th year it seems as good a time as any to look back on what has been learned and what it sees ahead in the prevention of prion diseases.

Set up by the province in 2005 in response to the disaster brought down on the cattle industry by BSE, the institute was given a mandate to fund research into transmissible spongiform encephalopathies (TSEs).

Besides BSE in cattle, APRI investigates chronic wasting disease (CWD) in wild and domestic elk and deer, scrapie in sheep and CJD in people, as well as other prion-like human neurodegenerative diseases, such as Alzheimer’s.

Kevin Keough
APRI executive director, Kevin Keough. photo: Supplied

In 2005, says APRI executive director Kevin Keough, there were no prion experts in Alberta and only a couple in Canada. So the first step was to recruit experts in the field to study how these brain cell proteins normally fold and how and why diseased ones change and propagate. Over time these investigations naturally branched out to include diagnostic, surveillance and control strategies, the socio-economic challenges of TSEs, and the disposal and uses of specified risk materials (SRM) rich in prions.

To date, the APRI has pledged $40.5 million on 103 projects by 48 researchers and three industry partners. The Alberta Livestock and Meat Agency (ALMA) has partnered on several. Another $4 million went to the universities of Alberta and Calgary.

The University of Alberta multiplied its APRI grant tenfold and founded the Centre for Prions and Protein Folding Diseases (Prion Centre) with three enhanced biosafety level (BSL2+) labs. It was certified in 2009 and a smaller version at the University of Calgary received certification this year.

The prion mystery

Dr. David Westaway, the prion centre’s director since 2006, offers an overview of the current state of the science on prions and prion-like diseases.

It’s now widely accepted that a gene determines the normal shape of cellular prions. As such it is subject to genetic mutation, meaning everyone carries the potential to develop misshapen prions. This is how sporadic forms of TSE disease pop up in about one in a million people and perhaps one in 10 million cattle, says Westaway.

The evidence to date suggests normal prion proteins are involved in the electrical activity and signalling mechanisms between brain cells. They also protect brain cells from stress and fatal chemical hyperactivity.

Dr. Stephen Moore’s team, when he was the bovine genomics chair at the U of A, found genes associated with susceptibility to BSE and others become activated when BSE is present. It also found 85 genes associated with CWD.

More recently, Dr. Stefanie Czub with the Canadian Food Inspection Agency (CFIA) at Lethbridge found a biomarker in urine for a gene associated with BSE.

Westaway and his team have taken this discovery to the next level by developing a potential non-invasive live test for prion disease. It has been validated on miniaturized samples using mouse models and is now being tested on samples from larger animals. It is slow work due to the long incubation period and slow progression of prion diseases in large animals.

Understanding the fundamental biology and chemistry of normal folding prions compared to rogue prions remains a priority. APRI-funded research has identified three proteins that regulate how other proteins fold and several protein-to-protein interactions associated with BSE. Researchers are moving these discoveries forward by investigating whether modifying certain protein activity could be one way to stop the progression of prion disease.

Scientists now know that there are at least three strains of BSE — classical, low type and high type (Canadian Cattlemen, October 2013). Czub is now investigating whether one strain could lead to another and whether sporadically formed prions differ from the other strains.

Others are looking at how CWD strains differ and whether some may be capable of jumping the species barrier. Much of the CWD research underway focuses on wild elk and deer to learn how the disease spreads and control measures such as oral vaccines.

In an article published in the January Journal of Clinical Investigation, Westaway’s team reported the body does have some natural protection against some TSEs. “It has always been believed that the body has no defence against prion diseases,” says Westaway. “We now know that there is a natural partially effective protective response that seems to apply to BSE, CWD and CJD, so we are trying to understand how we can make it more effective. We are now starting a second generation of experiments to hit (the disease process) from two directions.”

They’ll be using drugs already approved for other human diseases by Health Canada and the U.S. Food and Drug Administration.

Edmonton neurologist Dr. Valerie Sim is filming diseased prions growing in a Petri dish to understand how these diseases progress. The hope is she’ll identify opportune times during its development when the drugs could either halt the disease or render it less infective.

A second investigation is looking into what Westaway describes as a lock-and-key approach to decontaminate the infectious material. Some early research has been completed, but it’s a long-term goal because the shape of the key remains a mystery.

“No one has yet been able to solve what the surface of a prion looks like. Once that is known, it may be possible to synthesize a reverse molecule that, when locked with the infective protein, would deactivate the disease,” he explains.

The next frontier and subject of current studies is to understand what triggers a prion to change shape and, in turn, what can be done to prevent the change from occurring.

“We want to understand what goes wrong, stop it from happening, detect it in cases when it does happen, and dampen down the disease,” Westaway sums up.

The thorny side

The ruminant-to-ruminant feed ban has been in place since 1997. As a precautionary measure, CFIA removed all SRM from the food supply in July 2003. Then in 2007 the feed ban was enhanced to remove SRM from all animal feed, pet food and fertilizer, which had far-flung implications. Not only did it couple additional processing cost with a loss of salvage value of animals 30 months and over, it also resulted in a significant waste of protein.

According to the CFIA’s 2012 review of the enhanced feed ban, more than 90 per cent of SRM was going into landfills for the lack of economically feasible alternatives to recapture its value.

David Bressler, an associate professor of bioengineering and fermentation at the U of A, estimated that amounted to 5,000 metric tons per week back in 2011. With funding from APRI and ALMA his team developed a process for converting SRM into heavy-duty industrial plastics relying on thermal or caustic hydrolysis to meet the CFIA’s safety requirements for SRM.

Dr. Tim McAllister and colleagues at Ag Canada’s Lethbridge Research Centre have compiled a lengthy list of research on composting cattle mortalities as a viable method of destroying prions and other pathogens. APRI and ALMA funding has also allowed them to test whether plants absorb prions from soil contaminated with CWD prions. Results published earlier this year suggest that prions interact with roots of wheat plants, but if prions are transported to stems and leaves, the level is below what can be detected by current testing methods.

Other avenues of study

Tracking prions in plants

Thankfully for beef and dairy producers, there is no evidence anywhere in the world that BSE prion proteins can be picked up from the environment, only from contaminated feed.

The case isn’t that open and shut for chronic wasting disease (CWD). Dr. Tim McAllister, principal research scientist in ruminant nutrition and microbiology at Ag Canada’s Lethbridge Research Centre, says U.S. research has confirmed CWD prions left on the surface of plants by body fluids and decaying carcasses can persist for years and definitely act as a vector for transmitting CWD. This wasn’t a surprising result because of a known case where elk and deer became infected in a pasture that had been kept free of widlife after it was contaminated with CWD five years before. The same persistence has been demonstrated with scrapie in sheep.

BSE prions don’t seem to share the same trait. Supporting evidence for this comes from the United Kingdom where dairy cows didn’t get sick when placed on farms that had been depopulated because of BSE during the crisis there.

A second way plants could act as a vector for CWD prions would be if infectious proteins are absorbed by the roots and transported to the stems and leaves. McAllister’s team put this question to the test by soaking the roots of intact wheat plants grown in the lab for 24 hours in CWD-positive and CWD- negative elk brain homogenates that were either digested with proteinase K or left undigested. Protein extracts from the roots, stems and leaves were then tested for the presence of CWD prions. Only the digested CWD prions were detected interacting with the roots, leading to the conclusion that if CWD prions were transported to the stems and leaves, it was at a level undetectable by the three kinds of diagnostic tests commonly used for this purpose. The Canadian Food Inspection Agency has ask them to rerun this study with a more sensitive diagnostic test.

At this point, McAllister can say that the likelihood of CWD prions being taken up by plants in a natural setting is infinitesimally small, but may not be zero.

Prions have an affinity for organic matter and would likely be bound by the soil particles reducing the likelihood of plant uptake. On the other hand, the chance that they would be taken up likely increases in plants if the roots were damaged, such as the holes created by a nematode attack.

His team is now looking at whether uptake differs when roots are exposed to purified CWD prions, and hopes to investigate soil interactions with CWD prions.

All of the plant research is with CWD prions because experience and research has shown plants are a possible route of transmission for this disease. That is not the case with BSE prions.

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