Feature: Puzzling prions

When Andrew Hill first heard about the then newly-discovered prions as an undergraduate student in Wellington, he was fascinated. “I thought they sounded like science fiction – these things that nobody knew much about and they were incredibly hard to destroy.

It sounded like a neat thing to work on.” So, in 1992, when a PhD project came up at Imperial College in the UK in a group working on prions, he applied and was soon jetting off to start his research career in earnest.

“My PhD work in the 1990s was on a newly discovered human form of prion disease known as variant Creutzfeldt-Jakob disease [vCJD], which was linked to people who had been exposed to bovine spongiform encephalopathy [BSE, or mad cow disease] presumably through diet. So we did a lot of transmission studies and found a marker for this particular form of the disease that was used to develop an early diagnostic test.”

Hill’s PhD and subsequent postdoctoral research in London also showed that the biological and molecular transmission characteristics of vCJD were consistent with it being the human equivalent of BSE. This body of work put Hill’s name firmly on the newly chartered prion-disease map.

Prions are most famous for causing BSE in the UK and Europe in the late 1980s and early 1990s. Prion is actually a portmanteau coined in 1982 after their discovery, and is derived from a mixture of “protein” and “infection” to describe a proteinaceous infectious particle.

The normal prion protein (PrP) is actually expressed in all mammalian tissues, although its exact function there remains unclear but, in general, prion refers to the infectious unit that causes disease. This form is an abnormally folded form of the host-encoded PrP and is often denoted as PrP Sc (after its first association with the sheep prion disease, scrapie).

Unlike other infectious particles, such as viruses or bacteria, that have their own genome, prions have quite a unique structure that looks to be protein only, without a nucleic acid component. Thus, they must have a robust means of transferring between and propagating in the cells that they infect. One advantage they do hold in that regard is their unusually stable nature, being resistant to the usual range of protein-denaturing agents or conditions and making disposal and containment of prion infectious material difficult.

Prions are now known to cause a variety of progressive and invariably fatal neurological diseases including BSE in cattle, Creutzfeldt-Jakob disease and kuru in humans, and scrapie in sheep. “Prion diseases are unique in that there are three different ways that other individuals can contract the infection,” explains Hill. “They can inherit the abnormal prion protein through a gene mutation, the mutated form can arise randomly in a population or the abnormal protein can be acquired through an infectious process.”

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Common problem

Moving to Melbourne in 2000, Hill’s research on prions broadened to encompass other neurodegenerative disorders. All known prions induce the formation of an insoluble amyloid protein aggregate in the brain or other neural tissues.

It turned out that this pathogenic process involving a protein misfolding event that parallels the way in which amyloid precursor protein (APP) in the brain of Alzheimer’s sufferers is proteolytically processed into the Abeta peptide to form the neurotoxic plaques that characterise this most common cause of human dementia.

Thus, although prion diseases and other neurodegenerative disorders differ clinically and pathologically, they might share a common triggering event at the molecular level, with the associated possibility of common therapeutic strategies.

At the OzBio 2010 Conference, Hill will review the significance of his own and others work on prion disease and then focus on how the infectious prion units spread amongst cells and individuals. One of Hill’s major findings a few years ago was that mammalian cells could transfer a prion infection from one cell type to a different cell type via small membrane-coated vesicles called exosomes.

“The initial link between prions and exosomes was reported by a French group in 2004,” says Hill. “Then our finding in 2007 was very significant, given that prions have to travel around the body to reach the brain, and provided a means of prions entering a neuronal cell.”

In 2008, the group cemented this significance by showing that APP and the Abeta peptide could also be transported in association with exosomes. Interestingly, the enzymes that cleave APP into the plaque-forming Abeta peptide have also been identified in exosomes, indicating that cleavage of APP could occur within these vesicles.

Since then, Hill’s team has concentrated on the role that exosomes and their other components play in the pathogenesis of a range of neurodegenerative diseases. “Specifically, we have been looking at various properties of exosomes for their potential use in diagnostics because you can isolate exosomes from bodily fluids.

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“We were recently successful in isolating them from cerebrospinal fluid [CSF] and found both the normal and abnormal form of the prion protein on exosomes isolated from CSF. This was in a large animal model, and what it told us was that part of the normal PrP processing pathway is release from cells in association with exosomes. So even normal forms of the PrP get transferred around the body in this way.”

These studies together also suggested to Hill that exosomes isolated from CSF could be analysed as a novel way to detect abnormal forms of the prion protein early on in the disease pathogenesis.

“Early diagnosis is crucial for treating these prion diseases because even though the infection generally takes years to establish, once it reaches the brain the progression can be very rapid indeed and very devastating. With kuru, for example, the incubation time can be as long as 50 years from exposure to disease, but once taken hold the clinical course can be a matter of days to weeks.”

Disease signatures

“The other thing we are doing, which I think is quite exciting, is looking at the stack of microRNAs [miRNA] also present in these exosomes. One way that a cell might communicate with the outside world is by packaging up either some of their mRNAs that can be translated into proteins or a few miRNAs that can affect gene expression, and sending them via exosomes to a completely different cell.”

By characterising the miRNA palette of exosomes from normal and prion-infected cells to see if they have a different miRNA ‘signature’, Hill hopes to come across a new biomarker for disease testing.

“This is quite important in Australia particularly because people cannot donate blood if they lived in the UK or Europe during the 90s because of the BSE epidemic and the risk of transmitting the infection through blood products. So a diagnostic test for prions in blood could make a huge difference in the blood products field.”

This strategy is already being used successfully in the cancer field. Certain cancers including ovarian cancer and the highly malignant brain cancer, gliobastoma, can be diagnosed based on their specific exosomal miRNA signature.

Hill suggests that the approach could be equally successful for neurodegenerative disease. In addition to prion-carrying exosomes, other diseases such as Alzheimer’s might also be identifiable based on the miRNA signature of exosomes carrying the relevant misfolded protein. “This is all a new, but very exciting idea, and this whole field of exosomes and cell-to-cell transmission has really exploded in the last few years,” he says.

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“At the moment we are just completing a next-generation sequencing run of the RNA species associated with exosomes isolated from neuronal cells. There is no hard data yet and lots to do sift through and analyse all the sequence information to identify every miRNAs present in these vesicles, some of which could be novel. We would also like to look at the more fundamental biological question of how and why these miRNAs get packaged into the exosomes.

“Another very recent focus of the lab is looking at conserved regions of the prion proteins that are involved in the infectious process. We published a paper this year on two highly conserved regions of the protein that seem important for transmission – of course, both are located in fairly unstructured areas of the protein that nobody has really characterised in detail.

“However, we have some hints already and we have developed a functional assay of the effects of these domains on the protein’s ability to infect cells with prions. Such key regions could potentially be used in developing therapeutics. If we can find molecules that bind to these regions then we might be able to stop the transmission and thus the infectious disease. So this really is very basic science but the implications are very clinical in finding new therapeutic targets.”

Over the few short years since their emergence, prions have challenged quite a few dogmas in basic biology – a protein having one amino acid sequence but adopting different structures, and this whole protein-only based model of inheritance. And, of course, we don’t have all the answers yet about prions – far from it.

In an ideal world Hill would find out exactly what makes the prion protein infectious – why and how does it turn bad? He would also develop a differential diagnostic for Alzheimer’s and prion disease using the miRNA profiles of exosomes isolated from patients. Finally, we would understand why cells release these exosomes filled with specific baggage, and what is the contribution of the RNAs associated with the exosomes to the infected cell signalling.

Andrew Hill is an Associate Professor and Principal Research Fellow in the Department of Biochemistry and Molecular Biology at the University of Melbourne. After doing a BSc and Honours at Victoria University in Wellington, New Zealand, Hill completed his PhD at Imperial College in the UK on variant CJD in what was to become the MRC Prion Unit. This work led to a number of high profile publications in Nature and Science, and to the development of a diagnostic and classification system for human prion diseases. In 2000, Hill moved to Australia on a Wellcome Fellowship to work on Alzheimer’s disease and other neurodegenerative disorders with Colin Masters at the University of Melbourne. He established his own research group in 2003 in the same Department on an NHMRC RD Wright Fellowship, and, in 2005, was the first group moved into the new Bio21 Institute.