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Cambridge Infectious Diseases

An Interdisciplinary Research Centre at the University of Cambridge

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Flying high with infectious proteins!

An interview on prion diseases with Dr. Raymond Bujdoso and Dr. Alana Thackray, Department of Veterinary Medicine, Cambridge Infectious Diseases Interdisciplinary Centre.

A fly model of prion disease offers new opportunities for the study of neurodegenerative diseases. Dr. Raymond Bujdoso and Dr. Alana Thackray (Department of Veterinary Medicine) have successfully begun to use Drosophila as a new animal model for the study of prion diseases that will allow new approaches to the analysis of genetic modifiers and therapeutic agents for these devastating conditions

Prion diseases

Diagram of conformational change
Fig. 1. The change in conformation from health prion protein to the infectious form.

Prion diseases are fatal neurodegenerative conditions of humans and various other vertebrate species. These diseases include scrapie in sheep, bovine spongiform encephalopathy (BSE) in cattle and Creutzfeldt-Jakob disease (CJD) in humans. Collectively, these diseases may be inherited, arise sporadically or occur through environmental exposure to infectious prion material. All of these prion disease manifestations appear to share the same pathogenic mechanism, which involves the misfolding and aggregation of the normal host protein PrPC into a polymeric ß-sheet-rich abnormal isoform termed PrPSc(see Figure 1).

During the course of prion diseases, prion infectivity and PrPSc accumulate in the brains of prion-infected individuals, resulting in chronic neurodegeneration of the central nervous system. The conformational change of PrPC into PrPSc is fundamental to prion propagation and transmission of the abnormal prion protein has been known to occur both within and between mammalian species. Accordingly, prion diseases are considered a significant threat to public health through their potential for zoonotic transmission as evidenced by the BSE epizootic in UK cattle and subsequent emergence of a new variant form of CJD (vCJD) in humans.

While prion disease of ruminants and humans is relatively rare, interest in how prions spread between cells has risen because other, more common, human neurodegenerative conditions show prion-like phenomena. For example, Alzheimer’s, Parkinson’s and Huntington’s diseases appear to show an “infectious” spread of disease-specific misfolded protein through the brain of affected individuals. It is expected therefore that the study of prion diseases will make a significant contribution not only to the understanding of the molecular pathogenesis of these diseases, but also to that of common human neurodegenerative diseases that show prion-like phenomena. We don’t yet understand the exact molecular nature of the infectious prion agent. The only reliable method to measure prion infectivity and to analyse the genes and metabolic pathways that may affect PrPCmisfolding in prion-infected individuals has been through study in animal models, typically rodents. However, the mouse bioassay for prion infectivity can be time-consuming because of the slow development of murine prion disease and also requires a large number of animals.

Fly image
Fig. 2 Transgenic Drosophila expressing the PrP protein. The red eyes show the expression of the PrP gene through use of a tag.
A Drosophila model of prion disease

In a move aimed at producing a model of prion disease in a well-defined genetic host that is quicker and easier to use, and also with the aim of reducing the number of mammalian animals used in research, Dr. Raymond Bujdoso’s group in the Department of Veterinary Medicine have developed a new model for studying prion disease using the fruit fly. Drosophila melanogaster have long been used for neurodegenerative studies because in common with mammals they show conservation in fundamental elements of the nervous system and biochemical pathways, allowing fly models of neurodegenerative disease to identify ways of modifying or slowing the disease in the host species. The normal physiology and development of Drosophila is also well characterised, so as to allow the use of behavioural assays that could be used to detect prion-induced neurotoxicity in the live fly. An essential feature of this work is the ability to rapidly generate flies capable of expressing proteins usually found in other species (see Figure 2). Drosophila do not naturally express the protein PrPC, the healthy protein necessary for prion infection. By introducing the PrP gene from sheep into the Drosophila genome Dr. Bujdoso’s group have generated flies capable of expressing the ovine PrP protein and, as a consequence, a new and exciting model for the study of prion-induced neurodegeneration.

This novel transgenic model now allows us to see much more quickly which parts of the prion protein are involved in the neurodegenerative process and study the mechanism of prion-induced neurotoxicity. In order to do so, the next step was to determine whether these PrP transgenic flies were susceptible to prion infection. Larvae from parent transgenic flies were fed brain homogenate from a scrapie-infected sheep. The larvae pupate and hatch into adult Drosophila with, in theory, PrPSc in their brains. But how to find out if the infectious prion is present?

How far can a fly climb?

One of the primary clinical signs of prion infection is ataxia, or lack of voluntary coordination of muscle movements, which manifests itself as an impeded ability to move properly. To assess this in Drosophila, Dr. Bujdoso’s group measured the climbing ability of flies. Prion-exposed flies are placed in specially adapted plastic pipettes, which are used as vertical climbing columns. After time to acclimatise to the pipette, the flies are tapped to the bottom of the tube from where they begin to climb up the column. By measuring how far each fly travels up the pipette over a fixed period of time it is possible to work out the performance index for each group of flies tested. Although flies show a normal decrease in locomotor activity with age, this can be exacerbated by neurotoxicity. Dr. Bujdoso’s group found that the locomotor activity of prion-exposed flies expressing the PrP gene showed an accelerated decline over time, indicating PrPSc propagation and neurodegeneration in the fly brain (see Figure 3). They also showed that the prions were not toxic to Drosophila per se, since flies that did not express the PrP protein showed no accelerated decline in locomotor activity after exposure to the scrapie-infected brain homogenate.

So where does this research take us? Dr. Bujdoso’s group have started to examine the mechanism of prion-induced

Graph
Fig. 3. PrP transgenic (red) or control non-transgenic (blue) flies were fed scrapie free (open symbols) or scrapie infected (closed symbols) sheep brain homogenate at the larval stage. The performance index is shown for each genotype of fly per time point.
neurodegeneration within PrP transgenic Drosophila by comparing the function of neuronal proteostatic systems in normal and prion-exposed fly brains. Ultimately, this new animal model allows us to rapidly screen for factors that could alter the course of prion infection, such as genes that act as enhancers or suppressors of prion infection, i.e. to identify potential modifiers of the disease process. The genes identified and their products could, collectively, help establish breeding programmes to select for prion-resistant animals, be used to develop diagnostic markers, or identify potential therapeutic targets of prion diseases. Particular areas of interest for therapeutic intervention are those cellular systems that function to clear misfolded proteins, for example the unfolded protein response (UPR). Dr. Bujdoso’s group has already started collaborations with industry to screen for therapeutic interventions that can modulate the UPR.

Dr. Bujdoso’s group have recently received research funding from the National Centre for the Replacement, Refinement and Reduction of Animals in Research (NC3Rs) programme, which funds projects that work to replace, refine and reduce the use of animals in clinical research.  In addition, his group have received funding from PrionNet, a joint MRC/EPSRC/BBSRC initiative, to screen for potential therapeutic interventions effective against prion disease.

References

Thackray, A.M., et al. (2012) Prion-induced toxicity in PrP transgenic Drosophila. Experimental and Molecular Pathology. 92, 194-201

Thackray, A.M., et al. (2012) Ovine PrP transgenic Drosophila show reduced locomotor activity and decreased survival. Biochemical Journal. 444, 487-495