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Rabbit kidney epithelial cells expressing the normal prion protein of a given species (in green). Cell nuclei are labelled in blue. These cells are used to prepare protein extract competent for prion conversion and amplification by the method described in the present work. © VIM, Inra Jouy-en-Josas, Jérôme Chapuis

Prion diseases: toward further reduction of animal experimentation

INRA scientists have developed an efficient method to amplify in vitro prions of different species. Beside its interest to rapidly address scientific questions, such as the study of the molecular mechanisms underlying prion propagation, this procedure represents a step forward to reduce the use of animal models in the prion field. This work is published in the 7 July 2016 issue of the journal Scientific Reports.

Updated on 07/21/2016
Published on 07/07/2016

Prion diseases are found in humans (Creutzfeldt-Jakob Disease CJD) and in farm (sheep scrapie, Mad Cow disease) and wild animals with invariably fatal outcome. Prions are infectious proteinaceous particles devoid of nucleic acid (DNA or RNA). Like Dr Jekyll and Mr. Hyde, the prion protein exists in two different forms, the normal one involved in several biological functions, and the abnormal, misfolded and infectious one. Prion propagation occurs once the abnormal form interacts with the normal one. The consequence of such interaction is a change of the spatial architecture of the normal protein that becomes converted into the abnormal form. By a domino effect, the propagation of this phenomenon takes place progressively inside the nervous tissue leading to neuronal death and the appearance of “holes” into the brain (spongiosis).

A novel acellular system for prion studies

Since the beginning of prion studies, laboratory animals have been used as the main experimental models to measure prion infectivity in biological samples. One major inconvenience of this approach is the incubation time to disease which, in laboratory rodents, varies between 2 months and 2 years. Furthermore, the total number of animals used in these bioassays is very large. Some in vitro cell culture systems susceptible to prions have also been developed. However their sensitivity to prions of different species is limited.

Thanks to the Protein Misfolding Cyclic Amplification (PMCA) technique, researchers have used since the middle of the 2000s an experimental alternative that consists in directly searching for the presence of the abnormal prion protein in samples. The PMCA technique allows amplification of tiny amounts of abnormal prion protein by conversion of normal prion protein substrate contained in brain extracts of overexpresser transgenic mice. Thus, scientists obtain a sufficient quantity of abnormal prion protein to be detected by classical biochemical methods. Researchers from INRA Jouy-en-Josas and Toulouse have improved the PMCA technique by using protein extracts from cultured cells. First, they developed a cultured cell model allowing the expression of the normal prion protein of a given species (human, ovine, murine, hamster…) - see image below. Then they set up, optimized and adapted to high throughput a PMCA procedure based on the use of cell extracts as sole source of normal prion protein. They showed efficient amplification of prions from different species including the human variant of Creutzfeldt-Jakob disease. The sensitivity of this new method, called Cell-based PMCA is close to that obtained with mouse brain extracts. It is a rapid method (3-4 days) and its development should facilitate the study of prion diseases.

Applying this method to some prion protein mutants, the INRA scientists were able to show that prion glycosylation state (the sugars that are naturally associated with the prion protein) is not directly involved in the transmission of its infectious properties, the latter are enciphered within the intrinsic organization of the prion infectious particle. Furthermore, the Cell-based PMCA constitutes, in bioethical and practical terms, a promising step toward further reduction of animal experimentation. It also might pave the way to the development of new diagnostic methods of human prion diseases.

Rabbit kidney epithelial cells expressing the normal prion protein of a given species (in green). Cell nuclei are labelled in blue. These cells are used to prepare protein extract competent for prion conversion and amplification by the method described in the present work.. © VIM, Inra Jouy-en-Josas, Jérôme Chapuis
Rabbit kidney epithelial cells expressing the normal prion protein of a given species (in green). Cell nuclei are labelled in blue. These cells are used to prepare protein extract competent for prion conversion and amplification by the method described in the present work. © VIM, Inra Jouy-en-Josas, Jérôme Chapuis

Contact(s)
Scientific contact(s):

Press Relations:
INRA News Office (33 (0)1 42 75 91 86)
Associated Division(s):
Animal Health
Associated Centre(s):
Occitanie-Toulouse, Jouy-en-Josas

Reference

Mohammed Moudjou, Jérôme Chapuis, Mériem Mekrouti, Fabienne Reine, Laetitia Herzog, Pierre Sibille, Hubert Laude, Didier Vilette, Olivier Andréoletti, Human Rezaei, Michel Dron and Vincent Béringue. PrP glycosylation-independent amplification of prions using highly efficient, cell-based, protein misfolding cyclic amplification. Scientific Reports, 7 July 2016. DOI:10.1038/srep29116