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Prion : a « multifaceted » protein

Prion diseases are found in human (Creutzfeldt-Jakob Disease CJD) and in farm (sheep scrapie, Mad Cow disease) and wild animals with invariably fatal issue in all cases. 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 “dominoes falling 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 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 important. Some in vitro cell culture systems susceptible to prion have also been developed. However their sensitivity to prions of different species is limited.

Thanks to the Protein Misfolding Cyclic Amplification (PMCA) technique, researchers use since the middle of 2000 an experimental alternative that consists to directly search for the presence of the abnormal prion protein in samples. PMCA technique allows amplification of tiny amount 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 prion 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.