Molecular characterisation of human prion amplification in cell-free systems and diagnostic applications

Abstract

Prion diseases are a group of fatal neurodegenerative diseases associated with proteopathy occurring in humans and other mammals. The mechanism of prion replication is thought to be based on the induced misfolding of the host encoded prion protein and can be emulated in vitro by methodologies termed cell-free conversion (CFC) systems. Currently, the two most common implementation of CFC systems are the Protein Misfolding Cyclic Amplification (PMCA) reaction and the Real Time Quaking Induced Conversion (RT-QuIC) reaction. The overarching aim of this thesis is to describe and compare the human prion amplification in vitro by PMCA and RTQuIC and to extend the diagnostic applicability of the latter, which is currently employed for the diagnosis of sporadic Creutzfeldt-Jakob disease (sCJD). Therefore, in this thesis, PMCA and RT-QuIC differential amplification abilities were systematically investigated and, for the first time, PMCA and RT-QuIC reaction kinetics compared using a specifically defined set of conditions. In addition, the research performed in this thesis has shown that the sCJD cerebrospinal fluid and brain samples seed the conformational change of the full-length hamster recombinant prion protein in a similar way in RT-QuIC, and that the RT-QuIC analysis of sCJD urine samples is possible, however, it has low sensitivity and, therefore, a limited diagnostic potential.