Amplification of PrPSc From Fatal Familial Insomnia Brain Tissue using Wild-Type Human PrPC Brain Substrate

Human prion diseases are a group of neurodegenerative conditions classified as sporadic, familial or acquired, all of which are associated with misfolding and aggregation of the prion protein in the form of PrPSc. They comprise a wide range of clinical and neuropathological phenotypes including: Creutzfeldt–Jakob disease (CJD), Gerstmann-Straussler-Scheinker syndrome (GSS), fatal familial insomnia (FFI), among others. FFI is a rare inherited form closely associated with the D178N mutation in the prion protein gene (PRNP) linked with methionine (M) at position 129. Levels of detectable protease resistant prion protein (PrPres) are known to be very low in the brain in the majority of FFI cases.
The search of molecular biomarkers for the early detection of the human prion disease has led to the development of cell-free conversion systems as rapid diagnostic tools. A recent study reported the detection of PrPSc in the CSF of several FFI cases by real-time quaking-induced conversion or RT-QuIC1, suggesting that this method could provide a useful clinical diagnostic tool for genetic as well as sporadic human prion diseases. However, the biochemical properties of the RT-QuIC amyloid products have not been fully characterized.
Using an alternative in vitro conversion system, termed protein misfolding cyclic amplification or PMCA, we evaluated the amplification potential and biochemical properties (both pre- and post-amplification) of PrPSc from two FFI cases that had only very low levels of PrPres. A single round of PMCA using a wild-type human PrP (PRNP 129 MM) substrate resulted in amplification, as judged by increased detection of PrPres by Western blot analysis. Furthermore, the FFI PMCA reactions products retained the ability to seed a second round of PMCA. Moreover, the characteristic FFI PrPres type was conserved during the amplification process. However, the protease resistance of the PMCA product was greater than that of the PrPSc in the sample that was used to seed the reaction. The significance of this increase in protease resistance is unknown, but we note that the amplification process involved the production of wild-type PrPSc rather than the D178N mutant PrPSc present in the seed.