Modulation of PrP Aggregation and Cellular Prion Transfer

Abstract

Transmissible spongiform encephalopathies are fatal neurodegenerative diseases which affect many mammals, including humans. A unique feature involves the conversion of the prion protein (PrP) to an abnormal isoform. Previous studies indicate interconversion of PrP is associated with the binding of metals and glycosaminoglycans (GAGS) such as heparin. However, the impact of metals or heparin on the formation of disease-related PrP forms is not well understood. To determine whether metals and GAGs play a role in transfer of prion infection, a cell culture infection model was developed. Data from these studies show that prion infectivity was reduced with depletion of metals, but enhanced with the addition of heparin. Isothermal titration calorimetric experiments, a novel method to study heparin-PrP interactions, were performed to further classify heparin binding regions of PrP. Four sites were identified, and mutation analysis confirmed that at least two sites are located within the first 90 residues of the N-terminus. Finally, to address whether the effects seen in the cellular infection model were due to PrP-metal or PrP-GAG interactions, a aggregation method was developed to characterise recombinant PrP under physiologically relevant conditions. This is important to do as many in vitro prion conversion studies are performed in conditions not seen in vivo including extreme temperature or pH or in the presence of denaturants. The results of these studies showed that metals are able to significantly increase the conversion of PrP mutants containing deleted regions within the octameric repeat domain. In contrast, no significant promotional effect was seen with the heparin aggregation experiments. This result suggests a possible explanation of the cell culture data is that other cellular interactions besides PrP-heparin associations must be considered to fully account for these findings. This body of work establishes that PrP interactions with metals and glycosaminoglycans play a role in transmissible spongiform encephalopathies through prion conversion.

Date of Award	1 Mar 2010
Original language	English
Awarding Institution	University of Bath
Supervisor	David Brown (Supervisor)