Changes in protein structure and distribution observed at pre-clinical stages of scrapie pathogenesis.

Scrapie is a neurodegenerative disorder that involves the misfolding, aggregation and accumulation of the prion protein (PrP). The normal cellular PrP (PrP(C)) is rich in alpha-helical secondary structure, whereas the disease-associated pathogenic form of the protein (PrP(Sc)) has an anomalously high beta-sheet content. In this study, protein structural changes were examined in situ in the dorsal root ganglia from perorally 263K scrapie-infected and mock-infected hamsters using synchrotron Fourier Transform InfraRed Microspectroscopy (FTIRM) at four time points over the course of the disease (pre-clinical, 100 and 130 days post-infection (dpi); first clinical signs ( approximately 145 dpi); and terminal ( approximately 170 dpi)). Results showed clear changes in the total protein content, structure, and distribution as the disease progressed. At pre-clinical time points, the scrapie-infected animals exhibited a significant increase in protein expression, but the beta-sheet protein content was significantly lower than controls. Based on these findings, we suggest that the pre-clinical stages of scrapie are characterized by an overexpression of proteins low in beta-sheet content. As the disease progressed, the beta-sheet content increased significantly. Immunostaining with a PrP-specific antibody, 3F4, confirmed that this increase was partly - but not solely - due to the formation of PrP(Sc) in the tissue and indicated that other proteins high in beta-sheet were produced, either by overexpression or misfolding. Elevated beta-sheet was observed near the cell membrane at pre-clinical time points and also in the cytoplasm of infected neurons at later stages of infection. At the terminal stage of the disease, the protein expression declined significantly, likely due to degeneration and death of neurons. These dramatic changes in protein content and structure, especially at pre-clinical time points, emphasize the possibility for identifying other proteins involved in early pathogenesis, which are important for a further understanding of the disease.