NR AXVW
AU Sasaki,K.; Minaki,H.; Iwaki,T.
TI Time Course of the Development of PrP Aggregates in a Mouse Model of Prion Disease
QU International Conference - Prion 2007 (26.-28.9.2007) Edinburgh International Conference Centre, Edinburgh, Scotland, UK - Book of Abstracts: Protein Misfolding P01.43
IA http://www.prion2007.com/pdf/Prion Book of Abstracts.pdf
PT Konferenz-Poster
AB
Background: Abnormal prion protein (PrP) has a property of partial protease resistance, which is one of the grounds for the diagnosis of transmissible spongiform encephalopathies (TSEs), i.e. prion diseases. However, recent studies have revealed that protease-sensitive abnormal PrP (sPrPsc) also exists considerably in the TSE brains, and PrP oligomers have more intense infectivity and neurotoxicity than highly aggregated fibrils. Accordingly, protease-resistant PrP (rPrPsc or PrPres) might account for only a part of prions, which prompted us to establish another method for the detection of abnormal PrP.
Objectives: We designed the simplified gel filtration method to detect PrP aggregates and/or oligomers, and applied it to the study on a TSE mouse model to examine the time-course development of abnormal PrP.
Methods: Gel filtration spin columns, CHROMA SPIN (Clontech, USA), were used for size-exclusion fractionation of PrP without protease digestion. Serially corrected brain samples of NZW mice inoculated with the Fukuoka-1 strain intracranially were processed to whole brain homogenates in the buffer with detergents. Gel-fractionated samples were applied to western blotting to detect PrP in each molecular mass range and the densities were measured. Conventional protease-digestion assay was also performed to detect PrPres in the same samples.
Results: Mice inoculated with scrapie agent were died at around 4.0 months post inoculation. Spongiform change and abnormal PrP deposition detected by immunohistochemistry were apparently observed from 3.0 months post inoculation particularly in the thalamus. PrPres was drastically increased from 3.5 months, whereas the increase of PrP aggregates detected by the gel filtration spin column method became apparent from 3.0 months.
Discussion: Gel filtration assay under certain conditions with detergents is useful for the detection of abnormal, detergent-insoluble PrP aggregates. Application of spin columns provides the benefit of simple and safety handling in the closed system. In our study, the development of PrP aggregates preceded PrPres multiplication in the TSE mouse model. Because this assay does not include protease digestion process, the detected PrP aggregates in the early disease stage would be sPrPsc molecules. Protease-sensitive PrP aggregates may affect the pathological process in TSEs, thus we would have need to investigate the other properties of abnormal PrP than the protease resistance.
AD K. Sasaki, H. Minaki, T. Iwaki, Graduate School of Medical Sciences, Department of Neuropathology, Japan
SP englisch
PO Schottland