Development of oligomeric prion-protein aggregates in a mouse model of prion disease.

In prion diseases the normal cellular isoform of prion protein (PrP), denoted PrP(C), is converted into an abnormal, pathogenic isoform of PrP (PrP(Sc)). Diagnostic tools for prion diseases are conventionally based on the detection of protease-resistant PrP (PrP(res)) after proteinase K digestion. However, recent studies have revealed that protease-sensitive abnormal PrP (sPrP(Sc)) also exists in significant amounts in brains suffering from prion diseases. Here, we designed a simplified size-exclusion gel chromatography assay, using disposable spin columns to examine PrP aggregates in the course of the disease, without proteinase K digestion. Brain homogenates of NZW mice, inoculated intracranially with Fukuoka-1 strain, and which died at around 120 days post-inoculation, were assayed by this gel-fractionation method and eluted PrP molecules in each fraction were detected by western blot analysis. Oligomeric PrP molecules were well separated from monomers, as predicted. A conventional protease-digestion assay was also performed to detect PrP(res) and revealed that the ratio of PrP(res) to total PrP increased drastically from 105 days. However, the increase of PrP oligomers became significant from 90 days. These PrP oligomers in the early disease stage would, therefore, be sPrP(Sc) molecules that might affect the disease pathology, such as spongiform change and abnormal PrP deposition. We also observed that the resistance of PrP oligomers to proteinase K and insolubility in phosphotungstic acid precipitation increased with disease progression, which suggests that PrP oligomers are not clearly distinguished from cellular PrP or PrP(res) but may overlap in a continuous spectrum. Our study casts light on the ambiguity of the definition of PrP(Sc) and indicates that the abnormality of PrP molecules should be determined from various perspectives, more than protease resistance.