Development of oligomeric prion‐protein aggregates in a mouse model of prion disease

Abstract

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.

Localization of A11‐reactive oligomeric species in prion diseases

“…Both pore formation and local disruption of the membrane have been suggested as possible mechanisms. Interestingly, membrane permeabilization for cations also appears to be an intrinsic property of A11‐reactive oligomers, which accords with the reactivity of Prp Sc oligomers with the A11 antibody . As proposed for Prp Sc oligomers, both pore formation and membrane dielectric disruption have been suggested as the mechanisms of permeabilization by A11‐reactive oligomers.…”

“…In this study, we investigated in situ the presence of A11‐reactive species in sCJD and GSS. Given that the A11 antibody has been shown to bind Prp Sc oligomers but not monomers or fibrils in vitro , and as we have demonstrated overlapping immunostaining of Prp Sc and A11‐reactive species, we believe that A11 can be used to detect the in‐situ localization of Prp Sc oligomers.…”

“…The toxicity of Prp Sc oligomers seems to be coupled with the conformation recognized by the A11 antibody. Prp Sc oligomers are detected by the antibody, whereas fibrils and monomers are not . The addition of A11 antibody to culture medium abolishes the toxicity of Prp Sc oligomers in the human neuroblastoma cell line SH‐SY5Y, and although the addition of a preparation of Prp Sc to neuronal lysates has been shown to result in proteasomal inhibition, the preincubation of Prp Sc with the A11 antibody completely abolished this effect …”

Protease‐resistant PrP and PrP oligomers in the brain in human prion diseases after intraventricular pentosan polysulfate infusion

“…We performed the size‐exclusion gel chromatography assay using the spin‐column kit CHROMA SPIN‐200 (Clontech, San Francisco, CA, USA) that clearly separated oligomeric PrP from monomeric PrP 19,22 . The samples were first centrifuged at 120 g for 2 min, and the first fraction was collected in the collection tube.…”

Morphine Withdrawal Modifies Prion Protein Expression in Rat Hippocampus

“…It has been suggested that oligomeric forms of PrP C , such as dimers, could facilitate a more rapid conversion of PrP C Proteinase K (PK) sensitive into PrP Proteinase K resistant (PrP res ) [41]. Thus, PK sensitivity was assessed in hippocampal samples overexpressing PrP C (i.e.…”