Scrapie-infected murine neuroblastoma cells produce protease-resistant prion proteins

Abstract

Scrapie and Creutzfeldt-Jakob disease are transmissible, degenerative neurological diseases caused by prions. Considerable evidence argues that prions contain protease-resistant sialoglycoproteins, designated PrPSc, encoded by a cellular gene. The prion protein (PrP) gene also encodes a normal cellular protein designated PrPC. We established clonal cell lines which support the replication of mouse scrapie or Creutzfeldt-Jakob disease prions. Mouse neuroblastoma N2a cells were exposed to mouse scrapie prions and subsequently cloned. After limited proteinase K digestion, three PrP-immunoreactive proteins with apparent molecular masses ranging between 20 and 30 kilodaltons were detected in extracts of scrapie-infected N2a cells by Western (immuno-) blotting. The authenticity of these PrPSc molecules was established by using monospecific antiserum raised against a synthetic peptide corresponding to a portion of the prion protein. Those clones synthesizing PrPSc molecules possessed scrapie prion infectivity as measured by bioassay; clones without PrPsc failed to demonstrate infectivity. Detection of PrPSc molecules in scrapie-infected N2a cells supports the contention that PrPSc is a component of the infectious scrapie particle and opens new approaches to the study of prion diseases.

Green tea extracts interfere with the stress‐protective activity of PrPC and the formation of PrPSc

“…The screen was based on a collection of natural compounds with a documented activity to interfere with amyloid fibril formation in vitro (Porat et al 2006). We used scrapieinfected N2a (ScN2a) cells as a model to study propagation of PK-resistant PrP Sc and infectious scrapie-prions in rodent cells (Butler et al 1988;. A fast and robust method to determine the relative amount of PrP Sc is the detergent solubility assay.…”

Chemical chaperones interfere with the formation of scrapie prion protein.

“…After passage of the infected cells for several generations, the cells then were subcloned by limiting dilution. The ScN2a cells continued to form PrPSc as shown by the relative protease resistance of the PrP and to propagate prions as demonstrated by bioassays in mice (Butler et al, 1988). For steady-state labeling, cells were incubated with 200 lCi/ml [35S]methionine/cysteine (Amersham, >37 TBq/mmol) for 12-15 h in medium consisting of methionineand cysteine-free DME supplemented with 5% FCS and 5% complete MEM Eagle's medium.…”

Cytochalasin D enhances the accumulation of a protease‐resistant form of prion protein in ScN2a cells: involvement of PI3 kinase/Akt signalling pathway

“…N2a cells have been widely used to measure the infectivity of mouse-adapted scrapie prions in vitro (Vilette, 2008). However, ordinary N2a cells produce only low levels of PrPres after infection with limited prion strains (Butler et al, 1988). To obtain sufficient quantities of PrPres for biochemical analysis, prion-infected cells must be subcloned and the most highly infected sublines need to be selected and expanded (Race et al, 1988).…”