Immunolocalisation of PrP Sc in scrapie-infected N2a mouse neuroblastoma cells by light and electron microscopy

Abstract

The causative agent of transmissible spongiform encephalopathies (TSE) is PrP Sc, an infectious, misfolded isoform of the cellular prion protein (PrP C). The localisation and trafficking of PrP Sc and sites of conversion from PrP C to PrP Sc are under debate, particularly since most published work did not discriminate between PrP C and PrP Sc. Here we describe the localisation of PrP C and PrP Sc in a scrapie-infected neuroblastoma cell line, ScN2a, by light and electron microscopic immunolocalisation. After eliminating PrP C with proteinase K, PrP Sc was detected at the plasma membrane, endocytosed via clathrin-coated pits and delivered to early endosomes. Finally, PrP Sc was detected in late endosomes/lysosomes. As we detected PrP Sc at the cell surface, in early endosomes and in late endosomes/lysosomes, i.e. locations where PrP C is also present, our data imply that the conversion process could take place at the plasma membrane and/or along the endocytic pathway. Finally, we observed the release of PrP C/PrP Sc via exocytotic pathways, i.e. via exosomes and as an opaque electron-dense mass which may represent a mechanism of intercellular spreading of infectious prions.