Immunolocalisation of PrPSc in scrapie-infected N2a mouse neuroblastoma cells by light and electron microscopy.

The causative agent of transmissible spongiform encephalopathies (TSE) is PrPSc, an infectious, misfolded isoform of the cellular prion protein (PrPC). The localisation and trafficking of PrPSc and sites of conversion from PrPC to PrPSc are under debate, particularly since most published work did not discriminate between PrPC and PrPSc. Here we describe the localisation of PrPC and PrPSc in a scrapie-infected neuroblastoma cell line, ScN2a, by light and electron microscopic immunolocalisation. After eliminating PrPC with proteinase K, PrPSc was detected at the plasma membrane, endocytosed via clathrin-coated pits and delivered to early endosomes. Finally, PrPSc was detected in late endosomes/lysosomes. As we detected PrPSc at the cell surface, in early endosomes and in late endosomes/lysosomes, i.e. locations where PrPC 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 PrPC/PrPSc 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.