Identification of an Intracellular Site of Prion Conversion

Abstract

Prion diseases are fatal, neurodegenerative disorders in humans and animals and are characterized by the accumulation of an abnormally folded isoform of the cellular prion protein (PrPC), denoted PrPSc, which represents the major component of infectious scrapie prions. Characterization of the mechanism of conversion of PrPC into PrPSc and identification of the intracellular site where it occurs are among the most important questions in prion biology. Despite numerous efforts, both of these questions remain unsolved. We have quantitatively analyzed the distribution of PrPC and PrPSc and measured PrPSc levels in different infected neuronal cell lines in which protein trafficking has been selectively impaired. Our data exclude roles for both early and late endosomes and identify the endosomal recycling compartment as the likely site of prion conversion. These findings represent a fundamental step towards understanding the cellular mechanism of prion conversion and will allow the development of new therapeutic approaches for prion diseases.

Prion aggregates transfer through tunneling nanotubes in endocytic vesicles

“…As ScCAD do not undergo apoptosis upon scrapie infection, certain mechanisms must operate in order to maintain a balance between scrapie production and degradation that allows the stably infected cell line to continue without the heavy cell death observed in normal progression of the disease. Thus, the large percentage of PrP Sc found within lysosomes is consistent with continual degradation 13,19 of the misfolded conformer. Interestingly, while average lysosome number per cell is similar between CAD and ScCAD, we note a small but significant increase in average lysosome size in ScCAD (Fig.…”

“…14 In this study, we investigated the mechanism of PrP Sc transfer through TNTs within intracellular organelles and vesicles. Using a chronically infected neuronal cell line (ScCAD), 5,13 we demonstrate that endocytic organelles are present within TNTs and PrP Sc is colocalized with these vesicles, suggesting that the active transport of PrP Sc -containing vesicles in TNTs indeed occurs. Additionally, we demonstrate that PrP Sc increases both number of TNTs and transfer efficiency.…”

“…The co-localization of PrP Sc with markers for early endosomes and early recycling compartments is consistent with our previous work showing that the ERC is a site for conversion of PrP C to PrP Sc and does suggest a continual recycling of surface PrP wherein conversion might be occurring, thus maintaining the persistent infection of the ScCAD. 13 It has been proposed that the ERC is a major storage organelle for cholesterol 17 and since PrP is GPI-anchored, and found predominantly in cholesterol-rich lipid rafts 18 the redistribution to the ERC environment together with its cellular positioning might provide an explanation for the higher observed colocalization within TNTs. In addition, spreading PrP Sc within the ERC compartment where conversion occurs would provide the uninfected recipient cells with both the infectious seed and the propitious environment (the machinery for conversion) and thereby promote propagation of PrP Sc through the recycling of PrP C through this organelle.…”

“…5 We have previously characterized the subcellular compartmentalization of PrP C and PrP Sc in this cell line and showed that while PrP C was enriched at the plasma membrane (PM) and in the Golgi, PrP Sc was found predominantly in the endocytic pathway, in early endosomes and late endosomes/lysosomes and specifically enriched in recycling endosomes (ERC). 13 Thus, the infectious protein may well be transported in these organelles along TNTs. After guanidium thiocyanate denaturation to reveal PrP Sc epitopes (Fig.…”

“…1,11,12 Furthermore, we have demonstrated that the endocytic recycling compartment (ERC) is a major intracellular site of PrP Sc conversion. 13 Thus, a likely possibility is that the TNTmediated prion transfer occurs using active transport of prion-containing vesicles. 14 In this study, we investigated the mechanism of PrP Sc transfer through TNTs within intracellular organelles and vesicles.…”