Synthesis and trafficking of prion proteins in cultured cells.

Abstract

Scrapie prions are composed largely, if not entirely, of the scrapie prion protein (PrPSc) that is encoded by a chromosomal gene. Scrapie-infected mouse neuroblastoma (ScN2a) and hamster brain (ScHaB) cells synthesize PrPSc from the normal PrP isoform (PrPC) or a precursor through a posttranslational process. In pulse-chase radiolabeling experiments, we found that presence of brefeldin A (BFA) during both the pulse and the chase periods prevented the synthesis of PrPSc. Removal of BFA after the chase permitted synthesis of PrPSc to resume. BFA also blocked the export of nascent PrPC to the cell surface but did not alter the distribution of intracellular deposits of PrPSc. Under the same conditions, BFA caused the redistribution of the Golgi marker MG160 into the endoplasmic reticulum (ER). Using monensin as an inhibitor of mid-Golgi glycosylation, we determined that PrP traverses the mid-Golgi stack before acquiring protease resistance. About 1 h after the formation of PrPSc, its N-terminus was removed by a proteolytic process that was inhibited by ammonium chloride, chloroquine, and monensin, arguing that this is a lysosomal event. These results suggest that the ER is not competent for the synthesis of PrPSc and that the synthesis of PrPSc occurs during the transit of PrP between the mid-Golgi stack and lysosomes. Presumably, the endocytic pathway features in the synthesis of PrPSc.

Altered Glycosylated PrP Proteins Can Have Different Neuronal Trafficking in Brain but Do Not Acquire Scrapie-like Properties

“…7A). This observation is in accord with several results previously obtained both in vitro and in vivo with different detection techniques (41)(42)(43)(44)(45)(46)(47)(48).…”

Comparison of abnormal isoform of prion protein in prion-infected cell lines and primary-cultured neurons by PrPSc-specific immunostaining

“…This mAb is expected to react with full-length PrP Sc but not with the N-terminally truncated form of PrP Sc that is generated by endogenous enzymes after PrP Sc formation in the cells Taraboulos et al, 1992). The mAb 8D5 specifically immunoprecipitated PrP Sc from brain homogenates of prion-infected animals, similar to other PrP Sc -specific antibodies reported to date (Curin Serbec et al, 2004;Horiuchi et al, 2009;Korth et al, 1997;Paramithiotis et al, 2003;Ushiki-Kaku et al, 2010).…”

“…Recently, Godsave and colleagues reported that in RML strain-infected mouse brains, the majority of PrP Sc clusters were detected at the plasma membranes, including membrane invaginations and the site of cell-to-cell contact, whereas in the other subcellular regions such as synapses and intracellular vesicles, PrP Sc clusters were detected only occasionally by immuno-electron microscopic examination using an antibody that recognizes the N-terminal region of PrP (Godsave et al, 2013). The authors believed that PrP Sc detected by the anti-PrP N-terminus mAb Saf32 included nascent full-length PrP Sc , as the N-terminal region tends to be processed after generation of PrP Sc Taraboulos et al, 1992). Considered collectively, ultrastructural studies suggest the plasma membranes as the primary site for PrP Sc formation.…”

Structural changes of membrane-anchored native PrP C

“…In our earlier binding studies, significantly higher binding of PrP C to raft-like lipids was shown as compared to other lipids, but it was pointed out that binding of PrP C was not exclusive to rafts (25). The formation of PrP C clusters was described in cell-culture studies (32)(33)(34)(35). For example, highdensity PrP C clusters on the surface of primary culture neurons were observed (32).…”