Characterization of intracellular localization of PrPSc in prion-infected cells using a mAb that recognizes the region consisting of aa 119–127 of mouse PrP

Abstract

Generation of an abnormal isoform of the prion protein (PrPSc signals that were clustered at peri-nuclear regions appeared to be localized in an endocytic recycling compartment through which exogenously loaded transferrin, shiga and cholera toxin B subunits were transported. The granular PrP Sc signals at peri-nuclear regions were dispersed to the peripheral regions including the plasma membrane during incubation at 20 6C, at which temperature transport from the plasma membrane to peri-nuclear regions was impaired. Conversely, dispersed PrP Sc signals appeared to return to peri-nuclear regions within 30 min during subsequent incubation at 37 6C, following which PrP Sc at peri-nuclear regions appeared to redisperse again to peripheral regions over the next 30 min incubation. These results suggest that PrP Sc is dynamically transported along with the membrane trafficking machinery of cells and that at least some PrP Sc circulates between peri-nuclear and peripheral regions including the plasma membrane via an endocytic recycling pathway.

Prions disturb post-Golgi trafficking of membrane proteins

“…This is consistent with previous reports by other investigators using other prion-infected cells 8,25 . Instead, most PrP Sc was observed throughout endosomal compartments, being particularly abundant in recycling endosome in infected cells.…”

“…Instead, most PrP Sc was observed throughout endosomal compartments, being particularly abundant in recycling endosome in infected cells. Similar subcellular distribution of PrP Sc was shown by others 25 . Accumulation of PrP Sc in recycling endosome was also shown in neurons of RML prion-infected mice 37 .…”

“…To investigate the mechanism of prion infection-induced impairment of post-Golgi vesicular trafficking, we investigated subcellular localization of PrP Sc in infected cells by performing PrP Sc -specific IHC with anti-PrP monoclonal Ab mAb132. The mAb132 was demonstrated to recognize the residues 119-127 of mouse PrP, and specifically visualize PrP Sc under partially denatured conditions 25 . No signals were detected in uninfected N2aC24 and cured N2aC24L1-3 cells (Fig.…”

“…Cells on coverslips were fixed in 3% paraformaldehyde for 15 min, treated with 0.1 M glycine in PBS for 10 min and permeabilized with 0.1% Triton X-100 in PBS for 4 min at RT. To detect PrP Sc , the cells were treated with 5 M guanidinium thiocyanate for 10 min at RT 25 . After washing with PBS, the cells were incubated with the first Ab in 5% FBS in PBS and then with fluorescent secondary Ab.…”

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

“…Earlier studies suggested that PrP Sc is generated from mature PrP C expressed on the cell surface and that the PrP Sc generation occurs at cellular compartments on the endocytotic pathway, including the cholesterol-rich membrane microdomains called lipid rafts Naslavsky et al, 1997;Vey et al, 1996). Further cell biological studies within the following decade indicated the involvement of endocytic recycling compartments and endocytic recycling pathway in PrP Sc formation (Beranger et al, 2002;Marijanovic et al, 2009;Pimpinelli et al, 2005;Veith et al, 2009;Yamasaki et al, 2012). We recently reported that after the inoculation of prions, newly generated PrP Sc appeared on the cell surface, early endosomes, and late endosomes of N2a cells (Yamasaki et al, 2014a).…”