Cryo-Immunogold Electron Microscopy for Prions: Toward Identification of a Conversion Site

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Abstract
Prion diseases are caused by accumulation of an abnormally folded isoform (PrPSc) of the cellular prion protein (PrPC). The subcellular distribution of PrPScand the site of its formation in brain are still unclear. We performed quantitative cryo-immunogold electron microscopy on hippocampal sections from mice infected with the Rocky Mountain Laboratory strain of prions. Two antibodies were used: R2, which recognizes both PrPCand PrPSc; and F4–31, which only detects PrPCin undenatured sections. At a late subclinical stage of prion infection, both PrPCand PrPScwere detected principally on neuronal plasma membranes and on vesicles resembling early endocytic or recycling vesicles in the neuropil. The R2 labeling was approximately six times higher in the infected than the uninfected hippocampus and gold clusters were only evident in infected tissue. The biggest increase in labeling density (24-fold) was found on the early/recycling endosome-like vesicles of small-diameter neurites, suggesting these as possible sites of conversion. Trypsin digestion of infected hippocampal sections resulted in a reduction in R2 labeling of >85%, which suggests that a high proportion of PrPScmay be oligomeric, protease-sensitive PrPSc.