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The Journal of Neuroscience, November 19, 2008, 28(47):12489-12499; doi:10.1523/JNEUROSCI.4474-08.2008

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

Susan F. Godsave,1 Holger Wille,2,3 Pekka Kujala,1 Diane Latawiec,2,3 Stephen J. DeArmond,2,3,4 Ana Serban,2 Stanley B. Prusiner,2,3 and Peter J. Peters1

1Section of Tumor Biology, Netherlands Cancer Institute, 1066 CX Amsterdam, The Netherlands, 2Institute for Neurodegenerative Diseases, and Departments of 3Neurology and 4Pathology, University of California, San Francisco, San Francisco, California 94143

Correspondence should be addressed to Peter J. Peters, Netherlands Cancer Institute, Antoni van Leeuwenhoek Hospital, Plesmanlaan 121-H4, 1066 CX Amsterdam, The Netherlands. Email: p.peters{at}nki.nl

Prion diseases are caused by accumulation of an abnormally folded isoform (PrPSc) of the cellular prion protein (PrPC). The subcellular distribution of PrPSc and 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 PrPC and PrPSc; and F4–31, which only detects PrPC in undenatured sections. At a late subclinical stage of prion infection, both PrPC and PrPSc were 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 PrPSc may be oligomeric, protease-sensitive PrPSc.

Key words: prion; electron microscopy; trypsin; endosome; cryo-immunogold EM; plasma membrane

Received Sept. 16, 2008; accepted Oct. 5, 2008.

Correspondence should be addressed to Peter J. Peters, Netherlands Cancer Institute, Antoni van Leeuwenhoek Hospital, Plesmanlaan 121-H4, 1066 CX Amsterdam, The Netherlands. Email: p.peters{at}nki.nl






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