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A versatile prion replication assay in organotypic brain slices

Nature Neuroscience volume 11, pages 109–117 (2008)Cite this article

Abstract

Methods enabling prion replication ex vivo are important for advancing prion studies. However, few such technologies exist, and many prion strains are not amenable to them. Here we describe a prion organotypic slice culture assay (POSCA) that allows prion amplification and titration ex vivo under conditions that closely resemble intracerebral infection. Thirty-five days after contact with prions, mouse cerebellar slices had amplified the abnormal isoform of prion protein, PrPSc, >105-fold. This is quantitatively similar to amplification in vivo, but fivefold faster. PrPSc accumulated predominantly in the molecular layer, as in infected mice. The POSCA detected replication of prion strains from disparate sources, including bovines and ovines, with variable detection efficiency. Pharmacogenetic ablation of microglia from POSCA slices led to a 15-fold increase in prion titers and PrPSc concentrations over those in microglia-containing slices, as well as an increase in susceptibility to infection. This suggests that the extensive microglial activation accompanying prion diseases represents an efficacious defensive reaction.

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Figure 1: Prions in slice cultures.
Figure 2: Localization and impact of prion replication.
Figure 3: A broad range of strains is detectable by POSCA.
Figure 4: Microglial depletion in organotypic slice cultures.
Figure 5: Impact of microglial depletion on prion replication.

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Acknowledgements

We thank L. Rietschin and B. Gähwiler for help with slice cultures, D. Marino and A. Marcel for technical help, and C.J. Sigurdson and M. Glatzel for prion strains. A.A. is supported by grants from the Sixth European Union Framework program (TSEUR, LSHB-CT-2005-018805), the Swiss National Foundation, the National Competence Center on Neural Plasticity and Repair, the Stammbach Foundation, and the UK Department for Environment, Food and Rural Affairs. J.F. is supported by a grant from Zentrum für Neurowissenschaften Zurich, the Desiree and Niels Yde foundation, the Swiss Center of Transgenic Expertise, the Henny Sophie Clausen og møbelarkitekt Axel Clausens Foundation and the Ivan Nielsens Foundation, and F.L.H. was supported by US National Institutes of Health grant R01 NS046006 from the National Institute of Neurological Disorders and Stroke.

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Author notes

  1. Frank L Heppner

    Present address: Present address: Institute of Neuropathology, Charité - Universitätsmedizin Berlin, Augustenburger Platz 1, D-13353 Berlin, Germany.,

Authors and Affiliations

  1. Institute of Neuropathology, University of Zürich, Schmelzbergstrasse 12, Zürich, CH-8091, Switzerland

    Jeppe Falsig, Christian Julius, Ilan Margalith, Petra Schwarz, Frank L Heppner & Adriano Aguzzi

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Contributions

P.S. performed histoblots and transmissions of RML into tga20 and CD1 mice and C.J. performed SCEPAs. I.M. performed all experimental work in Figure 3 and in Supplementary Figure 7b. Development of POSCA, slice culture preparation, infections, western blots and all other experimental work were done by J.F. CD11b-HSVTK mice were generated by F.L.H. This study was conducted under the supervision of A.A. All authors contributed intellectually with practical or theoretical input.

Corresponding author

Correspondence to Adriano Aguzzi.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–7, Supplementary Table 1 (PDF 1288 kb)

Supplementary Video 1

Video microscopy of microglial depletion. Slices prepared from CD11b-HSVTK mice were treated with GCV for 8 days and IB4 was added to the cell culture medium. Images were recorded on a wide-field fluorescence microscope every 30 minutes for 5 days and the video is shown at a rate of 10 frames s−1. (MOV 2659 kb)

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Falsig, J., Julius, C., Margalith, I. et al. A versatile prion replication assay in organotypic brain slices. Nat Neurosci 11, 109–117 (2008). https://doi.org/10.1038/nn2028

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