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Regioselective Biolistic Targeting in Organotypic Brain Slices Using a Modified Gene Gun
Published on: October 24, 2014
A versatile prion replication assay in organotypic brain slices
Jeppe Falsig1, Christian Julius, Ilan Margalith
1Institute of Neuropathology, University of Zürich, Schmelzbergstrasse 12, Zürich, Switzerland.
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, PrP(Sc), >10(5)-fold. This is quantitatively similar to amplification in vivo, but fivefold faster. PrP(Sc) 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 PrP(Sc) 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.
Insights
A new prion organotypic slice culture assay (POSCA) amplifies prion protein (PrPSc) ex vivo, closely mimicking in vivo infection. This method offers a faster, effective tool for studying prion diseases and their potential treatments.
Area of Science:
- Neuroscience
- Infectious Diseases
- Biotechnology
Background:
- Prion diseases are fatal neurodegenerative disorders.
- Studying prions ex vivo is crucial but challenging due to limited methods.
- Existing methods often fail to support replication of diverse prion strains.
Purpose of the Study:
- To develop a novel ex vivo method for prion replication and titration.
- To establish a model that closely mimics intracerebral prion infection.
- To investigate the role of microglia in prion disease progression.
Main Methods:
- Development of a prion organotypic slice culture assay (POSCA).
- Infection of mouse cerebellar slices with prions.
- Quantification of abnormal prion protein (PrPSc) accumulation.
- Pharmacogenetic ablation of microglia to assess their role.
Main Results:
- POSCA achieved >10(5)-fold PrPSc amplification within 35 days, comparable to in vivo levels but fivefold faster.
- PrPSc accumulation occurred predominantly in the molecular layer, mirroring in vivo infection.
- The assay detected prion strains from bovine and ovine sources with variable efficiency.
- Microglia ablation increased prion titers by 15-fold, suggesting a defensive role for microglia.
Conclusions:
- POSCA is an effective ex vivo tool for prion amplification and titration.
- Microglial activation in prion diseases may represent an innate defense mechanism.
- This assay provides a valuable platform for studying prion pathogenesis and therapeutic interventions.
