Establishment of a cell-based anti-prion compound screen and analysis of host response to prion infection in cerebellar organotypic slice cultures

Abstract

Prions are unconventional infectious agents that cause always fatal neurodegenerative diseases termed prion disease or transmissible spongiform encephalopathies in mammals. Prion diseases are caused by an accumulation of the misfolded, aggregated host encoded prion protein (PrP). The normal, cellular, α-helix rich isoform (PrP^C) is converted into the disease-associated β-sheet rich pathogenic isoform (PrP^Sc). PrP^Sc can adopt multiple conformations that likely encipher prion strain characteristics. Currently, prion therapeutic clinical trials lack success and there is an urgent need for novel therapeutics. The aim of this study was to develop a cell-based assay for high content screening of large compound libraries with an automated microscope to identify compounds that might impair prion replication. Furthermore, identified compounds should be tested on prion infected organotypic slice cultures to test whether <em>in vitro</em> detected anti-prion compounds are also effective in a more complex neuronal environment. Additionally, two promising compounds, FeTMPyP and PIM-B31, identified by our collaboration partner Emiliano Biasini (University of Trento), were tested <em>ex vivo</em>. Beside this a comparative study of host response between <em>ex vivo </em>and<em> in vivo</em> should evaluate the transferability between the two systems, as this has not been was not shown until now. In the established screen 152 compounds were tested, 84 had an inhibitory effect on PrP^Sc accumulation in persistently infected N2a^22L cells and the seven strongest inhibitors were further validated by western blot analysis. The most promising candidate, PHA665752, was tested <em>ex vivo</em> and showed a reduction of PrP^Sc accumulation that was however not significant. FeTMPyP showed strong toxicity and PIM-B31 showed inconsistent results that depended on different concentration and strain-specificity. Beside this, pathway analysis of <em>ex vivo </em>and<em> in vivo</em> infected mouse cerebella with different strains at various time points was performed with DAVID 6.8, an online bioinformatics resource. Analysis of the 250 most significant differentially expressed genes revealed that several comparable pathways were changed due to prion infection in brain slices and brains. The calcium signaling pathways and neuroactive ligand-receptor pathways were deregulated the most by prion infection <em>ex vivo </em>as well as <em>in vivo</em>.