Characterization of Antiprion Activity and Differential Scrapie Permissiveness in an Ovine Microglia Culture System

Kelcey Dawn Dinkel

Prion diseases are transmissible neurodegenerative disorders that affect both humans and several animal species. A fundamental feature of prion pathogenesis is the conversion of the cellular form of the prion protein (PrPC) into a protease-resistant, disease-associated isoform (PrPSc or PrPD) which catalyzes further self-templated replication into nascent PrPSc. The stable, beta-sheet rich conformation of PrPSc facilitates aggregation within the central nervous system leading to slowly progressive neurological dysfunction and death. Despite extensive investigation, the mechanisms underlying this pathogenesis are not fully understood and treatments to prevent or cure disease do not exist. Due to the invariable lethality of prion diseases, the identification of antiprion compounds is essential. Further, the development of effective therapeutics depends on an understanding of prion pathogenesis, including permissiveness to infection. Elucidating factors affecting prion permissiveness can impart potential targets of inhibition and contribute to the development of cell culture models. While permissiveness to prion infection requires the cellular expression of PrPC, non-PrPC factors may also play a role. The aim of this work was to investigate antiprion activity and factors associated with permissiveness in an ovine microglia culture system. In the first study, we examined the inhibitory activity of DB772, a monocationic phenyl-furan-benzimidazole, by screening a structural library of 89 related compounds at 1 µM in scrapie-infected ovine microglia. Eleven compounds with inhibitory activity equivalent or higher than DB772 were identified, and further investigation suggested PrPC misfolding to PrPSc as a potential target for inhibition. A structure-activity relationship analysis revealed two structural elements associated with antiprion activity. In the second study, differential permissiveness to natural scrapie was examined in clones of ovine microglia and compared to rates of cell proliferation, levels of selected gene transcripts, and matrix metalloproteinase 2 (MMP2) activity. Significant correlations were observed between permissiveness and genes associated with cell proliferation, protein degradation, and heparin binding. Differences detected in MMP2 activity, while not correlated with permissiveness, suggested a multifactorial role of MMP2 in scrapie infection. Taken together, these findings suggest potential chemical structures and cell-based targets that may contribute to the development of effective therapeutics and highly permissive cell culture models.

Characterization of Antiprion Activity and Differential Scrapie Permissiveness in an Ovine Microglia Culture System

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