Prion processing and propagation in neuronal and dendritic cell culture models

Prion diseases are rare neurodegenerative diseases, associated with a conversion of a normal cellular protein, PrPC, into its misfolded and partial protease resistant isoform, PrPSc. These diseases affect both animals and humans and are unique since they can not only be genetic and sporadic, but also transmissible. When affecting the central nervous system (CNS), the prion diseases are lethal and characterized by accumulations of PrPSc, spongiform changes and astrogliosis. The cause of the neurodegeneration is not known, but has been shown to correlate with accumulations of misfolded PrPSc. The aim of this work is to study how the amount of PrPSc can be regulated in neurons and cells of the immune system to affect processing and spread of this protein. For this purpose we used cell-culture models of neuronal cells and antigen presenting cells. The immortalized hypothalamic GT1-1 cell line and primary cultures of mouse dorsal root ganglia (DRG) were used as a source of neuronal cells. Antigen presenting cells were represented by primary cultures of mouse bone marrow-derived CD11c+ dendritic cells. The results show that PrPSc can be degraded by cellular cysteine proteases in scrapie-infected GT1-1 cells (ScGT1-1). After incubation of ScGT1-1 with cysteine protease inhibitors, levels of PrPSc were increased as visualized by Western immunoblotting and immunofluorescence. Inhibitors of other protease families did not exert any effects on the PrPSc amount in these cells. Inhibition of both cathepsin B and cathepsin L by selective protease inhibitors and by siRNA increased the amount of PrPSc in ScGT1-1 cells. After blocking formation of PrPSc using pentosan polysulfate, the increase in PrPSc induced by the inhibitors was still evident showing that the inhibitors were acting on degradation, not formation of misfolded protein. Since IFN-gamma can affect the activity of cathepsins, the ScGT1-1 cells were exposed to this proinflammatory cytokine. IFN-gamma exposure lead to an increase in the activity of cathepsin L in the ScGT1-1 cells, visualized with the Magic red cathepsin L activity-assay. The increase in cathepsin L activity was followed by a decrease in the levels of PrPSc in the cells. Inhibitors of cathepsin B and cathepsin L could block this decrease of PrPSc in IFN-gamma treated cells. Combined treatment with IFN-gamma and pentosan polysulfate removed all trace of PrPSc in the cells, unlike after treatment with pentosan polysulfate alone, where still some remains of PrPSc could be seen. Bone marrow-derived dendritic cells (DCs) may be involved in the spread of prions in vivo. Cultured DCs did not express detectable levels of PrPC, and could therefore not form PrPSc. These cultured DCs were instead used as a model to study PrPSc degradation. After exposure of the DCs to PrPSc, the DCs were shown to degrade PrPSc. Inhibitors of cysteine proteases could block this degradation. The clearance of PrPSc occurred at acidic pH, implicating a role for the lysosomal proteases also in PrPSc degradation in this cell type. Finally, primary cultures of dorsal root ganglia, containing neurons, Schwann cells or satellite cells (glial cells) and macrophage-like cells were exposed to homogenates from scrapie-infected GT1-1 cells. PrPSc could be visualized by immunofluorescence in the glial cells and the macrophage-like cells. However, after prolonged exposure, the glial cells were cleared of PrPSc, but a few PrPSc labeled neurons were found in the cultures. After exposure to the homogenate of ScGT1-1 cells together with an inhibitor of cysteine and serine proteases, PrPSc remained in the glial cells and macrophage-like cells for extended periods of time. DRG neurons could also be cultivated together with DCs. Thus, primary cultures of dorsal root ganglion cells may serve as a model for studies on PrPSc processing and spread