Prion processing and propagation in neuronal and dendritic cell culture models

Prion diseases are rare neurodegenerative diseases, associated with aconversion of a normal cellular protein, PrPC, into its misfolded andpartial protease resistant isoform, PrPSc. These diseases affect bothanimals and humans and are unique since they can not only be genetic andsporadic, but also transmissible. When affecting the central nervoussystem (CNS), the prion diseases are lethal and characterized byaccumulations of PrPSc, spongiform changes and astrogliosis. The cause ofthe neurodegeneration is not known, but has been shown to correlatewith accumulations of misfolded PrPSc.The aim of this work is to study how the amount of PrPSc can be regulatedin neurons and cells of the immune system to affect processing and spreadof this protein. For this purpose we used cell-culture models of neuronalcells and antigen presenting cells. The immortalized hypothalamic GT1-1cell line and primary cultures of mouse dorsal root ganglia (DRG) wereused as a source of neuronal cells. Antigen presenting cells wererepresented by primary cultures of mouse bone marrow-derived CD11c+dendritic cells.The results show that PrPSc can be degraded by cellular cysteineproteases in scrapie-infected GT1-1 cells (ScGT1-1). After incubation ofScGT1-1 with cysteine protease inhibitors, levels of PrPSc were increasedas visualized by Western immunoblotting and immunofluorescence.Inhibitors of other protease families did not exert any effects on thePrPSc amount in these cells. Inhibition of both cathepsin B and cathepsinL by selective protease inhibitors and by siRNA increased the amount ofPrPSc in ScGT1-1 cells. After blocking formation of PrPSc using pentosanpolysulfate, the increase in PrPSc induced by the inhibitors was stillevident showing that the inhibitors were acting on degradation, notformation of misfolded protein.Since IFN-gamma can affect the activity of cathepsins, the ScGT1-1 cellswere exposed to this proinflammatory cytokine. IFN-gamma exposure lead toan increase in the activity of cathepsin L in the ScGT1-1 cells,visualized with the Magic red cathepsin L activity-assay. The increase incathepsin L activity was followed by a decrease in the levels of PrPSc inthe cells. Inhibitors of cathepsin B and cathepsin L could block thisdecrease of PrPSc in IFN-gamma treated cells. Combined treatment withIFN-gamma and pentosan polysulfate removed all trace of PrPSc in thecells, unlike after treatment with pentosan polysulfate alone, wherestill some remains of PrPSc could be seen.Bone marrow-derived dendritic cells (DCs) may be involved in the spreadof prions in vivo. Cultured DCs did not express detectable levels ofPrPC, 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 cellswere exposed to homogenates from scrapie-infected GT1-1 cells. PrPSccould be visualized by immunofluorescence in the glial cells and themacrophage-like cells. However, after prolonged exposure, the glial cellswere cleared of PrPSc, but a few PrPSc labeled neurons were found in thecultures. After exposure to the homogenate of ScGT1-1 cells together withan inhibitor of cysteine and serine proteases, PrPSc remained in theglial cells and macrophage-like cells for extended periods of time. DRGneurons could also be cultivated together with DCs. Thus, primarycultures of dorsal root ganglion cells may serve as a model for studieson PrPSc processing and spread.