In prion diseases, the cellular prion protein (PrP(C)) becomes misfolded into the pathogenic scrapie isoform (PrP(Sc)) responsible for prion infectivity. We show here that peptides derived from the prion protein N terminus have potent antiprion effects. These peptides are composed of a hydrophobic sequence followed by a basic segment. They are known to have cell-penetrating ability like regular cell-penetrating peptides (CPPs), short peptides that can penetrate cellular membranes. Healthy (GT1-1) and scrapie-infected (ScGT1-1) mouse neuronal hypothalamic cells were treated with various CPPs, including the prion protein-derived CPPs. Lysates were analyzed for altered protein levels of PrP(C) or PrP(Sc). Treatment with the prion protein-derived CPPs mouse mPrP(1-28) or bovine bPrP(1-30) significantly reduced PrP(Sc) levels in prion-infected cells but had no effect on PrP(C) levels in noninfected cells. Further, presence of prion protein-derived CPPs significantly prolonged the time before infection was manifested when infecting GT1-1 cells with scrapie. Treatment with other CPPs (penetratin, transportan-10, or poly-L-arginine) or prion protein-derived peptides lacking CPP function (mPrP(23-28,) mPrP(19-30,) or mPrP(23-50)) had no effect on PrP(Sc) levels. The results suggest a mechanism by which the signal sequence guides the prion protein-derived CPP into a cellular compartment, where the basic segment binds specifically to PrP(Sc) and disables formation of prions.

We have studied how prion infection may affect the Src kinase activity in three different neuronal cell lines, ScGT1 and ScN2a, where ScGT1 were generated in our laboratory. By immunoblotting, using clone 28 -a monoclonal antibody recognizing active Src, we have found a 32 ± 6.3% and 75 ± 7.7% elevation in Src activity in ScGT1 and ScN2a cells, respectively, compared to uninfected cells. Immunocomplex in vitro kinase assay confirmed the increased Src activity. The increased Src kinase activity in scrapie-infected cells was further shown to correlate to an increased level of Src protein.In addition, an important increase in the protein tyrosine phosphorylation signal was observed in ScGT1 and ScN2a cells, which was further shown to be Src-dependent, as treatment with PP2 -a Src family kinase specific inhibitor, reversed the protein tyrosine phosphorylation profile. Abnormal Src-kinase activation and subsequent protein tyrosine phosphorylation may be key elements in the neuropathology of the prion diseases.

The copper-binding cellular prion protein (PrP(C)) and the heparan sulphate (HS)-containing proteoglycan glypican-1 (Gpc-1) can both be attached to lipid rafts via their glycosylphosphatidylinositol anchors, and copper ions stimulate their cointernalization from the cell surface to endosomes. The prion protein controls cointernalization and delivers copper necessary for S-nitrosylation of conserved cysteines in the Gpc-1 core protein. Later, during recycling through endosomal compartments, nitric oxide can be released from the S-nitroso groups and catalyses deaminative degradation and release of the HS substituents. Here, by using confocal immunofluorescence microscopy, we show that normal PrP(C) and Gpc-1 colocalize inside GT1-1 cells. However, in scrapie-infected cells (ScGT1-1), Gpc-1 protein remained at the cell surface separate from the cellular prion protein. Scrapie infection stimulated Gpc-1 autoprocessing and the generated HS degradation products colocalized with intracellular aggregates of the disease-related scrapie prion protein isoform (PrP(Sc)). Coimmunoprecipitation experiments demonstrated an association between Gpc-1 and PrP(C) in uninfected cells, and between HS degradation products and PrP(Sc) in infected cells. Silencing of Gpc-1 expression or prevention of Gpc-1 autoprocessing elevated the levels of intracellular PrP(Sc) aggregates in infected cells. These results suggest a role for Gpc-1 autoprocessing in the clearance of PrP(Sc) from infected cells.

Prion infectivity is often linked to presence of the protease-resistant isoform of prion protein (PrP), PrP(res); therefore, it is of highest interest to have convenient methods for rapid detection of PrP(res) in the research laboratory. For detection of PrP(res) in model systems to confirm infectivity, there are several methods that can be applied. This chapter focuses on detection of PrP(res) by proteinase K digestion followed by Western blot, which is the only method that is both quantitative and qualitative. For large-scale screening of PrP(res) content in samples, the dot blot method offers a great advantage for detecting PrP(res), and this method is also thoroughly described in this chapter.