Antiprion properties of prion protein-derived cell-penetrating peptides.

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.

Involvement of glypican-1 autoprocessing in scrapie infection.

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.

Cell culture models to unravel prion protein function and aberrancies in prion diseases.

From an early stage of prion research, tissue cultures that could support and propagate the scrapie agent were sought after. The earliest attempts were explants from brains of infected mice, and their growth and morphological characteristics were compared with those from uninfected mice. Using the explant technique, several investigators reported increased cell growth in cultures established from scrapie-sick brain compared with cultures from normal mice. These are odd findings in the light of the massive neuronal cell death known to occur in scrapie-infected brains; however, the cell types responsible for the increased cell growth in the scrapie-explants most probably were not neuronal. The first successful cell culture established in this way, in which the scrapie agent was serially and continuously passaged beyond the initial explant, was in the scrapie mouse brain culture, which is still used today. This chapter describes the generation and use of chronically prion-infected cell lines as cell culture models of prion diseases. These cell lines have been crucial for the current understanding of the cell biology of both the normal (PrP(C)) and the pathogenic isoform (PrP(Sc)) of the prion protein. They also have been useful in the development of antiprion drugs, prospectively used for therapy of prion diseases, and they offer an alternative approach for transmission/infectivity assays normally performed by mouse bioassay. Cell culture models also have been used to study prion-induced cytopathological changes, which could explain the typical spongiform neurodegeneration in prion diseases.

Increased Src kinase level results in increased protein tyrosine phosphorylation in scrapie-infected neuronal cell lines.

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.

Changed iron regulation in scrapie-infected neuroblastoma cells.

Prion diseases are characterized by the conversion of the normal cellular prion protein PrP(C) into a pathogenic isoform, PrP(Sc). The mechanisms involved in neuronal cell death in prion diseases are largely unknown, but accumulating evidence has demonstrated oxidative impairment along with metal imbalances in scrapie-infected brains. In this study, we report changes in cellular iron metabolism in scrapie-infected mouse neuroblastoma N2a cells (ScN2a). We detected twofold lower total cellular iron and calcein-chelatable cytosolic labile iron pool (LIP) in ScN2a cells as compared to the N2a cells. We also measured in ScN2a cells significantly lower activities of iron regulatory proteins 1 and 2 (IRP1 and IRP2, respectively), regulators of cellular iron by sensing cytosolic free iron levels and controlling posttranscriptionally the expression of the major iron transport protein transferrin receptor 1 (TfR1) and the iron sequestration protein ferritin. IRP1 and IRP2 protein levels were decreased by 40% and 50%, respectively, in ScN2a cells. TfR1 protein levels were fourfold reduced and ferritin levels were threefold reduced in ScN2a cells. TfR1 and ferritin mRNA levels were significantly reduced in ScN2a cells. ScN2a cells responded normally to iron and iron chelator treatment with respect to the activities of IRP1 and IRP2, and biosynthesis of TfR1 and ferritin. However, the activities of IRP1 and IRP2, and protein levels of TfR1 and ferritin, were still significantly lower in iron-depleted ScN2a cells as compared to the N2a cells, suggesting lower need for iron in ScN2a cells. Our results demonstrate that scrapie infection leads to changes in cellular iron metabolism, affecting both total cellular and cytosolic free iron, and the activities and expression of major regulators of cellular iron homeostasis.

Increased susceptibility to oxidative stress in scrapie-infected neuroblastoma cells is associated with intracellular iron status.

The molecular mechanism of neurodegeneration in prion diseases remains largely uncertain, but one of the features of infected cells is higher sensitivity to induced oxidative stress. In this study, we have investigated the role of iron in hydrogen peroxide (H(2)O(2))-induced toxicity in scrapie-infected mouse neuroblastoma N2a (ScN 2 a) cells. ScN 2 a cells were significantly more susceptible to H(2)O(2) toxicity than N2a cells as revealed by cell viability (MTT) assay. After 2h exposure, significant decrease in cell viability in ScN 2 a cells was observed at low concentrations of extracellular H(2)O(2) (5-10 microM), whereas N2a cells were not affected. The increased H(2)O(2) toxicity in ScN 2 a cells may be related to intracellular iron status since ferrous iron (Fe(2+)) chelator 2,2'-bipyridyl (BIP) prevented H(2)O(2)-induced decrease in cell viability. Further, the level of calcein-sensitive labile iron pool (LIP) was significantly increased in ScN 2 a cells after H(2)O(2) treatment. Finally, the production of reactive oxygen species (ROS) was inhibited by 30% by iron chelators desferrioxamine (DFO) and BIP in ScN 2 a cells, whereas no significant effect of iron chelators on basal ROS production was observed in N2a cells. This study indicates that cellular resistance to oxidative stress in ScN 2 a cells is associated with intracellular status of reactive iron.

Increased levels of insulin and insulin-like growth factor-1 hybrid receptors and decreased glycosylation of the insulin receptor alpha- and beta-subunits in scrapie-infected neuroblastoma N2a cells.

We have previously shown that ScN2a cells (scrapie-infected neuroblastoma N2a cells) express 2-fold- and 4-fold-increased levels of IR (insulin receptor) and IGF-1R (insulin-like growth factor-1 receptor) respectively. In addition, the IR alpha- and beta-subunits are aberrantly processed, with apparent molecular masses of 128 and 85 kDa respectively, as compared with 136 and 95 kDa in uninfected N2a cells. Despite the 2-fold increase in IR protein, the number of (125)I-insulin-binding sites was slightly decreased in ScN2a cells [Ostlund, Lindegren, Pettersson and Bedecs (2001) Brain Res. 97, 161-170]. In order to determine the cellular localization of IR in ScN2a cells, surface biotinylation was performed, showing a correct IR trafficking and localization to the cell surface. The present study shows for the first time that neuroblastoma N2a cells express significant levels of IR-IGF-1R hybrid receptors, and in ScN2a cells the number of hybrid receptors was 2-fold higher than that found in N2a cells, potentially explaining the apparent loss of insulin-binding sites due to a lower affinity for insulin compared with the homotypic IR. Furthermore, the decreased molecular mass of IR subunits in ScN2a cells is not caused by altered phosphorylation or proteolytic processing, but rather by altered glycosylation. Enzymic deglycosylation of immunoprecipitated IR from N2a and ScN2a cells with endoglycosidase H, peptide N-glycosidase F and neuraminidase all resulted in subunits with increased electrophoretic mobility; however, the 8-10 kDa shift remained. Combined enzymic or chemical deglycosylation using anhydrous trifluoromethane sulphonic acid treatment ultimately showed that the IR alpha- and beta-subunits from ScN2a cells are aberrantly glycosylated. The increased formation of IR-IGF-1R hybrids in ScN2a cells may be part of a neuroprotective response to prion infection. The degree and functional significance of aberrantly glycosylated proteins in ScN2a cells remain to be determined.

Loss of lipopolysaccharide-induced nitric oxide production and inducible nitric oxide synthase expression in scrapie-infected N2a cells.

In scrapie-infected cells, the conversion of the cellular prion protein to the pathogenic prion has been shown to occur in lipid rafts, which are suggested to function as signal transduction platforms. Neuronal cells may respond to bacterial lipopolysaccharide (LPS) treatment with a sustained and elevated nitric oxide (NO) release. Because prions and the major LPS receptor CD14 are colocalized in lipid rafts, the LPS-induced NO production in scrapie-infected neuroblastoma cells was studied. This study shows that LPS induces a dose- and time-dependent increase in NO release in the murine neuroblastoma cell line N2a, with a 50-fold increase in NO production at 1 microg/ml LPS after 96 hr, as measured by nitrite in the medium. This massive NO release was not caused by activation of the neuronal NO synthase (nNOS), but by increased expression of the inducible NOS (iNOS) mRNA and protein. However, in scrapie-infected N2a cells (ScN2a), the LPS-induced NO production was completely abolished. The absence of LPS-induced NO production in ScN2a was due not to abolished enzymatic activity of iNOS but to a complete inhibition of the LPS-induced iNOS gene expression as measured by Western blot and RT-PCR. These results indicate that scrapie infection inhibits the LPS-mediated signal transduction upstream of the transcriptional step in the signaling cascade and may reflect the important molecular and cellular changes induced by scrapie infection.