Our goal has been to learn more about the pathogenesis of cell dysfunction in prion diseases using in vitro models. The recent observation on which this proposal is based is that plasma membrane fluidity of scrapie infected N2a cells, designated ScN2a, is 8 times less than in uninfected N2a cells. This correlated with a decreased affinity of bradykinin (Bk) receptors for its ligand. This change in the properties of the Bk receptor, in the absence of a decrease in the number of receptors, correlated with a 90% decrease in the IP3 response to Bk stimulation. Combined, these findings suggest a new hypothesis concerning the pathogenesis of cell dysfunction in prion diseases. Specifically, they suggest that the conversion of plasma membrane PrPC to PrPSc and the subsequent shunting of PrPSc into secondary lysosomes disrupts normal endosomal recycling and trafficking of PrPC and, perhaps, other membrane proteins and lipids and that this, in turn, results in an alteration of plasma membrane composition. This would account for the decreased responsiveness of Bk receptors and predicts that other plasma membrane signalling functions fail. While vacuolation is not a characteristic of ScN2a cells, it is a prominent feature in scrapie infected GT-1-trk9 cells (abbreviated Sc trk9). The Sc-trk9 cell line and its normal counterpart, designated trk9, are particularly relevant for studies of prion disease pathogenesis because they have the morphology of neurons (N2a and ScN2a cells are undifferentiated); because they express trkA; and because the manifestations of scrapie, including vacuolation and abnormal membrane collections appear to be reversed in Sc-trk9 cells in response to NGF. The overall strategy is to focus on the structure of the plasma membrane, trafficking of representative lipids and proteins, and endocytosis of selected membrane proteins in ScN2a and Sc-trk9 cells. Plasma membrane compositional analysis and measurements of trafficking/endocytosis parameters (destinations and amounts) will be assessed by a combination of biochemical/immunochemical measurements of cell fractions and qualitative and quantitative morphological measurements by immunogold electron microscopy and confocal/fluorescence microscopy. We believe these studies will be successful if we can demonstrate the following: (1) Plasma membrane fluidity and compositional abnormalities are present in both ScN2a and Sc-trk9 cells; (2) abnormalities of other relevant receptor mediated functions including transferrin receptor function, which is the """"""""gold standard"""""""" for endocytic recycling, and NGF signalling in the Sk-trk9/trk9 lines in addition to abnormalities of receptor mediated intracellular Ca2+ regulation; (3) abnormalities of trafficking of NGF and transferrin receptor as well as PrPC and selected lipids; and/or (4) we can modulate and/or ameliorate any or all of the abnormalities by treatment of Sc-trk9 cells of scrapie infected mice/hamsters with NGF.
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