The prion diseases occur following the conversion of the cellular prion protein (PrP C ) into disease-related isoforms (PrP Sc ). In this study, the role of the glycosylphosphatidylinositol (GPI) anchor attached to PrP C in prion formation was examined using a cell painting technique. PrP Sc formation in two prion-infected neuronal cell lines (ScGT1 and ScN2a cells) and in scrapie-infected primary cortical neurons was increased following the introduction of PrP C . In contrast, PrP C containing a GPI anchor from which the sialic acid had been removed (desialylated PrP C ) was not converted to PrP Sc . Furthermore, the presence of desialylated PrP C inhibited the production of PrP Sc within prioninfected cortical neurons and ScGT1 and ScN2a cells. The membrane rafts surrounding desialylated PrP C contained greater amounts of sialylated gangliosides and cholesterol than membrane rafts surrounding PrP C . Desialylated PrP C was less sensitive to cholesterol depletion than PrP C and was not released from cells by treatment with glimepiride. The presence of desialylated PrP C in neurons caused the dissociation of cytoplasmic phospholipase A 2 from PrP-containing membrane rafts and reduced the activation of cytoplasmic phospholipase A 2 . These findings show that the sialic acid moiety of the GPI attached to PrP C modifies local membrane microenvironments that are important in PrP-mediated cell signaling and PrP Sc formation. These results suggest that pharmacological modification of GPI glycosylation might constitute a novel therapeutic approach to prion diseases.

No abstract

The Arrhenius plot of the rate of V79 Chinese hamster cell inactivation due to hypothermia has a "break" around 7-10 degrees C with optimum storage temperature for unprotected cells being about 10 degrees C. Addition of the membrane lipid perturber, butylated hydroxytoluene, improves survival of cells when compared to controls at temperatures below this break but not above. Arrhenius plots of growth rates of the cells show breaks at 30 and 40 degrees C. Measurements of membrane fluidity by electron spin resonance or membrane polarization anisotropy by fluorescence spectrophotometry techniques as a function of temperature in these cells also reveal "breaks" centered around 8 and 30 degrees C. Hence, the changes in the rate of cell inactivation and growth as a function of temperature may be related to membrane lipid phase changes.

Although the cellular prion protein (PrP C ) is concentrated at synapses, the factors that target PrP C to synapses are not understood. Here we demonstrate that exogenous PrP C was rapidly targeted to synapses in recipient neurons derived from Prnp knock-out (0/0) mice. The targeting of PrP C to synapses was dependent upon both neuronal cholesterol concentrations and the lipid and glycan composition of its glycosylphosphatidylinositol (GPI) anchor. Thus, the removal of either an acyl chain or sialic acid from the GPI anchor reduced the targeting of PrP C to synapses. Isolated GPIs (derived from PrP C ) were also targeted to synapses, as was IgG conjugated to these GPIs. The removal of sialic acid from GPIs prevented the targeting of either the isolated GPIs or the IgG-GPI conjugate to synapses. Competition studies showed that pretreatment with sialylated GPIs prevented the targeting of PrP C to synapses. These results are consistent with the hypothesis that the sialylated GPI anchor attached to PrP C acts as a synapse homing signal.

No abstract