Functional Analysis of Mutants in the Transmembrane Region-1 and Octarepeat Region of Prion Protein

Abstract

The cellular prion protein (PrPPcP) is a glycosyl-phosphatidyl-inositol (GPI)-anchored 35 kDa glycoprotein located on the outer surface of the plasma membrane and plays an essential role in the pathogenesis of several inherited and transmissible neurodegenerative diseases such as Creutzfeldt-Jakob disease (CJD) and Gerstmann-Sträussler-Scheinker syndrome (GSSS). Despite being the subject of many recent studies, the physiological function of PrPPcP remains largely unresolved. Several candidate functions have been discussed, including binding and internalisation of copper or other metals, superoxide dismutase-like activity, signal transduction and regulation of cellular antioxidant activities. The transmembrane (TM1) region of PrPPcP (codons 110-135) should play a key role in PrPPcP function because of its high conservation throughout evolution. Moreover it contains an array of hydrophobic amino acids, and peptides derived from this region are neurotoxic.The aim of the present study was to try to elucidate a possible role of PrPPcP in the regulation mitochondrial membrane potential (), in the regulation of the basal level of endogenous reactive oxygen species (ROS) and in antioxidative defence. For this purpose transiently transfected mouse neuroblastoma cells overexpressing PrP, either as wild-type (wt) protein or as a deletion mutant lacking codons 114-121 (henceforth called Δ8TM1-PrP), were analysed. In addition a deletion mutant in the octarepeat region (octa-PrP) was studied. The results showed that wt-PrP and 8TM1-PrP have no impact on . Likewise, overexpression of PrP (wt or mutant) had no impact on endogenous ROS levels. However, under conditions of oxidative stress induced by H2O2 treatment of the cells, ROS levels were lower in cells transfected with wt-PrP or Δ8TM1-PrP expression plasmids.Increased phosphorylation of ERK2 (p42) and decreased phosphorylation of JNK1/JNK2/3 seemed to be linked to this protective effect of PrPc.Two other systems derived from transgenic Δ8TM1-PrP mouse brain (whole-brain or cerebellar granular neurones [CGN]) were also studied, but no biological impact of the transgene 8TM1-PrP was observable, most likely due to the low expression level.In conclusion, the protective effect of PrPPcP against oxidative stress implicates its octarepeat region but not its TM1 domain; it does not manifest as lowered basal ROS level; and it seems to involve the MAPK pathway, especially p42 and JNK1/JNK2/3.