Proximity of SCG10 and prion protein in membrane rafts

Abstract

The conversion of normal cellular prion protein (PrPC) into its pathogenic isoform (PrPSc) is an essential event in prion pathogenesis. In culture models, membrane rafts are suggested to play a critical role in PrPSc formation. To identify the candidate molecules capable of interacting with PrPC and facilitating PrPSc formation in membrane rafts, we applied a novel biochemical labeling method termed enzyme-mediated activation of radical sources. Enzyme-mediated activation of radical sources was applied to the Lubrol WX insoluble detergent-resistant membrane fractions from mouse neuroblastoma (N2a) cells in which the surface PrPC was labeled with HRP-conjugated anti-PrP antibody. Two-dimensional western blots of these preparations revealed biotinylated spots of approximately 20 kDa with an isoelectric point of 8.0-9.0. Liquid chromatography-tandem mass spectrometry analysis resulted in the identification of peptides containing SCG10, the neuron-specific microtubule regulator. Proximity of SCG10 and PrPC was confirmed using proximity ligation assay and co-immunoprecipitation assay. Transfection of persistently 22L prion-infected N2a cells with SCG10 small interfering RNA reduced SCG10 expression, but did not prevent PrPSc accumulation, indicating that SCG10 appears to be unrelated to PrPSc formation of 22L prion. Immunofluorescence and western blot analyses showed reduced levels of SCG10 in the hippocampus of prion-infected mice, suggesting a possible association between SCG10 levels and the prion neuropathogenesis. By applying a novel biochemical labeling method against detergent-resistant membrane fractions from mouse neuroblastoma cells, the neuron-specific microtubule-destabilization protein, SCG10 was identified as a novel candidate that is proximate to normal prion protein (PrP) in membrane rafts. SCG10 seemed unrelated to disease-related PrP formation under certain conditions, while there is a possible association between SCG10 levels and prion neuropathogenesis. Cover Image for this issue: doi: 10.1111/jnc.13310.

Keywords: N2a cell; SCG10; detergent-resistant membrane; enzyme-mediated activation of radical sources; membrane raft; prion protein.