Fig. 5
From: Structural and mechanistic aspects influencing the ADAM10-mediated shedding of the prion protein
Shedding plays a key role in PrPC homeostasis of a cell. a Representative western blot of N2a cells treated with the ADAM10 inhibitor GI254023X (GI; or DMSO as control). No sPrP is detected upon GI treatment yet re-probing the blot with POM2 Ab indicates release of PrP by other routes. b Quantification of relative amounts and (c) average size of exosomes released by N2a cells upon treatment with GI. Nanoparticle tracking analysis shows a significant increase in exosomal release (n = 4; p = 0.011) while the size of exosomes is not altered (n = 4). d After GI or DMSO treatment, cell lysates (Lys) and normalized amounts of exosomes (Exo) were analysed by western blot. Note that only mature ADAM10 is sorted into exosomes. Quantifications of relative exosomal PrPC load (e; n = 3; p = 0.0006) and PrPC levels in cell lysates (f; n = 3; p = 0.019) upon GI (or DMSO) treatment. PrPC signals were referred to flotillin signal for quantification. g Representative western blot of N2a cells treated with lysosomal inhibitor Leupeptin (Leupt.) or left untreated (untr.) showing levels of sAPPα, sPrP and released PrP in media supernatants as well as cell-associated fl-PrP and cytosolic proteins β-catenin, β-tubulin and β-actin in lysates. Quantification shows no alterations of relative PrP levels in lysates (h; n = 3) yet a significant increase in PrP shedding (i; n = 3; p = 0.018) caused by lysosomal inhibition. j Western blot analysis of brain homogenates of sortillin-1-deficient (Sort1 KO) and control mice (Sort1 WT). Sort1 KO mice present with elevated levels of both sPrP and fl-PrP. Quantification of relative levels of sPrP (referred to actin) is shown (n = 4; p = 0.0103). k Schematic drawing summarizing three compensatory arms in the regulation of cellular PrP levels: ADAM10-mediated shedding (orange arrow), exosomal release (green arrow) and lysosomal targeting and degradation (blue arrow). Scheme makes no claim to completeness as other important factors regulating PrPC levels, such as transcriptional/translational control, are not depicted here