Figure 1.
Cellular prion protein (PrPc) enables amyloid-β (Aβ) endocytosis and transcytosis across the blood–brain barrier. (A, B) [125I]-Aβ1−40 transcytosis across primary mouse brain capillary endothelial cells (pMBCECs) from PrPc wild-type (WT) and knockout (KO) mice was investigated in the presence of [14C]-inulin, a marker for paracellular diffusion, to determine the transcytosis quotient (TQ). Mouse monoclonal anti-PrPc antibody (Ab51.2) decreases the [125I]-Aβ1−40 TQ in the brain to blood (A) and blood to brain (B) direction to levels observed in PrPc KO pMBCECs. Simultaneous incubation of Ab51.2 and receptor-associated protein (RAP), a ligand binding inhibitor for lipoprotein receptors, did not further decrease Aβ transcytosis below pMBCECs treated with Ab51.2 alone or PrPc KO monolayers. Results represent mean+s.e.m. of three independent experiments in triplicate. *Statistically significant difference compared with control (P<0.05; one-way analysis of variance), n.s., not significant. (C) PrPc expression was analyzed in pMBCECs by immunoblotting. Brain homogenates and low density lipoprotein receptor-related protein 1 (LRP1)-deficient mouse embryonic fibroblast (MEF) cell lysate were included as controls. Mouse monoclonal anti-PrPc antibody (W226) detects strong expression levels in pMBCECs derived from WT animals, whereas LRP1-specific rabbit polyclonal 1704 antibody does not show any differences in LRP1 expression between both genotypes. Actin was used as loading control. (D) LRP1 was immunoprecipitated from pMBCECs and LRP1-deficient Chinese hamster ovary 13-5-1 cells transiently transfected with PrPc using 1704 antibody. Immunoblotting with W226 antibody shows that PrPc coimmunoprecipitates with LRP1 in pMBCECs. Tubulin was used as loading control, antibody light chain (LC). (E) Increasing concentrations of immobilized recombinant PrPc (rPrPc) were incubated with monomeric [125I]-Aβ1−40. Results represent mean±s.e.m. of three independent experiments in duplicate.