Figure 3.
Mechanism of IGF-I-induced transport of Aβ1-40 through choroid plexus cells. A, IGF-I does not modulate the association of Aβ with megalin. The amount of Aβ complexed with megalin did not change after exposure of rat choroid plexus cultures to IGF-I. Cells were immunoprecipitated with anti-Aβ and blotted with anti-megalin. B, Megalin in rat choroid plexus cells is constitutively associated with caveolin-1. Megalin coimmunoprecipitated and colocalized by immunocytochemical confocal analysis with caveolin-1. Scale bars, 10 μm. C, Megalin-mediated transport of Aβ across rat choroid plexus cells involves association of the Aβ/carrier complex with caveolin. Top blots, Caveolin-1 coimmunoprecipitates with exogenous Aβ in choroid plexus cultures. No association is seen in untreated cultures (Control). Middle blots, The amount of caveolin-1 present in the membrane (Mb) of choroid plexus cells is increased after addition of Aβ, whereas the cytoplasmic fraction (Cy) is not modified. Bottom blots, In cultured choroid plexus cells, caveolin-1 associates with albumin, an Aβ carrier (Carro et al., 2002), as determined by coimmunoprecipitation. Dig-Alb, Digoxigenin-labeled albumin. Right micrographs, Peri-membrane localization of caveolin-1 staining is apparent in control rat cultures. After adding Aβ, caveolin-1 colocalizes with it. Scalebars, 10 μm. D, IGF-I increases the amount of Aβ associated with caveolin-1. E, Megalin coimmunoprecipitates with Dab2 in choroid plexus epithelial cell cultures. Reciprocal coimmunoprecipation confirmed association of Dab2 with megalin. F, IGF-I induces transient Ser-dephosphorylation of Dab2 in rat choroid plexus cells. Total Dab2 levels remained unchanged. p, Phosphorylated. G, IGF-I induces transient decoupling of Dab2 and megalin. Note that the time course coincides with dephosphorylation of Dab2 by IGF-I. Total Dab2 levels remain constant. H, IGF-I-induced dephosphorylation of Dab2 is blocked by bisindolylamine (BIS), a PKC inhibitor, whereas wortmannin (Wort; which inhibits PI3K) or PD98059 (PD; which blocks MAPK activation) did not affect IGF-I action. I, Blockade of PKC activity with bisindolylamine (BIS) abrogates IGF-I-induced translocation of Aβ across the epithelial monolayer. Representative blots are shown throughout. A minimum of four experiments were performed for each condition shown in this figure. IP, Immunoprecipitation.