Figure 2.

Signaling pathways activated by TTR are megalin-dependent. TTR signaling activity was assessed by analyzing the phosphorylation levels of ERK (a, n=3–14), SRC (Tyr416) (b, n=6–9), Akt (Ser473) (c, n=3–9) and CREB (Ser133) (d, n=3–14) following stimulation of TTR KO cultured hippocampal neurons (7 DIV) with recombinant mouse TTR (55 μg/ml) for different time points. The results are the average±S.E.M. of 3–14 independent experiments. (e) FRET image and pseudocolor ratio images (FRET/Donor channels) of a representative TTR KO cultured hippocampal neuron expressing yellow Cameleon-Nano (YC-Nano15) stimulated with mouse TTR (55 μg/ml). Scale bar represents 20 μm and the FRET/Donor images were coded according to the indicated pseudocolor scale. (f) Time course of normalized FRET/Donor values in cell body of YC-Nano15-transfected TTR KO cultured hippocampal neurons (7 DIV) stimulated with two concentrations of mouse TTR (55 and 300 μg/ml), (two to three independent experiments, four to six neurons in each experiment). TTR administration was carried out at the '0 s' (line with dashes in the graph), but first time analyzed after stimulation is '0.33 s'. To address the role of megalin in TTR signaling activity, we used RAP, an LRP inhibitor and analyzed the phosphorylation levels of ERK (g, n=4–15), Akt (Ser473) (h, n=5–10) and CREB (Ser133) (I, n=3–12) following stimulation of TTR KO cultured hippocampal neurons (7 DIV) with recombinant mouse TTR (55 μg/ml) in the presence or absence of RAP (350 μg/ml, 30 min pre-incubation). The results are the average±S.E.M. of 3–15 independent experiments. TTR signaling activity was also addressed in megalin heterozygous TTR KO cultured hippocampal neurons (7 DIV) by analysis of the phosphorylation levels of ERK (j, n=3), Src (Tyr416) (l, n=4), Akt (Ser473) (m, n=3) and CREB (Ser133) (n, n=3) following stimulation with recombinant mouse TTR (55 μg/ml) for 30 min. The results are the average±S.E.M. of three to four independent experiments. (o) Time course of normalized FRET/Donor values in cell body of YC-Nano15-transfected TTR KO and megalin (+/−) TTR KO cultured hippocampal neurons stimulated with mouse TTR (55 μg/ml) (three independent experiments, five neurons in each experiment). (p) Same experimental design as in (o), but megalin (+/+) TTR KO littermate cultures were used and challenged with two concentrations of mouse TTR (55 and 300 μg/ml) (one independent experiment for each, five neurons in each experiment). Statistical analysis was performed using one-way ANOVA followed by Bonferroni's multiple comparison test performed for each condition or Student's unpaired t-test for two-groups only comparisons. ***P<0.001, **P<0.01, *P<0.05, n.s., not significant as compared with the control or as indicated