Figure 5.
Specific PrPc antibodies prevent soluble Aβ-induced Fyn activation in primary neurons. A, Quadruple-channel confocal immunofluorescence reveals that PrPc (labeled using the C20 antibody) and Aβ (labeled using the DW6 antiserum, a kind gift from Dr. Dominic Walsh) colocalize on neuronal dendritic spines (labeled using a PSD95 antibody) in 14-d-old primary cortical Tg2576 neurons. B, Quantification of colocalized voxels for each channel pair of interest. Representative 3D-rendered images used for voxel counts are shown in the left panel. Results for channel pairs are shown in the histogram. C, Triple-channel confocal imaging shows that Aβ42 species partly colocalize with pY416-Src and PrPc in Tg2576 primary neurons. D, Exogenous Aβ species purified from AD brain tissue colocalized with PrPc and Fyn at dendritic spines after application onto nontransgenic primary cortical neurons. E, F, Fyn activation induced by human oligomeric Aβ is partially blocked by the antibody 6D11 targeting the 95–105 domain of PrPc. Preincubation with the N terminus PrPc antibody 8B4 failed to inhibit Fyn phosphorylation. 6D11 (10 μg) and 8B4 (10 μg) were preincubated for 2 h onto cells before acute oAβ exposure (1 h at 37°C). 6D11 reduced Fyn activation in wild-type neurons acutely exposed to human-brain purified Aβ oligomers (E) and in Tg2576 neurons chronically exposed to low-n Aβ oligomers (F). Images were acquired using an oil-immersion 60 or 100× objective and processed with Imaris7.1 software. The arrowheads indicate examples of colocalization between Aβ/PrPc, while white asterisks correspond to colocalization between Aβ and PSD95 in absence of PrPc. Bars represent the mean ± SD (n > 3–6 dishes/experiment; independent experiments were performed in triplicate). *p < 0.05, ANOVA followed by Student's t test.