Figure 1.

Soluble Aβ_1–42 oligomers abrogate the PrP-dependent control over Fyn in PrP-expressing primary cortical neurons. Both the representative western blot (WB) obtained after probing PrP-expressing and PrP-KO cortical neurons (treated (+), or not (−), with Aβ_1–42 oligomers) with antibody to active (auto-phosphorylated, p) SFK or total Fyn (upper panel), and the densitometric analysis of the pSFK immuno-reactive bands normalized to the corresponding signal of total Fyn (lower panel), show that Aβ addition significantly relieves the restriction on Fyn activation observed in untreated PrP-expressing controls relative to PrP-KO counterparts (cntr, grey and black bars, respectively). Conversely, no impact of Aβ was observed in PrP-KO neurons (see text for the explanation of the higher Fyn activation in untreated PrP-KO neurons). After seven days in culture, primary cortical neurons (isolated from PrP-expressing and PrP-KO mice lines Tg46 and F10, respectively [57] (Authorization n. 13/2012), and cultured as previously described [46]), were incubated (1 h, 37 °C), or not, with Aβ_1–42 oligomers (5 µM, prepared and oligomerized as in [45]), and then lyzed using an ice-cold buffer containing glycerol (10% (w/v)), SDS (2% (w/v)), Tris/HCl (62.5 mM, pH 6.8), urea (1.8 M), Na₃VO₄ (5 mM), and cocktails of protease and phosphatase inhibitors (Roche). Total protein quantification, WB and densitometric analyses were accomplished as described elsewhere [45]. Here and after, values are expressed as mean ± SEM, and the number of replicates (i.e., different replicates from at least four primary cortical cultures prepared on different days) is indicated inside each bar diagram. * p < 0.05, ** p < 0.01 (Student’s t-test).