FIG. 4.

Increased oxidation of Akt1 and its increased affinity for PP2A are observed in synaptosomes of APP/PS1 mice. Aβ42 levels in cortical homogenates from different ages of WT and APP/PS1 mice measured using ELISA is depicted for ADL (1–1.5 months old) and YA (3–4 months old) mice (A). Immunoblotting provides evidence for increased accumulation of oligomeric Aβ in synaptosomes from ADL and YA APP/PS1 mice (B). Immunoreactive signals for PSD95 were used to normalize the blots. The leftmost panel with the synthetic Aβ standard is shown as a reference. Treatment of synaptosomes isolated from cortices of ADL and YA APP/PS1 mice with DCFH-DA leads to increased generation of DCF fluorescence when compared with that from age-matched controls (C). See Supplementary Fig. S4 also. Primary cortical neurons were treated with DCFH-DA and the resultant DCF fluorescence (green) was observed along with immunostaining for Homer1 (red; D). Increased colocalization of DCF with Homer1 is observed, indicating extensive ROS levels in neurites and terminals of APP/PS1 cortical neurons, whereas WT cortical neurons show very little staining for ROS (n = 7–11independent cultures). Scale bar is 20 μm. See Supplementary Fig. S5. Reduced Akt1 levels assayed as AMS derivatized protein were decreased in synaptosomes isolated from ADL (1 month old) APP/PS1 mice when compared with those from age-matched controls (E). Immunoprecipitated Akt1 from synaptosomes of 1 month (F) and 9 months (G) old APP/PS1 mice had increased affinity for the phosphatase, PP2A. Values are mean ± SEM (n = 4 mice) and * denotes values significantly different from corresponding controls (p < 0.05). ** denotes values significantly different from corresponding controls (p < 0.01).