Aged (22–24 months old) memory-impaired (AI; n = 9), aged memory-unimpaired (AU; n = 9), aged STEP knockout (KO; n = 9) and heterozygous (Het; n = 9), and young (Y; n = 34) adult (6 months old) mice were tested in the Morris water maze (MWM) (three-way ANOVA).

(A and B) During the acquisition phase of the MWM task (3 trials/day; 60 s; 30 min inter-trial interval), escape latency (A) and distance swam (B) before finding the hidden platform was longer for AI than AU and Y animals. ∗ or + indicates significant differences compared to Y or AU mice, respectively. Performance of STEP KO mice were similar to Y mice, and lower than AI mice on days 3 and 4 ($p < 0.05, $$p < 0.01). STEP Het mice had longer latency than Y animal on day 4 (&p < 0.05) but performed better than AI mice on days 3 and 4 (#p < 0.05).

(C) Swim speed was not significantly (n.s.) different between all aged groups but was decreased compared to Y mice (∗p < 0.05, two-way ANOVA, post hoc Tukey’s HSD test).

(D and E) During the probe trial performed 24 hr after the last acquisition day, the AI mice crossed the platform location less frequently than all the other groups (two-way ANOVA, Tukey’s HSD test) (D) and showed no preference for the target quadrant compared to the opposite one (three-way ANOVA) (E). # represents significant variations compared to AI for the target quadrant, and & indicates time differences for the target and opposite quadrants within each group.

(F) During the Y-maze, all groups, except the AI mice, spent more time in the new available arm than in the other arm previously explored during the training phase (three-way ANOVA).

(G) Representative immunoblots of non-phosphorylated STEP (non-pSTEP) at S221, STEP, pGluN2B Y1472, GluN2B, PSD-95, GAPDH, pERK1/2 Y204/187, ERK1/2 in synaptosomal (P2) fractions prepared from the dorsal hippocampus. The levels of pCREB S133 and CREB were measured in the nuclear fraction and BDNF levels in the cytosolic fraction.

(H) Densitometric quantification of changes expressed as the mean ratio of proteins (n = 9 in each group, two independent experiments). ∗ or + indicates significant variations compared to Y or AI mice, respectively.

(I) Representative immunoblots of polyubiquitinated STEP isolated from the P2 hippocampal fraction and probed with an anti-STEP antibody.

(J) The phosphatase activity of STEP immunoprecipitated from the P2 hippocampal fraction was assayed by using Fyn-phosphorylated GST-GluN2B as substrate. Dephosphorylation of GluN2B Y1472 was normalized to the initial level of Fyn-phosphorylated GST-GluN2B (input) not treated with immunoprecipitated STEP.