Figure 1. Aged memory impairment is associated in mice with higher STEP levels and is attenuated by genetic reduction of STEP.

Aged (22-24 mo old) memory-impaired (AI; n=9), aged memory-unimpaired (AU; n=9), aged STEP knock-out (KO; n=9) and heterozygous (Het; n=9), and young (Y; n=34) adult (6 mo old) mice were tested in the Morris water maze (MWM) (three-way ANOVA). During the acquisition phase of the MWM task (3 trials/day; 60 s; 30 m 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). During the probe trial performed 24 h 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 Y^204/187, 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. * or + indicates significant differences compared to Y or AU mice, respectively (n=9 in each group, two independent experiments. All histograms are presented as mean values ± s.e.m. *^,+,$,&,# P<0.05, **^,++,$$,## P<0.01, ***^,+++,###,&&& P<0.001. See also Figures S1, S2 and S3.