Recognition test of a displaced object 24 hours after Duvelisib (R enantiomer) training (B), shown as a ratio of time spent with the displaced object over all objects, show no difference in group preferences for the displaced object (F3,30 = 2.216). Test for recognition of a novel object shows differing group preferences for the novel object relative to the pre-existing objects (C), shown as a discrimination ratio for the novel object (F3,30 = 3.316). p < 0.05, p < 0.01, p < 0.001. Shown as mean s.e.m.Fig 4. Measure of protein phosphatases' activity following 6 weeks of respective housing conditions. (A) Absolute quantitation of free phosphates released in whole hippocampal fractions due to PP1 and PP2A activities (n = 6, 6, 6, 6 for Aged EE (solid red), Aged SH (red stripes), Young EE (solid blue), Young SH (blue stripes), respectively; F3,20 = 6.591; < 0.05 relative to both aged EE and young EE). (B) Free phosphates released due to calcineurin activity (F3,20 = 4.274; p < 0.05 relative to aged EE). p < 0.05, p < 0.01, p < 0.001. Shown as mean s.e.m.Similarly, calcineurin activity was significantly reduced in aged EE (2.00 0.26 nmol) compared to aged SH (2.85 0.16 nmol; p < 0.05) mice, but there was no difference in young (ANOVA, effect of `group', p < 0.05; Fig 4B). This data suggests that EE can reduce dysregulated PPs levels in the brains of aged animals, perhaps as effectively as transgenic inhibition of PPs or even pharmaceutical intervention [14,15].To assess the functional implications of increased PP activity with age, we performed iTRAQ quantification of trypsin-digested peptides from hippocampal nuclear protein extracts of nae aged and nae young mice. We identified 521 total peptides in aged and 1125 peptides in young with a confidence interval (C.I.) > 95% and significance at p < 0.05 (S1 Table), corresponding to 213 and 328 proteins, respectively. Of those, we identified, in total, 163 phosphorylated peptides (phosphopeptides) in aged and 197 phosphopeptides in young, with 94 phosphopeptides present in both groups (Fig 5A). This corresponded to 91 unique phosphorylated proteins in aged and 106 in young, with 45 present in both groups (Fig 5A).9542727 Overall, we identified fewer total peptides, fewer phosphopeptides, and fewer corresponding phosphorylated proteins in aged than in young mice. We then quantified the relative abundance of phosphopeptides common to both groups. From the 1646 total peptides identified in both aged and young (S1 Table), 233 unique phosphopeptides were identified (S2 Table). Of the 94 phosphopeptides identified in both groups, 67 were found to be unique and we were able to quantify the relative abundance of 56 phosphopeptides in aged relative to young (S3 Table). These 56 unique phosphopeptides correspond to 36 unique proteins present in both aged and young (Fig 5B). We performed gene ontology analysis on the 
71 unique phosphopeptides identified in aged but not in young (Table 1 and S4 Table), and on the 95 remaining phosphopeptides identified in young but not in aged (Table 1 and S5 Table).