*3.5. Memory Decline in Middle-Aged APPKINL-G-F Mice Is Accompanied by the Activation of Specific Ser Sites on IRS1 and Energy Depletion*

Next, we examined age-related alterations in the patterns of phosphorylation of IRS1 at Ser sites and its downstream components in the hippocampi of middle-aged APPKINL-G-F mice at 34–36 weeks of age. Consistently, body weight, blood glucose level, and plasma insulin concentration were comparable between middle-aged WT and APPKINL-G-F mice (Figure 5A, Figure S2A).

Although the onset of memory deficits was reported by 6 months in APPKINL-G-F mice [19], 6-month-old APPKINL-G-F mice did not display memory decline under our experimental conditions (data not shown). However, the water T-maze test demonstrated that middle-aged APPKINL-G-F mice exhibited memory decline after 34 weeks under our experimental conditions (Figure 5B). Meanwhile, both human/rat/mouse Aβ42 and human Aβ42 sandwich ELISA demonstrated increased levels of T-PER-extractable Aβ42 in the hippocampi of middle-aged APPKINL-G-F mice (right in Figure 5C, D) and young APPKINL-G-F mice, which were comparable to TBS-extractable Aβ42 levels in the brain and cortex of APPKINL-G-F mice [33].

While an age-related increase in the phosphorylation of IRS1 at mSer612 and mSer632/635 accompanied by sustained phosphorylation of IRS1 at T2DM-related mSer1097 site and of AMPK was observed in the hippocampi of middle-aged APPKINL-G-F mice showing memory decline (Figure 5E, Figure S4I, Table S1), there were no significant differences in phosphorylation levels at mSer307 site and of p70S6K between middle-aged WT and APPKINL-G-F mice (Figure 5E,F) owing to the age-related elevation of these factors (Figure 3D,E). Regardless of age, the activity of downstream components, such as Akt, GSK3β, aPKC ζ/λ, and JNKs, remained almost unchanged in the hippocampi of APPKINL-G-F mice (Figure 5F, Figure S3E, S4J and S5E). Our data suggest that the increased phosphorylation of hippocampal IRS1 at multiple Ser residues and persistent AMPK activation that corresponds to energy depletion accompanied by age-related elevation of Aβ42 levels are associated with the onset of memory decline in these mice.
