*3.4. Increased Phosphorylation of Hippocampal IRS1 at Ser Residues in Young Amyloid Precursor Protein (APP) Knock-In (APP KINL-G-F) Mice Occurs Prior to the Onset of Memory Decline*

To investigate whether AD-related activation of Ser residues on neural IRS1 emerges before or after the onset of memory decline, we employed a novel AD mouse model, APPKINL-G-F mice carrying a humanized Aβ sequence and three AD mutations, i.e., Swedish, Beyreuther/Iberian, and Arctic mutations, in the endogenous *App* gene [19]. First, we measured body weight and glucose metabolism in 12-week-old (young) APPKINL-G-F mice. There were no differences in terms of body weight, blood glucose level, and plasma insulin concentration between WT and APPKINL-G-F mice at 12 weeks of age (Figure 4A, Figure S2B). Next, we confirmed memory function as well as Aβ40 and Aβ42 levels in the hippocampi of young APPKINL-G-F mice. At 12 weeks of age, APPKINL-G-F mice exhibited normal memory function (Figure 4B).

Using monoclonal antibody (BNT77/BC05)-based sandwich ELISA for human/rat/mouse Aβ42, we successfully confirmed that T-PER-extractable Aβ42 level had already increased in young APP KINL-G-F mice (Figure 4C, right). A conspicuous increase in T-PER-extractable Aβ42 level in these mice was detected by human Aβ42 sandwich ELISA using monoclonal antibodies (BAN50/BC05) (Figure 4D, right). However, T-PER-extractable Aβ42 levels in both ELISA were comparable to the range of Tris-buffered saline (TBS)-extractable Aβ42 level in the brain and cortex of APPKINL-G-F mice reported by Saito et al. (2019) and Saito et al. (2014). Similarly, human/rat/mouse Aβ40 and human Aβ40 sandwich ELISA revealed the same range for Aβ40 level in the T-PER fractions of the hippocampi of young APPKINL-G-F mice (black bar on the left in Figure 4C,D and Figure 5C,D). Meanwhile, regardless of age, human/rat/mouse Aβ40 or human Aβ40 sandwich ELISA showed almost the same levels (white bar on the left in Figures 1D, 2C, 3C, 4C and 5C) or range (white bar on the left in Figures 4D and 5D) of T-PER-extractable Aβ40 in WT mice, respectively.

**Figure 4.** Activation of multiple Ser residues on hippocampal IRS1 unrelated to memory decline in young amyloid precursor protein (APP) knock-in (APPKINL-G-F) mice. (**A**) The graphs of body weight and blood glucose levels in young wild-type (WT) and APPKINL-G-F mice (12 weeks of age, n = 6 mice per group). (**B**) Evaluation of learning memory function in young WT (n = 5 mice per group) and APP KINL-G-F mice (n = 5 mice per group) using the water T-maze test. (**C**) Quantitative analysis of T-PER-extractable Aβ40 and Aβ42 levels in the hippocampi of young WT and APPKINL-G-F mice using the human/rat/mouse β amyloid (1–40 and 1–42) ELISA (12 weeks of age, n = 5 biologically independent samples per group). (**D**) Quantitative analysis of T-PER-extractable Aβ40 and Aβ42 levels in the hippocampi of young WT and APPKINL-G-F mice using the human β amyloid (1–40 and 1–42) ELISA (12 weeks of age, n = 5 biologically independent samples per group). (**E**) Western blot analysis of phosphorylated insulin receptor substrates 1 mouse Ser307 [p-IRS1 (mSer307)], p-IRS1 (mSer612), p-IRS1 (mSer632/635), p-IRS1 (mSer1097), IRS1, and ß-tubulin in the hippocampi of young WT and APP KINL-G-F mice (12 weeks of age, n = 6 biologically independent samples per group). Arrows indicate the p-IRS1 mSer612-corresponding band (lower band) and p-IRS1 mSer632/635-corresponding band (lower band) in (**E**). Quantitative analysis of the phosphorylation of IRS1 at mSer307, mSer612, mSer632/635, and mSer1097 normalized to the respective total protein contents. (**F**)Western blot analysis of phosphorylation levels of Akt Ser473, p70S6K Thr389, AMPK Thr172, and GSK3β Ser9 as well as total protein levels of Akt, p70S6K, AMPK, GSK3β, and ß-tubulin in the hippocampi of young WT and APPKINL-G-F mice (12 weeks of age, n = 6 biologically independent samples per group). Quantitative analysis of phosphorylation of Akt Ser473, p70S6K Thr389, AMPK Thr172, and GSK3β Ser9 normalized to the respective total protein contents. Results are presented as mean ± SEM, \* *p* < 0.05; \*\* *p* < 0.01.

**Figure 5.** Memory decline accompanied by sustained phosphorylation of IRS1 Ser residues in the hippocampus of middle-aged APPKINL-G-F mice. (**A**) The graphs of body weight and blood glucose levels in middle-aged wild-type (WT) and APPKINL-G-F mice (34–36 weeks of age, n = 5 mice per group). (**B**) Evaluation of learning memory function in middle-aged WT (n = 14) and APPKINL-G-F mice (n = 15 mice per group) using the water T-maze test. (**C**) Quantitative analysis of T-PER-extractable Aβ40 and Aβ42 levels in the hippocampi of middle-aged WT and APPKINL-G-F mice using the human/rat/mouse β amyloid (1–40 and 1–42) ELISA (34–36 weeks of age, n = 4 biologically independent samples per group). (**D**) Quantitative analysis of T-PER-extractable Aβ40 and Aβ42 levels in the hippocampi of middle-aged WT and APPKINL-G-F mice using the human β amyloid (1–40 and 1–42) ELISA (34–36 weeks of age, n = 4 biologically independent samples per group). (**E**) Western blot analysis of phosphorylated insulin receptor substrates 1 mouse Ser307 [p-IRS1 (mSer307)], p-IRS1 (mSer612), p-IRS1 (mSer632/635), p-IRS1 (mSer1097), IRS1, and ß-tubulin in the hippocampi of middle-aged WT and APPKINL-G-F mice (34–36 weeks of age, n = 6 biologically independent samples per group). Arrow indicates the p-IRS1 mSer612 -corresponding band (lower band) in (**E**). Quantitative analysis of the phosphorylation of IRS1 at mSer307, mSer612, mSer632/635, and mSer1097 normalized to total protein. (**F**) Western blot analysis of phosphorylation levels of Akt Ser473, p70S6K Thr389, AMPK Thr172, and GSK3β Ser9 as well as total protein levels of Akt, p70S6K, AMPK, GSK3β, and ß-tubulin in the hippocampi of middle-aged WT and APPKINL-G-F mice (34–36 weeks of age, n = 6 biologically independent samples per group). Quantitative analysis of the phosphorylation of Akt Ser473, p70S6K Thr389, AMPK Thr172, and GSK3β Ser9 normalized to the respective total protein contents. Results are presented as mean ± SEM, \* *p* < 0.05; \*\* *p* < 0.01.

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Subsequently, we investigated the phosphorylation levels of hippocampal IRS1 at Ser residues in young APPKINL-G-F mice. Importantly, the phosphorylation of hippocampal IRS1 at mSer307, mSer612, and mSer1097, but not at mSer632/635, significantly increased in 12-week-old APPKINL-G-F mice (Figure 4E, Figure S4G) displaying normal memory function (Figure 4B). In parallel to these increases, a concomitant elevation in the basal phosphorylation of AMPK, a metabolic energy sensor, with p70S6K activation and a slight decrease in basal JNK phosphorylation were observed in these mice, along with monotonous Akt, GSK3β, and aPKC ζ/λ activity (Figure 4F, Figure S3D, S4H and S5D). Thus, in young APPKINL-G-F mice exhibiting normal memory function, the increased phosphorylation of hippocampal IRS1 at multiple Ser sites accompanied by AMPK-related low energy conditions had already occurred in the presence of increased Aβ42 level, suggesting that the elevation of Aβ42 level and/or AMPK activation provokes the activation of Ser sites on IRS1 in brains of patients with AD prior to the onset of memory decline.
