3.4 I2-IR ligand LSL60101, but not donepezil, reduces Aβ plaques. By contrast, donepezil/LSL60101 attenuate Aβ pathology in 5XFAD mice.
The number of amyloid plaques in 5XFAD mice was assessed by histochemical staining with Thioflavin-S. Following treatment with LSL60101, there was a significant decrease in the total number and area of the plaques in 5XFAD mice when compared to the 5XFAD controls, demonstrating a neuroprotective function of I2-IR ligand regarding the senile plaque formation. 4-week-treatment with donepezil did not reduce the amyloid deposition significantly in 5XFAD mice (Figure 3a-3c). The protein levels of Aβ determined by WB tended to decrease in all treated groups without reaching significance (Figure 3d, 3f). Interestingly, alterations in the levels of proteins implicated in the APP processing showed complementary results in the combination of LSL60101 and donepezil treatment. In this line, the protein levels of C-terminal fragments (CTFs) were found significantly reduced in LSL60101 treated groups when compared to the 5XFAD controls while donepezil/LSL60101 treated group showed a higher decrease in CTFs compared with monotherapy (Figure 4a, 4g). The protein levels of phosphorylated amyloid precursor protein (p-APP) at Th668 were significantly decreased only for the combination of donepezil/LSL60101 treated animals (Figure 4b). Soluble APPβ (sAPPβ) levels were found significantly decreased in donepezil/LSL60101 treated group, whereas soluble APPα (sAPPα) levels were found significantly increased after combination treatment when compared to 5XFAD controls or donepezil treated mice (Figure 4c, 4d, 4g). No differences in the gene expression of APP were found among the groups (Figure 4h). Regarding the levels of BACE1(β-secretase) and ADAM10 (α-secretase), no significant differences were observed among the 5XFAD groups (Figures 4e-4g). However, when the gene expression and protein levels of enzymes implicated in amyloid degradation were studied, donepezil/LSL60101 treatment slightly increased gene expression of insulin-degrading enzyme (Ide ) and neprilysin(Nep ) (Figure 4i).