Fig 2: Identification of APPswe-overexpressing cell line. Five stable cell lines were obtained by viral infection and screening, four of which were infected by lentivirus containing the APPswe gene. The other cell line was infected with an empty vector. The expression levels of APP were determined by (A) real-time PCR (compared with the normal group, * p < 0.05, ** p < 0.01, n = 3) and (B) western blot (compared with the normal group, * p < 0.05, n = 4). (C) The levels of Aβ1-42 were measured by ELISA (compared with the normal group, ** p < 0.01, n = 6).

Fig 3: DOR activation down-regulated, but MOR activation up-regulated BACE1 expression for APP processing. BACE1 mRNA and proteins were measured in the PC12 cells in the conditions of normal control and Aβ1–42 injury (48 h). APP processing was evaluated by measuring APP full length protein and AICD. N = 3 in each group. *p < 0.05, **p<0.01 vs. (C) or (A). (A) DAMGO-induced alternations in BACE1 mRNA in physiological condition and AD injury. (B) DAMGO-induced upregulation of BACE1 protein in both physiological condition and AD injury, while DOR activation specifically attenuated BACE1 expression under AD injury. (C) APP cleavage was associated with DOR activation and inhibition, while MOR activation significantly accelerated APP processing. Note that DOR activation had no significant effect on BACE1 mRNA expression though it tended to induce a slight reduction in both physiological and Aβ1–42 oligomer conditions. However, it significantly reduced the level of BACE1 protein in AD condition (Aβ1–42 oligomer exposure), which could be largely reversed by DOR antagonist naltrindole. In contrast, MOR activation with DAMGO significantly increased the expression of both BACE1 mRNA and protein in both physiological and Aβ1–42 oligomer conditions. Consistently, the APP cleavage was seriously interfered by DOR activation in Aβ1–42 oligomer conditions, while DAMGO accelerated APP processing in both physiological and Aβ1–42 oligomer conditions with a significant decrease in APP full length protein and a large increase in AICD, the APP cleavage product.

Fig 4: Artesunate does not alter the levels of Aβ processing proteins and Aβ clearance enzymes in Picalm+/−; 5XFAD mice. A amyloid precursor protein (APP) abundance in cortex, B APP C-terminal fragment (APP-CTF) abundance in cortex. C β-secretase (BACE1) abundance in cortex, D soluble APP-β (sAPPβ) levels in cerebrospinal fluid (CSF), E γ–secretase activity as determined by the production of Notch intracellular domain (NICD) fragment from Notch protein, indicated by NICD abundance in cortex, F neprilysin abundance in cortex, and G insulin degrading enzyme (IDE) abundance in cortex of Picalm+/−; 5X FAD mice treated with artesunate or vehicle as in Fig. 2A. The relative abundance of proteins was normalized by the house-keeping gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH) protein. Single points per mouse indicated by circles, with boxes representing mean ± SD. A-E, G n = 8 per group; F n = 7–8 per group. ns = non-significant by two-tailed t-test. Full blots for A-C, E-G shown in supp. Fig. 5

Fig 5: NV-CUR ameliorates AD-related pathologies. (A) Immunofluorescence images of p-Tau205 (red) in the CA1 and DG regions of the brain. (B) Immunofluorescence images of Aβ (red) in the cortex of the brain. (C) Quantification of the fluorescence intensity of (A). (D) Quantification of the area of the Aβ plaques in (B). (E, F) Western blot detection and relative quantification of p-Tau and APP levels in the brain. (G) Nissl-stained images of different areas of the hippocampus (CA1, CA3, DG) in different treatment groups. Optical microscope, 20 × . (H) Quantitative analysis of Nissl staining. Data are presented as the means ± SEMs (n = 3–6), ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)