Curcumin decreases amyloid-beta peptide levels by attenuating the maturation of amyloid-beta precursor protein

Abstract

Alzheimer disease (AD) is a devastating neurodegenerative disease with no cure. The pathogenesis of AD is believed to be driven primarily by amyloid-beta (Abeta), the principal component of senile plaques. Abeta is an approximately 4-kDa peptide generated via cleavage of the amyloid-beta precursor protein (APP). Curcumin is a compound in the widely used culinary spice, turmeric, which possesses potent and broad biological activities, including anti-inflammatory and antioxidant activities, chemopreventative effects, and effects on protein trafficking. Recent in vivo studies indicate that curcumin is able to reduce Abeta-related pathology in transgenic AD mouse models via unknown molecular mechanisms. Here, we investigated the effects of curcumin on Abeta levels and APP processing in various cell lines and mouse primary cortical neurons. We show for the first time that curcumin potently lowers Abeta levels by attenuating the maturation of APP in the secretory pathway. These data provide a mechanism of action for the ability of curcumin to attenuate amyloid-beta pathology.

FIGURE 1.

Curcumin significantly decreases Aβ levels and the ratio of APPma:APPim in mouse primary cortical neurons in a dose-dependent manner. A and B, curcumin significantly decreases both Aβ40 and Aβ42 levels. Mouse primary cortical neurons (E18) were treated with different concentrations of curcumin and harvested after 24 h. Conditioned medium was used in ELISA analysis to detect the Aβ40 and Aβ42 levels, which were normalized to cell numbers. C and D, the curcumin treatment altered APP levels and decreased the ratio of APPma:APPim. In the Western blotting analysis, cell lysates were probed with the APP8717 antibody to reveal APP. β-Actin was used as the loading control. C, a representative gel showing full-length APP and β-actin. D, densitometry of C (n = 3 for each treatment group). Mean ± S.E. *, p < 0.05; **, p < 0.01.

FIGURE 2.

Curcumin significantly modulates APP processing in B104-APP751 cells in a dose-dependent manner. Stable rat neuroblastoma B104-APP751 cells were treated with different concentrations of curcumin for 24 h and were collected for Western blotting analysis. Cell lysates were probed with the APP8717 antibody to reveal APP. β-Actin was used as the loading control. The cell medium was probed with 6E10 to reveal sAPPα. A and B, a representative gel showing full-length APP, APP-C83, and sAPPα. C, densitometry of A. Curcumin significantly increased the levels of immature APP and total APP. It also markedly increased and then decreased the level of mature APP, as well as the ratio of APPma:APPim with increasing curcumin concentration. D, curcumin treatment significantly decreased the ratio of C83/APPtotal. E, curcumin treatment significantly decreased the ratio of sAPPα/APPtotal compared with control (n = 3 for each treatment group). Mean ± S.E.; *, p < 0.05; **, p < 0.01.

FIGURE 3.

Curcumin treatment alters APP metabolism in H4-APP751 cells and CHO-APP751 cells in a dose-dependent manner. Various cell models were treated with different concentrations of curcumin for 24 h and collected for Western blotting analysis. Cell lysates were probed with the APP8717 antibody to reveal APP. β-Actin was used as the loading control. A and B, the effects of curcumin treatment on H4-APP751 cells. Curcumin treatment had a trend to increase the level of mature APP, significantly increased the levels of immature APP and total APP, and markedly decreased the ratio of APPma/APPim. C and D, the effects of curcumin on CHO-APP751 cells. Curcumin significantly increased the levels of mature APP, immature APP, and total APP and had a trend to decrease the ratio of APPma:APPim with increasing concentration (n = 3 for each treatment group). Mean ± S.E.; *, p < 0.05; **, p < 0.01.

FIGURE 4.

Curcumin treatment alters the turnover rate of both mature and immature APP in H4-APP751 cells. H4-APP751 cells were treated with 20 μm curcumin for 24 h and then treated with 40 μg/ml cycloheximide for different time (0, 0.5, 1.5, and 3 h). Cell lysates were collected and utilized for Western blotting analysis. Cell lysates were probed with the APP8717 antibody to reveal APP. β-Actin was used as the loading control. A, a representative gel revealing the cycloheximide treatment of different time points with or without curcumin treatment. B and C, quantitative Western blot analysis for mature (B) and immature (C) APP levels. Immature APP levels in curcumin treatment were significantly higher at 0.5, 1.5, and 3 h, compared with the corresponding control treatment. D, quantitative Western blot analysis for total APP levels. E, the ratio of APPma:APPim was decreased in curcumin treatment compared with control at cycloheximide treatment of 0, 0.5, 1.5, and 3 h (n = 3 for each treatment group). Mean ± S.E. *, p < 0.05; **, p < 0.01.

FIGURE 5.

Curcumin treatment significantly increases cell surface APP levels in both H4-APP751 and CHO-APP751 cells. Cells were treated with 20 μm curcumin for 24 h and then subjected to biotinylation analysis to assess cell surface APP. Cell lysates were collected and utilized for Western blotting analysis. A and B, curcumin treatment markedly increased the level of cell surface APP in H4-APP751 cells compared with control treatment. C and D, curcumin treatment significantly increased the level of cell surface APP in CHO-APP751 cells compared with control (n = 3 for each treatment group). Mean ± S.E. *, p < 0.05; **, p < 0.01.

FIGURE 6.

Curcumin treatment affects APP metabolism at the endoplasmic reticulum. A, BFA disrupts APP maturation process at the Golgi complex and induces the generation of the intermediate APP in H4-APP751 cells. H4-APP751 cells were treated with or without 5 and 10 μg/ml BFA for 5 or 30 min. Cell lysates were collected and prepared for Western blot analysis. B and C, curcumin treatment markedly decreased the level of intermediate APP in the presence of BFA. H4-APP751 cells were treated with 5 μg/ml BFA in the presence or absence of 20 μm curcumin for 5 or 30 min (n = 3 for each treatment group). Cell lysates were collected and prepared for Western blot analysis, as described under “Experimental Procedures.”

FIGURE 7.

Curcumin treatment does not alter APLP2 protein levels. B104-APP751 and H4-APP751 cells were treated with different concentrations of curcumin for 24 h, and cell lysates were collected and utilized for Western blot analysis. Cell lysates were probed with the APLP2 antibody to reveal APLP2. β-Actin was used as the loading control. A and B, the effects of curcumin treatment on APLP2 in B104-APP751 cells. Curcumin treatment did not alter full-length APLP2 levels and did not alter levels of mature versus immature forms of APLP2. C and D, the effects of curcumin treatment on APLP2 in H4-APP751 cells. Curcumin treatment did not alter full-length APLP2 levels (n = 3 for each treatment group). Mean ± S.E. p > 0.05.

FIGURE 8.

Curcumin treatment does not decrease cell viability. A and B, H4-APP751 cells (A) or mouse primary cortical neurons (B) were treated with different concentrations of curcumin for 24 h and then subjected to alamarBlue analysis as described under “Experimental Procedures.” Curcumin treatment did not decrease cell viability in either cell type, compared with control (0 μm). The dose of 2.5 μm curcumin significantly increased cell viability by 29.3% in H4-APP751 cells compared with control (A) (p < 0.05) (n = 3 in each treatment group). Mean ± S.E. *, p < 0.05.