Curcumin Decreases Aβ Levels and Attenuates APP Maturation in Mouse Primary Cortical Neurons We first tested whether curcumin affects Aβ levels and/or APP processing and metabolism in mouse primary neuronal cells. Mouse primary neurons (E18) were prepared on poly-d-lysine-coated plates and treated with curcumin (0, 1, 2.5, 7.5, 10, and 20 μm). Cells were harvested after 24 h of treatment. Conditioned medium was applied to ELISA analysis to measure the Aβ40 and Aβ42 levels, which were normalized to cell number. Curcumin treatment decreased both Aβ40 and Aβ42 levels in a dose-dependent manner (Fig. 1, A and B). For example, 20 μm curcumin decreased Aβ40 and Aβ42 levels by 38.4% (p < 0.01), and 43.9% (p < 0.05), respectively, compared with the control treatment (0 μm) (Fig. 1, A and B). View larger version: Download as PowerPoint Slide 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. Next, we studied the effects of curcumin on APP metabolism and processing. Cell lysates from the previous experiment were subjected to quantitative Western blotting analysis with antibody, APP8717, targeted at the C terminus of APP. β-Actin was used to normalize loading variation between gel lanes. We assessed the effects of curcumin treatment on levels of total APP (APPma and APPim), mature and immature APP, as well as the APPma:APPim ratio. Curcumin did not significantly alter the levels of mature APP but increased levels of immature APP and total APP in comparison to control. For example, 10 μm curcumin treatment increased immature APP levels by 59.7% and increased total APP levels by 41.6% versus control (p < 0.05; Fig. 1, C and D). In addition, curcumin decreased the ratio of mature APP to immature APP compared with control. For example, 10 μm curcumin treatment decreased the ratio by 23.3% (p < 0.05) (Fig. 1, C and D). Overall, these data suggest that curcumin decreases Aβ levels by retarding APP maturation.

Curcumin Significantly Alters APP Maturation and Processing in Other Cell Types Next, we asked whether curcumin impairs APP maturation in other cell types. For this purpose, we used stably transfected rat neuroblastoma B104-APP751 cells, which were treated with different concentrations of curcumin for 24 h and then collected for Western blotting analysis with the antibody APP8717. In addition, the media was probed with antibody 6E10 to measure sAPPα levels. Curcumin significantly increased levels of mature APP at doses of 5 and 10 μm (by 60.0%; p < 0.05, compared with control) but revealed a trend toward decreased levels at 15 and 20 μm compared with control (Fig. 2, A and C). The level of immature APP was increased significantly by curcumin treatment (e.g. increased by 279.1% at 20 μm; p < 0.01). Additionally, curcumin treatment increased markedly the ratio of APPma/APPim at lower concentrations, but significantly decreased the ratio with increasing concentrations (Fig. 2, A and C). Specifically, the doses of 5 and 10 μm curcumin decreased the ratio by 56.3% (p < 0.01) and 82.1% (p < 0.01), respectively, compared with the control, whereas 15 and 20 μm curcumin markedly decreased the APPma/APPim ratio by 55.8% (p < 0.01) and 84.4% (p < 0.01), respectively, compared with the control. Finally, curcumin treatment significantly increased total APP levels in comparison to control treatment (e.g. increased by 137.7% at 20 μm; p < 0.01) (Fig. 2C). In contrast to what was observed in the primary neurons, lower doses of curcumin, e.g. 5 and 10 μm, increased APP maturation. However, at higher doses, e.g. 15 and 20 μm, these data recapitulate the data from the primary neurons showing that curcumin treatment retards APP maturation in rat neuroblastoma B104-APP751 cells. View larger version: Download as PowerPoint Slide 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. In this same experiment, we also assessed the effects of curcumin on APP processing by measuring levels of the APP proteolytic products, sAPPα and C83, the products of α-secretase cleavage of APP. We observed a trend toward decreased levels of C83 and sAPPα (Fig. 2, A and C–E) with curcumin treatment. We also observed a significant decrease in the ratio of C83/total APP as well as in the ratio of sAPPα/total APP, compared with the control treatment (Fig. 2, D–E), following curcumin treatment. For example, 20 μm curcumin decreased the ratio of C83/APPtotal by 74.3% (p < 0.01) (Fig. 2D) and decreased the ratio of sAPPα/total APP by 57.1% in comparison to control (p < 0.05) (Fig. 2E). Collectively, these data show that curcumin treatment attenuates α-secretase processing of APP in rat neuroblastoma B104-APP751 cells, consistent with attenuation of APP maturation. Next, we tested the effects of curcumin on APP metabolism and processing in human H4 neuroglioma cells stably transfected with APP751 (H4-APP751) and in Chinese hamster ovary cells stably transfected with APP751 (CHO-APP751). As observed with the rat neuroblastoma B104-APP751 cells, curcumin treatment led to an increase (trend) and then a decrease in the level of mature APP with increasing concentrations (p > 0.05) (Fig. 3A) in H4-APP751 cells. Curcumin treatment also significantly increased levels of immature APP compared with the control treatment (0 μm) (Fig. 3B). 20 μm curcumin markedly increased immature APP levels by 79.1% (p < 0.01; versus control) (Fig. 3B). Additionally, curcumin significantly decreased the ratio of mature APP to immature APP with increasing doses, compared with control. 20 μm curcumin markedly decreased the ratio of APPma:APPim by 42.2% (p < 0.01; compared with control). Curcumin treatment significantly increased levels of total APP (APPma and APPim) with increased concentration. 20 μm curcumin markedly increased total APP levels by 44.3% (p < 0.01; compared with control) (Fig. 3B). Curcumin treatment significantly increased levels of mature APP and immature APP, as well as total APP compared with the control in CHO-APP751 cells (Fig. 3, C–D). 20 μm curcumin elevated the level of immature APP by 65.0% (p < 0.05) and increased the levels of total APP by 59.2% (p < 0.05). Curcumin treatment also revealed a trend toward decrease in the ratio of APPma:APPim compared with control (p > 0.05) (Fig. 3D). We also measured the Aβ levels from curcumin treated samples in these three cell models. We found that curcumin significantly decreased both Aβ40 and Aβ42 levels in all cell models (supplemental Fig. 1, A–C). Collectively, these data recapitulated the effects of curcumin treatment on APP metabolism and processing in several different cell types. View larger version: Download as PowerPoint Slide 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.

Curcumin Alters the Turnover of Mature and Immature APP In the next step, we assessed the effects of curcumin on the rate of APP turnover using cycloheximide treatment. H4-APP751 cells were treated with 20 μm curcumin for 24 h and then treated with 40 μg/ml cycloheximide for various time intervals (0, 0.5, 1.5, and 3 h). Cell lysates were collected and utilized for quantitative Western blotting analysis. Curcumin markedly decreased the half-life of mature APP from 2.03 to 1.14 h (changing by 43.4%), increased the half-life of immature APP from 1.86 to 2.36 h (changing by 26.0%), and shortened total APP half-life from 1.94 to 1.87 h (Fig. 4, A–D). Curcumin also markedly decreased the ratio of APPma/APPim (e.g. decreased by 67.6% at 0.5 h; p < 0.05) (Fig. 4E). Thus, curcumin increased the stability of immature APP while decreasing the stability of mature APP, consistent with attenuated APP maturation. View larger version: Download as PowerPoint Slide 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.

Curcumin Increases the Level of Plasma Membrane APP Next, we tested the effects of curcumin on plasma membrane levels of APP. Stable H4-APP751 and CHO-APP751 cells were treated with 20 μm curcumin for 24 h and then subjected to biotinylation analysis to assess the levels of cell surface APP. Cell lysates were collected and utilized for Western blotting analysis. Curcumin significantly increased the levels of cell surface APP by 28.1% in H4-APP751 cells (p < 0.05) (Fig. 5, A and B) and markedly increased the level of cell surface APP by 133.7% in CHO-APP751 cells (p < 0.01) (Fig. 5, C and D), in comparison with control. We also studied whether curcumin may alter plasma membrane levels of APLP2, an APP homologue protein. We did not find any differences in the levels of APLP2 (amyloid precursor-like protein 2) between cells treated with curcumin versus control samples.3 In combination with prior observations of attenuated maturation of APP following treatment with curcumin, these data suggest that curcumin treatment may also lead to decreased endocytosis of APP, consistent with decreased Aβ levels. View larger version: Download as PowerPoint Slide 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.

Curcumin Decreases the Levels of Intermediate APP Induced by Brefeldin A Next, we investigated the mechanism by which curcumin may increase immature APP levels. Previous reports have shown that curcumin is a sarcoplasmic/endoplasmic reticulum calcium-ATPase inhibitor (31). It potentially may affect the functions of ER lumen chaperones, which are calcium binding proteins (32). We hypothesized that curcumin may affect APP metabolism at the level of the endoplasmic reticulum. BFA is an agent that disassembles the Golgi complex and redistributes proteins into the ER. BFA treatment has been shown to induce the buildup of an intermediate APP isoform (33). Thus, if curcumin decreased levels of intermediate APP induced by BFA, it would strongly suggest that the curcumin acts before the Golgi complex, likely at the ER. First, H4-APP751 cells were treated with 5 μg/ml BFA for 5 or 30 min and then harvested and subjected to Western blotting analysis. As expected, 5 μg/ml and 10 μg/ml BFA for 30 min induced the generation of intermediate APP (Fig. 6A). Then, H4-APP751 cells were treated with 20 μm curcumin ± 5 μg/ml BFA for 0.5 or 3 h. BFA markedly induced the generation of the intermediate APP at both 0.5 and 3 h, compared with control (Fig. 6, B–C). BFA-induced generation of intermediate APP was attenuated significantly in the presence of curcumin (at both 0.5 and 3 h; Fig. 6, B and C). These data suggest that curcumin affects APP metabolism in the secretory pathway at the level of the endoplasmic reticulum. View larger version: Download as PowerPoint Slide 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.”

Curcumin Does Not Alter APLP2 Levels To assess whether the effects of curcumin are specific to APP, we next measured the levels of APP homologue APLP2 following curcumin treatment. Stable B104-APP751 cells and H4-APP751 cells were treated with different concentrations of curcumin for 24 h, and cell lysates were collected and utilized for Western blotting analysis. APLP2 antibody has been reported previously(26, 27). β-Actin was used as the loading control. Curcumin treatment did not alter the levels of full length APLP2 in either B104-APP751 cells (p > 0.05) (Fig. 7, A and B) or H4-APP751 cells (p > 0.05) and did not alter levels of mature versus immature forms of APLP2 (Fig. 7, C and D). In addition, we found that curcumin treatment does not alter the levels of ADAM10 in mouse primary cortical neurons (supplemental Fig. 2). Thus, these data suggest that the effects of curcumin on APP levels and maturation are specific. View larger version: Download as PowerPoint Slide 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.