Modulation of Amyloid β-Induced Microglia Activation and Neuronal Cell Death by Curcumin and Analogues

Abstract

Alzheimer's disease (AD) is a progressive neurodegenerative disorder that is not restricted to the neuronal compartment but includes important interactions with immune cells, including microglia. Protein aggregates, common pathological hallmarks of AD, bind to pattern recognition receptors on microglia and trigger an inflammatory response, which contributes to disease progression and severity. In this context, curcumin is emerging as a potential drug candidate able to affect multiple key pathways implicated in AD, including neuroinflammation. Therefore, we studied the effect of curcumin and its structurally related analogues cur6 and cur16 on amyloid-β (Aβ)-induced microglia activation and neuronal cell death, as well as their effect on the modulation of Aβ aggregation. Primary cortical microglia and neurons were exposed to two different populations of Aβ42 oligomers (Aβ42Os) where the oligomeric state had been assigned by capillary electrophoresis and ultrafiltration. When stimulated with high molecular weight Aβ42Os, microglia released proinflammatory cytokines that led to early neuronal cell death. The studied compounds exerted an anti-inflammatory effect on high molecular weight Aβ42O-stimulated microglia and possibly inhibited microglia-mediated neuronal cell toxicity. Furthermore, the tested compounds demonstrated antioligomeric activity during the process of in vitro Aβ42 aggregation. These findings could be investigated further and used for the optimization of multipotent candidate molecules for AD treatment.

Keywords: Alzheimer’s disease; amyloid β oligomers; curcumin analogues; microglia activation; neuronal toxicity.

Figure 1

Chemical structures of curcumin and its analogues cur6 and cur16.

Scheme 1

Synthetic route for cur16. Reagents and conditions: (i) B₂O₃, EtOAc; (ii) B(n-BuO)₃; (iii) n-BuNH₂, 80 °C; (iv) HCl, 80 °C.

Figure 2

Effect of Aβ42 oligomers on microglia cell viability. Microglia were cultured overnight in medium containing 10% serum, which was replaced with serum-free medium before treatment with vehicle, LMW or HMW Aβ42Os for 24 (A) or 48 (B) h. At the end of incubation, cell viability was determined by LDH assay. Results are expressed as percentage of LDH release relative to control cells (dashed lines). Data are means ± SEM of 3 independent experiments, each performed in triplicate. *** p ˂ 0.001 versus control cells. Two-way ANOVA, followed by Tukey’s multiple comparison test.

Figure 3

Effect of Aβ42 oligomers on cytokine release from cortical microglia. Microglia were cultured overnight in medium containing 10% serum, which was replaced with serum-free medium before treatment with vehicle, or a serial dilution of LMW and HMW Aβ42Os for 6, 24, or 48 h. Supernatants were collected and analyzed for IL-1β (A–C) and TNF-α (D–F) release by ELISA. Data are means ± SEM (n = 3 in triplicate). ** p ˂ 0.01, and *** p ˂ 0.001 versus control cells. Two-way ANOVA, followed by Tukey’s multiple comparison test.

Figure 4

Effect of curcumin and its analogues on cytokine release from HMW Aβ42O-stimulated microglia and cell viability. Microglia were cultured overnight in medium containing 10% serum, which was replaced with serum-free medium before pretreatment with curcumin (cur), cur6, or cur16 (1–10 μM) followed by stimulation with HMW Aβ42Os (1:20 dilution) for 24 h. Supernatants were collected and analyzed for IL-1β (A–C) and TNF-α (D–F) release by ELISA. (G–I) Cell viability was also determined by MTT assay. Results are expressed as percentage of cell viability relative to control cells (dashed lines). Data are means ± SEM (n = 3 in triplicate). * p ˂ 0.05, ** p ˂ 0.01, and *** p ˂ 0.001 versus control cells; ° p ˂ 0.05, °° p ˂ 0.01, and °°° p ˂ 0.001 versus HMW Aβ42O-treated cells. One-way ANOVA, followed by Holm–Sidak’s multiple comparison test.

Figure 5

Effect of Aβ42 oligomers on neuronal cell toxicity. Primary cortical neurons were cultured from E17 rat embryos and after 7 days in vitro were exposed to a serial dilution of LMW or HMW Aβ42Os (1:200, 1:40, 1:20, and 1:10) for 24 (A), 48 (B), or 72 (C) h. At the end of incubation, cell viability was determined by LDH assay. Results are expressed as percentage of LDH release relative to control cells (dashed lines). Data are means ± SEM of 3 independent experiments, each performed in triplicate. * p ˂ 0.05 versus control cells. Two-way ANOVA, followed by Tukey’s multiple comparison test. (D) Primary neurons were treated with HMW Aβ42Os (1:10 dilution) for 24, 48, or 72 h. Cells were then processed for DAPI staining. Yellow arrows indicate cells with small, fragmented, and condensed nuclei. Experiments were performed 3 times and representative fluorescence microscopy images are shown. The scale bar is 10 µm. (E) Primary neurons were treated with LMW or HMW Aβ42Os (1:10 dilution) for 24, 48, or 72 h. Cells were then processed for βIII-tubulin immunostaining. Experiments were performed 3 times and representative fluorescence microscopy images are shown. The scale bar on the left is 50 µm. In the zoomed images, the scale bar is 10 µm. Quantification of neuritic beading, performed using ImageJ software, is shown at the bottom of the corresponding images. Data are means ± SEM from five random fields of 3 independent experiments. *** p ˂ 0.001 versus control cells. One-way ANOVA, followed by Holm–Sidak’s multiple comparison test.

Figure 6

Effect of microglia-conditioned media on neuronal cell toxicity. (A) The procedure of collecting microglia-conditioned media to stimulate primary cortical neurons. (B) Primary cortical neurons were cultured from E17 rat embryos and after 7 days in vitro were exposed for 24 h to conditioned media from microglia untreated (CM-CTR) or treated with 1:20 dilution of LMW (CM-LMW) or HMW (CM-HMW) Aβ42Os for 24 h. At the end of incubation, cell toxicity was determined by LDH assay. Results are expressed as percentage of LDH release relative to control cells (dashed line). Data are means ± SEM of 3 independent experiments, each performed in duplicate. ** p ˂ 0.01 versus control cells. One-way ANOVA, followed by Holm–Sidak’s multiple comparison test.

Figure 7

Effect of conditioned medium from microglia treated with curcumin and analogues on neuronal cell toxicity. Primary cortical neurons were cultured from E17 rat embryos and after 7 days in vitro were exposed for 24 h to conditioned media from microglia pretreated with 10 μM curcumin (cur), cur6, or cur16 before stimulation with HMW (CM-HMW; 1:20 dilution) Aβ42Os for 24 h. At the end of incubation, cell viability was determined by LDH assay. Results are expressed as percentage of LDH release relative to control cells (dashed line). Data are means ± SEM of 3 independent experiments, each performed in duplicate. ** p ˂ 0.01 versus control cells and ° p ˂ 0.05 versus CM-HMW. One-way ANOVA, followed by Holm–Sidak’s multiple comparison test.

Figure 8

Effect of curcumin and analogues on Aβ42 oligomerization. (A–C) Normalized area percentage plots of toxic HMW Aβ42Os from a 221 μM solution of Aβ42 monomer, in the absence (control) and in the presence of decreasing concentrations of curcumin (cur, A), cur6 (B) and cur16 (C). Data are expressed as mean ± standard deviation (n = 3). (D,E) Comparison among electrophoretic profiles of 221 μM Aβ42 control (black traces) and in the presence of 10 μM (D) and 1 μM (E) curcumin, cur6, and cur16. Peak annotation: * electroosmotic flow; 1 and 2: LMW Aβ42Os; 3 HMW Aβ42Os. Electropherograms are taken at 25 days from dissolution and are representative of n = 3.