Effect of Sublethal Copper Overload on Cholesterol De Novo Synthesis in Undifferentiated Neuronal Cells

Abstract

Although copper (Cu) is an essential trace metal for cells, it can induce harmful effects as it participates in the Fenton reaction. Involuntary exposure to Cu overload is much more common than expected and has been linked with neurodegeneration, particularly with Alzheimer's disease (AD) evidenced by a positive correlation between free Cu in plasma and the severity of the disease. It has been suggested that Cu imbalance alters cholesterol (Chol) homeostasis and that high membrane Chol promotes the amyloidogenic processing of the amyloid precursor protein (APP) secreting the β-amyloid (Aβ) peptide. Despite the wide knowledge on the effects of Cu in mature brain metabolism, the consequence of its overload on immature neurons remains unknown. Therefore, we used an undifferentiated human neuroblastoma cell line (SH-SY5Y) to analyze the effect of sublethal concentrations of Cu on 1- de novo Chol synthesis and membrane distribution; 2-APP levels in cells and its distribution in membrane rafts; 3-the levels of Aβ in the culture medium. Our results demonstrated that Cu increases reactive oxygen species (ROS) and favors Chol de novo synthesis in both ROS-dependent and independent manners. Also, at least part of these effects was due to the activation of 3-hydroxy-3-methyl glutaryl CoA reductase (HMGCR). In addition, Cu increases the Chol/PL ratio in the cellular membranes, specifically Chol content in membrane rafts. We found no changes in total APP cell levels; however, its presence in membrane rafts increases with the consequent increase of Aβ in the culture medium. We conclude that Cu overload favors Chol de novo synthesis in both ROS-dependent and independent manners, being at least in part, responsible for the high Chol levels found in the cell membrane and membrane rafts. These may promote the redistribution of APP into the rafts, favoring the amyloidogenic processing of this protein and increasing the levels of Aβ.

Figure 1

DCX and NeuN expression in SH-SY5Y cells and the brain homogenate (TH) obtained from Wistar rats. TH containing mature neurons among other cells was used as the positive control for NeuN expression.

Figure 2

Effect of Cu, Fe, and Zn treatment on cell viability. SH-SY5Y cells were cultured and treated for 24 h with increasing concentrations of CuSO₄, FeSO₄, and ZnSO₄. Cell viability was determined by the resazurin assay. Results were calculated using ANOVA and Dunnett’s multiple-comparison test and expressed mean ± SD percentage of control (n = 3 to 6 for each concentration used). Statistical differences are indicated as *p < 0.05 and **p < 0.01.

Figure 3

Determination of ROS generation in SH-SY5Y cells by flow cytometry. DCF-DA was used to test ROS production. SH-SY5Y cells were treated for 24 h with 200 μM CuSO₄ (light-gray bar), 200 μM FeSO₄ (gray bar), or 200 μM ZnSO₄ (almost white bar) for 24 h. Cells without metal addition (black bar) were used as control, and 500 μM TBH was used as the positive control of ROS generation (dark-gray bar). Data are expressed mean ± SD (n = 4) as the percentage of control. Significance of statistical difference was calculated using one-way ANOVA and Bonferroni’s multiple-comparison test and was indicated as ***p < 0.001 compared to the control and ^# compared to Cu treatment.

Figure 4

Lipid peroxidation (A) and cell death (B) in SH-SY5Y cells. Lipid peroxidation was determined by the TBARS assay (A), whereas PI staining was used to test and cell death (B) after 24 h of Cu treatment (200 μM of CuSO₄; gray bar). Untreated cells were used as control (black bar). Data expressed mean ± SD (n = 4) as the percentage of control.

Figure 5

Chol synthesized de novo. (A) SH-SY5Y cells were treated for 24 h with 200 μM CuSO₄ (light-gray bar), 200 μM FeSO₄ (gray bar), or 200 μM of ZnSO₄ (almost white bar) for 24 h. (B) SH-SY5Y cells were treated with 50 (lined light-gray bar), 200 (light-gray bar), and 400 μM (squared light-gray bar) CuSO₄. Cells without metal addition (black bar) were used as the control, and 500 μM TBH was used as the positive control of ROS generation (dark-gray bar). Data expressed mean ± SD (n = 4) percentage of control. Significant differences were detected using one-way ANOVA and Bonferroni’s multiple-comparison test and indicated as *** (p < 0,001), ** (p < 0.01), and * (p < 0.05) differences compared with control; # (p < 0.001) and ## (p < 0.01) differences compared with Cu; and (p < 0.01) differences compared with Zn.

Figure 6

HMGCR expression in SH-SY5Y homogenates. Cells after 24 h of treatment with (gray bar) or without (black bar) 200 μM CuSO₄ were collected, and HMGCR expression was measured by qRT-PCR. Data were calculated using the Mann–Whitney test and expressed as mean ± SD (n = 5) percentage of control. Statistical differences are indicated as *p < 0.05.

Figure 7

Effect of Cu overloads on the membrane (A) and raft (B) Chol levels. (A) % of Chol/PL ratio compared with control in SH-SY5Y membranes (n = 3) and (B) % of Chol compared with control in membrane rafts (fraction 3 and 4) (n = 3) after 24 h of treatment with (gray bar) or without (black bar) 200 μM CuSO₄. Results are expressed as mean ± SD and were calculated using the Mann–Whitney test (A) and two-way ANOVA and Bonferroni’s multiple-comparison test (B). Statistical differences are indicated as *p < 0.05 and **p < 0.01.

Figure 8

Effect of Cu treatment on APP levels. SH-SY5Y cells were treated for 24 h with or without 200 μM Cu and APP in the homogenate (A) and in membrane rafts (B). APP expression was normalized to β-actin and flotillin, respectively. Results were calculated using the Mann–Whitney test and expressed as the mean ± SD percentage of control for panel A (n = 4) and two-way ANOVA plus Bonferroni’s test and expressed as the mean ± SD percentage of control fraction 3 for panel B (n = 4). Statistical difference is indicated as ***p < 0.001.

Figure 9

Effect of Cu treatment on Aβ levels. SH-SY5Y cells were treated for 24 h with or without 200 μM Cu and Aβ in the culture medium. Results were calculated using the Mann–Whitney test and expressed as the mean ± SD percentage of control (n = 4). Statistical difference is indicated as *p < 0.05.

Figure 10

Proposed mechanism of toxicity of Cu eliciting Aβ release following ROS production. ROS are already shown to affect different pathways involved in Chol metabolism. Dark arrows show cellular signals described by other authors (referenced). The increased expression or concentrations of key components of these pathways are indicated by thick vertical arrows. Mechanisms involved in this article are represented as continuous red arrows.