Sex-dependent effect of sublethal copper concentrations on de novo cholesterol synthesis in astrocytes and their possible links to variations in cholesterol and amyloid precursor protein levels in neuronal membranes

Abstract

Background: Cholesterol (Cho) is an essential lipophilic molecule in cells; however, both its decrease and its increase may favor the development of neurological diseases such as Alzheimer's disease (AD). Although copper (Cu) is an essential trace metal for cells, the increased plasma concentration of its free form has been linked with AD development and severity. AD affects aged people, but its prevalence and severity are higher in women than in men. We have previously shown that Cu promotes Cho de novo synthesis in immature neurons as well as increased Cho in membrane rafts and Aβ levels in culture medium, but there are no results yet regarding sex differences in the effects of sublethal Cu exposure on Cho de novo synthesis.

Methods: We examined the potential sex-specific impact of sublethal Cu concentrations on de novo Cho synthesis in primary cultures of male and female astrocytes. We also explored whether this had any correlation with variations in Cho and APP levels within neuronal membrane rafts.

Results: Flow cytometry analysis demonstrated that Cu treatment leads to a greater increase in ROS levels in female astrocytes than in males. Furthermore, through RT-PCR analysis, we observed an upregulation of SREBP-2 and HMGCR. Consistently, we observed an increase in de novo Cho synthesis. Finally, western blot analysis indicated that the levels of ABCA1 increase after Cu treatment, accompanied by a higher release of radiolabeled Cho and an elevation in Cho and APP levels in neuronal membrane rafts. Importantly, all these results were significantly more pronounced in female astrocytes than in males.

Conclusions: Our findings confirm that Cu stimulates Cho synthesis in astrocytes, both in a ROS-dependent and -independent manner. Moreover, female astrocytes displayed elevated levels of HMGCR, and de novo Cho synthesis compared to males following TBH and Cu treatments. This corresponds with higher levels of Cho released into the culture medium and a more significant Cho and APP rise within neuronal rafts. We consider that the increased risk of AD in females partly arises from sex-specific responses to metals and/or exogenous substances, impacting key enzyme regulation in various biochemical pathways, including HMGCR.

Keywords: Astrocytes; Cholesterol; Copper; Neurons; Sexual differences.

Fig. 1

Cell viability. Astrocytes were cultured and treated for 24 h with increasing concentrations of CuSO₄. Cell viability was determined by resazurin assay. Results were calculated using ANOVA and Bonferroni’s multiple comparisons test, and data expressed mean ± SD percentage of control (n = 6 for each concentration used). Statistical differences are indicated as ***p < 0.01

Fig. 2

Cu treatment effect on oxidative stress in female and male astrocytes and cellular Cu content. A Cellular Cu concentration (µg/dL) measured by spectrophotometry (n = 6); B ROS levels were determined using DCF-DA by flow cytometry (n = 6); C SOD activity was measured by spectrophotometry (n = 6 to 8); and D Lipid peroxidation was determined by the TBARS assay (n = 8). All determinations were made after 24 h of Cu treatment (400 µM of CuSO₄; gray bar). Untreated cells were used as controls (black bar), TBH (500 µM) was used as a positive control of ROS production (dark gray bars), and NAC (10 µg/mL) was used as an antioxidant molecule to counteract Cu ROS generation to be used as a second negative control (light gray bars). Results were calculated using 2-way ANOVA plus Bonferroni’s multiple comparisons test, and data expressed mean ± SD, **p < 0.01 and ***p < 0.001 significant differences respect to the control of the same sex, ^#significant differences between female and male astrocytes with the same treatment (p < 0.05 for TBH,  p < 0.01 for Cu ROS and p < 0.1 for Cu SOD activity), and + p < 0.05 significant differences between Cu and Cu + NAC treatment of the same sex

Fig. 3

Cu effect on Cho de novo synthesis. A Expression levels of SREBP-2 determined by RT-qPCR (n = 4); B Expression levels of HMGCR determined by RT-qPCR (n = 4); C Cho de novo synthesis determined by HP-TLC followed by the intensity analysis of radioactive bands (n = 6–14). All determinations were made after 24 h of Cu treatment (400 µM of CuSO₄; gray bar). Untreated cells were used as controls (black bar), TBH (500 µM) was used as a positive control of ROS production (dark gray bars), and NAC (10 µg/mL) was used as an antioxidant molecule to counteract Cu ROS generation to be used as a second negative control (light gray bars). Results were calculated using 2-way ANOVA plus Bonferroni’s multiple comparisons test, and data expressed mean ± SD, *p < 0.05 and **p < 0.01 significant differences respect to the control of the same sex, and ^# significant differences between female vs male astrocytes with same treatment (p < 0.05 for SREBP-2, p < 0.01 for Cu HMGCR, p < 0.1 TBH HMGCR, p < 0.01 for Cu Cho de novo synthesis and p < 0.05 for TBH Cho de novo synthesis), + + p < 0.01 significant differences between Cu and Cu + NAC treatment of the same sex, and & p < 0.01 significant differences between Cu and TBH treatment of the same sex

Fig. 4

Effects of Cu on Cho release. A ABCA1 levels determined by western blot (n = 8); B Labeled Cho (¹⁴Cho) produced by de novo synthesis determined by HP-TLC followed by the analysis of intensity of radioactive bands (n = 8). All determinations were made after 24 h of Cu treatment (400 µM of CuSO₄; gray bar). Untreated cells were used as controls (black bar), TBH (500 µM) was used as a positive control of ROS production (dark gray bars), and NAC (10 µg/mL) was used as an antioxidant molecule to counteract Cu ROS generation to be used as a second negative control (light gray bars). Results were calculated using 2-way ANOVA plus Bonferroni’s multiple comparisons test, and data expressed mean ± SD, **p < 0.01 significant differences respect to the control of the same sex, ^# significant differences between Cu female vs Cu male (p < 0.05 for ABCA1 and p < 0.01 for ¹⁴Cho release), and ^&p < 0.01 significant differences between Cu and THB treatments in the same sex

Fig. 5

Effect of astrocyte-conditioned culture medium on Cho and APP levels in neuronal membrane rafts. A Cho levels in membrane rafts of neurons determined by HP-TLC followed by the analysis of intensity of bands (n = 12). B APP/flotillin ratio in neuronal membrane rafts determined by western blot (n = 12). All determinations were made after 24 h of neuronal primary culture exposure to conditioned astrocyte culture medium previously exposed or not (control; black bars) to 400 µM of CuSO₄ (gray bar). Results were calculated using 2-way ANOVA plus Bonferroni’s multiple comparisons test, and data expressed mean ± SD, **p < 0.01 and ***p < 0.001 significant differences between Cu vs control, ^#p < 0.01 significant differences between Cu female vs Cu male