Amyloid-ß peptides inhibit the expression of AQP4 and glutamate transporter EAAC1 in insulin-treated C6 glioma cells

Abstract

Astrocytic aquaporin 4 (AQP4) facilitates glutamate clearance via regulation of the glutamate transporter function, involved in the modulation of brain plasticity and cognitive function to prevent neurodegenerative disorders such as Alzheimer's disease (AD). In in vitro studies, the C6 rat glioma cell line is a widely applied aging model system to investigate changes in glial cells associated with aging or AD. However, the neurotoxicity mechanism whether AQP4 mediate glutamate uptake in Aβ-stimulated C6 cell remain uncertain. In this study, we examined the effects of Aβ on the expression of AQP4, Glu transporters, Glu uptake, and cell viability in insulin-treated C6 cells. Our results showed that the expression of AQP4 mRNA and protein was significantly enhanced by insulin in older cultures (passage 45), and the expression was inhibited by Aβ at 10 μM. In addition, the cell viability and glutamate uptake in Aβ-treated C6 cells were decreased in dose-dependent manners. GFAP showed similar changes in gene and protein expression patterns as AQP4, but no significant alterations were seen in GLAST expression. In C6 cells, the glutamate transport was found to be EAAC1, not GLT-1. EAAC1 expression was decreased by the treatment of Aβ. Taken together, our findings suggest that C6 cells may have astrocytic characteristics, and the astrocytic cytotoxicity induced by Aβ was mediated by reduction of glutamate uptake through AQP4/EAAC1 pathway in C6 cells. This indicates that C6 glioma cells could be used to study the roles of AQP4 on astrocyte function in AD.

Keywords: Alzheimer’s disease; Amyloid β; Aquaporin 4; Glutamate transporter excitatory amino acid carrier 1; Glutamate uptake.

Fig. 1

AQP4 expressions in C6 cells under various conditions, (A) effects of cell passages and insulin on the AQP4 protein expression, (B) the AQP4 protein expression in Aβ_1-42 or Aβ_25-35-treated C6 cells in the presence of insulin, (C) the AQP4 gene expression in Aβ_1-42 or Aβ_25-35-treated C6 cells in the presence of insulin. β-Actin was used as the internal control for Western blot and RT-PCR analysis. Results are presented as the means ± SDE of percentages calculated with respect to control levels of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 vs. insulin alone (n = 3).

Fig. 2

Effects of Aβ peptides on the cell viability (A), Glu uptake (B), and caspase-3 protein expression (C), in insulin-treated C6 cells (passage number 45). TGN-020 was used as an AQP4 inhibitor, and wortmannin used as an EAAC1 inhibitor in the insulin-treated C6 cells. Results are presented as the means ± SDE of percentages calculated with respect to control levels of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 vs. insulin alone (n = 3).

Fig. 3

Effects of Aβ peptides on Glu receptors and GFAP protein expressions in insulin-treated C6 cells (passage number 45). β-Actin was used as the internal control for Western blot analysis. Results are presented as the means ± SDE of percentages calculated with respect to control levels of three independent experiments. Results are presented as the means ± SDE of percentages calculated with respect to control levels of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 vs. insulin alone (n = 3).

Fig. 4

Effects of Aβ peptides on Glu receptors and GFAP gene expressions in insulin-treated C6 cells (passage number 45). β-Actin was used as the internal control for RT-PCR analysis. Results are presented as the means ± SDE of percentages calculated with respect to control levels of three independent experiments. Results are presented as the means ± SDE of percentages calculated with respect to control levels of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 vs. insulin alone (n = 3).

Fig. 5

Relationship between AQP4, EAAC1, and GFAP protein expressions in insulin-treated C6 cells (passage number 45). TGN-020 and wortmannin were used as AQP4 inhibitor and EAAC1 inhibitor, respectively, in insulin-treated C6 cells. Results are presented as the means ± SDE of percentages calculated with respect to control levels of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 vs. insulin alone (n = 3).