Brain-derived neurotrophic factor exerts neuroprotective actions against amyloid β-induced apoptosis in neuroblastoma cells

Abstract

Alzheimer's disease (AD) brains demonstrate decreased levels of brain-derived neurotrophic factor (BDNF) and increased levels of β-amyloid peptide (Aβ), which is neurotoxic. The present study assessed the impact of BDNF on the toxic effects of Aβ_25-35-induced apoptosis and the effects on BDNF-mediated signaling using the MTT assay, western blotting and reverse transcription quantitative polymerase chain reaction. Aβ_25-35 was found to induce an apoptosis, dose-dependent effect on SH-SY5Y neuroblastoma cells, which peaked at a concentration of 20 μM after 24 h. A combination of Aβ_25-35 and BDNF treatment increased the levels of Akt and decreased the level of glycogen synthase kinase-3β (GSK3β) in SH-SY5Y neuroblastoma cells. These findings indicated that BDNF administration exerted a neuroprotective effect against the toxicity of the Aβ_25-35-induced apoptosis in these cells, which was accompanied by phosphoinositide 3-kinase/Akt activation and GSK3β phosphorylation. The mechanisms and signaling pathways underlying neuronal degeneration induced by the Aβ peptide remain to be further elucidated.

Keywords: Akt; brain-derived neurotrophic factor; glycogen synthase kinase 3β; neuroblastoma cell; β-amyloid peptide.

Figure 1

Dose-dependent neurotoxicity of Aβ_25–35 peptides in human SH-SY5Y neuroblastoma cells. Increasing concentrations of Aβ_25–35 were added to the culture medium of cells, and the toxicity was estimated after 24 h using the MTT assay. Values represent the mean ± standard error of the mean for three different cultures, with n=3 dishes/culture for each concentration. ^*P<0.05 compared with the non-treated groups. Aβ, β-amyloid.

Figure 2

Neuroprotective effect of BDNF on SH-SY5Y neuroblastoma cells exposed to Aβ_25–35 (20 μM). Aβ_25–35 (20 μM) was added to cells in the presence of increasing concentrations of BDNF in the culture medium for 24 h, after which toxicity was estimated using the MTT assay. Values represent the mean ± standard error of the mean for three different cultures, with n=3 dishes/culture for each concentration. ^*P<0.05 compared with groups without BDNF treatment. Aβ, β-amyloid; BDNF, brain-derived neurotrophic factor.

Figure 3

Neuroprotective effect of BDNF on SH-SY5Y neuroblastoma cells exposed to Aβ_25–35 (20 μM). Aβ_25–35 (20 μM) was added to cells in the presence of BDNF (10 ng/ml) in medium for 24 h, after which toxicity was estimated by western blotting. Results showed that BDNF levels increased when cells were treated with Aβ_25–35 (20 μM) combined with BDNF (10 ng/ml). Aβ, β-amyloid; BDNF, brain-derived neurotrophic factor.

Figure 4

BDNF attenuates Aβ_25–35-induced apoptosis and inhibition of SH-SY5Y neuroblastoma cells. (A) Flow cytometry assay was performed to visualize the extent of programmed cell death in the control group. (B) Apoptosis of cells that were administered Aβ_25–35 (20 μM). (C) Apoptosis of cells that were administered Aβ_25–35 (20 μM) combined with BDNF (10 ng/ml). (D) Apoptosis of cells that were administered Aβ_25–35 (20 μM) combined with BDNF (10 ng/ml) and LY294002 (20 μM). M2 represents the population of cells with high annexin V binding. Aβ, β-amyloid; BDNF, brain-derived neurotrophic factor.

Figure 5

BDNF-mediated activation of Akt in SH-SY5Y neuroblastoma cells. Representative western blots showing protein levels of phosphorylated- and total Akt in SH-SY5Y cells. The western blots presented are representative of three independent experiments with similar results. ^*P<0.05 vs. the group treated only with Aβ_25–35 (20 μM). Aβ, β-amyloid; BDNF, brain-derived neurotrophic factor.

Figure 6

Levels of mRNA expression of (A) Akt and (B) GSK3β. mRNA expression was measured by reverse transcription quantitative polymerase chain reaction. Results are shown as the mean ± standard error of the mean of three independent experiments. ^*P<0.05 vs. the group treated only with Aβ_25–35 (20 μM). Aβ, β-amyloid; BDNF, brain-derived neurotrophic factor; GSK3β, glycogen synthase kinase-3β.

Figure 7

BDNF-mediated activation of GSK3β in SH-SY5Y neuroblastoma cells. Representative western blots showing the protein levels of phosphorylated (p-GSK3β) and total GKS3β in SH-SY5Y cells. The western blots presented represent three independent experiments with similar results. ^*P<0.05 vs. the group treated with only Aβ_25–35 (20 μM). Aβ, β-amyloid; BDNF, brain-derived neurotrophic factor; GSK3β, glycogen synthase kinase-3β.