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

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 Aβ25–35-induced apoptosis and the effects on BDNF-mediated signaling using the MTT assay, western blotting and reverse transcription quantitative polymerase chain reaction. Aβ25–35 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 Aβ25–35 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 Aβ25–35-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.


Introduction
Alzheimer's disease (AD) is a neurodegenerative disorder of the human brain and is associated with loss of memory and cognitive abilities (1). AD is characterized by amyloid plaques, neurofibrillary tangles and neuronal loss (2). In AD brains, there is also an abundance of two abnormal structures: Senile plaques composed of β-amyloid peptide (Aβ), that are deposited outside neuronal bodies, and neurofibrillary tangles, which are aggregates of hyperphosphorylated tau proteins that bind to microtubules within the neurons (3). Synaptic dysfunction in AD may be caused by accumulation of aggregated amyloid peptides (3). Aβ, a 40-42 amino acid peptide fragment of the Aβ precursor, has been shown to be a key pathological feature in the formation of senile plaques (4). Aβ peptides induce cell death, decrease survival rate, and increase inflammation, oxidative stress and neurotoxicity in in vitro models used to study AD (5,6).
Brain-derived neurotrophic factor (BDNF) is an important neurotrophin that has been extensively studied and that may play a role in the pathology of AD. BDNF is involved in the structural and functional plasticity of the brain (7), protects neurons in the brain against insults (8) and plays a role in neural development and maintenance of the central and peripheral neurons (9). Another important intracellular regulatory protein is glycogen synthase kinase-3β (GSK3β). This protein is phoshorylated by growth factor-stimulated signaling pathways (10). GSK3β is a protein kinase that also has regulatory effects on neuronal survival and plasticity. Previously, a study indicated that GSK3β may play a part in AD and that its deregulation may account for a number of the pathological hallmarks of AD (11).
In the present study, the impact of BDNF on the toxic effects of the Aβ-induced apoptosis was examined via the phosphoinositide 3-kinase (PI3K)/Akt signaling pathway in SH-SY5Y neuroblastoma cells.

Materials and methods
Cell culture. Human SH-SY5Y neuroblastoma cells were maintained in Dulbecco's modified Eagles's medium (DMEM) and F-12 (Gibco-BRL, Gaithersburg, MD, USA) supplemented with 10% fetal bovine serum (FBS; HyClone, Logan, UT, USA) in a humidified atmosphere of 5% CO 2 and 95% air at 37˚C. The media was replaced every two days. Prior to the experiments, the SH-SY5Y cells were plated in 96-well plates at a density of ~1.5x10 4 cells per well (for MTT) and in six-well plates at 8x10 6 cells per well (all other assays). For the experiments, the cells were incubated with agents for 24 h at 37˚C. For a single experiment, each treatment was performed in triplicate.
Cell proliferation and MTT assay. A cell survival analysis was performed according to the MTT (Cell Titer 96 Aqueous Cell Proliferation Assay kit; Promega, Madison, WI, USA) assay method. Briefly, the cells were cultured with Aβ (0-20 µM) or BDNF (0-30 ng/ml), and 10 µl of 4 mg/ml MTT solution was added to each well of the 96-well plate. The cells were subsequently incubated for 4 h in the dark. The absorbance was measured in a microplate reader at 490 nm, and the results were expressed as a percentage of the control. Western blotting. The cells were washed in fresh phosphatebuffered saline, homogenized in lysis buffer and centrifuged. Protein concentration was determined by the Bradford assay. The purified proteins were separated by polyacrylamide gel electrophoresis (SDS-PAGE) and the resolved proteins were transferred to a nitrocellulose membrane. Each membrane was incubated overnight with a 1:1000 dilution of the primary antibody at 4˚C. The membranes were treated with a 1:1000 dilution of peroxidase-conjugated secondary anti-rabbit or anti-mouse antibodies for 2 h. The proteins were detected using the enhanced chemiluminescence western blotting method (GE Healthcare, Piscataway, NJ, USA). Densitometric quantification of the bands was performed using ImageJ software version 1.29x (National Institutes of Health, Bethesda, MD, USA) (12).
Statistical analysis. Data were obtained from three separate cultures and expressed as the mean ± standard error of the mean. Statistical comparison was determined by an analysis of variance test with the Student's t-test as the post hoc test. P<0.05 was considered to indicate a statistically significant difference.
Effect of increasing BDNF concentrations on Aβ 25-35 -treated cells. The ability of BDNF to protect SH-SY5Y cells against  [25][26][27][28][29][30][31][32][33][34][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. Aβ 25-35 -induced toxicity using the MTT assay was examined (Fig. 2). BDNF was able to protect SH-SY5Y cells from 20 µM Aβ 25-35 -induced toxicity. This protective effect of BDNF was dose-dependent, and the effect was significant ≥10 ng/ml. In Fig. 3, the results from a western blot analysis are shown, indicating that BDNF levels increased following exposure of the cells to treatment with a combination of 20 µM Aβ 25-35 and 10 ng/ml BDNF.

BDNF reduces the apoptosis in SH-SY5Y cells.
To examine whether β 25-35 -induced cell death is apoptotic-like, the flow cytometry assay was performed (Fig. 4). In the control, apoptotic cells comprised 7.8% of the total number of cells. Following exposure to 20 µM Aβ 25-35 for 24 h, the number of apoptotic cells increased to 42.2%. This increase was prevented by addition of BDNF (10 ng/ml). To further investigate, LY294002 (20 µM), the inhibitor of Akt, was used. The apoptosis of cells increased to 34.6% following the use of LY294002 (20 µM).
The PI3K/Akt pathway is important for cell survival. PI3K enhances neuroprotection by regulating the level of phosphorylation and activation of Akt. Akt activity can be modulated by phosphorylation of either Thr308 or Ser473 (20). Phosphorylation of Thr308 has been reported to be stimulated by dopamine receptor activation, while   Aβ, β-amyloid; BDNF, brain-derived neurotrophic factor; GSK3β, glycogen synthase kinase-3β.

A B
phosphorylation of Ser473 is regulated by the activation of the N-methyl-D-aspartate receptor (21)(22)(23). Therefore, phosphorylation and activation of Akt may underlie the observed protective effect of BDNF. GSK3β is a substrate of Akt, which phosphorylates and inhibits GSK3β. Furthermore, GSK3β is subject to inhibitory regulation by growth factors that activate the PI3K/Akt pathway (24). Beaulieu et al (25) reported that activation of the PI3K/Akt pathway can increase the phosphorylation of GSK3β, thereby inhibiting the activity of the latter; thus, Akt may be a regulator of GSK3β. The present study indicated that the neuroprotective effect of BDNF against Aβ 25-35 toxicity was mediated by the inhibitory effect of Akt on GSK3β. In conclusion, the data showed that administration of BDNF exerts neuroprotective actions against the toxic effect of the Aβ 25-35 -induced apoptosis in SH-SY5Y cells, which involved PI3K/Akt activation and GSK3β phosphorylation. The mechanism and signaling pathways underlying neuronal degeneration induced by the Aβ peptide remain to be further elucidated.