Anti-depressant natural flavonols modulate BDNF and beta amyloid in neurons and hippocampus of double TgAD mice

Abstract

Increasing evidence suggests that depression may be both a cause and consequence of neurological disorders such as Alzheimer's disease (AD), and that anti-depressants could provide an alternative strategy to current AD therapies. Association of side effect and herbal-drug interaction with conventional anti-depressant and St. John's wort warrant investigating new anti-depressant drugs. Anti-depressant effects of ginkgo biloba extract (EGb 761) have been demonstrated in animal models of depression and in human volunteers. We report here that ginkgo flavonols quercetin and kaempferol stimulates depression-related signaling pathways involving brain-derived neurotrophic factor BDNF/phosphorylation of cyclic AMP response element binding protein CREB/postsynaptic density proteins PSD95, and reduces amyloid-beta peptide (Abeta) in neurons isolated from double transgenic AD mouse (TgAPPswe/PS1e9). In addition, enhanced BDNF expression and reduction of Abeta oligomers was confirmed in hippocampus of the double transgenic mice administered with flavonol, which correlates with cognitive improvement behaviors in these mice. The present results suggest that stimulating BDNF and reducing Abeta toxicity by natural flavonols provide a therapeutic implication for treatment of AD.

Copyright 2009 Elsevier Ltd. All rights reserved.

Figures

Figure 1

Flavonols stimulate levels of BDNF in neurons and hippocampus of double transgenic (TgAPP/PS1) mice. A. Levels of BDNF proteins in cortical neurons (14 DIV), derived from wild type (WT) or double transgenic mice (Tg) E14 embryos, incubated for 24 h with vehicle (Ctrl, 0.01% DMSO) or flavonols (flav, 50μg/ml). B. Levels of BDNF proteins in Tg neurons treated with increasing concentrations of flavonols (12.5, 25, 50μg/ml). Cell lysates were subjected to standard Western blotting using antibody to BDNF. Immunoreactive bands were analyzed from three independent experiments and expressed as percentage of vehicle control. C. Levels of phosphorylated CREB (pCREB) in WT and Tg neurons (14DIV) treated with or without flavonols (50μg/ml) for 24 h. D. Levels of BDNF and pCREB in hippocampus of 8-month old WT or Tg mice brain treated with or without 50 mg/kg flavonols for 4 month (three mice from each group). * p<0.05.

Figure 2

Flavonols stimulate glutamate-evoked phosphorylation of CREB in neurons derived from TgAPP/PS1 mice. A. Levels of phosphorylated CREB in neurons (14 DIV) from E14 embryos of WT or Tg mice treatment with (+) or without (−) glutamate (50μM) for 15min. Cell lysates were subjected to Western blotting using antibody specific to phosphorylated CREB. Immunoreactive bands were analyzed and expressed as percentage of WT control. B. Levels of pCREB in neurons of WT or Tg mice incubated with vehicle (0.01% DMSO) or flavonols (50μg/ml) for 24 h followed by treatment with (+) glutamate (50μM) for 15min. C. Pharmacological blockage of flavonol-enhanced pCREB after glutamate exposure. Neurons were incubated for 24 h with vehicle (DMSO) or 50μg/ml flavonols. 50μM H89, 50μM KN-93, 20μM PD98059 and 50μM APV were applied 1 h prior to glutamate exposure for 15 min. Cell lysates were subjected to Western blotting to detect pCREB. D. Changes of cell surface NMDA receptors NR1 and Postsynaptic proteins PSD95 in WT and Tg neurons (14 DIV). Tg neurons were treated with or without flavonols (flavon) for 24 h. Membrane proteins isolated by biotinylation were subjected to immunoblotting using antibodies to NR1 or PSD95. Immunoreactive bands were analyzed and expressed as percentage of vehicle control. All the results were obtained from three independent measurements and expressed as mean ± SD. *p<0.05.

Figure 3

Flavonols decrease intracellular and extracellular Aβ in transgenic neurons and hippocampus of Tg mice brain. A. Levels of Aβ in neurons derived from Tg mice brain (14 DIV) were incubated with vehicle (0.01% DMSO) or flavonols (50μg/ml) for 24 h. Cell lysates or cultural medium were subjected to Western blotting using monoclonal antibody 6E10 to detect Aβ species. Immunoreactive bands (7 kD for intracellular Aβ blots, the only band at ~ kD for medium Aβ blots) were analyzed and expressed percentage of vehicle control. B. Aβ levels in hippocampus of TgAPP/PS1 mice. 8-month old TgAPP/PS1 mice and age-matched wild type (WT) mice were oral administered with flavonols 50mg/kg or equivalent amount of vehicle daily for 4 months. The brains were removed and hippocampi were dissected. Equal amount homogenous protein was probed with antibody 6E10. The changes of immunoreactive Aβ were analyzed by densitometry and expressed as percentage of vehicle control. Results are obtained from 3 mice and expressed as mean ± SD. C. Immunohistochemical staining of Aβ in hippocampus of the Tg mice after treatment with flavonols (b and b′) for 4 months compared to the control group administered with vehicle (a and a′). (Bar: a & b= 250 μm, a′& b′=20 μm). Images represent at least five sections from three mice brains for each group.

Figure 4

Antidepressant-like activity of flavonols in animal behavioral models. A. Tail suspension test (TST) in 12-month old male WT and Tg mice. Drugs were administered by intraperitoneal injection daily for 7 days. The results represent mean ± SEM (n = 4–8). The data shows significant difference between WT untreated and flavonols treated hippocampus by ANOVA analysis (P= 0.0038). Post hoc Dunnett’s test shows significant difference for the treatment in comparison to control.* P< 0.05 and ** P<0.01. B. Locomotor activity was measured in the same mice tested for TST after one-day recovery. The assay was based on the distance traveled by each mouse in the chamber for 30 min. All results represent the mean ± SEM (n = 4–7). Data shows no significant difference by ANOVA analysis (P= 0.5940) between all groups. Post hoc Dunnett’s multiple comparison tests show no significant difference between the Wt control and any other group (P>0.05). C. Morris water maze assay in WT and Tg mice orally administrated with or without flavonols (50 mg/kg) for 4 month starting from 8-month old. Mice were trained twice every day during 5 consecutive days. The time needed to find the platform was recorded as latency. Results are expressed as mean ± SEM. One-way ANOVA is used to analyze the statistical difference between the groups on each day followed by post hoc dunntte’s multiple comparison test. On the fifth day, flavonol treated transgenic mice shows significant reduction in the latency time in comparison to vehicle-treated Tg mice (* P<0.05).