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Published as doi: 10.1096/fj.06-7649com.
 (The FASEB Journal. 2007;21:2400-2408.)
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EGb 761 enhances adult hippocampal neurogenesis and phosphorylation of CREB in transgenic mouse model of Alzheimer’s disease

Flaubert Tchantchou*, Yanan Xu*, Yanjue Wu*, Yves Christen{dagger} and Yuan Luo*,{ddagger},1

* Department of Pharmaceutical Sciences, School of Pharmacy,

{ddagger} Center for Integrative Medicine, School of Medicine, University of Maryland, Baltimore, Maryland, USA; and

{dagger} Ipsen, Paris, France

1Correspondence: Department of Pharmaceutical Sciences, School of Pharmacy, University of Maryland, Baltimore, MD 21201, USA. E-mail: yluo{at}rx.umaryland.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Standardized Ginkgo biloba extract EGb 761 exhibits beneficial effects to patients with Alzheimer’s disease (AD). It was previously demonstrated that EGb 761 inhibits amyloid beta (Aß) oligomerization in vitro, protects neuronal cells against Aß toxicity, and improves cognitive defects in a mouse model of AD (Tg 2576). In this study, the neurogenic potential of EGb 761 and its effect on cAMP response element binding protein (CREB) were examined in a double transgenic mouse model (TgAPP/PS1). EGb 761 significantly increases cell proliferation in the hippocampus of both young (6 months) and old (22 months) TgAPP/PS1 mice, and the total number of neuronal precursor cells in vitro in a dose-dependent manner. Furthermore, Aß oligomers inhibit phosphorylation of CREB and cell proliferation in the hippocampus of TgAPP/PS1 mice. Administration of EGb 761 reduces Aß oligomers and restores CREB phosphorylation in the hippocampus of these mice. The present findings suggest that 1) enhanced neurogenesis by EGb 761 may be mediated by activation of CREB, 2) stimulation of neurogenesis by EGb 761 may contribute to its beneficial effects in AD patients and improved cognitive functions in the mouse model of AD, and 3) EGb 761 has therapeutic potential for the prevention and improved treatment of AD.—Tchantchou, F., Xu, Y., Wu, Y., Christen, Y., Luo, Y. EGb 761 enhances adult hippocampal neurogenesis and phosphorylation of CREB in transgenic mouse model of Alzheimer’s disease.


Key Words: AD • dementia • neuronal cells • memory


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
ALZHEIMER’S DISEASE (AD) IS THE MOST COMMON cause of dementia among elderly people. This neurodegenerative disorder is believed to start with synaptic dysfunction and subsequent loss of neuronal cells (1) . Evidence of neurogenesis in the adult rodent’s brain (2 3 4 5) raised the hope that replacement of lost neurons could represent a therapeutic approach for management of AD (6) . It is well accepted that neurogenesis occurs throughout adulthood and is confined to two different areas of the brain: the subventricular zone (SVZ) and the dentate gyrus (DG) of the hippocampus (7) . As the epicenter of declarative memory in humans and cognition in many animal species (8 , 9) , the hippocampus is the principal focus of neuronal loss in AD, and thus is of special interest for the neuronal replacement therapeutic approach.

Neurogenesis can be regulated by several factors including age, stress, environmental enrichment, and pathological changes, as well as pharmacological agents (3 , 10 11 12 13) . A relationship between the status of neurogenesis in AD animal models and postmortem patient brains has not yet been defined (14 15 16 17 18) . A better understanding of neurogenesis regulation by pharmacological agents in a defined animal model of AD would strengthen the idea that the normalization of hippocampal neurogenesis could be an important therapeutic intervention in the treatment of AD. The amyloid beta peptide (Aß) and the transcription factor cyclic AMP response element binding protein (CREB) have important effects on the regulation of neurogenesis (19 20 21 22 23 24 25) .

EGb 761 is one of the most commonly prescribed drugs for dementia in many countries and a leading dietary supplement in the United States for memory enhancement (26 , 27) . Several clinical trials have proved its efficacy as a symptomatic treatment for AD (28 29 30 31) , and the clinical evaluation of EGb 761 as a preventive drug is currently under way (32) . The neuroprotective effects of EGb 761 in vitro have been indicated by substantial experimental evidence (26 , 33 34 35 36 37 38) , including improvement in cognitive impairment of transgenic AD mice (Tg 2576) (39) . The neuroprotective mechanism for EGb 761 in vivo has not yet been defined.

Considering the beneficial effects of EGb 761 on cognitive impairment in AD patients (28) and AD mice (39) , and its ability to inhibit Aß oligomerization in vitro (36 , 40) , the present study aimed to examine the neurogenic potential of EGb 761 in the hippocampus of a mouse model of AD (TgAPP/PS1) (41) . The secondary goal was to determine the impact of Aß deposition and/or CREB phosphorylation on neurogenesis after EGb 761 administration. Results from this study demonstrate that the reduction of neurogenesis in the hippocampus of the AD mice is normalized by EGb 761 administration, and that the neurogenic effect of EGb 761 is associated with Aß oligomerization and phosphorylation of CREB.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals and EGb 761 treatment
Heterozygote APPswe/PS1-{Delta}E9 transgenic (TgAPP/PS1) founder mice (42) were provided by Dr. Borchelt at Johns Hopkins University (Baltimore, MD, USA). These mice were maintained by cross-breeding with the wild-type (WT, C57BL/6J) breeders, and resulting offspring were genotyped and used at the age indicated. For in vivo studies, young (6 months) and old (22 months) TgAPP/PS1 and age-matched WT mice (3–4 in each treatment group) were fed for 1 month with a Purina "5001" basal diet (Dyets Inc., Bethlehem, PA, USA), supplemented with or without EGb 761 (Schwabe Pharmaceuticals, Karlsruhe, Germany) for an equivalent ad libitum daily intake of 100 mg/kg. For in vitro studies, day 14 WT mice embryonic brains were used. All animal handling and treatment were approved by the Institutional Animal Care and Use Committee.

BrdU injections and tissue preparation
The TgAPP/PS1 and the WT mice were intraperitonially injected with 75 mg/kg 5-bromo-2-deoxyuridine solution (BrdU; Sigma, St. Louis, MO, USA) daily during the last 7 days of treatment. One day after the last injection, animals were anesthetized with phenobarbital and perfused transcardially with 4% paraformaldehyde in phosphate buffer (PF). The brains were removed, stored overnight in 4% PF, and transferred into 30% sucrose. Coronal sections (30 µm) were cut on a Bright OTF5000 Cryostat (Jencons Scientific Inc., Bridgeville, PA, USA) and stored at –20°C in a cryoprotectant. For Western blotting, the hippocampus was dissected immediately after removal of the brain from non-BrdU-injected mice and stored at –80°C.

Primary cell culture and EGb 761/BrdU treatments
Brain hemispheres from embryonic 14-day-old (E14) WT mice were isolated, rinsed, chopped into small pieces, and suspended in 0.25% trypsin overnight at 4°C, followed by 20 min incubation at 37°C. Dissociated embryonic cells were seeded on coverslips coated with 0.1 µg/ml polylysine at a density of 105 cells/ml in Neurobasal/B-27 supplement (Invitrogen, Carlsbad, CA, USA). The cells were incubated at 37°C for 15 days. On the 11th day cells were cotreated in triplicate for 48 h with different concentrations of EGb 761, 1 µM Rolipram, a neurogenic enhancer (23) , or 50 µM H89, a PKA inhibitor (43) and 20 µg/ml BrdU in the growth medium. The cells were washed and maintained in the growth medium for 3 additional days without EGb 761 and BrdU before being fixed for immunostaining.

Immunofluorescence staining
For in vivo BrdU incorporation, floating sections (30 µm), prepared as described above, were incubated in 50% formamide/50% SSC buffer (0.3 M NaCl/0.03 M sodium citrate) at 65°C for 2 h. The sections were then rinsed in SSC and incubated in 2N HCl for 30 min at 37°C. The sections were blocked with 5% serum for 1 h, then incubated with primary antibodies against BrdU (at 1:600 dilutions, GeneTex, Inc., San Antonio, TX, USA) overnight at 4°C, followed by secondary antibody anti-rat ALEXA FLUOR 488 (1:400, Invitrogen-Molecular Probes) for 2 h. Sections were rinsed, transferred on slides, and cover slipped in an antifading agent (Gel/mount; Biomeda, Foster City, CA, USA). Fluorescent signals were detected with a confocal microscope (NIKON E-2000).

For phenotypic differentiation of BrdU incorporated cells, the sections were incubated with a primary antibody mixture containing antibodies against BrdU, glial fibrillary acidic protein (GFAP),and neuronal nuclei-specific protein (NeuN, 1:300, Chemicon International, Temecula, CA, USA) overnight at 4°C, followed by incubation with secondary antibodies. For double labeling of Aß oligomers and BrdU incorporation, the sections were incubated with a primary antibody mixture containing antibodies against BrdU and Aß oligomers (A11, 1:500, Biosource Inc., Camarillo, CA, USA), followed by incubation with secondary antibodies for 2 h. For double immunostaining for Aß and phospho-CREB, the sections were incubated with primary antibodies to human amyloid beta (6E10) and to phosphorylated CREB (1:500, Cell Signaling Solutions, Lake Placid, NY, USA). The secondary antibodies are anti-mouse ALEXA FLUOR 488 for Aß and anti-rabbit ALEXA FLUOR 594 for pCREB.

For immunostaining of cell proliferation in neuronal progenitor cells, cultured embryonic neuronal cells treated with or without EGb 761 in the presence of BrdU were costained for BrdU incorporation and for the expression of the neuron-specific marker (NeuN). Cells were fixed with 4% PF and blocked with normal serum. They were then incubated in a mixture of primary antibodies that contained antibodies against BrdU and NeuN overnight at 4°C, followed by the secondary antibodies at room temperature for 2 h. Cells were then rinsed and the coverslip from each Petri dish was transferred on a slide and examined under a fluorescence microscope.

Immunoblotting
Standard Western blotting analysis was performed. Hippocampus samples were homogenized in the lysis buffer containing: 50 mM HEPES, pH 7.5, 6 mM MgCl2, 1 mM EDTA, 75 mM sucrose, 2.5 mM benzamidine, 1 mM dithiothreitol, and 1% Triton X-100. Equal amounts of protein (20 µg) were resolved on a 12% SDS-AGE, then transferred to polyvinylidene difluoride membranes and blocked with 5% nonfat dry milk. This was followed by overnight incubation at 4°C with different primary antibodies, which included antibody against polysialic acid neural cell adhesion molecule (NCAM-180, Chemicon Inc., 1:500); phosphorylated CREB (pCREB, 1:1000); CREB (Santa Cruz, Inc., Santa Cruz, CA, USA; 1:500) or human Aß (6E10, Biosource Inc.; 1:750). The blots were then incubated with horseradish peroxidase-conjugated secondary antibodies (Santa Cruz Inc.; 1:5000). Immunoreactivities were detected by an enhanced chemilunescence kit (Amersham Biosciences, Piscataway, NJ, USA).

Quantitative and statistical analyses
Stereology: BrdU-labeled cells in the dentate gyrus of one section out of every seven were counted under high power on a Nikon E2000 microscope with a Magnifier digital camera, and the image was displayed on a computer monitor. BrdU incorporated cells were quantified on dentate gyros (DG) using IPLab analysis software (Scanalytic, Inc., Fairfax, VA, USA). The number of BrdU-positive cells in each subgroup of seven sections was obtained by multiplying the number of those cells found in the dentate gyrus of the section by seven, and the total number of BrdU-positive cells in whole brain’s dentate gyrus was expressed as the sum of those numbers in all subgroups of seven sections from the same brain (3 , 23) .

The immunoreactive bands from Western blots were scanned and the mean density was obtained with AlphaEase FC software (Alpha Innotech, San Leandro, CA, USA). The statistical significance of differences among mice genetic groups, age difference, and/or drug treatment was determined using either 3-way ANOVA or a Student’s t test.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
EGb 761 enhances cell proliferation in the hippocampus of transgenic mice (TgAPP/PS1)
To determine the neurogenic potential of EGb 761, cell proliferation levels were accessed in the dentate gyrus (DG) of the hippocampus of mice treated with or without EGb 761 by immunofluorescence of BrdU incorporated cells. As shown in images of BrdU incorporated cells in the DG of WT or Tg mice (Fig. 1A , a–h), the incorporation of BrdU was observed within the nuclei of dividing or divided cells (arrows). Statistically, Fig. 1B shows that EGb 761 treatment significantly enhanced cell proliferation in the DG of both young (6 months, filled column) and aged (22 months, open column) Tg mice compared with their untreated counterparts (young: Tg/EGb vs. Tg/Ctrl=4427.5±224.33 vs. 2520±63.9 respectively; P<0.05; aged: Tg/EGb vs. Tg/Ctrl=3091.66±274.35 vs. 1435±24.45; P<0.01). Similar effects of EGb 761 administration were also observed in the aged WT mice (22 months, aged=3721.66±294.45) compared with untreated age-matched controls (WT/Ctrl=2298.33±30.86; P<0.01). No significant difference was observed between levels of proliferating cells in the young (6 months) WT mice treated with or without EGb 761 (young WT/Ctrl vs. WT/EGb=5215±225.01 vs. 5553.33±262.95; P=0.3). These results highlight the fact that the EGb 761-enhanced cell proliferation is better manifested in older mice than in younger mice. In addition, it was also found that cell proliferation was significantly decreased with advanced age in WT (young WT/Ctrl vs. old WT/Ctrl 5215±225.01 vs. 2298.33±30.86, P<0.01) as well as in Tg mice (young Tg/Ctrl vs. old Tg/Ctrl=2520±63.90 vs. 1435±24.45, P<0.01). This phenomenon was greatly affected by the genotype. Cell proliferation levels were lower in untreated Tg mice (Tg/Ctrl) than WT age-matched controls (Wt/Ctrl, Young: 5215±225.01 vs. Tg/Ctrl 2520±63.90; Old: 2298.33±30.86 vs. 1435±24.45, P<0.01). Furthermore, the combination of age and genotype showed an augmented deleterious effect on cell proliferation (Young WT/Ctrl vs. Old Tg/Ctrl: 5215±225.01 vs. 1435±24.45, P<<0.01) (Fig. 1A , B).


Figure 1
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  		Figure 1. EGb 761 enhances cell proliferation in the DG of TgAPP/PS1 mice and in primary cultures of embryonic brain cells. A) Representative confocal images showing BrdU-positive cells (green) in the DG of young (6 months, a–d) and old (22 months, e–h), WT (a, b and e, f) and Tg (c, d and g, h) mice maintained on a dietary regiment supplemented with (a, c and e, g) or without (b, d and f, h) 100 mg/kg of EGb 761 for 1 month and intraperitonially injected with BrdU (75 mg/kg/daily) for 7 consecutive days. B) Quantification of BrdU incorporated cells from fluorescence images presented in panel A. BrdU-positive cells were counted in both DG of one brain section taken out of every consecutive seven, and were expressed as the mean ± SE of total number of BrdU-positive cells/DG of 3–4 WT or Tg mice per treatment category (see Materials and Methods for serology). **P < 0.01 (3-way ANOVA). C) Representative immuno-fluorescence images of BrdU (green) and neuronal marker NeuN (red) from the primary culture of WT embryonic brain cells. A range of EGb 761 concentrations (0, 60, 80, 100, or 120 µg/ml) and 20 µM BrdU were coincubated with isolated WT mice embryonic brain cells for 48 h, followed by immunostaining. As controls, the cells were treated in triplicate with Rolipram (1 µM), which is known to stimulate CREB phosphorylation, or a protein kinase A inhibitor (PKAI or H89) that blocks CREB phosphorylation (50 µM) for 48 h intermitted by a change of culture medium containing the treatment. D) Quantification of BrdU-positive cells in primary culture of embryonic brain cells untreated (control) or treated with EGb 761. Values are mean ± SE. *P < 0.05, **P < 0.01 (3-way ANOVA) compared with values from control (three cultures per condition and five field captured per culture). Scale bars = 200 µm.

Enhanced neurogenesis by EGb 761 in neural progenitor cell cultures is concentration dependent and is blocked by H89, an inhibitor of CREB phosphorylation
Pharmacological evaluation of the neurogenic effects of EGb 761 in TgAPP/PS1 mice is time-consuming. To efficiently assess the dose-response effects of EGb 761 on neurogenesis, primary cultures of embryonic brain cells were treated with a range of EGb 761 concentrations (0, 60, 80, 100, and 120 µg/ml), followed by an evaluation of the number of proliferating cells. Representative images of costaining for BrdU and NeuN in cultures treated with or without EGb 761 are shown in Fig. 1C . The number of newly formed neuronal cells significantly increased in response to increasing concentrations of EGb 761 (Fig. 1D ), starting from 80 µg/ml (P<0.05), (P<<0.01 for 100 and 120 µg/ml EGb 761). Cultures treated with 60 µg/ml EGb 761 compared with untreated controls showed no significant neurogenic difference (P=0.07). To further validate these data, we conducted an experiment with a "positive control" using rolipram, which is known to stimulate CREB phosphorylation and subsequent neurogenesis in the animal model and in neuronal cells (44) . Cells treated with rolipram (1 µM) for 48 h significantly enhanced neurogenesis compared with untreated controls. In contrast, EGb-761 (100 µg/ml) -induced nerurogenesis was blocked in cells cotreated with H89 (50 µM for 48 h), which has been reported to block neurogenesis by blocking its upstream transcription factor CREB phosphorylation via inhibition of kinase PKA (43) . These results further confirm the specific effects of EGb 761 on neurogenesis and implicate the involvement of CREB activation.

The majority of the proliferating cells in adult hippocampus are neurons
To determine the proportion of BrdU incorporated cells that were neurons vs. glial cells, coimmunostaining was preformed. Among the 230 BrdU immuno-positive cells examined (from 16 brain sections, 8 for each genotypes), >95% of the newly formed cells were found to differentiate into neurons, and <1% into glial cells (Fig. 2 A). The differentiation of newly formed cells into neurons or glial cells was not affected by EGb 761 treatment, genotype, or age (data not shown).


Figure 2
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  		Figure 2. Characterization of proliferating cell type. A) Immunofluorescent images of the DG of a WT mouse showing BrdU-positive cells (green) colocalizing with NeuN-positive cells (red) and not GFAP-positive cells (blue) in the SGZ, GCL, or H of the DG. Scale bars = 100 µm. B) Representative immunoblots showing levels of NCAM proteins in homogenates of hippocampus of mice treated with or without EGb 761. C) Quantification of NCAM level from the immunoblots of panel B. Results for NCAM (three mice per condition) are expressed as mean density ± SE. **P < 0.01 (Student’s t test).

Next, the expression pattern of the major isomer of the immature neuronal marker NCAM-180 was examined by Western blotting (Fig. 2B ). Quantitative analysis shows a similar pattern (Fig. 2C ), as observed with neurogenesis (Fig. 1B ). A significant increase of NCAM-180 was observed in EGb 761-treated young (6 months, open columns) and aged (22 months, filled columns) Tg mice (Tg/EGb) compared with untreated age-matched controls (Tg/Ctrl, P<0.01). This increase in the expression of NCAM was also present in the aged WT mice maintained on an EGb 761-supplemented diet (open columns, WT/Ctrl vs. WT/EGb, P<0.01). However, no significant difference was found in young (6 months) WT mice treated with or without EGb 761 (filled columns, WT/Ctrl vs. WT/EGb, P=0.5). Moreover, there was a significant decrease of NCAM in the Tg mice (Tg/Ctrl) compared with wild type (WT/Ctrl), and this decrease was augmented with advanced age (22 months, P<0.01), suggesting that expression of the transgenes affects the expression of the immature neuron marker NCAM-180 (Fig. 2B, C ).

Aß oligomers impair cell proliferation and inhibit phosphorylation of CREB in the hippocampus of the transgenic mice
Aß deposition has been shown to impair neurogenesis in cortical neurons and in AD mice (15 , 18) . Next we examined the impact of Aß oligomers on cell proliferation by double labeling BrdU and Aß oligomers using specific antibody A11 (45) . Apparently, Aß oligomers impair cell proliferation, as illustrated in Fig. 3 A: the disruption of the paired division pattern of cells located in close proximity to Aß suggests that the division of cells associated with Aß oligomers was impaired. Those away from the Aß oligomers show a paired or complete division pattern. Subsequently, the effect of Aß aggregation on pCREB expression in the hippocampus of Tg mice was assessed. Figure 3B demonstrates that the aggregation of Aß completely blocks pCREB in the same regions of the DG of the hippocampus (arrows). Furthermore, levels of pCREB and CREB expression in proliferating cells were evaluated by double labeling. Quantitative analysis shows that compared with nonproliferating cells, BrdU-positive cells exhibit a 3-fold increase in pCREB (Fig. 3C , open bars P<0.01), but not CREB (Fig. 3C, P =0.2, filled bars). This suggests that activity of CREB rather than CREB protein synthesis plays a role in mediating cell proliferation.


Figure 3
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  		Figure 3. Colocalization of Aß with BrdU or with pCREB in brain sections of the Tg mice. A) Representative immuno-fluorescence image of the hilus zone of the DG of Tg mice showing that the physical association between Aß (red) and proliferating cells (green) prevents their division. B) Representative image of the DG of the Tg mouse demonstrating that Aß plaques (green) suppress pCREB (red) at their proximity. Scale bars = 200 µm. C) Quantitative analysis of pCREB and CREB levels in proliferating cells compared with respective levels in nonproliferating cells (25 cells per condition) in the DG of WT mice. Results for each protein were expressed as mean fluorescence intensity ± SE. **P < 0.01 (Student’s t test).

EGb 761 reduces Aß oligomers and restores pCREB levels in the hippocampus of transgenic mice
Given the impact of Aß aggregation on cell proliferation and pCREB expression, the effect of EGb 761 treatment on these two proteins was assessed in the brains of Tg mice (12 months old). As demonstrated in Fig. 4 A, B, EGb 761 treatment for 1 month (Tg/EGb) significantly decreases Aß oligomers at a molecular size of ~21 kDa compared with untreated controls (Tg/Ctrl, P=0.02). The reduced pCREB levels in the Tg mice (Tg/Ctrl) compared with that of WT control mice (Wt/Ctrl) was partially restored by EGb 761 treatment vs. EGb 761 untreated WT mice of the same age (P<0.01, Fig. 4C, D ). In WT mice, EGb 761 treatment only induced a moderate increase in pCREB levels when compared with untreated control mice of the same age (P>0.05; Fig. 4C, D ). There was no significant difference in levels of CREB expression in WT and Tg mice treated with or without EGb 761 (P>>0.05, Fig. 4D ). These results suggest that the neurogenic effects of EGb 761 include a possible association between pCREB expression and beta amyloid aggregation.


Figure 4
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  		Figure 4. Aß oligomers and phosphorylation of CREB in the hippocampus of TgAPP/PS1 mice. A) Representative Aß immunoblot (6E10) showing Aß species in the hippocampus of the WT or Tg mice (12 months old) treated with or without EGb 761. B) Quantification of 21 kDa Aß oligomers represented in panel A. C) Immunoblots showing levels of pCREB or CREB in the hippocampus of WT or Tg (12 months old) treated with or without EGb 761. D) Quantitation of pCREB and CREB levels (three per condition) represented in panel C. Results for each protein are expressed as mean density ± SE. **P < 0.01 (Student’s t test).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Neurogenesis is considered to be a neuronal replacement therapeutic approach for neurodegenerative disorders, including AD. In this study, we tested the hypothesis that the beneficial effect of EGb 761 on dementia in humans (28) and on cognitive impairment of AD mice (39) is mediated by enhanced adult neurogenesis in their hippocampus. The present results provide evidence to support this hypothesis by demonstrating that 1) EGb 761 enhances adult neurogenesis in hippocampal neuronal cells of the AD mice (TgAPP/PS1); and 2) neurogenesis by EGb 761 is associated with enhanced phosphorylation of CREB as well as reduced Aß oligomers in the AD mice.

Previous studies by Stackman and colleagues demonstrated that chronic administration of EGb 761 to the Tg2576 mice enhanced spatial learning and cognition (39) . Since Tg2576 mice share the same transgene and cognitive deficits as the TgAPP/PS1 mice used in the present study, EGb 761-induced neurogenesis (Fig. 1) may provide a mechanism through which EGb 761 enhances cognitive performance (39) . Along with observations of increased Aß deposition with advanced age and its association with an increased loss of neuronal cells (18) , these findings underscore the ability of EGb 761 to induce neurogenesis as compensation for the cell loss seen in AD. This notion is further supported by the observation of a similar increase in the pattern of NCAM found in the hippocampus of Tg mice treated with EGb 761 (Fig. 2B, C ). The dose dependency of EGb 761 on neurogenesis assessed by double labeling of embryonic stem cells validates its specificity and provides a tool for initial screening. A good correlation has been reported between neurogenesis in rodent embryonic culture and in the adult hippocampus by Aß and neurogenesis agents (15 , 46) .

The physical association between proliferating cells and toxic Aß oligomers obtained by double labeling for BrdU and Aß oligomers is striking. EGb 761 has been reported to inhibit Aß oligomerization both in solution (36 , 40) and in a transgenic C. elegans model of Alzheimer’s disease (47) in contrast to observations showing no effect of EGb 761 on soluble and fibril Aß levels in the mouse model of AD (39) . An explanation could be that the toxic Aß oligomers and nontoxic monomers are both present in the soluble fraction (48) and that there is equilibrium between the oligomers and the monomers (49) . EGb 761 may prevent oligomerization or shift the equilibrium from the toxic oligomeric conformation to the nontoxic monomers, which would not affect the total level of soluble Aß as determined by ELISA (39) .

The immnuno-histofluorescence double labeling for Aß and pCREB presented in Fig. 3B demonstrates that cells of the GLC, SGZ, and the hilus zone, which are immuno-negative to Aß, are immuno-positive to pCREB antibodies, suggesting that Aß inhibits CREB activation. These observations are consistent with those reported by Vitolo and colleagues (43) , who demonstrated that treatment of hippocampal neurons with Aß (1 µM) inhibited CREB phosphorylation via the inactivation of protein kinase A, which is an upstream kinase for CREB. Furthermore, they showed that down-regulation of protein kinase A and of CREB phosphorylation by Aß could be reversed by rolipram, which enhances the cAMP signaling pathway. Alternatively, PS-1 mutant present in these mice could directly or indirectly down-regulate CREB-CBP signaling as well (50) . To this end, EGb 761 has been reported to increase a small {alpha}APP release, a nontoxic, nonamyloidogenic metabolite of APP, through a PKC-independent manner in hippocampi and cortices of EGb761-treated rats (51) . They proposed that the benefit of EGb761 in clinical studies is underscored by directly affecting the release of the nonamyloidogenic metabolite (51) .

The statistically significant increase in pCREB expression in Tg, but not WT, mice treated with EGb 761 observed in this study is consistent with our previous findings in WT mice (52) . Most important, we observed by double labeling for BrdU incorporation and pCREB expression a 3-fold increase in pCREB levels in BrdU-positive cells compared with BrdU negative ones, indicating that the increased neurogenesis due to EGb 761 treatment would be mediated by an increase in pCREB expression. Taking into account the neurogenic regulating properties of pCREB and its association with Aß and long-term potentiation (53) , it is possible that the enhanced neurogenesis by EGb 761 is associated with its inhibitory effect on Aß oligomerization (Fig. 4A , B). This effect may be augmented by some of EGb 761’s constituents directly regulating the CREB-mediated signaling pathway (38) .

Although other downstream effector of CREB activation by EGb 761 is unknown, it could be multiple (54 , 55) . pCREB has been known to also mediate important neurosurvival signaling pathway by increasing the expression of Bcl-2 and spinophilin (56 , 57) . We previous reported, by microarrary assay, an up-regulation of Bcl-2 in the neuronal cells treated with EGb 761 (37) that would be mediated via CREB activation. Studies using specific pharmacological modulators and/or inhibitors would allow revealing specific mechanism of CREB activation by EGb 761. Primary hippocampal cell culture might provide a better means for pharmacological manipulation and mechanistic studies.

Considering the deleterious effect of the association between aging and Aß in the DG and the role of newly generated neurons in hippocampal-dependent learning and memory (58) , the neurogenic enhancing property of EGb 761 and the potential mechanisms suggested by this study qualify this top-selling dietary supplement to be a therapeutic candidate in the management of Alzheimer’s dementia.


   ACKNOWLEDGMENTS
 
We thank Julie Rich of animal facility for technical assistance, Dr. Ashiwel Undie for helping with the statistical analysis, and Dr. Julie Smith and Miss Marishka Brown for editing and comment on the manuscript. This study is supported by National Institutes of Health grant R01AT001928–03A1 (Y.L.) from the National Center for Complementary and Alternative Medicine, and by IPSEN, Paris, France.

Received for publication October 30, 2006. Accepted for publication February 15, 2007.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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