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Comparative Study
. 2006 Dec 13;26(50):13102-13.
doi: 10.1523/JNEUROSCI.3448-06.2006.

Amyloid-beta-induced pathological behaviors are suppressed by Ginkgo biloba extract EGb 761 and ginkgolides in transgenic Caenorhabditis elegans

Yanjue Wu  1 , Zhixin Wu, Peter Butko, Yves Christen, Mary P Lambert, William L Klein, Christopher D Link, Yuan Luo
Affiliations
Comparative Study

Amyloid-beta-induced pathological behaviors are suppressed by Ginkgo biloba extract EGb 761 and ginkgolides in transgenic Caenorhabditis elegans

Yanjue Wu et al. J Neurosci. .

Abstract

Amyloid-beta (Abeta) toxicity has been postulated to initiate synaptic loss and subsequent neuronal degeneration seen in Alzheimer's disease (AD). We previously demonstrated that the standardized Ginkgo biloba extract EGb 761, commonly used to enhance memory and by AD patients for dementia, inhibits Abeta-induced apoptosis in neuroblastoma cells. In this study, we use EGb 761 and its single constituents to associate Abeta species with Abeta-induced pathological behaviors in a model organism, Caenorhabditis elegans. We report that EGb 761 and one of its components, ginkgolide A, alleviates Abeta-induced pathological behaviors, including paralysis, and reduces chemotaxis behavior and 5-HT hypersensitivity in a transgenic C. elegans. We also show that EGb 761 inhibits Abeta oligomerization and Abeta deposits in the worms. Moreover, reducing oxidative stress is not the mechanism by which EGb 761 and ginkgolide A suppress Abeta-induced paralysis because the antioxidant L-ascorbic acid reduced intracellular levels of hydrogen peroxide to the same extent as EGb 761, but was not nearly as effective in suppressing paralysis in the transgenic C. elegans. These findings suggest that (1) EGb 761 suppresses Abeta-related pathological behaviors, (2) the protection against Abeta toxicity by EGb 761 is mediated primarily by modulating Abeta oligomeric species, and (3) ginkgolide A has therapeutic potential for prevention and treatment of AD.

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Figures

Figure 1.
Figure 1.
Chemical structures of the ginkgolides (A), the bilobalide (B), and three flavonol glycosides (C). The ginkgolides and bilobalide are specific to the G. biloba tree. Ginkgolides A, B, C, J, and bilobalide, but not ginkgolide M, are constituents of EGb 761. The flavonol glycosides quercetin, kaempferol, and isorhamnetin are present in trace amounts in EGb 761 (DeFeudis, 1998).
Figure 2.
Figure 2.
Effect of EGb 761 and its constituents on Aβ-induced paralysis in muscle Aβ strain CL4176. A, Representative images of Aβ-induced paralysis in the transgenic CL4176 strain without transgene expression (no Aβ), untreated with EGb 761 (Ctrl; left panel), and in the transgenic CL4176 strain (muscle Aβ strain) fed with (EGb; right) or without EGb 761 (Ctrl; middle) at 36 h after temperature upshift. B, Time course of paralysis assays in CL4176 fed with different drugs. Synchronized eggs of CL4176 C. elegans were maintained at 16°C, on the 35 × 10 mm culture plates (∼35 eggs/plate) containing vehicle (Ctrl), EGb 761 (100 μg/ml), or CR (139 μg/ml). The hatched worms were grown for 38 h at 16°C followed by upshifting the temperature to 23°C to induce the transgene expression. The paralysis was scored at 60 min intervals. Data are expressed as percentage of nonparalyzed worms from at least three independent assays of 100 worms in each experiment. Paralysis in the transgenic strain CL4176 is attributable to Aβ expression compared with the control strain CL1175, which does not express Aβ transgene (filled triangles). C, Time course of paralysis assay in CL4176 worms fed with ginkgolide A or J (10 μg/ml for each). Paralysis was scored at 30 min intervals. Data were obtained from three different experiments with 100 worms in each group. Error bars indicate SEM. D, An illustrative diagram indicating the time duration of temperature upshift for expressing Aβ transgene, onset of paralysis, detectable Aβ, and EGb protective effect at different treatment regimens.
Figure 3.
Figure 3.
Assays for chemotaxis behavior and serotonin sensitivity in neuronal Aβ-expressing strain CL2355. A, Schematic diagram of the chemotaxis assay. Synchronized L4 worms treated with or without drugs (20 worms for each assay) were placed in the center of an assay plate (100 × 15 mm) containing 1 μl of attractant (0.1% benzaldelhyde) plus 1 μl of 1 m sodium azide at the edge of the plate on one side (black dot), and 1 μl of control odorant (100% ethanol, *) plus 1 μl of 1 m sodium azide on the opposite edge of the plate (star). After 1 h incubation at room temperature, CI was scored as follows: CI = (number of worms at attractant − number of worms at control)/total number of worms. In each experiment, at least 60 worms from each group were analyzed. B, Chemotaxis behavior in neuronal strain CL2355 (neuronal Aβ strain) was reduced compared with the transgenic control strain CL2122 (no Aβ strain). Feeding with 100 μg/ml EGb 761 for 4 d alleviated this descent in the transgenic strain, but not in the control strains (n = 4; *p < 0.05). Error bars indicate SEM. C, Serotonin hypersensitivity in neuronal-expressing Aβ strain (CL2355) is normalized by EGb 761 and its constituents. Strain CL2355 were fed with EGb 761 (100 μg/ml), vehicle (Ctrl; 100 μg/ml), ginkgolides (GA, GB, GC, GJ) (10 μg/ml each), BB (10 μg/ml), quercetin (QC) (10 μg/ml), kaempferol (KA; 10 μg/ml) l-ascorbic acid (VC) (100 μg/ml), and CR (139 μg/ml) for 4 d. The worms were collected at 36 h after temperature upshift to 23°C. 5-HT sensitivity assay was conducted in 96-well plate containing 200 μl of 1 mm 5-HT solution. Percentage of active worms is calculated as follows: number of the active worms (after placed into serotonin solution for 5 min and still kept moving for 5 s)/total worms. A total of 60 worms of each group was used for each time (n = 3; **p < 0.01).
Figure 4.
Figure 4.
Aβ species in the transgenic C. elegans. A, Representative Western blot (BL) of Aβ species in the transgenic C. elegans CL4176 fed with or without EGb 761 (EGb), l-ascorbic acid (VC), and CR. The CL4176 worms maintained at 16°C were fed with a vehicle (Ctrl), EGb 761 (100 μg/ml), or l-ascorbic acid (100 μg/ml), Congo red (139 μg/ml) at day 1 of age for 72 h. The worms were collected, and equal amounts of protein were loaded on each gel lane and immunoblotted with an anti-Aβ antibody (6E10). The arrow indicates changes of Aβ oligomers and the monomers in the worms after the treatment. B, Quantification of immunoreactive Aβ oligomers (the bend at 20 kDa; filled bars) and Aβ monomers (dashed bars) by Gel Documentary System Fluochem SP (Alpha Innotech, San Leandro, CA). Data are expressed as mean density of an indicated band from three independent experiments. Error bars represent SEM. *p < 0.05 C, Aβ species in transgenic C. elegans CL4176 fed with or without EGb 761 (100 μg/ml), GA, GB, GC, GJ, BB, and a flavonol diglycosides quercetin (QC) (10 μg/ml for each component). Aβ oligomers (top) and the protein loading control actin (bottom) are indicated. Note that immunoreactive Aβ monomers (arrow on the right) are enhanced in worms fed with GA. D, Quantification of immunoreactive Aβ oligomers (solid bars) and Aβ monomers (dashed bars) from three independent experiments. Error bars represent SEM. *p < 0.05; **p < 0.01; # p < 0.05 (4 kDa monomer). E, Immunoblot using antibody A11 (anti-oligomers) in the C. elegans strains CL4176 fed with EGb 761 (100 μg/ml) or vehicle (Ctrl) for 4 d. The blot represents three independent experiments. F, Immunoblot using NU-4 (anti-Aβ oligomers) antibody in C. elegans strains CL4176 and CL2355 (-Tg) fed with EGb 761 (100 μg/ml) or vehicle (Ctrl). The blot represents two independent experiments. G, An integrated myo-3/GFP strain (CL2179), as a control for CL4176, was fed with (bottom panel) or without (top) EGb 761 in the same way as CL4176 in A. GFP fluorescence was examined at 36 h after temperature upshift, under a 20× objective on the fluorescence microscope (E800; Nikon). The images were processed by IPLab software 3.7 (Scanalytics, Fairfax, VA). No difference in GFP fluorescence density and intensity were observed in the two groups.
Figure 5.
Figure 5.
Aβ deposits in transgenic C. elegans CL2006 fed with or without drugs. A, Representative images of background fluorescence in a C. elegans without staining (a), with thioflavin S staining in the wild type (b), or in the transgenic strain CL2006 fed with (d) or without EGb 761 (c). Synchronized CL2006 worms were maintained at 20°C on NGM agar plates seeded with E. coli as the food source. The worms were fed with EGb 761 or Congo red for 2 d starting at day 4 of age. At the end of the treatment, the nematodes were fixed in 4% paraformaldehyde, permeabilized, and then stained with 0.125% thioflavin S. β-Amyloid deposits were examined using a fluorescence microscope attached to a digital camera. The numbers of deposits (arrowheads) were scored in the worm head, which is separated from the body by pharyngeal bulb (arrows). B, Quantitative analysis of β-amyloid deposits in the transgenic C. elegans CL2006 fed with different chemicals for 48 h (EGb 761, 100 μg/ml; the flavonoid fraction of EGb 761, 100 μg/ml; Congo red, 139 μg/ml). The quantity is expressed as mean number of β-amyloid deposits/anterior area of the worm (n = 24 for each analysis). Error bars indicate SEM. *p < 0.05; **p < 0.01.
Figure 6.
Figure 6.
Levels of ROS in transgenic C. elegans. A, Levels of H2O2 in the C. elegans fed with different concentration of EGb 761 (0, 25, 50, 100 μg/ml). Age-synchronized groups of transgenic C. elegans strain CL4176 and the control strain CL1175 maintained at 16°C for 38 h were temperature upshifted to 23°C for 38 h followed by the DCF assay for H2O2 described in Materials and Methods. Statistical significance is observed in the worms fed with 100 μg/ml EGb 761 (25 μg/ml, p = 0.13; 50 μg/ml, p = 0.09; 100 μg/ml, p = 0.03; n = 3; total of 120 worms in each group). B, Levels of H2O2 in CL4176 worms fed with vehicle (Ctrl), EGb 761 (EGb), l-ascorbic acid (VC), or CR from 1 d of age until 3 d of age. C, Levels of H2O2 in the C. elegans fed with different constituents of EGb 761 (ginkgolides GA, GB, GC, GJ). At least 60 animals from each group were analyzed for the levels of H2O2. Results are expressed as percentage of fluorescence (%DCF) relative to vehicle-treated controls, which is set as 100%. Statistical significance is represented as follows: *p < 0.05; **p < 0.01. Error bars indicate SEM.
Figure 7.
Figure 7.
Correlation analysis between paralysis (PT50) and 20 kDa Aβ oligomers (A) or with levels of H2O2 (B). Correlation analysis was performed with the GraphPad Prism 4.0a, using a one-tailed Pearson test. The linear regression line along with the 95% confidence interval is shown only for illustrative purposes.
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