Inhibition of amyloid-beta aggregation and caspase-3 activation by the Ginkgo biloba extract EGb761

Abstract

Standardized extract from the leaves of the Ginkgo biloba tree, labeled EGb761, has been used in clinical trials for its beneficial effects on brain functions, particularly in connection with age-related dementias and Alzheimer's disease (AD). Substantial experimental evidence indicates that EGb761 protects against neuronal damage from a variety of insults, but its cellular and molecular mechanisms remain unknown. Using a neuroblastoma cell line stably expressing an AD-associated double mutation, we report that EGb761 inhibits formation of amyloid-beta (Abeta) fibrils, which are the diagnostic, and possibly causative, feature of AD. The decreased Abeta fibrillogenesis in the presence of EGb761 was observed both in the conditioned medium of this Abeta-secreting cell line and in solution in vitro. In the cells, EGb761 significantly attenuated mitochondrion-initiated apoptosis and decreased the activity of caspase 3, a key enzyme in the apoptosis cell-signaling cascade. These results suggest that (i) neuronal damage in AD might be due to two factors: a direct Abeta toxicity and the apoptosis initiated by the mitochondria; and (ii) multiple cellular and molecular neuroprotective mechanisms, including attenuation of apoptosis and direct inhibition of Abeta aggregation, underlie the neuroprotective effects of EGb761.

Figures

Fig 1.

In vitro Aβ fibrillogenesis in the presence or absence of EGb761. (A) Thioflavin T fluorescence assay. Aβ (46 μM) was incubated in the absence (control) or presence of 100 μg/ml of EGb761 (EGb), or 29 μg/ml of bilobalide (BB) or ginkgolides A, B, C, and J (GA, GB, GC, and GJ, respectively) for 96 h at room temperature. The whole extract EGb761 was tested in four independent experiments, the difference was statistically significant: P = 0.0014 by the unpaired two-tailed t test. The individual components were only tested once, therefore no variability or statistical significance is given. (B) Electron microscopy. Aβ peptide was incubated overnight either alone (a) or with 100 μg/ml of EGb761 (b) and examined by electron microscopy. (Bar = 100 nm.) The results were qualitatively reproduced in three independent experiments. (C) Immunoblotting of Aβ species using the antibody 6E10. Lane 1, freshly prepared Aβ; lanes 2 and 3, Aβ incubated without or with EGb761 for 24 h, respectively; lanes 4 and 5, Aβ incubated without or with EGb761 for 8 days, respectively. Arrows indicate Aβ monomers (Aβ) or oligomers (nAβ).

Fig 2.

Aβ fibrillogenesis in mutant neuroblastoma cells in the presence or absence of EGb761. (A) Immunoelectron microscopy of Aβ fibrils in culture media. (a) In vitro aggregated Aβ immunolabeled with the antibody 4G8; (b) the wt cell culture medium collected and immunolabeled with 4G8; (c) the swe/Δ9 cell culture medium collected and probed with 4G8; (d) the culture medium collected from swe/Δ9 cells treated with EGb761 for 48 h and probed with 4G8. a–d represent areas of several grids in three separate experiments. (B) Immunoblotting of Aβ species from the culture media of neuroblastoma cells: purified Aβ (lane 1); wt untreated (wt, lane 2) or treated with 100 μg/ml of EGb761 for 48 h (wt + EGb, lane 3); mutant cell line untreated (swe/Δ9, lane 4) or treated with 100 μg/ml of EGb761 for 48 h (swe/Δ9 + EGb, lane 5). Arrows indicate Aβ monomer (Aβ) or aggregated oligomers (nAβ). The blot represents three independent experiments.

Fig 3.

Mitochondrion-sensitive Aβ cytotoxicity in the mutant cells untreated or treated with EGb761. (A) Representative fluorescence staining for apoptosis in the swe/Δ9 cells with and without drug treatments. Mitochondrial integrity was probed by using Mitosensor (see Materials and Methods). Mitochondria of healthy cells exhibit red fluorescence, cells undergoing apoptosis exhibit green fluorescence. (a) Mutant cells (swe/Δ9) stimulated with 1 μM butyric acid for 12 h to express the transgene; (b) unstimulated wt N2a cells (wt); (c) swe/Δ9 cells pretreated with EGb761 for 48 h; (d) swe/Δ9 cells pretreated with vitamin E (VE) for 48 h. Similar results were obtained in three experiments. (B) Cell death assay by using quantitative trypan blue exclusion. wt or mutant (swe/Δ9) neuroblastoma cells were treated with 100 μg/ml of EGb761 for 48 h and stimulated with 1 μM butyric acid to express the transgene. Results are mean ± SEM (n = 6). EGb761 had a significant effect on cell death in the mutant cell line (P = 0.0015), but not the wt (P = 0.098).

Fig 4.

Caspase-3 activity in neuroblastoma cells in the presence or absence of EGb761. (A) Caspase-3 enzymatic activity assay in the N2a cells (wt), and the swe/Δ9 (swe/Δ9) cells alone, or treated with EGb761 for 48 h. Data are expressed as percentage of the maximum caspase-3 activity in swe/Δ9 cells, which, in average, was equivalent to 1.6 pmol/mg of protein/min. *, statistical significance (P < 0.05, n = 4) by unpaired t test. (B) Representative Western blots of caspase-3 activation in the control and the mutant cells. Lane 1: unstimulated N2a cells; lane 2: N2a cells stimulated with 1 μM butyric acid for 12 h; lane 3: mutant cells stimulated with butyric acid for 12 h; lane 4: mutant cells treated with 100 μg/ml of EGb761 for 48 h before stimulation with butyric acid; lane 5: N2a wt cells treated with 0.1 μM Aβ for 12 h; lane 6: N2a cells treated with 100 μg/ml of EGb761 for 48 h before treatment with 0.1 μM Aβ for 12 h. Arrows indicate cleaved (activated) caspase 3 at about 17 kD. The lower blot is an immunoblot of actin indicating that same amount of proteins were loaded in each lane. Results are representative of two independent experiments.