Protection against beta-amyloid induced abnormal synaptic function and cell death by Ginkgolide J

Abstract

A new Ginkgo biloba extract P8A (TTL), 70% enriched with terpene trilactones, prevents A beta(1-42) induced inhibition of long-term potentiation in the CA1 region of mouse hippocampal slices. This neuroprotective effect is attributed in large part to ginkgolide J that completely replicates the effect of the extract. Ginkgolide J is also capable of inhibiting cell death of rodent hippocampal neurons caused by A beta(1-42). This beneficial and multi-faceted mode of action of the ginkgolide makes it a new and promising lead in designing therapies against Alzheimer's disease.

Figure 1

Structures of native terpene trilactones from Ginkgo biloba extract and a reduced derivative, GA-triether.

Figure 2. Aβ-induced LTP impairment in the CA1 region of hippocampal slices and its reversal by P8A

P8A reverses Aβ-induced LTP impairment in the CA1 region of hippocampal slices (n = 8 slices from 8 mice for P8A, 6 slices from 6 mice for Aβ, and 6 slices from 6 mice for vehicle). Both Aβ and P8A did not affect baseline transmission in experiments where no tetanus was applied (n = 3 slices from 3 mice both for P8A, Aβ and vehicle). The horizontal bar indicates the period during which Aβ (200 nM) and P8A (200 μg/ml) were added to the bath solution. The arrows indicate the time at which the theta-burst stimulation was applied in this and the following figures. Every fourth recording point is shown for clarity in this and the following figures. Values represent mean ± SEM (Two-way ANOVA F(1, 12)=10.64, P<0.01 for P8A + Aβ vs. Aβ alone).

Figure 3. Effect of individual ginkgolides and bilobalide on Aβ-induced LTP impairment in CA1 region of hippocampal slices

A) Summary graph showing that 20 min treatment with 1 μM GJ, GA and GA-triether rescues LTP impairment in slices treated with 200 nM Aβ_1-42 for 20 min prior to LTP induction (n = 9 slices from 8 mice for GJ + Aβ, 8 slices from 8 mice for GA + Aβ and 8 slices from 7 mice for GA-triether + Aβ, 6 slices from 6 mice for Aβ alone). Similar concentrations of GJ, GA and GA-triether did not affect baseline transmission in experiments where no tetanus was applied (n = 4 slices from 4 mice for each group; data not shown). Two way ANOVA: F(1, 13)=12.08, P<0.01 for GJ + Aβ vs. Aβ alone; F(1, 12)=10.02, P<0.01 for GA + Aβ vs. Aβalone; F(1, 12) = 5.83, P<0.05 for GA-triether + Aβ vs. Aβ alone). B) A dose-response curve for GJ effect on Aβ-induced LTP impairment indicates that GJ has its maximal beneficial effect at 1 μM (n = 12 slices from 12 mice for each point). Nonlinear regression analysis was used to fit curves for different concentrations using GraphPad Prism software (GraphPad Software Inc., San Diego, CA). C) Summary graph showing that 20 min treatment with 5 μM (but not 1 μM) GB re-establishes normal LTP in slices treated with 200 nM Aβ_1-42 for 20 min prior to LTP induction (n = 12 slices from 10 mice for 5 μM and 8 slices from 8 mice for 1 μM). Both 5 and 1 μM concentrations GB did not affect baseline transmission in experiments where no tetanus was applied (n = 4 slices from 4 mice for each group; data not shown). Two way ANOVA: F(1, 16)=9.47, P<0.01 for 5 μM GB + Aβ vs. Aβ alone; and F(1, 12)=1.85, P>0.05 for 1 μM GB + Aβ vs. Aβ. Alone. D) Summary graph showing that 20 min treatment with the ginkgolides GC as well as the bilobalide BB does not rescue LTP impairment in slices treated with 200 nM Aβ for 20 min prior to LTP induction (n = 9 slices from 7 mice for GC, and 7 slices from 7 mice for BB). Both GC and BB did not affect baseline transmission in experiments where no tetanus was applied (n = 4 for each group; data not shown). The horizontal bar indicates the period during which these drugs were added to the bath solution. Experiments were interleaved with each other. Two way ANOVA: F(1, 13)=0.98, P>0.05 for GC + Aβ vs. Aβ alone and F(1, 11)=0.78, P>0.05 for BB + Aβ vs. Aβ alone. Every fourth recording point is shown for clarity.

Figure 4. Residual potentiation during the last 10 min, following administration of 1 μM ginkgo derivatives

Residual levels of potentiation were averaged during the last 10 min of LTP. P8A, GJ, GA and GA-triether rescued the LTP impairment (Student-Newman-Keuls multiple comparison test, p<0.05 for Aβ vs P8A + Aβ, Aβ vs GJ + Aβ, Aβ vs GA + Aβ, and Aβ vs GA-triether + Aβ, *). Vehicle-no tetanus = 100.0% ± 4.69. Tetanus + vehicle = 246.32 ± 24.0. Tetanus + Aβ = 148.85% ± 9.5. Tetanus + P8A = 246.33% ± 24.1. Tetanus + GJ = 252.25% ± 29.9. Tetanus + GA = 214.72% ± 26.4. Tetanus + GA-triether = 200.23% ± 22.9. Tetanus + GB = 173.45% ± 24.0. Tetanus + GC = 163.31% ± 29.3. Tetanus + BB = 131.09% ± 11.3.

Figure 5. P8A and GJ protect cultured hippocampal neurons treated with oligomeric Aβ peptide against cell death

Hippocampal cultures were treated by adding 10 μM Aβ1-42 in its oligomeric form with or without P8A at 50 μg/ml, or alternatively each one of the ginkgolides (GA, GJ) at a concentration of 1 μM. After 24 hrs the number of viable cells was assessed using cell nuclei counting. Aβ induced death of ~50% of the cell population. Both the P8A and GJ, but no GA or any of the other ginkgolides were consistently able to prevent cell death (Student-Newman-Keuls multiple comparison test, p<0.01 for Aβ vs P8A + Aβ, **; p<0.05 for Aβ vs GJ + Aβ, *). CTRL = Control, 100%. Aβ = 48.5% ± 2.4. P8A + Aβ = 76.0 ± 7.9. GJ + Aβ = 70.7 ± 1.4. GA + Aβ = 50.5 ± 5.7. GB ± Aβ = 48.5 ± 7.0. GC ± Aβ = 45.5 ± 9.0. BB ± Aβ = 46.5 ± 10.0. Values represent mean ± SEM of three consecutive experiments. Each experiment was performed in triplicate.