Prolonged exposure of cortical neurons to oligomeric amyloid-β impairs NMDA receptor function via NADPH oxidase-mediated ROS production: protective effect of green tea (-)-epigallocatechin-3-gallate

Abstract

Excessive production of Aβ (amyloid β-peptide) has been shown to play an important role in the pathogenesis of AD (Alzheimer's disease). Although not yet well understood, aggregation of Aβ is known to cause toxicity to neurons. Our recent study demonstrated the ability for oligomeric Aβ to stimulate the production of ROS (reactive oxygen species) in neurons through an NMDA (N-methyl-D-aspartate)-dependent pathway. However, whether prolonged exposure of neurons to aggregated Aβ is associated with impairment of NMDA receptor function has not been extensively investigated. In the present study, we show that prolonged exposure of primary cortical neurons to Aβ oligomers caused mitochondrial dysfunction, an attenuation of NMDA receptor-mediated Ca2+ influx and inhibition of NMDA-induced AA (arachidonic acid) release. Mitochondrial dysfunction and the decrease in NMDA receptor activity due to oligomeric Aβ are associated with an increase in ROS production. Gp91ds-tat, a specific peptide inhibitor of NADPH oxidase, and Mn(III)-tetrakis(4-benzoic acid)-porphyrin chloride, an ROS scavenger, effectively abrogated Aβ-induced ROS production. Furthermore, Aβ-induced mitochondrial dysfunction, impairment of NMDA Ca2+ influx and ROS production were prevented by pre-treatment of neurons with EGCG [(-)-epigallocatechin-3-gallate], a major polyphenolic component of green tea. Taken together, these results support a role for NADPH oxidase-mediated ROS production in the cytotoxic effects of Aβ, and demonstrate the therapeutic potential of EGCG and other dietary polyphenols in delaying onset or retarding the progression of AD.

Figure 1. Neuronal toxicity of oligomeric Aβ

Oligomeric Aβ was prepared as indicated in the Materials and methods section. (A) The sample from a typical preparation was analysed by Western blot showing the presence of trimers and tetramers as well as monomers after aggregation in PBS at 4°C for 24 h. Positions of molecular mass markers are shown on the left in kDa. (B) Aβ was aggregated for different time periods (0, 4, 12 and 24 h) at 4°C and then 1 μM was incubated with rat primary cortical neurons for 24 h. Results depict mitochondrial function using the MTT assay. (C) Neurons were treated with or without Aβ (1 μM) for 24 h followed by assay of LDH release as an indication of a loss in membrane integrity. (D) Neurons were treated with or without oligomeric Aβ (1 μM) for 24 h and neuronal viability was determined using the AlarmaBlue™ assay. (E) Rat primary cortical neurons were incubated with or without oligomeric Aβ_1-42 (1 μM) or Aβ_42-1 (1 μM) for 0, 8, 16 or 24 h, and mitochondrial function was measured using the MTT assay. Results are mean±S.E.M. values for three independent experiments and are analysed by one-way ANOVA followed by Bonferroni's multiple comparison tests.*P<0.05 and ***P<0.001, as compared with control.

Figure 2. Prolonged exposure of neurons to Aβ impairs NMDA receptor-mediated Ca²⁺ influx

Primary cortical neurons were cultured on coverslips and exposed to oligomeric Aβ_1-42 (1 μM) or Aβ_42-1 (1 μM) for 16 h. Neurons were labelled with Fluo-4-AM for 30 min before stimulation with NMDA (50 μM) for 30 s in ACSF medium containing nimopridine (1 μM) without Mg²⁺, and then followed by perfusion with ACSF medium with Mg²⁺. Average fluorescence of Fluo-4 was measured as described in the Materials and methods section. (A) Representative curves depicting NMDA-induced Ca²⁺ influx with or without oligomeric Aβ for 16 h. (B) Representative fluorescent images of neurons taken before and during (30 s) NMDA stimulation. (C) Bar graphs to depict NMDA-induced Ca²⁺ influx as determined by calculating the area under the curve using the GraphPad Prism Software 4. Results are means±S.E.M. for three independent experiments and are analysed by one-way ANOVA followed by Bonferroni's multiple comparison tests.* P<0.05, as compared with control.

Figure 3. Prolonged exposure of neurons to Aβ inhibits NMDA receptor-mediated AA release

Neurons were treated with Aβ (1 μM) for 0, 4, 12 and 24 h, and subsequently labelled with [1-¹⁴C]AA for the final 4 h. After removing free [1-¹⁴C]AA, neurons were stimulated with NMDA (100 μM) for 30 min and [1-¹⁴C]AA release from phospholipids into the medium was measured after subtracting background release. Results are means±S.E.M. for three independent experiments and are analysed by one-way ANOVA followed by Bonferroni's multiple comparison tests; *P<0.05 and **P<0.01, as compared with control.

Figure 4. Prolonged exposure to Aβ induces ROS accumulation in neurons

(A) Neurons were pre-treated with Aβ (1 μM) for 30 min or 16 h, and then loaded with DHE. In each field, DHE fluorescent neurons and the corresponding bright field images were recorded. (B) Average fluorescent intensity of neurons was calculated. Results are means±S.E.M. for three independent experiments and analysed by one-way ANOVA followed by Bonferroni's multiple comparison tests. **P<0.01 as compared with control.

Figure 5. NADPH oxidase inhibitor, gp91ds-tat, inhibits ROS production and protects neurons against Aβ-induced cytotoxicity

(A) Rat primary cortical neurons were treated with gp91ds-tat (1 μM) for 30 min before exposure to Aβ (1 μM) for 16 h and followed by ROS determination as described for Figure 4. Results represent the average fluorescent intensity of DHE in neurons and are means±S.E.M. for three independent experiments. Two-way ANOVA revealed a significant interaction (P = 0.0072) and significant effects of Aβ (P = 0.0039) and gp91ds-tat (P = 0.0180). Bonferroni post-tests showed a significant difference between control and Aβ (**P<0.01) and between Aβ treatment without versus with gp91ds-tat (∧P<0.05). (B) Neurons were treated with gp91ds-tat (1 μM) for 30 min before exposure to Aβ (1 μM) for 16 h followed by the MTT assay. Results are means±S.E.M. for three independent experiments. Two-way ANOVA revealed a significant interaction (P = 0.0320) and significant effects of Aβ (P = 0.0160) and gp91ds-tat (P = 0.0433). Bonferroni post-tests showed a significant difference between control and Aβ (*P<0.05) and between Aβ treatment without versus with gp91ds-tat (∧P<0.05). (C) Neurons were incubated with gp91ds-tat (1 μM) for 30 min before exposure to Aβ (1 μM) for 16 h, and NMDA (50 μM)-induced Ca²⁺ influx was determined as described in Figure 2 Results are means±S.E.M. for three independent experiments. Two-way ANOVA revealed a significant interaction (P = 0.0167) and significant effects of Aβ (P = 0.0095) and gp91ds-tat (P = 0.0027). Bonferroni post-tests showed a significant difference between control and Aβ (*P<0.05) and between Aβ treatment without versus with gp91ds-tat (ˆˆP<0.01).

Figure 6. EGCG protects neurons against Aβ-induced cytotoxicity

(A) Rat primary cortical neurons were treated with EGCG (10 μM) for 30 min before exposure to Aβ (1 μM) for 16 h, and followed by determination of mitochondrial function using the MTT assay. Results are means±S.E.M. for three independent experiments. Two-way ANOVA revealed a significant interaction (P = 0.0139), and significant effects of Aβ (P = 0.0149) and EGCG (P = 0.0172). Bonferroni post-tests showed a significant difference between control and Aβ (**P<0.01) and between Aβ treatment without versus with EGCG (∧P<0.05). (B) Neurons were incubated with EGCG (10 μM) for 30 min before exposure to Aβ (1 μM) for 16 h, followed by determination of NMDA-induced Ca²⁺ influx as described in Figure 2. Analysis by two-way ANOVA revealed a significant interaction between EGCG and Aβ (P = 0.032), and a significant effect of Aβ (P = 0.027). Bonferroni post tests showed a significant difference between control and Aβ (*P<0.05). (C) Neurons were treated with EGCG (10 μM) or MnTBAP (20 μM) for 30 min before exposure to Aβ (1 μM) for 16 h, followed by determination of ROS as described in Figure 4. Results are means±S.E.M. for three independent experiments. Two-way ANOVA revealed a significant interaction between Aβ and EGCG (P = 0.0109), and significant effects of Aβ (P = 0.0075) and EGCG (P = 0.0184). A significant interaction between Aβ and MnTBAP (P = 0.0183) was also found. Bonferroni post-tests showed a significant difference between control and Aβ (**P<0.01) and between Aβ treatment without versus with EGCG or MnTBAP (ˆˆP<0.01).

Figure 7. A scheme depicting possible mechanisms for neuronal impairment due to prolonged treatment with Aβ and possible sites for the protective effects of NADPH oxidase inhibitor and EGCG