Specific inhibition of NADPH oxidase 2 modifies chronic epilepsy

Abstract

Recent work by us and others has implicated NADPH oxidase (NOX) enzymes as main producers of reactive oxygen species (ROS) following a brain insult such as status epilepticus, contributing to neuronal damage and development of epilepsy. Although several NOX isoforms have been examined in the context of epilepsy, most attention has focused on NOX2. In this present study, we demonstrate the effect of gp91ds-tat, a specific competitive inhibitor of NOX2, in in vitro epileptiform activity model as well as in temporal lobe epilepsy (TLE) model in rats. We showed that in in vitro seizure model, gp91ds-tat modulated Ca²⁺ oscillation, prevented epileptiform activity-induced ROS generation, mitochondrial depolarization, and neuronal death. Administration of gp91ds-tat 1 h after kainic acid-induced status epilepticus significantly decreased the expression of NOX2, as well as the overall NOX activity in the cortex and the hippocampus. Finally, we showed that upon continuous intracerebroventricular administration to epileptic rats, gp91ds-tat significantly reduced the seizure frequency and the total number of seizures post-treatment compared to the scrambled peptide-treated animals. The results of the study suggest that NOX2 may have an important effect on modulation of epileptiform activity and has a critical role in mediating seizure-induced NOX activation, ROS generation and oxidative stress in the brain, and thus significantly contributes to development of epilepsy following a brain insult.

Keywords: NOX2; Reactive oxygen species; Status epilepticus; Temporal lobe epilepsy; gp91ds-tat.

Graphical abstract

Fig. 1

Gp91 ds-tat modulated Ca²⁺oscillatory signal in neurons. (A) Representative image of synchronous Ca²⁺ oscillatory signal in neurons, indicating epileptiform activity induced by replacement of artificial CSF (aCSF) with low Mg2+ aCSF (indicated by arrow) before, during and after washout of gp91 (5 μM) to the coverslip solution. (B–C) Bar graphs summarizing the effect of gp91 (5 μM) on frequency (B) and the amplitude of Ca²⁺ oscillations recorded from rat neuronal cultures (n = 7 experiments) at 13–17 DIV. (D–E) Bar graphs summarizing the effect of scrambled gp91 ds-tat (control; 5 μM) on frequency (D) and the amplitude of Ca²⁺ oscillations (n = 7 experiments). Data are expressed as mean ± SEM. ***P < 0.001 relative to low-Mg²⁺ condition, by one-way ANOVA with Tukey's post hoc test. ns = not significant.

Fig. 2

Selective inhibition of NOX2 promotes neuroprotection in-vitro. (A) Normalized Rh-123 fluorescence of neurons 5, 10, 20 and 30 min stimulated with either aCSF (n = 6 experiments (exp.), or low Mg²⁺ (n = 6 exp.) and treated acutely either with gp91ds-tat peptide (5 μM; n = 6 exp.) or with scrambled peptide (5 μM). (B) Normalized rates of ROS generation in neurons at 2, 10, and 15 min in aCSF, low Mg²⁺ aCSF, and gp91ds-tat (5 μM) or scrambled peptide-treated neurons in low-Mg²⁺ condition (n = 5 exp for all groups). (C) The percentage of neuronal cell death in cultures following 2 h exposure to aCSF (n = 7), low Mg²⁺ (n = 7), and treatment with either gp91ds-tat (5 μM; n = 7 exp.) or with scrambled peptide (5 μM). Data (Mean ± S.E.M.) were analyzed by either Two-way ANOVA followed by Dunnett's post hoc test (A and B) or one-way ANOVA (C) followed by Tukey's post hoc test. *P < 0.05, **P < 0.01 and ***P < 0.001 versus low Mg²⁺ condition.

Fig. 3

Gp91 ds-tat suppresses mRNA expression of NOX2 and inhibits the NOX activity in the brain following SE. (A) Schematic diagram of experimental design and procedures to determine the NOX2 mRNA expression and NOX activity in the study. (B–C) NOX2 mRNA expression in the cortex (B, n = 6) and the hippocampus (C, n = 6) of gp91ds-tat (400 ng/kg) or its scrambled peptide treated rats following SE (relative to sham group). (D–E) Normalized NOX activity in the cortex (D; n = 6) and the hippocampus (E, n = 6) of gp91ds-tat (or its scrambled peptide) treated rats following SE (relative to sham group). Data are expressed as mean ± SEM. *P < 0.05, **P < 0.01 and ***P < 0.001 versus kainic acid group, by one-way ANOVA with Bonferroni post hoc test.

Fig. 4

Selective therapy with gp91ds-tat inhibits seizure progression and modifies chronic epilepsy. (A) Schematic illustration of experimental setup and timeline. (B) Representative seizures from animals subjected to KA-SE and treated with gp91ds-tat (black) or its scrambled peptide control (red). Scale bar: 10 s and 0.5 mV. (C). Normalized seizure frequency (per week, mean ± SEM) in animals recorded for 3-weeks of baseline, then treated (indicated with arrow) either gp91 ds-tat (800 ng/kg/day for 2 weeks; n = 9) or gp91ds-tat scrambled peptide (800 ng/kg/day for 2 weeks; n = 9). (D) Normalized (to baseline) cumulative number of seizures of animals in C. (E) Total number of seizures after treatment, normalized to baseline (pre-treatment). In C: *P < 0.05, by generalized log-linear mixed model followed by Sidak's post hoc test. In E: **P < 0.01, Student's t-test). . (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)