Mitochondrial oxidative stress and epilepsy in SOD2 deficient mice: attenuation by a lipophilic metalloporphyrin

Abstract

Epileptic seizures are a common feature associated with inherited mitochondrial diseases. This study investigated the role of mitochondrial oxidative stress in epilepsy resulting from mitochondrial dysfunction using cross-bred mutant mice lacking mitochondrial manganese superoxide dismutase (MnSOD or SOD2) and a lipophilic metalloporphyrin catalytic antioxidant. Video-EEG monitoring revealed that in the second to third week of postnatal life (P14-P21) B6D2F2 Sod2(-/-) mice exhibited frequent spontaneous motor seizures providing evidence that oxidative stress-induced mitochondrial dysfunction may contribute to epileptic seizures. To confirm the role of mitochondrial oxidative stress in epilepsy a newly developed lipophilic metalloporphyrin, AEOL 11207, with high potency for catalytic removal of endogenously generated reactive oxygen species was utilized. AEOL 11207-treated Sod2(-/-) mice showed a significant decrease in both the frequency and duration of spontaneous seizures but no effect on seizure severity. A significant increase in the average lifespan of AEOL 11207-treated Sod2(-/-) mice compared to vehicle-treated Sod2(-/-) mice was also observed. Indices of mitochondrial oxidative stress and damage (aconitase inactivation, 3-nitrotyrosine formation, and depletion of reduced coenzyme A) and ATP levels affecting neuronal excitability were significantly attenuated in the brains of AEOL 11207-treated Sod2(-/-) mice compared to vehicle-treated Sod2(-/-) mice. The occurrence of epileptic seizures in Sod2(-/-) mice and the ability of catalytic antioxidant therapy to attenuate seizure activity, mitochondrial dysfunction, and ATP levels suggest that ongoing mitochondrial oxidative stress can contribute to epilepsy associated with mitochondrial dysfunction and disease.

Figure 1. Behavioral seizure parameters in Sod2^−/− mice

The average duration (A) and number (B)of spontaneous behavioral seizures monitored by digital recordings from vehicle or AEOL11207 treated Sod2^−/− mice from 17 to 20 days old. Bars represent mean or mean + S.E.M, *p < 0.05, compared to the same treatment of 17 and 18 day old mice and # p < 0.05 compared to age-matched vehicle-treated mice. Data were analyzed by one way ANOVA (Figure 1A) n=46 mice (23 each vehicle- or AEOL 11207-treated mice).

Figure 2. Spontaneous electrographic seizure parameters in Sod2^−/− mice

Dural EEG recording from a P18 (A) Sod2^+/+ mouse showing no seizure activity as a result of electrode implantation, (B) vehicle-treated Sod2^−/− mouse showing a period of continuous seizure activity, (C) vehicle-treated Sod2^−/− mouse showing a period of isolated seizure activity, (D) AEOL 11207-treated Sod2^−/− mouse showing the attenuation of seizure activity. (E) Averaged inter-seizure interval and (F) seizure duration of electrographically recorded spontaneous seizures in vehicle or AEOL 11207-treated Sod2^−/− mice. n=5 per treatment group. Bars represent mean + S.E.M, **p<0.01; Mann-Whitney non-parametric test.

Figure 3. Neuropathological damage in Sod2^−/− mice

Panel 1: Representative H&E and Fluoro-Jade B staining images in the parietal cortex of P15–16 Sod2^+/+ or Sod2^−/− mice receiving either vehicle or AEOL 11207 treatment. H&E staining (A, B, C) and Fluoro-Jade B staining (D, E, F). Control (A, D), vehicle- (B, E) and AEOL 11207-treated (C, F). The insets on the upper right corner of each picture are the enlarged image from the white rectangle. Panel 2: Quantitative analysis of Fluoro-Jade B fluorescence in the parietal cortex of P15–16 Sod2^+/+ or Sod2^−/− mice receiving either vehicle or AEOL 11207 treatment. The Fluoro-Jade B positive signal in a given area of parietal cortex from both hemispheres of each animal was estimated with Image J. Bars represent mean + S.E.M, *p<0.01 vs. Sod2^+/+ mice with same treatment; #p<0.05 vs. vehicle-treated Sod2^−/− mice; two way ANOVA, n=6 mice per group.

Figure 4

Markers of oxidative stress in Sod2^−/− mice were attenuated by AEOL 11207 treatment. (A) CoASH, (B) CoASSG, (C) aconitase activity, and (D) 3-NT levels in mitochondrial fractions of P15–16 Sod2^+/+ or Sod2^−/− mice receiving either vehicle or AEOL 11207 treatment. Bars represent mean + S.E.M, *p<0.01 vs. Sod2^+/+ mice with same treatment; #p<0.05 vs. vehicle-treated Sod2^−/− mice; two-way ANOVA, n=6–12 mice per group.

Figure 5

AEOL 11207 partially rescued altered bioenergetics and mitochondrial ETC complexes in Sod2^−/− mice. (A) complex I activity, (B) complex II activity and (C) ATP levels from Sod2^+/+ plus vehicle or AEOL 11207-treated Sod2^−/− mice. Bars represent mean + S.E.M, *p<0.01 vs. Sod2^+/+ mice with same treatment; #p<0.05 vs. vehicle-treated Sod2^−/− mice; two way ANOVA, n=5–8 mice per group.

Figure 6

Survival curve of Sod2^−/− mice treated with AEOL 11207 or vehicle. Lifespan from 72 vehicle and 21 AEOL 11207 treated Sod2^−/− mice was analyzed by a Kaplan-Meier survival curve. *p<0.01 vehicle vs AEOL 11207.