Mitochondrial DNA damage and impaired base excision repair during epileptogenesis

Abstract

Oxidative stress and mitochondrial dysfunction are acute consequences of status epilepticus (SE). However, the role of mitochondrial oxidative stress and genomic instability during epileptogenesis remains unknown. Using the kainate animal model of temporal lobe epilepsy, we investigated oxidative mitochondrial DNA (mtDNA) damage and changes in the mitochondrial base excision repair pathway (mtBER) in the rat hippocampus for a period of 3 months after SE. Acute seizure activity caused a time-dependent increase in mitochondrial, but not nuclear 8-hydroxy-2-deoxyguanosine (8-OHdG/2dG) levels and a greater frequency of mtDNA lesions. This was accompanied by increased mitochondrial H2O2 production and a transient decrease in mtDNA repair capacity. The mtBER proteins 8-oxoguanine glycosylase (Ogg1) and DNA polymerase gamma (Pol gamma) demonstrated elevated expression at mRNA and protein levels shortly after SE and this was followed by a gradual improvement in mtDNA repair capacity. Recurrent seizures associated with the chronic phase of epilepsy coincided with the accumulation of mtDNA damage, increased mitochondrial H2O2 levels, decreased expression of Ogg1 and Pol gamma and impaired mtDNA repair capacity. Together, increased oxidative mtDNA damage, mitochondrial H2O2 production and alterations in the mtBER pathway provide evidence for mitochondrial oxidative stress in epilepsy and suggest that mitochondrial injury may contribute to epileptogenesis.

Fig. 1

The development of spontaneous seizures in kainate-treated rats. Rats were treated with kainate (12mg/kg; s.c) to initiate SE and seizures scored as described in Materials and Methods. Behavioral observations of seizure activity were monitored for 6hr a week and rats that suffered ≥ 2 spontaneous seizures were considered to be epileptic. Saline treated controls did not show any seizure activity or modulation of oxidative stress indices throughout the study. For each animal A) the most severe motor seizure score and B) length of each seizure was determined at the indicated time point after kainate administration. Bars represent mean ± SEM, n=3–4 per time point.

Fig. 2

A comparison of 8-OHdG generation in the mitochondrial and nuclear genomes following SE. Hippocampal tissue was harvested at the indicated time points, DNA extracted and HPLC performed as described in Materials and Methods. DNA damage was expressed as the ratio of oxidized DNA base (8-OHdG) to non-oxidized base (2-dG) in mtDNA and nDNA. Bars represent mean ± SEM. *P<0.05 mtDNA vs. saline treated controls, #P<0.05 mtDNA vs. nDNA, one way ANOVA, n= 6 per group.

Fig. 3

A comparison of mitochondrial and nuclear DNA damage during epileptogenesis. Hippocampal tissue was harvested at the indicated time points, DNA extracted and QPCR performed. The graph represents the number of DNA lesions per 10kb in a mitochondrial or nuclear gene fragment. Data was normalized to 250bp fragments amplified from saline treated controls. A) mtDNA and nDNA lesions per 10kb at the indicated time points after kainate treatment. B and C) Agarose gels derived from QPCR analysis of hippocampal tissue after kainate treatment. Lane 1, CON; lane 2, 24h; lane 3; Lane 4, 48h; Lane 5, 96h; Lane 6, 7d; Lane 7, 21d; Lane 8, 3m. Bars represent mean ± SEM. *P<0.05; **P<0.01, vs. saline treated controls; #P<0.05 mtDNA vs. nDNA, one way ANOVA, n= 6 per group.

Fig. 4

Expression of Ogg1 and Pol γ at mRNA and protein levels during epileptogenesis. To assess the induction of the BER pathway during epileptogenesis, the levels of both Ogg1 and Pol γ at an mRNA and protein level were monitored as described in Materials and Methods. A) mRNA expression at the indicated time points was determined by real-time PCR performed on a primer/probe set specific to Ogg1 and Pol γ. B) Protein expression of Ogg1 and Pol γ at the indicated time points was determined via immunoblot analysis. C and D) Immunoblots of Ogg1 and Pol γ, respectively from hippocampal tissue after kainate treatment. Lane 1, CON; lane 2, 24h; lane 3; Lane 4, 48h; Lane 5, 96h; Lane 6, 7d; Lane 7, 21d; Lane 8, 3m. Bars represent mean ± SEM. **P<0.01, vs. saline treated controls, one way ANOVA, n= 3–5 per group.

Fig. 5

A comparison of mtDNA repair capacity in isolated mitochondria during epileptogenesis. Hippocampal mitochondria were freshly isolated at the indicated time points after kainate administration and treated with H₂O₂ (50μM) for 30min and either harvested immediately or allowed to repair for 30min. The data was normalized to the respective 30min DNA damage levels and repair efficiency expressed as percentage repair. Bars represent mean ± SEM. *P<0.05, vs. saline treated controls, one way ANOVA, n= 4 per group.

Fig. 6

Mitochondrial oxidative stress during epileptogenesis. To assess the state of mitochondrial oxidative stress in the hippocampus during epileptogenesis the levels of A) H₂O₂ production and B) mitochondrial aconitase activity were monitored as described in Materials and Methods. H₂O₂ production was expressed as percentage mitochondrial H₂O₂ change compared to saline treated controls. Aconitase activity was expressed as activity/g of protein. Bars represent mean ± SEM. *P<0.05; **P<0.01, vs. saline treated controls, one way ANOVA, n= 3–4 per group.

Fig. 7

The relationship betweens seizures, mtDNA damage, mtBER expression and H₂O₂ production. Indices of the mitochondrial oxidative burden measured during this study illustrate how mitochondrial oxidative stress and mtDNA damage could play a role in the progression from acute seizure activity to chronic epilepsy. Acute seizure activity causes preferential oxidative damage to the mtDNA and the mtBER is induced to attempt to repair mtDNA lesions. However, there is a failure in the mtBER during the chronic phase of epilepsy, which may hinder the repair of mtDNA damage and contribute to increased mitochondrial oxidative stress. The increased mitochondrial oxidative stress burden and genomic instability may render the brain more susceptible to subsequent chronic epileptic seizures. The (X) represents the mean seizure score and the other data points represent the mean percentage change of mtDNA Damage, Ogg1, Pol γ and H₂O₂ production compared to controls.