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- MATERIALS AND METHODS
Summary: Purpose: Kindled seizures are widely used to model epileptogenesis, but the molecular mechanisms underlying the attainment of kindling status are largely unknown. Recently we showed that achievement of kindling status in the Sprague–Dawley rat is associated with a critical developmental interval of 25 ± 1 days; the identification of this long, well-defined developmental interval for inducing kindling status makes possible a dissection of the cellular and genetic events underlying this phenomenon and its relation to normal and pathologic brain function.
Methods: By using proteomics on cerebral tissue from our new rat kindling model, we undertook a global analysis of protein expression in kindled animals. Some of the identified proteins were further investigated by using immunohistochemistry.
Results: We report the identification of a modified variant of the Rieske iron-sulfur protein, a component of the mitochondrial cytochrome bc1 complex, whose isoelectric point is shifted toward more alkaline values in the hippocampus of kindled rats. By immunohistochemistry, the Rieske protein is well expressed in the hippocampus, except in the CA1 subfield, an area of selective vulnerability to seizures in humans and animal models. We also noted an asymmetric, selective expression of the Rieske protein in the subgranular neurons of the dorsal dentate gyrus, a region implicated in neurogenesis.
Conclusions: These results indicate that the Rieske protein may play a role in the response of neurons to seizure activity and could give important new insights into the molecular pathogenesis of epilepsy.
Epilepsy is a chronic disorder characterized by recurrent seizures occurring over long periods. The most studied model of chronic limbic epilepsy is kindled seizures, a condition achieved by the repeated administration of an initially subconvulsant stimulus that ultimately results in the generation of seizure activity (1). Although the attainment of the kindling criterion is known to require time to develop, the precise developmental period has not been identified.
We recently reported that optimal achievement of the kindling criterion in the Sprague–Dawley rat is associated with a critical interstimulus interval of 24–26 days. Specifically, a high proportion (>90%) of animals reached full kindling status after only two subconvulsant doses of pentylenetetrazole (PTZ; 30 mg/kg) given 25 days apart; we dubbed this model the critical time window kindling model (2). The identification of this long, well-defined developmental interval for inducing kindling status makes possible a dissection of the cellular and genetic events underlying this phenomenon and its relation to normal and pathologic brain function.
With this model, we have shown that the high-susceptibility period is associated with increased expression of a protease [tissue plasminogen activator (tPA)], a structural protein [microtubule-associated protein 1B (MAP1B)] and an axonal growth-associated protein (GAP43) in the hippocampus. Although this work suggests that the persistent expression of several classes of brain plasticity–associated proteins may be required for the maintenance of kindling status, it seemed likely that a number of other proteins might be involved, the identification of which could further our understanding of the molecular mechanisms that underlie the pathogenesis of epilepsy. Based on a proteomic analysis of the brains of PTZ-treated rats, we here report, for the first time, that a modification of the Rieske iron-sulfur protein in hippocampal neurons is strongly dependent on the development of kindling status.
- Top of page
- MATERIALS AND METHODS
In this work, we have shown that seizure activity in rats leads to pronounced alterations in the Rieske iron-sulfur protein in specific brain regions. Specifically, a shift toward a more alkaline pI range in the isoelectric point of the Rieske protein occurs in kindled rats. Work is in progress to determine whether the change in the overall charge of the protein is due to a posttranslational modification of the protein.
The Rieske protein is a high-potential iron-sulfur cluster [(2Fe-2S)](ISP) that is part of the mitochondrial cytochrome bc1 complex (ubiquinol:cytochrome c reductase, or complex III), a multifunctional membrane protein complex that catalyzes electron transfer from ubiquinol to cytochrome c but also is involved in proton translocation, peptide processing, and superoxide generation (6). Mutations affecting mitochondrial oxidative phosphorylation complexes have been linked to a large number of clinical phenotypes. For instance, impaired complex III activity due to a gene deletion in cytochrome b is associated with oxidative phosphorylation deficits in parkinsonism (7), and a stop-codon mutation in the cytochrome b gene causes a form of mitochondrial encephalomyopathy (8).
Posttraumatic epilepsy has been modeled by the intracerebral deposition of iron compounds such as hemosiderin, which occurs as a consequence of the extravasation of erythrocytes after injury (9,10). Abnormally high iron levels have been found in patients with epilepsy (11). Likewise, it has been proposed that elevated circulating iron is a risk factor for epilepsy (12). Interestingly, partial seizures have been reported in Bull Terriers with augmented serum iron levels (13). Furthermore, intracortical or amygdalar injection of Fe (III) salts or heme compounds into the rodent brain induces recurrent seizures and epileptic discharges in the EEGs (14–16).
The most conspicuous histopathologic changes occur in the hippocampus, a region heavily implicated in the development of seizures in both humans and animal models. The CA1 subfields of the hippocampal formation in particular have been recognized as being extremely susceptible to damage after seizure activity in humans (17–19) and in rats (20), especially when seizures are accompanied by hypoxia (i.e., a low-energy state) (21,22). The selective reduction of Rieske protein levels in the CA1 region in our rat model lends support to the hypothesis that low-energy states may facilitate seizure development. Conversely, the Rieske protein may have a protective role for regions that are rich in this protein and known to be less prone to damage, such as CA3 and CA2. Equally important is the asymmetric expression of the Rieske protein in the subgranular zone of the dentate gyrus, a region known to be involved in neurogenesis (23). However, it is not clear at this time how the Rieske protein can be related to neurogenesis. The dorsoventral asymmetry in the hippocampus has been documented after seizure activity in mice (24) and seems to be important for the acquisition of spatial memory (25,26). Furthermore, the modified form of the Rieske protein appears to have caused a reduction in mitochondrial activity, the consequence of which would be an accumulation of bodily adipose tissue in kindled rats. This hypothesis is supported by the gain of weight in rats subjected repeatedly to episodes of seizure activity.
Our results are in line with a recent report demonstrating that spontaneous seizure activity induced by pilocarpine treatment of rats causes a decline in the activities of complex I and complex IV in CA1 and CA3, but not in the dentate gyrus (27). Furthermore, positron emission tomography demonstrates hypometabolism in the epileptogenic zone of patients with temporal lobe epilepsy that correlates with impaired oxidative metabolism in the CA3 region (28).