Abstracts

Status epilepticus (SE) is a life-threatening condition charecterized by continuous seizure activity. Previous studies have documented an increase in calcineurin (CaN) activity following SE, as well as an SE-induced increase in the post-synaptic concentration of CaN (Kurz et al. J Neurochem,2001 78:304-15 and Neurobiol Dis,2003 14:483-93). The role of these changes in CaN in the pathology of SE remains unclear. This study examines a potential CaN-dependent modification of the dendritic cytoskeleton involving cofilin activation. CaN has been shown to indirectly induce dephosphorylation of cofilin, an actin depolymerization factor. The ensuing actin depolymerization can lead to a number of physiological changes that are of interest in SE., Pilocarpine injection was used to induce SE in adult male Sprague-Dawley rats. When appropriate, animals were administered 10 mg/kg cyclosporin A or 2.5 mg/kg FK506 i.v. 3h prior to SE. Animals were monitored by video-EEG. At specfic times after the initiation of seizure activity brain tissue was harvested and separated into subcellular fractions by differential centrifugation. Cofilin phosphorylation and total cofilin were assayed by western analysis. CaN activity was assayed via an RII-phosphopeptide based assay. Actin polymerization was assayed both by histochemical means and by determining the ratio of G-to F-actin after separating the two with a solubilization/centrifugation protocol., The immunoreactivity of phosphocofilin decreased significantly in both hippocampal and cortical synaptosomal samples after SE. Hippocampal and cortical phosphocofilin were decreased to approximately 20% of control after 1h of SE (n=10, p[lt]0.001). Total cofilin immunoreactivity remained unchanged after 1h of SE, indicating that the loss of phosphocofilin was likely due to dephosphorylation rather than overall loss of cofilin. This dephosphorylation occured as early as 10 min after SE onset, a timeframe that correlates with the timing of the increase in SPM CaN. Cofilin dephosphorylation could be partially blocked by the injection of CaN inhibitors prior to SE, indicating that this process is partially CaN-dependent. Cofilin activation could be further demonstrated by increased actin-cofilin binding on coimmunoprecipitation, which could also be blocked by CaN inhibitors. Finally, SE induced a significant CaN-dependent depolymerization of neuronal actin., The data demonstrate a significant, CaN-dependent activation of cofilin in SE and a subsequent CaN-mediated depolymerization of actin. This research could be applied to explain SE-induced modulation and loss of dendritic spines, as well as changes in receptor localization. Further research into this area may provide useful insights into the pathology of SE and epileptogenic mechanisms., (Supported by the Epilepsy Foundation through the American Epilepsy Society and the Lennox Trust Fund (JEK) and by R01-NS39770 (SBC).)