Abstracts

Rationale: Abnormal activation of the mammalian target of rapamycin (mTOR) signaling pathway is associated with the development of seizures and epilepsy in human and animal models of cortical dysplasia. Studies have shown that pharmacological inhibition of mTOR signaling attenuates epileptiform activity, suggesting a role for the mTOR pathway in the regulation of neuronal excitability. However, the underlying molecular mechanisms are unknown. There is emerging evidence for mTOR-dependent regulation of protein translation and surface expression of various potassium channels including Kv1.1 (Raab-Graham et al, 2006). Kv1.1 is mainly localized in the axon initial segment (AIS) where it plays a critical role in repolarization during an action potential. Aberrant levels of Kv1.1 have been linked to neuronal hyperexcitability and epilepsy. In the current study, we evaluated whether excessive mTOR signaling due to loss of PTEN, a negative regulator of the pathway, contributes to altered protein levels and localization of Kv1.1.Methods: Western blotting was used to measure the protein levels of Kv1.1 in whole hippocampal homogenates of 6 week-old neuronal subset-specific conditional PTEN knockout (NS-PTEN KO) and wildtype (WT) mice. In parallel, we also evaluated Kv1.1 levels in animals that were treated with rapamycin (10 mg/kg, i.p. injections) or vehicle for 5 days/week during postnatal weeks 4 and 5. Immunostaining was used to evaluate Kv1.1 localization in coronal brain sections from NS-PTEN KO mice and in PTEN^{floxed/floxed} primary hippocampal neuronal cultures transduced with RFP-Cre lentivirus to delete PTEN. Results: We found a significant increase in the protein levels of Kv1.1 in the hippocampus of NS-PTEN KO compared to WT mice. Following rapamycin treatment, Kv1.1 levels in the NS-PTEN KO mice were restored to WT levels (p<0.05, n=7-16 per group). Staining for Kv1.1 revealed an increase in the distribution of Kv1.1 in somatic regions of dentate granule cells in the hippocampus of NS-PTEN KO compared to WT mice. Similarly, there was increased somatic staining for Kv1.1 coupled with weak staining in the AIS in cultured hippocampal neurons lacking PTEN compared to the control (non-transduced) neurons.Conclusions: Excessive mTOR signaling in hippocampal neurons is associated with increased protein levels and mislocalization of Kv1.1 to the soma. These data suggest Kv1.1 remodeling may be one candidate mechanism that contributes to neuronal hyperexcitability and epilepsy in the NS-PTEN KO mice. Future studies will investigate the functional relevance of altered Kv1.1 protein levels and localization due to mTOR hyperactivation in this model.