Abstracts

Rationale: Hyperactivation of the mechanistic target of rapamycin (mTOR) pathway has been demonstrated in human cortical dysplasia (CD), which is a prevalent cause of intractable pediatric epilepsy. Previous studies in our lab have shown that pharmacological inhibition of mTOR attenuates epilepsy in the neuron subset-specific Pten knockout (NS-Pten KO) mouse model of CD. However, the underlying mechanisms remain elusive. Given that emerging research has suggested a role for mTOR in the regulation of ion channels, which are critical modulators of neuronal excitability, we investigated whether mTOR-associated dysregulation of ion channel expression (mRNA and protein levels) occurs in NS-Pten KO mice. In the present study, we focus on the Kv1 subfamily of voltage-gated potassium channels (Kv1.1, Kv1.2, and Kv1.4) that are predominantly found in axons and nerve terminals of mammalian neurons where they modulate action potentials and regulate neuronal excitability.Methods: Western blotting was used to measure protein levels of Kv1.1, Kv1.2, and Kv1.4 in whole hippocampal homogenates from 2, 4, 6, and 8 week-old NS-Pten KO and wildtype (WT) mice. Real-time quantitative PCR was performed to determine Kv1.1 mRNA levels in 8 week-old NS-Pten KO and WT hippocampal tissue. A cohort of NS-Pten KO and WT mice were treated with the mTOR inhibitor rapamycin (10 mg/kg i.p., 5 days/week) or vehicle either during postnatal weeks 4 and 5 (early treatment) or during postnatal weeks 9 and 10 (late treatment). Western blotting was performed to evaluate the effects of rapamycin on Kv1.1, Kv1.2, and Kv1.4 protein levels.Results: Kv1.1 protein levels aberrantly increased with age in NS-Pten KO mice in parallel to previously reported increases in mTOR dysregulation and epileptiform activity (p<0.001, n=6-15 mice/group). No changes in Kv1.2 and Kv1.4 protein levels were observed between NS-Pten KO and WT mice. By 6-8 weeks of age, we found a significant increase in Kv1.1 protein levels in NS-Pten KO compared to WT mice (p<0.001, n=6-8 mice/group). However, no changes in mRNA expression were found between the two genotypes (n=7-8 mice/group). Administration of rapamycin treatment at an age before Kv1.1 protein levels were dysregulated (early treatment) in NS-Pten KO mice prevented the aberrant increase in Kv1.1 levels (p<0.01, n=7-16 mice/group). Administration of rapamycin treatment at an age after Kv1.1 protein levels were dysregulated (late treatment) in NS-Pten KO mice restored the aberrant increase in Kv1.1 levels to WT levels (p<0.05, n=6-14 mice/group). Rapamycin treatment had no effects on Kv1.2 and Kv1.4 protein levels.Conclusions: Our results demonstrate the presence of altered Kv1.1 protein levels in association with mTOR hyperactivation in NS-Pten KO mice, and provide support for mTOR-associated ion channel dysregulation as a candidate mechanism underlying epilepsy in this model. Future studies will further explore the potential mechanisms by which mTOR regulate Kv1.1 protein levels and investigate the functional relevance of altered Kv1.1 protein levels in NS-Pten KO mice.