Abstracts

Rationale: The mammalian target of rapamycin (mTOR) signaling pathway has been the focus of intense investigative efforts to understand molecular and cellular mechanisms of epileptogenesis. Protein synthesis, cellular growth and proliferation, and synaptic plasticity are regulated by mTOR through its control of downstream effectors such as S6K, S6, and 4EBP1. In addition, mTOR is influenced by multiple upstream factors that either activate or inhibit mTOR via PI3K/PTEN/Akt, ERK, AMPK, and REDD. Recently, it has been shown that the ketogenic diet (KD) reduced the expression of pS6 and pAkt, which are markers of mTOR pathway activation, in normal rat hippocampus, and prevented increases in pS6 seen after kainate-induced status epilepticus [Epilepsia 2011; 52(3):e7]. Whether similar changes would be seen in a genetic model of developmental epilepsy is unknown. Here, we examined the role of mTOR signaling in epileptic Kcna1-null (KO) mice fed either a standard diet (SD) or KD.Methods: Spontaneously epileptic KO mice were generated using heterozygous breeding pairs, and at P20, either a KD (Bio-serv F3666) or SD was administered ad libitum for 2 weeks. After 7-10 days of treatment, each animal underwent 72 hrs of continuous video-EEG monitoring. For Western blots, hippocampal lysates were collected from four groups of mice: (1) SD-fed wild type (WT); (2) SD-fed KO; (3) KD-fed WT; and KD-fed KO mice. Samples (n=4 per group) were probed with antibodies against upstream effectors [e.g., PTEN (phosphatase and tensin homolog), Phosphor (P)-Akt, and P-AMPK] and downstream effectors [e.g., P-mTOR, P-S6, and P-4EBP1].Results: SD-fed KO mice exhibited a mean daily tonic-clonic seizure frequency of 6.0 0.5, and this effect was significantly reduced by KD treatment (p<0.05). Phosphorylation of S6, mTOR, and AMPK was increased in the hippocampus of SD-fed KO mice (34%, 27%, and 34% increase, respectively, as compared to SD-fed WT mice), whereas phosphorylation of these proteins was reduced in those animals fed the KD (46%, 20%, and 40% decrease, respectively, as compared to SD-fed KO mice). No significant differences in 4EBP1 and Akt expression were seen between SD-fed WT and SD-fed KO groups. However, KD-fed WT and KO mice showed a significant reduction in the expression levels of both 4EBP1 and Akt. No changes in PTEN expression were found in any of the experimental groups. Conclusions: Our data indicate that: (1) activation of the mTOR pathway appears to be an important feature in spontaneously epileptic Kcna1-null mice; (2) KD treatment consistent with a previous report reduces several markers of mTOR pathway activation; (3) abnormal mTOR signaling may contribute to epileptogenesis in the Kcna1-null mouse model; and (4) the broad neuroprotective effects of the KD, as exemplified in an increasing number of recent reports, may be in part a consequence of mTOR inhibition. Supported by the Barrow Neurological Foundation and the National Institutes of Health NINDS RO1 (NS070261).