Abstracts

Rationale: The ketogenic diet (KD) is an effective treatment for pediatric epilepsy, and although it has been in use for nearly a century, its mechanisms of action remain poorly-understood. Unlike other treatments for epilepsy, there is evidence that the KD possesses antiepileptogenic as well as anticonvulsant properties. mTOR is a protein kinase that regulates numerous cellular functions including growth, proliferation, survival, and synaptic plasticity. mTOR is activated by PI3K/Akt signaling in the presence of nutrients and growth factors, and inhibited by AMPK in the setting of energy starvation. Dysregulated mTOR signaling has been implicated in epileptogenesis in models of genetic and acquired epilepsies including tuberous sclerosis complex (TSC) and kainic acid (KA)-induced status epilepticus (SE). Given the ability of mTOR to integrate nutrient and energy signals, we investigated the effects of the KD on mTOR pathway signaling in normal animals as well as rodent models of epilepsy. Methods: Sprague Dawley rats were given ad libitum access to KD with 6:1 ratio of fat to carbohydrate protein (F3666; Bioserv) or standard diet (SD) beginning at P21. For the KA model, rats were injected with 15mg/kg KA i.p. to induce SE, and started on KD or SD after resolution of SE. For TSC experiments, conditional GFAP Tsc1 knockout mice (Tsc1^GFAPCKO) and littermate controls were weaned to KD or SD at P21. mTOR activity was assessed using western blot (WB) for phosphorylation of its downstream target S6 (pS6) compared to total ribosomal protein S6. Upstream signaling was evaluated by WB for phospho Akt (pAkt), total Akt, phospho AMPK (pAMPK), and total AMPK. Tissue was harvested for WB after two weeks of KD or SD in normal rat and TSC experiments, and 1, 7, or 21 days after SE in the KA model. Results: In normal rats, KD reduced pS6 and pAkt in hippocampus (24% and 14%, respectively, p<0.05 for both) and liver (45% and 54%, p<0.05 for both), but not neocortex. KD increased pAMPK only in liver (p<0.001). Tsc1^GFAPCKO mice on SD had higher pS6 (p<0.01) and lower pAkt (p<0.05) than controls. KD decreased pS6 and pAkt in combined neocortex and hippocampus of control mice (22% and 19%, p<0.05 for both), but not Tsc1^GFAPCKO mice. There was no effect of KD on brain pAMPK in control or Tsc1^GFAPCKO mice. KA-induced SE increased hippocampal pS6 acutely and at 7 days, with return to baseline by 21 days, and KD blocked this elevation at 7 days. Conclusions: KD inhibited mTOR activity in hippocampus and liver of normal rats, most likely via decreased Akt signaling in both regions, as well as increased AMPK signaling in liver. KD also inhibited mTOR in control mouse brain. In contrast, KD had no effect on mTOR hyperactivation in the TSC model, possibly due to an inability of KD to bypass the genetic inactivation of Tsc1. However, in the KA model, KD blocked the SE-induced mTOR activation. As pharmacological inhibition of mTOR by rapamycin prevents epilepsy in some models, these results suggest that the KD may also have antiepileptogenic actions via inhibition of the mTOR pathway.