The Ketogenic Diet Attenuates Both Hyperactivity in mTOR pathway and Astrogliosis Through Regulation of AMPK signaling in the Epileptic Brain

Abstract

BackgroundThe high‐fat, low carbohydrate ketogenic diet (KD) is a proven therapy for patients with medically refractory epilepsy. The metabolic changes induced by the KD mimics the physiological alteration of fasting and calorie restriction – wherein ketone levels are elevated (ketosis). Both the KD and its metabolic substrates are involved in modulating anti‐seizure effects, but the molecular target of these beneficial effects have yet to be fully elucidated. One potential molecular explanation links the mammalian target of rapamycin (mTOR) signaling pathway. Disruptions in the mTOR pathway may explain in part susceptibility to and pathogenesis of epilepsy. The primary purpose of this study was to investigate the role of metabolic changes in mTOR signaling pathway in the Kcna1‐null mouse model of epilepsy and whether its mechanistic effects are linearly correlated with ketosis.MethodsSpontaneously epileptic Kcna1‐null (KO) mice were generated using heterozygous breeding pairs. At P21‐ 23, mice were treated with the KD and either standard diet (SD) or SD with subcutaneous administration of BHB through osmotic mini‐pumps for 2–3 weeks. Blood from tail clippings were used to measure BHB and glucose levels. EEG electrodes were implanted through parasaggital burr holes in either wild type (WT) mice or KO mice around P28. Following a 3‐day recovery period, seizure activity was assessed over 72 continuous hours. After over 2 weeks of treatment, hippocampal lysates were collected from: (1) SD‐fed WT; (2) SD‐fed KO; (3) KD‐fed WT; (4) KD‐fed KO; (5) saline‐treated WT; (6) saline‐treated KO; (7) BHB‐treated WT; and (8) BHB–treated KO mice. Samples were probed with antibodies against upstream modulators of mTOR, either phosphorylated ‐ or total –AMPK, and downstream effectors of mTOR, phospho‐ or total ‐ S6, as well as a downstream effectors of AMPK, either glial fibrillary acidic protein (GFAP) or Tubulin. Total RNA obtained from hippocampus or whole brain using TRIzol was reverse transcribed into cDNA by RT‐PCR and then the single‐stranded cDNA was amplified by PCR with primers for GFAP and β‐actin.ResultsUsing western blot analysis, mTOR signaling was significantly increased in KO mice accompanied with spontaneous epileptic discharge, and levels of upstream pAMPK were decreased in these animals compared to control and KD‐fed KO mice. KO mice showed a significant enhancement in the downstream markers pS6 and p4EBP1, whereas this effect was strongly suppressed by the KD treatment. KD treatment led to a reduction in pS6 in kainic acid (KA)‐treated control mice. KA resulted in a gradual increase of GFAP. In contrast, at 24 hours post KA injection, KD‐fed mice exhibited a significant decrease in expression level of GFAP.ConclusionCollectively, either the KD or ketone not only reduces the severity of seizures in two different mouse models, but also attenuates mTOR signaling pathways. Further, our finding that phospho‐S6 is reduced by KD treatment suggests a mechanistic link between mTOR inhibition and decreases in astrogliosis, both mechanisms that could contribute to the anti‐seizure and neuroprotective effects of the KD. The results provide evidence for the functional protective link between the KD and its primary metabolic substrate, β‐hydroxybutyrate, in spontaneously epileptic Kcna1‐null mice. Finally, the therapeutic effects of BHB and the KD may be in part inhibitory action of glial cell activation through modulation of mTOR signaling pathway Effects of the ketogenic diet on seizure‐evoked upregulation of mTOR signaling pathways in the hippocampus of Kcna1‐null mice. (A & D)