In contrast to other metabolism-based therapies that protect in acute seizure tests, rapamycin has limited acute anticonvulsant effects in normal mice. Significance: The efficacy of rapamycin as an acute anticonvulsant agent may be limited. Furthermore, the combined pattern of acute seizure test results places rapamycin in a third category distinct from both fasting and the ketogenic diet, and which is more similar to drugs acting on sodium channels.
Citation: Hartman AL, Santos P, Dolce A, Hardwick JM (2012) The mTOR Inhibitor Rapamycin Has Limited Acute Anticonvulsant Effects in Mice. PLoS ONE 7(9): e45156. doi:10.1371/journal.pone.0045156 Editor: Ulrike Schmidt, Max Planck Institute of Psychiatry, Germany Received May 14, 2012; Accepted August 13, 2012; Published September 12, 2012 Copyright: ?2012 Hartman et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: The project described was supported by Grant Numbers K08NS070931 (ALH) and RO1NS037402 (JMH) from the National Institute of Neurological Disorders and Stroke. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institute of Neurological Disorders and Stroke or the National Institutes of Health. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist.
Introduction
Epilepsy affects 0.5?% of the US population but ,20?0% of patients do not respond to the two initial medications prescribed [1,2,3,4]. One underutilized option for this population is metabolism-based therapy through dietary or pharmacologic interventions, particularly if the patient does not have a surgically resectable lesion [5]. The most commonly used metabolism-based therapy is the high-fat, low carbohydrate ketogenic diet. The efficacy of the ketogenic diet in children was shown in a randomized controlled trial showing a robust 75% decrease in patient seizures over three months [6]. Small molecules that potentially target the same pathways are being investigated for antiseizure effects, including agents that act on nutrient-sensing mechanisms such as the mTOR-containing TORC1 complex. In cell culture models, depletion of glucose and specific amino acids suppresses mTOR serine-threonine kinase activity, leading to reduced protein translation and induction of autophagy [7]. Mutations in TSC1/2, genes that normally suppress mTOR, are responsible for tuberous sclerosis complex, which includes seizures, tubers, subependymal giant cell tumors, autism, behavior problems, and other systemic complications [8]. In Tsc1- or Ptendeficient mice that have increased mTOR activity and chronic spontaneous seizures, sustained treatment with the mTOR inhibitor rapamycin decreased seizure frequency [9,10,11]. Furthermore, the rapamycin analog everolimus restricted tumor growth and decreased seizure frequency in a clinical trial of patients with tuberous sclerosis complex [12]. Inhibitors of mTOR may improve seizure control in other chronic epilepsy models where the underlying cause of epilepsy is not due to mutations in the TOR pathway.
For example,rapamycin suppressed behavioral spasms in the doxorubicin/ lipopolysaccharide/p-chlorophenylalanine model of infantile spasms [13]. Rapamycin also decreased susceptibility to kainic acid-induced seizures in P13 rats exposed to graded hypoxia at P10 [14]. In addition, rapamycin protected against spontaneous seizures that recur for several months following one-time kainic acid- or pilocarpine-induced status epilepticus in rats [15,16]. Collectively, these reports with chronic models support the general opinion that rapamycin protects by inducing long-term cellular changes [17]. Rapamycin also protected against seizures when administered after the initial induction of status epilepticus in the pilocarpine rat model [16], raising the possibility that rapamycin also may act acutely to inhibit seizure activity [18]. However, rapamycin failed to protect when the same post-treatment model of pilocarpine-induced status epilepticus was applied to mice [19] and it did not protect against seizures during the first 48 hours after a hypoxic insult in P10 rats, challenging the idea that rapamycin has acute antiseizure effects. Similarly, attempts to study the short-term effects of rapamycin in vitro also have not provided strong support for acute effects of rapamycin. Short-term exposure of neurons in vitro to rapamycin did not alter neuronal firing under baseline conditions, and it had limited benefits under conditions of provoked neuronal firing [20,21]. One way to determine if rapamycin acutely suppresses seizure activity is to compare it to known anticonvulsants. Rapamycin has not been systematically tested in a battery of acute seizure tests like those used routinely to screen candidate therapeutics in preclinical trials [22]. Using similar tests, we found that rapamycin has a limited acute anticonvulsant effect. Furthermore, rapamycin exposure for #6 h (defined here as a `short’ exposure) has a profile (i.e., a combination of positive and negative seizure test results) that is comparable to drugs that suppress voltage-gated sodium channel activity. Even when tested for longer times (3?3 days), rapamycin still has an acute seizure test profile that does not match the profiles of either the ketogenic diet or another dietary antiseizure intervention, intermittent fasting. Thus, the anticonvulsant mechanisms of rapamycin may be distinct from other metabolism-based therapies. Because of the adverse effects of rapamycin and related drugs in patients [23], finding an explanation for how mTOR inhibition protects against seizures could help facilitate the design of more specific agents and minimize side effects.
