Elsevier

Experimental Neurology

Volume 280, June 2016, Pages 1-12
Experimental Neurology

Impact of rapamycin on status epilepticus induced hippocampal pathology and weight gain

https://doi.org/10.1016/j.expneurol.2016.03.015Get rights and content

Highlights

  • Rapamycin prevents pathological weight gain in epileptic rodents.

  • Rapamycin reduces mossy fiber sprouting in epilepsy.

  • Rapamycin does not block ectopic granule cell accumulation or somatic hypertrophy.

  • Rapamycin does not block hippocampal inflammatory changes.

  • Rapamycin does not prevent hilar mossy cell death.

Abstract

Growing evidence implicates the dentate gyrus in temporal lobe epilepsy (TLE). Dentate granule cells limit the amount of excitatory signaling through the hippocampus and exhibit striking neuroplastic changes that may impair this function during epileptogenesis. Furthermore, aberrant integration of newly-generated granule cells underlies the majority of dentate restructuring. Recently, attention has focused on the mammalian target of rapamycin (mTOR) signaling pathway as a potential mediator of epileptogenic change. Systemic administration of the mTOR inhibitor rapamycin has promising therapeutic potential, as it has been shown to reduce seizure frequency and seizure severity in rodent models. Here, we tested whether mTOR signaling facilitates abnormal development of granule cells during epileptogenesis. We also examined dentate inflammation and mossy cell death in the dentate hilus. To determine if mTOR activation is necessary for abnormal granule cell development, transgenic mice that harbored fluorescently-labeled adult-born granule cells were treated with rapamycin following pilocarpine-induced status epilepticus. Systemic rapamycin effectively blocked phosphorylation of S6 protein (a readout of mTOR activity) and reduced granule cell mossy fiber axon sprouting. However, the accumulation of ectopic granule cells and granule cells with aberrant basal dendrites was not significantly reduced. Mossy cell death and reactive astrocytosis were also unaffected. These data suggest that anti-epileptogenic effects of mTOR inhibition may be mediated by mechanisms other than inhibition of these common dentate pathologies. Consistent with this conclusion, rapamycin prevented pathological weight gain in epileptic mice, suggesting that rapamycin might act on central circuits or even peripheral tissues controlling weight gain in epilepsy.

Introduction

Status epilepticus (SE) selectively disrupts the integration of adult-generated hippocampal granule cells (Parent et al., 2006, Jessberger et al., 2007, Walter et al., 2007, Kron et al., 2010, Santos et al., 2011). Changes include mossy fiber axon sprouting, formation of hilar basal dendrites and ectopic migration of newly-generated cells. These phenomena have been implicated in promoting hyperexcitability in the temporal lobe and may facilitate epileptogenesis (Jung et al., 2004, Jung et al., 2006, Cho et al., 2015). Hilar ectopic granule cells, for example, are more excitable in rodent epilepsy models (Zhan et al., 2010, Althaus et al., 2015) and can exhibit both spontaneous and evoked bursting (Scharfman et al., 2000, Cameron et al., 2011), distinguishing them from normal granule cells. In addition to ectopically-located cells, granule cells with mossy fiber axon sprouting to the dentate inner molecular layer and hilar-projecting basal dendrites mediate the formation functional granule cell to granule cell synapses (Okazaki et al., 1999, Thind et al., 2008). These synaptic connections may mediate increased excitatory flow through the dentate gyrus. Despite the evidence that these abnormalities may contribute to epileptogenesis and associated comorbidities, no current FDA-approved treatments can prevent the aberrant integration of adult-generated neurons in epilepsy.

Recently, the mTOR pathway has emerged as a promising molecular target that may mediate aberrant granule cell integration. mTOR signaling in the dentate is enhanced in chemical, injury-induced and genetic models of epilepsy (Wong, 2013, LaSarge and Danzer, 2014), suggesting the pathway could be involved in many different forms of the disease. Treatment with rapamycin, an inhibitor of mTOR complex 1, has been shown to reduce seizures (Zeng et al., 2009, Huang et al., 2010, van Vliet et al., 2012), prevent mossy fiber sprouting (Zeng et al., 2009, Huang et al., 2010, Buckmaster et al., 2009, Buckmaster and Lew, 2011, Tang et al., 2012, van Vliet et al., 2012, Heng et al., 2013, Shima et al., 2015), mitigate cell loss (Zeng et al., 2009, van Vliet et al., 2012, Guo et al., 2013, Butler et al., 2015) and reduce reactive astrogliosis (Shima et al., 2015) in rodent modes of acquired epilepsy. Conversely, recent work from our lab demonstrates that genetically enhancing mTOR signaling by PTEN deletion in adult-generated hippocampal granule cells causes mossy fiber axon sprouting, formation of hilar basal dendrites and ectopic cell migration (Pun et al., 2012). We hypothesized, therefore, that mTOR hyperactivation underlies the cellular abnormalities evident in the hippocampus following status epilepticus.

To test our hypothesis we utilized rapamycin to directly inhibit mTOR activity in mice following pilocarpine-induced status epilepticus. A genetic fate-mapping strategy was used to determine the effectiveness of rapamycin at mitigating cellular abnormalities among adult-generated granule cells. In addition, mossy cell survival and reactive astrocytosis were examined in the animals, since these changes have also been implicated in epileptogenesis. These experiments provide new insights into the role of mTOR signaling in the formation of epilepsy-induced anatomical changes within the dentate gyrus.

Section snippets

Animals

All procedures involving animals were approved by the Institutional Animal Care and Use Committee of the Cincinnati Children's Hospital Research Foundation and conform to NIH guidelines for the care and use of animals. Two animal breeding schemes were followed: (1) In order to generate Gli1-CreERT2::GFP mice on a pure C57BL/6NCrl background for the study, hemizygous Gli1-CreERT2 mice (Ahn and Joyner, 2004, Ahn and Joyner, 2005) on a C57BL/6NCrl background were crossed to homozygous CAG-CAT-EGFP

Results

To induce the development of epilepsy, mice were treated with the cholinergic agonist pilocarpine to provoke status epilepticus (SE), which was allowed to proceed for 3 h before seizures were mitigated by treatment with diazepam. Control animals received saline instead of pilocarpine. Studies were begun using C57BL/6NCrl mice. Because of high mortality during the 24 h period following SE, however, mice on a 50:50 background of C57BL/6NCrl::FVB N (B6/FVB) were also used. B6/FVB mice showed

Discussion

mTOR signaling is enhanced during the development of acquired temporal lobe epilepsy (Fig. 1), and rapamycin treatment has been shown to reduce seizures frequency in a variety of epilepsy models, including kainate-SE (Zeng et al., 2009), pilocarpine-SE (Huang et al., 2010), angular bundle electrical stimulation (van Vliet et al., 2012), neonatal hypoxia (Talos et al., 2012), traumatic brain injury (Guo et al., 2013), models of TSC (Zeng et al., 2008, Goto et al., 2011, Zeng et al., 2011) and

Conflict of interests

The authors declare no competing financial interests.

Acknowledgments

This work was supported by the National Institute of Neurological Disorders and Stroke (SCD, Award Numbers R01NS065020 and R01NS062806). 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. We thank Keri Kaeding for assistance with earlier versions of this manuscript. We thank the CCHMC Confocal Core for providing access the 3024 Nikon

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