Deletion of <Emphasis Type="Italic">mTOR</Emphasis> in Reactive Astrocytes Suppresses Chronic Seizures in a Mouse Model of Temporal Lobe Epilepsy

Germline and somatic mutations in key genes of the mammalian target of rapamycin (mTOR) pathway have been identified in seizure-associated disorders. mTOR mutations lead to aberrant activation of mTOR signaling, and, although affected neurons are critical for epileptogenesis, the role of mTOR activation in glial cells remains poorly understood. We previously reported a consistent activation of the mTOR pathway in astrocytes in the epileptic foci of temporal lobe epilepsy. In this study, it was demonstrated that mTOR deletion from reactive astrocytes prevents increases in seizure frequency over the disease course. By using a tamoxifen-inducible mTOR conditional knockout system and kainic acid, a model was developed that allowed astrocyte-specific mTOR gene deletion in mice with chronic epilepsy. Animals in which mTOR was deleted from 44 % of the astrocyte population exhibited a lower seizure frequency compared with controls. Down-regulation of mTOR significantly ameliorated astrogliosis in the sclerotic hippocampus but did not rescue mossy fiber sprouting. In cultured astrocytes, the mTOR pathway modulated the stability of the astroglial glutamate transporter 1 (Glt1) and influenced the ability of astrocytes to remove extracellular glutamate. Taken together, these data indicate that astrocytes with activated mTOR signaling may provide conditions that are favorable for spontaneous recurrent seizures.     

Regulation of cell death and epileptogenesis by the mammalian target of rapamycin (mTOR): A double-edged sword?

Identification of cell signaling mechanisms mediating seizure-related neuronal death and epileptogenesis is important for developing more effective therapies for epilepsy. The mammalian target of rapamycin (mTOR) pathway has recently been implicated in regulating neuronal death and epileptogenesis in rodent models of epilepsy. In particular, kainate-induced status epilepticus causes abnormal activation of the mTOR pathway, and the mTOR inhibitor, rapamycin, can decrease the development of neuronal death and chronic seizures in the kainate model. Here, we discuss the significance of these findings and extend them further by identifying upstream signaling pathways through which kainate status epilepticus activates the mTOR pathway and by demonstrating limited situations where rapamycin may paradoxically increase mTOR activation and worsen neuronal death in the kainate model. Thus, the regulation of seizure-induced neuronal death and epileptogenesis by mTOR is complex and may have dual, opposing effects depending on the physiological and pathological context. Overall, these findings have important implications for designing potential neuroprotective and antiepileptogenic therapies that modulate the mTOR pathway.     

Changes in TWIK-related Acid Sensitive K+-1 and -3 Channel Expressions from Neurons to Glia in the Hippocampus of Temporal Lobe Epilepsy Patients and Experimental Animal Model

In the present study, we analyzed expressions of tandem of P domains in a Weak Inwardly rectifying K⁺ channel (TWIK)-related Acid-Sensitive K⁺ (TASK) channel-1 and -3 in the hippocampus of patients with temporal lobe epilepsy (TLE) and in rat model. In the control human subjects, TASK-1, and -3 immunoreactivity was observed in pyramidal neurons and dentate granule cells. In TLE patients, TASK-1 and -3 immunoreactivity was rarely observed in neurons. However, TASK-1 immunoreactivity was observed in astrocytes, and TASK-3 immunoreactivity was detected in both astrocytes and microglia. In the rat hippocampus, TASK-1 immunoreactivity was observed in astrocytes within normal and epileptic hippocampus. The alterations in TASK-3 immunoreactivity in the rat hippocampus were similar to those in the human hippocampus. These findings reveal that TASK-1 and -3 are differentially expressed in the normal and epileptic hippocampus, and suggest that TASK channels may contribute to the properties of the epileptic hippocampus.     

The Recurrent Mossy Fiber Pathway of the Epileptic Brain

The dentate gyrus is believed to play a key role in the pathogenesis of temporal lobe epilepsy. In normal brain the dentate granule cells serve as a high-resistance gate or filter, inhibiting the propagation of seizures from the entorhinal cortex to the hippocampus. The filtering function of the dentate gyrus depends in part on the near absence of monosynaptic connections among granule cells. In humans with temporal lobe epilepsy and in animal models of temporal lobe epilepsy, dentate granule cells form an interconnected synaptic network associated with loss of hilar interneurons. This recurrent mossy fiber pathway mediates reverberating excitation that can reduce the threshold for granule cell synchronization. Factors that augment activity in this pathway include modest increases in [K⁺]_o; loss of GABA inhibition; short-term, frequency-dependent facilitation (frequencies of 1–2 Hz); feedback activation of kainate autoreceptors; and release of zinc from recurrent mossy fiber boutons. Factors that diminish activity include short-term, frequency-dependent depression (frequencies <1 Hz); feedback activation of type II metabotropic glutamate receptors; and the potential release of GABA, neuropeptide Y, adenosine, and dynorphin from recurrent mossy fiber boutons. The axon sprouting and reactive synaptogenesis that follow seizure-related brain damage can also create or strengthen recurrent excitation in other brain regions. These changes are expected to facilitate participation of these regions in seizures. Thus, reactive processes that are often considered important for recovery of function after most brain injuries probably contribute to neurological dysfunction in epilepsy.     

Protective Effect of Resveratrol against Kainate-induced Temporal Lobe Epilepsy in Rats

Resveratrol (Res) is a phytoalexin produced naturally by several plants, which has multi functional effects such as neuroprotection, anti-inflammatory, and anti-cancer. The present study was to evaluate a possible anti-epileptic effect of Res against kainate-induced temporal lobe epilepsy (TLE) in rat. We performed behavior monitoring, intracranial electroencepholography (IEEG) recording, histological analysis, and Western blotting to evaluate the anti-epilepsy effect of Res in kainate-induced epileptic rats. Res decreased the frequency of spontaneous seizures and inhibited the epileptiform discharges. Moreover, Res could protect neurons against kainate-induced neuronal cell death in CA1 and CA3a regions and depressed mossy fiber sprouting, which are general histological characteristics both in TLE patients and animal models. Western blot revealed that the expression level of kainate receptors (KARs) in hippocampus was reduced in Res-administrated rats compared to that in epileptic ones. These results suggest that Res is a potent anti-epilepsy agent, which protects against epileptogenesis and progression of the kainate-induced TLE animal. The authors Z. Wu and Q. Xu contributed equally to this work.     

Down-Regulation of CRMP-1 in Patients with Epilepsy and a Rat Model

The Collapsin Response Mediator Protein-1 (CRMP-1) is a brain specific protein identified as a signaling molecule of Semaphorin-3A and act as axon repellent guidance factor in nervous system. Recent studies indicated that axon guidance molecules may play a role in synaptic reorganization in the adult brain and thereby promote epileptogenesis. This study aimed to investigate expression pattern of CRMP-1 in epileptogenesis. Using double immunofluorescence labeling, immunohistochemistry and western blot analysis, we looked into the CRMP-1 expression in temporal neocortex from patients with temporal lobe epilepsy (TLE) and histological normal temporal neocortex from the controls. We also studied the expression pattern of CRMP-1 in hippocampus and adjacent cortex of a TLE rat model on 6, 24, 72 h, 1, 2 weeks, 1 month, and 2 months post-seizure, and from control rats. CRMP-1 was mainly expressed in the neuronal cytoplasm in the temporal lobe of intractable TLE patients, which was co-expressed with -2. CRMP-1 expression was downregulated in temporal neocortical of TLE patients. In addition, in pilocarpine-induced animal model of epilepsy, CRMP-1 dynamically decreased in a range of 2 months. Thus, our results indicate that CRMP-1 may be involved in the development of TLE.     

Conditional deletion of TrkB but not BDNF prevents epileptogenesis in the kindling model

Epileptogenesis is the process whereby a normal brain becomes epileptic. We hypothesized that the neurotrophin brain-derived neurotrophic factor (BDNF) activates its receptor, TrkB, in the hippocampus during epileptogenesis and that BDNF-mediated activation of TrkB is required for epileptogenesis. We tested these hypotheses in Synapsin-Cre conditional BDNF(-/-) and TrkB(-/-) mice using the kindling model. Despite marked reductions of BDNF expression, only a modest impairment of epileptogenesis and increased hippocampal TrkB activation were detected in BDNF(-/-) mice. In contrast, reductions of electrophysiological measures and no behavioral evidence of epileptogenesis were detected in TrkB(-/-) mice. Importantly, TrkB(-/-) mice exhibited behavioral endpoints of epileptogenesis, tonic-clonic seizures. Whereas TrkB can be activated, and epileptogenesis develops in BDNF(-/-) mice, the plasticity of epileptogenesis is eliminated in TrkB(-/-) mice. Its requirement for epileptogenesis in kindling implicates TrkB and downstream signaling pathways as attractive molecular targets for drugs for preventing epilepsy.     

Important role of matrix metalloproteinase 9 in epileptogenesis

Temporal lobe epilepsy (TLE) is a devastating disease in which aberrant synaptic plasticity plays a major role. We identify matrix metalloproteinase (MMP) 9 as a novel synaptic enzyme and a key pathogenic factor in two animal models of TLE: kainate-evoked epilepsy and pentylenetetrazole (PTZ) kindling-induced epilepsy. Notably, we show that the sensitivity to PTZ epileptogenesis is decreased in MMP-9 knockout mice but is increased in a novel line of transgenic rats overexpressing MMP-9. Immunoelectron microscopy reveals that MMP-9 associates with hippocampal dendritic spines bearing asymmetrical (excitatory) synapses, where both the MMP-9 protein levels and enzymatic activity become strongly increased upon seizures. Further, we find that MMP-9 deficiency diminishes seizure-evoked pruning of dendritic spines and decreases aberrant synaptogenesis after mossy fiber sprouting. The latter observation provides a possible mechanistic basis for the effect of MMP-9 on epileptogenesis. Our work suggests that a synaptic pool of MMP-9 is critical for the sequence of events that underlie the development of seizures in animal models of TLE.     

Metformin Plus Caloric Restriction Show Anti-epileptic Effects Mediated by mTOR Pathway Inhibition

Caloric restriction (CR) has anti-epileptic effects in different animal models, at least partially due to inhibition of the mechanistic or mammalian target of rapamycin (mTOR) signaling pathway. Adenosine monophosphate-activated protein kinase (AMPK) inhibits mTOR cascade function if energy levels are low. Since hyper-activation of mTOR participates in epilepsy, its inhibition results in beneficial anti-convulsive effects. A way to attain this is to activate AMPK with metformin. The effects of metformin, alone or combined with CR, on the electrical kindling epilepsy model and the mTOR cascade in the hippocampus and the neocortex were studied. Combined metformin plus CR beneficially affected many kindling aspects, especially those relating to generalized convulsive seizures. Therefore, metformin plus CR could decrease measures of epileptic activity in patients with generalized convulsive seizures. Patients that are obese, overweight or that have metabolic syndrome in addition to having an epileptic disease are an ideal population for clinical trials to test the effectiveness of metformin plus CR.     

Decreased Astroglial Monocarboxylate Transporter 4 Expression in Temporal Lobe Epilepsy

Efflux of monocaroxylates like lactate, pyruvate, and ketone bodies from astrocytes through monocarboxylate transporter 4 (MCT4) supplies the local neuron population with metabolic intermediates to meet energy requirements under conditions of increased demand. Disruption of this astroglial-neuron metabolic coupling pathway may contribute to epileptogenesis. We measured MCT4 expression in temporal lobe epileptic foci excised from patients with intractable epilepsy and in rats injected with pilocarpine, an animal model of temporal lobe epilepsy (TLE). Cortical MCT4 expression levels were significantly lower in TLE patients compared with controls, due at least partially to MCT4 promoter methylation. Expression of MCT4 also decreased progressively in pilocarpine-treated rats from 12 h to 14 days post-administration. Underexpression of MCT4 in cultured astrocytes induced by a short hairpin RNA promoted apoptosis. Knockdown of astrocyte MCT4 also suppressed excitatory amino acid transporter 1 (EAAT1) expression. Reduced MCT4 and EAAT1 expression by astrocytes may lead to neuronal hyperexcitability and epileptogenesis in the temporal lobe by reducing the supply of metabolic intermediates and by allowing accumulation of extracellular glutamate.     

Vigabatrin Inhibits Seizures and mTOR Pathway Activation in a Mouse Model of Tuberous Sclerosis Complex

Epilepsy is a common neurological disorder and cause of significant morbidity and mortality. Although antiseizure medication is the first-line treatment for epilepsy, currently available medications are ineffective in a significant percentage of patients and have not clearly been demonstrated to have disease-specific effects for epilepsy. While seizures are usually intractable to medication in tuberous sclerosis complex (TSC), a common genetic cause of epilepsy, vigabatrin appears to have unique efficacy for epilepsy in TSC. While vigabatrin increases gamma-aminobutyric acid (GABA) levels, the precise mechanism of action of vigabatrin in TSC is not known. In this study, we investigated the effects of vigabatrin on epilepsy in a knock-out mouse model of TSC and tested the novel hypothesis that vigabatrin inhibits the mammalian target of rapamycin (mTOR) pathway, a key signaling pathway that is dysregulated in TSC. We found that vigabatrin caused a modest increase in brain GABA levels and inhibited seizures in the mouse model of TSC. Furthermore, vigabatrin partially inhibited mTOR pathway activity and glial proliferation in the knock-out mice in vivo, as well as reduced mTOR pathway activation in cultured astrocytes from both knock-out and control mice. This study identifies a potential novel mechanism of action of an antiseizure medication involving the mTOR pathway, which may account for the unique efficacy of this drug for a genetic epilepsy.     

Lovastatin modulates glycogen synthase kinase-3β pathway and inhibits mossy fiber sprouting after pilocarpine-induced status epilepticus

This study was undertaken to assay the effect of lovastatin on the glycogen synthase kinase-3 beta (GSK-3β) and collapsin responsive mediator protein-2 (CRMP-2) signaling pathway and mossy fiber sprouting (MFS) in epileptic rats. MFS in the dentate gyrus (DG) is an important feature of temporal lobe epilepsy (TLE) and is highly related to the severity and the frequency of spontaneous recurrent seizures. However, the molecular mechanism of MFS is mostly unknown. GSK-3β and CRMP-2 are the genes responsible for axonal growth and neuronal polarity in the hippocampus, therefore this pathway is a potential target to investigate MFS. Pilocarpine-induced status epilepticus animal model was taken as our researching material. Western blot, histological and electrophysiological techniques were used as the studying tools. The results showed that the expression level of GSK-3β and CRMP-2 were elevated after seizure induction, and the administration of lovastatin reversed this effect and significantly reduced the extent of MFS in both DG and CA3 region in the hippocampus. The alteration of expression level of GSK-3β and CRMP-2 after seizure induction proposes that GSK-3β and CRMP-2 are crucial for MFS and epiletogenesis. The fact that lovastatin reversed the expression level of GSK-3β and CRMP-2 indicated that GSK-3β and CRMP-2 are possible to be a novel mechanism of lovatstain to suppress MFS and revealed a new therapeutic target and researching direction for studying the mechanism of MFS and epileptogenesis.     

Phosphorylation of PTEN and Akt in astrocytes of the rat hippocampus following transient forebrain ischemia

To ascertain whether the PTEN (phosphatase and tensin homolog deleted on chromosome 10)/Akt signaling pathway is activated during ischemic brain injury, we investigated the expression and phosphorylation of PTEN and Akt by immunohistochemistry in the rat hippocampus after transient forebrain ischemia. Weak immunoreactivity for PTEN and its phosphorylated form (p-PTEN) was constitutively expressed in hippocampal neurons and astrocytes of the control rats, but their upregulation was detected mainly in reactive astrocytes in the ischemic hippocampus. Increased immunoreactivity for PTEN and p-PTEN occurred specifically in astrocytes by day 1 and was sustained for more than 2 weeks. The spatiotemporal activation of Akt in the ischemic hippocampus mirrored that of p-PTEN expression. Post-ischemic activation of Akt, revealed by phosphorylated Akt (p-Akt) immunoreactivity, was first detected at day 1 and was maintained for at least 2 weeks. Double-labeling experiments revealed that the cells expressing PTEN, p-PTEN, or p-Akt were reactive astrocytes expressing glial fibrillary acidic protein. These results demonstrate the increased phosphorylation of PTEN and Akt in reactive astrocytes of the post-ischemic hippocampus, suggesting that the PTEN/Akt pathway is involved in the astroglial reaction in the rat hippocampus after transient forebrain ischemia.This research was supported by Korea Science and Engineering Foundation (R01-2002-000-00334-0(2002)).     

Antioxidant Ability and Lipid Peroxidation in the Hippocampus with Epileptogenesis Induced by Fe3+ Injection into the Amygdaloid Body of Rats

To analyze antioxidant ability and lipid peroxidation in the hippocampus of rats in an interictal state of FeCl₃-induced epileptogenesis, the hippocampal eliminating decay ratio of exogenously applied nitroxide radical (3-carbamoyl-2,2,5,5-tetramethylpyrrolidine-1-oxyl (carbamoyl-PROXYL)) by electron paramagnetic resonance (EPR) spectroscopy, and the thiobarbituric reactive substances (TBARS) level in the hippocampus were measured. The prolonged half-life of electron paramagnetism of carbamoyl-PROXYL in the hippocampus of rats with chronic FeCl₃-induced epileptogenesis revealed decreased antioxidant ability, which supports the vulnerability against oxidative stress. In addition, TBARS level (marker of lipid peroxidation) was increased in the hippocampus of rats injected with FeCl₃ compared with that of control. This study revealed that repetitive seizures resulted in the decreased hippocampal antioxidant ability with lipid peroxidation and explained the regional vulnerability to oxidative stress in the limbic system with epileptogenesis.     

The interaction between early life epilepsy and autistic-like behavioral consequences: a role for the mammalian target of rapamycin (mTOR) pathway

Early life seizures can result in chronic epilepsy, cognitive deficits and behavioral changes such as autism, and conversely epilepsy is common in autistic children. We hypothesized that during early brain development, seizures could alter regulators of synaptic development and underlie the interaction between epilepsy and autism. The mammalian Target of Rapamycin (mTOR) modulates protein translation and is dysregulated in Tuberous Sclerosis Complex, a disorder characterized by epilepsy and autism. We used a rodent model of acute hypoxia-induced neonatal seizures that results in long term increases in neuronal excitability, seizure susceptibility, and spontaneous seizures, to determine how seizures alter mTOR Complex 1 (mTORC1) signaling. We hypothesized that seizures occurring at a developmental stage coinciding with a critical period of synaptogenesis will activate mTORC1, contributing to epileptic networks and autistic-like behavior in later life. Here we show that in the rat, baseline mTORC1 activation peaks during the first three postnatal weeks, and induction of seizures at postnatal day 10 results in further transient activation of its downstream targets phospho-4E-BP1 (Thr37/46), phospho-p70S6K (Thr389) and phospho-S6 (Ser235/236), as well as rapid induction of activity-dependent upstream signaling molecules, including BDNF, phospho-Akt (Thr308) and phospho-ERK (Thr202/Tyr204). Furthermore, treatment with the mTORC1 inhibitor rapamycin immediately before and after seizures reversed early increases in glutamatergic neurotransmission and seizure susceptibility and attenuated later life epilepsy and autistic-like behavior. Together, these findings suggest that in the developing brain the mTORC1 signaling pathway is involved in epileptogenesis and altered social behavior, and that it may be a target for development of novel therapies that eliminate the progressive effects of neonatal seizures.     

Brain-derived neurotrophic factor (BDNF)-induced synthesis of early growth response factor 3 (Egr3) controls the levels of type A GABA receptor alpha 4 subunits in hippocampal neurons

Altered function of gamma-aminobutyric acid type A receptors (GABA(A)Rs) in dentate granule cells of the hippocampus has been associated with temporal lobe epilepsy (TLE) in humans and in animal models of TLE. Such altered receptor function (including increased inhibition by zinc and lack of modulation by benzodiazepines) is related, in part, to changes in the mRNA levels of certain GABA(A)R subunits, including alpha4, and may play a role in epileptogenesis. The majority of GABA(A)Rs that contain alpha4 subunits are extra-synaptic due to lack of the gamma2 subunit and presence of delta. However, it has been hypothesized that seizure activity may result in expression of synaptic receptors with altered properties driven by an increased pool of alpha4 subunits. Results of our previous work suggests that signaling via protein kinase C (PKC) and early growth response factor 3 (Egr3) is the plasticity trigger for aberrant alpha4 subunit gene (GABRA4) expression after status epilepticus. We now report that brain derived neurotrophic factor (BDNF) is the endogenous signal that induces Egr3 expression via a PKC/MAPK-dependent pathway. Taken together with the fact that blockade of tyrosine kinase (Trk) neurotrophin receptors reduces basal GABRA4 promoter activity by 50%, our findings support a role for BDNF as the mediator of Egr3-induced GABRA4 regulation in developing neurons and epilepsy and, moreover, suggest that BDNF may alter inhibitory processing in the brain by regulating the balance between phasic and tonic inhibition.     

Human Fetal Brain-Derived Neural Stem/Progenitor Cells Grafted into the Adult Epileptic Brain Restrain Seizures in Rat Models of Temporal Lobe Epilepsy

Cell transplantation has been suggested as an alternative therapy for temporal lobe epilepsy (TLE) because this can suppress spontaneous recurrent seizures in animal models. To evaluate the therapeutic potential of human neural stem/progenitor cells (huNSPCs) for treating TLE, we transplanted huNSPCs, derived from an aborted fetal telencephalon at 13 weeks of gestation and expanded in culture as neurospheres over a long time period, into the epileptic hippocampus of fully kindled and pilocarpine-treated adult rats exhibiting TLE. In vitro, huNSPCs not only produced all three central nervous system neural cell types, but also differentiated into ganglionic eminences-derived γ-aminobutyric acid (GABA)-ergic interneurons and released GABA in response to the depolarization induced by a high K⁺ medium. NSPC grafting reduced behavioral seizure duration, afterdischarge duration on electroencephalograms, and seizure stage in the kindling model, as well as the frequency and the duration of spontaneous recurrent motor seizures in pilocarpine-induced animals. However, NSPC grafting neither improved spatial learning or memory function in pilocarpine-treated animals. Following transplantation, grafted cells showed extensive migration around the injection site, robust engraftment, and long-term survival, along with differentiation into β-tubulin III⁺ neurons (∼34%), APC-CC1⁺ oligodendrocytes (∼28%), and GFAP⁺ astrocytes (∼8%). Furthermore, among donor-derived cells, ∼24% produced GABA. Additionally, to explain the effect of seizure suppression after NSPC grafting, we examined the anticonvulsant glial cell-derived neurotrophic factor (GDNF) levels in host hippocampal astrocytes and mossy fiber sprouting into the supragranular layer of the dentate gyrus in the epileptic brain. Grafted cells restored the expression of GDNF in host astrocytes but did not reverse the mossy fiber sprouting, eliminating the latter as potential mechanism. These results suggest that human fetal brain-derived NSPCs possess some therapeutic effect for TLE treatments although further studies to both increase the yield of NSPC grafts-derived functionally integrated GABAergic neurons and improve cognitive deficits are still needed.