Canonical Wnt activator Chir99021 prevents epileptogenesis in the intrahippocampal kainate mouse model of temporal lobe epilepsy.

The Wnt signaling pathway mediates the development of dentate granule cell neurons in the hippocampus. These neurons are central to the development of temporal lobe epilepsy and undergo structural and physiological remodeling during epileptogenesis, which results in the formation of epileptic circuits. The pathways responsible for granule cell remodeling during epileptogenesis have yet to be well defined, and represent therapeutic targets for the prevention of epilepsy. The current study explores Wnt signaling during epileptogenesis and for the first time describes the effect of Wnt activation using Wnt activator Chir99021 as a novel anti-epileptogenic therapeutic approach. Focal mesial temporal lobe epilepsy was induced by intrahippocampal kainate (IHK) injection in wild-type and POMC-eGFP transgenic mice. Wnt activator Chir99021 was administered daily, beginning 3 h after seizure induction, and continued up to 21-days. Immature granule cell morphology was quantified in the ipsilateral epileptogenic zone and the contralateral peri-ictal zone 14 days after IHK, targeting the end of the latent period. Bilateral hippocampal electrocorticographic recordings were performed for 28-days, 7-days beyond treatment cessation. Hippocampal behavioral tests were performed after completion of Chir99021 treatment. Consistent with previous studies, IHK resulted in the development of epilepsy after a 14 day latent period in this well-described mouse model. Activation of the canonical Wnt pathway with Chir99021 significantly reduced bilateral hippocampal seizure number and duration. Critically, this effect was retained after treatment cessation, suggesting a durable antiepileptogenic change in epileptic circuitry. Morphological analyses demonstrated that Wnt activation prevented pathological remodeling of the primary dendrite in both the epileptogenic zone and peri-ictal zone, changes in which may serve as a biomarker of epileptogenesis and anti-epileptogenic treatment response in pre-clinical studies. These findings were associated with improved object location memory with Chir99021 treatment after IHK. This study provides novel evidence that canonical Wnt activation prevents epileptogenesis in the IHK mouse model of mesial temporal lobe epilepsy, preventing pathological remodeling of dentate granule cells. Wnt signaling may therefore play a key role in mesial temporal lobe epileptogenesis, and Wnt modulation may represent a novel therapeutic strategy in the prevention of epilepsy.

The Glycolysis Inhibitor 2-Deoxy-D-Glucose Exerts Different Neuronal Effects at Circuit and Cellular Levels, Partially Reverses Behavioral Alterations and does not Prevent NADPH Diaphorase Activity Reduction in the Intrahippocampal Kainic Acid Model of Temporal Lobe Epilepsy.

Temporal lobe epilepsy is the most drug-resistant type with the highest incidence among the other focal epilepsies. Metabolic manipulations are of great interest among others, glycolysis inhibitors like 2-deoxy D-glucose (2-DG) being the most promising intervention. Here, we sought to investigate the effects of 2-DG treatment on cellular and circuit level electrophysiological properties using patch-clamp and local field potentials recordings and behavioral alterations such as depression and anxiety behaviors, and changes in nitric oxide signaling in the intrahippocampal kainic acid model. We found that epileptic animals were less anxious, more depressed, with more locomotion activity. Interestingly, by masking the effect of increased locomotor activity on the parameters of the zero-maze test, no altered anxiety behavior was noted in epileptic animals. However, 2-DG could partially reverse the behavioral changes induced by kainic acid. The findings also showed that 2-DG treatment partially suppresses cellular level alterations while failing to reverse circuit-level changes resulting from kainic acid injection. Analysis of NADPH-diaphorase positive neurons in the CA1 area of the hippocampus revealed that the number of positive neurons was significantly reduced in dorsal CA1 of the epileptic animals and 2-DG treatment did not affect the diminishing effect of kainic acid on NADPH-d+ neurons in the CA1 area. In the control group receiving 2-DG, however, an augmented NADPH-d+ cell number was noted. These data suggest that 2-DG cannot suppress epileptiform activity at the circuit-level in this model of epilepsy and therefore, may fail to control the seizures in temporal lobe epilepsy cases.

The Neuroprotective Effect of miR-136 on Pilocarpine-Induced Temporal Lobe Epilepsy Rats by Inhibiting Wnt/ß-Catenin Signaling Pathway.

Objective: To explore the effect of miR-136 on temporal lobe epilepsy (Ep) and its mechanism of action. Methods: 30 male rats were injected intraperitoneally with 30 mg/kg pilocarpine to construct a rat temporal lobe epilepsy model, and they were randomly divided into 5 groups (n = 6 per group): control group, Ep group, agomir NC group, miR-136 agomir group, and miR-136+LiCl group. The brain tissues of the rats were collected 7 days after the treatment. The expression of miR-136 in the hippocampus tissue was detected by qRT-PCR. H&E and Nissl staining were used to observe the histopathological changes and neuron damage in the hippocampus tissue. IL-1ß, IL-6, and TNF-α levels in the hippocampus tissue were detected by ELISA. Flow cytometry was used to detect the apoptosis rate in the hippocampus tissue. Western blot was used to detect the expression levels of c-Caspase-3, Bcl-2, ß-catenin, Cyclin D1, and c-myc protein in the hippocampus. Results: The expression of miR-136 was significantly downregulated in the hippocampus tissue of epileptic rats. After overexpression of miR-136, the number of seizures and the duration of epilepsy in rats were significantly reduced. At the same time, hippocampal tissue damage was improved considerably, and the degree of neuronal damage decreased. Overexpression of miR-136 also significantly reduced the apoptosis rate in the hippocampus tissue and inhibited the levels of inflammatory factors. Meanwhile, miR-136 downregulates the expression of Wnt/ß-catenin signaling pathway-related proteins. However, Wnt pathway activator LiCl could destroy the protective effect of miR-136. Conclusion: miR-136 could exert its neuroprotective influence on temporal lobe epilepsy rats by inhibiting the Wnt/ß-catenin signaling pathway.

Scopolamine prevents aberrant mossy fiber sprouting and facilitates remission of epilepsy after brain injury.

Prevention or modification of acquired epilepsy in patients at risk is an urgent, yet unmet, clinical need. Following acute brain insults, there is an increased risk of mesial temporal lobe epilepsy (mTLE), which is often associated with debilitating comorbidities and reduced life expectancy. The latent period between brain injury and the onset of epilepsy may offer a therapeutic window for interfering with epileptogenesis. The pilocarpine model of mTLE is widely used in the search for novel antiepileptogenic treatments. Recent biochemical studies indicated that cholinergic mechanisms play a role in the epileptogenic alterations induced by status epilepticus (SE) in this and other models of mTLE, which prompted us to evaluate whether treatment with the muscarinic antagonist scopolamine during the latent period after SE is capable of preventing or modifying epilepsy and associated behavioral and cognitive alterations in female Sprague-Dawley rats. First, in silico pharmacokinetic modeling was used to select a dosing protocol by which M-receptor inhibitory brain levels of scopolamine are maintained during prolonged treatment. This protocol was verified by drug analysis in vivo. Rats were then treated twice daily with scopolamine over 17 days after SE, followed by drug wash-out and behavioral and video/EEG monitoring up to ~6 months after SE. Compared to vehicle controls, rats that were treated with scopolamine during the latent period exhibited a significantly lower incidence of spontaneous recurrent seizures during periods of intermittent recording in the chronic phase of epilepsy, less behavioral excitability, less cognitive impairment, and significantly reduced aberrant mossy fiber sprouting in the hippocampus. The present data may indicate that scopolamine exerts antiepileptogenic/disease-modifying activity in the lithium-pilocarpine rat model, possibly involving increased remission of epilepsy as a new mechanism of disease-modification. For evaluating the rigor of the present data, we envision a study that more thoroughly addresses the gender bias and video-EEG recording limitations of the present study.

Development of an antiepileptogenesis drug screening platform: Effects of everolimus and phenobarbital.

OBJECTIVE: The kainic acid (KA)-induced status epilepticus (SE) model in rats is a well-defined model of epileptogenesis. This model closely recapitulates many of the clinical and pathological characteristics of human temporal lobe epilepsy (TLE) that arise following SE or another neurological insult. Spontaneous recurrent seizures (SRS) in TLE can present after a latent period following a neurological insult (traumatic brain injury, SE event, viral infection, etc.). Moreover, this model is suitable for preclinical studies to evaluate the long-term process of epileptogenesis and screen putative disease-modifying/antiepileptogenic agents. The burden of human TLE is highly variable, similar to the post-KA SE rat model. In this regard, this model may have broad translational relevance. This report thus details the pharmacological characterization and methodological refinement of a moderate-throughput drug screening program using the post-KA-induced SE model of epileptogenesis in male Sprague Dawley rats to identify potential agents that may prevent or modify the burden of SRS. Specifically, we sought to demonstrate whether our protocol could prevent the development of SRS or lead to a reduced frequency/severity of SRS. METHODS: Rats were administered either everolimus (2-3 mg/kg po) beginning 1, 2, or 24 h after SE onset, or phenobarbital (60 mg/kg ip) beginning 1 h after SE onset. All treatments were administered once/day for 5-7 days. Rats in all studies (n = 12/treatment dose/study) were then monitored intermittently by video-electroencephalography (2 weeks on, 2 weeks off, 2 weeks on epochs) to determine latency to onset of SRS and disease burden. RESULTS: Although no adverse side effects were observed in our studies, no treatment significantly modified disease or prevented the presentation of SRS by 6 weeks after SE onset. SIGNIFICANCE: Neither phenobarbital nor everolimus administered at several time points after SE onset prevented the development of SRS. Nonetheless, we demonstrate a practical and moderate-throughput screen for potential antiepileptogenic agents in a rat model of TLE.

PKA-RIIß autophosphorylation modulates PKA activity and seizure phenotypes in mice.

Temporal lobe epilepsy (TLE) is one of the most common and intractable neurological disorders in adults. Dysfunctional PKA signaling is causally linked to the TLE. However, the mechanism underlying PKA involves in epileptogenesis is still poorly understood. In the present study, we found the autophosphorylation level at serine 114 site (serine 112 site in mice) of PKA-RIIß subunit was robustly decreased in the epileptic foci obtained from both surgical specimens of TLE patients and seizure model mice. The p-RIIß level was negatively correlated with the activities of PKA. Notably, by using a P-site mutant that cannot be autophosphorylated and thus results in the released catalytic subunit to exert persistent phosphorylation, an increase in PKA activities through transduction with AAV-RIIß-S112A in hippocampal DG granule cells decreased mIPSC frequency but not mEPSC, enhanced neuronal intrinsic excitability and seizure susceptibility. In contrast, a reduction of PKA activities by RIIß knockout led to an increased mIPSC frequency, a reduction in neuronal excitability, and mice less prone to experimental seizure onset. Collectively, our data demonstrated that the autophosphorylation of RIIß subunit plays a critical role in controlling neuronal and network excitabilities by regulating the activities of PKA, providing a potential therapeutic target for TLE.

TMEM2 binds to CSNK2A3 to inhibit HBV infection via activation of the JAK/STAT pathway.

To investigate mechanisms that TMEM2 activation inhibits hepatitis B virus (HBV) infection in hepatocarcinoma (HCC) cells, co-immunoprecipitation (Co-IP) and mass spectrometry were used in screening interacting proteins for TMEM2. Levels of casein kinase 2 subunit α3 (CSNK2A3) in HCC cells were found to be inhibited or overexpressed using siRNAs and pcDNA3.1-CSNK2A3, respectively. Effect of CSNK2A3 expression on cell proliferation was analyzed using MTS, while its effect on HBV infection was measured using ddPCR and IHC. Western blotting and JAK inhibitor ruxolitinib were also used to determine whether TMEM2-regulated CSNK2A3 expression and HBV infection were affected by JAK-STAT signaling. Co-IP and mass spectrometry results showed that CSNK2A3 interacts with TMEM2. Moreover, overexpression of CSNK2A3 significantly inhibited cell proliferation, while inhibition of CSNK2A3 promoted proliferation of HCC cells. In addition, overexpression of CSNK2A3 was observed to significantly enhance HBV infection, while siRNA knockdown of CSNK2A3 inhibited HBV infection. Notably, effect of CSNK2A3 overexpression on HBV infection was suppressed by TMEM2 overexpression. Further mechanistic analyses have revealed that TMEM2 could antagonize the effects of CSNK2A3 on cell proliferation and HBV infection via JAK-STAT pathway activation. In conclusion, TMEM2 has been determined to bind to CSNK2A3 to inhibit HBV infection via activation of the JAK-STAT pathway.

In vivo CRISPRa decreases seizures and rescues cognitive deficits in a rodent model of epilepsy.

Epilepsy is a major health burden, calling for new mechanistic insights and therapies. CRISPR-mediated gene editing shows promise to cure genetic pathologies, although hitherto it has mostly been applied ex vivo. Its translational potential for treating non-genetic pathologies is still unexplored. Furthermore, neurological diseases represent an important challenge for the application of CRISPR, because of the need in many cases to manipulate gene function of neurons in situ. A variant of CRISPR, CRISPRa, offers the possibility to modulate the expression of endogenous genes by directly targeting their promoters. We asked if this strategy can effectively treat acquired focal epilepsy, focusing on ion channels because their manipulation is known be effective in changing network hyperactivity and hypersynchronziation. We applied a doxycycline-inducible CRISPRa technology to increase the expression of the potassium channel gene Kcna1 (encoding Kv1.1) in mouse hippocampal excitatory neurons. CRISPRa-mediated Kv1.1 upregulation led to a substantial decrease in neuronal excitability. Continuous video-EEG telemetry showed that AAV9-mediated delivery of CRISPRa, upon doxycycline administration, decreased spontaneous generalized tonic-clonic seizures in a model of temporal lobe epilepsy, and rescued cognitive impairment and transcriptomic alterations associated with chronic epilepsy. The focal treatment minimizes concerns about off-target effects in other organs and brain areas. This study provides the proof-of-principle for a translational CRISPR-based approach to treat neurological diseases characterized by abnormal circuit excitability.

Prevention and treatment of temporal lobe epilepsy: lessons from hepatitis B story!

Temporal lobe epilepsy (TLE) is the most common type of drug-resistant epilepsy and hippocampal sclerosis (HS) is the most common pathological substrate of TLE. Considering the significant consequences of uncontrolled seizures (e.g. increased morbidity and mortality), epilepsy prevention remains a necessity that potentially could save many lives. Human herpes virus-6 (HHV-6) has been linked to TLE in humans. The relationship between HHV-6 and HS-TLE could be attributed to a neuro-inflammatory cascade triggered by the infection, involving direct neuronal damage and production of several pro-inflammatory cytokines under certain conditions that are still incompletely understood. Hepatitis B virus (HBV) infection is another chronic viral infection with a life-long latency. HBV infection is linked to various clinical conditions, including liver cirrhosis. There are currently three ways to fight HBV infection and its consequences; primary prevention (by vaccination), secondary prevention (by drug therapy), and tertiary prevention (by liver transplantation). Considering the similarities between the natural histories of HHV-6 and HBV infections, and also the successful strategies which are currently available to fight HBV infection and its long-term consequences, here, we propose three strategies to fight HHV-6 and its possible long-term consequence (i.e. HS-TLE): Primary prevention: by developing vaccines to prevent HHV-6 infection; Secondary prevention: by considering trials of antiviral drugs to treat HHV-6 infection, when it happens in the childhood to hopefully prevent its long-term consequences; and, Tertiary prevention: by stem cell therapy for drug-resistant epilepsy.

Selective inhibition of mTORC1/2 or PI3K/mTORC1/2 signaling does not prevent or modify epilepsy in the intrahippocampal kainate mouse model.

Dysregulation of the PI3K/Akt/mTOR pathway has been implicated in several brain disorders, including epilepsy. Rapamycin and similar compounds inhibit mTOR. complex 1 and have been reported to decrease seizures, delay seizure development, or prevent epileptogenesis in different animal models of genetic or acquired epilepsies. However, data for acquired epilepsy are inconsistent, which, at least in part, may be due to the poor brain penetration and long brain persistence of rapamycin and the fact that it blocks only one of the two cellular mTOR complexes. Here we examined the antiepileptogenic or disease-modifying effects of two novel, brain-permeable and well tolerated 1,3,5-triazine derivatives, the ATP-competitive mTORC1/2 inhibitor PQR620 and the dual pan-PI3K/mTORC1/2 inhibitor PQR530 in the intrahippocampal kainate mouse model, in which spontaneous seizures develop after status epilepticus (SE). Following kainate injection, the two compounds were administered over 2 weeks at doses previously been shown to block mTORC1/2 or PI3K/mTORC1/2 in the mouse brain. When spontaneous seizures were recorded by continuous (24/7) video-EEG recording starting 6 weeks after termination of treatment, no effects on incidence or frequency of seizures were observed. Drug treatment suppressed the epilepsy-induced activation of the PI3K/Akt/mTOR pathway in the hippocampus, but granule cell dispersion in the dentate gyrus was not prevented. When epilepsy-associated behavioral alterations were determined 12-14 weeks after kainate, mice pretreated with PQR620 or PQR530 exhibited reduced anxiety-related behavior in the light-dark box, indicating a disease-modifying effect. Overall, the data indicate that mTORC1/C2 or PI3K/mTORC1/C2 inhibition may not be an antiepileptogenic strategy for SE-induced epilepsy.

Excitation, but not inhibition, of the fastigial nucleus provides powerful control over temporal lobe seizures.

KEY POINTS: On-demand optogenetic inhibition of glutamatergic neurons in the fastigial nucleus of the cerebellum does not alter hippocampal seizures in a mouse model of temporal lobe epilepsy. In contrast, on-demand optogenetic excitation of glutamatergic neurons in the fastigial nucleus successfully inhibits hippocampal seizures. With this approach, even a single 50 ms pulse of light is able to significantly inhibit seizures. On-demand optogenetic excitation of glutamatergic fastigial neurons either ipsilateral or contralateral to the seizure focus is able to inhibit seizures. Selective excitation of glutamatergic nuclear neurons provides greater seizure inhibition than broadly exciting nuclear neurons without cell-type specificity. ABSTRACT: Temporal lobe epilepsy is the most common form of epilepsy in adults, but current treatment options provide limited efficacy, leaving as many as one-third of patients with uncontrolled seizures. Recently, attention has shifted towards more closed-loop therapies for seizure control, and on-demand optogenetic modulation of the cerebellar cortex was shown to be highly effective at attenuating hippocampal seizures. Intriguingly, both optogenetic excitation and inhibition of cerebellar cortical output neurons, Purkinje cells, attenuated seizures. The mechanisms by which the cerebellum impacts seizures, however, are unknown. In the present study, we targeted the immediate downstream projection of vermal Purkinje cells - the fastigial nucleus - in order to determine whether increases and/or decreases in fastigial output can underlie seizure cessation. Though Purkinje cell input to fastigial neurons is inhibitory, direct optogenetic inhibition of the fastigial nucleus had no effect on seizure duration. Conversely, however, fastigial excitation robustly attenuated hippocampal seizures. Seizure cessation was achieved at multiple stimulation frequencies, regardless of laterality relative to seizure focus, and even with single light pulses. Seizure inhibition was greater when selectively targeting glutamatergic fastigial neurons than when an approach that lacked cell-type specificity was used. Together, these results suggest that stimulating excitatory neurons in the fastigial nucleus may be a promising approach for therapeutic intervention in temporal lobe epilepsy.

Long-term chemogenetic suppression of spontaneous seizures in a mouse model for temporal lobe epilepsy.

OBJECTIVE: More than one-third of patients with temporal lobe epilepsy (TLE) continue to have seizures despite treatment with antiepileptic drugs, and many experience severe drug-related side effects, illustrating the need for novel therapies. Selective expression of inhibitory Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) allows cell-type-specific reduction of neuronal excitability. In this study, we evaluated the effect of chemogenetic suppression of excitatory pyramidal and granule cell neurons of the sclerotic hippocampus in the intrahippocampal mouse model (IHKA) for temporal lobe epilepsy. METHODS: Intrahippocampal IHKA mice were injected with an adeno-associated viral vector carrying the genes for an inhibitory DREADD hM4Di in the sclerotic hippocampus or control vector. Next, animals were treated systemically with different single doses of clozapine-N-oxide (CNO) (1, 3, and 10 mg/kg) and clozapine (0.03 and 0.1 mg/kg) and the effect on spontaneous hippocampal seizures, hippocampal electroencephalography (EEG) power, fast ripples (FRs) and behavior in the open field test was evaluated. Finally, animals received prolonged treatment with clozapine for 3 days and the effect on seizures was monitored. RESULTS: Treatment with both CNO and clozapine resulted in a robust suppression of hippocampal seizures for at least 15 hours only in DREADD-expressing animals. Moreover, total EEG power and the number of FRs were significantly reduced. CNO and/or clozapine had no effects on interictal hippocampal EEG, seizures, or locomotion/anxiety in the open field test in non-DREADD epileptic IHKA mice. Repeated clozapine treatment every 8 hours for 3 days resulted in almost complete seizure suppression in DREADD animals. SIGNIFICANCE: This study shows the potency of chemogenetics to robustly and sustainably suppress spontaneous epileptic seizures and pave the way for an epilepsy therapy in which a systemically administered exogenous drug selectively modulates specific cell types in a seizure network, leading to a potent seizure suppression devoid of the typical drug-related side effects.

2-Deoxyglucose protects hippocampal neurons against kainate-induced temporal lobe epilepsy by modulating monocyte-derived macrophages (mo-MΦ) and progranulin production in the hippocampus.

Inflammation is an important factor in the pathology of epilepsy with the hallmarks of resident microglia activation and infiltration of circulating monocytes in the damaged area. In the case of recovery and tissue repair, some monocytes change to macrophages (mo-MΦ) to enhance tissue repair. 2-deoxyglucose (2DG) is an analog of glucose capable of protecting the brain, and progranulin is a neurotrophic factor produced mainly by microglia and has an inflammation modulator effect. This study attempted to evaluate if one of the neuroprotective mechanisms of 2-DG is comprised of increasing monocyte-derived macrophages (mo-MΦ) and progranulin production. Status epilepticus (SE) was induced by i.c.v. injection of kainic acid (KA).2DG (125/mg/kg/day) was administered intraperitoneally. Four days later, animals were sacrificed. Their brain sections were then stained with Cresyl violet and Fluoro-Jade B to count the number of necrotic and degenerating neurons in CA3 and Hilus of dentate gyrus of the hippocampus. Lastly, immunohistochemistry was used to detect CD11b + monocyte, macrophage cells, and Progranulin level was evaluated by Western blotting. The histological analysis showed that 2DG can reduce the number of necrotic and degenerating neurons in CA3 and Hilar areas. Following KA administration, a great number of cD11b+ cells with monocyte morphology were observed in the hippocampus. 2DG not only reduced cD11b+ monocyte cells but was able to convert them to cells with the morphology of macrophages (mo-MΦ). 2DG also caused a significant increase in progranulin level in the hippocampus. Because macrophages and microglia are the most important sources of progranulin, it appears that 2DG caused the derivation of monocytes to macrophages and these cells produced progranulin with a subsequent anti-inflammation effect. In summary, it was concluded that 2DG is neuroprotective and probably one of its neuroprotective mechanisms is by modulating monocyte-derived macrophages by progranulin production.

Positive modulation of mGluR5 attenuates seizures and reduces TNF-α+ macrophages and microglia in the brain in a murine model of virus-induced temporal lobe epilepsy.

Viral encephalitis markedly increases the risk for the development of epilepsy. The Theiler's murine encephalomyelitis virus (TMEV)-induced model of seizures/epilepsy is a murine model of both viral-induced seizures/epilepsy and human Temporal Lobe Epilepsy. The inflammatory cytokines interleukin (IL)-6 and tumor necrosis factor (TNF)-α have been shown to play a role in seizure development in the TMEV-induced model of seizures/epilepsy, and infiltrating macrophages along with microglia have been shown to be major producers of these cytokines. The metabotropic glutamate receptor 5 (mGluR5) is a G-protein coupled receptor that has been shown to reduce IL-6 and TNF-α and to provide neuroprotection in other disease models. Therefore, we hypothesized that stimulation of mGluR5 would not only reduce seizures but attenuate IL-6 and TNF-α production in microglia and macrophages in the TMEV model. We found that pharmacological stimulation of mGluR5 with the selective positive allosteric modulator VU0360172 not only reduced acute seizure outcomes, but also reduced the percent of microglia and macrophages producing TNF-α 3 days post infection. Furthermore, treatment with VU0360172 did not alter the level of viral antigen, compared to controls, showing that this treatment does not compromise viral clearance. These results establish that mGluR5 may represent a therapeutic target in the TMEV-induced model of seizures/epilepsy.

ERG3 potassium channel-mediated suppression of neuronal intrinsic excitability and prevention of seizure generation in mice.

KEY POINTS: ERG3 channels have a high expression level in the central nervous system. Knockdown of ERG3 channels enhances neuronal intrinsic excitability (caused by decreased fast afterhyperpolarization, shortened delay time to the generation of an action potential and enhanced summation of somatic excitatory postsynaptic potentials) in hippocampal CA1 pyramidal neurons and dentate gyrus granule cells. The expression of ERG3 protein is reduced in human and mouse hippocampal epileptogenic foci. Knockdown of ERG3 channels in hippocampus enhanced seizure susceptibility, while mice treated with the ERG channel activator NS-1643 were less prone to epileptogenesis. The results provide strong evidence that ERG3 channels have a crucial role in the regulation of neuronal intrinsic excitability in hippocampal CA1 pyramidal neurons and dentate gyrus granule cells and are critically involved in the onset and development of epilepsy. ABSTRACT: The input-output relationship of neuronal networks depends heavily on the intrinsic properties of their neuronal elements. Profound changes in intrinsic properties have been observed in various physiological and pathological processes, such as learning, memory and epilepsy. However, the cellular and molecular mechanisms underlying acquired changes in intrinsic excitability are still not fully understood. Here, we demonstrate that ERG3 channels are critically involved in the regulation of intrinsic excitability in hippocampal CA1 pyramidal neurons and dentate gyrus granule cells. Knock-down of ERG3 channels significantly increases neuronal intrinsic excitability, which is mainly caused by decreased fast afterhyperpolarization, shortened delay time to the generation of an action potential and enhanced summation of somatic excitatory postsynaptic potentials. Interestingly, the expression level of ERG3 protein is significantly reduced in human and mouse brain tissues with temporal lobe epilepsy. Moreover, ERG3 channel knockdown in hippocampus significantly enhanced seizure susceptibility, while mice treated with the ERG channel activator NS-1643 were less prone to epileptogenesis. Taken together, our results suggest ERG3 channels play an important role in determining the excitability of hippocampal neurons and dysregulation of these channels may be involved in the generation of epilepsy. ERG3 channels may thus be a novel therapeutic target for the prevention of epilepsy.

Protective effect of compound Danshen (Salvia miltiorrhiza) dripping pills alone and in combination with carbamazepine on kainic acid-induced temporal lobe epilepsy and cognitive impairment in rats.

CONTEXT: Temporal lobe epilepsy (TLE) is resistant to antiepileptic drugs (AEDs) and is associated with cognitive impairment. The modern Chinese medicine, compound Danshen dripping pills (CDDP), is clinically effective in treating epilepsy and improving cognitive impairment. OBJECTIVE: This study evaluated the protective effects of CDDP alone and in combination with carbamazepine (CBZ) on kainic acid-induced TLE and cognitive impairment in rats. MATERIALS AND METHODS: Sprague-Dawley rats were randomly divided into five groups: control (sham operated), model, CDDP, CBZ and combined. A TLE model was then created via bilateral intrahippocampal injection of 0.35 µg kainic acid (KA). Rats received CDDP (85 mg/kg), CBZ (100 mg/kg) or combined (85 mg/kg CDDP +100 mg/kg CBZ) via intragastric administration for 90 d, respectively. Seizure intensity, apoptosis and glial cell line-derived neurotrophic factor (GDNF) were measured. Furthermore, the improvement in cognitive impairment and hippocampal neuronal damage was evaluated. RESULTS: CDDP combined with CBZ significantly decreased seizure severity and frequency (p < 0.05) and ameliorated cognitive impairment (p < 0.05). The model group showed a significant reduction of neurons and Bcl-2/Bax expression in the hippocampus CA3 area (p < 0.01), the combined groups significantly reversed these change (p < 0.01). GDNF expression in the combined groups showed a clear increase over the model group (p < 0.05). CONCLUSION: These findings support the use of CDDP as an adjuvant drug for the treatment of TLE and cognitive deficit. Its mechanism might be related to an anti-apoptosis effect and up-regulation of GDNF.

Epigenetic Histone Deacetylation Inhibition Prevents the Development and Persistence of Temporal Lobe Epilepsy.

Epilepsy is a chronic brain disease characterized by repeated unprovoked seizures. Currently, no drug therapy exists for curing epilepsy or disease modification in people at risk. Despite several emerging mechanisms, there have been few studies of epigenetic signaling in epileptogenesis, the process whereby a normal brain becomes progressively epileptic because of precipitating factors. Here, we report a novel role of histone deacetylation as a critical epigenetic mechanism in epileptogenesis. Experiments were conducted using the histone deacetylase (HDAC) inhibitor sodium butyrate in the hippocampus kindling model of temporal lobe epilepsy (TLE), a classic model heavily used to approve drugs for treatment of epilepsy. Daily treatment with butyrate significantly inhibited HDAC activity and retarded the development of limbic epileptogenesis without affecting after-discharge signal. HDAC inhibition markedly impaired the persistence of seizure expression many weeks after epilepsy development. Moreover, subchronic HDAC inhibition for 2 weeks resulted in a striking retardation of epileptogenesis. HDAC inhibition, unexpectedly, also showed erasure of the epileptogenic state in epileptic animals. Finally, butyrate-treated animals exhibited a powerful reduction in mossy fiber sprouting, a morphologic index of epileptogenesis. Together these results underscore that HDAC inhibition prevents the development of TLE, indicating HDAC's critical signaling role in epileptogenesis. These findings, therefore, envisage a unique novel therapy for preventing or curing epilepsy by targeting the epigenetic HDAC pathway.

Agomelatine protects against neuronal damage without preventing epileptogenesis in the kainate model of temporal lobe epilepsy.

Recent studies about the novel antidepressant agomelatine, which is a mixed MT1 and MT2 melatonin receptor agonist and 5HT2C serotonin receptor antagonist possessing an anticonvulsant and neuroprotective action, suggest that it may have potential to contribute against epileptogenesis and epilepsy-induced memory impairment. In order to ascertain whether protection of some brain structures could suppress epileptogenesis, in the present study, we evaluated the effect of chronic post-status treatment with agomelatine on epileptogenesis, behavioral and neuronal damage induced by kainate acid (KA) status epilepticus (SE). Agomelatine/vehicle treatment (40mg/kg, i.p.) started one hour after SE and continued up to 10weeks in Wistar rats. Latency for onset of spontaneous motor seizures (SMS) and their frequency was detected by a 24-h video-recording. Locomotor activity, anxiety and hippocampus-dependent spatial memory in open field (OF), elevated plus maze (EPM), light-dark test (LDT) and radial arm maze (RAM) test, respectively, were evaluated during the last two weeks after SE. Agomelatine significantly decreased the latency for onset of SMS and increased the seizure frequency during the 2nd and the 3rd week of treatment. The MT1 and MT2 receptor agonist and serotonin 5HT2C receptor antagonist exacerbated the KA-induced hyperlocomotion and impulsive behavior and it was unable to prevent spatial memory impairment of epileptic rats. However, agomelatine induced a neuroprotection in the dorsal hippocampus, specifically in the CA1, septal CA2 and partially in the CA3c region, the hilus of the dentate gyrus, piriform cortex and septo-temporal and temporal basolateral amygdala. Our findings suggest that the beneficial impact against SE-induced neuronal loss exerted by agomelatine is not crucial for the suppression of epileptogenesis and its deleterious consequences in KA model of temporal lobe epilepsy.

Deletion of mTOR 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.