HB-EGF activates EGFR to induce reactive neural stem cells in the mouse hippocampus after seizures.

Hippocampal seizures mimicking mesial temporal lobe epilepsy cause a profound disruption of the adult neurogenic niche in mice. Seizures provoke neural stem cells to switch to a reactive phenotype (reactive neural stem cells, React-NSCs) characterized by multibranched hypertrophic morphology, massive activation to enter mitosis, symmetric division, and final differentiation into reactive astrocytes. As a result, neurogenesis is chronically impaired. Here, using a mouse model of mesial temporal lobe epilepsy, we show that the epidermal growth factor receptor (EGFR) signaling pathway is key for the induction of React-NSCs and that its inhibition exerts a beneficial effect on the neurogenic niche. We show that during the initial days after the induction of seizures by a single intrahippocampal injection of kainic acid, a strong release of zinc and heparin-binding epidermal growth factor, both activators of the EGFR signaling pathway in neural stem cells, is produced. Administration of the EGFR inhibitor gefitinib, a chemotherapeutic in clinical phase IV, prevents the induction of React-NSCs and preserves neurogenesis.

ArcKR expression modifies synaptic plasticity following epileptic activity: Differential effects with in vitro and in vivo seizure-induction protocols.

OBJECTIVES: Pathological forms of neural activity, such as epileptic seizures, modify the expression pattern of multiple proteins, leading to persistent changes in brain function. One such protein is activity-regulated cytoskeleton-associated protein (Arc), which is critically involved in protein-synthesis-dependent synaptic plasticity underlying learning and memory. In the present study, we have investigated how the expression of ArcKR, a form of Arc in which the ubiquitination sites have been mutated, resulting in slowed Arc degradation, modifies group I metabotropic glutamate receptor-mediated long-term depression (G1-mGluR-LTD) following seizures. METHODS: We used a knock-in mice line that express ArcKR and two hyperexcitation models: an in vitro model, where hippocampal slices were exposed to zero Mg2+, 6 mM K+; and an in vivo model, where kainic acid was injected unilaterally into the hippocampus. In both models, field excitatory postsynaptic potentials (fEPSPs) were recorded from the CA1 region of hippocampal slices in response to Schaffer collateral stimulation and G1-mGluR-LTD was induced chemically with the group 1 mGluR agonist DHPG. RESULTS: In the in vitro model, ArcKR expression enhanced the effects of seizure activity and increased the magnitude of G1-mGluR LTD, an effect that could be blocked with the mGluR5 antagonist MTEP. In the in vivo model, fEPSPs were significantly smaller in slices from ArcKR mice and were less contaminated by population spikes. In this model, the amount of G1-mGluR-LTD was significantly less in epileptic slices from ArcKR mice as compared to wildtype (WT) mice. SIGNIFICANCE: We have shown that expression of ArcKR, a form of Arc in which degradation is reduced, significantly modulates the magnitude of G1-mGluR-LTD following epileptic seizures. However, the effect of ArcKR on LTD depends on the epileptic model used, with enhancement of LTD in an in vitro model and a reduction in the kainate mouse model.

Inhibition of Neuron-Restrictive Silencing Factor (REST/NRSF) Chromatin Binding Attenuates Epileptogenesis.

The mechanisms by which brain insults lead to subsequent epilepsy remain unclear. Insults including trauma, stroke, infections, and long seizures (status epilepticus, SE) increase the nuclear expression and chromatin binding of the neuron-restrictive silencing factor/RE-1 silencing transcription factor (NRSF/REST). REST/NRSF orchestrates major disruption of the expression of key neuronal genes, including ion channels and neurotransmitter receptors, potentially contributing to epileptogenesis. Accordingly, transient interference with REST/NRSF chromatin binding after an epilepsy-provoking SE suppressed spontaneous seizures for the 12 d duration of a prior study. However, whether the onset of epileptogenesis was suppressed or only delayed has remained unresolved. The current experiments determined if transient interference with REST/NRSF chromatin binding prevented epileptogenesis enduringly or, alternatively, slowed epilepsy onset. Epileptogenesis was elicited in adult male rats via systemic kainic acid-induced SE (KA-SE). We then determined if decoy, NRSF-binding-motif oligodeoxynucleotides (NRSE-ODNs), given twice following KA-SE (1) prevented REST/NRSF binding to chromatin, using chromatin immunoprecipitation, or (2) prevented the onset of spontaneous seizures, measured with chronic digital video-electroencephalogram. Blocking NRSF function transiently after KA-SE significantly lengthened the latent period to a first spontaneous seizure. Whereas this intervention did not influence the duration and severity of spontaneous seizures, total seizure number and seizure burden were lower in the NRSE-ODN compared with scrambled-ODN cohorts. Transient interference with REST/NRSF function after KA-SE delays and moderately attenuates insult-related hippocampal epilepsy, but does not abolish it. Thus, the anticonvulsant and antiepileptogenic actions of NRSF are but one of the multifactorial mechanisms generating epilepsy in the adult brain.

Synergistic neuroprotective action of prolactin and 17ß-estradiol on kainic acid-induced hippocampal injury and long-term memory deficit in ovariectomized rats.

PURPOSE: The neuroprotective actions of the ovarian hormone 17ß-estradiol (E2) against different brain lesions have been constantly confirmed in a variety of models including kainic acid (KA) lesions. Similarly, the pituitary hormone prolactin (PRL), traditionally associated with lactogenesis, has recently been linked to a large diversity of functions, including neurogenesis, neuroprotection, and cognitive processes. While the mechanisms of actions of E2 as regards its neuroprotective and behavioral effects have been extensively explored, the molecular mechanisms of PRL related to these roles remain under investigation. The current study aimed to investigate whether the simultaneous administration of PRL and a low dose of E2 prevents the KA-induced cognitive deficit and if this action is associated with changes in hippocampal neuronal density. METHODS: Ovariectomized (OVX) rats were treated with saline, PRL, and/or E2 in the presence or absence of KA. Neuroprotection was assessed by Nissl staining and neuron counting. Memory was evaluated with the novel object recognition test (NOR). RESULTS: On their own, both PRL and E2 prevented short- and long-term memory deficits in lesioned animals and exerted neuroprotection against KA-induced excitotoxicity in the hippocampus. Interestingly, the combined hormonal treatment was superior to either of the treatments administered alone as regards improving both memory and neuronal survival. CONCLUSION: Taken together, these results point to a synergic effect of E2 and PRL in the hippocampus to produce their behavioral, proliferative, and neuroprotective effects.

Sorbs2 regulates seizure activity by influencing AMPAR-mediated excitatory synaptic transmission in temporal lobe epilepsy.

Temporal lobe epilepsy (TLE), the most common type of drug-resistant epilepsy, severely affects quality of life. However, the underlying mechanism of TLE remains unclear and deserves further exploration. Sorbs2, a key synaptic regulatory protein, plays an important role in the regulation of synaptic transmission in the mammalian brain. In this study, we aimed to investigate the expression pattern of Sorbs2 in a kainic acid (KA)-induced TLE mouse model and in patients with TLE to further determine whether Sorbs2 is involved in seizure activity and to explore the potential mechanism by which Sorbs2 affects seizures in this TLE mouse model. First, we found that the expression of Sorbs2 was obviously increased in the hippocampus and cortex of a TLE mouse model and in the temporal cortex of TLE patients, indicating an abnormal expression pattern of Sorbs2 in TLE. Importantly, subsequent behavioral analyses and local field potential (LFP) analyses of a TLE mouse model demonstrated that the downregulation of hippocampal Sorbs2 could prolong the latency to spontaneous recurrent seizures (SRSs) and protect against SRSs. We also found that the knockdown of Sorbs2 in the hippocampus could decrease excitatory synaptic transmission in pyramidal neurons (PNs) in the hippocampal CA1 region and reduce the expression levels of the AMPAR subunits GluA1 and GluA2. Thus, we speculated that Sorbs2 may promote epileptogenesis and the development of TLE by affecting AMPAR-mediated excitatory synaptic transmission in PNs in the CA1 region. Therefore, reducing the expression of hippocampal Sorbs2 could restrain epileptogenesis and the development of TLE.

Small-molecule caspase-1 inhibitor CZL80 terminates refractory status epilepticus via inhibition of glutamatergic transmission.

Status epilepticus (SE), a serious and often life-threatening medical emergency, is characterized by abnormally prolonged seizures. It is not effectively managed by present first-line anti-seizure medications and could readily develop into drug resistance without timely treatment. In this study, we highlight the therapeutic potential of CZL80, a small molecule that inhibits caspase-1, in SE termination and its related mechanisms. We found that delayed treatment of diazepam (0.5 h) easily induces resistance in kainic acid (KA)-induced SE. CZL80 dose-dependently terminated diazepam-resistant SE, extending the therapeutic time window to 3 h following SE, and also protected against neuronal damage. Interestingly, the effect of CZL80 on SE termination was model-dependent, as evidenced by ineffectiveness in the pilocarpine-induced SE. Further, we found that CZL80 did not terminate KA-induced SE in Caspase-1-/- mice but partially terminated SE in IL1R1-/- mice, suggesting the SE termination effect of CZL80 was dependent on the caspase-1, but not entirely through the downstream IL-1ß pathway. Furthermore, in vivo calcium fiber photometry revealed that CZL80 completely reversed the neuroinflammation-augmented glutamatergic transmission in SE. Together, our results demonstrate that caspase-1 inhibitor CZL80 terminates diazepam-resistant SE by blocking glutamatergic transmission. This may be of great therapeutic significance for the clinical treatment of refractory SE.

Visually or auditorily induced seizures involve the activation of nonhippocampal brain areas and hippocampal removal does not alleviate seizures in a mouse model of temporal lobe epilepsy.

OBJECTIVE: Several studies have attributed epileptic activities in temporal lobe epilepsy (TLE) to the hippocampus; however, the participation of nonhippocampal neuronal networks in the development of TLE is often neglected. Here, we sought to understand how these nonhippocampal networks are involved in the pathology that is associated with TLE disease. METHODS: A kainic acid (KA) model of temporal lobe epilepsy was induced by injecting KA into dorsal hippocampus of C57BL/6J mice. Network activation after spontaneous seizure was assessed using c-Fos expression. Protocols to induce seizure using visual or auditory stimulation were developed, and seizure onset zone (SOZ) and frequency of epileptic spikes were evaluated using electrophysiology. The hippocampus was removed to assess seizure recurrence in the absence of hippocampus. RESULTS: Our results showed that cortical and hippocampal epileptic networks are activated during spontaneous seizures. Perturbation of these networks using visual or auditory stimulation readily precipitates seizures in TLE mice; the frequency of the light-induced or noise-induced seizures depends on the induction modality adopted during the induction period. Localization of SOZ revealed the existence of cortical and hippocampal SOZ in light-induced and noise-induced seizures, and the development of local and remote epileptic spikes in TLE occurs during the early stage of the disease. Importantly, we further discovered that removal of the hippocampi does not stop seizure activities in TLE mice, revealing that seizures in TLE mice can occur independent of the hippocampus. SIGNIFICANCE: This study has shown that the network pathology that evolves in TLE is not localized to the hippocampus; rather, remote brain areas are also recruited. The occurrence of light-induced or noise-induced seizures and epileptic discharges in epileptic mice is a consequence of the activation of nonhippocampal brain areas. This work therefore demonstrates the fundamental role of nonhippocampal epileptic networks in generating epileptic activities with or without the hippocampus in TLE disease.

Chemogenetic approaches reveal dual functions of microglia in seizures.

Microglia are key players in maintaining brain homeostasis and exhibit phenotypic alterations in response to epileptic stimuli. However, it is still relatively unknown if these alterations are pro- or anti-epileptic. To unravel this dilemma, we employed chemogenetic manipulation of microglia using the artificial Gi-Dreadd receptor within a kainic acid (KA) induced murine seizure model. Our results indicate that acute Gi-Dreadd activation with Clozapine-N-Oxide can reduce seizure severity. Additionally, we observed increased interaction between microglia and neuronal soma, which correlated with reduced neuronal hyperactivity. Interestingly, prolonged activation of microglial Gi-Dreadds by repeated doses of CNO over 3 days, arrested microglia in a less active, homeostatic-like state, which associated with increased neuronal loss after KA induced seizures. RNAseq analysis revealed that prolonged activation of Gi-Dreadd interferes with interferon ß signaling and microglia proliferation. Thus, our findings highlight the importance of microglial Gi signaling not only during status epilepticus (SE) but also within later seizure induced pathology.

Small-molecule positive allosteric modulation of homomeric kainate receptors GluK1-3: development of screening assays and insight into GluK3 structure.

The kainate receptors GluK1-3 (glutamate receptor ionotropic, kainate receptors 1-3) belong to the family of ionotropic glutamate receptors and are essential for fast excitatory neurotransmission in the brain, and are associated with neurological and psychiatric diseases. How these receptors can be modulated by small-molecule agents is not well understood, especially for GluK3. We show that the positive allosteric modulator BPAM344 can be used to establish robust calcium-sensitive fluorescence-based assays to test agonists, antagonists, and positive allosteric modulators of GluK1-3. The half-maximal effective concentration (EC50) of BPAM344 for potentiating the response of 100 µm kainate was determined to be 26.3 µm for GluK1, 75.4 µm for GluK2, and 639 µm for GluK3. Domoate was found to be a potent agonist for GluK1 and GluK2, with an EC50 of 0.77 and 1.33 µm, respectively, upon co-application of 150 µm BPAM344. At GluK3, domoate acts as a very weak agonist or antagonist with a half-maximal inhibitory concentration (IC50) of 14.5 µm, in presence of 500 µm BPAM344 and 100 µm kainate for competition binding. Using H523A-mutated GluK3, we determined the first dimeric structure of the ligand-binding domain by X-ray crystallography, allowing location of BPAM344, as well as zinc-, sodium-, and chloride-ion binding sites at the dimer interface. Molecular dynamics simulations support the stability of the ion sites as well as the involvement of Asp761, Asp790, and Glu797 in the binding of zinc ions. Using electron microscopy, we show that, in presence of glutamate and BPAM344, full-length GluK3 adopts a dimer-of-dimers arrangement.

Domoic acid affects brain morphology and causes behavioral alterations in two fish species.

Domoic acid (DA) produces neurotoxic damage in seabirds and marine mammals when they are exposed to this potent neurotoxin. Other vertebrates are also susceptible to DA intoxication including humans. However, neurobehavioral affectations have not been detected in fish when naturally exposed to DA but only when it is administered intraperitoneally. Therefore, the current idea is that fish are less sensitive to DA acquired under ecologically relevant routes of exposure. Here, we show that oral consumption of DA induces neurobehavioral and histopathological alterations in the brain and heart of totoaba (Totoaba macdonaldi) and striped bass (Morone saxatilis). Lesions were found in both species in the optic tectum and cerebellum after exposure for 7 days to a diet containing 0.776 µgDA g-1. The affectations prevailed chronically. Also, we found that cardiac tissue exhibits lesions and focal atrium melanism. Although affectations of the brain and heart tissue were evident, excitotoxic signs like those described for other vertebrates were not observed. However, the use of standardized behavioral tests (dark/light and antipredator avoidance tests) permitted the detection of behavioral impairment of fish after DA exposure. Pathological and associated behavioral alterations produced by DA can have relevant physiological consequences but also important ecological implications.

Effect of intranasal and oral administration of levetiracetam on the temporal and spatial distributions of SV2A in the KA-induced rat model of SE.

To investigate the effectiveness of nasal delivery of levetiracetam (LEV) on the distributions of synaptic vesicle protein 2 isoform A (SV2A) in epileptic rats with injection of kainic acid (KA) into amygdala. A total of 138 rats were randomly divided into four groups, including the Sham surgery group, the epilepsy group (EP), and the LEV oral administration (LPO) and nasal delivery (LND) groups. The rat intra-amygdala KA model of epilepsy was constructed. Pathological changes of rat brain tissue after status epilepticus (SE) were detected using haematoxylin and eosin staining. Expression of SV2A in rat hippocampus after SE was evaluated using the western blotting analysis. Expression and distribution of SV2A in rat hippocampus after SE were detected based on immunofluorescence staining. The EP group showed evident cell loss and tissue necrosis in the CA3 area of hippocampus, whereas the tissue damage in both LPO and LND groups was significantly reduced. Western blotting analysis showed that the expressions of SV2A in the hippocampus of both EP and LND groups were significantly decreased 1 week after SE, increased to the similar levels of the Sham group in 2 weeks, and continuously increased 4 weeks after SE to the level significantly higher than that of the Sham group. Results of immunofluorescence revealed largely the same expression patterns of SV2A in the CA3 area of hippocampus as those in the entire hippocampus. Our study revealed the same antiepileptic and neuronal protective effects by the nasal and oral administrations of LEV, without changing the expression level of SV2A.

Morphological alterations of the neuronal Golgi apparatus upon seizures.

AIMS: Epilepsy is one of the most common chronic neurological disorders, affecting around 50 million people worldwide, but its underlying cellular and molecular events are not fully understood. The Golgi is a highly dynamic cellular organelle and can be fragmented into ministacks under both physiological and pathological conditions. This phenomenon has also been observed in several neurodegenerative disorders; however, the structure of the Golgi apparatus (GA) in human patients suffering from epilepsy has not been described so far. The aim of this study was to assess the changes in GA architecture in epilepsy. METHODS: Golgi visualisation with immunohistochemical staining in the neocortex of adult patients who underwent epilepsy surgery; 3D reconstruction and quantitative morphometric analysis of GA structure in the rat hippocampi upon kainic acid (KA) induced seizures, as well as in vitro studies with the use of Ca2+ chelator BAPTA-AM in primary hippocampal neurons upon activation were performed. RESULTS: We observed GA dispersion in neurons of the human neocortex of patients with epilepsy and hippocampal neurons in rats upon KA-induced seizures. The structural changes of GA were reversible, as GA morphology returned to normal within 24 h of KA treatment. KA-induced Golgi fragmentation observed in primary hippocampal neurons cultured in vitro was largely abolished by the addition of BAPTA-AM. CONCLUSIONS: In our study, we have shown for the first time that the neuronal GA is fragmented in the human brain of patients with epilepsy and rat brain upon seizures. We have shown that seizure-induced GA dispersion can be reversible, suggesting that enhanced neuronal activity induces Golgi reorganisation that is involved in aberrant neuronal plasticity processes that underlie epilepsy. Moreover, our results revealed that elevated cytosolic Ca2+ is indispensable for these KA-induced morphological alterations of GA in vitro.

PDI augments kainic acid-induced seizure activity and neuronal death by inhibiting PP2A-GluA2-PICK1-mediated AMPA receptor internalization in the mouse hippocampus.

Protein disulfide isomerase (PDI) is a redox-active enzyme and also serves as a nitric oxide donor causing S-nitrosylation of cysteine residues in various proteins. Although PDI knockdown reduces α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor (AMPAR)-mediated neuronal activity, the underlying mechanisms are largely unknown. In the present study, we found that under physiological condition PDI knockdown increased CaMKII activity (phosphorylation) in the mouse hippocampus. However, PDI siRNA inhibited protein phosphatase (PP) 2A-mediated GluA2 S880 dephosphorylation by increasing PP2A oxidation, independent of S-nitrosylation. PDI siRNA also enhanced glutamate ionotropic receptor AMPA type subunit 1 (GluA1) S831 and GluA2 S880, but not GluA1 S845 and GluA2 Y869/Y873/Y876 phosphorylations, concomitant with the enhanced protein interacting with C kinase 1 (PICK1)-mediated AMPAR internalization. Furthermore, PDI knockdown attenuated seizure activity and neuronal damage in response to kainic acid (a non-desensitizing agonist of AMPAR). Therefore, these findings suggest that PDI may regulate surface AMPAR expression through PP2A-GluA2-PICK1 signaling pathway, and that PDI may be one of the therapeutic targets for epilepsy via AMPAR internalization without altering basal neurotransmission.

Principal neurons in the olfactory cortex mediate bidirectional modulation of seizures.

Although the piriform cortex (PC) has been previously implicated as a critical node for seizure generation and propagation, the underlying neural mechanism has remained unclear. Here, we found increased excitability in PC neurons during amygdala kindling acquisition. Optogenetic or chemogenetic activation of PC pyramidal neurons promoted kindling progression, whereas inhibition of these neurons retarded seizure activities induced by electrical kindling in the amygdala. Furthermore, chemogenetic inhibition of PC pyramidal neurons alleviated the severity of kainic acid-induced acute seizures. These results demonstrate that PC pyramidal neurons bidirectionally modulate seizures in temporal lobe epilepsy, providing evidence for the efficacy of PC pyramidal neurons as a potential therapeutic target for epileptogenesis. KEY POINTS: While the piriform cortex (PC) is an important olfactory centre critically involved in olfactory processing and plays a crucial role in epilepsy due to its close connection with the limbic system, how the PC regulates epileptogenesis is largely unknown. In this study, we evaluated the neuronal activity and the role of pyramidal neurons in the PC in the mouse amygdala kindling model of epilepsy. PC pyramidal neurons are hyperexcited during epileptogenesis. Optogenetic and chemogenetic activation of PC pyramidal neurons significantly promoted seizures in the amygdala kindling model, whereas selective inhibition of these neurons produced an anti-epileptic effect for both electrical kindling and kainic acid-induced acute seizures. The results of the present study indicate that PC pyramidal neurons bidirectionally modulate seizure activity.

GKLF, a transcriptional activator of Txnip, drives microglia activation in kainic acid-induced murine models of epileptic seizures.

Neuroinflammation is a major component of epilepsy. Gut-enriched Kruppel-like factor (GKLF), a transcription factor of Kruppel-like factor family, has been reported to promote microglia activation and mediate neuroinflammation. However, the role of GKLF in epilepsy remains poorly characterized. This study focused on the function of GKLF in neuron loss and neuroinflammation in epilepsy and the molecular mechanism underlying microglia activation induced by GKLF upon lipopolysaccharides (LPS) treatment. An experimental epileptic model was induced by an intraperitoneal injection of 25 mg/kg kainic acid (KA). Lentivirus vectors (Lv) carrying Gklf CDS or short hairpin RNA targeting Gklf (shGKLF) was injected into the hippocampus, resulting in Gklf overexpression or knockdown in the hippocampus. BV-2 cells were co-infected with Lv-shGKLF or/and Lv carrying thioredoxin interacting protein (Txnip) CDS for 48 h and treated with 1 µg/mL LPS for 24 h. Results showed that GKLF enhanced KA-induced neuronal loss, pro-inflammatory cytokine secretion, activation of NOD-like receptor protein-3 (NLRP3) inflammasomes and microglia, and TXNIP expression in the hippocampus. GKLF inhibition showed negative effects on LPS-induced microglia activation, as evidenced by reduced pro-inflammatory cytokine secretion and activation of NLRP3 inflammasomes. GKLF bound to Txnip promoter and increased TXNIP expression in LPS-activated microglia. Interestingly, Txnip overexpression reversed the inhibitory effect of Gklf knockdown on microglia activation. These findings indicated that GKLF was involved in microglia activation via TXNIP. This study demonstrates the underlying mechanism of GKLF in the pathogenesis of epilepsy and uncovers that GKLF inhibition may be a therapeutic strategy for epilepsy treatment.

GSDMD knockdown exacerbates hippocampal damage and seizure susceptibility by crosstalk between pyroptosis and apoptosis in kainic acid-induced temporal lobe epilepsy.

BACKGROUND: Neuronal loss is a vital pathological feature of temporal lobe epilepsy (TLE). However, the exact mechanism of neuronal loss in TLE is not fully understood. Pyroptosis, a novel form of programmed cell death (PCD), has been considered a contributor to the pathogenesis of TLE. However, recent studies have implicated extensive molecular crosstalk among pyroptosis, apoptosis, and necroptosis in various diseases, and they can be transformed to each other according to different contexts. This study aimed to investigate whether gasdermin D (GSDMD)-mediated pyroptosis is involved in the pathogenesis of TLE and whether crosstalk exists in the process of the modulation of pyroptosis. METHODS: The TLE model was established by intra-amygdala injection of kainic acid. The Racine score and local field potential (LFP) recordings were used to assess seizure severity. Western blotting and immunofluorescence were applied to detect the levels and cellular localization of GSDMD. The neuronal loss and type of neuronal death in the bilateral hippocampus were assessed by Nissl staining and flow cytometry analysis. The underlying crosstalk among pyroptosis, apoptosis, and necroptosis was explored by western blot and verified by VX765. RESULTS: GSDMD was significantly upregulated and mainly expressed within the neurons of the hippocampus in the TLE model. Inhibition of pyroptosis by GSDMD knockdown triggered caspase-3-mediated apoptosis, leading to excess neuronal loss and deterioration of epileptic behaviors. Blocking caspase-1 markedly inhibited caspase-3-mediated apoptosis and improved epileptic behaviors under GSDMD knockdown. CONCLUSIONS: Our results demonstrate that GSDMD-mediated pyroptosis is involved in the pathogenesis of TLE. However, inhibition of GSDMD triggers caspase-1-mediated crosstalk between pyroptosis and apoptosis, which exacerbates neuronal loss and seizure susceptibility. Therefore, the complex crosstalk among different forms of PCD should be considered when a potential molecular target in the single PCD pathway is modulated. On the other hand, along with further studies of molecular crosstalk among the PCD pathways, taking advantage of crosstalk to attenuate neuronal loss may provide new insight for the clinical therapy of TLE.

Repurposing of cholesterol-lowering agents in status epilepticus: A neuroprotective effect of simvastatin.

The increase of cholesterol synthesis after a status epilepticus may lead to excitotoxic processes, neuronal loss and favor the appearance of spontaneous epileptic seizures. Lowering cholesterol content could be a neuroprotective strategy. Here, we evaluated the protective effect of simvastatin administrated daily for 14 days, after the induction of a status epilepticus by intrahippocampal injection of kainic acid in mice. The results were compared to those obtained from mice showing a kainic acid-induced status epilepticus, treated daily with a saline solution, and from mice injected with a control phosphate-buffered solution without any status epilepticus. We first assessed the antiseizure effects of simvastatin by performing video-electroencephalographic recordings during the first three hours after kainic acid injection and continuously between the fifteenth and the thirty-first days. Mice treated with simvastatin had significantly fewer generalized seizures during the first three hours without a significant effect on generalized seizures after two weeks. There was a trend for fewer hippocampal electrographic seizures after two weeks. Secondly, we evaluated the neuroprotective and anti-inflammatory effects of simvastatin by measuring the fluorescence of neuronal and astrocyte markers on the thirtieth day after status onset. We found that simvastatin reduced CA1 reactive astrocytosis, demonstrated by a significant 37% decrease in GFAP-positive cells, and that simvastatin prevented the neuronal loss in CA1, demonstrated by a significant 42% increase in the NeuN-positive cells, as compared to the findings in mice with kainic acid-induced status epilepticus treated by a saline solution. Our study confirms the interest of cholesterol-lowering agents, and in particular simvastatin, in status epilepticus and paves the way for a clinical pilot study to prevent neurological sequelae after status epilepticus. This paper was presented at the 8th London-Innsbruck Colloquium on Status Epilepticus and Acute Seizures held in September 2022.

Alpha-Pinene Exerts Antiseizure Effects by Preventing Oxidative Stress and Apoptosis in the Hippocampus in a Rat Model of Temporal Lobe Epilepsy Induced by Kainate.

Oxidative stress and apoptosis following seizures play pivotal roles in the consequences of repeated seizures. Beneficial effects of alpha-pinene (APN) have been reported in some experimental models of neurodegenerative diseases. However, its neuroprotective efficacy in a rat model of temporal lobe epilepsy (TLE) induced by kainic acid (KA) has remained unexplored. We aimed to explore the possible antiseizure effects of APN pretreatment and underlying molecular mechanisms in a rat model of TLE induced by KA. TLE was induced in male Wistar rats by intracerebroventricular injection of KA. APN at a dose of 50 mg/kg/day was intraperitoneally injected for 2 weeks before induction of TLE. One day after the induction of TLE, behavioral expressions of seizure were recorded and scored using Racine's scale. Furthermore, the hippocampal levels of oxidative stress markers, B-cell lymphoma 2 (Bcl2), BCL2-associated X protein (BAX), and c-Jun N-terminal kinase (JNK) protein levels were also assessed. Histopathological assessment in the hippocampus was performed with Nissl staining 5 days following induction of TLE. The results revealed that APN pretreatment alleviated epileptic seizures, diminished oxidative stress indicators, blocked the mitochondrial apoptotic pathway via decreasing BAX and raising BCL2 protein levels in the hippocampus at least partly through inhibiting JNK activity, and decreased neuronal death in the CA3 and hilus regions. These findings reveal that APN pretreatment mitigates KA-induced seizures by blocking oxidative stress and neuronal damage factors. It can be concluded that APN has a potent potential to be considered an antiseizure medication, but it needs further investigation.

Investigating the mechanism of antiepileptogenic effect of apigenin in kainate temporal lobe epilepsy: possible role of mTOR.

Clarifying the underlying mechanisms of epileptogenesis is important in preventing the progression of chronic epilepsy. In epilepsy, the mTOR (mammalian target of rapamycin) pathway plays a critical role in mediating the mechanism of epileptogenesis. In this study, we investigate whether apigenin can exert antiepileptogenic effects through the inhibition of mTOR in the kainate model of epilepsy. For assessing the antiepileptogenic effect of apigenin in kainic acid (KA)-induced temporal lobe epilepsy (TLE) model, apigenin at a dose of 50 mg/kg was administrated by gavage for 6 days. An intracranial electroencephalogram (iEEG) was performed to confirm the establishment of status epilepticus. BrdU was used to detect neurogenesis in the CA3, and dentate gyrus and mossy fiber sproutings were assessed by Timm staining. The expression of mTOR was quantified via western blot. We found that apigenin-pretreatment had a significant inhibitory effect on neural cell death, spontaneous seizure spikes, aberrant neurogenesis, mTOR hyperactivity, and aberrant mossy fiber sprouting. Overall, these results suggest that apigenin has an antiepileptogenic effect and may be a useful target for inhibiting mTOR hyperactivity in epilepsy.

Characterization of the anticonvulsant effect of dapsone on metabolic activity assessed by [18F]FDG -PET after kainic acid-induced status epilepticus in rats.

BACKGROUND: Development of effective drugs for epilepsy are needed, as nearly 30 % of epileptic patients, are resistant to current treatments. This study is aimed to characterize the anticonvulsant effect of dapsone (DDS), in the kainic acid (KA)-induced Status Epilepticus (SE) by recording the brain metabolic activity with an [18F]FDG-PET analysis. METHODS: Wistar rats received KA (10 mg/kg, i.p., single dose) to produce sustained seizures. [18F]FDG-PET and electroencephalographic (EEG) studies were then performed. DDS or vehicle were administered 30 min before KA. [18F]FDG uptake and EEG were evaluated at baseline, 2 and 25 h after KA injection. Likewise, caspase-8, 3 hippocampal activities and Fluoro-Jade B neuronal degeneration and Hematoxylin-eosin staining were measured 25 h after KA. RESULTS: PET data evaluated at 2 h showed hyper-uptake of [18F]FDG in the control group, which was decreased by DDS. At 25 h, hypo-uptake was observed in the control group and higher values due to DDS effect. EEG spectral power was increased 2 h after KA administration in the control group during the generalized tonic-clonic seizures, which was reversed by DDS, correlated with [18F]FDG-PET uptake changes. The values of caspases-8 activity decreased 48 and 43 % vs control group in the groups treated with DDS (12.5 y 25 mg/kg respectively), likewise; caspase-3 activity diminished by 57 and 53 %. Fewer degenerated neurons were observed due to DDS treatments. CONCLUSIONS: This study pinpoints the anticonvulsant therapeutic potential of DDS. Given its safety and effectiveness, DDS may be a viable alternative for patients with drug-resistant epilepsy.