Subicular Caspase-1 Contributes to Pharmacoresistance in Temporal Lobe Epilepsy.

OBJECTIVE: Unidentified mechanisms largely restrict the viability of effective therapies in pharmacoresistant epilepsy. Our previous study revealed that hyperactivity of the subiculum is crucial for the genesis of pharmacoresistance in temporal lobe epilepsy (TLE), but the underlying molecular mechanism is not clear. METHODS: Here, we examined the role of subicular caspase-1, a key neural pro-inflammatory enzyme, in pharmacoresistant TLE. RESULTS: We found that the expression of activated caspase-1 in the subiculum, but not the CA1, was upregulated in pharmacoresistant amygdaloid-kindled rats. Early overexpression of caspase-1 in the subiculum was sufficient to induce pharmacoresistant TLE in rats, whereas genetic ablation of caspase-1 interfered with the genesis of pharmacoresistant TLE in both kindled rats and kainic acid-treated mice. The pro-pharmacoresistance effect of subicular caspase-1 was mediated by its downstream inflammasome-dependent interleukin-1ß. Further electrophysiological results showed that inhibiting caspase-1 decreased the excitability of subicular pyramidal neurons through influencing the excitation/inhibition balance of presynaptic input. Importantly, a small molecular caspase-1 inhibitor CZL80 attenuated seizures in pharmacoresistant TLE models, and decreased the neuronal excitability in the brain slices obtained from patients with pharmacoresistant TLE. INTERPRETATION: These results support the subicular caspase-1-interleukin-1ß inflammatory pathway as a novel alternative mechanism hypothesis for pharmacoresistant TLE, and present caspase-1 as a potential target. ANN NEUROL 2021;90:377-390.

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.

The matrix metalloproteinase inhibitor IPR-179 has antiseizure and antiepileptogenic effects.

Matrix metalloproteinases (MMPs) are synthesized by neurons and glia and released into the extracellular space, where they act as modulators of neuroplasticity and neuroinflammatory agents. Development of epilepsy (epileptogenesis) is associated with increased expression of MMPs, and therefore, they may represent potential therapeutic drug targets. Using quantitative PCR (qPCR) and immunohistochemistry, we studied the expression of MMPs and their endogenous inhibitors tissue inhibitors of metalloproteinases (TIMPs) in patients with status epilepticus (SE) or temporal lobe epilepsy (TLE) and in a rat TLE model. Furthermore, we tested the MMP2/9 inhibitor IPR-179 in the rapid-kindling rat model and in the intrahippocampal kainic acid mouse model. In both human and experimental epilepsy, MMP and TIMP expression were persistently dysregulated in the hippocampus compared with in controls. IPR-179 treatment reduced seizure severity in the rapid-kindling model and reduced the number of spontaneous seizures in the kainic acid model (during and up to 7 weeks after delivery) without side effects while improving cognitive behavior. Moreover, our data suggest that IPR-179 prevented an MMP2/9-dependent switch-off normally restraining network excitability during the activity period. Since increased MMP expression is a prominent hallmark of the human epileptogenic brain and the MMP inhibitor IPR-179 exhibits antiseizure and antiepileptogenic effects in rodent epilepsy models and attenuates seizure-induced cognitive decline, it deserves further investigation in clinical trials.

Upregulation of lactate dehydrogenase A in a chronic model of temporal lobe epilepsy.

The ketogenic diet treatment is effective for drug-resistant epilepsy. Because its antiepileptic effect is associated with lactate dehydrogenase (LDH), drug development is possible by targeting LDH enzymes. Seizures in rodent models are suppressed by inhibiting LDH; however, it remains unclear whether LDH in the brain is changed by seizures. In the present study, we examined the expression of LDH subunits (LDHA and LDHB) in a chronic model of temporal lobe epilepsy, in which seizures were induced by the microinjection of kainate into the mouse hippocampus. Using Western blot analyses, we found that LDHA expression was increased in the hippocampus of the chronic seizure model, whereas LDHB expression was not. Lactate levels in the hippocampus were also increased in this seizure model, suggesting elevated LDH enzymatic activities. Furthermore, the inhibition of LDHA suppressed spontaneous paroxysmal discharges in vivo in the chronic seizure model. In conclusion, our results show that chronic seizures increase LDHA, and conversely, the inhibition of LDHA suppresses seizures, which supports LDHA as a molecular target for the development of new antiepileptic drugs.

JNK Isoforms Are Involved in the Control of Adult Hippocampal Neurogenesis in Mice, Both in Physiological Conditions and in an Experimental Model of Temporal Lobe Epilepsy.

Neurogenesis in the adult dentate gyrus (DG) of the hippocampus allows the continuous generation of new neurons. This cellular process can be disturbed under specific environmental conditions, such as epileptic seizures; however, the underlying mechanisms responsible for their control remain largely unknown. Although different studies have linked the JNK (c-Jun-N-terminal-kinase) activity with the regulation of cell proliferation and differentiation, the specific function of JNK in controlling adult hippocampal neurogenesis is not well known. The purpose of this study was to analyze the role of JNK isoforms (JNK1/JNK2/JNK3) in adult-hippocampal neurogenesis. To achieve this goal, we used JNK-knockout mice (Jnk1-/-, Jnk2-/-, and Jnk3-/-), untreated and treated with intraperitoneal injections of kainic acid (KA), as an experimental model of epilepsy. In each condition, we identified cell subpopulations at different stages of neuronal maturation by immunohistochemical specific markers. In physiological conditions, we evidenced that JNK1 and JNK3 control the levels of one subtype of early progenitor cells (GFAP+/Sox2+) but not the GFAP+/Nestin+ cell subtype. Moreover, the absence of JNK1 induces an increase of immature neurons (Doublecortin+; PSA-NCAM+ cells) compared with wild-type (WT). On the other hand, Jnk1-/- and Jnk3-/- mice showed an increased capacity to maintain hippocampal homeostasis, since calbindin immunoreactivity is higher than in WT. An important fact is that, after KA injection, Jnk1-/- and Jnk3-/- mice show no increase in the different neurogenic cell subpopulation analyzed, in contrast to what occurs in WT and Jnk2-/- mice. All these data support that JNK isoforms are involved in the adult neurogenesis control.

Astrocytes and Glutamine Synthetase in Epileptogenesis.

The cellular, molecular, and metabolic mechanisms that underlie the development of mesial temporal lobe epilepsy are incompletely understood. Here we review the role of astrocytes in epilepsy development (a.k.a. epileptogenesis), particularly astrocyte pathologies related to: aquaporin 4, the inwardly rectifying potassium channel Kir4.1, monocarboxylate transporters MCT1 and MCT2, excitatory amino acid transporters EAAT1 and EAAT2, and glutamine synthetase. We propose that inhibition, dysfunction or loss of astrocytic glutamine synthetase is an important causative factor for some epilepsies, particularly mesial temporal lobe epilepsy and glioblastoma-associated epilepsy. We postulate that the regulatory mechanisms of glutamine synthetase as well as the downstream effects of glutamine synthetase dysfunction, represent attractive, new targets for antiepileptogenic interventions. Currently, no antiepileptogenic therapies are available for human use. The discovery of such interventions is important as it will fundamentally change the way we approach epilepsy by preventing the disease from ever becoming manifest after an epileptogenic insult to the brain.

Astrocytic glutamine synthetase is expressed in the neuronal somatic layers and down-regulated proportionally to neuronal loss in the human epileptic hippocampus.

Human mesial temporal lobe epilepsy (MTLE) features subregion-specific hippocampal neurodegeneration and reactive astrogliosis, including up-regulation of the glial fibrillary acidic protein (GFAP) and down-regulation of glutamine synthetase (GS). However, the regional astrocytic expression pattern of GFAP and GS upon MTLE-associated neurodegeneration still remains elusive. We assessed GFAP and GS expression in strict correlation with the local neuronal number in cortical and hippocampal surgical specimens from 16 MTLE patients using immunohistochemistry, stereology and high-resolution image analysis for digital pathology and whole-slide imaging. In the cortex, GS-positive (GS+) astrocytes are dominant in all neuronal layers, with a neuron to GS+ cell ratio of 2:1. GFAP-positive (GFAP+) cells are widely spaced, with a GS+ to GFAP+ cell ratio of 3:1-5:1. White matter astrocytes, on the contrary, express mainly GFAP and, to a lesser extent, GS. In the hippocampus, the neuron to GS+ cell ratio is approximately 1:1. Hippocampal degeneration is associated with a reduction of GS+ astrocytes, which is proportional to the degree of neuronal loss and primarily present in the hilus. Up-regulation of GFAP as a classical hallmark of reactive astrogliosis does not follow the GS-pattern and is prominent in the CA1. Reactive alterations were proportional to the neuronal loss in the neuronal somatic layers (stratum pyramidale and hilus), while observed to a lesser extent in the axonal/dendritic layers (stratum radiatum, molecular layer). We conclude that astrocytic GS is expressed in the neuronal somatic layers and, upon neurodegeneration, is down-regulated proportionally to the degree of neuronal loss.

Neuroprotective Effects of the Absence of JNK1 or JNK3 Isoforms on Kainic Acid-Induced Temporal Lobe Epilepsy-Like Symptoms.

The activation of c-Jun-N-terminal kinases (JNK) pathway has been largely associated with the pathogenesis and the neuronal death that occur in neurodegenerative diseases. Altogether, this justifies why JNKs have become a focus of screens for new therapeutic strategies. The aim of the present study was to identify the role of the different JNK isoforms (JNK1, JNK2, and JNK3) in apoptosis and inflammation after induction of brain damage. To address this aim, we induced excitotoxicity in wild-type and JNK knockout mice (jnk1 -/- , jnk2 -/- , and jnk3 -/- ) via an intraperitoneal injection of kainic acid, an agonist of glutamic-kainate-receptors, that induce status epilepticus.Each group of animals was divided into two treatments: a single intraperitoneal dose of saline solution, used as a control, and a single intraperitoneal dose (30 mg/kg) of kainic acid. Our results reported a significant decrease in neuronal degeneration in the hippocampus of jnk1 -/- and jnk3 -/- mice after kainic acid treatment, together with reduced or unaltered expression of several apoptotic genes compared to WT treated mice. In addition, both jnk1 -/- and jnk3 -/- mice exhibited a reduction in glial reactivity, as shown by the lower expression of inflammatory genes and a reduction of JNK phosphorylation. In addition, in jnk3 -/- mice, the c-Jun phosphorylation was also diminished.Collectively, these findings provide compelling evidence that the absence of JNK1 or JNK3 isoforms confers neuroprotection against neuronal damage induced by KA and evidence, for the first time, the implication of JNK1 in excitotoxicity. Accordingly, JNK1 and/or JNK3 are promising targets for the prevention of cell death and inflammation during epileptogenesis.

Tissue Expressions of Soluble Human Epoxide Hydrolase-2 Enzyme in Patients with Temporal Lobe Epilepsy.

OBJECTIVE: We sought to simply demonstrate how levels of soluble human epoxide hydrolase-2 show changes in both temporal the cortex and hippocampal complex in patients with temporal lobe epilepsy. METHODS: A total of 20 patients underwent anterior temporal lobe resection due to temporal lobe epilepsy. The control group comprised 15 people who died in traffic accidents or by falling from a height, and their autopsy findings were included. Adequately sized temporal cortex and hippocampal samples were removed from each patient during surgery, and the same anatomic structures were removed from the control subjects during the autopsy procedures. Each sample was stored at -80°C as rapidly as possible until the enzyme assay. RESULTS: The temporal cortex in the epilepsy patients had a significantly higher enzyme level than did the temporal cortex of the control group (P = 0.03). Correlation analysis showed that as the enzyme level increases in the temporal cortex, it also increases in the hippocampal complex (r2 = 0.06, P = 0.00001). More important, enzyme tissue levels showed positive correlations with seizure frequency in both the temporal cortex and hippocampal complex in patients (r2 = 0.7, P = 0.00001 and r2 = 0.4, P = 0.003, respectively). The duration of epilepsy was also positively correlated with the hippocampal enzyme level (r2 = 0.06, P = 0.00001). CONCLUSIONS: Soluble human epoxy hydrolase enzyme-2 is increased in both lateral and medial temporal tissues in temporal lobe epilepsy. Further studies should be conducted as inhibition of this enzyme has resulted in a significant decrease in or stopping of seizures and attenuated neuroinflammation in experimental epilepsy models in the current literature.

Down-regulation of adenylate kinase 5 in temporal lobe epilepsy patients and rat model.

Adenylate kinase 5 (AK5) is one member of the AK family and plays a critical role in maintaining cellular homeostasis. Different from the other AKs, AK5 is almost exclusively expressed in the brain. However, its exact biological functions remain unclear. The aim of the present study is to explore the expression pattern of AK5 in patients with refractory epilepsy and in a chronic pilocarpine-induced epileptic rat model. Using Western blot, immunofluorescence and immunoprecipitation analysis, we found that AK5 protein was mainly expressed in neurons, demonstrated by colocalization with the dendritic marker, MAP2, which were similar to the corresponding controls. However, the expression of AK5 decreased remarkably in epileptic patients and experimental rats. Furthermore, immunoprecipitation analysis showed that the interaction of AK5 with copine VI (CPNE6, a brain specific protein) increased in epileptic patients and rat models. Our results are the first to indicate that the expression of AK5 in epileptic brain tissue may play important roles in epilepsy, especially refractory epilepsy.

Epigenetic Suppression of GADs Expression is Involved in Temporal Lobe Epilepsy and Pilocarpine-Induced Mice Epilepsy.

Recent studies have shown that histone acetylation is involved with the regulation of enzyme glutamate decarboxylases (GADs), including GAD67 and GAD65. Here, we investigated the histone acetylation modifications of GADs in the pathogenesis of epilepsy and explored the therapeutic effect of a novel second-generation histone deacetylase inhibitor (HDACi) JNJ-26481585 in epilepsy animals. We revealed the suppression of GADs protein and mRNA level, and histone hypoacetylation in patients with temporal lobe epilepsy and pilocarpine-induced epilepsy mice model. Double-immunofluorescence also indicated that the hypoacetyl-H3 was located in hippocampal GAD67/GAD65 positive neurons in epilepsy mice. JNJ-26481585 significantly reversed the decrease of the GAD67/GAD65 both protein and mRNA levels, and the histone hypoacetylation of GABAergic neurons in epilepsy mice. Meanwhile, single-cell real-time PCR performed in GFP-GAD67/GAD65 transgenic mice demonstrated that JNJ-26481585 induced increase of GAD67/GAD65 mRNA level in GABAergic neurons. Furthermore, JNJ-26481585 significantly alleviated the epileptic seizures in mice model. Together, our findings demonstrate inhibition of GADs gene via histone acetylation plays an important role in the pathgenesis of epilepsy, and suggest JNJ-26481585 as a promising therapeutic strategy for epilepsy.

Increased Expression of Matrix Metalloproteinase-9 in Patients with Temporal Lobe Epilepsy.

AIM: The molecular mechanism of epileptogenesis in temporal lobe epilepsy is still unclear. Experimental studies have suggested that matrix metalloproteinases have important roles in this process, but human studies are limited. The aim of this study was to assess the expression of MMP-9, MMP-2 and their tissue inhibitors (TIMP-1 and TIMP-2) in patients with temporal lobe epilepsy with hippocampal sclerosis (TLE-HS). MATERIAL AND METHODS: The tissue samples from temporal neocortex and hippocampus were obtained from patients with temporal lobe epilepsy with hippocampal sclerosis who had undergone anterior temporal lobectomy for recurrent medically resistant seizures. Immunohistochemical methods were used to determine the expression of MMP-9, MMP-2 and their tissue inhibitors. Tissue samples were also analyzed with transmission electron microscopy. RESULTS: The immunoreactivity for MMP-9 both in hippocampal and temporal neocortical neurons was stronger than that of MMP-2. Additionally, there was a mild reaction for its tissue inhibitor TIMP-1 as with TIMP-2. The TEM analysis of the hippocampus revealed that there was apparent ultra-structural damage on the pericarya and neuropil of some neurons. There was obvious damage in the mitochondria and the nuclear membrane. CONCLUSION: The preliminary results of this study revealed that MMP-9 may have a role in patients with drug resistant TLE-HS.

Rapid changes in expression of class I and IV histone deacetylases during epileptogenesis in mouse models of temporal lobe epilepsy.

A prominent role of epigenetic mechanisms in manifestation of epilepsy has been proposed. Thus altered histone H3 and H4 acetylation has been demonstrated in experimental models of temporal lobe epilepsy (TLE). We now investigated changes in the expression of the class I and class IV histone deacetylases (HDAC) in two complementary mouse TLE models. Unilateral intrahippocampal injection of kainic acid (KA) induced a status epilepticus lasting 6 to 24h, development of spontaneous limbic seizures (2 to 3 days after KA injection) and chronic epilepsy, as revealed by telemetric recordings of the EEGs. Mice were killed at different intervals after KA injection and expression of HDAC mRNAs was investigated by in situ hybridization. We observed marked decreases in the expression of HDACs 1, 2 and 11 (by up to 75%) in the granule cell and pyramidal cell layers of the hippocampus during the acute status epilepticus (2 to 6h after KA injection). This was followed by increased expression of all class I HDAC mRNAs in all principal cell layers of the hippocampus after 12 to 48 h. In the chronic phase, 14 and 28 days after KA, only modest increases in the expression of HDAC1 mRNA were observed in granule and pyramidal cells. Immunohistochemistry using an antibody detecting HDAC2 revealed results consistent with the mRNA data and indicates also expression in glial cells on the injection side. Similar changes as seen in the KA model were observed after a pilocarpine-induced status epilepticus except that decreases in HDACs 2, 3 and 8 were also seen at the chronic 28 day interval. The prominent decreases in HDAC expression during status epilepticus are consistent with the previously demonstrated increased expression of numerous proteins and with the augmented acetylation of histone H4. It is suggested that respective putative gene products could facilitate proconvulsive as well as anticonvulsive mechanisms. The increased expression of all class I HDACs during the "silent phase", on the other hand, may be related to decreased histone acetylation, which could cause a decrease in expression of certain proteins, a mechanism that could also promote epileptogenesis. Thus, addressing HDAC expression may have a therapeutic potential in interfering with a status epilepticus and with the manifestation of TLE.

Inhibition of glutamine synthetase in the central nucleus of the amygdala induces anhedonic behavior and recurrent seizures in a rat model of mesial temporal lobe epilepsy.

The prevalence of depression and suicide is increased in patients with mesial temporal lobe epilepsy (MTLE); however, the underlying mechanism remains unknown. Anhedonia, a core symptom of depression that is predictive of suicide, is common in patients with MTLE. Glutamine synthetase, an astrocytic enzyme that metabolizes glutamate and ammonia to glutamine, is reduced in the amygdala in patients with epilepsy and depression and in suicide victims. Here, we sought to develop a novel model of anhedonia in MTLE by testing the hypothesis that deficiency in glutamine synthetase in the central nucleus of the amygdala (CeA) leads to epilepsy and comorbid anhedonia. Nineteen male Sprague-Dawley rats were implanted with an osmotic pump infusing either the glutamine synthetase inhibitor methionine sulfoximine [MSO (n=12)] or phosphate buffered saline [PBS (n=7)] into the right CeA. Seizure activity was monitored by video-intracranial electroencephalogram (EEG) recordings for 21days after the onset of MSO infusion. Sucrose preference, a measure of anhedonia, was assessed after 21days. Methionine sulfoximine-infused rats exhibited recurrent seizures during the monitoring period and showed decreased sucrose preference over days when compared with PBS-infused rats (p<0.01). Water consumption did not differ between the PBS-treated group and the MSO-treated group. Neurons were lost in the CeA, but not the medial amygdala, lateral amygdala, basolateral amygdala, or the hilus of the dentate gyrus, in the MSO-treated rats. The results suggest that decreased glutamine synthetase activity in the CeA is a possible common cause of anhedonia and seizures in TLE. We propose that the MSO CeA model can be used for mechanistic studies that will lead to the development and testing of novel drugs to prevent seizures, depression, and suicide in patients with TLE.

Effects of site-specific infusions of methionine sulfoximine on the temporal progression of seizures in a rat model of mesial temporal lobe epilepsy.

Glutamine synthetase (GS) in astrocytes is critical for metabolism of glutamate and ammonia in the brain, and perturbations in the anatomical distribution and activity of the enzyme are likely to adversely affect synaptic transmission. GS is deficient in discrete regions of the hippocampal formation in patients with mesial temporal lobe epilepsy (MTLE), a disorder characterized by brain glutamate excess and recurrent seizures. To investigate the role of site-specific inhibition of GS in MTLE, we chronically infused the GS inhibitor methionine sulfoximine (MSO) into one of the following areas of adult laboratory rats: (1) the angular bundle, n=6; (2) the deep entorhinal cortex (EC), n=7; (3) the stratum lacunosum-moleculare of CA1, n=7; (4) the molecular layer of the subiculum, n=10; (5) the hilus of the dentate gyrus, n=6; and (6) the lateral ventricle, n=6. Twelve animals were infused with phosphate buffered saline (PBS) into the same areas to serve as controls. All infusions were unilateral, and animals were monitored by continuous video-intracranial EEG recordings for 3 weeks to capture seizure activity. All animals infused with MSO into the entorhinal-hippocampal area exhibited recurrent seizures that were particularly frequent during the first 3 days of infusion and that continued to recur for the entire 3 week recording period. Only a fraction of animals infused with MSO into the lateral ventricle had recurrent seizures, which occurred at a lower frequency compared with the other MSO infused group. Infusion of MSO into the hilus of the dentate gyrus resulted in the highest total number of seizures over the 3-week recording period. Infusion of MSO into all brain regions studied, with the exception of the lateral ventricle, led to a change in the composition of seizure severity over time. Low-grade (stages 1-3) seizures were more prevalent early during infusion, while severe (stages 4-5) seizures were more prevalent later. Thus, the site of GS inhibition within the brain determines the pattern and temporal evolution of recurrent seizures in the MSO model of MTLE.

Effects of JIP3 on epileptic seizures: Evidence from temporal lobe epilepsy patients, kainic-induced acute seizures and pentylenetetrazole-induced kindled seizures.

JNK-interacting protein 3 (JIP3), also known as JNK stress-activated protein kinase-associated protein 1 (JSAP1), is a scaffold protein mainly involved in the regulation of the pro-apoptotic signaling cascade mediated by c-Jun N-terminal kinase (JNK). Overexpression of JIP3 in neurons in vitro has been reported to lead to accelerated activation of JNK and enhanced apoptosis response to cellular stress. However, the occurrence and the functional significance of stress-induced modulations of JIP3 levels in vivo remain elusive. In this study, we investigated the expression of JIP3 in temporal lobe epilepsy (TLE) and in a kainic acid (KA)-induced mouse model of epileptic seizures, and determined whether down-regulation of JIP3 can decrease susceptibility to seizures and neuron damage induced by KA. We found that JIP3 was markedly increased in TLE patients and a mouse model of epileptic seizures; mice underexpressing JIP3 through lentivirus bearing LV-Letm1-RNAi showed decreased susceptibility, delayed first seizure and decreased seizure duration response to the epileptogenic properties of KA. Subsequently, a decreased activation of JNK following seizure induction was observed in mice underexpressing JIP3, which also exhibited less neuronal apoptosis in the CA3 region of the hippocampus, as assessed three days after KA administration. We also found that mice underexpressing JIP3 exhibited a delayed pentylenetetrazole (PTZ)-induced kindling seizure process.

Regulation of Cu-Zn superoxide dismutase on SCN2A in SH-SY5Y cells as a potential therapy for temporal lobe epilepsy.

In order to evaluate SCN2A as a candidate gene for epileptic susceptibility and the use of a Cu-Zn superoxide dismutase (SOD) supplement as a potential therapy for epilepsy, SCN2A expression in the cortex and the correlation between SCN2A and Cu-Zn SOD in SH-SY5Y cells were examined. SCN2A expression and the concentration of Cu-Zn SOD in the cerebral cortexes of patients with primary and secondary temporal lobe epilepsy and normal brain cortex tissues were detected. By transfecting SH-SY5Y cells, the expression of SCN2A and the concentration of Cu-Zn SOD was analyzed and the single-cell patch clamp technique was employed in order to investigate the changes in sodium ion levels following SCN2A knockdown. SCN2A level restoration was also investigated with a Cu-Zn SOD supplement using an expression study and evaluated the changes in sodium ion levels following SCN2A knockdown. SCN2A expression and Cu-Zn SOD concentration decreased in the epileptic cerebral cortex. Following SCN2A knockdown, the concentration of Cu-Zn SOD declined and the si-SCN2A vector group showed a repeated discharge. Furthermore, the Cu-Zn SOD concentration was capable of restoring the expression of SCN2A following SCN2A knockdown in SH-SY5Y cells and the overexpression of Cu-Zn SOD prevented the repeated discharge caused by si-SCN2A. The results indicated that there is a low expression of SCN2A and Cu-Zn SOD in the epileptic cerebral cortex and provided novel insights into potential therapies for temporal lobe epilepsy.

Increased metalloprotease activity in the epileptogenic lesion--Lobectomy reduces metalloprotease activity and urokinase-type uPAR circulating levels.

Inflammation influences the pathogenesis of seizures by boosting neuronal degeneration of temporal lobe epilepsy with hippocampal sclerosis (TLE-HS). This work aimed to determine the activity of metalloproteases (MMPs) in brain tissue fragments of TLE-HS patients and the effect of lobectomy on circulating inflammatory biomarkers. Surgical fragments (n=4) from epileptogenic focus (EF) e perilesion area (PL), and control hippocampus from autopsy (n=5) were processed for glial protein (GFAP), activated microglia (IB4) immunohistochemistry, and metalloprotease activity (MMP-2, -9). Perilesional area showed GFAP positive cells with morphology of activate astrocyte and reactive gliosis nearby the lesion. In the lesion foci, astrocytes had altered cytoarchitecture with disorganized stroma suggestive of necrosis, and numerous mononuclear cells with few projections and morphological characteristics of activate microglia. Analysis of MMP-9 and MMP-2 in the sera before and after hippocampectomy confirmed the inflammatory pattern of TLE-HS, with high MMP-9 activity; high MMP-9/TIMP-1 and urokinase uPAR plasma levels before lobectomy but low after surgery. Maintenance of MMP-2 activity indicates persistent tissue remodeling in both groups. The present work shows that patients with chronic and medically intractable TLE-HS that undergone amigdalo-hippocampectomy for removal of epileptogenic lesion had a clinical enduring benefit of lack seizure recurrence for up to a year, and consistent reduction of proteases (MMP-9 and uPAR) activation that participate as important inflammatory epileptogenic inducers.

Correlation between platelet and brain PLA(2) activity.

The phospholipase A2 (PLA2) enzymes have been implicated in several neuropsychiatry disorders and activity alterations have been described in brain and platelet. Since brain tissue is not readily available for the measurement of PLA2 activity, it would be of interest to test directly whether PLA2 activities in both tissues are correlated. We performed this task assessing PLA2 activity in platelets and hippocampus collected simultaneously from 19 patients undergoing temporal lobectomy for treatment of refractory epilepsy. Our findings suggest that total PLA2 activity in platelets may reflect the total activity of the enzyme in the brain (rs=0.59, p=0.008). However in our sample no correlations were found between the subgroups of the enzyme in brain and in platelets. This lack of correlations may be due to different effects of drug treatment on the PLA2 subtypes. In face of the difficulty to obtain brain tissues from living patients, further studies with larger drug-free samples are warranted to clarify whether the use of platelets is a reliable strategy to reflect the subtypes of PLA2 activity in the brain.