calpain and Seizures

To explore the effect of SRC activation on spontaneously recurrent seizures and to investigate the underlying mechanisms of NR2B phosphorylation.. C57BL/6 mice were injected intrahippocampally with kainic acid (KA, 0.4 μg/25 g) to induce status epilepticus (SE). Saracatinib(STB) was used as an SRC inhibitor. Spontaneously recurrent seizures were monitored from day 7 to day 14 after the KA injection. Nissl's stain and NeuN were used to detect neuron loss and Timm stain was used to evaluate mossy fibre sprouting 14 days after KA injection. We also investigated the effect of SRC on full-length expression of NR2B. MDL28170 was used to inhibit calpain activity. Western blotting and qPCR were performed to verify phosphorylation levels and expression of SRC and NR2B 24 h after KA injection.. The duration of status epileptics in the SRC inhibitor group decreased significantly compared to the KA group 24 h after the injection of KA (P < 0.05). The application of the SRC inhibitor significantly reduced the degree of contralateral mossy fibre sprouting (P < 0.05) and improved the degree of neuron loss (P < 0.01) compared to the epilepsy group. Full-length NR2B levels in the ipsilateral hippocampus decreased in the epilepsy group (P < 0.01) compared to the sham group, and it further decreased in the STB inhibitor group (P < 0.01). The effect of the STB inhibitor was counteracted by simultaneous inhibition of SRC activity and calpain activation, while the level of full-length NR2B increased compared to the KA+STB group(P < 0.01). Reduction of NR2B cleavage by MDL28170 significantly increased the duration of epileptic status compared to the KA group (P < 0.05).. Our data indicated that the early application of SRC inhibitors exerted protective effects on seizure severity, loss of neurons, and sprouting of mossy fibres in KA-induced mouse epilepsy. Seizure severity attenuation due to SRC inhibition was associated with the decrease of NR2B in both the phosphorylation and full-length forms.

Topics: Animals; Calpain; Epilepsy; Epilepsy, Temporal Lobe; Hippocampus; Kainic Acid; Mice; Mice, Inbred C57BL; Phosphorylation; Seizures

Calpain has been proposed to play a critical role in the development of epilepsy. Here we used conditional calpain-2 knock-out (C2CKO) mice in a C57/Bl6 background and a selective calpain-2 inhibitor to analyze the role of calpain-2 in epilepsy. Neurodegeneration was evident in various hippocampal subfields, in particular in mossy cells in the hilus of the dentate gyrus (DG) in C57/Bl6 mice 7 days after kainic acid (KA)-induced seizures. Calpain-2 activation was still observed in mossy cells 7 days after seizures. Calpain activation, astroglial and microglial activation, neurodegeneration, and cognitive impairment were absent in C2CKO mice and in C57/Bl6 mice treated with a selective calpain-2 inhibitor for 7 days after seizure initiation. Levels of the potassium chloride cotransporter 2 (KCC2) were decreased in mossy cells 7 days after seizures and this decrease was prevented by calpain-2 deletion or selective inhibition. Our results indicate that prolonged calpain-2 activation plays a critical role in neuropathology following seizures. A selective calpain-2 inhibitor could represent a therapeutic treatment for seizure-induced neuropathology.

Topics: Animals; Calpain; Epilepsy; Hippocampus; Mice; Mice, Inbred C57BL; Mice, Knockout; Seizures

Epileptogenesis is the processes by which a normal brain transforms and becomes capable of generate spontaneous seizures. In acquired epilepsy, it is thought that epileptogenesis can be triggered by a brain injury but the understanding of the cellular or molecular changes unraveling is incomplete. In the CA1 region of hippocampus less GABAergic activity precede the appearance of spontaneous seizures and calpain overactivation has been detected after chemoconvulsant-induced status epilepticus (SE). Inhibition of calpain overactivation following SE ameliorates seizure burden, suggesting a role for calpain dysregulation in epileptogenesis. The current study analyzed if GABAergic proteins (i.e., gephyrin, the vesicular GABA transporter and the potassium chloride co-transporter 2) undergo calpain-dependent cleavage during epileptogenesis. A time-dependent generation of break down products (BDPs) for these proteins was observed in the CA1 region of hippocampus after pilocarpine-induced SE. Generation of these BDPs was partially blocked by treatment with the calpain inhibitor MDL-28170. These findings suggest that calpain-dependent loss of GABAergic proteins might promote the erosion of inhibitory drive and contribute to hyperexcitability during epileptogenesis.

Topics: Animals; CA1 Region, Hippocampal; Calpain; Male; Membrane Proteins; Neurons; Pilocarpine; Rats; Rats, Sprague-Dawley; Seizures; Status Epilepticus; Vesicular Inhibitory Amino Acid Transport Proteins

Epilepsy is a brain disorder characterized by a predisposition to suffer epileptic seizures. Acquired epilepsy might be the result of brain insults like head trauma, stroke, brain infection, or status epilepticus (SE) when one of these triggering injuries starts a transformative process known as epileptogenesis. There is some data to suggest that, during epileptogenesis, seizures themselves damage the brain but there is no conclusive evidence to demonstrate that spontaneous recurrent seizures themselves injure the brain. Our recent evidence indicates that calpain overactivation might be relevant for epileptogenesis. Here, we investigated if spontaneous recurrent seizures that occur during an early period of epileptogenesis show any correlation with the levels of calpain activation and/or expression. In addition, we also investigated a possible association between the occurrence of spontaneous seizures and increased levels of cell death, gliosis and inflammation (typical markers associated with epileptogenesis). We found that the number of spontaneous seizures detected prior to sample collection was correlated with altered calpain activity and expression. Moreover, the levels of hippocampal neurodegeneration were also correlated with seizure occurrence. Our findings suggest that, at least during early epileptogenesis, there is a correlation between seizure occurrence, calpain activity and neurodegeneration. Thus, this study opens the possibility that aberrant calpain reactivation by spontaneous seizures might contribute to the manifestation of future spontaneous seizures.

Topics: Animals; Calpain; Cell Death; Encephalitis; Epilepsy; Gliosis; Hippocampus; Male; Microglia; Neurons; Rats, Sprague-Dawley; Seizures

In this study, we used the pilocarpine model of epilepsy to evaluate the involvement of calpain dysregulation on epileptogenesis. Detection of spectrin breakdown products (SBDPs, a hallmark of calpain activation) after induction of pilocarpine-induced status epilepticus (SE) and before appearance of spontaneous seizure suggested the existence of sustained calpain activation during epileptogenesis. Acute treatment with a cell permeable inhibitor of calpain, MDL-28170, resulted in a partial but significant reduction on seizure burden. The reduction on seizure burden was associated with a limited reduction on the generation of SBDPs but was correlated with a reduction in astrocytosis, microglia activation and cell sprouting. Together, these observations provide evidence for the role of calpain in epileptogenesis. In addition, provide proof-of-principle for the use of calpain inhibitors as a novel strategy to prevent epileptic seizures and its associated pathologies.

Topics: Animals; Anticonvulsants; Calpain; Cerebral Cortex; Dipeptides; Disease Models, Animal; Dose-Response Relationship, Drug; Epilepsy, Temporal Lobe; Glycoproteins; Hippocampus; Inflammation; Male; Neurodegenerative Diseases; Neurons; Pilocarpine; Random Allocation; Rats, Sprague-Dawley; Seizures

A robust increase in the functional expression of the neuronal K-Cl cotransporter KCC2 during CNS development is necessary for the emergence of hyperpolarizing ionotropic GABAergic transmission. BDNF-TrkB signaling has been implicated in the developmental up-regulation of KCC2 and, in mature animals, in fast activity-dependent down-regulation of KCC2 function following seizures and trauma. In contrast to the decrease in KCC2 expression observed in the adult hippocampus following trauma, seizures in the neonate trigger a TrkB-dependent up-regulation of neuronal Cl(-) extrusion capacity associated with enhanced surface expression of KCC2. Here, we show that this effect is transient, and impaired in the hippocampus of Bdnf(-/-) mice. Notably, however, a complete absence of BDNF does not compromise the increase in KCC2 protein or K-Cl transport functionality during neuronal development. Furthermore, we present data indicating that the functional up-regulation of KCC2 by neonatal seizures is temporally limited by calpain activity.

Topics: Animals; Animals, Newborn; Blotting, Western; Brain-Derived Neurotrophic Factor; Calpain; Chlorides; Disease Models, Animal; Hippocampus; K Cl- Cotransporters; Kainic Acid; Mice, Knockout; Neurons; Seizures; Status Epilepticus; Symporters; Tissue Culture Techniques; Up-Regulation

TO determine neuroprotective properties of levetiracetam and simvastatin using rats with pilocaroine-induced epilepsy.. Epileptic rat models were randomly divided into 4 groups, each being exposed to saline, simvastatin, levetiracetam, or levetiracetam + simvastatin. Brain tissues of the rats were examined. Nissl staining was used to determine pilocarpine-induced neuronal loss in CA1 and CA3 of hippocampus. Western blot was used to detect calpain-1 expression of hippocampus.. Severe cell death was found 24 h after seizures, with a level significantly higher than the controls. Compared with the saline-treated cells, simvastatin did not decrease severe cell death (P>0.05), but levetiracetam and levetiracetam+simvastatin decreased severe cell death 24 h after seizures (P<0.05). No significant differences were found between those treated with levetiracetam and those with levetiracetam+simvastatin. Compared with controls, overexpressed calpain-1 was found in the rats 24 h after seizures, which indicates that calpain-1 may be involved in the pathophysiological mechanisms of epilepsy. Compared with those treated with pilocarpine + saline, simvastatin, levetiracetam and levetiracetam + simvastatin reduced the level of calpain-1 24 h after seizures (P<0.05).. Levetiracetam, not simvastatin, possesses neuroprotective properties, through changing calpain-1 expression levels. But levetiracetam plus simvastatin treatment does not have advantages over the choice of monotherapy. Simvastain does not possess neuroprotective properties at the early stage of epilepsy.

Topics: Animals; Calpain; Disease Models, Animal; Epilepsy; Hippocampus; Levetiracetam; Pilocarpine; Piracetam; Rats; Seizures; Simvastatin

An experimental model based on kainic acid (KA) injections replicates many phenomenological features of human temporal lobe epilepsy, the most common type of epilepsy in adults. Taurine, 2-aminoethanesulfonic acid, present in high concentrations in many invertebrate and vertebrate systems, is believed to serve several important biological functions. In addition, it is believed to have a neuroprotective role against several diseases. In the present study, an experimental mouse model based on taurine pretreatment prior to KA administration has been improved to study whether taurine has a neuroprotective effect against KA-induced behavior and cell damage. Under different treatments tested, taurine's most neuroprotective effects were observed with intraperitoneal taurine injection (150 mg/kg dosage) 12 hr before KA administration. Thus, a reduction in or total absence of seizures, together with a reduction in or even disappearance of cellular and molecular KA-derived effects, was detected in mice pretreated with taurine compared with those treated only with KA. Moreover, the use of tritiated taurine revealed taurine entry into the brain, suggesting possible changes in intracellular:extracellular taurine ratios and the triggering of pathways related to neuroprotective effects.

Topics: Analysis of Variance; Animals; Anticonvulsants; Brain; Calpain; Cell Death; Chromatography, High Pressure Liquid; Disease Models, Animal; Epilepsy; Immunoblotting; Immunohistochemistry; Injections, Intraperitoneal; Kainic Acid; Male; Mice; Neuroglia; Neuroprotective Agents; Proto-Oncogene Proteins c-fos; Seizures; Taurine

Calpains are a family of calcium-dependent proteases with abundant expression in the CNS, and potent in cleaving some synaptic components. Assessment of calpain activity by its fluorescent substrate, Boc-Leu-Met-CMAC, revealed that cultured neurons display a significant level of constitutive enzyme activity. Notably, calpain activity differs in distinct neuronal populations, with a significantly higher level of activity in GABAergic cells. Using selectively-enriched cultures of fast-spiking GABAergic interneurons, we show that calpain activity partially contributes to the post-translational down regulation of SNAP-25, a calpain substrate, in differentiated GABA cells. In addition, we demonstrate that SNAP-25 is cleaved by calpain in response to acute seizures induced by intraperitoneal kainate injection in vivo. These data indicate that calpains in neurons are active even at physiological calcium concentrations and that different levels of calpain activation in selected neuron subtypes may contribute to the pattern of synaptic protein expression.

Topics: Animals; Brain-Derived Neurotrophic Factor; Calcium; Calpain; Cells, Cultured; Coumarins; Dipeptides; Embryo, Mammalian; Excitatory Amino Acid Agonists; gamma-Aminobutyric Acid; Glutamic Acid; Hippocampus; Kainic Acid; Mice; Mice, Inbred C57BL; Neurons; Patch-Clamp Techniques; Protein Isoforms; Rats; Seizures; Synaptosomal-Associated Protein 25; Synaptosomes

The molecular mechanisms mediating degeneration in response to neuronal insults, including damage evoked by prolonged seizure activity, show substantial variability across laboratories and injury models. Here we investigate the extent to which the proportion of cell death occurring by apoptotic vs. necrotic mechanisms may be shifted by changing the temporal parameters of the insult. In initial studies with continuous seizures (status epilepticus, SE), signs of apoptotic degeneration were most clearly observed when SE occurred following a long latency (>86 min) after injection of kainic acid as compared with a short-latency SE (<76 min). Therefore, in this study we directly compared short- with long-latency SE for the expression of molecular markers for apoptosis and necrosis in an especially vulnerable brain region (rhinal cortex). Molecular markers of apoptosis (DNA fragmentation, cleavage of ICAD, an inhibitor of "caspase-activated DNase" (CAD), and prevalence of a caspase-generated fragment of alpha-spectrin) were detected following long-latency SE. Short-latency SE resulted in expression of predominantly necrotic features of cell death, such as "non-ladder" pattern of genomic DNA degradation, prevalence of a calpain-generated alpha-spectrin fragment, and absence of ICAD cleavage. Silver staining revealed no significant difference in the extent and spatial distribution of degeneration between long- or short-latency SE. These data indicate that the latency to onset of SE determines the extent to which apoptotic or necrotic mechanisms contribute to the degeneration following SE. The presence of a long latency period, during which multiple brief seizure episodes may occur, favors the occurrence of apoptotic cell death. It is possible that the absence of such "preconditioning" period in short-latency SE favors predominantly necrotic profile.

Topics: Animals; Apoptosis; Apoptosis Regulatory Proteins; Boron Compounds; Calpain; Caspase 3; Caspases; Deoxyribonucleases; Disease Models, Animal; DNA Fragmentation; Kainic Acid; Male; Necrosis; Nerve Degeneration; Proteins; Rats; Rats, Sprague-Dawley; Seizures; Spectrin; Status Epilepticus; Time

Topics: Animals; Blotting, Western; Brain; Calpain; Enzyme Activation; Hippocampus; Immunohistochemistry; In Vitro Techniques; Rats; Rats, Sprague-Dawley; Receptors, AMPA; Receptors, Glutamate; Receptors, N-Methyl-D-Aspartate; Recombinant Proteins; Seizures; Substrate Specificity; Synaptic Membranes

The cellular distribution of calpain activation and glutamate receptor 1 (GluR1) subunits of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors and their alterations following kainic acid-induced seizure were evaluated during postnatal development using antibodies specific for spectrin breakdown product and the C-terminus of GluR1 subunits. In the first postnatal week, most brain regions exhibited high levels of calpain activity that progressively decreased during the following weeks. The highest levels of spectrin breakdown product immunoreactivity were observed in the somata and proximal dendrites of hippocampal pyramidal cells, non-pyramidal neurons in stratum oriens, and cortical neurons. In general, during the first two postnatal weeks, kainic acid treatment induced a decrease in spectrin breakdown product immunoreactivity in neuronal cell bodies and an increase in dendritic fields. Obvious elevation in spectrin breakdown product immunoreactivity in selective non-pyramidal cells in stratum oriens started at postnatal day 14, and was further evidenced by postnatal day 21. Likewise, massive calpain activation in subpopulations of neurons in some thalamic nuclei, amygdala, and pyriform cortex was observed after the third postnatal week. GluR1 subunits were highly expressed throughout the forebrain in the first postnatal week, further increased during the second postnatal week, decreased thereafter, and reached adult levels after postnatal day 21. In cortex, intense GluR1 immunostaining was found in the somata and proximal processes of pyramidal and non-pyramidal neurons, with the non-pyramidal neurons in layers IV through VI exhibiting the densest immunolabelling. In the first two postnatal weeks, the somata of hippocampal pyramidal neurons exhibited intense GluR1 immunostaining that became more dendritic in the subsequent developmental period. While hilar cells exhibited a similar developmental pattern as CA regions, the molecular layer of dentate gyrus exhibited weak immunoreactivity from postnatal day 7 to postnatal day 14. The early increase in GluR1 immunoreactivity in hippocampal pyramidal layer following kainic acid treatment occurred throughout the developmental period, while the later decrease in CA regions, amygdala, and pyriform cortex was observed only in postnatal day 21 animals. The combined immunocytochemical studies of spectrin breakdown product localization and GluR1 expression indicate that calpain activation might play

Topics: Animals; Brain; Brain Chemistry; Calpain; Cell Count; Cerebral Cortex; Excitatory Amino Acid Agonists; Immunohistochemistry; Kainic Acid; Rats; Rats, Sprague-Dawley; Seizures; Spectrin