metallothionein and Seizures

The hippocampus, a component of the limbic system, is a prominent subcortical structure, which not only contains high concentrations of zinc, but also exhibits regional variations in this essential element, with concentrations being highest in the hilar region and lowest in the fimbria. For example, the concentration of zinc in the mossy fiber axons has been estimated to approach 300-350 microM. Both zinc and pyridoxal phosphate (PLP) deficiency and excess have been reported to produce epileptiform seizures, which are blocked by gamma-aminobutyric acid (GABA). The proposed mechanism is that at physiological concentrations zinc stimulates the activity of the hippocampal pyridoxal kinase (50% stimulation at 1.7 x 10(-7) M), enhancing the formation of PLP, whereas in pharmacological doses zinc inhibits the activity of glutamate decarboxylase (GAD) directly (50% inhibition at 6.5 X 10(-4) M) by preventing the binding of PLP to HoloGAD. Furthermore, recent studies have shown that two forms of GAD are found in the rat brain. One form (GAD A) does not require PLP for maximal activity, while another form (GAD B) does. Furthermore, the ratio between GAD A and GAD B is nonuniform throughout brain areas, and the hippocampus contains twice as much GAD B (the PLP-requiring GAD) as GAD A. Although the hippocampus is a common target of exogenous neurotoxic agents, "free" zinc in greater than physiological concentrations should be considered an endogenous central neurotoxin. For example, iontophoretically applied zinc in the frontoparietal cortex enhances and prolongs the firing rate of neurons in urethane-anesthetized rat. In addition, zinc (50-500 microM) significantly depresses the paired-pulse potentation in the hippocampal CA3 subfield. Moreover, zinc selectively blocks the action of N-methyl-D-aspartate on cortical neurons and enhances the quisqualate receptor-mediated injury. Finally zinc competitively inhibits the calcium-dependent release of transmitter by inhibiting the entry of Ca2+ into the nerve terminals. Since zinc in a concentration of 300-350 microM could not possibly remain "unbound" in the hippocampus, we searched for and identified a metallothionein-like protein (MT) in the bovine hippocampus, which produces two isoforms on reverse-phase HPLC and lacks aromatic amino acids, but possesses metallomercaptide bonds. We believe that the hippocampal metallothionein, by donating zinc to an extensive number of zinc-activated, PLP-mediated biochemical reaction

Topics: Animals; Brain; Enzyme Activation; gamma-Aminobutyric Acid; Hippocampus; Metallothionein; Pyridoxal Kinase; Pyridoxal Phosphate; Seizures; Synapses; Zinc

Transgenic mice overexpressing spermine oxidase (SMO) in the cerebral cortex (Dach-SMO mice) showed increased vulnerability to excitotoxic brain injury and kainate-induced epileptic seizures. To investigate the mechanisms by which SMO overexpression leads to increased susceptibility to kainate excitotoxicity and seizure, in the cerebral cortex of Dach-SMO and control mice we assessed markers for astrocyte proliferation and neuron loss, and the ability of kainate to evoke glutamate release from nerve terminals and astrocyte processes. Moreover, we assessed a possible role of astrocytes in an in vitro model of epileptic-like activity in combined cortico-hippocampal slices recorded with a multi-electrode array device. In parallel, as the brain is a major metabolizer of oxygen and yet has relatively feeble protective antioxidant mechanisms, we analyzed the oxidative status of the cerebral cortex of both SMO-overexpressing and control mice by evaluating enzymatic and non-enzymatic scavengers such as metallothioneins. The main findings in the cerebral cortex of Dach-SMO mice as compared to controls are the following: astrocyte activation and neuron loss; increased oxidative stress and activation of defense mechanisms involving both neurons and astrocytes; increased susceptibility to kainate-evoked cortical epileptogenic activity, dependent on astrocyte function; appearance of a glutamate-releasing response to kainate from astrocyte processes due to activation of Ca(2+)-permeable AMPA receptors in Dach-SMO mice. We conclude that reactive astrocytosis and activation of glutamate release from astrocyte processes might contribute, together with increased reactive oxygen species production, to the vulnerability to kainate excitotoxicity in Dach-SMO mice. This mouse model with a deregulated polyamine metabolism would shed light on roles for astrocytes in increasing vulnerability to excitotoxic neuron injury.

Topics: Animals; Aspartic Acid; Astrocytes; Benzodiazepines; Biogenic Polyamines; Calcium; Cerebral Cortex; Enzyme Induction; Genetic Predisposition to Disease; Gliosis; Hippocampus; Kainic Acid; Male; Metallothionein; Mice; Mice, Neurologic Mutants; Mice, Transgenic; Nerve Tissue Proteins; Neurons; Neurotoxins; Oxidative Stress; Oxidoreductases Acting on CH-NH Group Donors; Polyamine Oxidase; Receptors, AMPA; Recombinant Fusion Proteins; Seizures; Synaptosomes; Up-Regulation

The divalent cation zinc is associated with cortical plasticity. However, the mechanism of zinc in the pathophysiology of cortical injury-associated neurobehavioral damage following neonatal seizures is uncertain. We have previously shown upregulated expression of ZnT-3; MT-3 in hippocampus of neonatal rats submitted to flurothyl-induced recurrent seizures, which was restored by pretreatment with ketogenic diet (KD). In this study, utilizing a novel "twist" seizure model by coupling early-life flurothyl-induced seizures with later exposure to penicillin, we further investigated the long-term effects of KD on cortical expression of zinc homeostasis-related genes in a systemic scale. Ten Sprague-Dawley rats were assigned each averagely into the non-seizure plus normal diet (NS + ND), non-seizure plus KD (NS + KD), recurrent seizures plus normal diet (RS + ND) and recurrent seizures plus KD (RS + KD) group. Recurrent seizures were induced by volatile flurothyl during P9-P21. During P23-P53, rats in NS + KD and RS + KD groups were dieted with KD. Neurological behavioral parameters of brain damage (plane righting reflex, cliff avoidance reflex, and open field test) were observed at P43. At P63, we examined seizure threshold using penicillin, then the cerebral cortex were evaluated for real-time RT-PCR and western blot study. The RS + ND group showed worse performances in neurological reflex tests and reduced latencies to myoclonic seizures induced by penicillin compared with the control, which was concomitant with altered expressions of ZnT-7, MT-1, MT-2, and ZIP7. Specifically, there was long-term elevated expression of ZIP7 in RS + ND group compared with that in NS + ND that was restored by chronic ketogenic diet (KD) treatment in RS + KD group, which was quite in parallel with the above neurobehavioral changes. Taken together, these findings indicate that the long-term altered expression of the metal transporter ZIP7 in adult cerebral cortex might correlate with neurobehavioral damage and reduced seizure threshold following recurrent neonate seizures and further highlights ZIP7 as a candidate for therapeutic target of KD for the treatment of neonatal seizure-induced long-term brain damage.

Topics: Animals; Brain Injuries; Cation Transport Proteins; Cerebral Cortex; Diet, Ketogenic; Female; Flurothyl; Gene Expression Regulation; Hippocampus; Male; Metallothionein; Nerve Tissue Proteins; Rats; Rats, Sprague-Dawley; Seizures

Metallothioneins I and II (MTI/II) are metal-binding proteins overexpressed in response to brain injury. Recently, we have designed a peptide, termed EmtinB, which is modeled after the beta-domain of MT-II and mimics the biological effects of MTI/II in vitro. Here, we demonstrate the neuroprotective effect of EmtinB in the in vitro and in vivo models of kainic acid (KA)-induced neurotoxicity. We show that EmtinB passes the blood-brain barrier and is detectable in plasma for up to 24 hr. Treatment with EmtinB significantly attenuates seizures in C57BL/6J mice exposed to moderate (20 mg/kg) and high (30 mg/kg) KA doses and tends to decrease mortality induced by the high KA dose. Histopathological evaluation of hippocampal (CA3 and CA1) and cortical areas of mice treated with 20 mg/kg KA shows that EmtinB treatment reduces KA-induced neurodegeneration in the CA1 region. These findings establish EmtinB as a promising target for therapeutic development.

Topics: Animals; Blood-Brain Barrier; Cells, Cultured; Cerebral Cortex; Disease Models, Animal; Dose-Response Relationship, Drug; Epilepsy, Temporal Lobe; Hippocampus; Intercellular Signaling Peptides and Proteins; Kainic Acid; Male; Metallothionein; Mice; Mice, Inbred C57BL; Nerve Degeneration; Neuroprotective Agents; Neurotoxins; Peptides; Rats; Rats, Wistar; Seizures

The role of interleukin-6 in hippocampal tissue damage after injection with kainic acid, a rigid glutamate analogue inducing epileptic seizures, has been studied by means of interleukin-6 null mice. At 35mg/kg, kainic acid induced convulsions in both control (75%) and interleukin-6 null (100%) mice, and caused a significant mortality (62%) only in the latter mice, indicating that interleukin-6 deficiency increased the susceptibility to kainic acid-induced brain damage. To compare the histopathological damage caused to the brain, control and interleukin-6 null mice were administered 8.75mg/kg kainic acid and were killed six days later. Morphological damage to the hippocampal field CA1-CA3 was seen after kainic acid treatment. Reactive astrogliosis and microgliosis were prominent in kainic acid-injected normal mice hippocampus, and clear signs of increased oxidative stress were evident. Thus, the immunoreactivity for inducible nitric oxide synthase, peroxynitrite-induced nitration of proteins and byproducts of fatty acid peroxidation were dramatically increased, as was that for metallothionein I+II, Mn-superoxide dismutase and Cu/Zn-superoxide dismutase. In accordance, a significant neuronal apoptosis was caused by kainic acid, as revealed by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling and interleukin-1beta converting enzyme/Caspase-1 stainings. In kainic acid-injected interleukin-6 null mice, reactive astrogliosis and microgliosis were reduced, while morphological hippocampal damage, oxidative stress and apoptotic neuronal death were increased. Since metallothionein-I+II levels were lower, and those of inducible nitric oxide synthase higher, these concomitant changes are likely to contribute to the observed increased oxidative stress and neuronal death in the interleukin-6 null mice. The present results demonstrate that interleukin-6 deficiency increases neuronal injury and impairs the inflammatory response after kainic acid-induced seizures.

Topics: Animals; Apoptosis; Blood-Brain Barrier; Encephalitis; Epilepsy; Excitatory Amino Acid Agonists; Gene Expression; Gliosis; Hippocampus; In Situ Nick-End Labeling; Interleukin-6; Kainic Acid; Macrophages; Metallothionein; Mice; Mice, Knockout; Microglia; Nerve Degeneration; Oxidative Stress; Seizures; Superoxide Dismutase

Soman, a potent acetylcholinesterase inhibitor, induces status epilepticus in rats followed by conspicuous neuropathology, most prominent in piriform cortex and the CA3 region of the hippocampus. Cholinergic seizures originate in striatal-nigral pathways and with fast-acting agents (soman) rapidly spread to limbic related areas and finally culminate in a full-blown status epilepticus. This leads to neurochemical changes, some of which may be neuroprotective whereas others may cause brain damage. Pretreatment with lithium sensitizes the brain to cholinergic seizures. Likewise, other agents that increase limbic hyperactivity may sensitize the brain to cholinergic agents. The hyperactivity associated with the seizure state leads to an increase in intracellular calcium, cellular edema and metal delocalization producing an oxidative stress. These changes induce the synthesis of stress-related proteins such as heat shock proteins, metallothioneins and heme oxygenases. We show that soman-induced seizures cause a depletion in tissue glutathione and an increase in tissue 'catalytic' iron, metallothioneins and heme oxygenase-1. The oxidative stress induces the synthesis of stress-related proteins, which are indicators of 'stress' and possibly provide neuroprotection. These findings suggest that delocalization of iron may catalyze Fenton-like reactions, causing progressive cellular damage via free radical products.

Topics: Animals; Chemical Warfare Agents; Glutathione; Heat-Shock Proteins; Heme Oxygenase (Decyclizing); Heme Oxygenase-1; Iron; Male; Metallothionein; Neuroprotective Agents; Oxidative Stress; Rats; Rats, Wistar; Seizures; Soman

Global hypoxia preconditioning provides neuroprotection against a subsequent, normally damaging challenge. While the mechanistic pathways are unknown, changes in the expression of stress-related proteins are implicated. Hypoxia preconditioning attenuates the brain edema and neuropathology associated with kainic acid-induced status epilepticus in a protein synthesis-dependent manner when a kainic acid challenge is given up to one week post-preconditioning. Kainic acid initiates a glutamate-driven status epilepticus causing a Ca2+ and oxidative stress, resulting in injury to the piriform cortex and hippocampus. Stress-related gene expression [e.g. metallothioneins (MTs), heme oxygenase-1 (HO-1)] is enhanced during seizures in vulnerable brain areas, (e.g. piriform cortex). This study explores the effects of hypoxia preconditioning on expression of MT-1, MT-2 and HO-1 before and after kainic acid-induced seizures. Analysis of MT-1, MT-2 and HO-1 expression, through Western and Northern blotting, indicates that there is a variable pattern of induction and suppression of these two genes following hypoxia preconditioning alone as well as after kainic acid-induced seizures compared to non-preconditioned animals. These findings suggest that hypoxia preconditioning induces an adaptive response that prevents kainic acid seizure-associated neuropathology even when robust seizures occur. This may involve a variety of stress-related proteins, working in concert, each with their own individual expression profiles. Induction of this type of neuroprotection pharmacologically, or through preconditioning, will provide a better understanding of the stress response in brain.

Topics: Animals; Cerebral Cortex; Disease Models, Animal; Heme Oxygenase (Decyclizing); Heme Oxygenase-1; Hippocampus; Hypoxia; Kainic Acid; Male; Metallothionein; Rats; Rats, Wistar; Seizures; Time Factors

Metallothioneins (MTs) are major zinc binding proteins in the CNS that could be involved in the control of zinc metabolism as well as in protection against oxidative stress. Mice lacking MT-I and MT-II (MT-I + II deficient) because of targeted gene inactivation were injected with kainic acid (KA), a potent convulsive agent, to examine the neurobiological importance of these MT isoforms. At 35 mg/kg KA, MT-I + II deficient male mice showed a higher number of convulsions and a longer convulsion time than control mice. Three days later, KA-injected mice showed gliosis and neuronal injury in the hippocampus. MT-I + II deficiency decreased both astrogliosis and microgliosis and potentiated neuronal injury and apoptosis as shown by terminal deoxynucleotidyl transferase-mediated in situ end labelling (TUNEL), detection of single stranded DNA (ssDNA) and by increased interleukin-1beta-converting enzyme (ICE) and caspase-3 levels. Histochemically reactive zinc in the hippocampus was increased by KA to a greater extent in MT-I + II-deficient compared with control mice. KA-induced seizures also caused increased oxidative stress, as suggested by the malondialdehyde (MDA) and protein tyrosine nitration (NITT) levels and by the expression of MT-I + II, nuclear factor-kappaB (NF-kappaB), and Cu/Zn-superoxide dismutase (Cu/Zn-SOD). MT-I + II deficiency potentiated the oxidative stress caused by KA. Both KA and MT-I + II deficiency significantly affected the expression of MT-III, granulocyte-macrophage colony stimulating factor (GM-CSF) and its receptor (GM-CSFr). The present results indicate MT-I + II as important for neuron survival during KA-induced seizures, and suggest that both impaired zinc regulation and compromised antioxidant activity contribute to the observed neuropathology of the MT-I + II-deficient mice.

Topics: Animals; Apoptosis; Astrocytes; Caspase 1; Caspase 3; Caspases; DNA, Single-Stranded; Epilepsy; Excitatory Amino Acid Agonists; Female; Gene Expression Regulation, Enzymologic; Glial Fibrillary Acidic Protein; Granulocyte-Macrophage Colony-Stimulating Factor; Hippocampus; In Situ Nick-End Labeling; Kainic Acid; Male; Malondialdehyde; Metallothionein; Metallothionein 3; Mice; Mice, Inbred Strains; Mice, Knockout; Microglia; Nerve Degeneration; Nerve Tissue Proteins; NF-kappa B; Nitrogen; Oxidative Stress; Receptors, Granulocyte-Macrophage Colony-Stimulating Factor; Seizures; Superoxide Dismutase; Tyrosine; Zinc

Kainic acid-induced seizures in the rat brain cause severe brain damage that is thought to result, in part, from oxidative stress. In this study, we examine the consequences of systemic administration of kainic acid on expression of several genes that encode proteins thought to play roles in protection from oxidative stress, including metallothionein-I, and -III. Kainic acid causes an increase in metallothionein-I and heme oxygenase-I mRNAs, as well as an increase in c-fos, heat shock protein-70, and interleukin-1 beta mRNAs. The induction of these mRNAs is seizure dependent, and is greater in brain areas with extensive damage (e.g. piriform cortex) than in areas with minimal damage (e.g. frontal cortex and cerebellum). In contrast, little or no change in mRNA for metallothionein-III, manganese superoxide dismutase, copper-zinc superoxide dismutase, glutathione-s-transferase ya subunit or glutathione peroxidase occur. The prolonged and robust concordant induction of the metallothionein-I and heme oxygenase-I genes may reflect the oxidative stress produced by kainic acid-induced seizures. In addition, the induction of interleukin-1 beta gene expression suggests an inflammatory response in brain regions damaged by kainic acid-induced seizures. Delineating the regulation of genes associated with oxidative and inflammatory responses can contribute to a fuller understanding of seizures and associated brain damage.

Topics: Animals; Base Sequence; Gene Expression; Genes, fos; Heme Oxygenase (Decyclizing); HSP70 Heat-Shock Proteins; Interleukin-1; Kainic Acid; Male; Metallothionein; Molecular Sequence Data; Oxidative Stress; Rats; Rats, Wistar; RNA, Messenger; Seizures