Oh-Shin Kwon

Epileptogenic roles of astroglial death and regeneration in the dentate gyrus of experimental temporal lobe epilepsy

Recent studies have demonstrated that blockade of neuronal death in the hippocampus cannot prevent epileptogenesis in various epileptic models. These reports indicate that neurodegeneration alone is insufficient to cause epilepsy, and that the role of astrocytes in epileptogenesis should be reconsidered. Therefore, the present study was designed to elucidate whether altered morphological organization or the functionalities of astrocytes induced by status epilepticus (SE) is responsible for epileptogenesis. Glial responses (reactive microgliosis followed by astroglial death) in the dentate gyrus induced by pilocarpine‐induced SE were found to precede neuronal damage and these alterations were closely related to abnormal neurotransmission related to altered vesicular glutamate and GABA transporter expressions, and mossy fiber sprouting in the dentate gyrus. In addition, newly generated astrocytes showed down‐regulated expressions of glutamine synthase, glutamate dehydrogenase, and glial GABA transporter. Taken together, our findings suggest that glial responses after SE may contribute to epileptogenesis and the acquisition of the properties of the epileptic hippocampus. Thus, we believe that it is worth considering new therapeutic approaches to epileptogenesis involving targeting the inactivation of microglia and protecting against astroglial loss.

Cinnamate Supplementation Enhances Hepatic Lipid Metabolism and Antioxidant Defense Systems in High Cholesterol-Fed Rats

This study investigated the effect of cinnamate, a phenolic compound found in cinnamon bark and other plant materials, on lipid metabolism and antioxidant enzyme activities in rats fed a high cholesterol diet. Three groups of rats were given a diet containing 1 g of cholesterol/kg for 6 weeks. The control group only received the high cholesterol diet, whereas the other two groups received a diet supplemented with lovastatin or cinnamate (0.1 g/100 g of diet). The plasma high-density lipoprotein-cholesterol levels were significantly higher in the cinnamate group than in either the control or lovastatin groups, and the atherogenic index was significantly lower in rats with cinnamate supplementation. Supplementation with cinnamate resulted in significantly lower hepatic cholesterol and triglyceride levels. Accumulation of hepatic lipid droplets was higher in the control group than in the rats supplemented with either cinnamate or lovastatin. Hepatic 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) reductase activity was significantly lower in the cinnamate group compared with the other groups, whereas only acyl-CoA:cholesterol acyltransferase activity was significantly lower in the lovastatin group compared with the control group. Cinnamate supplementation resulted in higher catalase and glutathione peroxidase activities, while hepatic thiobarbituric acid-reactive substances were significantly lower in both the cinnamate and lovastatin groups. The fecal acidic sterol was higher in the lovastatin group than in the control or cinnamate groups. These results suggest that dietary cinnamate inhibits hepatic HMG-CoA reductase activity, resulting in lower hepatic cholesterol content, and suppresses lipid peroxidation via enhancement of hepatic antioxidant enzyme activities.

Transduced human PEP-1–heat shock protein 27 efficiently protects against brain ischemic insult

Reactive oxygen species contribute to the development of various human diseases. Ischemia is characterized by both significant oxidative stress and characteristic changes in the antioxidant defense mechanism. Heat shock protein 27 (HSP27) has a potent ability to increase cell survival in response to oxidative stress. In the present study, we have investigated the protective effects of PEP‐1–HSP27 against cell death and ischemic insults. When PEP‐1–HSP27 fusion protein was added to the culture medium of astrocyte and primary neuronal cells, it rapidly entered the cells and protected them against cell death induced by oxidative stress. Immunohistochemical analysis revealed that, when PEP‐1–HSP27 fusion protein was intraperitoneally injected into gerbils, it prevented neuronal cell death in the CA1 region of the hippocampus in response to transient forebrain ischemia. Our results demonstrate that transduced PEP‐1–HSP27 protects against cell death in vitro and in vivo, and suggest that transduction of PEP‐1–HSP27 fusion protein provides a potential strategy for therapeutic delivery in various human diseases in which reactive oxygen species are implicated, including stroke.

Gastrodin decreases immunoreactivities of γ-aminobutyric acid shunt enzymes in the hippocampus of seizure-sensitive gerbils

Gastrodin is one of the natural compound isolated from Gastrodia elata and has known anticonvulsant effects, although the exact pharmacological principles of this natural compound and its effects on other aspects of γ‐aminobutyric acid (GABA) metabolism in vivo have not been explored. Therefore, in the present study, the effects of gastrodin on GABA metabolism in the gerbil hippocampus were examined, in an effort to identify the antiepileptic characteristics of this substance. Gastrodin reduced the seizure score in the treated group, although the immunoreactivities of GABA synthetic enzymes and GABA transporters were unaltered in gastrodin‐treated animals. Interestingly, in the gastrodin‐treated group, GABA transaminase (GABA‐T) immunoreactivity in the hippocampus, particularly in neurons, was significantly decreased. In the gastrodin‐treated group, both succinic semialdehyde dehydrogenase (SSADH) and succinic semialdehyde reductase (SSAR) immunoreactivities in the hippocampus was also decreased significantly, which stood in contrast to the nontreated group, in which strong SSADH and SSAR immunoreactivities were detected. From the neuroanatomical viewpoint, these findings suggest that gastrodin may cause the elevation of GABA concentration by inhibiting the GABA shunt.

Human pyridoxal phosphatase. Molecular cloning, functional expression, and tissue distribution.

Pyridoxal phosphatase catalyzes the dephosphorylation of pyridoxal 5′-phosphate (PLP) and pyridoxine 5′-phosphate. A human brain cDNA clone was identified to the PLP phosphatase on the basis of peptide sequences obtained previously. The cDNA predicts a 296-amino acid protein with a calculated Mr of 31698. The open reading frame is encoded by two exons located on human chromosome 22q12.3, and the exon-intron junction contains the GT/AG consensus splice site. In addition, a full-length mouse PLP phosphatase cDNA of 1978 bp was also isolated. Mouse enzyme encodes a protein of 292 amino acids with Mr of 31512, and it is localized on chromosome 15.E1. Human and mouse PLP phosphatase share 93% identity in protein sequence. A BLAST search revealed the existence of putative proteins in organism ranging from bacteria to mammals. Catalytically active human PLP phosphatase was expressed in Escherichia coli, and characteristics of the recombinant enzyme were similar to those of erythrocyte enzyme. The recombinant enzyme displayed Km and kcat values for pyridoxal of 2.5 μm and 1.52 s–1, respectively. Human PLP phosphatase mRNA is differentially expressed in a tissue-specific manner. A single mRNA transcript of 2.1 kb was detected in all human tissues examined and was highly abundant in the brain. Obtaining the molecular properties for the human PLP phosphatase may provide new direction for investigating metabolic pathway involving vitamin B6.

Spatiotemporal characteristics of astroglial death in the rat hippocampo‐entorhinal complex following pilocarpine‐induced status epilepticus

Recently we reported that astroglial loss and subsequent gliogenesis in the dentate gyrus play a role in epileptogenesis following pilocarpine‐induced status epilepticus (SE). In the present study we investigated whether astroglial damages in the hippocampo‐entorhinal complex following SE are relevant to pathological or electrophysiological properties of temporal lobe epilepsy. Astroglial loss/damage was observed in the entorhinal cortex and the CA1 region at 4 weeks and 8 weeks after SE, respectively. These astroglial responses in the hippocampo‐entorhinal cortex were accompanied by hyperexcitability of the CA1 region (impairment of paired‐pulse inhibition and increase in excitability ratio). Unlike the dentate gyrus and the entorhinal cortex, CA1 astroglial damage was protected by conventional anti‐epileptic drugs. α‐Aminoadipic acid (a specific astroglial toxin) infusion into the entorhinal cortex induced astroglial damage and changed the electrophysiological properties in the CA1 region. Astroglial regeneration in the dentate gyrus and the stratum oriens of the CA1 region was found to originate from gliogenesis, while that in the entorhinal cortex and stratum radiatum of the CA1 region originated from in situ proliferation. These findings suggest that regional specific astroglial death/regeneration patterns may play an important role in the pathogenesis of temporal lobe epilepsy. J. Comp. Neurol. 511:581–598, 2008.

The temporal alteration of GAD67/GAD65 ratio in the gerbil hippocampal complex following seizure

In the present study, the distribution of glutamic acid decarboxylase (GAD) isoforms in the hippocampus of the Mongolian gerbil and its association with different sequelae of spontaneous seizure were investigated to identify the roles of balance of GAD isoforms in the epileptogenesis and the recovery mechanisms in these animals. The GAD67/GAD65 ratio in the hippocampus of pre-seizure seizure sensitive (SS) gerbil was approximately 3.5-fold higher as compared to seizure resistant (SR) gerbil. Following seizure, this ratio shifted to the level of SR gerbils up to 12 h postical. Therefore, the mismatched GAD67/GAD65 ratio (imbalance of GAD isoform expressions) in the hippocampus of SS gerbil implies that GABAergic neurons may be highly activated in order to regulate the increased neuronal excitability. In addition, the alteration in this ratio after seizure may be the compensatory response for reduction of epileptic activity in this animal.

Transduced human PEP-1-catalase fusion protein attenuates ischemic neuronal damage.

Antioxidant enzymes are considered to have beneficial effects against various diseases mediated by reactive oxygen species (ROS). Ischemia is characterized by both oxidative stress and changes in the antioxidant defense system. Catalase (CAT) and superoxide dismutase (SOD) are major antioxidant enzymes by which cells counteract the deleterious effects of ROS. To investigate the protective effects of CAT, we constructed PEP-1-CAT cell-permeative expression vectors. When PEP-1-CAT fusion proteins were added to the culture medium of neuronal cells, they rapidly entered the cells and protected them against oxidative stress-induced neuronal cell death. Immunohistochemical analysis revealed that PEP-1-CAT prevented neuronal cell death in the hippocampus induced by transient forebrain ischemia. Moreover, we showed that the protective effect of PEP-1-CAT was observed in neuronal cells treated with PEP-1-SOD. Therefore, we suggest that transduced PEP-1-CAT and PEP-1-SOD fusion proteins could be useful as therapeutic agents for various human diseases related to oxidative stress, including stroke.

The altered expression of GABA shunt enzymes in the gerbil hippocampus before and after seizure generation.

In the present study, the distribution of succinic semialdehyde dehydrogenase (SSADH) and succinic semialdehyde reductase (SSAR) in the hippocampus of the Mongolian gerbil and its association with various sequelae of spontaneous seizure were investigated in order to identify the roles of GABA shunt in the epileptogenesis and the recovery mechanisms in these animals. Both SSADH and SSAR immunoreactivities in the GABAergic neurons were significantly higher in the pre-seizure groups of seizure sensitive (SS) gerbil as compared to those seen in the seizure resistant (SR) gerbils. The distributions of both SSADH and SSAR immunoreactivities in the hippocampus showed significant differences after the on-set of seizure. At 3 h postictal, when compared to the pre-seizure group of SS gerbils, a decline in the immunoreactivities in the perikarya was observed. At 12 h after seizure on-set, the densities of both SSADH and SSAR immunoreactivities were begun to recover to the pre-seizure level of SS gerbils. These results suggest that the GABAergic neurons in the hippocampal complex of the SS gerbil may be highly activated. In addition, the imbalance of GABA shunt expressions in the GABAergic neurons may imply a malfunction of the metabolism of GABAergic neurons in the SS gerbils, and this defect may trigger seizure on-set. Therefore, the initiation of seizure, at least in gerbils, may be the result of a malfunction in GABA shunt in the GABAergic neurons.

Effects of GABAergic transmissions on the immunoreactivities of calcium binding proteins in the gerbil hippocampus.

Although reduced calcium binding protein (CBP) immunoreactivities in the epileptic hippocampus have been well established, it has been controversial that these changes may directly indicate neuronal degeneration. In the present study, therefore, we investigated CBP expressions in the gerbil hippocampus following treatment with γ‐aminobutyric acid (GABA) receptor antagonists in order to assess whether altered CBP expressions are the result of either abnormal excitation or indicative of neuronal damage/degeneration. Seizure‐sensitive (SS) gerbils showed a loss/decline of CBP immunoreactivities in some hippocampal neurons as compared with seizure‐resistant (SR) gerbils. In muscimol (GABAA receptor agonist) treated SS gerbils, expression levels of CBP were enhanced as compared with saline‐treated SS gerbils. Bicuculline (a GABAA receptor antagonist) treatment markedly reduced CBP immunoreactivities in hippocampal neurons of the SR gerbil. Baclofen (a GABAB receptor agonist) treatment increased CBP immunoreactivities in the hippocampus of SS gerbils, although its effect was lower than that of muscimol treatment. Moreover, phaclofen (GABAB receptor antagonist) treated SR gerbil showed reduction in calbindin D‐28K immunoreactivity, not parvalbumin immunoreactivity, in the hippocampus. These findings therefore suggest that reduced CBP immunoreactivities may be the consequence of abnormal discharge caused by loss of GABAergic inhibition rather than an indication of the neuronal damage/degeneration. J. Comp. Neurol. 485:153–164, 2005.