To clarify the antiepileptic mechanisms of valproate (VPA), we determined the effects of acute and sub-acute administrations of VPA on ryanodine receptor (RyR)-associated hippocampal releases of GABA and glutamate using microdialysis, as well expression of mRNA and protein of RyR subtypes in the rat hippocampus. Acute administration of therapeutic-relevant VPA did not affect the hippocampal extracellular levels of GABA or glutamate, whereas sub-acute administration increased GABA level without affecting that of glutamate. Perfusion with ryanodine increased the hippocampal extracellular level of glutamate (ryanodine concentration range: 1-1000μM) concentration-dependently; however, that of GABA was increased by 1-100μM ryanodine concentration-dependently but the stimulatory effects of 1000μM ryanodine on GABA release was not observed. Both acute and sub-acute administrations of therapeutic-relevant VPA inhibited ryanodine-induced responses of hippocampal extracellular glutamate level without affecting that of GABA. Especially, both acute and sub-acute administrations of VPA prevented the breakdown of GABA release induced by 1000μM ryanodine. Sub-acute administration of therapeutically-relevant dose VPA weakly increased RyR mRNA expression but we could not detect the changes of RyR protein expression in rat hippocampus. These results suggest that VPA inhibited the neurotransmitter release associated with RyR without affecting the expression of RyR protein. Therefore, the antiepileptic action of VPA seems to be mediated, at least in part, by an increase in basal GABA release and inhibition of RyR-associated glutamate release.

The transgenic rat strain S284L-TG harbors the S284L mutant of the neuronal nicotinic acetylcholine receptor alpha4 subunit gene (CHRNA4), which is responsible for human autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE). S284L-TG rats have epileptic seizure phenotypes during slow-wave sleep, similar to those in NFLE. We previously demonstrated that γ-aminobutyric acid (GABA)ergic action of these rats was suppressed before the onset of ADNFLE seizures, and that glutamate release in the epileptic focus lesion was increased at the onset of epilepsy. Here, mRNA analysis revealed that Cl(-)-accumulating Na-K-2Cl cotransporter 1 (NKCC1) levels were increased and Cl(-)-extruding K-Cl cotransporter 1 and 2 (KCC1 and KCC2) levels were decreased at the onset of ADNFLE seizures in S284L-TG rat frontal cortexes, which perturbed the GABAergic inhibitory system. The reversal potentials (EGABA) of GABAA receptor-mediated currents in cortical layer V pyramidal neurons of S284L-TG rats also changed their polarity from hyperpolarization to depolarization, and S284L-TG miniature excitatory postsynaptic currents (mEPSCs), but not miniature inhibitory postsynaptic currents (mIPSCs), significantly increased in both amplitude and frequency. Administration of 25mg/kg/day furosemide before, but not after, the onset of interictal discharges prevented idiopathic epileptic activity, reversed the depolarizing shift of EGABA and increased mEPSC amplitude to normal levels. These data indicate that early treatment with an agent that normalizes pathogenesis has a prophylactic effect on epilepsy. We propose a strategy for prophylactic medication against idiopathic epilepsy through the suppression of epileptogenesis and/or ictogenesis.

One of the best-characterized causative factors of Alzheimer's disease (AD) is the generation of amyloid-β peptide (Aβ). AD subjects are at high risk of epileptic seizures accompanied by aberrant neuronal excitability, which in itself enhances Aβ generation. However, the molecular linkage between epileptic seizures and Aβ generation in AD remains unclear.

The Japanese Schizophrenia Sib-Pair Linkage Group (JSSLG) is a multisite collaborative study group that was organized to create a national resource for affected sib pair (ASP) studies of schizophrenia in Japan. We used a high-density single-nucleotide-polymorphism (SNP) genotyping assay, the Illumina BeadArray linkage mapping panel (version 4) comprising 5,861 SNPs, to perform a genomewide linkage analysis of JSSLG samples comprising 236 Japanese families with 268 nonindependent ASPs with schizophrenia. All subjects were Japanese. Among these families, 122 families comprised the same subjects analyzed with short tandem repeat markers. All the probands and their siblings, with the exception of seven siblings with schizoaffective disorder, had schizophrenia. After excluding SNPs with high linkage disequilibrium, we found significant evidence of linkage of schizophrenia to chromosome 1p21.2-1p13.2 (LOD=3.39) and suggestive evidence of linkage to 14q11.2 (LOD=2.87), 14q11.2-q13.2 (LOD=2.33), and 20p12.1-p11.2 (LOD=2.33). Although linkage to these regions has received little attention, these regions are included in or partially overlap the 10 regions reported by Lewis et al. that passed the two aggregate criteria of a meta-analysis. Results of the present study--which, to our knowledge, is the first genomewide analysis of schizophrenia in ASPs of a single Asian ethnicity that is comparable to the analyses done of ASPs of European descent--indicate the existence of schizophrenia susceptibility loci that are common to different ethnic groups but that likely have different ethnicity-specific effects.

Linear ubiquitin chain assembly complex (LUBAC), composed of SHARPIN (SHANK-associated RH domain-interacting protein), HOIL-1L (longer isoform of heme-oxidized iron-regulatory protein 2 ubiquitin ligase-1), and HOIP (HOIL-1L interacting protein), forms linear ubiquitin on nuclear factor-κB (NF-κB) essential modulator (NEMO) and induces NF-κB pathway activation. SHARPIN expression and LUBAC formation were significantly reduced in the livers of mice 24 h after the injection of either carbon tetrachloride (CCl₄) or acetaminophen (APAP), both of which produced the fulminant hepatitis phenotype. To elucidate its pathological significance, hepatic SHARPIN expression was suppressed in mice by injecting shRNA adenovirus via the tail vein. Seven days after this transduction, without additional inflammatory stimuli, substantial inflammation and fibrosis with enhanced hepatocyte apoptosis occurred in the livers. A similar but more severe phenotype was observed with suppression of HOIP, which is responsible for the E3 ligase activity of LUBAC. Furthermore, in good agreement with these in vivo results, transduction of Hepa1-6 hepatoma cells with SHARPIN, HOIL-1L, or HOIP shRNA adenovirus induced apoptosis of these cells in response to tumor necrosis factor-α (TNFα) stimulation. Thus, LUBAC is essential for the survival of hepatocytes, and it is likely that reduction of LUBAC is a factor promoting hepatocyte death in addition to the direct effect of drug toxicity.

Neurodevelopmental disorders (NDDs) often associate with epilepsy or craniofacial malformations. Recent large-scale DNA analyses identified hundreds of candidate genes for NDDs, but a large portion of the cases still remain unexplained. We aimed to identify novel candidate genes for NDDs.

Dynamic changes in intracellular free Ca(2+) concentration play a crucial role in various neural functions. The inositol 1,4,5-trisphosphate (IP3) receptor (IP3R) and the ryanodine (Ry) receptor (RyR) are involved in Ca(2+)-induced Ca(2+)-release (CICR). Recent studies have shown that type 3 IP3R is highly expressed in rat hippocampal neurons after kainic acid (KA)-induced seizures and that dantrolene, a RyR antagonist, reduces KA-induced neuronal cell death. We investigated the RyR-associated effects of CICR agents on basal and K(+)-evoked releases of GABA and glutamate in rat hippocampus and the changes in expression of mRNA for RyRs in mouse brain after KA-induced seizures. The stimulatory effect of Ry on releases of GABA and glutamate was concentration-dependent in a biphasic manner. The inflection point in concentration-response curves for Ry on GABA release was lower than that for glutamate in both basal and K(+)-evoked conditions, suggesting that hyperactivation of RyR-associated CICR produces the imbalance between GABAergic and glutamatergic transmission. Following KA-induced seizures, transient up-regulation of brain-type RyR mRNA was observed in the hippocampal CA3 region and striatum, and signals for c-Fos mRNA increased transiently in the hippocampus, dentate gyrus and deeper layers of the neocortex. Thereafter, some dead neurons with single-stranded DNA (ssDNA) immunoreactive fragmented nuclei appeared in these areas. These findings suggest that intracellular Ca(2+) release via the RyR might be one of the mechanisms involved in KA-induced neuronal cell death.

AP-3 is a member of the adaptor protein (AP) complex family that regulates the vesicular transport of cargo proteins in the secretory and endocytic pathways. There are two isoforms of AP-3: the ubiquitously expressed AP-3A and the neuron-specific AP-3B. Although the physiological role of AP-3A has recently been elucidated, that of AP-3B remains unsolved. To address this question, we generated mice lacking mu3B, a subunit of AP-3B. mu3B-/- mice suffered from spontaneous epileptic seizures. Morphological abnormalities were observed at synapses in these mice. Biochemical studies demonstrated the impairment of gamma-aminobutyric acid (GABA) release because of, at least in part, the reduction of vesicular GABA transporter in mu3B-/- mice. This facilitated the induction of long-term potentiation in the hippocampus and the abnormal propagation of neuronal excitability via the temporoammonic pathway. Thus, AP-3B plays a critical role in the normal formation and function of a subset of synaptic vesicles. This work adds a new aspect to the pathogenesis of epilepsy.

Mutations in GABRG2, which encodes the γ2 subunit of GABAA receptors, can cause both genetic epilepsy with febrile seizures plus (GEFS+) and Dravet syndrome. Most GABRG2 truncating mutations associated with Dravet syndrome result in premature termination codons (PTCs) and are stably translated into mutant proteins with potential dominant-negative effects. This study involved search for mutations in candidate genes for Dravet syndrome, namely SCN1A, 2A, 1B, 2B, GABRA1, B2, and G2. A heterozygous nonsense mutation (c.118C>T, p.Q40X) in GABRG2 was identified in dizygotic twin girls with Dravet syndrome and their apparently healthy father. Electrophysiological studies with the reconstituted GABAA receptors in HEK cells showed reduced GABA-induced currents when mutated γ2 DNA was cotransfected with wild-type α1 and β2 subunits. In this case, immunohistochemistry using antibodies to the α1 and γ2 subunits of GABAA receptor showed granular staining in the soma. In addition, microinjection of mutated γ2 subunit cDNA into HEK cells severely inhibited intracellular trafficking of GABAA receptor subunits α1 and β2, and retention of these proteins in the endoplasmic reticulum. The mutated γ2 subunit-expressing neurons also showed impaired axonal transport of the α1 and β2 subunits. Our findings suggested that different phenotypes of epilepsy, e.g., GEFS+ and Dravet syndrome (which share similar abnormalities in causative genes) are likely due to impaired axonal transport associated with the dominant-negative effects of GABRG2.

We generated a transgenic rat strain with a missense mutation in V286L (V286L-TG), in the gene encoding the neuronal nicotinic acetylcholine receptor β2 subunit (CHRNB2) found in patients with autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE). To confirm that V286L-TG rats exhibit seizures similar to those observed in humans, gene expression patterns and behavioral phenotypes were analyzed. In situ hybridization using a V286L Chrnb2-selective probe indicated that the transgene was expressed at higher levels in the cortex, hippocampus, and cerebellum of V286L-TG than wild-type littermates (non-TG). Spontaneous epileptic seizures with ictal discharges in electroencephalograms were detected in 45% of V286L-TG rats and the frequency of seizures was 0.73 times a week. This seizure type is similar to "paroxysmal arousals" that are observed in human ADNFLE. V286L-TG rats displayed nicotine-induced abnormal motor activity including seizures in comparison to non-TGs. Response time following nicotine administration occurred faster in V286L-TG than in non-TG rats. V286L-TG rats demonstrated spontaneous epileptic seizures, which are similar to human ADNFLE, and also showed a higher sensitivity to nicotine administration. Thus, the V286L-TG rat model could be a valuable tool for developing novel mechanism-driven treatment strategies for epilepsy and provide a better understanding of ADNFLE.

A study was conducted to establish the bioequivalence between different sustained-release formulations of valproic acid (Depakene R and Selenica R), which were developed in Japan.

Recent advances in DNA microarray technology have enabled the identification of small alterations throughout the genome. We used standard karyotype analysis, followed by DNA microarray analysis and PCR to precisely map the chromosomal 4p deletion and determine the deletion breakpoints in the genome of an epileptic patient. The karyotype of the patient was 46,XY,del(4)(p15.2p15.3) as determined by G-banding analysis. We used a high-density oligonucleotide genotyping array to estimate the size of the deletion (4.5 Mb) and to locate the breakpoints within a 9-kb region on one side of the deletion and a 100-kb region on the other side. We amplified by PCR and sequenced the genomic region encompassing the breakpoints, and mapped the deletion to regions extending from 21648457 to 26164287 and from 26164505 to 26167493, respectively (chromosome 4 of NCBI Homo sapiens Genome Build 35.1). The deletion involves 18 genes, one of which (CCKAR) is partially deleted.

Sex hormone-binding globulin (SHBG) is a serum protein released mainly by the liver, and a low serum level correlates with a risk for metabolic syndrome including diabetes, obesity, and cardiovascular events. However, the underlying molecular mechanism(s) linking SHBG and metabolic syndrome remains unknown. In this study, using adipocytes and macrophages, we focused on the in vitro effects of SHBG on inflammation as well as lipid metabolism. Incubation with 20 nM SHBG markedly suppressed lipopolysaccharide- (LPS-) induced inflammatory cytokines, such as MCP-1, TNFα, and IL-6 in adipocytes and macrophages, along with phosphorylations of JNK and ERK. Anti-inflammatory effects were also observed in 3T3-L1 adipocytes cocultured with LPS-stimulated macrophages. In addition, SHBG treatment for 18 hrs or longer significantly induced the lipid degradation of differentiated 3T3-L1 cells, with alterations in its corresponding gene and protein levels. Notably, these effects of SHBG were not altered by coaddition of large amounts of testosterone or estradiol. In conclusion, SHBG suppresses inflammation and lipid accumulation in macrophages and adipocytes, which might be among the mechanisms underlying the protective effect of SHBG, that is, its actions which reduce the incidence of metabolic syndrome.