Pentylenetetrazole-induced seizure is attenuated in serine racemase knockout mice

Pentylenetetrazole-induced seizure is attenuated in serine racemase knockout mice

e294 Abstracts / Neuroscience Research 71S (2011) e108–e415 severe muscle atrophy compared with SBMA mice. There was no aggregation of mutant AR in ...

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e294

Abstracts / Neuroscience Research 71S (2011) e108–e415

severe muscle atrophy compared with SBMA mice. There was no aggregation of mutant AR in Purkinje cells, liver or pituitary gland of SBMA mice, where Hsf-1 was expressed at a high level. However, in Hsf-1 knocked-out SBMA mice, mutant AR accumulated in these tissues. In the spinal anterior horn and cerebral cortex of Hsf-1 knocked-out SBMA mice, the accumulation of mutant AR increased through Hsp70 down-regulation. By contrast, the reduction of Hsf-1 did not enhance the pathogenic AR aggregation in the skeletal muscle where the nuclear uptake of nuclear transcription factor Y subunit alpha (NF-YA) is up-regulated. Conclusions: These results suggest that Hsf-1 contributes to the determination of the pathological lesion selectivity in SBMA. Furthermore, the degree of nuclear translocation of NF-YA also influences the aggregation of the pathogenic AR in a tissue-specific manner.

consistent with the results of our previous immunohistochemical analysis. We furthermore found that some parvalbumin-negative cells, which were also negative for T-box brain 1 (TBR1), a marker for pyramidal cells, expressed green fluorescent proteins. Although such parvalbumin-negative cells are currently under characterization, our data might implicate that the altered functions of neocortical and hippocampal parvalbumin-positive interneurons are the molecular basis of human epilepsies associated with SCN1A mutations. Research fund: KAKENHI 21791020, Takeda Science Foundation, Japan Epilepsy Research Foundation.

doi:10.1016/j.neures.2011.07.1281

P3-r17 Electrophysiological properties of variant voltagegated calcium channels in patients with epilepsy

P3-r15 Induction of astrocyte differentiation from human induced pluripotent stem cells carrying mutant SOD1 Shiho Kitaoka 1,2 , Kayoko Tsukita 1,2 , Kazutoshi Takahashi 1,2 , Keisuke Okita 1 , Takayuki Kondo 1,3 , Katsuhiro Yoshikawa 3 , Satoko Yamawaki 3 , Motoko Naitoh 3 , Shigehiko Suzuki 3 , Yuishin Izumi 4 , Ryuji Kaji 4 , Hiroshi Takuma 5 , Akira Tamaoka 5 , Mitsuya Morita 6 , Imaharu Nakano 6 , Akihiro Kawata 7 , Tatsutoshi Nakahata 1 , Ryosuke Takahashi 3 , Shinya Yamanaka 1 , Haruhisa Inoue 1,2 1

CiRA, Kyoto Univ., Kyoto, Japan 2 CREST, JST, Kawaguchi, Japan 3 Kyoto Univ Grad Sch Med, Kyoto, Japan 4 Tokushima Univ Fac Med, Tokushima, Japan 5 Dept Neurol, Med, Tsukuba Univ, Tsukuba, Japan 6 Jichi Medical Univ 7 Tokyo Metropolitan Neurological Hospital, Tokyo, Japan Accumulating evidence, explored by ALS mice model studies, supports the idea that the non-cell autonomous toxicity of astrocytes expressing mutant superoxide dismutase 1 (SOD1) contributes to motor neuron degeneration in amyotrophic lateral sclerosis (ALS). However, it is still enigmatic whether human ALS astrocytes haboring mutant SOD1 also has the same characteristic effect on motor neurons. Here, we describe the generation of induced pluripotent stem cells (iPSCs) from familial ALS patients and the differentiation of disease-specific iPSCs into astrocytes to establish an ALS model in vitro. To generate patient-specific iPSCs, dermal fibroblasts were obtained by biopsy from patients carrying mutant SOD1. The fibroblasts were reprogrammed by retrovirus vectors or episomal vectors to eliminate the differences in iPSC lines caused by reprogramming methods. These iPSC lines expressed ESC markers such as Nanog and SSEA-4, and had pluripotency to differentiate into cells of all three germ layers in vitro and in vivo. DNA sequencing revealed that mutated alleles of iPSCs are identical to the original specimen. All iPSCs preserve normal karyotype. Disease-specific iPSCs were differentiated into astrocytes expressing astrocyte markers such as glial fibrillary acidic protein (GFAP) and S100. Furthermore, we already reported these iPSCs were differentiated into spinal motor neurons. These results indicated that disease-specific iPSCs might provide an ALS model system in vitro. Research fund: KAKENHI (22700377). doi:10.1016/j.neures.2011.07.1282

P3-r16 Scn1a-GFP BAC transgenic mouse lines showed predominant Nav 1.1 expression in parvalbumin-positive interneurons in neocortex and hippocampus Ikuo Ogiwara 1 Yamakawa 1

, Emi Mazaki 1 , Shigeyoshi Itohara 2 , Kazuhiro

1

Lab. Neurogenetics, RIKEN BSI, Wako, Saitama, Japan 2 Lab. Behavior Genetics, RIKEN BSI, Wako, Saitama, Japan

Mutations of human voltage-gated sodium channel ␣1 gene, SCN1A, encoding Nav 1.1 are associated with a wide spectrum childhood epilepsies including Dravet syndrome (or severe myoclonic epilepsy in infancy, SMEI), generalized epilepsy febrile seizure plus (GEFS+) and their subtypes. We have previously shown that, in mouse neocortex and hippocampus at around weaning period, Nav1.1 was predominantly localized at axon initial segments and somata of interneurons, majority of which were immunopositive for parvalbumin (Ogiwara et al., Journal of Neuroscience, 2007, 27:5903–14). In order to examine the Scn1a expression pattern in mouse brain in further detail, we generated and characterized the BAC transgenic mouse lines that express green fluorescent proteins under the mouse Scn1a promoters. We found that, in neocortex and hippocampus, cells with relatively strong fluorescence were largely immunopositive for parvalbumin. These results are

doi:10.1016/j.neures.2011.07.1283

Iori Ohmori 1 , Mamoru Ouchida 2 , Haijiao Wang 1 , Yuichiro Kitagawa 1 , Hiroyuki Michiue 1 , Teiichi Nishiki 1 , Hideki Matsui 1 1 Dept. of Physiology, Grad. Sch. of Med. Dent. and Pharm. Sci, Okayama Univ., Okayama, Japan 2 Dept. of Molecular Genetics, Grad. Sch. of Med. Dent. and Pharm. Sci, Okayama Univ., Okayama, Japan

Mutations in the CACNA1A gene have been linked to familial hemiplegic migraine type 1 (FHM-1), episodic ataxia type 2 (EA-2), spinocerebellar ataxia type 6, and combination of epilepsy and ataxia. We conducted a mutational analysis of all coding exons of the CACNA1A gene in patients with epilepsy. Selected variant CACNA1A channels were studied using heterologous expression of recombinant human Cav2.1 and whole-cell patch-clamp recording. Full-length human CACNA1A (Cav2.1) cDNA in pcDNA1.1 and rabbit ␣2␦ subunit cDNA expression vector, pKCR␣2␦, were kindly provided by Prof. T. Tanabe (Tokyo Medical and Dental University, Tokyo, Japan). Variant Cav2.1 channels were coexpressed transiently with accessory ␤4 and ␣2␦ subunits in HEK293 cells. The present study detected nine variants including six novel variants of the CACNA1A gene in the 48 patients. Furthermore, in heterologous expression experiments, four of the five variants exhibited a predicted gain-of-function effect on channel activity in comparison to wild type (WT) Cav2.1. The majority of EA2-related CACNA1A mutations exhibited either a complete or almost complete loss of function, whereas FHM1-related Cacna1a mutation showed that a gain-of-function effect in synaptic transmission. Electrophysiological properties of variant Cav2.1 channels in some epileptic patients were similar to those in patients with FHM-1. Research fund: KAKENHI 21390312. doi:10.1016/j.neures.2011.07.1284

P3-r18 Pentylenetetrazole-induced seizure is attenuated in serine racemase knockout mice Ran Inoue 1 , Tomomi Harai 1,2 , Yuko Fujita 3 , Ayumi Tanaka 1 , Mao Horio 3 , Kenji Hashimoto 3 , Hisashi Mori 1

1 Dept Mol Neurosci, Grad. Sch. of Med. and Pharm., Univ. of Toyama, Toyama, Japan 2 Dept. Pediatrics, Grad. Sch. of Med. and Pharm., Univ. of Toyama, Toyama, Japan 3 Center for Forensic Mental Health, Chiba Univ. Chiba, Japan

Epilepsy is one of the most common neurological disorders of the central nervous system (CNS), which affects up to 1% of the world population. However, the neurochemical basis or mechanism of epilepsy remains to be elucidated. Growing evidence has shown that the N-methyl-D-aspartate (NMDA)-type glutamate receptor, which plays roles in excitatory neurotransmission and synaptic plasticity in the CNS, may be involved in the initiation or spread of epileptic neuronal hyperactivity and direct antagonists of the NMDA receptor exhibit antiepileptic effects. However, the severe adverse effect of NMDA receptor antagonists owing to the direct and widespread functional blocking of NMDA receptors has limited their clinical application. The mammalian brain contains high level of D-serine, which acts as a coagonist at the glycine site of the NMDA receptor. We have produced serine racemase (SR) knockout (KO) mice and reported that SR is the major enzyme for D-serine production in the mouse brain. We also found a reduced neurotoxicity induced by injection of NMDA and A␤1–42 into the forebrain in SR KO mice. Thus, we hypothesized that D-serine may also play a role in the pathogenesis of epilepsy related to NMDA receptor overactivation. In the present study, we investigated the influence of D-serine deficiency on pentylenetetrazol (PTZ)induced convulsive seizures by using SR-KO mice. We found that comparing to WT mice, SR-KO mice manifested less severity of tonic–clonic seizures and had a significantly reduced number of c-fos immunopositive cells labeled in cerebral cortex. Further, the activation of astrocytes and the increase in extracellular glutamate induced by PTZ in dentate gyrus (DG) of WT mice were inhibited in SR-KO mice. Our findings indicate an attenuating effect of

Abstracts / Neuroscience Research 71S (2011) e108–e415

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D-serine deficiency on tonic–clonic seizures induced by PTZ, and suggest SR as a target for development of new therapeutic strategy for epilepsy. Research fund: Grant-in-Aid for Young Scientists (B) 22700332.

of IP3 Rs. These results suggest that myoclonin1 regulates IP3 Rs-mediated ER-Ca2+ homeostasis by interacting with the C-terminal of IP3 Rs. Research fund: KAKENHI 22791154.

doi:10.1016/j.neures.2011.07.1285

doi:10.1016/j.neures.2011.07.1287

P3-r19 Phenotype analysis of transgenic rat harboring a missense mutation V286L of Chrnb2 identified in autosomal dominant nocturnal frontal lobe epilepsy

P3-r21 Enhancement of seizure susceptibility of Noda epileptic rat (NER) by zinc chelators

Yuko Shiba 1 , Junko Yamada 1 , Keisuke Migita 1 , Fumiaki Mori 2 , Kouichi Wakabayashi 2 , Sunao Kaneko 3 , Motohiro Okada 4 , Shinichi Hirose 5 , Shinya Ueno 1 1

Dept. Neurophysiol., Hirosaki Univ. Grad. Sch. Med., Hirosaki Japan 2 Dept. Neuropathology, Hirosaki Univ. Grad. Sch. Med., Hirosaki Japan 3 Dept. Neuropsychiatry, Hirosaki Univ. Grad. Sch. Med., Hirosaki Japan 4 Dept. Psychiatry, Mie Univ. Grad. Sch. Med., Tsu Japan 5 Dept. Pediatrics, Fukuoka Univ., Fukuoka, Japan Mutations of gene encoding ␣4, ␤2 subunit of nACh receptors cause autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE). We generated transgenic rat strains that harbor a missense mutation V286L, which had been identified in CHRNB2 in human ADNFLE. To investigate the pathogenesis of ADNFLE, we compared phenotypes between V286L transgenic rats (V286L-TG) and littermates (LM). V286L-TG at 8-10 weeks old developed a distinct spontaneous epileptic seizure type: paroxysmal arousals, brief episodes characterized by sudden frightened expression. The onset of ictal discharges was synchronized with seizure behaviors. The frequencies of this seizure were once a week. LM did not show any seizure and abnormality of electroencephalogram. Responses to nicotine of V286L-TG exhibited the higher sensitivity than those of LM. The intraperitoneal injection of nicotine (1 mg/kg) to V286L-TG but no LM caused seizure onset, straub tail, and gasping, which were associated the abnormal electroencephalogram. These findings indicate that mutation of ␤2 nACh R is involved in the epileptic phenotype. doi:10.1016/j.neures.2011.07.1286

P3-r20 Epilepsy protein myoclonin1 regulates endoplasmic reticulum calcium homeostasis Toshimitsu Suzuki 1 , Kripamoy Aguan 1 , Hideaki Mizuno 2 , Takeshi Nakamura 4 , Ikuyo Inoue 1 , Katsuhiko Mikoshiba 3,4 , Atsushi Miyawaki 2 , Kazuhiro Yamakawa 1 1

Lab. for Neurogenetics, RIKEN BSI, Saitama, Japan 2 Lab. for Cell Function Dynamics, RIKEN BSI, Saitama, Japan 3 Lab. for Developmental Neurobiology, RIKEN BSI, Saitama, Japan 4 Calcium Oscillation Project, ICORP, JST, Saitama, Japan

Juvenile myoclonic epilepsy (JME) accounts for 10–12% of all epilepsies and is characterized by adolescent onset myoclonic jerks on awakening, grand mal seizures, and less frequent absence seizures. By genetic linkage and mutation analyses, we found disease-associated missense mutations of EFHC1 gene on chromosome 6p12-p11 in JME patients (Suzuki et al., Nat. Genet., 2004). Subsequently other groups reported additional EFHC1 mutations not only in JME but also in other types of idiopathic epilepsies. Whereas most of the genes for idiopathic epilepsies encode ion-channels, EFHC1 encodes non-ion channel protein myoclonin1 that is composed of three consecutive DM10 domains and an EF-hand calcium-binding motif at the C terminus, and expresses in multiple tissues. We generated Efhc1-deficient mice and elucidated that reduction or loss of function of myoclonin1 in mouse showed epileptic phenotypes (Suzuki et al., Hum. Mol. Genet., 2009), and this further confirmed that EFHC1 is the gene for epilepsy. Previously we have shown that over-expression of myoclonin1 in primary cultured neuron activates R-type voltage-dependent calcium channel (Cav 2.3) resulting excessive intracellular Ca2+ influx and rapid cell-death, and both of these effects were compromised in myoclonin1 mutant proteins (Suzuki et al., 2004). The pharmacological blocking of Cav 2.3 marginally prolonged the survivability but did not completely block myoclonin1 induced neuronal cell-death. Thus, it is likely that myoclonin1 might modulate intracellular Ca2+ signaling and homeostasis. Here we report that loss of function of myoclonin1 alters the levels of Ca2+ stored in the ER and IP3 -induced Ca2+ release activity in living cells. Myoclonin1 is well co-expressed with Ip3 r1 at choroid plexus and ependymal cells in mouse brain. In addition, myoclonin1 binds to C-terminal

Masashi Iida , Atsushi Takeda, Masaki Ando, Syunsuke Takada, Masatoshi Nakamura, Naoto Oku Dept. of Med. Biochem. Grad. Sch. of Pharm. Sci., Univ. of Shizuoka, Japan Zinc homeostasis in the brain is associated with the etiology and manifestation of epileptic seizures. Approximately 80% of the total brain zinc exists as zinc metalloproteins and the rest is histochemically reactive (chelatable) zinc as revealed by Timm’s sulfide-silver staining method. Chelatable zinc predominantly exists in the presynaptic vesicle, is released from glutamatergic neuron terminals, and serves as a neuromodulator. Chelatable zinc may negatively modulate glutamate release, while positively modulating GABAergic neurotransmitter system. However, the relationship between the manifestation of epileptic seizures and the action of chelatable zinc is poorly understood. Adult Noda epileptic rat (NER) (>12week-old) exhibits spontaneous generalized tonic–clonic seizure about once a day. NER reveals seizure susceptibility to pentylenetetrazol, tossing, and transcorneal electroshock, but not to tactile, photic, and acoustic stimuli and transauricular electroshock. To pursue the involvement of chelatable zinc in seizure susceptibility, in the present study, the effect of zinc chelators on seizure susceptibility was examined using adult NER. Clioquinol (5-chloro-7-iodo-8-hydroxyquinoline; CQ) has a relatively weak affinity for zinc (Kd , approximately 1 × 10−7 M) compared to tetrakis-(2pyridylmethyl)ethylendediamine (TPEN, Kd = 2.6 × 10−16 M). Both chelators are lipophilic, transported into the brain and modulate chelatable zinc pools. CQ and TEPN were i.p. injected into NER and the incidence of tonic–clonic seizure was observed for 6 h. The incidence was markedly increased (CQ, 30–100 mg/kg; TEPN, 1 mg/kg). CQ (30 mg/kg)-potentiated seizure susceptibility was significantly ameliorated by co-injection with aminooxyacetic acid (AOAA, 5–20 mg/kg) or phenobarbital (20 mg/kg), which are known as anticonvulsant drugs enhancing GABAergic activity. These results suggest that the loss of chelatable zinc enhances seizure susceptibility of NER. doi:10.1016/j.neures.2011.07.1288

P3-s01 FosB and/or 2FosB regulate proliferation of adult hippocampal neural progenitor cells and suppress spontaneous epileptic seizures Noriko Yutsudo 1 , Takashi Kamada 2 , Hiroko Nomaru 1 , Yoko H. Ohnishi 1 , Yoshinori N. Ohnishi 1 , Kosuke Kajitani 1 , Kunihiko Sakumi 1 , Hiroshi Shigeto 2 , Yusaku Nakabeppu 1 1

Div. Neurofunc. Genomics, MIB, Kyushu Univ., Fukuoka, Japan 2 Dept. Neurol., Kyushu Univ., Fukuoka, Japan

Jun and Fos, the major components of the AP-1 transcription factor family, are induced by various intra- or extracellular signals that trigger cell proliferation or differentiation. Among fos family genes, only the fosB gene produces several isoforms (FosB, FosB, 2FosB). Their expression is highly induced in the adult hippocampus after transient forebrain ischemia, and seizures caused by electric stimulation or kainate. To understand the significance of these induction, we constructed fosB-null mice and fosBd/d mice which express only FosB and 2FosB but not FosB. We found that basal and kainate-induced proliferation of neural progenitor cells in the adult dentate gyrus decreased significantly in fosB-null mice compared to that in wild-type and fosBd/d mice. Moreover, 85% of fosB-null mice older than 13-week old exhibited spontaneous epileptic seizures originated in the hippocampus and most of them had abnormality in the DG structure. In contrast, fosBd/d and wild-type mice exhibited no abnormality. Furthermore, microarray analysis of the hippocampus revealed that expression levels of several genes involved in neurogenesis and epilepsy were significantly altered in fosB-null mice. These data suggest that FosB and/or 2FosB regulate proliferation of neural progenitor cells in the adult DG and suppress spontaneous epileptic seizures in the adult brain. Research fund: KAKENHI (18300124, 19390114, 22221004), Global COE Program. doi:10.1016/j.neures.2011.07.1289