研究等業績 - その他 - 沼田　朋大

SGLT2阻害薬イプラグリフロジンは乳癌細胞増殖を抑制する

小松志保, 野見山崇, 沼田朋大, 川波賢子, 濱口百合子, 高橋貴理子, 井上隆司, 柳瀬敏彦

糖尿病(Web)   60 ( Suppl )   2017年

J-GLOBAL

Allosteric activation of membrane-bound glutamate receptors using coordination chemistry within living cells

Shigeki Kiyonaka, Ryou Kubota, Yukiko Michibata, Masayoshi Sakakura, Hideo Takahashi, Tomohiro Numata, Ryuji Inoue, Michisuke Yuzaki, Itaru Hamachi

NATURE CHEMISTRY ( NATURE PUBLISHING GROUP )  8 ( 10 ) 958 - 967   2016年10月

The controlled activation of proteins in living cells is an important goal in protein-design research, but to introduce an artificial activation switch into membrane proteins through rational design is a significant challenge because of the structural and functional complexity of such proteins. Here we report the allosteric activation of two types of membrane-bound neurotransmitter receptors, the ion-channel type and the G-protein-coupled glutamate receptors, using coordination chemistry in living cells. The high programmability of coordination chemistry enabled two His mutations, which act as an artificial allosteric site, to be semirationally incorporated in the vicinity of the ligand-binding pockets. Binding of Pd(2,2'-bipyridine) at the allosteric site enabled the active conformations of the glutamate receptors to be stabilized. Using this approach, we were able to activate selectively a mutant glutamate receptor in live neurons, which initiated a subsequent signal-transduction pathway.

DOI PubMed

Discovery of allosteric modulators for GABA(A) receptors by ligand-directed chemistry

Kei Yamaura, Shigeki Kiyonaka, Tomohiro Numata, Ryuji Inoue, Itaru Hamachi

NATURE CHEMICAL BIOLOGY ( NATURE PUBLISHING GROUP )  12 ( 10 ) 822 - +   2016年10月

The fast inhibitory actions of gamma-aminobutyric acid (GABA) are mainly mediated by GABA(A) receptors (GABA(A)Rs) in the brain. The existence of multiple ligand-binding sites and a lack of structural information have hampered the efficient screening of drugs capable of acting on GABA(A)Rs. We have developed semisynthetic fluorescent biosensors for orthosteric and allosteric GABA(A)R ligands on live cells via coupling of affinity-based chemical labeling reagents to a bimolecular fluorescence quenching and recovery system. These biosensors were amenable to the high-throughput screening of a chemical library, leading to the discovery of new small molecules capable of interacting with GABA(A)Rs. Electrophysiological measurements revealed that one hit, 4,4',4 ''-(4-propyl-[1H]-pyrazole-1,3,5-triyl) trisphenol (PPT), was a novel negative allosteric modulator capable of strongly suppressing GABA-induced chloride currents. Thus, these semisynthetic biosensors represent versatile platforms for screening drugs to treat GABA(A)R-related neurological disorders, and this strategy can be extended to structurally complicated membrane proteins.

DOI PubMed

Cold sensitivity of TRPA1 is unveiled by the prolyl hydroxylation blockade-induced sensitization to ROS

Takahito Miyake, Saki Nakamura, Meng Zhao, Kanako So, Keisuke Inoue, Tomohiro Numata, Nobuaki Takahashi, Hisashi Shirakawa, Yasuo Mori, Takayuki Nakagawa, Shuji Kaneko

NATURE COMMUNICATIONS ( NATURE PUBLISHING GROUP )  7   12840   2016年09月

Mammalian transient receptor potential ankyrin 1 (TRPA1) is a polymodal nociceptor that plays an important role in pain generation, but its role as a cold nociceptor is still controversial. Here, we propose that TRPA1 can sense noxious cold via transduction of reactive oxygen species (ROS) signalling. We show that inhibiting hydroxylation of a proline residue within the N-terminal ankyrin repeat of human TRPA1 by mutation or using a prolyl hydroxylase (PHD) inhibitor potentiates the cold sensitivity of TRPA1 in the presence of hydrogen peroxide. Inhibiting PHD in mice triggers mouse TRPA1 sensitization sufficiently to sense cold-evoked ROS, which causes cold hypersensitivity. Furthermore, this phenomenon underlies the acute cold hypersensitivity induced by the chemotherapeutic agent oxaliplatin or its metabolite oxalate. Thus, our findings provide evidence that blocking prolyl hydroxylation reveals TRPA1 sensitization to ROS, which enables TRPA1 to convert ROS signalling into cold sensitivity.

DOI PubMed

Identification of MMP1 as a novel risk factor for intracranial aneurysms in ADPKD using iPSC models

Tomonaga Ameku, Daisuke Taura, Masakatsu Sone, Tomohiro Numata, Masahiro Nakamura, Fumihiko Shiota, Taro Toyoda, Satoshi Matsui, Toshikazu Araoka, Tetsuhiko Yasuno, Shin-Ichi Mae, Hatasu Kobayashi, Naoya Kondo, Fumiyo Kitaoka, Naoki Amano, Sayaka Arai, Tomoko Ichisaka, Norio Matsuura, Sumiko Inoue, Takuya Yamamoto, Kazutoshi Takahashi, Isao Asaka, Yasuhiro Yamada, Yoshifumi Ubara, Eri Muso, Atsushi Fukatsu, Akira Watanabe, Yasunori Sato, Tatsutoshi Nakahata, Yasuo Mori, Akio Koizumi, Kazuwa Nakao, Shinya Yamanaka, Kenji Osafune

SCIENTIFIC REPORTS ( NATURE PUBLISHING GROUP )  6   30013   2016年07月

Cardiovascular complications are the leading cause of death in autosomal dominant polycystic kidney disease (ADPKD), and intracranial aneurysm (ICA) causing subarachnoid hemorrhage is among the most serious complications. The diagnostic and therapeutic strategies for ICAs in ADPKD have not been fully established. We here generated induced pluripotent stem cells (iPSCs) from seven ADPKD patients, including four with ICAs. The vascular cells differentiated from ADPKD-iPSCs showed altered Ca2+ entry and gene expression profiles compared with those of iPSCs from non-ADPKD subjects. We found that the expression level of a metalloenzyme gene, matrix metalloproteinase (MMP) 1, was specifically elevated in iPSC-derived endothelia from ADPKD patients with ICAs. Furthermore, we confirmed the correlation between the serum MMP1 levels and the development of ICAs in 354 ADPKD patients, indicating that high serum MMP1 levels may be a novel risk factor. These results suggest that cellular disease models with ADPKD-specific iPSCs can be used to study the disease mechanisms and to identify novel disease-related molecules or risk factors.

DOI PubMed

Ablation of the N-type calcium channel ameliorates diabetic nephropathy with improved glycemic control and reduced blood pressure

Shoko Ohno, Hideki Yokoi, Kiyoshi Mori, Masato Kasahara, Koichiro Kuwahara, Junji Fujikura, Masaki Naito, Takashige Kuwabara, Hirotaka Imamaki, Akira Ishii, Moin A. Saleem, Tomohiro Numata, Yasuo Mori, Kazuwa Nakao, Motoko Yanagita, Masashi Mukoyama

SCIENTIFIC REPORTS ( NATURE PUBLISHING GROUP )  6   27192   2016年06月

Pharmacological blockade of the N- and L-type calcium channel lessens renal injury in kidney disease patients. The significance of specific blockade of alpha 1 subunit of N-type calcium channel, Ca(v)2.2, in diabetic nephropathy, however, remains to be clarified. To examine functional roles, we mated Ca(v)2.2(-/-) mice with db/db (diabetic) mice on the C57BLKS background. Ca(v)2.2 was localized in glomeruli including podocytes and in distal tubular cells. Diabetic Ca(v)2.2(-/-) mice significantly reduced urinary albumin excretion, glomerular hyperfiltration, blood glucose levels, histological deterioration and systolic blood pressure (SBP) with decreased urinary catecholamine compared to diabetic Ca(v)2.2(+/+) mice. Interestingly, diabetic heterozygous Ca(v)2.2(+/-) mice also decreased albuminuria, although they exhibited comparable systolic blood pressure, sympathetic nerve activity and creatinine clearance to diabetic Ca(v)2.2(+/+) mice. Consistently, diabetic mice with cilnidipine, an N-/L-type calcium channel blocker, showed a reduction in albuminuria and improvement of glomerular changes compared to diabetic mice with nitrendipine. In cultured podocytes, depolarization-dependent calcium responses were decreased by.-conotoxin, a Ca(v)2.2-specific inhibitor. Furthermore, reduction of nephrin by transforming growth factor-beta (TGF-beta) in podocytes was abolished with.-conotoxin, cilnidipine or mitogen-activated protein kinase kinase inhibitor. In conclusion, Ca(v)2.2 inhibition exerts renoprotective effects against the progression of diabetic nephropathy, partly by protecting podocytes.

DOI PubMed

"TRP inflammation" relationship in cardiovascular system

Tomohiro Numata, Kiriko Takahashi, Ryuji Inoue

SEMINARS IN IMMUNOPATHOLOGY ( SPRINGER HEIDELBERG )  38 ( 3 ) 339 - 356   2016年05月

Despite considerable advances in the research and treatment, the precise relationship between inflammation and cardiovascular (CV) disease remains incompletely understood. Therefore, understanding the immunoinflammatory processes underlying the initiation, progression, and exacerbation of many cardiovascular diseases is of prime importance. The innate immune system has an ancient origin and is well conserved across species. Its activation occurs in response to pathogens or tissue injury. Recent studies suggest that altered ionic balance, and production of noxious gaseous mediators link to immune and inflammatory responses with altered ion channel expression and function. Among plausible candidates for this are transient receptor potential (TRP) channels that function as polymodal sensors and scaffolding proteins involved in many physiological and pathological processes. In this review, we will first focus on the relevance of TRP channel to both exogenous and endogenous factors related to innate immune response and transcription factors related to sustained inflammatory status. The emerging role of inflammasome to regulate innate immunity and its possible connection to TRP channels will also be discussed. Secondly, we will discuss about the linkage of TRP channels to inflammatory CV diseases, from a viewpoint of inflammation in a general sense which is not restricted to the innate immunity. These knowledge may serve to provide new insights into the pathogenesis of various inflammatory CV diseases and their novel therapeutic strategies.

DOI PubMed

Distinct pharmacological and molecular properties of the acid-sensitive outwardly rectifying (ASOR) anion channel from those of the volume-sensitive outwardly rectifying (VSOR) anion channel

Kaori Sato-Numata, Tomohiro Numata, Ryuji Inoue, Yasunobu Okada

PFLUGERS ARCHIV-EUROPEAN JOURNAL OF PHYSIOLOGY ( SPRINGER )  468 ( 5 ) 795 - 803   2016年05月

Expressed by many cell types, acid-sensitive outwardly rectifying (ASOR) anion channels are known to be activated by extracellular acidification and involved in acidotoxic necrotic cell death. In contrast, ubiquitously expressed volume-sensitive outwardly rectifying (VSOR) anion channels are activated by osmotic cell swelling and involved in cell volume regulation and apoptotic cell death. Distinct inhibitors to distinguish ASOR from VSOR anion channels have not been identified. Although leucine-rich repeats containing 8A (LRRC8A) was recently found to be an essential component of VSOR anion channels, the possibility of an LRRC8 family member serving as a component of ASOR anion channels has not been examined. In this study, we explored the effects of 12 known VSOR channel inhibitors and small interfering RNA (siRNA)-mediated knockdown of LRRC8 family members on ASOR and VSOR currents in HeLa cells. Among these inhibitors, eight putative VSOR blockers, including 4-(2-butyl-6,7-dichlor-2-cyclopentylindan-1-on-5-yl) oxobutyric acid (DCPIB) and 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB), were totally ineffective at blocking ASOR channel activity, whereas suramin, R-(+)-[(2-n-butyl-6,7-dichloro-2-cyclopentyl-2,3-dihydro-1-oxo-1H-inden-5-yl)oxy] acetic acid (DIOA), arachidonic acid, and niflumic acid were found to be effective ASOR anion channel antagonists. In addition, gene-silencing studies showed that no LRRC8 family members are essentially involved in ASOR anion channel activity, whereas LRRC8A is involved in VSOR anion channel activity in HeLa cells.

DOI PubMed

ADPKD特異的iPS細胞を用いた脳動脈瘤合併に関与する新規リスク因子の同定

天久 朝廷, 田浦 大輔, 曽根 正勝, 沼田 朋大, 塩田 文彦, 豊田 太郎, 佐藤 泰憲, 浅香 勲, 武曾 惠理, 深津 敦司, 乳原 善文, 小泉 昭夫, 森 泰生, 中尾 一和, 長船 健二

日本腎臓学会誌 ( (一社)日本腎臓学会 )  58 ( 3 ) 284 - 284   2016年05月

Different Contribution of Redox-Sensitive Transient Receptor Potential Channels to Acetaminophen-Induced Death of Human Hepatoma Cell Line

Heba Badr, Daisuke Kozai, Reiko Sakaguchi, Tomohiro Numata, Yasuo Mori

FRONTIERS IN PHARMACOLOGY ( FRONTIERS MEDIA SA )  7 ( FEB ) 19   2016年02月

Acetaminophen (APAP) is a safe analgesic antipyretic drug at prescribed doses. Its overdose, however, can cause life-threatening liver damage. Though, involvement of oxidative stress is widely acknowledged in APAP-induced hepatocellular death, the mechanism of this increased oxidative stress and the associated alterations in Ca2+ homeostasis are still unclear. Among members of transient receptor potential (TRP) channels activated in response to oxidative stress, we here identify that redox-sensitive TRPV1, TRPC1, TRPM2, and TRPM7 channels underlie Ca2+ entry and downstream cellular damages induced by APAP in human hepatoma (HepG2) cells. Our data indicate that APAP treatment of HepG2 cells resulted in increased reactive oxygen species (ROS) production, glutathione (GSH) depletion, and Ca2+ entry leading to increased apoptotic cell death. These responses were significantly suppressed by pretreatment with the ROS scavengers N-acetyl-L-cysteine (NAC) and 4,5-dihydroxy-1,3-benzene disulfonic acid disodium salt monohydrate (Tiron), and also by preincubation of cells with the glutathione inducer Dimethylfumarate (DMF). TRP subtype targeted pharmacological blockers and siRNAs strategy revealed that suppression of either TRPV1, TRPC1, TRPM2, or TRPM7 reduced APAP-induced ROS formation, Ca2+ influx, and cell death; the effects of suppression of TRPV1 or TRPC1, known to be activated by oxidative cysteine modifications, were stronger than those of TRPM2 or TRPM7. Interestingly, TRPV1 and TRPC1 were labeled by the cysteine-selective modification reagent, 5,5'-dithiobis (2-nitrobenzoic acid)-2-biotin (DTNB-2Bio), and this was attenuated by pretreatment with APAP, suggesting that APAP and/or its oxidized metabolites act directly on the modification target cysteine residues of TRPV1 and TRPC1 proteins. In human liver tissue, TRPV1, TRPC1 TRPM2, and TRPM7 channels transcripts were localized mainly to hepatocytes and Kupffer cells. Our findings strongly suggest that APAP-induced Ca2+ entry and subsequent hepatocellular death are regulated by multiple redox-activated cation channels, among which TRPV1 and TRPC1 play a prominent role.

DOI PubMed

TRPM2 channels in alveolar epithelial cells mediate bleomycin-induced lung inflammation

Ryo Yonezawa, Shinichiro Yamamoto, Miki Takenaka, Yukiko Kage, Takaharu Negoro, Takahiro Toda, Masayuki Ohbayashi, Tomohiro Numata, Yasuko Nakano, Toshinori Yamamoto, Yasuo Mori, Masakazu Ishii, Shunichi Shimizu

FREE RADICAL BIOLOGY AND MEDICINE ( ELSEVIER SCIENCE INC )  90   101 - 113   2016年01月

Lung inflammation is a major adverse effect of therapy with the antitumor drug bleomycin (BLM). Transient receptor potential melastatin 2 (TRPM2) is a Ca2+-permeable channel that is activated by oxidative stress through the production of ADP-ribose. We herein investigated whether TRPM2 channels contributed to BLM-induced lung inflammation. The intratracheal instillation of BLM into wild-type (WT) mice increased the number of polymorphonuclear leukocytes (PMNs) and inflammatory cytokine levels in the lung. Increases in inflammatory markers in WT mice were markedly reduced in trpm2 knockout (KO) mice, which demonstrated that the activation of TRPM2 channels was involved in BLM-induced lung inflammation. The expression of TRPM2 mRNA was observed in alveolar macrophages, alveolar epithelial cells, and lung fibroblasts. Actually, TRPM2 protein was expressed in lung tissues. Of these, TRPM2 channels in epithelial cells were activated by the addition of H2O2 following a BLM pretreatment, resulting in the secretion of macrophage inflammatory protein-2 (MIP-2). The H2O2-induced activation of TRPM2 by the BLM pretreatment was blocked by the poly(ADP-ribose) polymerase (PARP) inhibitors PJ34 and 3-aminobenzamide. The accumulation of poly(ADP-ribose) in the nucleus, a marker for ADP-ribose production, was strongly induced by H2O2 following the BLM pretreatment. Furthermore, administration of PRAP inhibitors into WT mice markedly reduced recruitment of inflammatory cells and MIP-2 secretion induced by BLM instillation. These results suggest that the induction of MIP-2 secretion through the activation of TRPM2 channels in alveolar epithelial cells is an important mechanism in BLM-induced lung inflammation, and the TRPM2 activation is likely to be mediated by ADP-ribose production via PARP pathway. TRPM2 channels may be new therapeutic target for BLM-induced lung inflammation. (C) 2015 Elsevier Inc. All rights reserved.

DOI PubMed

Allosteric activation of glutamate receptors by on-cell coordination chemistry

Kiyonaka, Ryou Kubota, Yukiko Michibata, Masayoshi Sakakura, Hideo Takahashi, Tomohiro Numata, Ryuji Inoue, Michisuke Yuzaki, Itaru Hamachi

Nature Chemistry   8 ( 10 ) 958 - 967   2016年

DOI

Optical control of neuronal firing via photo-induced electron transfer in donor–acceptor conjugates.

Yuta Takano, Tomohiro Numata, Kazuto Fujishima, Kazuaki Miyake, Kazuya Nakao, Wesley David Grove, Ryuji Inoue, Mineko Kengaku, Shigeyoshi Sakaki, Yasuo Mori, Tatsuya Murakami, Hiroshi Imahori

Chemical Science ( ROYAL SOC CHEMISTRY )  7 ( 5 ) 3331 - 3337   2016年

A series of porphyrin-fullerene linked molecules has been synthesized to evaluate the effects of substituents and molecular structures on their charge-separation yield and the lifetime of a final charge-separated state in various hydrophilic environments. The selected high-performance molecule effectively achieved depolarization in a plasma cell membrane by visible light as well as two-photon excitation using a near-infrared light laser. Moreover, it was revealed that the depolarization can trigger neuronal firing in rat hippocampal neurons, demonstrating the potential and versatility for controlling cell functions using light.

DOI PubMed

アセトアミノフェンに誘導されるヒト肝ガン細胞死における酸化還元感受性TRPチャネルの役割

ヘバ バドゥー, 香西大輔, 坂口怜子, 沼田朋大, 森泰生

日本薬理学会近畿部会プログラム・要旨集   130th   2016年

J-GLOBAL

Sensitization of H2O2-induced TRPM2 activation and subsequent interleukin-8 (CXCL8) production by intracellular Fe2+ in human monocytic U937 cells

Shunichi Shimizu, Ryo Yonezawa, Takaharu Negoro, Shinichiro Yamamoto, Tomohiro Numata, Masakazu Ishii, Yasuo Mori, Takahiro Toda

INTERNATIONAL JOURNAL OF BIOCHEMISTRY & CELL BIOLOGY ( PERGAMON-ELSEVIER SCIENCE LTD )  68   119 - 127   2015年11月

Transient receptor potential melastatin 2 (TRPM2) is an oxidative stress-sensitive Ca2+-permeable channel. In monocytes/macrophages, H2O2-induced TRPM2 activation causes cell death and/or production of chemokines that aggravate inflammatory diseases. However, relatively high concentrations of H2O2 are required for activation of TRPM2 channels in vitro. Thus, in the present study, factors that sensitize TRPM2 channels to H2O2 were identified and subsequent physiological responses were examined in U937 human monocytes. Temperature increase from 30 degrees C to 37 degrees C enhanced H2O2-induced TRPM2-mediated increase in intracellular free Ca2+ ([Ca2+](i)) in TRPM2-expressing HEK 293 cells (TRPM2/HEK cells). The H2O2-induced TRPM2 activation enhanced by the higher temperature was dramatically sensitized by intracellular Fe2+-accumulation following pretreatment with FeSO4. Thus intracellular Fe2+-accumulation sensitizes H2O2-induced TRPM2 activation at around body temperature. Moreover, intracellular Fe2+-accumulation increased poly(ADP-ribose) levels in nuclei by H2O2 treatment, and the sensitization of H2O2-induced TRPM2 activation were almost completely blocked by poly(ADP-ribose) polymerase inhibitors, suggesting that intracellular Fe2+-accumulation enhances H2O2-induced TRPM2 activation by increase of ADP-ribose production through poly(ADP-ribose) polymerase pathway. Similarly, pretreatment with FeSO4 stimulated H2O2-induced TRPM2 activation at 37 degrees C in U937 cells and enhanced H2O2-induced ERK phosphorylation and interleukin-8 (CXCL8) production. Although the addition of H2O2 to cells under conditions of intracellular Fe2*-accumulation caused cell death, concentration of H2O2 required for CXCL8 production was lower than that resulting in cell death. These results indicate that intracellular Fe2+-accumulation sensitizes TRPM2 channels to H2O2 and subsequently produces CXCL8 at around body temperature. It is possible that sensitization of H2O2-induced TRPM2 channels by Fe2+ may implicated in hemorrhagic brain injury via aggravation of inflammation, since Fe2+ is released by heme degradation under intracerebral hemorrhage. (C) 2015 Elsevier Ltd. All rights reserved.

DOI PubMed

Thermosensitive Ion Channel Activation in Single Neuronal Cells by Using Surface-Engineered Plasmonic Nanoparticles

Hirotaka Nakatsuji, Tomohiro Numata, Nobuhiro Morone, Shuji Kaneko, Yasuo Mori, Hiroshi Imahori, Tatsuya Murakami

ANGEWANDTE CHEMIE-INTERNATIONAL EDITION ( WILEY-V C H VERLAG GMBH )  54 ( 40 ) 11725 - 11729   2015年09月

Controlling cell functions using external photo-responsive nanomaterials has enormous potential for the development of cell-engineering technologies and intractable disease therapies, but the former currently requires genetic modification of the target cells. We present a method using plasma-membrane-targeted gold nanorods (pm-AuNRs) prepared with a cationic protein/lipid complex to activate a thermosensitive cation channel, TRPV1, in intact neuronal cells. Highly localized photothermal heat generation mediated by the pm-AuNRs induced Ca2+ influx solely by TRPV1 activation. In contrast, the use of previously reported cationic AuNRs that are coated with a conventional synthetic polymer also led to photoinduced Ca2+ influx, but this influx resulted from membrane damage. Our method provides an optogenetic platform without the need for prior genetic engineering of the target cells and might be useful for novel TRPV1-targeted phototherapeutic approaches.

DOI PubMed

A screening of TRPC channels activators identifies novel neurotrophic compounds

Sawamura Seishiro, Hatano Masahiko, Sasaki Hajime, Takada Yoshinori, Kuwamura Takashi, Numata Tomohiro, Kiyonaka Shigeki, Hino Kyosuke, Kawamura Tetsuya, Tanikawa Jun, Nakagawa Hiroshi, Nagata Ryu, Inoue Ryuji, Mori Yasuo

JOURNAL OF PHARMACOLOGICAL SCIENCES ( JAPANESE PHARMACOLOGICAL SOC )  128 ( 3 ) S90 - S90   2015年07月

Characterization of redox-sensitive TRP channels in acetaminophen-induced death of human hepatoma cell line

Elsaid Heba A, Numata Tomohiro, Mori Yasuo

JOURNAL OF PHARMACOLOGICAL SCIENCES   128 ( 3 ) S178   2015年07月

Involvement of TRPA1 activation through distinct modifications in oxaliplatin-induced acute peripheral neuropathy

Nakamura Saki, Miyake Takahito, Zhao Meng, Hamano Satoshi, Inoue Keisuke, Takahashi Nobuaki, Numata Tomohiro, Shirakawa Hisashi, Mori Yasuo, Nakagawa Takayuki, Kaneko Shuji

JOURNAL OF PHARMACOLOGICAL SCIENCES   128 ( 3 ) S232   2015年07月

Compromised maturation of GABAergic inhibition underlies abnormal network activity in the hippocampus of epileptic Ca2+ channel mutant mice, tottering

Akito Nakao, Takafumi Miki, Ken Shimono, Hiroaki Oka, Tomohiro Numata, Shigeki Kiyonaka, Kaori Matsushita, Hiroo Ogura, Tetsuhiro Niidome, Jeffrey L. Noebels, Minoru Wakamori, Keiji Imoto, Yasuo Mori

PFLUGERS ARCHIV-EUROPEAN JOURNAL OF PHYSIOLOGY ( SPRINGER )  467 ( 4 ) 737 - 752   2015年04月

Cholinergically induced network activity is a useful analogue of theta rhythms involved in memory processing or epileptiform activity in the hippocampus, providing a powerful tool to elucidate the mechanisms of synchrony in neuronal networks. In absence epilepsy, although its association with cognitive impairments has been reported, the mechanisms underlying hippocampal synchrony remain poorly investigated. Here we simultaneously recorded electrical activities from 64 sites in hippocampal slices of Ca(V)2.1 Ca2+ channel mutant tottering (tg) mice, a well-established mouse model of spontaneous absence epilepsy, to analyze the spatiotemporal pattern of cholinergically induced hippocampal network activity. The cholinergic agonist carbachol induced oscillatory discharges originating from the CA3 region. In tg/tg mice, this hippocampal network activity was characterized by enhanced occupancy of discharges of relatively high frequency (6-10 Hz) compared to the wild type. Pharmacological analyses of slices, patch clamp electrophysiological characterization of isolated neurons, and altered patterns of hippocampal GABA(A) receptor subunit and Cl- transporter messenger RNA (mRNA) transcript levels revealed that this abnormality is attributable to a developmental retardation of GABAergic inhibition caused by immature intracellular Cl- regulation. These results suggest that the inherited Ca(V)2.1 Ca2+ channel mutation leads to developmental abnormalities in Cl- transporter expression and GABA(A) receptor compositions in hippocampal neurons and that compromised maturation of GABAergic inhibition contributes to the abnormal synchrony in the hippocampus of tg absence epileptic mice.

DOI PubMed