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論文 - 三澤　日出巳

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X-ray fluoroscopy-based kinematic analysis of quadrupedal locomotion in slow and fast fatigue-resistant motor neuron-deleted mice.

Ono, A., Inomata, D., Ohgaki, L., Koyama, T., Maeno, A., Misawa, H. Ogihara, N.

Muscle & Nerve 71 257 - 264 2024年12月

研究論文（学術雑誌）, 共著, 責任著者, 査読有り

NDRG1 upregulation by ubiquitin proteasome system dysfunction aggravates neurodegeneration.

Hoshino, T., Mukai, A., Yamashita, H., Misawa, H., Urushitani, M., Tashiro, Y., Matsuzawa, S. Takahashi, R.

Mol. Brain 17 77 2024年10月

研究論文（学術雑誌）, 共著, 査読有り

Type selective ablation of postnatal slow and fast fatigue-resistant motor neurons in mice induces late onset kinetic and postural tremor following fiber-type transition and myopathy.

Misawa, H., Kamishima, K., Koyama, T., Ohgaki, L., Morisaki, Y., Yamanaka, K., Itohara, S., Sawano, S., Mizunoya, W. Ogihara, N.

Exp. Neurol. 376 114772 2024年04月

研究論文（学術雑誌）, 共著, 筆頭著者, 責任著者, 査読有り

Suppression of neuroinflammation by coffee component pyrocatechol via inhibition of NF-κB in microglia.

Murata, T., Tago, K., Miyata, K., Moriwaki, Y., Misawa, H., Kobata, K., Nakazawa, Y., Tamura, H. Funakoshi-Tago, M.

Int. J. Mol. Sci. 25 316 2023年12月

研究論文（学術雑誌）, 共著, 査読有り

Depletion of perivascular macrophages delays ALS disease progression by ameliorating blood-spinal cord barrier impairment in SOD1G93A mice

Adachi K, Miyata K, Chida Y, Hirose M, Morisaki Y, Yamanaka K, Misawa H

Front Cell Neurosci 17 1291673 2023年11月

研究論文（学術雑誌）, 共著, 最終著者, 責任著者, 査読有り

LAG-3 expression in microglia regulated by IFN-γ/STAT1 pathway and metalloproteases

Morisaki Y, Ohshima M, Suzuki H, Misawa H

Front Cell Neurosci 17 1308972 2023年11月

研究論文（学術雑誌）, 共著, 最終著者, 責任著者, 査読有り

GTS-21 enhances regulatory T cell development from T cell receptor-activated human CD4+ T cells exhibiting varied levels of CHRNA7 and CHRFAM7A expression

Mashimo M, Fujii T, Ono S, Moriwaki Y, Misawa H, Azami T, Kasahara T, Kawashima K

Int J Mol Sci 24 12257 2023年07月

研究論文（学術雑誌）, 共著, 査読有り

Lysophosphatidic acid stimulates pericyte migration via LPA receptor 1

Yonezu Y, Tanabe S, Misawa H, Muramatsu R

Biochem Biophys Res Commun 618 61 - 66 2022年08月

研究論文（学術雑誌）, 共著, 査読有り

Involvement of neuronal and muscular Trk-fused gene (TFG) defects in the development of neurodegenerative diseases.

Yamamotoya T, Hasei S, Akasaka Y, Ohata Y, Nakatsu Y, Kanna M, Fujishiro M, Sakoda H, Ono H, Kushiyama A, Misawa H, Asano T

Scientific reports 12 （ 1 ） 1966 2022年02月

研究論文（学術雑誌）, 共著, 査読有り

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Regulation of immune functions by non-neuronal acetylcholine (Ach) via muscarinic and nicotinic ach receptors

Mashimo M., Moriwaki Y., Misawa H., Kawashima K., Fujii T.

International Journal of Molecular Sciences （International Journal of Molecular Sciences） 22 （ 13 ） 2021年07月

研究論文（学術雑誌）, 共著, 査読有り, ISSN 16616596

Acetylcholine (ACh) is the classical neurotransmitter in the cholinergic nervous system. However, ACh is now known to regulate various immune cell functions. In fact, T cells, B cells, and macrophages all express components of the cholinergic system, including ACh, muscarinic, and nicotinic ACh receptors (mAChRs and nAChRs), choline acetyltransferase, acetylcholinesterase, and choline transporters. In this review, we will discuss the actions of ACh in the immune system. We will first briefly describe the mechanisms by which ACh is stored in and released from immune cells. We will then address Ca2+ signaling pathways activated via mAChRs and nAChRs on T cells and B cells, highlighting the importance of ACh for the function of T cells, B cells, and macrophages, as well as its impact on innate and acquired (cellular and humoral) immunity. Lastly, we will discuss the effects of two peptide ligands, secreted lymphocyte antigen-6/urokinase-type plasminogen activator receptor-related peptide-1 (SLURP-1) and hippocampal cholinergic neurostimulating peptide (HCNP), on cholinergic activity in T cells. Overall, we stress the fact that ACh does not function only as a neurotransmitter; it impacts immunity by exerting diverse effects on immune cells via mAChRs and nAChRs.

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Competitive inhibition of the high-affinity choline transporter by tetrahydropyrimidine anthelmintics

Okuda T., Nomura Y., Konishi A., Misawa H.

European Journal of Pharmacology （European Journal of Pharmacology） 898 173986 2021年05月

研究論文（学術雑誌）, 共著, 査読有り, ISSN 00142999

The high-affinity choline transporter CHT1 mediates choline uptake, the rate-limiting and regulatory step in acetylcholine synthesis at cholinergic presynaptic terminals. CHT1-medated choline uptake is specifically inhibited by hemicholinium-3, which is a type of choline analog that acts as a competitive inhibitor. Although the substrate choline and the inhibitor hemicholinium-3 are well-established ligands of CHT1, few potent ligands other than choline analogs have been reported. Here we show that tetrahydropyrimidine anthelmintics, known as nicotinic acetylcholine receptor agonists, act as competitive inhibitors of CHT1. A ligand-dependent trafficking assay in cell lines expressing human CHT1 was designed to search for CHT1 ligands from a collection of biologically active compounds. We found that morantel as well as other tetrahydropyrimidines, pyrantel and oxantel, potently inhibits the high-affinity choline uptake activity of CHT1 in a competitive manner similar to the inhibitor hemicholinium-3. They also inhibit the high-affinity choline transporter from the nematode Caenorhabditis elegans. Finally, tetrahydropyrimidines potently inhibit the high-affinity choline uptake in rat brain synaptosomes at a low micromolar level, resulting in the inhibition of acetylcholine synthesis. The rank order of potency in synaptosomes is as follows: morantel > pyarantel > oxantel (Ki = 1.3, 5.7, and 8.3 μM, respectively). Our results reveal that tetrahydropyrimidine anthelmintics are novel CHT1 ligands that inhibit the high-affinity choline uptake for acetylcholine synthesis in cholinergic neurons.

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Stagnation of glymphatic interstitial fluid flow and delay in waste clearance in the SOD1-G93A mouse model of ALS

Hirose M., Asano M., Watanabe-Matsumoto S., Yamanaka K., Abe Y., Yasui M., Tokuda E., Furukawa Y., Misawa H.

Neuroscience Research （Neuroscience Research） 171 74 - 82 2021年

研究論文（学術雑誌）, 共著, 査読有り, ISSN 01680102

© 2020 The Author(s) Overexpression and mislocalization of aquaporin-4 (AQP4) in the SOD1G93A mouse model of amyotrophic lateral sclerosis (ALS) have previously been reported. However, how alterations of AQP4 affect interstitial bulk flow in the brain and spinal cord, the so-called glymphatic system, is unclear. Here, we report an enhanced accumulation of disease-associated SOD1 species including SOD1 oligomers in SOD1G93A;AQP4−/− mice compared with SOD1G93A mice during ALS disease progression, as analyzed by sandwich ELISA. By directly injecting SOD1 oligomers into the spinal cord parenchyma, we observed a significantly larger delay in clearance of biotinylated or fluorescent-labeled SOD1 oligomers in AQP4−/− mice than in wild-type mice. Furthermore, when we injected the fluorescent-labeled tracer protein ovalbumin into the cisterna magna and analyzed the tracer distribution in the cervical spinal cord, approximately 35 % processing ability was found to be reduced in SOD1G93A mice compared to wild-type mice. These results suggest that the glymphatic system is abnormal and that waste clearance is delayed in SOD1G93A mice.

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Endogenous neurotoxin-like protein Ly6H inhibits alpha7 nicotinic acetylcholine receptor currents at the plasma membrane

Moriwaki Y., Kubo N., Watanabe M., Asano S., Shinoda T., Sugino T., Ichikawa D., Tsuji S., Kato F., Misawa H.

Scientific Reports 10 （ 1 ） 11996 - 11996 2020年12月

研究論文（学術雑誌）, 共著, 査読有り

© 2020, The Author(s). α7 nicotinic acetylcholine receptors (nAChRs) are widely expressed in the central nervous system and regarded as potential therapeutic targets for neurodegenerative conditions, such as Alzheimer’s disease and schizophrenia. Yet, despite the assumed pathophysiological importance of the α7 nAChR, molecular physiological characterization remains poorly advanced because α7 nAChR cannot be properly folded and sorted to the plasma membranes in most mammalian cell lines, thus preventing the analyses in heterologous expression system. Recently, ER-resident membrane protein NACHO was discovered as a strong chaperone for the functional expression of α7 nAChR in non-permissive cells. Ly6H, a brain-enriched GPI-anchored neurotoxin-like protein, was reported as a novel modulator regulating intracellular trafficking of α7 nAChR. In this study, we established cell lines that stably and robustly express surface α7 nAChR by introducing α7 nAChR, Ric-3, and NACHO cDNA into HEK293 cells (Triple α7 nAChR/RIC-3/NACHO cells; TARO cells), and re-evaluated the function of Ly6H. We report here that Ly6H binds with α7 nAChRs on the cell membrane and modulates the channel activity without affecting intracellular trafficking of α7 nAChR.

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Lysophosphatidic acid stimulates pericyte migration via LPA receptor 1.

Yonezu, Y., Tanabe, S., Misawa, H., Muramatsu, R.

Biochem. Biophys. Res. Commun. 618 61 - 66 2020年08月

研究論文（学術雑誌）, 共著, 査読有り

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Behavioral and electrophysiological evidence for a neuroprotective role of aquaporin-4 in the 5xFAD transgenic mice model

Abe Y., Ikegawa N., Yoshida K., Muramatsu K., Hattori S., Kawai K., Murakami M., Tanaka T., Goda W., Goto M., Yamamoto T., Hashimoto T., Yamada K., Shibata T., Misawa H., Mimura M., Tanaka K.F., Miyakawa T., Iwatsubo T., Hata J.I., Niikura T., Yasui M.

Acta Neuropathologica Communications 8 （ 1 ） 67 - 67 2020年05月

研究論文（学術雑誌）, 共著, 査読有り

© 2020 The Author(s). Aquaporin-4 (AQP4) has been suggested to be involved in the pathogenesis of neurodegenerative diseases including Alzheimer's disease (AD), which may be due to the modulation of neuroinflammation or the impairment of interstitial fluid bulk flow system in the central nervous system. Here, we show an age-dependent impairment of several behavioral outcomes in 5xFAD AQP4 null mice. Twenty-four-hour video recordings and computational analyses of their movement revealed that the nighttime motion of AQP4-deficient 5xFAD mice was progressively reduced between 20 and 36 weeks of age, with a sharp deterioration occurring between 30 and 32 weeks. This reduction in nighttime motion was accompanied by motor dysfunction and epileptiform neuronal activities, demonstrated by increased abnormal spikes by electroencephalography. In addition, all AQP4-deficient 5xFAD mice exhibited convulsions at least once during the period of the analysis. Interestingly, despite such obvious phenotypes, parenchymal amyloid β (Aβ) deposition, reactive astrocytosis, and activated microgliosis surrounding amyloid plaques were unchanged in the AQP4-deficient 5xFAD mice relative to 5xFAD mice. Taken together, our data indicate that AQP4 deficiency greatly accelerates an age-dependent deterioration of neuronal function in 5xFAD mice associated with epileptiform neuronal activity without significantly altering Aβ deposition or neuroinflammation in this mouse model. We therefore propose that there exists another pathophysiological phase in AD which follows amyloid plaque deposition and neuroinflammation and is sensitive to AQP4 deficiency.

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Minireview: Divergent roles of α7 nicotinic acetylcholine receptors expressed on antigen-presenting cells and CD4+ T cells in the regulation of T cell differentiation

Mashimo M., Fujii T., Ono S., Moriwaki Y., Misawa H., Kawashima K.

International Immunopharmacology 82 106306 - 106306 2020年05月

研究論文（学術雑誌）, 共著, 査読有り, ISSN 15675769

© 2020 Elsevier B.V. α7 nAChRs expressed on immune cells regulate antigen-specific antibody and proinflammatory cytokine production. Using spleen cells from ovalbumin (OVA)-specific T cell receptor transgenic DO11.10 mice and the α7 nAChR agonist GTS-21, investigation of (1) antigen processing-dependent and (2) -independent, antigen presenting cell (APC)-dependent, naïve CD4+ T cell differentiation, as well as (3) non-specific APC-independent, anti-CD3/CD28 mAbs-induced CD4+ T cell differentiation, revealed the differential roles of α7 nAChRs expressed on T cells and APCs in the regulation of CD4+ T cell differentiation. GTS-21 suppressed OVA-induced antigen processing- and APC-dependent differentiation into regulatory T cells (Tregs) and effector T cells (Th1, Th2 and Th17) without affecting OVA uptake or cell viability. By contrast, GTS-21 upregulated OVA peptide-induced antigen processing-independent T cell differentiation into all lineages. During anti-CD3/CD28 mAbs-induced T cell differentiation in the presence of polarizing cytokines, GTS-21 promoted wild-type T cell differentiation into all lineages, but did not affect α7 nAChR-deficient T cell differentiation. These results demonstrate (1) that α7 nAChRs on APCs downregulate T cell differentiation by inhibiting antigen processing and thereby interfering with antigen presentation; and (2) that α7 nAChRs on T cells upregulate differentiation into Tregs and effector T cells. Thus, the divergent roles of α7 nAChRs on APCs and T cells likely regulate the intensity of immune responses. These findings suggest the possibility of using α7 nAChR agonists to harvest greater numbers of Tregs and Th1 and Th2 cells for adoptive immune therapies for treatment of autoimmune diseases and cancers.

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Oxidative misfolding of Cu/Zn-superoxide dismutase triggered by non-canonical intramolecular disulfide formation

Anzai I., Tokuda E., Handa S., Misawa H., Akiyama S., Furukawa Y.

Free Radical Biology and Medicine （Free Radical Biology and Medicine） 147 187 - 199 2020年02月

研究論文（学術雑誌）, 共著, 査読有り, ISSN 08915849

© 2019 Elsevier Inc. Misfolded Cu/Zn-superoxide dismutase (SOD1) is a pathological species in a subset of amyotrophic lateral sclerosis (ALS). Oxidative stress is known to increase in affected spinal cords of ALS and is thus considered to cause damages on SOD1 leading to the misfolding and aggregation. Despite this, it still remains elusive what triggers misfolding of SOD1 under oxidizing environment. Here, we show that a thiol group of Cys111 in SOD1 is oxidized to a sulfenic acid with hydrogen peroxide and reveal that further dissociation of the bound metal ions from the oxidized SOD1 allows another free Cys residue (Cys6) to nucleophilically attack the sulfenylated Cys111. As a result, an intra-molecular disulfide bond forms between Cys6 and Cys111. Such an abnormal SOD1 with the non-canonical disulfide bond was conformationally extended with significant cytotoxicity as well as high propensity to aggregate. Taken together, we propose a new model of SOD1 misfolding under oxidizing environment, in which formation of the non-canonical intramolecular disulfide bond plays a pivotal role.

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Distinct roles of α7 nAChRs in antigen-presenting cells and CD4+ T cells in the regulation of T cell differentiation

Mashimo M., Komori M., Matsui Y., Murase M., Fujii T., Takeshima S., Okuyama H., Ono S., Moriwaki Y., Misawa H., Kawashima K.

Frontiers in Immunology （Frontiers in Immunology） 10 （ MAY ） 1102 - 1102 2019年

研究論文（学術雑誌）, 共著, 査読有り

Copyright © 2019 Mashimo, Komori, Matsui, Murase, Fujii, Takeshima, Okuyama, Ono, Moriwaki, Misawa and Kawashima. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. It is now apparent that immune cells express a functional cholinergic system and that α7 nicotinic acetylcholine receptors (α7 nAChRs) are involved in regulating T cell differentiation and the synthesis of antigen-specific antibodies and proinflammatory cytokines. Here, we investigated the specific function α7 nAChRs on T cells and antigen presenting cells (APCs) by testing the effect of GTS-21, a selective α7 nAChR agonist, on differentiation of CD4+ T cells from ovalbumin (OVA)-specific TCR transgenic DO11.10 mice activated with OVA or OVA peptide323−339 (OVAp). GTS-21 suppressed OVA-induced antigen processing-dependent development of CD4+ regulatory T cells (Tregs) and effector T cells (Th1, Th2, and Th17). By contrast, GTS-21 up-regulated OVAp-induced antigen processing-independent development of CD4+ Tregs and effector T cells. GTS-21 also suppressed production of IL-2, IFN-γ, IL-4, IL-17, and IL-6 during OVA-induced activation but, with the exception IL-2, enhanced their production during OVAp-induced activation. In addition, during antigen-nonspecific, APC-independent anti-CD3/CD28 antibody-induced CD4+ polyclonal T cell activation in the presence of respective polarizing cytokines, GTS-21 promoted development of all lineages, which indicates that GTS-21 also acts via α7 nAChRs on T cells. These results suggest 1) that α7 nAChRs on APCs suppress CD4+ T cell activation by interfering with antigen presentation through inhibition of antigen processing; 2) that α7 nAChRs on CD4+ T cells up-regulate development of Tregs and effector T cells; and that α7 nAChR agonists and antagonists could be potentially useful agents for immune response modulation and enhancement.

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Identification of mesothelioma-specific sialylated epitope recognized with monoclonal antibody SKM9-2 in a mucin-like membrane protein HEG1.

Reiko Matsuura, Hiroyuki Kaji, Azusa Tomioka, Takashi Sato, Hisashi Narimatsu, Yasuhiro Moriwaki, Hidemi Misawa, Kohzoh Ima, Shoutaro Tsuji.

Scientific Reports 8 14251 2018年09月

研究論文（学術雑誌）, 共著, 査読有り

The anti-mesothelioma mAb SKM9-2 recognizes the sialylated protein HEG homolog 1 (HEG1). HEG1 is a 400 kDa mucin-like membrane protein found on mesothelioma. SKM9-2 can detect mesothelioma more specifically and sensitively than other antibodies against current mesothelioma markers; therefore, SKM9-2 would be likely useful for the precise detection and diagnosis of malignant mesothelioma. In the present study, we investigated the epitope of SKM9-2. We analyzed the binding of SKM9-2 to truncated HEG1 and candidate epitope-fused glycosylphosphatidylinositol-anchor proteins. The epitope of SKM9-2 was identified as an O-glycosylated region, 893-SKSPSLVSLPT-903, in HEG1. An alanine scanning assay of the epitope showed that SKM9-2 bound to a simple epitope in HEG1, and the SKxPSxVS sequence within the epitope was essential for SKM9-2 recognition. Mass spectrometry analysis and lectin binding analysis of soluble epitope peptides indicated that the SKM9-2 epitope, in which Ser897 was not glycosylated, contained two disialylated core 1 O-linked glycan-modified serine residues, Ser893 and Ser900. Neuraminidase treatment analysis also confirmed that the epitope in mesothelioma cells contained a similar glycan modification. The specific detection of mesothelioma with SKM9-2 can thus be performed by the recognition of sialylated glycan modification in the specific region of HEG1.

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Dissociation of blood-brain barrier disruption and disease manifestation in an aquaporin-4-deficient mouse model of amyotrophic lateral sclerosis

Watanabe-Matsumoto Saori, Moriwaki Yasuhiro, Okuda Takashi, Ohara Shinji, Yamanaka Koji, Abe Yoichiro, Yasui Masato, Misawa Hidemi

Neuroscience Research 133 48 - 57 2018年08月

研究論文（学術雑誌）, 共著, 査読有り, ISSN 0168-0102

Aquaporin-4 (AQP4) is abundantly expressed in the central nervous system and is involved in the water balance in the cellular environment. Previous studies have reported that AQP4 expression is upregulated in rat models of amyotrophic lateral sclerosis (ALS), a fatal disease that affects motor neurons in the brain and spinal cord. In this study, we report that astrocytic AQP4 overexpression is evident during the course of disease in the spinal cord of an ALS mouse model, as well as in tissue from patients with ALS. AQP4 overexpression appears to be specifically associated with ALS because it was not induced by other experimental manipulations that produced acute or chronic gliosis. In order to examine the contribution of AQP4 to ALS disease development, we crossed AQP4 knockout mice with a mouse model of ALS. Significant improvement in blood-brain barrier (BBB) permeability was observed in the AQP4-deficient ALS mouse model. However, the time to disease onset and total lifespan were reduced in the AQP4-deficient ALS mouse model. The contradictory results suggest that changes in AQP4 may be context-dependent and further studies are required to understand the precise contribution of brain water balance in ALS.

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SIMPLE binds specifically to PI4P through SIMPLE-like domain and participates in protein trafficking in the trans-Golgi network and/or recycling endosomes

Moriwaki Yasuhiro, Ohno Yuho, Ishii Tomohiro, Takamura Yuki, Kita Yuko, Watabe Kazuhiko, Sango Kazunori, Tsuji Shoutaro, Misawa Hidemi

PLoS One 13 （ 6 ） e0199829 2018年06月

研究論文（学術雑誌）, 共著, ISSN 1932-6203

Small integral membrane protein of the lysosome/late endosome (SIMPLE) is a 161-amino acid cellular protein that contains a characteristic C-terminal domain known as the SIMPLE-like domain (SLD), which is well conserved among species. Several studies have demonstrated that SIMPLE localizes to the trans-Golgi network (TGN), early endosomes, lysosomes, multivesicular bodies, aggresomes and the plasma membrane. However, the amino acid regions responsible for its subcellular localization have not yet been identified. The SLD resembles the FYVE domain, which binds phosphatidylinositol (3)-phosphate (PI3P) and determines the subcellular localization of FYVE domain-containing proteins. In the present study, we have found that SIMPLE binds specifically to PI4P through its SLD. SIMPLE colocalized with PI4P and Rab11, a marker for recycling endosomes (REs, organelles enriched in PI4P) in both the IMS32 mouse Schwann cell line and Hela cells. Sucrose density-gradient centrifugation revealed that SIMPLE co-fractionated with syntaxin-6 (a TGN marker) and Rab11. We have also found that SIMPLE knockdown impeded recycling of transferrin and of transferrin receptor. Our overall results indicate that SIMPLE may regulate protein trafficking physiologically by localizing to the TGN and/or REs by binding PI4P.

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A copper-deficient form of mutant Cu/Zn-superoxide dismutase as an early pathological species in amyotrophic lateral sclerosis

Tokuda Eiichi, Nomura Takao, Ohara Shinji, Watanabe Seiji, Yamanaka Koji, Morisaki Yuta, Misawa Hidemi, Furukawa Yoshiaki

Biochimica et Biophysica Acta - Molecular Basis of Disease 1864 （ 6 ） 2119 - 2130 2018年06月

研究論文（学術雑誌）, 共著, 査読有り, ISSN 0925-4439

Dominant mutations in the gene encoding copper and zinc-binding superoxide dismutase (SOD1) cause amyotrophic lateral sclerosis (ALS). Abnormal accumulation of misfolded SOD1 proteins in spinal motoneurons is a major pathological hallmark in SOD1-related ALS. Dissociation of copper and/or zinc ions from SOD1 has been shown to trigger the protein aggregation/oligomerization in vitro, but the pathological contribution of such metal dissociation to the SOD1 misfolding still remains obscure. Here, we tested the relevance of the metal-deficient SOD1 in the misfolding in vivo by developing a novel antibody (anti-apoSOD), which exclusively recognized mutant SOD1 deficient in metal ions at its copper-binding site. Notably, anti-apoSOD-reactive species were detected specifically in the spinal cords of the ALS model mice only at their early pre-symptomatic stages but not at the end stage of the disease. The cerebrospinal fluid as well as the spinal cord homogenate of one SOD1-ALS patient also contained the anti-apoSOD-reactive species. Our results thus suggest that metal-deficiency in mutant SOD1 at its copper-binding site is one of the earliest pathological features in SOD1-ALS.

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Innate immune adaptor TRIF deficiency accelerates disease progression of ALS mice with accumulation of aberrantly activated astrocytes

Komine Okiru, Yamashita Hirofumi, Fujimori-Tonou Noriko, Koike Masato, Jin Shijie, Moriwaki Yasuhiro, Endo Fumito, Watanabe Seiji, Uematsu Satoshi, Akira Shizuo, Uchiyama Yasuo, Takahashi Ryosuke, Misawa Hidemi, Yamanaka Koji

Cell Death and Differentiation 1 - 17 2018年03月

研究論文（学術雑誌）, 共著, 査読有り, ISSN 1350-9047

There is compelling evidence that glial-immune interactions contribute to the progression of neurodegenerative diseases. The adaptive immune response has been implicated in disease processes of amyotrophic lateral sclerosis (ALS), but it remains unknown if innate immune signaling also contributes to ALS progression. Here we report that deficiency of the innate immune adaptor TIR domain-containing adaptor inducing interferon-β (TRIF), which is essential for certain Toll-like receptor (TLR) signaling cascades, significantly shortens survival time and accelerates disease progression of ALS mice. While myeloid differentiation factor 88 (MyD88) is also a crucial adaptor for most TLR signaling pathways, MyD88 deficiency had only a marginal impact on disease course. Moreover, TRIF deficiency reduced the number of natural killer (NK), NK-T-lymphocytes, and CD8-T cells infiltrating into the spinal cord of ALS mice, but experimental modulation of these populations did not substantially influence survival time. Instead, we found that aberrantly activated astrocytes expressing Mac2, p62, and apoptotic markers were accumulated in the lesions of TRIF-deficient ALS mice, and that the number of aberrantly activated astrocytes was negatively correlated with survival time. These findings suggest that TRIF pathway plays an important role in protecting a microenvironment surrounding motor neurons by eliminating aberrantly activated astrocytes.

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Selective elimination of slow motor neurons in mice progressively induces a kinetic tremor that resembles patients with essential tremor

Ohgaki Lisa, Inomata Daijiro, Koyama Tenkei, Yamanaka Tomoyuki, Itohara Shigeyoshi, Misawa Hidemi

日本薬理学会年会要旨集（公益社団法人日本薬理学会） 2018 （ 0 ） PO4 - 1-82 2018年

VAChT-Cre is a Cre-driver mouse line that can direct DNA recombination in postnatal FR-type (fast-twitch fatigue resistant) or S-type (slow-twitch fatigue resistant) motor neurons (hereinafter referred to as ¨slow motor neurons¨) innervating type-I or -IIa muscle fibers (Fig. 1; Misawa et al., genesis, 54, 568-572, 2016). In order to express cytotoxic substances selectively in slow motor neurons, VAChT-Cre mice were crossbred with NSE-DTA mice in which diphtheria toxin A (DTA) was expressed after Cre-mediated loxP-site excision. The resultant VAChT-Cre;NSE- DTA mice were born normally and grow healthy, but started voluntary shaking around 20 weeks of age. Reduced body weight and hunched back posture in the spine (kyphosis) were observed around this age. Muscular atrophy was evident in ¨red muscle¨ including the soleus muscle, however ¨white muscle¨ such as the extensor digitorum longus muscle was normal. The tremor was kinetic in nature and became progressively worsen with ageing. The tremor was most conspicuous in head and neck.Tremor frequencies and amplitudes were analyzed using an accelerometer (glued to hanging box) followed by fast Fourier transformation (FFT). The power spectrum analysis revealed that the tremor amplitude peaked at 18- to 22-Hz, which was different from tremor frequency induced by a typical tremor-inducing drug harmaline (peaked at 11- to 15-Hz), implying different causes for the tremor oscillation. The observed tremor phenotype in VAChT-Cre;NSE-DTA mice is reminiscent of essential tremor (ET) in humans: the most frequent movement disorders affecting elderly people with unknown cause. Possible mechanisms and sites of tremor origin in VAChT-Cre;NSE-DTA mice and it's relation to ET will be discussed.

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Roles for α7 nicotinic acetylcholine receptors on naïve CD4+ T cells and antigen-presenting cells in regulation of differentiation

Kawashima Koichiro, Mashimo Masato, Fujii Takeshi, Moriwaki Yasuhiro, Misawa Hidemi, Ono Shiro

日本薬理学会年会要旨集（公益社団法人日本薬理学会） 2018 （ 0 ） PO4 - 3-25 2018年

Immune cells such as T cells, B cells, macrophages and dendritic cells have the ability to synthesize acetylcholine (ACh) by choline acetyltransferase (ChAT). Activation of T cells via the T-cell receptor (TCR)-mediated pathways, and macrophages and DCs by LPS enhances ChAT expression and increase ACh synthesis. Expression of various subtypes of muscarinic and nicotinic ACh receptors (nAChRs) in immune cells suggests a role for ACh from the immune cells in regulation of immune function. In fact, α7 nAChR activation down-regulates the synthesis and release of TNF-α in LPS-stimulated macrophages. 　We investigated the effect of α7 nAChR agonist GTS-21 on naïve CD4+ T cell differentiation. Spleen cells from ovalbumin (OVA)-TCR transgenic DO11.10 mice were cultured in the presence or absence of GTS-21and activated with either OVA or OVA-323-339-peptide (OVA-p) for 5 days. Cultured cells were stained with specific fluorescent antibodies against respective markers, and the differentiation into Treg and effector T cells (Th1, Th2 and Th17) was estimated by cytofluorometry. GTS-21 suppressed T cell differentiation into both Treg and T effector cells when activated with OVA, but enhanced the differentiation into Treg and T effector cells when activated with OVA-p. The results suggest that GTS-21 down-regulates antigen presentation by inhibiting either uptake of OVA or processing in the antigen-presenting cells (APCs). However, once T cell differentiation is activated by OVA-p bound to the surface MHC class II molecules, GTS-21 facilitates the differentiation by stimulating α7 nAChRs in the T cells. Secondly, we investigated the effect of GTS-21 on OVA-FITC uptake by APCs, and found no effect on FITC-uptake, suggesting a role for α7 nAChRs in regulation of OVA processing. Thirdly, to confirm the direct effects of GTS-21 on CD4+ T cells, spleen cells from α7 nAChR-deficient (α7-KO) or the wild type (WT) mice were activated independently of APCs by anti-CD3/CD28 mAbs in the presence of GTS-21. GTS-21 facilitated the differentiation into Tregs only in the WT but not in the α7-KO, confirming the role for α7 nAChRs in T cell differentiation. In conclusion, α7 nAChRs play a role in T cell differentiation by acting on APCs and T cells.

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Myelinating cocultures of rodent stem cell line-derived neurons and immortalized Schwann cells

Ishii Tomohiro, Kawakami Emiko, Endo Kentaro, Misawa Hidemi, Watabe Kazuhiko

Neuropathology 37 （ 5 ） 475 - 481 2017年10月

研究論文（学術雑誌）, 共著, 査読有り, ISSN 0919-6544

Myelination is one of the most remarkable biological events in the neuron-glia interactions for the development of the mammalian nervous system. To elucidate molecular mechanisms of cell-to-cell interactions in myelin synthesis in vitro, establishment of the myelinating system in cocultures of continuous neuronal and glial cell lines are desirable. In the present study, we performed co-culture experiments using rat neural stem cell-derived neurons or mouse embryonic stem (ES) cell-derived motoneurons with immortalized rat IFRS1 Schwann cells to establish myelinating cultures between these cell lines. Differentiated neurons derived from an adult rat neural stem cell line 1464R or motoneurons derived from a mouse ES cell line NCH4.3, were mixed with IFRS1 Schwann cells, plated, and maintained in serum-free F12 medium with B27 supplement, ascorbic acid, and glial cell line-derived neurotrophic factor. Myelin formation was demonstrated by electron microscopy at 4 weeks in cocultures of 1464R-derived neurons or NCH4.3-derived motoneurons with IFRS1 Schwann cells. These in vitro coculture systems utilizing the rodent stable stem and Schwann cell lines can be useful in studies of peripheral nerve development and regeneration.

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Expression and function of the cholinergic system in immune cells

Fujii Takeshi, Mashimo Masato, Moriwaki Yasuhiro, Misawa Hidemi, Ono Shiro, Horiguchi Kazuhide, Kawashima Koichiro

Frontiers in Immunology 8 （ SEP ） 1085 2017年09月

研究論文（学術雑誌）, 共著, 査読有り

T and B cells express most cholinergic system components-e.g., acetylcholine (ACh), choline acetyltransferase (ChAT), acetylcholinesterase, and both muscarinic and nicotinic ACh receptors (mAChRs and nAChRs, respectively). Using ChAT^BAC-eGFP transgenic mice, ChAT expression has been confirmed in T and B cells, dendritic cells, and macrophages. Moreover, T cell activation via T-cell receptor/CD3-mediated pathways upregulates ChAT mRNA expression and ACh synthesis, suggesting that this lymphocytic cholinergic system contributes to the regulation of immune function. Immune cells express all five mAChRs (M₁-M₅). Combined M₁/M₅ mAChR-deficient (M₁/M₅-KO) mice produce less antigen-specific antibody than wild-type (WT) mice. Furthermore, spleen cells in M₁/M₅-KO mice produce less tumor necrosis factor (TNF)-a and interleukin (IL)-6, suggesting M₁/M₅ mAChRs are involved in regulating pro-inflammatory cytokine and antibody production. Immune cells also frequently express the α2, α5, α6, α7, α9, and α10 nAChR subunits. α7 nAChR-deficient (α7-KO) mice produce more antigen-specific antibody than WT mice, and spleen cells from α7-KO mice produce more TNF-α and IL-6 than WT cells. This suggests that α7 nAChRs are involved in regulating cytokine production and thus modulate antibody production. Evidence also indicates that nicotine modulates immune responses by altering cytokine production and that α7 nAChR signaling contributes to immunomodulation through modification of T cell differentiation. Together, these findings suggest the involvement of both mAChRs and nAChRs in the regulation of immune function. The observation that vagus nerve stimulation protects mice from lethal endotoxin shock led to the notion of a cholinergic anti-inflammatory reflex pathway, and the spleen is an essential component of this anti-inflammatory reflex. Because the spleen lacks direct vagus innervation, it has been postulated that ACh synthesized by a subset of CD4+ T cells relays vagal nerve signals to α7 nAChRs on splenic macrophages, which downregulates TNF-α synthesis and release, thereby modulating inflammatory responses. However, because the spleen is innervated solely by the noradrenergic splenic nerve, confirmation of an anti-inflammatory reflex pathway involving the spleen requires several more hypotheses to be addressed. We will review and discuss these issues in the context of the cholinergic system in immune cells.

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Formation and spreading of TDP-43 aggregates in cultured neuronal and glial cells demonstrated by time-lapse imaging

Ishii Tomohiro, Kawakami Emiko, Endo Kentaro, Misawa Hidemi, Watabe Kazuhiko

PLoS One 12 （ 6 ） e0179375 2017年06月

研究論文（学術雑誌）, 共著, 査読有り, ISSN 1932-6203

TAR DNA-binding protein 43 (TDP-43) is a main constituent of cytoplasmic aggregates in neuronal and glial cells in cases of amyotrophic lateral sclerosis and frontotemporal lobar degeneration. We have previously demonstrated that adenovirus-transduced artificial TDP-43 cytoplasmic aggregates formation is enhanced by proteasome inhibition in vitro and in vivo. However, the relationship between cytoplasmic aggregate formation and cell death remains unclear. In the present study, rat neural stem cell lines stably transfected with EGFP- or Sirius-expression vectors under the control of tubulin beta III, glial fibrillary acidic protein, or 2′,3′-cyclic nucleotide 3′-phosphodiesterase promoter were differentiated into neurons, astrocytes, and oligodendrocytes, respectively, in the presence of retinoic acid. The differentiated cells were then transduced with adenoviruses expressing DsRedtagged human wild type and C-terminal fragment TDP-43 under the condition of proteasome inhibition. Time-lapse imaging analyses revealed growing cytoplasmic aggregates in the transduced neuronal and glial cells, followed by collapse of the cell. The aggregates remained insoluble in culture media, consisted of sarkosyl-insoluble granular materials, and contained phosphorylated TDP-43. Moreover, the released aggregates were incorporated into neighboring neuronal cells, suggesting cell-to-cell spreading. The present study provides a novel tool for analyzing the detailed molecular mechanisms of TDP-43 proteinopathy in vitro.

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Physiological functions of the cholinergic system in immune cells

Fujii Takeshi, Mashimo Masato, Moriwaki Yasuhiro, Misawa Hidemi, Ono Shiro, Horiguchi Kazuhide, Kawashima Koichiro

Journal of Pharmacological Sciences 134 （ 1 ） 1 - 21 2017年05月

研究論文（学術雑誌）, 共著, ISSN 1347-8613

T and B cells, macrophages and dendritic cells (DCs) all express most of the components necessary for a functional cholinergic system. This includes choline acetyltransferase (ChAT), muscarinic and nicotinic acetylcholine (ACh) receptors (mAChRs and nAChRs, respectively) and acetylcholinesterase (AChE). Immunological activation of T cells up-regulates cholinergic activity, including ChAT and AChE expression. Moreover, toll-like receptor agonists induce ChAT expression in DCs and macrophages, suggesting cholinergic involvement in the regulation of immune function. Immune cells express all five M₁–M₅ mAChR subtypes and several nAChR subtypes, including α7. Modulation of antigen-specific antibody and pro-inflammatory cytokine production in M₁/M₅ mAChR gene-knockout (KO) and α7 nAChR-KO mice further support the idea of a non-neuronal cholinergic system contributing to the regulation of immune function. Evidence also suggests that α7 nAChRs are involved in suppressing DC and macrophage activity, leading to suppression of T cell differentiation into effector T cells. These findings suggest the possibility that immune function could be modulated by manipulating immune cell cholinergic activity using specific agonists and antagonists. Therefore, a fuller understanding of the immune cell cholinergic system should be useful for the development of drugs and therapeutic strategies for the treatment of inflammation-related diseases and cancers.

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麻酔下マウスの脳局所血流に及ぼす橈骨神経電気刺激の影響

内田さえ、鍵谷方子、堀田晴美、渡辺さおり、三澤日出巳

自律神経 54 26 - 33 2017年04月

研究論文（学術雑誌）, 共著, 査読有り

HEG1 is a novel mucin-like membrane protein that serves as a diagnostic and therapeutic target for malignant mesothelioma

Tsuji Shoutaro, Washimi Kota, Kageyama Taihei, Yamashita Makiko, Yoshihara Mitsuyo, Matsuura Rieko, Yokose Tomoyuki, Kameda Yoichi, Hayashi Hiroyuki, Morohoshi Takao, Tsuura Yukio, Yusa Toshikazu, Sato Takashi, Togayachi Akira, Narimatsu Hisashi, Nagasaki Toshinori, Nakamoto Kotaro, Moriwaki Yasuhiro, Misawa Hidemi, Hiroshima Kenzo, Miyagi Yohei, Imai Kohzoh

Scientific Reports 7 45768 2017年03月

研究論文（学術雑誌）, 共著, 査読有り, ISSN 2045-2322

The absence of highly specific markers for malignant mesothelioma (MM) has served an obstacle for its diagnosis and development of molecular-targeting therapy against MM. Here, we show that a novel mucin-like membrane protein, sialylated protein HEG homolog 1 (HEG1), is a highly specific marker for MM. A monoclonal antibody against sialylated HEG1, SKM9-2, can detect even sarcomatoid and desmoplastic MM. The specificity and sensitivity of SKM9-2 to MM reached 99% and 92%, respectively; this antibody did not react with normal tissues. This accurate discrimination by SKM9-2 was due to the recognition of a sialylated O-linked glycan with HEG1 peptide. We also found that gene silencing of HEG1 significantly suppressed the survival and proliferation of mesothelioma cells; this result suggests that HEG1 may be a worthwhile target for function-inhibition drugs. Taken together, our results indicate that sialylated HEG1 may be useful as a diagnostic and therapeutic target for MM.

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A misfolded dimer of Cu/Zn-superoxide dismutase leading to pathological oligomerization in amyotrophic lateral sclerosis

Anzai Itsuki, Tokuda Eiichi, Mukaiyama Atsushi, Akiyama Shuji, Endo Fumito, Yamanaka Koji, Misawa Hidemi, Furukawa Yoshiaki

Protein Science 26 （ 3 ） 484 - 496 2017年03月

研究論文（学術雑誌）, 共著, ISSN 0961-8368

Misfolding of mutant Cu/Zn-superoxide dismutase (SOD1) is a pathological hallmark in a familial form of amyotrophic lateral sclerosis. Pathogenic mutations have been proposed to monomerize SOD1 normally adopting a homodimeric configuration and then trigger abnormal oligomerization of SOD1 proteins. Despite this, a misfolded conformation of SOD1 leading to the oligomerization at physiological conditions still remains ambiguous. Here, we show that, around the body temperature (∼37°C), mutant SOD1 maintains a dimeric configuration but lacks most of its secondary structures. Also, such an abnormal SOD1 dimer with significant structural disorder was prone to irreversibly forming the oligomers crosslinked via disulfide bonds. The disulfide-crosslinked oligomers of SOD1 were detected in the spinal cords of the diseased mice expressing mutant SOD1. We hence propose an alternative pathway of mutant SOD1 misfolding that is responsible for oligomerization in the pathologies of the disease.

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Immunochemical characterization on pathological oligomers of mutant Cu/Zn-superoxide dismutase in amyotrophic lateral sclerosis

Tokuda Eiichi, Anzai Itsuki, Nomura Takao, Toichi Keisuke, Watanabe Masahiko, Ohara Shinji, Watanabe Seiji, Yamanaka Koji, Morisaki Yuta, Misawa Hidemi, Furukawa Yoshiaki

Molecular Neurodegeneration 12 （ 1 ） 2017年01月

研究論文（学術雑誌）, 共著, 査読有り, ISSN 1750-1326

Background: Dominant mutations in Cu/Zn-superoxide dismutase (SOD1) gene cause a familial form of amyotrophic lateral sclerosis (SOD1-ALS) with accumulation of misfolded SOD1 proteins as intracellular inclusions in spinal motor neurons. Oligomerization of SOD1 via abnormal disulfide crosslinks has been proposed as one of the misfolding pathways occurring in mutant SOD1; however, the pathological relevance of such oligomerization in the SOD1-ALS cases still remains obscure. Methods: We prepared antibodies exclusively recognizing the SOD1 oligomers cross-linked via disulfide bonds in vitro. By using those antibodies, immunohistochemical examination and ELISA were mainly performed on the tissue samples of transgenic mice expressing mutant SOD1 proteins and also of human SOD1-ALS cases. Results: We showed the recognition specificity of our antibodies exclusively toward the disulfide-crosslinked SOD1 oligomers by ELISA using various forms of purified SOD1 proteins in conformationally distinct states in vitro. Furthermore, the epitope of those antibodies was buried and inaccessible in the natively folded structure of SOD1. The antibodies were then found to specifically detect the pathological SOD1 species in the spinal motor neurons of the SOD1-ALS patients as well as the transgenic model mice. Conclusions: Our findings here suggest that the SOD1 oligomerization through the disulfide-crosslinking associates with exposure of the SOD1 structural interior and is a pathological process occurring in the SOD1-ALS cases.

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Differential roles of NF-Y transcription factor in ER chaperone expression and neuronal maintenance in the CNS

Yamanaka, T., Tosaki, A., Miyazaki, H., Kurosaka, M., Koike M., Uchiyama Y., Maity, S.N., Misawa, H., Takahashi, R., Shimogori, T., Hattori, N. and Nukina, N.

Sci. Rep. 6 34575 2016年09月

研究論文（学術雑誌）, 査読有り

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Reappraisal of VAChT-Cre: Preference in slow motor neurons innervating type I or IIa muscle fibers

Misawa Hidemi, Inomata Daijiro, Kikuchi Miseri, Maruyama Sae, Moriwaki Yasuhiro, Okuda Takashi, Nukina Nobuyuki, Yamanaka Tomoyuki

Genesis 54 （ 11 ） 568 - 572 2016年09月

研究論文（学術雑誌）, 共著, 査読有り, ISSN 1526-954X

VAChT-Cre.Fast and VAChT-Cre.Slow mice selectively express Cre recombinase in approximately one half of postnatal somatic motor neurons. The mouse lines have been used in various studies with selective genetic modifications in adult motor neurons. In the present study, we crossed VAChT-Cre lines with a reporter line, CAG-Syp/tdTomato, in which synaptophysin-tdTomato fusion proteins are efficiently sorted to axon terminals, making it possible to label both cell bodies and axon terminals of motor neurons. In the mice, Syp/tdTomato fluorescence preferentially co-localized with osteopontin, a recently discovered motor neuron marker for slow-twitch fatigue-resistant (S) and fast-twitch fatigue-resistant (FR) types. The fluorescence did not preferentially co-localize with matrix metalloproteinase-9, a marker for fast-twitch fatigable (FF) motor neurons. In the neuromuscular junctions, Syp/tdTomato fluorescence was detected mainly in motor nerve terminals that innervate type I or IIa muscle fibers. These results suggest that the VAChT-Cre lines are Cre-drivers that have selectivity in S and FR motor neurons. In order to avoid confusion, we have changed the mouse line names from VAChT-Cre.Fast and VAChT-Cre.Slow to VAChT-Cre.Early and VAChT-Cre.Late, respectively. The mouse lines will be useful tools to study slow-type motor neurons, in relation to physiology and pathology.

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Selective expression of osteopontin in ALS-resistant motor neurons is a critical determinant of late phase neurodegeneration mediated by matrix metalloproteinase-9

Morisaki Yuta, Niikura Mamiko, Watanabe Mizuho, Onishi Kosuke, Tanabe Shogo, Moriwaki Yasuhiro, Okuda Takashi, Ohara Shinji, Murayama Shigeo, Takao Masaki, Uchida Sae, Yamanaka Koji, Misawa Hidemi

Scientific Reports 6 27354 2016年06月

研究論文（学術雑誌）, 共著, 査読有り, ISSN 2045-2322

Differential vulnerability among motor neuron (MN) subtypes is a fundamental feature of amyotrophic lateral sclerosis (ALS): fast-fatigable (FF) MNs are more vulnerable than fast fatigue-resistant (FR) or slow (S) MNs. The reason for this selective vulnerability remains enigmatic. We report here that the extracellular matrix (ECM) protein osteopontin (OPN) is selectively expressed by FR and S MNs and ALS-resistant motor pools, whereas matrix metalloproteinase-9 (MMP-9) is selectively expressed by FF MNs. OPN is secreted and accumulated as extracellular granules in ECM in three ALS mouse models and a human ALS patient. In SOD1 G93A mice, OPN/MMP-9 double positivity marks remodeled FR and S MNs destined to compensate for lost FF MNs before ultimately dying. Genetic ablation of OPN in SOD1 G93A mice delayed disease onset but then accelerated disease progression. OPN induced MMP-9 up-regulation via αvβ3 integrin in ChAT-expressing Neuro2a cells, and also induced CD44-mediated astrocyte migration and microglial phagocytosis in a non-cell-autonomous manner. Our results demonstrate that OPN expressed by FR/S MNs is involved in the second-wave neurodegeneration by up-regulating MMP-9 through αvβ3 integrin in the mouse model of ALS. The differences in OPN/MMP-9 expression profiles in MN subsets partially explain the selective MN vulnerability in ALS.

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Neuregulin 1 confers neuroprotection in SOD1-linked amyotrophic lateral sclerosis mice via restoration of C-boutons of spinal motor neurons

Lasiene, J., Komine, O., Fujimori-Tonou, N., Powers, B., Endo, F., Watanabe, S., Shijie, J., Ravits, J., Horner, P., Misawa, H., Yamanaka, K.

Acta Neuropathol. Commun. 4 15 2016年02月

研究論文（学術雑誌）, 共著, 査読有り

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IL-22/STAT3-induced increases in SLURP1 expression within psoriatic lesions exerts antimicrobial effects against Staphylococcus aureus

Moriwaki Yasuhiro, Takada Kiyoko, Nagasaki Toshinori, Kubo Natsuki, Ishii Tomohiro, Kose Kazuaki, Kageyama Taihei, Tsuji Shoutaro, Kawashima Koichiro, Misawa Hidemi

PLoS One 10 （ 10 ） e0140750 2015年10月

研究論文（学術雑誌）, 共著, 査読有り

Background SLURP1 is the causal gene for Mal de Meleda (MDM), an autosomal recessive skin disorder characterized by diffuse palmoplantar keratoderma and transgressive keratosis. Moreover, although SLURP1 likely serves as an important proliferation/differentiation factor in keratinocytes, the possible relation between SLURP1 and other skin diseases, such as psoriasis and atopic dermatitis, has not been studied, and the pathophysiological control of SLURP1 expression in keratinocytes is largely unknown. Objectives Our aim was to examine the involvement of SLURP1 in the pathophysiology of psoriasis using an imiquimod (IMQ)-induced psoriasis model mice and normal human epidermal keratinocytes (NHEKs). Results SLURP1 expression was up-regulated in the skin of IMQ-induced psoriasis model mice. In NHEKs stimulated with the inflammatory cytokines IL-17, IL-22 and TNF-α, which are reportedly expressed in psoriatic lesions, SLURP1 mRNA expression was significantly upregulated by IL-22 but not the other two cytokines. The stimulatory effect of IL-22 was completely suppressed in NHEKs treated with a STAT3 inhibitor or transfected with siRNA targeting STAT3. Because IL-22 induces production of antimicrobial proteins in epithelial cells, the antibacterial activity of SLURP1 was assessed against Staphylococcus aureus (S. aureus), which is known to be associated with disease severity in psoriasis. SLURP1 significantly suppressed the growth of S. aureus. Conclusions These results indicate SLURP1 participates in pathophysiology of psoriasis by regulating keratinocyte proliferation and differentiation, and by suppressing the growth of S. aureus.

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Transcriptional regulation of SLURP2, a psoriasis-associated gene, is under control of IL-22 in the skin

Moriwaki Yasuhiro, Takada Kiyoko, Tsuji Shoutaro, Kawashima Koichiro, Misawa Hidemi

International Immunopharmacology 29 （ 1 ） 71 - 75 2015年02月

研究論文（学術雑誌）, 共著, 査読有り, ISSN 1567-5769

A novel nicotinic acetylcholine (ACh) receptor (nAChR)-mediated transduction pathway, regulating keratinocyte function, has been elucidated in studies of secreted mammalian Ly6/urokinase plasminogen activator receptor-related protein (SLURP)-1 and -2. SLURPs are members of Ly6/neurotoxin superfamily (Ly6SF) of proteins containing the unique three-finger domain in their three-dimensional structure. Some endogenously expressed Ly6SF proteins (such as LYNX1, SLURP-1, and SLURP-2) modulate the function of nAChR, either as allosteric and/or orthosteric modulators, or as antagonists. Although the expression and functions of SLURP-1 and SLURP-2 in keratinocytes are well documented, the expression and the modes of action of LYNX1 in keratinocytes are unknown. Additionally, a particular hybrid transcript, LYNX1-SLURP2, which contains both LYNX1 and SLURP-2 sequences, with unknown function, has been reported. Furthermore, although SLURP2 is a gene strongly induced in psoriatic skin lesions, the mechanisms controlling SLURP2 expression are largely unknown. To better understand the function of nAChRs in keratinocytes, we investigated the expression profiles of LYNX1, LYNX1-SLURP-2, and SLURP-2 in keratinocytes under various inflammatory conditions. We found that keratinocytes express LYNX1 and SLURP2, but not LYNX1-SLURP2, at mRNA and protein levels. IL-22 treatment increased SLURP2 expression in keratinocytes, but this effect was completely abolished by IFN-γ. Furthermore, the IL-22-induced up-regulation of SLURP2 was completely suppressed by the inhibitor or siRNA for STAT3, a major transcriptional factor downstream of IL-22. These findings provide new insights into the nAChR-mediated regulatory mechanism of SLURP-2 expression in keratinocytes.

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Non-neuronal cholinergic system in regulation of immune function with a focus on α7 nAChRs

Kawashima Koichiro, Fujii Takeshi, Moriwaki Yasuhiro, Misawa Hidemi, Horiguchi Kazuhide

International Immunopharmacology 29 （ 1 ） 127 - 134 2015年02月

研究論文（学術雑誌）, 査読有り, ISSN 1567-5769

In 1929, Dale and Dudley described the first reported natural occurrence of acetylcholine (ACh) in an animal's body. They identified this ACh in the spleens of horses and oxen, which we now know suggests possible involvement of ACh in the regulation of lymphocyte activity and immune function. However, the source and function of splenic ACh were left unexplored for several decades. Recent studies on the source of ACh in the blood revealed ACh synthesis catalyzed by choline acetyltransferase (ChAT) in CD4⁺ T cells. T and B cells, macrophages and dendritic cells (DCs) all express all five muscarinic ACh receptor subtypes (mAChRs) and several subtypes of nicotinic AChRs (nAChRs), including α7 nAChRs. Stimulation of these mAChRs and nAChRs by their respective agonists causes functional and biochemical changes in the cells. Using AChR knockout mice, we found that M₁/M₅ mAChR signaling up-regulates IgG₁ and pro-inflammatory cytokine production, while α7 nAChR signaling has the opposite effect. These findings suggest that ACh synthesized by T cells acts in an autocrine/paracrine fashion at AChRs on various immune cells to modulate immune function. In addition, an endogenous allosteric and/or orthosteric α7 nAChR ligand, SLURP-1, facilitates functional development of T cells and increases ACh synthesis via up-regulation of ChAT mRNA expression. SLURP-1 is expressed in CD205⁺ DCs residing in the tonsil in close proximity to T cells, macrophages and B cells. Collectively, these findings suggest that ACh released from T cells along with SLURP-1 regulates cytokine production by activating α7 nAChRs on various immune cells, thereby facilitating T cell development and/or differentiation, leading to immune modulation.

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SIRT1 overexpression ameliorates a mouse model of SOD1-linked amyotrophic lateral sclerosis via HSF1/HSP70i chaperone system

Watanabe Seiji, Ageta-Ishihara Natsumi, Nagatsu Shinji, Takao Keizo, Komine Okiru, Endo Fumito, Miyakawa Tsuyoshi, Misawa Hidemi, Takahashi Ryosuke, Kinoshita Makoto, Yamanaka Koji

Molecular Brain 7 （ 1 ） 62 2014年08月

研究論文（学術雑誌）, 共著, 査読有り

Background: Dominant mutations in superoxide dismutase 1 (SOD1) cause degeneration of motor neurons in a subset of inherited amyotrophic lateral sclerosis (ALS). The pathogenetic process mediated by misfolded and/or aggregated mutant SOD1 polypeptides is hypothesized to be suppressed by protein refolding. This genetic study is aimed to test whether mutant SOD1-mediated ALS pathology recapitulated in mice could be alleviated by overexpressing a longevity-related deacetylase SIRT1 whose substrates include a transcription factor heat shock factor 1 (HSF1), the master regulator of the chaperone system. Results: We established a line of transgenic mice that chronically overexpress SIRT1 in the brain and spinal cord. While inducible HSP70 (HSP70i) was upregulated in the spinal cord of SIRT1 transgenic mice (PrP-Sirt1), no neurological and behavioral alterations were detected. To test hypothetical benefits of SIRT1 overexpression, we crossbred PrP-Sirt1 mice with two lines of ALS model mice: A high expression line that exhibits a severe phenotype (SOD1^G93A-H) or a low expression line with a milder phenotype (SOD1^G93A-L). The Sirt1 transgene conferred longer lifespan without altering the time of symptomatic onset in SOD1^G93A-L. Biochemical analysis of the spinal cord revealed that SIRT1 induced HSP70i expression through deacetylation of HSF1 and that SOD1^G93A-L/PrP-Sirt1 double transgenic mice contained less insoluble SOD1 than SOD1^G93A-L mice. Parallel experiments showed that Sirt1 transgene could not rescue a more severe phenotype of SOD1^G93A-H transgenic mice partly because their HSP70i level had peaked out. Conclusions: The genetic supplementation of SIRT1 can ameliorate a mutant SOD1-linked ALS mouse model partly through the activation of the HSF1/HSP70i chaperone system. Future studies shall include testing potential benefits of pharmacological enhancement of the deacetylation activity of SIRT1 after the onset of the symptom.

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Selective disruption of acetylcholine synthesis in subsets of motor neurons

Lecomte Marie José, Bertolus Chloé, Santamaria Julie, Bauchet Anne Laure, Herbin Marc, Saurini Françoise, Misawa Hidemi, Maisonobe Thierry, Pradat Pierre François, Nosten-Bertrand Marika, Mallet Jacques, Berrard Sylvie

Neurobiology of Disease 65 102 - 111 2014年05月

ISSN 0969-9961

Motor neuron diseases are characterized by the selective chronic dysfunction of a subset of motor neurons and the subsequent impairment of neuromuscular function. To reproduce in the mouse these hallmarks of diseases affecting motor neurons, we generated a mouse line in which ~. 40% of motor neurons in the spinal cord and the brainstem become unable to sustain neuromuscular transmission. These mice were obtained by conditional knockout of the gene encoding choline acetyltransferase (ChAT), the biosynthetic enzyme for acetylcholine. The mutant mice are viable and spontaneously display abnormal phenotypes that worsen with age including hunched back, reduced lifespan, weight loss, as well as striking deficits in muscle strength and motor function. This slowly progressive neuromuscular dysfunction is accompanied by muscle fiber histopathological features characteristic of neurogenic diseases. Unexpectedly, most changes appeared with a 6-month delay relative to the onset of reduction in ChAT levels, suggesting that compensatory mechanisms preserve muscular function for several months and then are overwhelmed. Deterioration of mouse phenotype after ChAT gene disruption is a specific aging process reminiscent of human pathological situations, particularly among survivors of paralytic poliomyelitis. These mutant mice may represent an invaluable tool to determine the sequence of events that follow the loss of function of a motor neuron subset as the disease progresses, and to evaluate therapeutic strategies. They also offer the opportunity to explore fundamental issues of motor neuron biology.

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SLURP-1, an endogenous α7 nicotinic acetylcholine receptor allosteric ligand, is expressed in CD205⁺ dendritic cells in human tonsils and potentiates lymphocytic cholinergic activity

Fujii Takeshi, Horiguchi Kazuhide, Sunaga Hiroshi, Moriwaki Yasuhiro, Misawa Hidemi, Kasahara Tadashi, Tsuji Shoutaro, Kawashima Koichiro

Journal of Neuroimmunology 267 （ 1-2 ） 43 - 49 2014年02月

研究論文（学術雑誌）, 共著, 査読有り, ISSN 0165-5728

Immune cells often express various nicotinic ACh receptor (nAChR) subtypes, including α7 nAChRs, as well as mRNA encoding secreted lymphocyte antigen-6/urokinase-type plasminogen activator receptor-related peptide (SLURP)-1, an endogenous α7 nAChR allosteric ligand. We detected SLURP-1 immunoreactivity in CD205⁺ dendritic cells (DCs) residing in human tonsils. Phytohemagglutinin (PHA, 10μg/ml), a T cell activator, attenuated cell proliferation and increased the ACh content of MOLT-3 human leukemic T cells compared with the vehicle control. Methyllycaconitine (MLA, 100nM), a specific α7 nAChR antagonist, abolished all effects elicited by PHA. Recombinant (r)SLURP-1 (0.5μg/ml) attenuated peripheral blood mononuclear cell proliferation and increased ChAT gene expression and the ACh content in MOLT-3 cells compared with the control, all of which were abolished by MLA. This suggests SLURP-1 activates cholinergic transmission by potentiating ACh synthesis and its action at α7 nAChRs, thereby facilitating functional development of T cells. These findings support the notion that SLURP-1 acts as a key modulator of immune responses.

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Effect of secreted lymphocyte antigen-6/urokinase-type plasminogen activator receptor-related peptide-1 (SLURP-1) on airway epithelial cells

Narumoto Osamu, Niikura Yuichi, Ishii Satoshi, Morihara Hirofumi, Okashiro Saki, Nakahari Takashi, Nakano Takashi, Matsumura Hitoshi, Shimamoto Chikao, Moriwaki Yasuhiro, Misawa Hidemi, Yamashita Naohide, Nagase Takahide, Kawashima Koichiro, Yamashita Naomi

Biochemical and Biophysical Research Communications 438 （ 1 ） 175 - 179 2013年08月

研究論文（学術雑誌）, 共著, ISSN 0006-291X

Acetylcholine (ACh) exerts various anti-inflammatory effects through α7 nicotinic ACh receptors (nAChRs). We have previously shown that secreted lymphocyte antigen-6/urokinase-type plasminogen activator receptor-related peptide-1 (SLURP-1), a positive allosteric modulator of α7 nAChR signaling, is down-regulated both in an animal model of asthma and in human epithelial cells treated with an inflammatory cytokine related to asthma. Our aim of this study was to explore the effect of SLURP-1, signal through α7 nAChR, in the pathophysiology of airway inflammation. Cytokine production was examined using human epithelial cells. Ciliary beat frequency of murine trachea was measured using a high speed camera. The IL-6 and TNF-α production by human epithelial cells was augmented by siRNA of SLURP-1 and α7 nicotinic ACh receptor. The cytokine production was also dose-dependently suppressed by human recombinant SLURP-1 (rSLURP-1). The ciliary beat frequency and amplitude of murine epithelial cells were augmented by PNU282987, a selective α7 nAChR agonist. Those findings suggested that SLURP-1 and stimulus through α7 nicotinic ACh receptors actively controlled asthmatic condition by stimulating ciliary beating and also by suppressing airway inflammation.

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Loss of TDP-43 causes age-dependent progressive motor neuron degeneration

Iguchi Yohei, Katsuno Masahisa, Niwa Jun Ichi, Takagi Shinnosuke, Ishigaki Shinsuke, Ikenaka Kensuke, Kawai Kaori, Watanabe Hirohisa, Yamanaka Koji, Takahashi Ryosuke, Misawa Hidemi, Sasaki Shoichi, Tanaka Fumiaki, Sobue Gen

Brain 136 （ 5 ） 1371 - 1382 2013年05月

研究論文（学術雑誌）, 共著, ISSN 0006-8950

Amyotrophic lateral sclerosis is a devastating, progressive neurodegenerative disease that affects upper and lower motor neurons. Although several genes are identified as the cause of familial cases, the pathogeneses of sporadic forms, which account for 90% of amyotrophic lateral sclerosis, have not been elucidated. Transactive response DNA-binding protein 43 a nuclear protein regulating RNA processing, redistributes to the cytoplasm and forms aggregates, which are the histopathological hallmark of sporadic amyotrophic lateral sclerosis, in affected motor neurons, suggesting that loss-of-function of transactive response DNA-binding protein 43 is one of the causes of the neurodegeneration. To test this hypothesis, we assessed the effects of knockout of transactive response DNA-binding protein 43 in mouse postnatal motor neurons using Cre/loxp system. These mice developed progressive weight loss and motor impairment around the age of 60 weeks, and exhibited degeneration of large motor axon, grouped atrophy of the skeletal muscle, and denervation in the neuromuscular junction. The spinal motor neurons lacking transactive response DNA-binding protein 43 were not affected for 1 year, but exhibited atrophy at the age of 100 weeks; whereas, extraocular motor neurons, that are essentially resistant in amyotrophic lateral sclerosis, remained preserved even at the age of 100 weeks. Additionally, ultra structural analysis revealed autolysosomes and autophagosomes in the cell bodies and axons of motor neurons of the 100-week-old knockout mice. In summary, the mice in which transactive response DNA-binding protein 43 was knocked-out specifically in postnatal motor neurons exhibited an age-dependent progressive motor dysfunction accompanied by neuropathological alterations, which are common to sporadic amyotrophic lateral sclerosis. These findings suggest that transactive response DNA-binding protein 43 plays an essential role in the long term maintenance of motor neurons and that loss-of-function of this protein seems to contribute to the pathogenesis of amyotrophic lateral sclerosis.

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Loss of TDP-43 causes age-dependent progressive motor neuron degeneration

Iguchi Y, Katsuno M, Niwa J, Takagi S, Ishigaki S, Ikenaka K, Kawai K, Watanabe H, Yamanaka K, Takahashi R, Misawa H, Sasaki S, Tanaka F, Sobue G.

Brain 136 （ Pt 5 ） 1371-1382 2013年05月

研究論文（学術雑誌）, 共著, 査読有り

The missing link between long-term stimulation of nicotinic receptors and the increases of acetylcholine release and vasodilation in the cerebral cortex of aged rats

Uchida Sae, Hotta Harumi, Misawa Hidemi, Kawashima Koichiro

Journal of Physiological Sciences 63 （ 2 ） 95 - 101 2013年

研究論文（学術雑誌）, 共著, ISSN 1880-6546

In adult rats (4-9 months), chronic nicotine infusion increases the basal level of acetylcholine (ACh) release in the cerebral cortex and enhances responses of cortical ACh release and cortical vasodilation elicited by nucleus basalis of Meynert (NBM) stimulation. In the present study, we examined whether these effects of nicotine are detected in aged rats. Aged rats (27-30 months) received sustained subcutaneous nicotine (100 μg/kg/h) or saline for 14 days. Under urethane anesthesia, ACh release and regional blood flow in the parietal cortex were measured. The basal level of ACh release in the cerebral cortex was not changed by chronic nicotine. In addition, the magnitudes of ACh release and vasodilation by NBM stimulation were similar between the saline-treated and nicotine-treated groups. The lack of an effect of chronic nicotine in aged rats may be due to a decrease in nicotinic receptors in the cerebral cortex during aging (Nordberg et al., J Neurosci Res 31:103-111, 1992).

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Motor neuron-specific disruption of proteasomes, but not autophagy, replicates amyotrophic lateral sclerosis

Tashiro Yoshitaka, Urushitani Makoto, Inoue Haruhisa, Koike Masato, Uchiyama Yasuo, Komatsu Masaaki, Tanaka Keiji, Yamazaki Maya, Abe Manabu, Misawa Hidemi, Sakimura Kenji, Ito Hidefumi, Takahashia Ryosuke

Journal of Biological Chemistry 287 （ 51 ） 42984 - 42994 2012年12月

研究論文（学術雑誌）, 共著, 査読有り, ISSN 0021-9258

Evidence suggests that protein misfolding is crucially involved in the pathogenesis of amyotrophic lateral sclerosis (ALS). However, controversy still exists regarding the involvement of proteasomes or autophagy in ALS due to previous conflicting results. Here, we show that impairment of the ubiquitin-proteasome system, but not the autophagy-lysosome system in motor neurons replicates ALS in mice. Conditional knock-out mice of the proteasome subunit Rpt3 in a motor neuron-specific manner (Rpt3-CKO) showed locomotor dysfunction accompanied by progressive motor neuron loss and gliosis. Moreover, diverse ALS-linked proteins, including TAR DNA-binding protein 43 kDa (TDP-43), fused in sarcoma (FUS), ubiquilin 2, and optineurin were mislocalized or accumulated in motor neurons, together with other typical ALS hallmarks such as basophilic inclusion bodies. On the other hand, motor neuron-specific knock-out of Atg7, a crucial component for the induction of autophagy (Atg7-CKO), only resulted in cytosolic accumulation of ubiquitin and p62, and no TDP-43 or FUS pathologies or motor dysfunction was observed. These results strongly suggest that proteasomes, but not autophagy, fundamentally govern the development of ALS in which TDP-43 and FUS proteinopathy may play a crucial role. Enhancement of proteasome activity may be a promising strategy for the treatment of ALS.

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Klf4 regulates the expression of slurp1, which functions as an immunomodulatory peptide in the mouse cornea

Swamynathan Sudha, Buela Kristine Ann, Kinchington Paul, Lathrop Kira L., Misawa Hidemi, Hendricks Robert L., Swamynathan Shivalingappa K.

Investigative Ophthalmology and Visual Science 53 （ 13 ） 8433 - 8446 2012年12月

研究論文（学術雑誌）, 共著, 査読有り, ISSN 0146-0404

Purpose. The secreted Ly6/uPAR-related protein-1 (Slurp1), associated with the hyperkeratotic disorder mal de Meleda, is abundantly expressed in corneas. Here, we examine its corneal expression and functions. Methods. Gene expression was quantified by quantitative PCR (qPCR), immunoblots, and immunofluorescent staining. Effect of Kruppel-like factor 4 (Klf4) on Slurp1 promoter was evaluated by chromatin immunoprecipitation (ChIP) and transient transfections. Adenoviral vectors were used to express Slurp1 in corneas. Leukocytic infiltration in bacterial lipopolysaccharide (LPS)-, herpes simplex virus type 1 (HSV-1)-, or adenovirus (serotype 5)-treated mouse corneas was characterized by flow cytometry. Results. Corneal expression of Slurp1 increased sharply upon mouse eyelid opening, concurrent with the elevated expression of Klf4. Slurp1 was significantly decreased in Klf4 conditional null (Klf4CN) corneas that displayed elevated expression of cytokines and cytokine receptors, as well as neutrophil influx consistent with a proinflammatory environment. In additional models of corneal inflammation, Slurp1 expression was abrogated within 24 hours of LPS injection or HSV-1 or adenoviral infection, accompanied by a predominantly neutrophilic infiltrate. Neutrophilic infiltration was enhanced in HSV-1-infected Klf4CN corneas lacking Slurp1. SLURP1 promoter activity was stimulated by KLF4, suppressed by IL-4, IL-13, and TNFα, and unperturbed by IFN-γ. Slurp1 downregulation and neutrophil influx were comparable in HSV-1-infected wild-type (WT) and Ifng-/- mouse corneas. Mouse corneas infected with Slurp1-expressing adenoviral vectors displayed reduced signs of inflammation and restricted neutrophilic infiltration compared with those infected with control vectors. Conclusions. Klf4 regulates the expression of Slurp1, a key immunomodulatory peptide that is abundantly expressed in healthy corneas and is downregulated in proinflammatory conditions.

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Critical roles of acetylcholine and the muscarinic and nicotinic acetylcholine receptors in the regulation of immune function

Kawashima Koichiro, Fujii Takeshi, Moriwaki Yasuhiro, Misawa Hidemi

Life Sciences 91 （ 21-22 ） 1027 - 1032 2012年11月

研究論文（学術雑誌）, 共著, 査読有り, ISSN 0024-3205

Lymphocytes express both muscarinic and nicotinic acetylcholine (ACh) receptors (mAChRs and nAChRs, respectively), and stimulation of mAChRs and nAChRs produces various biochemical and functional changes. Although it has been postulated that parasympathetic cholinergic nerves directly innervate immune cells, no evidence has supported this hypothesis. We measured ACh in the blood of various animal species and determined its localization in T cells using a sensitive and specific radioimmunoassay. Furthermore, we showed that T cells express choline acetyltransferase (ChAT), an ACh synthesizing enzyme. Immunological T cell activation enhances ACh synthesis through the up-regulation of ChAT expression, suggesting lymphocytic cholinergic activity is related to immunological activity. Most immune cells such as T cells, B cells, and monocytes express all five subtypes of mAChRs (M₁-M₅), and various subunits of the nAChR, such as α3, α5, α7, α9, and α10. Studies on serum antibody production in M₁ and M ₅ combined mAChR gene knockout (KO) mice immunized with ovalbumin (OVA) revealed that M₁/M₅ mAChRs up-regulate TNF-α, IFN-γ and IL-6 production in spleen cells, leading to an elevation of serum anti-OVA specific IgG₁. In contrast, studies of nAChR α7 subunit gene KO mice immunized with OVA show that α7 nAChRs down-regulate these proinflammatory cytokines, thereby leading to a reduction of anti-OVA specific IgG₁. Taken together, these findings demonstrate that both mAChRs and nAChRs modulate production of cytokines, such as TNF-α, resulting in a modification of antibody production. These findings support the notion that a non-neuronal cholinergic system is involved in the regulation of immune cell function.

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Localization of acetylcholine-related molecules in the retina: implication of the communication from photoreceptor to retinal pigment epithelium

Matsumoto Hidetaka, Shibasaki Koji, Uchigashima Motokazu, Koizumi Amane, Kurachi Masashi, Moriwaki Yasuhiro, Misawa Hidemi, Kawashima Koichiro, Watanabe Masahiko, Kishi Shoji, Ishizaki Yasuki

PLoS One 7 （ 8 ） e42841 2012年08月

研究論文（学術雑誌）, 共著, 査読有り

It has been long speculated that specific signals are transmitted from photoreceptors to the retinal pigment epithelium (RPE). However, such signals have not been identified. In this study, we examined the retinal expression and localization of acetylcholine-related molecules as putative candidates for these signals. Previous reports revealed that α7 nicotinic acetylcholine receptors (nAChRs) are present in the microvilli of RPE cells that envelope the tips of photoreceptor outer segments (OS). Secreted mammalian leukocyte antigen 6/urokinase-type plasminogen activator receptor-related protein-1 (SLURP-1) is a positive allosteric modulator of the α7 nAChR. Therefore, we first focused on the expression of SLURP-1. SLURP-1 mRNA was expressed in the outer nuclear layer, which is comprised of photoreceptor cell bodies. SLURP-1 immunoreactivity co-localized with rhodopsin and S-opsin in photoreceptor OS, while choline acetyltransferase (ChAT) and high affinity choline transporter (CHT-1) were also expressed in photoreceptor OS. Immunoelectron microscopy identified that the majority of SLURP-1 was localized to the plasma membranes of photoreceptor OS. These results provide evidence that SLURP-1 is synthesized in photoreceptor cell bodies and transported to photoreceptor OS, where SLURP-1 may also be secreted. Our findings suggest that photoreceptor OS communicate via neurotransmitters such as ACh and SLURP-1, while RPE cells might receive these signals through α7 nAChRs in their microvilli.

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Reconciling neuronally and nonneuronally derived acetylcholine in the regulation of immune function

Kawashima Koichiro, Fujii Takeshi, Moriwaki Yasuhiro, Misawa Hidemi, Horiguchi Kazuhide

Annals of the New York Academy of Sciences 1261 （ 1 ） 7 - 17 2012年07月

研究論文（学術雑誌）, 共著, 査読有り, ISSN 0077-8923

Immune cells, including lymphocytes, express muscarinic and nicotinic acetylcholine (ACh) receptors (mAChRs and nAChRs, respectively), and agonist stimulation of these AChRs causes functional and biochemical changes in the cells. The origin of the ACh that acts on immune cell AChRs has remained unclear until recently, however. In 1995, we identified choline acetyltransferase mRNA and protein in human T cells, and found that immunological T cell activation potentiated lymphocytic cholinergic transmission by increasing ACh synthesis and AChR expression. We also found that M₁/M₅ mAChR signaling upregulates IgG₁ and proinflammatory cytokine production, whereas α7 nAChR signaling has the opposite effect. These findings suggest that ACh synthesized by T cells acts as an autocrine and/or paracrine factor via AChRs on immune cells to modulate immune function. In addition, a recently discovered endogenous allosteric α7 nAChR ligand, SLURP-1, also appears to be involved in modulating normal T cell function.

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ニコチン受容体の機能

内田さえ、三澤日出巳

Clinical Neuroscience 30 （ 6 ） 267-270 2012年06月

研究論文（学術雑誌）, 共著

Osteopontin is an alpha motor neuron marker in the mouse spinal cord

Misawa Hidemi, Hara Mayumi, Tanabe Shogo, Niikura Mamiko, Moriwaki Yasuhiro, Okuda Takashi

Journal of Neuroscience Research 90 （ 4 ） 732 - 742 2012年04月

研究論文（学術雑誌）, 共著, 査読有り, ISSN 0360-4012

Motor neurons (MNs) are designated as alpha/gamma and fast/slow based on their target sites and the types of muscle fibers innervated; however, few molecular markers that distinguish between these subtypes are available. Here we report that osteopontin (OPN) is a selective marker of alpha MNs in the mouse spinal cord. OPN was detected in approximately 70% of postnatal choline acetyltransferase (ChAT)-positive MNs with relatively large somas, but not in those with smaller somas. OPN ⁺/ChAT ⁺ MNs were also positive for NeuN, an alpha MN marker, but were negative for Err3, a gamma MN marker. The size distribution of OPN ⁺/ChAT ⁺ cells was nearly identical to that of NeuN ⁺/ChAT ⁺ alpha MNs. Group Ia proprioceptive terminals immunoreactive for vesicular glutamate transporter-1 were selectively detected on the OPN ⁺/ChAT ⁺ cells. OPN staining was also detected at motor axon terminals at neuromuscular junctions, where the OPN ⁺ terminals were positive or negative for SV2A, a marker distinguishing fast/slow motor endplates. Finally, retrograde labeling following intramuscular injection of fast blue indicated that OPN is expressed in both fast and slow MNs. Collectively, our findings show that OPN is an alpha MN marker present in both the soma and the endplates of alpha MNs in the postnatal mouse spinal cord.

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The in vivo contribution of motor neuron TrkB receptors to mutant SOD1 motor neuron disease

Zhai Jinbin, Zhou Weiguo, Li Jian, Hayworth Christopher R., Zhang Lei, Misawa Hidemi, Klein Rudiger, Scherer Steven S., Balice-Gordon Rita J., Kalb Robert Gordon

Human Molecular Genetics 20 （ 21 ） 4116 - 4131 2011年11月

研究論文（学術雑誌）, 共著, 査読有り, ISSN 0964-6906

Brain-derived neurotrophic factor (BDNF) and its receptor tropomyosin-related kinase B (TrkB) are widely expressed in the vertebrate nervous system and play a central role in mature neuronal function. In vitro BDNF/TrkB signaling promotes neuronal survival and can help neurons resist toxic insults. Paradoxically, BDNF/TrkB signaling has also been shown, under certain in vitro circumstances, to render neurons vulnerable to insults. We show here that in vivo conditional deletion of TrkB from mature motor neurons attenuates mutant superoxide dismutase 1 (SOD1) toxicity. Mutant SOD1 mice lacking motor neuron TrkB live a month longer than controls and retain motor function for a longer period, particularly in the early phase of the disease. These effects are subserved by slowed motor neuron loss, persistence of neuromuscular junction integrity and reduced astrocytic and microglial reactivity within the spinal cord. These results suggest that manipulation of BDNF/TrkB signaling might have therapeutic efficacy in motor neuron diseases.

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Substrate-induced internalization of the high-affinity choline transporter

Okuda Takashi, Konishi Asami, Misawa Hidemi, Haga Tatsuya

Journal of Neuroscience 31 （ 42 ） 14989 - 14997 2011年10月

研究論文（学術雑誌）, 共著, 査読有り, ISSN 0270-6474

Cholinergic neurons are endowed with a high-affinity choline uptake system for efficient synthesis of acetylcholine at the presynaptic terminals. The high-affinity choline transporter CHT1 is responsible for choline uptake, the rate-limiting step in acetylcholine synthesis. However, endogenous physiological factors that affect CHT1 expression or function and consequently regulate the acetylcholine synthesis rate are essentially unknown. Here we demonstrate that extracellular substrate decreases the cell-surface expression of CHT1 in rat brain synaptosomes, primary cultures from the basal forebrain, and mammalian cell lines transfected with CHT1. Extracellular choline rapidly decreases cell-surface CHT1 expression by accelerating its internalization, a process that is mediated by a dynamin-dependent endocytosis pathway in HEK293 cells. Specific inhibitor hemicholinium-3 decreases the constitutive internalization rate and thereby increases cell-surface CHT1 expression. We also demonstrate that the constitutive internalization of CHT1 depends on extracellular pH in cultured cells. Our results collectively suggest that the internalization of CHT1 is induced by extracellular substrate, providing a novel feedback mechanism for the regulation of acetylcholine synthesis at the cholinergic presynaptic terminals.

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Sustained subcutaneous infusion of nicotine enhances cholinergic vasodilation in the cerebral cortex induced by stimulation of the nucleus basalis of Meynert in rats

Uchida Sae, Hotta Harumi, Misawa Hidemi, Kawashima Koichiro

European Journal of Pharmacology 654 （ 3 ） 235 - 240 2011年03月

研究論文（学術雑誌）, 共著, 査読有り, ISSN 0014-2999

The present study examined the effects of sustained nicotine exposure on the cholinergic vasodilative system originating in the nucleus basalis of Meynert (NBM) and projecting to the cerebral cortex in rats. Rats received sustained subcutaneous nicotine (100 μg/kg/h) for 14 days. Under urethane anesthesia, the vasodilation response and acetylcholine release in the parietal cortex induced by electrical stimulation of the NBM (10-200 μA) were measured. The basal level of acetylcholine release was significantly higher in nicotine-treated rats than in saline-treated control rats. In the control rats, both the acetylcholine release and blood flow were increased by NBM stimulation in a stimulus intensity-dependent manner, and a threshold of 50 μA. In nicotine-treated rats, the threshold intensity of NBM stimulation producing increases in acetylcholine release and blood flow was reduced to 20 μA. The stimulus intensity-dependent acetylcholine release and vasodilation by NBM stimulation were significantly larger in nicotine-treated rats than in control rats. We conclude that sustained subcutaneous infusion of nicotine enhances cholinergic vasodilative system in the cerebral cortex originating in the NBM.

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Induced loss of ADAR2 engenders slow death of motor neurons from Q/R site-unedited GluR2

Hideyama Takuto, Yamashita Takenari, Suzuki Takeshi, Tsuji Shoji, Higuchi Miyoko, Seeburg Peter H., Takahashi Ryosuke, Misawa Hidemi, Kwak Shin

Journal of Neuroscience 30 （ 36 ） 11917 - 11925 2010年09月

研究論文（学術雑誌）, 共著, 査読有り, ISSN 0270-6474

GluR2 is a subunit of the AMPA receptor, and the adenosine for the Q/R site of its pre-mRNA is converted to inosine (A-to-I conversion) by the enzyme called adenosine deaminase acting on RNA 2 (ADAR2). Failure of A-to-I conversion at this site affects multiple AMPA receptor properties, including the Ca ²⁺ permeability of the receptor-coupled ion channel, thereby inducing fatal epilepsy in mice (Brusa et al., 1995; Feldmeyer et al., 1999). In addition, inefficient GluR2 Q/R site editing is a disease-specific molecular dysfunction found in the motor neurons of sporadic amyotrophic lateral sclerosis (ALS) patients (Kawahara et al., 2004). Here, we generated genetically modified mice (designated as AR2) in which the ADAR2 gene was conditionally targeted in motor neurons using the Cre/loxP system. These AR2 mice showed a decline in motor function commensurate with the slow death of ADAR2-deficient motor neurons in the spinal cord and cranial motor nerve nuclei. Notably, neurons in nuclei of oculomotor nerves, which often escape degeneration in ALS, were not decreased in number despite a significant decrease in GluR2 Q/R site editing. All cellular and phenotypic changes in AR2 mice were prevented when the mice carried endogenous GluR2 alleles engineered to express edited GluR2 without ADAR2 activity (Higuchi et al., 2000). Thus, loss of ADAR2 activity causes AMPA receptor-mediated death of motor neurons.

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Down-regulation of secreted lymphocyte antigen-6/urokinase-type plasminogen activator receptor-related peptide-1 (SLURP-1), an endogenous allosteric α7 nicotinic acetylcholine receptor modulator, in murine and human asthmatic conditions

Narumoto Osamu, Horiguchi Kazuhide, Horiguchi Satomi, Moriwaki Yasuhiro, Takano-Ohmuro Hiromi, Shoji Shunsuke, Misawa Hidemi, Yamashita Naohide, Nagase Takahide, Kawashima Koichiro, Yamashita Naomi

Biochemical and Biophysical Research Communications 398 （ 4 ） 713 - 718 2010年08月

研究論文（学術雑誌）, 共著, 査読有り, ISSN 0006-291X

Whereas acetylcholine (ACh) acts as a bronchoconstrictor and stimulator of mucus secretion from bronchial epithelium, it acts via α7 nicotinic Ach receptors (nAChRs) on macrophages in the airways to exert anti-inflammatory effects by reducing synthesis of pro-inflammatory cytokines, such as tumor necrosis factor-α (TNF-α). Moreover, the effects of ACh are modified by secreted ly-6/urokinase-type plasminogen activator receptor-related peptide-1 (SLURP-1), a positive allosteric modulator of α7 nAChR signaling. Our aim was to explore the roles played by SLURP-1 in the pathophysiology of asthma by assessing SLURP-1 expression in the OVA-sensitized murine asthma model and in cultured human bronchial epithelial cells. Using real-time PCR we found that expression of SLURP-1 mRNA is down-regulated in the lungs of asthmatic model mice, as compared to healthy mice. In addition, immunohistochemical studies confirmed the diminished expression of SLURP-1 in the bronchioles of asthmatic mice, and showed it was due to extensive metaplasia of mucus-secreting cells and the concomitant loss of ciliated epithelial cells. Expression of SLURP-1 mRNA and protein was also significantly down-regulated in human epithelial cells stimulated with the pro-inflammatory cytokine interleukin-13 (IL-13), which is related to asthmatic condition. Thus SLURP-1 appears to be down-regulated in both an animal model of asthma and human epithelial cells treated with an inflammatory cytokine related to asthma. Those findings suggest that diminished expression of SLURP-1 in asthma attenuates its negative regulation of airway inflammation, and that perhaps changes in SLURP-1 expression could serve as a marker of airway damage in asthma.

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miRNA malfunction causes spinal motor neuron disease

Haramati Sharon, Chapnik Elik, Sztainberg Yehezkel, Eilam Raya, Zwang Raaya, Gershoni Noga, McGlinn Edwina, Heiser Patrick W., Wills Anne Marie, Wirguin Itzhak, Rubin Lee L., Misawa Hidemi, Tabin Clifford J., Brown Robert, Chen Alon, Hornstein Eran

Proceedings of the National Academy of Sciences of the United States of America 107 （ 29 ） 13111 - 13116 2010年07月

研究論文（学術雑誌）, 共著, 査読有り, ISSN 0027-8424

Defective RNA metabolism is an emerging mechanism involved in ALS pathogenesis and possibly in other neurodegenerative disorders. Here, we show that microRNA (miRNA) activity is essential for long-term survival of postmitotic spinal motor neurons (SMNs) in vivo. Thus, mice that do not process miRNA in SMNs exhibit hallmarks of spinal muscular atrophy (SMA), including sclerosis of the spinal cord ventral horns, aberrant end plate architecture, and myofiber atrophy with signs of denervation. Furthermore, a neurofilament heavy subunit previously implicated in motor neuron degeneration is specifically up-regulated in miRNA-deficient SMNs. We demonstrate that the heavy neurofilament subunit is a target of miR-9, a miRNA that is specifically down-regulated in a genetic model of SMA. These data provide evidence for miRNA function in SMN diseases and emphasize the potential role of miR-9-based regulatory mechanisms in adult neurons and neurodegenerative states.

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Acetylcholine synthesis and release in NIH3T3 cells coexpressing the high-affinity choline transporter and choline acetyltransferase

Fujii Takeshi, Masai Manabu, Misawa Hidemi, Okuda Takashi, Takada-Takatori Yuki, Moriwaki Yasuhiro, Haga Tatsuya, Kawashima Koichiro

Journal of Neuroscience Research 87 （ 13 ） 3024 - 3032 2009年10月

研究論文（学術雑誌）, 共著, 査読有り, ISSN 0360-4012

Acetylcholine (ACh) is known to be a key neurotransmitter in the central and peripheral nervous systems, but it is also produced in a variety of non-neuronal tissues and cells, including lymphocytes, placenta, amniotic membrane, vascular endothelial cells, keratinocytes, and epithelial cells in the digestive and respiratory tracts. To investigate contribution made by the high-affinity choline transporter (CHT1) to ACh synthesis in both cholinergic neurons and nonneuronal cells, we transfected rat CHT1 cDNA into NIH3T3ChAT cells, a mouse fibroblast line expressing mouse choline acetyltransferase (ChAT), to establish the NIH3T3ChAT 112-1 cell line, which stably expresses both CHT1 and ChAT. NIH3T3ChAT 112-1 cells showed increased binding of the CHT1 inhibitor [³H]hemicholinium-3 (HC-3) and greater [ ³H]choline uptake and ACh synthesis than NIH3T3ChAT 103-1 cells, a CHT1-negative control cell line. HC-3 significantly inhibited ACh synthesis in NIH3T3ChAT 112-1 cells but did not affect synthesis in NIH3T3ChAT 103-1 cells. ACh synthesis in NIH3T3ChAT 112-1 cells was also reduced by amiloride, an inhibitor of organic cation transporters (OCTs) involved in low-affinity choline uptake, and by procaine and lidocaine, two local anesthetics that inhibit plasma membrane phospholipid metabolism. These results suggest that CHT1 plays a key role in ACh synthesis in NIH3T3ChAT 112-1 cells and that choline taken up by OCTs or derived from the plasma membrane is also utilized for ACh synthesis in both cholinergic neurons and nonneuronal cholinergic cells, such as lymphocytes.

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Expression of SLURP-1, an endogenous α7 nicotinic acetylcholine receptor allosteric ligand, in murine bronchial epithelial cells

Horiguchi Kazuhide, Horiguchi Satomi, Yamashita Naomi, Irie Kaoru, Masuda Junya, Takano-Ohmuro Hiromi, Himi Toshiyuki, Miyazawa Mai, Moriwaki Yasuhiro, Okuda Takashi, Misawa Hidemi, Ozaki Hiroshi, Kawashima Koichiro

Journal of Neuroscience Research 87 （ 12 ） 2740 - 2747 2009年09月

研究論文（学術雑誌）, 共著, 査読有り, ISSN 0360-4012

Mammalian secreted lymphocyte antigen-6/urokinase-type plasminogen activator receptor-related peptide-1 (SLURP-1) is a positive allosteric ligand for α7 nicotinic acetylcholine (ACh) receptors (α7 nAChRs) that potentiates responses to ACh and elicits proapoptotic activity in human keratinocytes. Mutations in the gene encoding SLURP-1 have been detected in patients with Mal de Meleda, a rare autosomal recessive skin disorder characterized by transgressive palmoplantar keratoderma. On the basis of these findings, SLURP-1 is postulated to be involved in regulating tumor necrosis factor-α (TNFα) release from keratinocytes and macrophages via α7 nAChR-mediated pathways. In the present study, we assessed SLURP-1 expression in lung tissue from C57BL/6J mice to investigate the functions of SLURP-1 in pulmonary physiology and pathology. Immunohistochemical and in situ hybridization analyses revealed expression of SLURP-1 protein and mRNA, respectively, exclusively in ciliated bronchial epithelial cells. This was supported by Western blotting showing the presence of the 9.5-kDa SLURP-1 protein in whole-lung tissue and trachea. In addition, high-affinity choline transporter (CHT1) was detected in apical regions of bronchial epithelial cells and in neurons located in the lamina propria of the bronchus, suggesting that bronchial epithelial cells are able to synthesize both SLURP-1 and ACh. We also observed direct contact between F4/80-positive macrophages and bronchial epithelial cells and the presence of invading macrophages in close proximity to CHT1-positive nerve elements. Collectively, these results suggest that SLURP-1 contributes to the maintenance of bronchial epithelial cell homeostasis and to the regulation of TNF-α release from macrophages in bronchial tissue.

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Primary sensory neuronal expression of SLURP-1, an endogenous nicotinic acetylcholine receptor ligand

Moriwaki Yasuhiro, Watanabe Yosuke, Shinagawa Tomoe, Kai Miho, Miyazawa Mai, Okuda Takashi, Kawashima Koichiro, Yabashi Atsuko, Waguri Satoshi, Misawa Hidemi

Neuroscience Research 64 （ 4 ） 403 - 412 2009年08月

研究論文（学術雑誌）, 共著, 査読有り, ISSN 0168-0102

Secreted mammalian Ly6/urokinase plasminogen activator receptor-related protein-1 (SLURP-1) is a recently identified, endogenous ligand of the α7 subunit of nicotinic acetylcholine receptors. SLURP-1 is also the causative gene for an autosomal recessive palmoplantar keratoderma, Mal de Meleda. Although the function of SLURP-1 in keratinocyte development and differentiation has been extensively studied, little is known about its role in the nervous system. In the present study, we analyzed SLURP-1 expression in the spinal cord of rats, as a number of studies suggest spinal nicotinic acetylcholine receptors are important modulators of pain transmission. We detected intense SLURP-1 immunoreactivity in the dorsal horn of the spinal cord, especially in lamina I and outer II. In dorsal root ganglia, SLURP-1 immunoreactivity was detected in small- to medium-sized neurons, where in situ hybridization also revealed the presence of SLURP-1 mRNA. Fluorescent labeling of SLURP-1 partially overlapped that of calcitonin-gene related peptide (CGRP) or substance P (SP) in both the spinal cord dorsal horn and glabrous skin, and electron microscopic analysis revealed colocalization of SLURP-1 with SP or CGRP, in large synaptic vesicles in terminals within the superficial layer of the spinal cord. Finally, sciatic nerve axotomy reduced levels of SLURP-1 immunoreactivity in parallel with that of SP and CGRP in the ipsilateral superficial dorsal horn. These findings suggest that SLURP-1 is expressed in a subset of primary peptidergic sensory neurons.

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Role of GluR1 in activity-dependent motor system development

Zhang Lei, Schessl Joachim, Werner Markus, Bonnemann Carsten, Xiong Guoxiang, Mojsilovic-Petrovic Jelena, Zhou Weiguo, Cohen Akiva, Seeburg Peter, Misawa Hidemi, Jayaram Aditi, Personius Kirkwood, Hollmann Michael, Sprengel Rolf, Kalb Robert

Journal of Neuroscience 28 （ 40 ） 9953 - 9968 2008年10月

研究論文（学術雑誌）, 共著, 査読有り, ISSN 0270-6474

Activity-dependent specification of neuronal architecture during early postnatal life is essential for refining the precision of communication between neurons. In the spinal cord under normal circumstances, the AMPA receptor subunit GluR1 is expressed at high levels by motor neurons and surrounding interneurons during this critical developmental period, although the role it plays in circuit formation and locomotor behavior is unknown. Here, we show that GluR1 promotes dendrite growth in a non-cell-autonomous manner in vitro and in vivo. The mal-development of motor neuron dendrites is associated with changes in the pattern of interneuronal connectivity within the segmental spinal cord and defects in strength and endurance. Transgenic expression of GluR1 in adult motor neurons leads to dendrite remodeling and supernormal locomotor function. GluR1 expression by neurons within the segmental spinal cord plays an essential role in formation of the neural network that underlies normal motor behavior.

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Aberrant trafficking of the high-affinity choline transporter in AP-3-deficient mice

Misawa Hidemi, Fujigaya Hirofumi, Nishimura Takashi, Moriwaki Yasuhiro, Okuda Takashi, Kawashima Koichiro, Nakata Kazuko, Ruggiero Alicia M., Blakely Randy D., Nakatsu Fubito, Ohno Hiroshi

European Journal of Neuroscience 27 （ 12 ） 3109 - 3117 2008年06月

研究論文（学術雑誌）, 共著, 査読有り, ISSN 0953-816X

The high-affinity choline transporter (CHT) is expressed in cholinergic neurons and efficiently transported to axon terminals where it controls the rate-limiting step in acetylcholine synthesis. Recent studies have shown that the majority of CHT is unexpectedly localized on synaptic vesicles (SV) rather than the presynaptic plasma membrane, establishing vesicular CHT trafficking as a basis for activity-dependent CHT regulation. Here, we analyse the intracellular distribution of CHT in the adaptor protein-3 (AP-3)-deficient mouse model mocha. In the mocha mouse, granular structures in cell bodies are intensely labelled with CHT antibody, indicating possible deficits in CHT trafficking from the cell body to the axon terminal. Western blot analyses reveal that CHT on SV in mocha mice is decreased by 30% compared with wild-type mice. However, no significant difference in synaptosomal choline uptake activity is detected, consistent with the existence of a large reservoir pool for CHT. To further characterize CHT trafficking, we established a PC12D-CHT cell line. In this line, CHT is found associated with a subpopulation of synaptophysin-positive synaptic-like microvesicles (SLMV). The amounts of CHT detected on SLMV are greatly reduced by treating the cell with agents that halt AP-dependent membrane trafficking. These results demonstrate that APs have important functions for CHT trafficking in neuronal cells.

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L347P PINK1 mutant that fails to bind to Hsp90/Cdc37 chaperones is rapidly degraded in a proteasome-dependent manner

Moriwaki Yasuhiro, Kim Yeon Jeong, Ido Yukari, Misawa Hidemi, Kawashima Koichiro, Endo Shogo, Takahashi Ryosuke

Neuroscience Research 61 （ 1 ） 43 - 48 2008年05月

研究論文（学術雑誌）, 共著, 査読有り, ISSN 0168-0102

Mutation of PTEN-induced kinase 1 (PINK1), which encodes a putative mitochondrial serine/threonine kinase, leads to PARK6, an autosomal recessive form of familial Parkinson's disease. Although the precise function(s) of PINK1 protein is unknown, the recessive inheritance of this form of Parkinson's disease suggests loss of PINK1 function is closely associated with its pathogenesis. Here we report that PINK1 forms a complex with the molecular chaperones Hsp90 and Cdc37/p50 within cells, which appears to enhance its stability. When cells were treated with an Hsp90 inhibitor (geldanamycin or novobiocin), levels of PINK1 were greatly diminished, reflecting its rapid degradation via ubiquitin-proteasome pathway. Similarly, the half-life of a pathogenic PINK1 mutant (L347P) that did not interact with Hsp90 or Cdc37/p50 was only 30 min, whereas that of wild-type PINK1 was 1 h. These results strongly suggest that Hsp90 and Cdc37 are binding partners of PINK1 which regulate its stability.

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Astrocytes as determinants of disease progression in inherited amyotrophic lateral sclerosis

Yamanaka Koji, Chun Seung Joo, Boillee Severine, Fujimori-Tonou Noriko, Yamashita Hirofumi, Gutmann David H., Takahashi Ryosuke, Misawa Hidemi, Cleveland Don W.

Nature Neuroscience 11 （ 3 ） 251 - 253 2008年03月

研究論文（学術雑誌）, 共著, 査読有り, ISSN 1097-6256

Dominant mutations in superoxide dismutase cause amyotrophic lateral sclerosis (ALS), an adult-onset neurodegenerative disease that is characterized by the loss of motor neurons. Using mice carrying a deletable mutant gene, diminished mutant expression in astrocytes did not affect onset, but delayed microglial activation and sharply slowed later disease progression. These findings demonstrate that mutant astrocytes are viable targets for therapies for slowing the progression of non-cell autonomous killing of motor neurons in ALS.

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Renal defects associated with improper polarization of the CRB and DLG polarity complexes in MALS-3 knockout mice

Olsen Olav, Funke Lars, Long Jia Fu, Fukata Masaki, Kazuta Toshinari, Trinidad Jonathan C., Moore Kimberly A., Misawa Hidemi, Welling Paul A., Burlingame Alma L., Zhang Mingjie, Bredt David S.

Journal of Cell Biology 179 （ 1 ） 151 - 164 2007年10月

研究論文（学術雑誌）, 共著, 査読有り, ISSN 0021-9525

Kidney development and physiology require polarization of epithelia that line renal tubules. Genetic studies show that polarization of invertebrate epithelia requires the crumbs, partition-defective-3, and discs large complexes. These evolutionarily conserved protein complexes occur in mammalian kidney; however, their role in renal development remains poorly defined. Here, we find that mice lacking the small PDZ protein mammalian LIN-7c (MALS-3) have hypomorphic, cystic, and fibrotic kidneys. Proteomic analysis defines MALS-3 as the only known core component of both the crumbs and discs large cell polarity complexes. MALS-3 mediates stable assembly of the crumbs tight junction complex and the discs large basolateral complex, and these complexes are disrupted in renal epithelia from MALS-3 knockout mice. Interestingly, MALS-3 controls apico-basal polarity preferentially in epithelia derived from metanephric mesenchyme, and defects in kidney architecture owe solely to MALS expression in these epithelia. These studies demonstrate that defects in epithelial cell polarization can cause cystic and fibrotic renal disease.

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Enhanced serum antigen-specific IgG₁ and proinflammatory cytokine production in nicotinic acetylcholine receptor α7 subunit gene knockout mice

Fujii Yoshihito X., Fujigaya Hirofumi, Moriwaki Yasuhiro, Misawa Hidemi, Kasahara Tadashi, Grando Sergei A., Kawashima Koichiro

Journal of Neuroimmunology 189 （ 1-2 ） 69 - 74 2007年09月

研究論文（学術雑誌）, 共著, 査読有り, ISSN 0165-5728

Human and murine immune cells such as mononuclear leukocytes consisting of mainly T and B cells, bone marrow derived dendritic cells (DCs) and macrophages all express various nicotinic acetylcholine (ACh) receptor (nAChR) subunits. Activated T cells and DCs have the ability to synthesize ACh by choline acetyltransferase, suggesting the role of non-neuronal cholinergic system expressed in immune cells in the regulation of immune cell function. Stimulation of human leukemic T and B cell lines with nicotine causes a transient Ca²⁺-signaling that is antagonized by α-bungarotoxin, suggesting the involvement of α7 subunit. Furthermore, α7 nAChRs have been shown to negatively regulate synthesis and release of tumor necrosis factor (TNF)-α in macrophages. These findings suggest that immune cell function is regulated by its own non-neuronal cholinergic system, at least in part, via α7 nAChR-mediated pathways. In the present study, we tested the role of α7 nAChRs in the regulation of immune function by measuring total serum and antigen-specific IgG₁ and IgM, and production of TNF-α, gamma interferon (IFN-γ) and interleukin (IL)-6 in activated spleen cells of nAChR α7 subunit gene knockout (α7 KO) and wild-type C57BL/6J mice immunized with ovalbumin (OVA). We found that serum levels of total and anti-OVA-specific IgG₁ were significantly elevated in α7 KO mice, though there were no significant differences in serum levels of total and anti-OVA-specific IgM between the two genotypes. Production of TNF-α, IFN-γ and IL-6 in spleen cells was significantly facilitated in α7 KO mice. Expression of AChE mRNA was not different between the two genotypes. These results suggest that α7 nAChRs are involved in the regulation of cytokine production, through which modulates TNF-α, IFN-γ and IL-6 productions, leading to modification of antibody production, but are not involved in expression of cholinergic components in immune cells.

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Diminished antigen-specific IgG₁ and interleukin-6 production and acetylcholinesterase expression in combined M₁ and M₅ muscarinic acetylcholine receptor knockout mice

Fujii Yoshihito X., Tashiro Ayako, Arimoto Kumiko, Fujigaya Hirofumi, Moriwaki Yasuhiro, Misawa Hidemi, Fujii Takeshi, Matsui Minoru, Kasahara Tadashi, Kawashima Koichiro

Journal of Neuroimmunology 188 （ 1-2 ） 80 - 85 2007年08月

研究論文（学術雑誌）, 共著, 査読有り, ISSN 0165-5728

Immunological activation of T cells enhances synthesis of acetylcholine (ACh) and transcription of choline acetyltransferase (ChAT), M₅ muscarinic ACh receptor (mAChR) and acetylcholinesterase (AChE). Stimulation of mAChRs on T and B cells causes oscillating Ca²⁺-signaling and up-regulation of c-fos expression; moreover, M₁ mAChRs play a crucial role in the differentiation of CD8⁺ T cells into cytolytic T lymphocytes. Collectively, these findings suggest that immune cell function is regulated by its own cholinergic system. Bearing that in mind, we tested whether immune function can be regulated via mAChR-mediated pathways by immunizing combined M₁ and M₅ mAChR knockout (M₁/M₅ KO) and wild-type (WT) C57BL/6JJcl mice with ovalbumin (OVA) and measuring serum IgG₁ and IgM 1 wk later. We found that serum levels of total and anti-OVA-specific IgG₁ were significantly lower in M₁/M₅ KO than WT mice, though there was no difference in serum levels of total and anti-OVA-specific IgM between the two genotypes. Secretion of interleukin (IL)-6 from activated spleen cells was significantly reduced in M₁/M₅ KO mice, whereas there was no significant change in gamma interferon secretion. Expression of AChE mRNA was significantly reduced in activated spleen cells from M₁/M₅ KO mice. These results suggest that M₁ and/or M₅ mAChRs are involved in regulating cytokine (e.g., IL-6) production, leading to modulation of antibody class switching from IgM to IgG₁, but are not involved in the initial generation of the antibody response. They also support the notion that a non-neuronal cholinergic system is involved in regulating immune cell function.

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Ubiquitous expression of acetylcholine and its biological functions in life forms without nervous systems

Kawashima Koichiro, Misawa Hidemi, Moriwaki Yasuhiro, Fujii Yoshihito X., Fujii Takeshi, Horiuchi Yoko, Yamada Tomoya, Imanaka Tadayuki, Kamekura Masahiro

Life Sciences 80 （ 24-25 ） 2206 - 2209 2007年05月

研究論文（学術雑誌）, 共著, 査読有り, ISSN 0024-3205

Using a radioimmunoassay (RIA) with high specificity and sensitivity (1 pg/tube) for acetylcholine (ACh), we have been able to measure the ACh content in samples from the bacteria, archaea and eucarya domains of the universal phylogenetic tree. We found detectable levels of ACh to be ubiquitous in bacteria (e.g., Bacillus subtilis), archaea (e.g., Thermococcus kodakaraensis KOD1), fungi (e.g., shiitake mushroom and yeast), plants (e.g., bamboo shoot and fern) and animals (e.g., bloodworm and lugworm). The levels varied considerably, however, with the highest ACh content detected in the top portion of bamboo shoot (2.9 μmol/g), which contained about 80 times that found in rat brain. In addition, using the method of Fonnum, various levels of ACh-synthesizing activity also were detected, a fraction of which was catalyzed by a choline acetyltransferase (ChAT)-like enzyme (sensitive to bromoACh, a selective ChAT inhibitor) in T. kodakaraensis KOD1 (15%), bamboo shoot (91%) and shiitake mushroom (51%), bloodworm (91%) and lugworm (81%). Taken together, these findings demonstrate the ubiquitous expression of ACh and ACh-synthesizing activity among life forms without nervous systems, and support the notion that ACh has been expressed and may be active as a local mediator and modulator of physiological functions since the early beginning of life.

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Expression and function of genes encoding cholinergic components in murine immune cells

Kawashima Koichiro, Yoshikawa Ken, Fujii Yoshihito X., Moriwaki Yasuhiro, Misawa Hidemi

Life Sciences 80 （ 24-25 ） 2314 - 2319 2007年05月

研究論文（学術雑誌）, 共著, ISSN 0024-3205

It is now evident that acetylcholine (ACh) synthesized by choline acetyltransferase (ChAT) and released from T cells during antigen presentation binds to muscarinic and nicotinic ACh receptors (mAChRs and nAChRs, respectively) on T and B cells or dendritic cells, leading to modulation of their function. In the present study, we used reverse transcription-polymerase chain reaction (RT-PCR) to investigate whether mononuclear leukocytes (MNLs), bone marrow-derived dendritic cells (DCs) and macrophages from C57BL/6J mice express components of the cholinergic system. Expression of ChAT mRNA was detected in MNLs activated with ConA and DCs stimulated with LPS, but not in resting MNLs and DCs or in resting and stimulated macrophages. MNLs, DCs and macrophages all expressed mRNAs encoding the five mAChR subtypes (M₁-M₅) and the nAChR α2, α5, α6, α7, α10 and β2 subunits. Expression of VIP mRNA was detected in MNLs and macrophages, but not in DCs. MNLs, DCs and macrophages all expressed VIP receptor-1 (VPAC1) and -2 (VPAC2) mRNAs, as well as mRNAs encoding secreted mammalian Ly-6/urokinase-type plasminogen activator receptor-related protein (SLURP)-1 and SLURP-2, two endogenous nAChR ligands. These results suggest that the lymphocytic cholinergic system is activated by ACh via mAChR- and nAChR-mediated pathways during antigen presentation between T cells and DCs or macrophages, leading to modulation of immune cell function. Moreover, VIP released from both postganglionic cholinergic neurons and immune cells may play a role in the cholinergic anti-inflammatory reflex, acting via VPAC1 and VPAC2 on immune cells.

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Immune system expression of SLURP-1 and SLURP-2, two endogenous nicotinic acetylcholine receptor ligands

Moriwaki Yasuhiro, Yoshikawa Ken, Fukuda Hiromi, Fujii Yoshihito X., Misawa Hidemi, Kawashima Koichiro

Life Sciences 80 （ 24-25 ） 2365 - 2368 2007年05月

研究論文（学術雑誌）, 共著, 査読有り, ISSN 0024-3205

A novel transduction pathway via which apoptosis of keratinocytes is regulated through nicotinic acetylcholine (ACh) receptors (nAChRs) has emerged in studies of secreted mammalian Ly6/urokinase plasminogen-type activator receptor-related protein-1 and-2 (SLURP-1 and SLURP-2, respectively). SLURP-1 reportedly binds to α7 nAChRs and enhances the amplitude of macroscopic currents induced by ACh, leading to facilitation of apoptosis, whereas SLURP-2 binds to α3 nAChRs and prevents apoptosis. These observations prompted us to test whether SLURPs are expressed in immune cells and are involved in the regulation of immune function. We initially used reverse transcription-polymerase chain reaction analysis to characterize the expression profiles of SLURP mRNAs in several murine tissues and organs. Although SLURP-1 mRNA was not expressed in the pancreas, all other tissues and organs tested, including spleen and thymus, expressed both SLURP-1 and SLURP-2 mRNAs. Expression of both mRNAs also was detected in T and B cells, bone marrow-derived dendritic cells (DCs) and macrophages. Moreover, as in keratinocytes, stimulation of MOLT-3 human leukemic T cells with recombinant human SLURP-1 evoked intracellular Ca²⁺ signaling. These results suggest that both SLURP-1 and SLURP-2 are expressed in various immune cells and organs, and that not only ACh but also SLURPs may be involved in regulating lymphocyte function via nAChR-mediated pathways.

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科学を志す者と「清兵衛と瓢箪」.

三澤日出巳.

内藤財団時報（/内藤記念科学振興財団） 78 30 2006年09月

研究論文（学術雑誌）, 単著

Conditional knockout of Mn superoxide dismutase in postnatal motor neurons reveals resistance to mitochondrial generated superoxide radicals

Misawa Hidemi, Nakata Kazuko, Matsuura Junko, Moriwaki Yasuhiro, Kawashima Koichiro, Shimizu Takahiko, Shirasawa Takuji, Takahashi Ryosuke

Neurobiology of Disease 23 （ 1 ） 169 - 177 2006年07月

研究論文（学術雑誌）, 共著, 査読有り, ISSN 0969-9961

Mitochondrial dysfunction and oxidative damage are implicated in the pathogenesis of neurodegenerative disease. Mice deficient in the mitochondrial form of superoxide dismutase (SOD2) die during embryonic or early postnatal development, precluding analysis of a pathological role for superoxide in adult tissue. Here, we generated postnatal motor neuron-specific SOD2 knockouts by crossing mice with floxed SOD2 alleles to VAChT-Cre transgenic mice in which Cre expression is restricted to postnatal somatomotor neurons. SOD2 immunoreactivity was specifically lost in a subset of somatomotor neurons resulting in enhanced superoxide production. Yet extensive histological examination revealed no signs of oxidative damage in animals up to 1 year after birth. However, disorganization of distal nerve axons following injury was accelerated in SOD2-deficient motor neurons. These data demonstrate that postnatal motor neurons are surprisingly resistant to oxidative damage from mitochondrial-derived superoxide radicals, but that such damage may sensitize axons to disorganization following nerve injury.

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Neurotransmitter release regulated by a MALS-liprin-α presynaptic complex

Olsen Olav, Moore Kimberly A., Fukata Masaki, Kazuta Toshinari, Trinidad Jonathan C., Kauer Fred W., Streuli Michel, Misawa Hidemi, Burlingame Alma L., Nicoll Roger A., Bredt David S.

Journal of Cell Biology 170 （ 7 ） 1127 - 1134 2005年09月

研究論文（学術雑誌）, 共著, 査読有り, ISSN 0021-9525

Synapses are highly specialized intercellular junctions organized by adhesive and scaffolding molecules that align presynaptic vesicular release with postsynaptic neurotransmitter receptors. The MALS/Veli-CASK-Mint-1 complex of PDZ proteins occurs on both sides of the synapse and has the potential to link transsynaptic adhesion molecules to the cytoskeleton. In this study, we purified the MALS protein complex from brain and found liprin-α as a major component. Liprin proteins organize the presynaptic active zone and regulate neurotransmitter release. Fittingly, mutant mice lacking all three MALS isoforms died perinatally with difficulty breathing and impaired excitatory synaptic transmission. Excitatory postsynaptic currents were dramatically reduced in autaptic cultures from MALS triple knockout mice due to a presynaptic deficit in vesicle cycling. These findings are consistent with a model whereby the MALS-CASK-liprin-α complex recruits components of the synaptic release machinery to adhesive proteins of the active zone.

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Calcium-permeable AMPA receptors promote misfolding of mutant SOD1 protein and development of amyotrophic lateral sclerosis in a transgenic mouse model

Tateno Minako, Sadakata Hisako, Tanaka Mika, Itohara Shigeyoshi, Shin Ryong Moon, Miura Masami, Masuda Masao, Aosaki Toshihiko, Urushitani Makoto, Misawa Hidemi, Takahashi Ryosuke

Human Molecular Genetics 13 （ 19 ） 2183 - 2196 2004年10月

研究論文（学術雑誌）, 共著, 査読有り, ISSN 0964-6906

Mutant Cu/Zn-superoxide dismutase (SOD1) protein aggregation has been suggested as responsible for amyotrophic lateral sclerosis (ALS), although the operative mediating factors are as yet unestablished. To evaluate the contribution of motoneuronal Ca²⁺ -permeable (GluR2 subunit-lacking) α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)-type glutamate receptors to SOD1-related motoneuronal death, we generated chat-GluR2 transgenic mice with significantly reduced Ca²⁺ -permeability of these receptors in spinal motoneurons. Crossbreeding of the hSOD1^G93A transgenic mouse model of ALS with chat-GluR2 mice led to marked delay of disease onset (19.5%), mortality (14.3%) and the pathological hallmarks such as release of cytochrome c from mitochondria, induction of cox2 and astrogliosis. Subcellular fractionation analysis revealed that unusual SOD1 species first accumulated in two fractions dense with neurofilaments/ glial fibrillary acidic protein/nuclei and mitochondria long time before disease onset, and then concentrated into the former fraction by disease onset. All these processes for unusual SOD1 accumulation were considerably delayed by GluR2 overexpression. Ca²⁺ -influx through atypical motoneuronal AMPA receptors thus promotes a misfolding of mutant SOD1 protein and eventual death of these neurons.

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Ultrastructural localization of high-affinity choline transporter in the rat neuromuscular junction

Nakata Kazuko, Okuda Takashi, Misawa Hidemi

Synapse 53 （ 1 ） 53 - 56 2004年07月

研究論文（学術雑誌）, 共著, 査読有り, ISSN 0887-4476

In cholinergic neurons, Na⁺- and Cl^--dependent, hemicholinium-3-sensitive, high-affinity choline uptake system is thought to be the rate-limiting step in acetylcholine (ACh) synthesis. The system is highly regulated by neuronal activity; the choline uptake is increased by a condition in which ACh release is favored. Here we analyzed the ultrastructural localization of the high-affinity choline transporter (CHT) in the rat neuromuscular junctions with two separate antibodies. The majority (>90%) of immunogold labeling of CHT was observed on synaptic vesicles rather than the presynaptic plasma membrane. Less than 5% of the gold-silver particles were associated with the plasma membrane, and more than 70% of such particles were localized within or in close vicinity to presynaptic active zones. Our morphological data support the recent hypothesis that trafficking of CHT from synaptic vesicles to the plasma membrane couples neuronal activity and choline uptake.

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c-Ret regulates cholinergic properties in mouse sympathetic neurons

Burau K., Stenull I., Huber K., Misawa H., Berse B., Unsicker K., Ernsberger U.

European Journal of Neuroscience 20 （ 2 ） 353 - 362 2004年07月

研究論文（学術雑誌）, 共著, 査読有り, ISSN 0953-816X

The search for signalling systems regulating development of noradrenergic and cholinergic sympathetic neurons is a classical problem of developmental neuroscience. While an essential role of bone morphogenetic proteins for induction of noradrenergic properties is firmly established, factors involved in the development of cholinergic traits in vivo are still enigmatic. Previous studies have shown that the c-ret receptor and cholinergic properties are coexpressed in chick sympathetic neurons. Using in situ hybridization we show now that a loss-of-function mutation of the c-ret receptor in mice dramatically reduces numbers of cells positive for choline acetyltransferase (ChAT) and the vesicular acetylcholine transporter (VAChT) in stellate ganglia of homozygous newborn animals. The number of neurons positive for tyrosine hydroxylase (TH) mRNA, the rate-limiting enzyme of noradrenaline synthesis, is reduced to a smaller degree and expression levels are not detectably altered. Already at embryonic day 16 (E16), ChAT and VAChT-positive cells are affected by the c-ret mutation. At E14, however, ChAT and VAChT mRNAs are detectable at low levels and no difference is observed between wildtype and mutant mice. Our data suggest that c-ret signalling is necessary for the maturation of cholinergic sympathetic neurons but dispensable for de novo induction of ChAT and VAChT expression.

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The LIM homeobox gene, L3/Lhx8, is necessary for proper development of basal forebrain cholinergic neurons

Mori Tetsuji, Yuxing Zhang, Takaki Hiromi, Takeuchi Mayumi, Iseki Ken, Hagino Seita, Kitanaka Jun Ichi, Takemura Motohiko, Misawa Hidemi, Ikawa Masahito, Okabe Masaru, Wanaka Akio

European Journal of Neuroscience 19 （ 12 ） 3129 - 3141 2004年06月

研究論文（学術雑誌）, 共著, 査読有り, ISSN 0953-816X

Basal forebrain cholinergic neurons (BFCNs) are involved in cognitive functions such as learning and memory, and are affected in several neurodegenerative diseases (e.g. Alzheimer's disease). Despite their importance, the molecular mechanisms of their development are not fully elucidated. A recent report demonstrated that some BFCNs in adult rat are positive for L3/Lhx8, a LIM homeobox transcription factor. To examine the function of L3/Lhx8 in the development of BFCNs, we generated L3/Lhx8 gene-disrupted mice. In these mice, cells expressing cholinergic neuron markers, such as choline acetyltransferase, vesicular acetylcholine transporter and p75 low-affinity NGF receptor, were markedly reduced in the basal forebrain, whereas other cholinergic neurons including brain stem and spinal motor neurons expressed the markers. Neurotransmitter phenotypes other than cholinergic in the basal forebrain appeared intact. From these results, we suggested that L3/Lhx8 has a pivotal and specific role in the development and/or maintenance of BFCNs.

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Comparative study of gene expression of cholinergic system-related molecules in the human spinal cord and term placenta

Oda Y., Muroishi Y., Misawa H., Suzuki S.

Neuroscience 128 （ 1 ） 39 - 49 2004年

研究論文（学術雑誌）, 共著, 査読有り, ISSN 0306-4522

By reverse transcription-polymerase chain reaction, Southern blot analysis, direct sequencing, and immunohistochemistry, we studied the expression of cholinergic neuronal markers (choline acetyltransferase [ChAT], vesicular acetylcholine transporter [VAChT], and a high-affinity choline transporter [CHT1]), and gene regulatory molecules (repressor element-1 silencing transcription factor/neuron-restrictive silencer factor [REST/NRSF] and CoREST) in the human spinal cord and term placenta, both of which are well known to contain cells synthesizing acetylcholine. H-type, M-type, N2-type, and R-type ChAT mRNAs, VAChT mRNA, and CHT1 mRNA were detected in the spinal cord, but only H-type, M-type, and N2-type ChAT mRNAs, in the term placenta. REST/NRSF and CoREST were detected in the spinal cord and the placenta, but the amounts of both mRNAs were greater in the placenta than in the spinal cord. Further microdissection analyses revealed that the placental trophoblastic cells contained more REST/NRSF and CoREST transcripts than the spinal large motor neurons. Large motor neurons in the anterior horn of the spinal cord were immunohistochemically stained for ChAT and VAChT. In the placenta, stromal fibroblasts, endothelial cells, and trophoblastic cells of the chorionic villi were positively stained with anti-ChAT antibody but not with anti-VAChT antibody. These findings suggest that transcriptions of the R-type ChAT and VAChT mRNAs are coordinately suppressed in the human term placenta, which might be regulated in part by a REST/NRSF complex that binds to a consensus sequence of repressor element 1/neuron-restrictive silencer element (RE1/NRSE) in the 5′ region upstream from exon R, whereas transcriptions of the H-type, M-type, and N2-type ChAT mRNAs might be independent of control by RE1/NRSE. It is possible that at least two separate regulatory mechanisms of gene expression are present for the human cholinergic gene locus, which might be selected by different combinations of DNA motifs and binding proteins to function in neuronal and non-neuronal cells.

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The crucial role of caspase-9 in the disease progression of a transgenic ALS mouse model

Inoue Haruhisa, Tsukita Kayoko, Iwasato Takuji, Suzuki Yasuyuki, Tomioka Masanori, Tateno Minako, Nagao Masahiro, Kawata Akihiro, Saido Takaomi C., Miura Masayuki, Misawa Hidemi, Itohara Shigeyoshi, Takahashi Ryosuke

EMBO Journal 22 （ 24 ） 6665 - 6674 2003年12月

研究論文（学術雑誌）, 共著, 査読有り, ISSN 0261-4189

Mutant copper/zinc superoxide dismutase (SOD1)-overexpressing transgenic mice, a mouse model for familial amyotrophic lateral sclerosis (ALS), provides an excellent resource for developing novel therapies for ALS. Several observations suggest that mitochondria-dependent apoptotic signaling, including caspase-9 activation, may play an important role in mutant SOD1-related neurodegeneration. To elucidate the role of caspase-9 in ALS, we examined the effects of an inhibitor of X chromosome-linked inhibitor of apoptosis (XIAP), a mammalian inhibitor of caspase-3, -7 and -9, and p35, a baculoviral broad caspase inhibitor that does not inhibit caspase-9. When expressed in spinal motor neurons of mutant SOD1 mice using transgenic techniques, XIAP attenuated disease progression without delaying onset. In contrast, p35 delayed onset without slowing disease progression. Moreover, caspase-9 was activated in spinal motor neurons of human ALS subjects. These data strongly suggest that caspase-9 plays a crucial role in disease progression of ALS and constitutes a promising therapeutic target.

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Hyperproliferation of synapses on spinal motor neurons of Duchenne muscular dystrophy and myotonic dystrophy patients

Nagao Masahiro, Kato Shuichi, Hayashi Hideaki, Misawa Hidemi

Acta Neuropathologica 106 （ 6 ） 557 - 560 2003年12月

研究論文（学術雑誌）, 共著, 査読有り, ISSN 0001-6322

Synapses on the motor neurons of patients with Duchenne muscular dystrophy (DMD) and myotonic dystrophy (MD) were studied immunohistochemically using antibodies against synaptobrevin and synaptophysin. Some motor neurons showed hyperproliferation of synapses on the soma and the proximal dendrites. Hyperproliferated synapses were non-cholinergic, because they were not identified with antibodies against vesicular acetylcholine transporter. Regeneration of motor nerve terminals in patients with muscular diseases may affect reorganization of the subsets of central synapses.

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VAChT-Cre.Fast and VAChT-Cre.Slow: Postnatal expression of Cre recombinase in somatomotor neurons with different onset

Misawa Hidemi, Nakata Kazuko, Toda Kyoko, Matsuura Junko, Oda Yoshio, Inoue Haruhisa, Tateno Minako, Takahashi Ryosuke

Genesis 37 （ 1 ） 44 - 50 2003年09月

研究論文（学術雑誌）, 共著, 査読有り, ISSN 1526-954X

The cholinergic gene locus (CGL) consists of the genes encoding the choline acetyltransferase (ChAT) and the vesicular acetylcholine transporter (VAChT). To establish a cholinergic-specific Cre-expressing mouse, we constructed a transgene expression vector (VAChT-Cre) with 11.3 kb human CGL in which a Cre-IRES-EGFP unit was inserted in the VAChT open reading frame. The activity of Cre, whose expression was driven by the VAChT promoter, was examined by crossing a reporter mouse (CAG-CAT-Z) in which expression of LacZ is activated upon Cre-mediated recombination. Transgenic lines with the VAChT-Cre construct displayed the restricted Cre expression in a subset of cholinergic neurons in the somatomotor nuclei and medial habenular nucleus, but absent in visceromotor and other central and peripheral cholinergic neurons. Cre expression was first observed at postnatal day 7 and later detected in -40-60% of somatomotor neurons. Based on the onset of Cre expression, we generated two mouse lines (two alleles; VAChT-Cre.Fast and VAChT-Cre.Slow) in which Cre expression reaches maximal levels fast and slow, respectively. The use of VAChT-Cre mice should allow us to deliver Cre to a subset of postnatal motor neurons, thereby bypassing lethality and facilitating analysis of gene function in adult motor neurons.

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Identification of a monogenic locus (jams1) causing juvenile audiogenic seizures in mice

Misawa Hidemi, Sherr Elliott H., Lee David J., Chetkovich Dane M., Tan Andrew, Schreiner Christoph E., Bredt David S.

Journal of Neuroscience 22 （ 23 ） 10088 - 10093 2002年12月

研究論文（学術雑誌）, 共著, 査読有り, ISSN 0270-6474

Epilepsy is a debilitating disease with a strong genetic component. Positional cloning has identified a few genes for rare monogenic epilepsy syndromes; however, the genetics of common human epilepsies are too complex to be analyzed easily by current techniques. Mouse models of epilepsy can further this analysis by eliminating genetic background heterogeneity and enabling the production of sufficient numbers of offspring. Here, we report that Black Swiss mice have a heretofore unrecognized specific susceptibility to audiogenic seizures. These seizures are characterized by wild running, loss of righting reflex, and tonic flexion and extension, and are followed by a postictal period. The susceptibility to these seizures is developmentally regulated, peaking at 21 d of age and nearly disappearing by adulthood. Interestingly, both the susceptibility to seizures and their developmental regulation appear unrelated to hearing thresholds in the Black Swiss strain and backcrossed progeny. Genetic mapping and linkage analysis of hybrid mice localize the seizure gene, jams1 (juvenile audiogenic monogenic seizures), to a 1.6 ± 0.5 centimorgan (cM) region on mouse chromosome 10, delimited by the gene basigin (Bsg) and marker D10Mit140. Interestingly, the majority of the critical region is syntenic to a region on human chromosome 19p13.3 implicated in a familial form of juvenile febrile convulsions. Cloning the gene for audiogenic seizures in these mice may provide important insight into the fundamental mechanisms for developmentally regulated human epilepsy syndromes.

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Distribution of the high-affinity choline transporter in the central nervous system of the rat

Misawa H., Nakata K., Matsuura J., Nagao M., Okuda T., Haga T.

Neuroscience 105 （ 1 ） 87 - 98 2001年07月

研究論文（学術雑誌）, 共著, 査読有り, ISSN 0306-4522

In cholinergic nerve terminals, Na⁺- and Cl^--dependent, hemicholinium-3-sensitive, high-affinity choline uptake is thought to be the rate-limiting step in acetylcholine synthesis. The high-affinity choline transporter cDNA responsible for the activity was recently cloned. Here we report production of a highly specific antibody to the high-affinity choline transporter and distribution of the protein in the CNS of the rat. The antibody stained almost all known cholinergic neurons and their terminal fields. High-affinity choline transporter-immunoreactive cell bodies were demonstrated in the olfactory tubercle, basal forebrain complex, striatum, mesopontine complex, medial habenula, cranial nerve motor nuclei, and ventral horn and intermediate zone of the spinal cord. Noticeably, high densities of high-affinity choline transporter-positive axonal fibers and puncta were encountered in many brain regions such as cerebral cortex, hippocampus, amygdala, striatum, several thalamic nuclei, and brainstem. Transection of the hypoglossal nerve resulted in a loss of high-affinity choline transporter immunoreactivity in neurons within the ipsilateral hypoglossal motor nucleus, which paralleled a loss of immunoreactivity to choline acetyltransferase. The antibody also stained brain sections from human and mouse, suggesting cross-reactivity. These results confirm that the high-affinity choline transporter is uniquely expressed in cholinergic neurons and is efficiently transported to axon terminals. The antibody will be useful to investigate possible changes in cholinergic cell bodies and axon terminals in human and rodents under various pathological conditions.

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Cellular mRNA expression of the transcription factor NGFI-B suggests a gene regulatory role in striatal opiate-peptide neurons

Bäckman Cristina, Hoffer Barry J., Misawa Hidemi, Morales Marisela

Brain Research 903 （ 1-2 ） 26 - 32 2001年06月

研究論文（学術雑誌）, 共著, 査読有り, ISSN 0006-8993

Previous studies have shown that NGFI-B mRNA is highly expressed in the adult striatum. In the present study we analyzed the anatomical distribution of NGFI-B mRNA within this brain region as well as the degree of co-existence of NGFI-B with different striatal markers in the adult brain. NGFI-B mRNA levels were found to be significantly higher within the dorsomedial portion of the striatum as compared to the ventrolateral striatum. This distribution pattern was maintained throughout the rostro-caudal axis of the striatum. Double in situ hybridization studies showed that striatal NGFI-B mRNA colocalized with a subset of preproenkephalin and prodynorphin positive spiny neurons within the dorsomedial striatum; 22-28% of all opiate-peptide positive cells co-expressed NGFI-B mRNA. NGFI-B did not colocalize with striatal aspiny interneurons expressing choline acetyl transferase mRNA or those containing the calcium-binding protein parvalbumin. The pattern of NGFI-B mRNA expression within different striatal spiny projecting neurons suggests that this transcription factor may have a direct effect on the function of different striatal efferent pathways.

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Contrasting Localizations of MALS/LIN-7 PDZ Proteins in Brain and Molecular Compensation in Knockout Mice

Misawa Hidemi, Kawasaki Yoshimi, Mellor Jack, Sweeney Neal, Jo Kiwon, Nicoll Roger A., Bredt David S.

Journal of Biological Chemistry 276 （ 12 ） 9264 - 9272 2001年03月

研究論文（学術雑誌）, 共著, 査読有り, ISSN 0021-9258

Proteins containing PDZ (postsynaptic density-95, discs large, zonula occludens) domains play a general role in recruiting receptors and enzymes to specific synaptic sites. In Caenorhabditis elegans, a complex of three PDZ proteins, LIN-2/7/10, mediates basolateral targeting of a receptor tyrosine kinase. Homologs of these LIN proteins have also been identified in higher organisms, and here we analyze the MALS/Veli (mammalian LIN-7/vertebrate homolog of LIN-7) proteins in brain. Immunohistochemical staining and in situ hybridization show that MALS occur differentially in discrete populations of neurons throughout the brain. Most neurons express only one MALS protein, although some cells contain two or even all three MALS isoforms. At the subcellular level, MALS proteins are found in both dendritic and axonal locations, suggesting that they may regulate processes at both pre- and postsynaptic sites. Targeted disruption of MALS-1 and MALS-2 does not yield a detectable phenotype, and hippocampal synaptic function and plasticity are intact in the MALS-1/2 double knockouts. Interestingly, MALS-3 protein is dramatically induced in the MALS-1/2 double knockouts, implying that dynamic changes in protein expression may play an important regulatory role for this family of synaptic PDZ proteins.

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A novel function of synapsin II in neurotransmitter release

Sugiyama Takashi, Shinoe Toru, Ito Yoko, Misawa Hidemi, Tojima Takuro, Ito Etsuro, Yoshioka Tohru

Molecular Brain Research 85 （ 1-2 ） 133 - 143 2000年12月

研究論文（学術雑誌）, 共著, 査読有り, ISSN 0169-328X

Although synapsin has been localized to presynaptic structures, its function remains poorly understood. In the present study, we investigated the presynaptic function of synapsin II using a synaptic vesicle recycling process using synapsin-II-overexpressing NG108-15 cells. Western blot analysis with antibodies for synaptic-vesicle-associated protein indicated that the number of synaptic vesicles was approximately doubled in synapsin II transfectants as reported previously. In differentiated synapsin-II-overexpressing and control cells, the application of high potassium induced strong intracellular calcium elevation along neurites and varicosities after differentiation and a weak calcium rise in the cell bodies. The uptake and release of the fluorescent dye FM1-43 revealed that synaptic vesicle recycling in synapsin-II-transfected cells occurred with the same kinetics in the cell body and neuritic varicosities. Furthermore, the area labeled with FM1-43 fluorescence in the synapsin-II-transfected cells was approximately twice as much as in control cells after stimulation, and ATP released after synaptic vesicle fusion with the plasma membrane in synapsin-II-expressing cells was significantly elevated relative to controls. The number of synaptic vesicles paralleled the amount of transmitter released from the cells leading to the conclusion that the number of releasable synaptic vesicles were increased by synapsin II transfection into NG108-15 cells, suggesting that synapsin II may have a role in the regulation of synaptic vesicle number in presynapse-like structures in NG108-15 cells.

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Aberrant splicing of human Cu/Zn superoxide dismutase (SOD1) RNA transcripts

Kawata A., Kato S., Shimizu T., Hayashi H., Hirai S., Misawa H., Takahashi R.

NeuroReport 11 （ 12 ） 2649 - 2653 2000年08月

研究論文（学術雑誌）, 共著, 査読有り, ISSN 0959-4965

Two abnormal SOD 1 mRNAs, exon 2-skipping and exon 2 and 3-skipping species, were identified from occipital brain tissue of sporadic amyotrophic lateral sclerosis (ALS) patients carrying no mutations in the SOD 1 gene. Both transcripts were ubiquitously expressed in non-neuronal as well as neuronal tissues from a subject without neurological diseases. The expression pattern did not show disease specificity or lesional selectivity associated with ALS. Transient expression studies revealed weak expression of the proteins derived from the exon 2-skipping SOD 1 cDNA in a cell-free translation system but not in cells. The putative abnormal SOD 1 protein may accumulate and exert toxic effects on motor neurons in ALS when the proteolytic system is disturbed by aging or some causal factors. (C) 2000 Lippincott Williams and Wilkins.

Transfection analysis of functional roles of complexin I and II in the exocytosis of two different types of secretory vesicles

Itakura Makoto, Misawa Hidemi, Sekiguchi Mariko, Takahashi Seiichi, Takahashi Masami

Biochemical and Biophysical Research Communications 265 （ 3 ） 691 - 696 1999年11月

研究論文（学術雑誌）, 共著, 査読有り, ISSN 0006-291X

Classical neurotransmitters such as γ-aminobutyric acid and glutamate are released from synaptic nerve terminals by exocytosis of synaptic vesicles. PC12 cells also have SSVs capable of storing acetylcholine (ACh). A novel method to examine the effect of transient transfection of any gene of interest on the exocytosis of SSVs was developed. The transfection of choline acetyltransferase (ChAT) into PC12 cells which have lost ACh synthesizing activity resulted in the accumulation of a substantial amount of ACh. Synthesized ACh was released in Ca²⁺-dependent manner. Release was thought to occur by an exocytosis of SSVs because: (1) release was abolished by treating the cells with vesamicol, a specific inhibitor of the vesicular ACh transporter (VAChT) localizing specifically in SSVs; and (2) the release was further increased by cotransfecting rat VAChT with the ChAT. By means of this method, we showed that overexpression of complexin I or II with ChAT markedly suppressed high-K⁺-dependent ACh release of SSVs.

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Constitutive expression of mRNA for the same choline acetyltransferase as that in the nervous system, an acetylcholine-synthesizing enzyme, in human leukemic T-cell lines

Fujii Takeshi, Tajima Sonoko, Yamada Shin, Watanabe Yoshihiro, Sato Kazuo Z., Matsui Marika, Misawa Hidemi, Kasahara Tadashi, Kawashima Koichiro

Neuroscience Letters 259 （ 2 ） 71 - 74 1999年01月

研究論文（学術雑誌）, 共著, 査読有り, ISSN 0304-3940

Both muscarinic and nicotinic acetylcholine (ACh) receptors are known to be present on the surface of lymphocytes. We have shown that variable amounts of ACh are detectable in the blood of various mammals including humans, and a major portion of blood ACh is localized in circulating mononuclear leukocytes in humans. In order to investigate which types of blood cell are the source of ACh in human blood, expression of mRNA for choline acetyltransferase (CHAT, EC 2.3.1.6), which catalyzes ACh synthesis, was analyzed using human leukemic cell lines as models of lymphocytes and the reverse transcription- polymerase chain reaction (RT-PCR) method. We observed that mRNA for the same ChAT as that in the nervous system is expressed constitutively in all the T- cell lines tested, but not in B-, pre-lymphoma or monocytic cell lines. Furthermore, only T-cell lines showed high ACh-synthesizing activities and intracellular ACh contents. These results suggest that the major portion of ACh in the circulating blood originates from T-lymphocytes.

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Identification and transgenic analysis of a murine promoter that targets cholinergic neuron expression

Naciff Jorge M., Behbehani Michael M., Misawa Hidemi, Dedman John R.

Journal of Neurochemistry 72 （ 1 ） 17 - 28 1999年

研究論文（学術雑誌）, 共著, ISSN 0022-3042

Choline acetyltransferase (ChAT) is a specific phenotypic marker of cholinergic neurons. Previous reports showed that different upstream regions of the ChAT gene are necessary for cell type-specific expression of reporter genes in cholinergic cell lines. The identity of the mouse ChAT promoter region controlling the establishment, maintenance, and plasticity of the cholinergic phenotype in vivo is not known. We characterized a promoter region of the mouse ChAT gene in transgenic mice, using β-galactosidase (LacZ) as a reporter gene. A 3,402-bp segment from the 5'-untranslated region of the mouse ChAT gene (from -3,356 to +46, +1 being the translation initiation site) was sufficient to direct the expression of LacZ to selected neurons of the nervous system; however, it did not provide complete cholinergic specificity. A larger fragment (6,417 bp, from -6,371 to +46) of this region contains the requisite regulatory elements that restrict expression of the LacZ reporter gene only in cholinergic neurons of transgenic mice. This 6.4-kb DNA fragment encompasses 633 bp of the 5'- flanking region of the mouse vesicular acetylcholine transporter (VAChT), the entire open reading frame of the VAChT gene, contained within the first intron of the ChAT gene, and sequences upstream of the start coding sequences of the ChAT gene. This promoter will allow targeting of specific gene products to cholinergic neurons to evaluate the mechanisms of diseases characterized by dysfunction of cholinergic neurons and will be valuable in design strategies to correct those disorders.

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Loss of cholinergic synapses on the spinal motor neurons of amyotrophic lateral sclerosis

Nagao Masahiro, Misawa Hidemi, Kato Shuichi, Hirai Shunsaku

Journal of Neuropathology and Experimental Neurology 57 （ 4 ） 329 - 333 1998年04月

研究論文（学術雑誌）, 共著, 査読有り, ISSN 0022-3069

The expression of vesicular acetylcholine transporter (VAChT) was examined immunohistochemically in the cholinergic synapses on the spinal motor neuron of the patient with sporadic amyotrophic lateral sclerosis (SALS). VAChT immunoreactive synapses were depleted on surviving motor neurons in SALS, while synaptophysin immunoreactivity was undiminished on the same neurons. This discrepancy suggests that in SALS, loss of cholinergic input on lower motor neurons is an early event, and may be part of the cause of death of those motor neurons.

Acetylcholine synthesis and muscarinic receptor subtype mRNA expression in T-lymphocytes

Kawashima K., Fujii T., Watanabe Y., Misawa H.

Life Sciences 62 （ 17-18 ） 1701 - 1705 1998年03月

研究論文（学術雑誌）, 共著, 査読有り, ISSN 0024-3205

We used a sensitive and specific radioimmunoassay for acetylcholine (ACh), and detected significant amounts of ACh in the blood of various mammals, including humans. About 60% of human blood ACh was localized in mononuclear leukocytes. Human leukemic T-cell lines, used as T-lymphocyte models, contained both ACh and choline acetyltransferase (ChAT) activity. Furthermore, ChAT mRNA and protein were detected in the T-cell line MOLT-3. Phytohemagglutinin, a T-cell activator, increased both synthesis and release of ACh by MOLT-3 cells. Muscarinic receptor subtype mRNA expression was confirmed in various T-cell lines. These findings indicate that ACh synthesized by ChAT in T-lymphocytes acts on the muscarinic receptors on lymphocytes in autocrine and/or paracrine pathways and suggest that ACh in blood functions as a modulator of T-cell-dependent immune responses.

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Induction of choline acetyltransferase mRNA in human mononuclear leukocytes stimulated by phytohemagglutinin, a T-cell activator

Fujii Takeshi, Yamada Shin, Watanabe Yoshihiro, Misawa Hidemi, Tajima Sonoko, Fujimoto Kazuko, Kasahara Tadashi, Kawashima Koichiro

Journal of Neuroimmunology 82 （ 1 ） 101 - 107 1998年02月

研究論文（学術雑誌）, 共著, 査読有り, ISSN 0165-5728

The induction of mRNA for choline acetyltransferase (CHAT), which catalyzes acetylcholine (ACh) synthesis was investigated in human mononuclear leukocytes (MNL) stimulated by phytohemagglutinin (PHA), a T-cell activator, using the reverse transcription-polymerase chain reaction. Stimulation of MNL by PHA induced the expression of ChAT mRNA, and potentiated ACh synthesis. ChAT mRNA induction required more time than the induction of interleukin-2 mRNA. Expression of the gene encoding the vesicular ACh transporter, which mediates ACh transport in cholinergic neurons, was not observed in PHA- stimulated MNL, suggesting that the mechanisms controlling ACh release from T-lymphocytes differ from those in cholinergic neurons. These findings demonstrate that activation of T-lymphocytes up-regulates ACh synthesis in the blood, and suggest that ACh plays an important role as a neuroimmunomodulator besides its role as a neurotransmitter.

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Changes of expression levels of choline acetyltransferase and vesicular acetylcholine transporter mRNAs after transection of the hypoglossal nerve in adult rats

Matsuura Junko, Ajiki Kyoko, Ichikawa Tomoyuki, Misawa Hidemi

Neuroscience Letters 236 （ 2 ） 95 - 98 1997年10月

研究論文（学術雑誌）, 共著, 査読有り, ISSN 0304-3940

Acetylcholine, synthesized in the cytoplasm of cholinergic neurons by choline acetyltransferase (CHAT), is packaged in synaptic vesicles by vesicular acetylcholine transporter (VAChT). The entire VAChT gene has been reported to be located within the first intron of the ChAT gene. In order to examine whether or not ChAT and VAChT transcription may be coordinately regulated, the levels of ChAT and VAChT mRNAs in hypoglossal neurons were analyzed by in situ hybridization following transection of the hypoglossal nerve in adult rats. After unilateral transection, the levels of expression of ChAT and VAChT mRNAs were dramatically reduced in the ipsilateral hypoglossal nucleus I week after the surgery. However the expression of both mRNAs gradually recovered thereafter. These results suggest that the transcription of the two cholinergic genes is tightly linked in motor neurons.

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Evidence for active acetylcholine metabolism in human amniotic epithelial cells

Sakuragawa Norio, Misawa Hidemi, Ohsugi Keiko, Kakishita Kouji, Ishii Takashi, Thangavel Ramasamy, Tohyama Jun, Elwan Mohamed, Yokoyama Yasunobu, Okuda Osamu, Arai Hajime, Ogino Ikuko, Sato Kiyoshi

Neuroscience Letters 232 （ 1 ） 53 - 56 1997年08月

研究論文（学術雑誌）, 共著, 査読有り, ISSN 0304-3940

Human amniotic epithelial (HAE) cells have been used for allotransplantation in patients with lysosomal storage disease due to lack of expression of HLA antigens. Previously, we have reported the expression of differentiation markers for both neural stem cells, and neuron and glial cells. In the present study, we investigated the presence of choline acetyltransferase (CHAT) and acetylcholine (ACh) in HAE cells using different experimental approaches. Cultured HAE cells showed strong immunoreactivity against ChAT antibody. ChAT activity in primary cells was 24.9 ±8.5 pmol/mg protein/h. Using HPLC with electrochemical detection, ACh was detected in both cell incubation media and cell pellets indicating that these cells synthesize and release ACh in a time-dependent manner. Additional confirmation of this hypothesis was gained from the data obtained from RT- PCR and Western blot analyses which revealed the expression of ChAT mRNA and ChAT protein, respectively, in HAE cells. Results of the present study suggest that HAE cells can possibly be applied for intracerebral allografting to treat neurologic diseases in which cholinergic neurons are damaged.

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Localization of two cholinergic markers, choline acetyltransferase and vesicular acetylcholine transporter in the central nervous system of the rat

Ichikawa Tomoyuki, Ajiki Kyoko, Matsuura Junko, Misawa Hidemi

Journal of Chemical Neuroanatomy 13 （ 1 ） 23 - 39 1997年06月

研究論文（学術雑誌）, 共著, 査読有り, ISSN 0891-0618

Choline acetyltransferase (ChAT) and vesicular acetylcholine transporter (VAChT) are proteins that are required for cholinergic neurotransmission. Present knowledge concerning the organization of cholinergic structures has been derived primarily from immunohistochemistry for ChAT. In the present study, we investigated the distribution of mRNAs and the corresponding proteins for ChAT and VAChT by in situ hybridization histochemistry and immunohistochemistry. The patterns of distribution of perikarya containing ChAT mRNA, ChAT protein, VAChT mRNA and VAChT protein were similar in most regions, and co-localization in the same neuron of mRNAs for ChAT and VAChT, that of ChAT mRNA and ChAT protein, and that of VAChT mRNA and VAChT protein were demonstrated. However, in the cerebral cortex and hypothalamus, ChAT-immunoreactive perikarya were present, but they did not contain mRNAs for ChAT and VAChT, and VAChT protein. On the other hand, in the cerebellum, Purkinje cell bodies contained VAChT mRNA and VAChT protein, but they did not contain either ChAT mRNA or ChAT protein. Axon bundles were clearly revealed by immunohistochemistry for ChAT, but they were not detected by that for VAChT. Both ChAT and VAChT antibodies revealed preterminal axons and terminal-like structures. In the forebrain, they were present in the olfactory bulb, nucleus of the lateral olfactory tract, olfactory tubercle, lateral septal nucleus, amygdala, hippocampus, neocortex, caudate-putamen, thalamus and median eminence of the hypothalamus. In the brainstem, they were localized in the superior colliculus, interpeduncular nucleus and some cranial nerve motor nuclei, and further in the ventral horn of the spinal cord. These results indicate strongly that ChAT and VAChT are expressed in most of the cholinergic neurons, and that immunohistochemistry for VAChT is as useful to detect cholinergic terminal fields as that for ChAT.

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Characterization of the 5′-noncoding region of the mouse choline acetyltransferase gene in transgenic mice

Naciff J. M., Behbehani M. M., Misawa H., Dedman J. R.

FASEB Journal 11 （ 3 ） 1997年

研究論文（学術雑誌）, 共著, 査読有り, ISSN 0892-6638

Choline acetyltransferase (ChAT) is a specific phenotypic marker of cholinergic neurons. Previous reports showed that different upstream regions of the ChAT gene are necessary for cell-type specific expression of reporter genes in cholinergic cell lines. The identity of the mouse ChAT promoter region controlling the establishment, maintenance, and plasticity of the cholinergic phenotype in vivo is not known. We characterized the promoter region of the mouse ChAT gene in transgenic mice, using β-galactosidase (β-gal) as a reporter. A 3401-bp segment from the 5′-untranslated region of the mouse ChAT gene was sufficient to direct the expression of β-gal to selected neurons of the nervous system (NS); it did not provide cholinergic specificity A larger fragment (∼6 Kb) of this region encompassing most of the coding sequence for the putative mouse vesicular acetylcholine transporter (VAChT) contained within the first intron of the ChAT gene, directs correct developmental and tissue-specific expression to cholinergic cells in transgenic mice. This fragment targeted β-gal expression only to cholinergic regions of the NS of transgenic mice, including basal forebrain, striatum, cortex, brain stem, and exclusively to α-motor neurons in the ventral horn of the spinal cord. This ChAT promoter will allow targeting of specific gene products to cholinergic neurons, in order to develop animal models for human diseases where injury and degeneration of chotinergic neurons is involved.

Human choline acetyltransferase mRNAs with different 5'-region produce a 69-kDa major translation product

Misawa H., Matsuura J., Oda Y., Takahashi R., Deguchi T.

Molecular Brain Research 44 （ 2 ） 323 - 333 1997年

研究論文（学術雑誌）, 共著, 査読有り, ISSN 0169-328X

Choline acetyltransferase (ChAT, EC 2.3.1.6) is the biosynthetic enzyme for acetylcholine. We have previously shown that multiple ChAT mRNA species with different 5'-noncoding regions are expressed in the rat and mouse. However, the diversity of ChAT mRNA species in human has not completely been elucidated. In this work N1- and N2-type ChAT cDNAs were cloned from a human brain cDNA library and the N-exon located in the human ChAT gene. Polymerase chain reaction analysis indicates that four species of ChAT mRNAs (R-, N1-, N2- and M-types) are produced in human brain and spinal cord. In all human transcripts, the ATG initiation codon in the rat, mouse and pig was replaced by ACG, which does not serve as an initiation codon for translation. In vitro translation and mammalian expression analyses revealed that N1-, N2- and R-type mRNAs give rise to a single 69 kDa enzyme, while M-type mRNA produces both 82 and 69 kDa enzymes. The translation efficiency of M-type mRNA was lower than that of the other mRNA species. Moreover, the translation efficiency of human ChAT mRNAs was considerably lower than that of rat ChAT mRNA, suggesting that the ATG codons for human ChAT are unfavorable for translation initiation compared with the initiation codon for rat ChAT. These results provide rational explanations for the previous reports that human ChAT protein purified from the brain acid placenta had 66-70 kDa molecular mass, and that ChAT activity in a single motor neuron of human was far lower than that of other vertebrates. Sequencing of monkey ChAT gene showed that the initiation ATG in rodent ChAT was also replaced by ACA in the monkey.

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Molecular characterization of the mouse vesicular acetylcholine transporter gene

Naciff Jorge M., Misawa Hidemi, Dedman John R.

NeuroReport 8 （ 16 ） 3467 - 3473 1997年

研究論文（学術雑誌）, 共著, 査読有り, ISSN 0959-4965

The organization of the mouse choline acetyltransferase (ChAT) gene has been previously analyzed. Here we show that the first intron of the mouse ChAT gene contains an uninterrupted open reading frame. It is in the same transcriptional orientation as ChAT and encodes the vesicular acetylcholine (ACh) transporter (VAChT), the protein responsible for the translocation of cytoplasmic ACh into synaptic vesicles. The sequence of this transporter is very similar to the VAChT from rat and human (99% and 95% identity, respectively). Reverse transcription-polymerase chain reaction (RT-PCR) analysis showed expression of mouse VAChT mRNA in spinal cord, brain (excluding the cerebellum) and brain stem, but not in peripheral tissues such as liver and kidney. Transgenic mouse analysis revealed that the 5'-flanking region of the mouse ChAT gene encompasses regulatory elements that allowed elevated expression of VAChT in the cholinergic system of transgenic mice.

A subtype of κ-opioid receptor mediates inhibition of high-affinity GTPase inherent in G_i1 in Guinea pig cerebellar membranes

Ueda Hiroshi, Misawa Hidemi, Fukushima Nobuyuki, Katada Toshiaki, Ui Michio, Satoh Masamichi

Journal of Neurochemistry 66 （ 2 ） 845 - 851 1996年02月

研究論文（学術雑誌）, 共著, 査読有り, ISSN 0022-3042

The κ-opioid receptor agonists including U-50,488H and dynorphin A (1-17) in ranges of 0.1-100 nM inhibited the hydrolysis of GTP to GDP (P_i release) inherent in GTP-binding proteins (G proteins) in guinea pig cerebellar membranes. U-50,488H inhibited only high-affinity GTPase activity, not low-affinity activity. The action of this agonist was found to be biphasic, and there was no inhibition at concentrations >1 μM. The inhibition was abolished by pretreatment with preactivated pertussis toxin (PTX) at concentrations >1 μg/ml but not with preactivated cholera toxin (30 μg/ml). Similar blockade of κ-receptor-mediated inhibition was also observed when membranes were pretreated with a low concentration (8 μM) of N-ethylmaleimide (NEM) at low temperature (4°C), which alkylates the cysteine residue to be ADP-ribosylated by PTX; but this treatment caused no significant change in κ-agonist binding. When purified G_i1, but not G_o, was reconstituted into membranes pretreated with NEM, the κ-receptor-mediated inhibition was recovered. These findings suggest that a subtype of κ-opioid receptor is coupled to inhibition of intrinsic activity of G_i1.

A subtype of opioid κ-receptor is coupled to inhibition of Gil-mediated phospholipase C activity in the guinea pig cerebellum

Misawa Hidemi, Ueda Hiroshi, Katada Toshiaki, Ui Michio, Satoh Masamichi

FEBS Letters 361 （ 1 ） 106 - 110 1995年03月

研究論文（学術雑誌）, 共著, 査読有り, ISSN 0014-5793

PLC activity was stimulated either by 1-100 μM of GTP or by 100-3,000 μM Ca²⁺ in lysed synaptosomal membranes of the guinea pig cerebellum. The κ-opioid receptor agonist selectively inhibited the PLC activity stimulated by 100 μM GTP, but not by 100-3,000 μM Ca²⁺. Pretreatment of membranes with PTX abolished such a κ-agonist-induced inhibition of PLC activity. The reconstitution of Gi1, but not of Go purified from porcine brains with PTX-treated membranes showed a complete recovery of the κ-agonist-inhibition of PLC activity. These findings suggest that a novel subtype κ-receptor mediates inhibition of PLC through inhibiting the intrinsic activity of PTX-substrate G-proteins.

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cDNA cloning and chromosomal localization of the human ciliary neurotrophic factor gene

Yokoji Hidehiro, Ariyama Takeshi, Takahashi Ryosuke, Inazawa Johji, Misawa Hidemi, Deguchi Takeo

Neuroscience Letters 185 （ 3 ） 175 - 178 1995年02月

研究論文（学術雑誌）, 共著, 査読有り, ISSN 0304-3940

Full-length cDNA for human ciliary neurotrophic factor (CNTF) was isolated from a human sciatic nerve cDNA library. Sequence analysis revealed that the longest cDNA was comprised of a 48-bp 5′-untranslated region, a 600-bp coding region and a 1207-bp 3′-untranslated region containing four ATTA pentamer motifs and a polyadenylation sequence. The transcription starting point was assigned at 81 bp upstream of the initiation methionine by 5′ RACE analysis. Using the cDNA and genomic DNA fragment including the entire intron region as mixed probes, the human CNTF gene was localized to the long arm of chromosome 11 at region q12 by fluorescence in situ hybridization.

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Coordinate expression of vesicular acetylcholine transporter and choline acetyltransferase in sympathetic superior cervical neurones

Misawa H., Takahashi R., Deguchi T.

NeuroReport 6 （ 7 ） 965 - 968 1995年

研究論文（学術雑誌）, 共著, 査読有り, ISSN 0959-4965

The neurotransmitter acetylcholine is synthesized by choline acetyltransferase (ChAT) and transported into synaptic vesicles by the vesicular acetylcholine transporter (VAChT). Recently it has been reported that the entire coding region of VAChT mRNA is located in the first intron of the ChAT gene. In this study, ChAT and VAChT mRNAs were analysed in cultured sympathetic neurones. Cholinergic differentiation factor/leukaemia inhibitory factor and ciliary neurotrophic factor induced strong expression of ChAT and VAChT mRNAs in parallel. RT-PCR analysis of ChAT mRNAs revealed that five types of ChAT transcripts which differed in the 5' non coding regions were increased. RT-PCR analysis of VAChT mRNA indicated that the cytokines induced only VAChT mRNA species which did not contain the R-exon, and not those containing the R-exon. The results indicate that ChAT and VAChT expressions are coordinately but differentially regulated in cultured sympathetic neurones.

Discrete acetylcholine release from neuroblastoma or hybrid cells overexpressing choline acetyltransferase into the neuromuscular synaptic cleft

Zhong Zhen Guo, Kimura Yasuhiro, Noda Mami, Misawa Hidemi, Higashida Haruhiro

Neuroscience Research 22 （ 1 ） 81 - 88 1995年

研究論文（学術雑誌）, 共著, 査読有り, ISSN 0168-0102

Neuroblastoma (clones NS-20Y, N1E-115, and Neuro2A) and neuroblastoma × glioma hybrid (NG108-15) cells were transfected with mouse choline acetyltransferase (ChAT) complementary DNA (cDNA) or vector DNA alone and stably transformed cell lines were established to examine their ability to secrete acetylcholine (ACh). Membrane potentials were recorded from either presynaptic neuroblastoma and hybrid cells or postsynaptic myotubes in co-culture. After transformation with ChAT, synapses were formed and miniature end-plate potentials (MEPPs) were recorded in myotubes co-cultured with Neuro2A and N1E-115 cells, while parental and mock-transfected control cells totally lacked this ability. The rate of synapse formation and/or MEPP frequency was higher in transformed NG108-15 hybrid and NS-20Y cells than that in the control cells. Action potentials of NS-20Y, Neuro2A or NG108-15 cells overexpressing ChAT were able to evoke end-plate potentials in myotubes, though the average quantum content of these cells was 0.04-0.14, which is as low as the control value. The results show that increased concentrations of ACh by ChAT cDNA transfection reveal a masked property in vesicular ACh release from Neuro2A and N1E-115 cells with no endogenous ChAT activity, or modify their secretory capacity upwardly from NG108-15 and NS-20Y cells with endogenous activity.

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Overexpression of choline acetyltransferase reconstitutes discrete acetylcholine release in some but not all synapse formation-defective neuroblastoma cells

Zhong ZG G., Misawa H., Furuya S., Kimura Y., Noda M., Yokoyama S., Higashid H.

Journal of Physiology - Paris 89 （ 3 ） 137 - 145 1995年

研究論文（学術雑誌）, 共著, 査読有り, ISSN 0928-4257

Secretion of acetylcholine (ACh) in neuroblastoma cells overexpressing choline acetyltransferase (ChAT) was examined. With transient transfection of ChAT cDNA, neuroblastoma cells, which have no endogenous ChAT and either adhere to myotubes or not, failed to form functional synapses, and thus no evidence for release of ACh was detected. Stable neuroblastoma cell lines overexpressing ChAT accumulated ACh inside the cell, and slowly released ACh to the outside of the cell in a calcium-independent fashion. However, after co-culturing them with rat muscle cells, these transformed cells adhered to myotubes and ACh was secreted in a discrete fashion into the synaptic cleft efficiently in some neuroblastoma cell lines but rather inefficiently in another cell line. The results show that the latent secretion machinery of ChAT overexpressing neuroblastoma cells either is competent or possess defect(s) in ACh release.

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A null mutation in the human CNTF gene is not causally related to neurological diseases

Takahashi Ryosuke, Yokoji Hidehiro, Misawa Hidemi, Hayashi Michiyuki, Hu Jianguo, Deguchi Takeo

Nature Genetics 7 （ 1 ） 79 - 84 1994年05月

研究論文（学術雑誌）, 共著, 査読有り, ISSN 1061-4036

We report a null mutation in the human ciliary neurotrophic factor gene (CNTF). The mutated allele shows a G to A transition producing a new splice acceptor site and the resulting mRNA species codes for an aberrant protein. Analysis of tissue samples and transfection of CNTF minigenes into cultured cells demonstrates that the mutated allele expresses only the mutated mRNA species. In 391 Japanese people tested, 61.9% were normal homozygotes, 35.8% heterozygotes and 2.3% mutant homozygotes. The distribution of the three genotypes is similar in healthy and neurological disease subjects, indicating that human CNTF deficiency is not causally related to neurological diseases.

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Calcium-independent release of acetylcholine from stable cell lines expressing mouse choline acetyltransferase cDNA

Misawa Hidemi, Takahashi Ryosuke, Deguchi Takeo

Journal of Neurochemistry 62 （ 2 ） 465 - 470 1994年02月

研究論文（学術雑誌）, 共著, 査読有り, ISSN 0022-3042

Stably transfected cells expressing mouse choline acetyltransferase (ChAT) cDNA were established, and the synthesis and release of acetylcholine (ACh) were examined. A cDNA clone coding for mouse ChAT was inserted into an expression vector (pEF321) containing a promoter for human elongation factor 1α to construct pEFmChAT. Neuronal (NG108-15, NS20Y, N1E115, and Neuro2A) and nonneuronal cell lines (L cells and NIH3T3) were transfected with pEFmChAT, and the cell lines that stably expressed high ChAT activity were selected. These cells expressed the 66-kDa ChAT protein and accumulated ACh mostly in the cytosol. The concentration of intracellular ACh in the cells increased upon raising the choline level in the medium. The cells continuously released ACh in a Ca²⁺-independent fashion. Neither high K⁺ nor calcium ionophore stimulated release of ACh from the cells.

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Transcriptional regulation of choline acetyltransferase gene by cyclic AMP

Misawa Hidemi, Takahashi Ryosuke, Deguchi Takeo

Journal of Neurochemistry 60 （ 4 ） 1383 - 1387 1993年04月

研究論文（学術雑誌）, 共著, 査読有り, ISSN 0022-3042

The effect of cyclic AMP on the gene expression of cholinc acetyltransferase (ChAT) was studied in NG108-15, mouse neuroblastoma and rat glioma hybrid cell lines. Addition of dibutyryl cyclic AMP to the culture medium increased both the ChAT mRNA level and ChAT activity twofold. Polymerase chain reaction analysis of the ChAT mRNA indicated that, among the multiple mRNA species, M-type mRNA was transcribed most efficiently, with or without the addition of dibutyryl cyclic AMP. The 5́ region of the mouse ChAT gene was ligated to the bacterial chloramphenicol acetyltransferase gene, and the expression of chloramphenicol acetyltransferase activity was determined by transaction analysis. Cyclic AMP derivatives enhanced the reporter gene expression in both transiently and stably transfected cells. DNA deletion analysis indicated that the intron region downstream of the M-type exon is necessary for the cyclic AMP responsiveness, and that cyclic AMP derivatives increase ChAT gene transcription mainly from M-type promoter. These results suggest that a cis-acting DNA element that confers the cyclic AMP responsiveness of the ChAT gene is present in the intron downstream of the M-type exon.

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Multiple mRNA species of choline acetyltransferase from rat spinal cord

Kengaku Mineko, Misawa Hidemi, Deguchi Takeo

Molecular Brain Research 18 （ 1-2 ） 71 - 76 1993年

研究論文（学術雑誌）, 共著, 査読有り, ISSN 0169-328X

A cDNA library directed by a specific primer was constructed from the rat spinal cord and screened with ³²P-labeled rat choline acetyltransferase cDNA which was recently isolated in this laboratory. Sequence analysis of 29 clones indicated that there are four types of cDNA (R1-, R2-, N1- and M-types). The nucleotide sequences in these cDNAs were identical in the coding region and the first 38 bp of the 5′-noncoding region, but differed in the 5′-noncoding region upstream of -38 bp. The R1-type was identical to the cDNA previously cloned from the rat spinal cord. The M and N1-type cDNAs both had sequences homologous to that of the cDNA previously obtained from the mouse spinal cord. Polymerase chain reaction analysis confirmed the presence of these 4 types of mRNA and found another type (N2-type) of transcript. The numbers of cDNA clones isolated and the relative amounts of polymerase chain reaction products for each type of mRNA suggested that the most abundant transcript was M-type. Sequencing of the genomic clone containing the 5′-region of choline acetyltransferase mRNA revealed that these five types of mRNA species were transcribed from three different promoter regions and produced by differential splicing of the 5′-noncoding exons.

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Gene expression of mouse choline acetyltransferase: Alternative splicing and identification of a highly active promoter region

Misawa Hidemi, Ishii Kayoko, Deguchi Takeo

Journal of Biological Chemistry 267 （ 28 ） 20392 - 20399 1992年10月

研究論文（学術雑誌）, 共著, 査読有り, ISSN 0021-9258

Seven types of mRNA for choline acetyltransferase that differ in the 5′-noncoding region were identified in the mouse spinal cord by cDNA cloning and polymerase chain reaction. Among these transcripts, the M-type mRNA corresponding to the previously cloned mouse cDNA was most abundant in the spinal cord of mouse. A mouse genomic DNA clone containing the 5′-region of choline acetyltransferase mRNA was isolated and sequenced. Comparison of the sequences between the cDNAs and the genomic DNA revealed that the different mRNA species were transcribed from different promoter regions and produced by differential splicing. Two murine cholinergic cell lines, NS20Y and NG108-15, were shown to express the M-type mRNA almost exclusively, and were therefore used to study transcription of M-type mRNA. Fragments of the 5′-region of choline acetyltransferase gene were ligated with chloramphenicol acetyltransferase reporter gene and introduced into cultured cells. The fragment from -2752 to +46, which contained the M-type exon, a TATA-box like element upstream of the M-type exon, and the downstream intron, induced a significant expression of CAT activity in neuronal but not in non-neuronal cell lines. This result indicates that this region of choline acetyltransferase gene contains elements that regulate neuron-specific expression of choline acetyltransferase activity. However, there was no parallel correlation between reporter gene expression in the transfected cells and intrinsic choline acetyltransferase activity in these neuronal cell lines. Possible mechanisms that would explain this observation are discussed.

κ-Opioid agonist inhibits phospholipase C, possibly via an inhibition of G-protein activity

Misawa Hidemi, Ueda Hiroshi, Satoh Masamichi

Neuroscience Letters 112 （ 2-3 ） 324 - 327 1990年05月

研究論文（学術雑誌）, 共著, ISSN 0304-3940

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Functional reconstitution of purified G_i and G_o with μ-opioid receptors in guinea pig striatal membranes pretreated with micromolar concentrations of N-ethylmaleimide

Ueda Hiroshi, Misawa Hidemi, Katada Toshiaki, Ui Michio, Takagi Hiroshi, Satoh Masamichi

Journal of Neurochemistry 54 （ 3 ） 841 - 848 1990年

研究論文（学術雑誌）, 共著, 査読有り, ISSN 0022-3042

Functional coupling between μ-opioid receptors and GTP-binding regulatory proteins (G proteins) was investigated in reconstituted membranes of the guinea pig striatum. Selective μ-opioid agonists stimulated low-K_m GTPase in striatal membranes, in a Na⁺-dependent manner. The same μ-opioid agonist {[D-Ala²,N-Me-Phe⁴,Gly⁵-ol]-enkephaIin (DAGO)} caused no stimulation when the membranes were exposed to islet-activating protein (IAP; pertussis toxin). There was also no DAGO stimulation in preparations pretreated with a lower concentration (5 μM) of N-ethylmaleimide (NEM), which abolished the ADP-ribosylation of purified G_i( (the G protein that mediates inhibition of adenylate cyclase) and G_o (a G protein of unknown function purified from bovine brain) by IAP. In addition, as the NEM treatment caused no change in the μ-agonist binding, NEM could probably substitute for IAP in inactivating native G proteins, without exhibiting effects on the receptor binding in mem-branes. The μ-agonist stimulation of low-K_m GTPase activity in NEM-treated membranes was recovered by reconstitution with purified G_i or G_o. The μ-agonist stimulation of low-K_m GTPase was additive when G_i and G_o were simultaneously reconstituted in NEM-treated membranes in amounts of 0.5 pmol/assay, which was required for maximal recovery, in either reconstitution experiment. The present findings provide the first evidence that the μ-opioid receptor may exist in at least two different forms, separately coupled to G₁ or G_o.

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The kyotorphin (tyrosine-arginine) receptor and a selective reconstitution with purified G(i), measured with GTPase and phospholipase C assays

Ueda H., Yoshihara Y., Misawa H., Fukushima N., Katada T., Ui M., Takagi H., Satoh M.

Journal of Biological Chemistry 264 （ 7 ） 3732 - 3741 1989年

研究論文（学術雑誌）, 共著, 査読有り, ISSN 0021-9258

We attempted to identify the kyotorphin receptor and the post receptor mechanisms mediated by GTP-binding proteins (G-proteins), using reconstitution techniques. The specific binding of [³H]kyotorphin in rat brain membranes was composed of high affinity (K(d) = 0.34 nM) and low affinity (K(d) = 9.07 nM) binding. As the high affinity binding disappeared in the presence of guanosine 5'-O-(3-thiotriphosphate) and MgCl₂, we investigated the kyotorphin receptor-mediated changes in membrane G-protein activity by measuring low K(m) GTPase activity. Kyotorphin produced a stimulation of low K(m) GTPase, and this stimulation was antagonized by Leu-Arg, a synthetic dipeptide which showed a potent displacement of [³H]kyotorphin binding, yet in itself had no effect on the low K(m) GTPase. The kyotorphin stimulation of low K(m) GTPase was abolished by pretreating membranes with islet-activating protein, pertussis toxin, and was recovered by reconstitution with purified G-protein, G(i), but not with G(o). Similar evidence of selective coupling of kyotorphin receptor to G(i) was obtained with the phospholipase C assay. Kyotorphin-induced stimulation of phospholipase C was also abolished by islet-activating protein-treatment and recovered by reconstitution with G(i) but not with G(o). These findings indicate that specific high and low affinity kyotorphin receptors exist in the rat brain and that the kyotorphin receptor is functionally coupled to stimulation of phospholipase C, through G(i). This study provides the first evidence of a selective involvement of G(i) in the receptor-mediated activation of phospholipase C.

The specific opioid κ-agonist U-50,488H inhibits low Km GTPase

Ueda Hiroshi, Misawa Hidemi, Fukushima Nobuyuki, Takagi Hiroshi

European Journal of Pharmacology 138 （ 1 ） 129 - 132 1987年06月

研究論文（学術雑誌）, 共著, 査読有り, ISSN 0014-2999

DAGO, a specific opioid μ-agonists stimulated the low Km GTPase activity of membranes of the guinea-pig striatum at 1-1000 nM and this effect was antagonized by naloxone 10-100 nM. On the other hand, U-50,488H, a specific opioid κ-agonist inhibited the low Km GTPase activity of membranes of the guinea-pig cerebellum at 1-100 nM. As this effect was antagonized by MR2266 but not by naloxone, the findings suggest that opioid μ- and κ-receptor stimulation is probably linked to the stimulation and inhibition of low Km GTPase, respectively.

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Purified opioid μ-receptor is of a different molecular size than δ- and κ-receptors

Ueda Hiroshi, Harada Hitoshi, Misawa Hidemi, Nozaki Masakatsu, Takagi Hiroshi

Neuroscience Letters 75 （ 3 ） 339 - 344 1987年04月

研究論文（学術雑誌）, 共著, 査読有り, ISSN 0304-3940

Opioid μ-receptors were solubilized from rat brains with 0.5% Triton X-100 in the presence of 0.32 M sucrose and then purified with 6-succinyl morphine-Affi-Gel 102 column. Purified materials showed a major band with mol.wt. 58,000 Da and a minor band with mol.wt. 41,000 Da. Affinity cross linking with [³H]DAGO, a selective μ-opioid agonist, to the neural membranes and purified materials revealed that opioid μ-binding protein has a mol.wt. of 58,000 Da, a value totally different from that of δ- (mol.wt. 18,500 Da) and κ- (mol.wt. 36,600 Da) binding protein.

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