研究者詳細 - 三澤　日出巳

論文 - 三澤　日出巳

分割表示 118 件中 61 - 80 件目／全件表示 >>

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.

Access to Document (DOI)

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.

Access to Document (DOI)

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.

Access to Document (DOI)

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.

Access to Document (DOI)

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.

Access to Document (DOI)

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.

Access to Document (DOI)

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.

Access to Document (DOI)

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.

Access to Document (DOI)

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.

Access to Document (DOI)

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.

Access to Document (DOI)

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.

Access to Document (DOI)

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.

Access to Document (DOI)

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.

Access to Document (DOI)

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.

Access to Document (DOI)

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.

Access to Document (DOI)

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.

Access to Document (DOI)

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.

Access to Document (DOI)

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.

Access to Document (DOI)

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.

Access to Document (DOI)

　前のページ - 次のページ　

三澤 日出巳

論文 - 三澤 日出巳

三澤　日出巳

論文 - 三澤　日出巳