研究者詳細 - 三澤　日出巳

論文 - 三澤　日出巳

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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.

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