Details of a Researcher - Misawa, Hidemi

Papers - Misawa, Hidemi

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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 2014.08

Research paper (scientific journal), Joint Work, Accepted

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 2014.05

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 2014.02

Research paper (scientific journal), Joint Work, Accepted, 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 2013.08

Research paper (scientific journal), Joint Work, 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 2013.05

Research paper (scientific journal), Joint Work, 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 2013.05

Research paper (scientific journal), Joint Work, Accepted

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

Research paper (scientific journal), Joint Work, 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 2012.12

Research paper (scientific journal), Joint Work, Accepted, 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 2012.12

Research paper (scientific journal), Joint Work, Accepted, 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 2012.11

Research paper (scientific journal), Joint Work, Accepted, 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 2012.08

Research paper (scientific journal), Joint Work, Accepted

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 2012.07

Research paper (scientific journal), Joint Work, Accepted, 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 2012.06

Research paper (scientific journal), Joint Work

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 2012.04

Research paper (scientific journal), Joint Work, Accepted, 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 2011.11

Research paper (scientific journal), Joint Work, Accepted, 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 2011.10

Research paper (scientific journal), Joint Work, Accepted, 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 2011.03

Research paper (scientific journal), Joint Work, Accepted, 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 2010.09

Research paper (scientific journal), Joint Work, Accepted, 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 2010.08

Research paper (scientific journal), Joint Work, Accepted, 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 2010.07

Research paper (scientific journal), Joint Work, Accepted, 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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