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論文 - 三澤　日出巳

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

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

PLoS One 13 （ 6 ） e0199829 2018年06月

研究論文（学術雑誌）, 共著, ISSN 1932-6203

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

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

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

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

研究論文（学術雑誌）, 共著, 査読有り, ISSN 0925-4439

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

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

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

Cell Death and Differentiation 1 - 17 2018年03月

研究論文（学術雑誌）, 共著, 査読有り, ISSN 1350-9047

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

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

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

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

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

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

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

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

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

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

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

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

研究論文（学術雑誌）, 共著, 査読有り, ISSN 0919-6544

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

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Expression and function of the cholinergic system in immune cells

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

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

研究論文（学術雑誌）, 共著, 査読有り

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

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

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

PLoS One 12 （ 6 ） e0179375 2017年06月

研究論文（学術雑誌）, 共著, 査読有り, ISSN 1932-6203

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

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Physiological functions of the cholinergic system in immune cells

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

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

研究論文（学術雑誌）, 共著, ISSN 1347-8613

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

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麻酔下マウスの脳局所血流に及ぼす橈骨神経電気刺激の影響

内田さえ、鍵谷方子、堀田晴美、渡辺さおり、三澤日出巳

自律神経 54 26 - 33 2017年04月

研究論文（学術雑誌）, 共著, 査読有り

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

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

Scientific Reports 7 45768 2017年03月

研究論文（学術雑誌）, 共著, 査読有り, ISSN 2045-2322

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

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

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

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

研究論文（学術雑誌）, 共著, ISSN 0961-8368

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

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

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

Molecular Neurodegeneration 12 （ 1 ） 2017年01月

研究論文（学術雑誌）, 共著, 査読有り, ISSN 1750-1326

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

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

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

Sci. Rep. 6 34575 2016年09月

研究論文（学術雑誌）, 査読有り

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

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

Genesis 54 （ 11 ） 568 - 572 2016年09月

研究論文（学術雑誌）, 共著, 査読有り, ISSN 1526-954X

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

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

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

Scientific Reports 6 27354 2016年06月

研究論文（学術雑誌）, 共著, 査読有り, ISSN 2045-2322

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

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

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

Acta Neuropathol. Commun. 4 15 2016年02月

研究論文（学術雑誌）, 共著, 査読有り

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

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

PLoS One 10 （ 10 ） e0140750 2015年10月

研究論文（学術雑誌）, 共著, 査読有り

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

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

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

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

研究論文（学術雑誌）, 共著, 査読有り, ISSN 1567-5769

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

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

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

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

研究論文（学術雑誌）, 査読有り, ISSN 1567-5769

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

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