Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system (CNS). In MS, a long disease duration is known to be a strong risk factor for converting the clinical course of the disease from relapse remitting MS to secondary progressing MS. There is a hypothesis that long sustained demyelination may exhaust neurons, however, pathological changes induced in neurons following demyelination remain unknown. Cuprizone administration can induce and sustain demyelination in the mouse CNS. We examined pathological changes in mice following long sustained demyelination caused by up to 34-week cuprizone administration. Twelve-week cuprizone administration induced severe demyelination in the cerebral cortex, corpus callosum and deep cerebellar nuclei. Demyelination persisted up to 34 weeks, as shown by myelin basic protein immunohistochemistry. In contrast, cuprizone administration developed demyelination in the striatum by week 34. In these demyelinated regions, no neuronal loss was observed. However, in the striatum and deep cerebellar nuclei, cuprizone-induced demyelination changed the intracellular distribution of parvalbumin (PV). Furthermore, in the striatum, there was an increase in PV in the demyelinated axons and most PV immunoreactivity did not co-localize with SMI32 immunoreactivity in mice with 34-week cuprizone administration. Further, mice with 34-week cuprizone administration showed motor coordination dysfunction in the balance beam test. However, 12-week withdrawal from the cuprizone diet induced remyelination in the regions and motor coordination dysfunction recovered. These results indicate that 34-week cuprizone administration induces and sustains demyelination and results in reversible motor coordination dysfunction. The change of intracellular PV distribution suggests that PV may protect demyelinated axons by Ca2+ buffering. This model may be useful to investigate pathological and behavioral changes following demyelination in the CNS.

DOI： 10.1016/j.neuint.2019.03.018

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Wiley  

Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system (CNS), and experimental autoimmune encephalomyelitis (EAE) is a well-established animal model of the disease. Here, we examined the pathophysiological role of Kallikrein 6 (Klk6), a serine protease produced by oligodendrocytes (OLs), in EAE using Klk6 knockout (Klk6-/-) mice. Compared with Klk6+/+ (wild-type) mice, Klk6-/- mice showed milder EAE symptoms, including delayed onset and milder paralysis. Loss of Klk6 suppressed matrix metalloprotease-9 expression and diminished the infiltration of peripheral inflammatory cells into the CNS by decreasing blood-brain barrier (BBB) permeability and reducing expression levels of inflammatory cytokines, chemokines and their receptors. Scanning electron microscopic analysis revealed demyelination characterized by myelin detachment from the axons in the early phase of EAE progression (days 3-7) in Klk6+/+ mice but not in Klk6-/- mice. Interestingly, anti-MOG (myelin oligodendrocyte glycoprotein) autoantibody was also detected in the cerebrospinal fluid (CSF) and spinal cord on day 3 after MOG immunization. Furthermore, treatment of primary cultured OLs with anti-MOG autoantibody induced oligodendroglial morphological changes and increases in myelin basic protein and Klk6 expression. We also developed a novel enzyme-linked immunoabsorbent assay method for detecting activated KLK6 in human CSF. In human autopsy brain samples, expression of active KLK6 was detected in OLs using an antibody that specifically recognizes the protein's activated form. Taken together, our findings demonstrate that Klk6 secreted by OLs plays a critical role in the pathogenesis of EAE/MS and that it might serve as a potential therapeutic target for MS.

DOI： 10.1002/glia.23249

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Springer Science and Business Media LLC  

Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system (CNS). The release of inflammatory cytokines and pro-oxidant molecules from microglia has been shown to play a key role in the pathology of MS. Thus, suppression of microglial cell activation is an attractive therapeutic option. Yokukansan, a traditional Japanese herbal medicine, has been shown to suppress microglial activity in the CNS. However, whether or not yokukansan reduces demyelination observed in the CNS during MS remains unknown. In this study, female C57BL/6 mice were fed a diet containing 0.2% cuprizone (bis-cyclohexanone oxaldihydrazone) to induce demyelination in the corpus callosum. We investigated whether or not yokukansan reduces cuprizone-induced demyelination using immunohistochemical analyses. Furthermore, we examined the in vitro anti-inflammatory effects of yokukansan on LPS-stimulated BV2 cells, a murine microglial cell line. Luxol fast blue staining and immunostaining for myelin basic protein demonstrated that yokukansan reduces demyelination of the corpora callosa of cuprizone-fed mice. In addition, yokukansan significantly decreased the number of activated microglial cells in the corpora callosa of cuprizone-fed mice. Furthermore, treatment with 500 μg/ml yokukansan suppressed the expression of interleukin-1β and inducible nitric-oxide synthase mRNA and protein in LPS-stimulated BV2 cells. These findings suggest that yokukansan reduces demyelination owing to anti-inflammatory effects on microglia. As yokukansan has few adverse effects, yokukansan has the potential to be a novel option to treat MS.

DOI： 10.1007/s11064-017-2400-z

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Other Link： http://link.springer.com/content/pdf/10.1007/s11064-017-2400-z.pdf

Language：English   Publishing type：Research paper (scientific journal)   Publisher：PERGAMON-ELSEVIER SCIENCE LTD  

Microglia are essential in developmental processes and maintenance of neuronal homeostasis. Experimental axotomy of motor neurons results in neurodegeneration, and microglia in motor nuclei become activated and migrate towards injured neurons. However, whether these activated microglia are protective or destructive to neurons remains controversial. In the present study, we transected the hypo-glossal nerve in BALB/c mice, causing activating transcription factor 3 (ATF3) and growth associated protein 43 (GAP43) induction, and partial neuronal death. Inhibition of microglial accumulation by minocycline administration impaired microglial accumulation, decreased GAP43 mRNA expression, and reduced motor neuron survival. Expression of ATF3 contributed to nerve regeneration, and increased within 6 h after axotomy, prior to microglial migration. Further, microglial contact with neuronal cell bodies was associated with neuronal ATF3 expression. Colchicine administration blocked lesion-induced ATF3 transcription in axotomized neurons and microglial accumulation. In addition, perineuronal microglia-derived ciliary neurotrophic factor (CNTF) increased, indicating that perineuronal microglia in the hypoglossal nucleus protect axotomized motor neurons by releasing trophic factors. We also observed that microglia secrete CNTF and that neurons have CNTFR alpha and can respond to it in vitro. CNTF promote neurite elongation and neuronal survival of primary cultured neurons. Microglia make contact through unknown neuronal signals that are possibly regulated by ATF3 in hypoglossal nucleus. Moreover, they play important roles in regenerating motor neurons and are potential new therapeutic targets for motor neuron diseases. (C) 2017 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.neuint.2017.05.007

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY  

DOI： 10.1111/exd.13204

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：PUBLIC LIBRARY SCIENCE  

Oligodendrocytes myelinate axons and form myelin sheaths in the central nervous system. The development of therapies for demyelinating diseases, including multiple sclerosis and leukodystrophies, is a challenge because the pathogenic mechanisms of disease remain poorly understood. Primate pluripotent stem cell-derived oligodendrocytes are expected to help elucidate the molecular pathogenesis of these diseases. Oligodendrocytes have been successfully differentiated from human pluripotent stem cells. However, it is challenging to prepare large amounts of oligodendrocytes over a short amount of time because of manipulation difficulties under conventional primate pluripotent stem cell culture methods. We developed a proprietary dissociated monolayer and feeder-free culture system to handle pluripotent stem cell cultures. Because the dissociated monolayer and feeder-free culture system improves the quality and growth of primate pluripotent stem cells, these cells could potentially be differentiated into any desired functional cells and consistently cultured in large-scale conditions. In the current study, oligodendrocyte progenitor cells and mature oligodendrocytes were generated within three months from monkey embryonic stem cells. The embryonic stem cell-derived oligodendrocytes exhibited in vitro myelinogenic potency with rat dorsal root ganglion neurons. Additionally, the transplanted oligodendrocyte progenitor cells differentiated into myelin basic protein-positive mature oligodendrocytes in the mouse corpus callosum. This preparative method was used for human induced pluripotent stem cells, which were also successfully differentiated into oligodendrocyte progenitor cells and mature oligodendrocytes that were capable of myelinating rat dorsal root ganglion neurons. Moreover, it was possible to freeze, thaw, and successfully re-culture the differentiating cells. These results showed that embryonic stem cells and human induced pluripotent stem cells maintained in a dissociated monolayer and feeder-free culture system have the potential to generate oligodendrocyte progenitor cells and mature oligodendrocytes in vitro and in vivo. This culture method could be applied to prepare large amounts of oligodendrocyte progenitor cells and mature oligodendrocytes in a relatively short amount of time.

DOI： 10.1371/journal.pone.0171947

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：IMPACT JOURNALS LLC  

One of the major adverse effects of topical glucocorticoids is cutaneous atrophy often followed by development of resistance to steroids (tachyphylaxis). Previously we showed that after two weeks, interfollicular mouse keratinocytes acquired resistance to anti-proliferative effects of glucocorticoid fluocinolone acetonide (FA). One of the top genes activated by FA during tachyphylaxis was Klk6 encoding kallikrein-related peptidase 6, known to enhance keratinocyte proliferation. KLK6 was also strongly induced by chronic glucocorticoids in human skin. Double immunostaining showed that KLK6(+) keratinocytes, localized in suprabasal layer of mouse skin, were frequently adjacent to proliferating 5-bromo-2'-deoxyuridine-positive basal keratinocytes. We used KLK6 knockout (KO) mice to evaluate KLK6 role in skin regeneration after steroid-induced atrophy. KLK6 KOs had thinner epidermis and decreased keratinocyte proliferation. The keratinocytes in wild type and KLK6 KO epidermis were equally sensitive to acute anti-proliferative effect of FA. However, the development of proliferative resistance during chronic treatment was reduced in KO epidermis. This was not due to the changes in glucocorticoid receptor (GR) expression or function as GR protein level and induction of GR-target genes were similar in wild type and KLK6 KO skin. Overall, these results suggest a novel mechanism of epidermal regeneration after glucocorticoid-induced atrophy via KLK6 activation.

DOI： 10.18632/oncotarget.9926

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Language：Japanese   Publishing type：Research paper (scientific journal)  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL  

DOI： 10.1111/exd.12794

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：PERGAMON-ELSEVIER SCIENCE LTD  

Heparan sulfate proteoglycans (HSPGs) have important functions in development of the central nervous system; however, their functions in nerve injury are not yet fully understood. We previously reported the expression of syndecan-1, a type of HSPG, in cranial motor neurons after nerve injury, suggesting the importance of syndecan-1 in the pathology of motor nerve injury. In this study, we examined the expression of syndecan-1, a type of HSPG, in primary sensory neurons after nerve injury in mice. Sciatic nerve axotomy strongly induced the expression of syndecan-1 in a subpopulation of injured dorsal root ganglion (DRG) neurons, which were small in size and had CGRP-or isolectin B4-positive fibers. Syndecan-1 was also distributed in the dorsal horn of the spinal cord ipsilateral to the axotomy, and located on the membrane of axons in lamina II of the dorsal horn. Not only sciatic nerve axotomy, infraorbital nerve axotomy also induced the expression of syndecan-1 in trigeminal ganglion neurons. Moreover, syndecan-1 knockdown in cultured DRG neurons induced a shorter neurite extension. These results suggest that syndecan-1 expression in injured primary sensory neurons may have functional roles in nerve regeneration and synaptic plasticity, resulting in the development of neuropathic pain. (C) 2015 IBRO. Published by Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.neuroscience.2015.05.033

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL  

Microglia are generally considered the immune cells of the central nervous system. Recent studies have demonstrated that under specific polarization conditions, microglia develop into two different phenotypes, termed M1-like and M2-like microglia. However, the phenotypic characteristics of M1-like- and M2-like-polarized microglia and the mechanisms that regulate polarization are largely unknown. In this study, we characterized lipopolysaccharide-treated M1-like and IL-4-treated M2-like microglia and investigated the mechanisms that regulate phenotypic switching. The addition of M2-like microglial conditioned medium (CM) to primary neurons resulted in an increase in neurite length when compared with neurons treated with M1-like microglial CM, possibly because of the enhanced secretion of neurotrophic factors by M2-like microglia. M1-like microglia were morphologically characterized by larger soma, whereas M2-like microglia were characterized by long processes. M2-like microglia exhibited greater phagocytic capacity than M1-like microglia. These features switched in response to polarization cues. We found that expression of interferon regulatory factor 7 (IRF7) increased during the M2-like to M1-like switch in microglia in vitro and in vivo. Knockdown of IRF7 using siRNA suppressed the expression of M1 marker mRNA and reduced phosphorylation of STAT1. Our findings suggest that IRF7 signaling may play an important role in microglial polarization switching. GLIA 2015;63:595-610

DOI： 10.1002/glia.22770

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：PERGAMON-ELSEVIER SCIENCE LTD  

Demyelination and axonal damage are responsible for neurological deficits in multiple sclerosis (MS), an inflammatory demyelinating disease of the central nervous system. However, the pathology of axonal damage in MS is not fully understood. In this study, histological analysis of morphological changes of axonal organelles during demyelination in murine models was investigated by scanning electron microscopy (SEM) using an osmium-maceration method.
In cuprizone-induced demyelination, SEM showed typical morphology of demyelination in the corpus callosum of mouse brain. In contrast, SEM displayed variations in ultrastructural abnormalities of myelin structures and axonal organelles in spinal cord white matter of experimental autoimmune encephalomyelitis (EAE) mice, an animal model of MS. Myelin detachment and excessive myelin formation were observed as typical morphological myelin abnormalities in EAE. In addition, well-developed axoplasmic reticulum-like structures and accumulated mitochondria were observed in tortuous degenerating/degenerated axons and the length of mitochondria in axons of EAE spinal cord was shorter compared with naive spinal cord. Immunohistochemistry also revealed dysfunction of mitochondrial fusion/fission machinery in EAE spinal cord axons. Moreover, the number of Y-shaped mitochondria was significantly increased in axons of the EAE spinal cord. Axonal morphologies in myelin basic proteindeficient shiverer mice were similar to those in EAE. However, shiverer mice had "tortuous" (S-curve shaped mitochondria) and larger mitochondria compared with wild-type and EAE mice. Lastly, analysis of human MS patient autopsied brains also demonstrated abnormal myelin structures in demyelinating lesions.
These results indicate that morphological abnormalities of myelin and axonal organelles play important role on the pathogenesis of axonal injury in demyelinating diseases. (C) 2015 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.neuint.2015.01.002

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：BENTHAM SCIENCE PUBL LTD  

Several studies have reported differences in physiological and pathological phenotypes between different strains of experimental mice, such as environment-based behavior, skin damage, damage in response to toxins and nervous system injury. However, the mechanisms underlying these differences have not yet been fully elucidated. We have been studying the function of kallikrein-related peptidases (KLKs), serine proteases known to serve a variety of functions. In this study, we focused on differences in KLKs between C57BL/6 mice and 129 mice. Among 13 KLKs genes examined, 12 KLKs showed differences in the mRNA coding region sequence and 7 KLKs showed different deduced amino acid sequences of their proteins when comparing C57BL/6 and 129 mice. KLK6 protein from 129 mice had six amino acid differences compared with that from C57BL/6 mice. KLK6 protein from 129 mice showed reduced SDS-PAGE mobility compared with that from C57BL/6 mice. Moreover, recombinant KLK6 protein from 129 mice had a higher optimum pH and >15 times higher hydrolytic enzymatic activity for several substrates than that from C57BL/6 mice. These results suggest that KLKs may contribute to the genetic basis of the differences between mouse strains. Koichi Murakami

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Authorship：Lead author   Language：Japanese   Publishing type：Research paper (scientific journal)  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL  

Remyelination is disrupted in demyelinating diseases such as multiple sclerosis, but the underlying pathogenetic mechanisms are unclear. In this study, we employed the murine cuprizone model of demyelination, in which remyelination occurs after removal of the toxin from the diet, to examine the cellular and molecular changes during demyelination and remyelination. Microglia accumulated in the corpus callosum during weeks 2-4 of the cuprizone diet, and these cells remained activated 2weeks after the change to the normal diet. To examine the role of microglia in remyelination, mice were treated with minocycline to inactivate these cells after cuprizone-induced demyelination. Minocycline treatment reduced the number of CC1-positive oligodendrocytes, as well as levels of myelin basic protein (MBP) and CNPase in the remyelination phase. The expression of CNTF mRNA in the corpus callosum increased after 4weeks on the cuprizone diet and remained high 2weeks after the change to the normal diet. Minocycline suppressed CNTF expression during the remyelination phase on the normal diet. Primary culture experiments showed that CNTF was produced by microglia in addition to astrocytes. In vitro, CNTF directly affected the differentiation of oligodendrocytic cells. These findings suggest that minocycline reduces remyelination by suppressing CNTF expression by microglia after cuprizone-induced demyelination.

DOI： 10.1111/jnc.12289

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Language：Japanese   Publisher：日本脳科学会  

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Oligodendrocytes are important for not only nerve conduction but also central nervous system (CNS) development and neuronal survival in a variety of conditions. Kallikrein-related peptidase 6 (KLK6) is expressed in oligodendrocytes in the CNS and its expression is changed in several physiological and pathological conditions, especially following spinal cord injury (SCI) and experimental autoimmune encephalomyelitis. In this study, we investigated the functions of KLK6 in oligodendrocyte lineage cell development and the production of myelin proteins using KLK6-deficient (KLK6-/-) mice. KLK6-/- mice were born without apparent defects and lived as long as wild-type (WT) mice. There was no significant difference in the numbers of oligodendrocyte precursor cells and mature oligodendrocytes in the adult naive spinal cord between WT and KLK6-/- mice. However, there were fewer mature oligodendrocytes in the KLK6-/- spinal cord than in the WT spinal cord at postnatal day 7 (P7). Expression of myelin basic protein (MBP) and oligodendrocyte-specific protein/claudin-11, major myelin proteins, was also decreased in the KLK6-/- spinal cord compared with the WT spinal cord at P7-21. Moreover, after SCI, the amount of MBP in the damaged spinal cords of KLK6-/- mice was significantly less than that in the damaged spinal cords of WT mice. These results indicate that KLK6 plays a functional role in oligodendrocyte development and the expression of myelin proteins. © 2013 IBRO.

DOI： 10.1016/j.neuroscience.2012.12.073

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Oligodendrocytes are important for not only nerve conduction but also central nervous system (CNS) development and neuronal survival in a variety of conditions. Kallikrein-related peptidase 6 (KLK6) is expressed in oligodendrocytes in the CNS and its expression is changed in several physiological and pathological conditions, especially following spinal cord injury (SCI) and experimental autoimmune encephalomyelitis. In this study, we investigated the functions of KLK6 in oligodendrocyte lineage cell development and the production of myelin proteins using KLK6-deficient (KLK6-/-) mice. KLK6-/- mice were born without apparent defects and lived as long as wild-type (WT) mice. There was no significant difference in the numbers of oligodendrocyte precursor cells and mature oligodendrocytes in the adult naive spinal cord between WT and KLK6-/- mice. However, there were fewer mature oligodendrocytes in the KLK6-/- spinal cord than in the WT spinal cord at postnatal day 7 (P7). Expression of myelin basic protein (MBP) and oligodendrocyte-specific protein/claudin-11, major myelin proteins, was also decreased in the KLK6-/- spinal cord compared with the WT spinal cord at P7-21. Moreover, after SCI, the amount of MBP in the damaged spinal cords of KLK6-/- mice was significantly less than that in the damaged spinal cords of WT mice. These results indicate that KLK6 plays a functional role in oligodendrocyte development and the expression of myelin proteins. © 2013 IBRO.

DOI： 10.1016/j.neuroscience.2012.12.073

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Axonal injury and demyelination are observed in demyelinating diseases such as multiple sclerosis. However, pathological changes that underlie these morphologies are not fully understood. We examined in vivo morphological changes using a new histological technique, scanning electron microscopy (SEM) with osmium maceration method to observe three-dimensional structures such as myelin and axons in the spinal cord. Myelin basic protein-deficient shiverer mice and mice with experimental autoimmune encephalomyelitis (EAE) were used to visualize how morphological changes in myelin and axons are induced by dysmyelination and demyelination.SEM revealed following morphological changes during dysmyelination of shiverer mice. First, enriched mitochondria and well-developed sER in axons were observed in shiverer, but not in wild-type mice. Second, the processes from some perinodal glial cells ran parallel to internodes of axons in addition to the process that covered the nodal region of the axon in shiverer mice. Last, this technique left myelin and axonal structures undisturbed. Moreover, SEM images showed clear variations in the ultrastructural abnormalities of myelin and axons in the white matter of the EAE spinal cord. This technique will be a powerful tool for identifying the mechanisms underlying the pathogenesis in demyelination. © 2013 Elsevier Ireland Ltd and the Japan Neuroscience Society.

DOI： 10.1016/j.neures.2013.01.009

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Axonal injury and demyelination are observed in demyelinating diseases such as multiple sclerosis. However, pathological changes that underlie these morphologies are not fully understood. We examined in vivo morphological changes using a new histological technique, scanning electron microscopy (SEM) with osmium maceration method to observe three-dimensional structures such as myelin and axons in the spinal cord. Myelin basic protein-deficient shiverer mice and mice with experimental autoimmune encephalomyelitis (EAE) were used to visualize how morphological changes in myelin and axons are induced by dysmyelination and demyelination.SEM revealed following morphological changes during dysmyelination of shiverer mice. First, enriched mitochondria and well-developed sER in axons were observed in shiverer, but not in wild-type mice. Second, the processes from some perinodal glial cells ran parallel to internodes of axons in addition to the process that covered the nodal region of the axon in shiverer mice. Last, this technique left myelin and axonal structures undisturbed. Moreover, SEM images showed clear variations in the ultrastructural abnormalities of myelin and axons in the white matter of the EAE spinal cord. This technique will be a powerful tool for identifying the mechanisms underlying the pathogenesis in demyelination. © 2013 Elsevier Ireland Ltd and the Japan Neuroscience Society.

DOI： 10.1016/j.neures.2013.01.009

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER IRELAND LTD  

Heparan sulfate proteoglycans play important roles in embryogenesis, including the development of the central nervous system. However, their function in nerve regeneration is not yet understood. We previously reported that nerve injury induces the expression of heparan sulfate glycosaminoglycans and syndecan-1, a heparan sulfate proteoglycan, in injured hypoglossal motor neurons. In this study, we examined the expression of syndecan family members, including syndecan-1, in injured hypoglossal motor neurons after hypoglossal nerve axotomy. We could not detect any changes in expression after axotomy, except for syndecan-1. The expression of syndecan-1 was markedly increased on post-operative day 7. Syndecan-1 was localized not only in the cell bodies of hypoglossal motor neurons, but also in the injured hypoglossal nerve, and it accumulated in the terminals of regenerating fibers. Similarly, facial nerve axotomy and vagus nerve axotomy induced the expression of syndecan-1 in the facial nucleus, dorsal nucleus of vagus and ambiguous nucleus, respectively. However, sciatic nerve axotomy induced very little syndecan-1 expression in injured spinal motor neurons. These results suggest that syndecan-1 may have a crucial role in the survival of injured motor neurons and in nerve regeneration after injury. Our observations also reveal the diversity of peripheral motor neurons. (C) 2012 Elsevier Ireland Ltd. All rights reserved.

DOI： 10.1016/j.neulet.2012.08.043

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATURE PUBLISHING GROUP  

DOI： 10.1038/jid.2012.18

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Language：Japanese   Publisher：(一社)日本解剖学会  

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DOI： 10.1016/j.neulet.2012.08.043

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DOI： 10.1038/jid.2012.18

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Language：English   Publisher：日本神経化学会  

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Authorship：Lead author   Language：Japanese   Publishing type：Research paper (scientific journal)  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WALTER DE GRUYTER & CO  

Klk8 is a tryptic serine protease with limited substrate specificity. Klk8 mRNA is expressed in many developing organs, whereas its expression is confined to limited regions, including the hippocampus, in adults. In the hippocampus, Klk8 is involved in activity-dependent synaptic changes such as long-term potentiation, which was found to be suppressed in Klk8 knockout (KO) mice. Oligodendrocytes only expressed Klk8 mRNA after injury to the central nervous system. The epidermis of the skin is one of the tissues that exhibits a high level of KLK8 expression. Klk8 might be involved in desquamation through the degradation of adhesive molecules that connect layers of the epidermis. Klk8 might thus be involved in tissue development and rearrangement.

DOI： 10.1515/BC.2010.034

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DOI： 10.1515/BC.2010.034

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL  

Transgenic and disease model mice have been used to investigate the molecular mechanisms of demyelinating diseases. However, less attention has been given to elucidating changes in nerve conduction in these mice. We established an experimental system to measure the response latency of cortical neurons and examined changes in nerve conduction in cuprizone-induced demyelinating mice and in myelin basic protein-deficient shiverer mice. Stimulating and recording electrodes were placed in the right and left sensori-motor cortices, respectively. Electrical stimulation of the right cortex evoked antidromic responses in left cortical neurons with a latency of 9.38 +/- 0.31 ms (n = 107; mean +/- SEM). While response latency was longer in mice at 7 days and 4 weeks of cuprizone treatment (12.35 +/- 0.35 ms, n = 102; 11.72 +/- 0.29 ms, n = 103, respectively), response latency at 7 days and 4 weeks after removal of cuprizone was partially restored (10.72 +/- 0.45 ms, n = 106; 10.27 +/- 0.34 ms, n = 107, respectively). Likewise, electron microscopy showed cuprizone-induced demyelination in the corpus callosum and nearly complete remyelination after cuprizone removal. We also examined whether the myelin abnormalities in shiverer mice affected their response latencies. But there were no significant differences in response latencies in shiverer (9.83 +/- 0.24 ms, n = 103) and wild-type (9.33 +/- 0.22 ms, n = 112) mice. The results of these electrophysiological assessments imply that different demyelinating mechanisms, differentially affecting axon conduction, are present in the cuprizone-treated and shiverer mice, and may provide new insights to understanding the pathophysiology of demyelination in animal models in the CNS.

DOI： 10.1111/j.1460-9568.2008.06474.x

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL  

Transgenic and disease model mice have been used to investigate the molecular mechanisms of demyelinating diseases. However, less attention has been given to elucidating changes in nerve conduction in these mice. We established an experimental system to measure the response latency of cortical neurons and examined changes in nerve conduction in cuprizone-induced demyelinating mice and in myelin basic protein-deficient shiverer mice. Stimulating and recording electrodes were placed in the right and left sensori-motor cortices, respectively. Electrical stimulation of the right cortex evoked antidromic responses in left cortical neurons with a latency of 9.38 +/- 0.31 ms (n = 107; mean +/- SEM). While response latency was longer in mice at 7 days and 4 weeks of cuprizone treatment (12.35 +/- 0.35 ms, n = 102; 11.72 +/- 0.29 ms, n = 103, respectively), response latency at 7 days and 4 weeks after removal of cuprizone was partially restored (10.72 +/- 0.45 ms, n = 106; 10.27 +/- 0.34 ms, n = 107, respectively). Likewise, electron microscopy showed cuprizone-induced demyelination in the corpus callosum and nearly complete remyelination after cuprizone removal. We also examined whether the myelin abnormalities in shiverer mice affected their response latencies. But there were no significant differences in response latencies in shiverer (9.83 +/- 0.24 ms, n = 103) and wild-type (9.33 +/- 0.22 ms, n = 112) mice. The results of these electrophysiological assessments imply that different demyelinating mechanisms, differentially affecting axon conduction, are present in the cuprizone-treated and shiverer mice, and may provide new insights to understanding the pathophysiology of demyelination in animal models in the CNS.

DOI： 10.1111/j.1460-9568.2008.06474.x

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：PERGAMON-ELSEVIER SCIENCE LTD  

Previous studies indicated that the expression of neuropsin, a serine protease, is induced in mature oligodendrocytes after injury to the CNS. The pathophysiology of spinal cord injury (SCI) involves primary and secondary mechanisms, the latter contributing further to permanent losses of function. To explore the role of neuropsin after SCI, histochemical and behavioral analyses were performed in wild-type (WT) and neuropsin-deficient (neuropsin(-/-)) mice using a crush injury model, a well-characterized and consistently reproducible model of SCI. In situ hybridization revealed that neuropsin mRNA expression was induced in the spinal cord white matter from WT mice after crush SCI, peaking at day 4. Neuropsin(-/-) mice showed attenuated demyelination, oligodendrocyte death, and axonal damage after SCI. Although axonal degeneration in the corticospinal tract was obvious caudal to the lesion site in both strains of mice after SCI, the number of surviving nerve fibers caudal to the lesion was significantly larger in neuropsin(-/-) mice than WT mice. Behavioral analysis revealed that the recovery at days 10-42 was significantly improved in neuropsin(-/-) mice compared with WT mice in spite of the severe initial hindlimb impairments due to SCI in both strains. These observations suggest that neuropsin is involved in the secondary phase of the pathogenesis of SCI mediated by demyelination, oligodendrocyte death, and axonal degeneration. (c) 2007 IBRO. Published by Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.neuroscience.2007.05.037

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：PERGAMON-ELSEVIER SCIENCE LTD  

Previous studies indicated that the expression of neuropsin, a serine protease, is induced in mature oligodendrocytes after injury to the CNS. The pathophysiology of spinal cord injury (SCI) involves primary and secondary mechanisms, the latter contributing further to permanent losses of function. To explore the role of neuropsin after SCI, histochemical and behavioral analyses were performed in wild-type (WT) and neuropsin-deficient (neuropsin(-/-)) mice using a crush injury model, a well-characterized and consistently reproducible model of SCI. In situ hybridization revealed that neuropsin mRNA expression was induced in the spinal cord white matter from WT mice after crush SCI, peaking at day 4. Neuropsin(-/-) mice showed attenuated demyelination, oligodendrocyte death, and axonal damage after SCI. Although axonal degeneration in the corticospinal tract was obvious caudal to the lesion site in both strains of mice after SCI, the number of surviving nerve fibers caudal to the lesion was significantly larger in neuropsin(-/-) mice than WT mice. Behavioral analysis revealed that the recovery at days 10-42 was significantly improved in neuropsin(-/-) mice compared with WT mice in spite of the severe initial hindlimb impairments due to SCI in both strains. These observations suggest that neuropsin is involved in the secondary phase of the pathogenesis of SCI mediated by demyelination, oligodendrocyte death, and axonal degeneration. (c) 2007 IBRO. Published by Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.neuroscience.2007.05.037

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

Kallikrein type serine proteases, KLK8/neuropsin, KLK6, and KLK7, have been implicated in the proliferation and differentiation of epidermal keratinocytes and in the pathogenesis of psoriasis. However, their mechanistic roles in these processes remain largely unknown. We applied 12-O-tetradecanoylphorbol-13-acetate on the wild type (WT) and the Klk8 gene-disrupted (Klk8(-/-)) mouse skin, inducing keratinocyte proliferation similar to the human psoriatic lesion. Klk8 mRNA as well as Klk6 and Klk7 mRNA were up-regulated after 12-O-tetradecanoylphorbol-13-acetate application in the WT mice. In contrast, Klk8(-/-) mice showed minimum increases of Klk6 and Klk7 transcripts, the proteins, and enzymatic activities. Relative to the WT, the Klk8(-/-) skin showed less proliferation and an increase in the number of cell layers in the stratum corneum. However, overexpression of Klk8 by adenovirus vector in knock-out keratinocytes did not result in an increase in Klk6 or Klk7 mRNA. The inefficient cleavage of adhesion molecules DSG1 and CDSN in Klk8(-/-) skin contributes to a delay in corneocyte shedding, resulting in the hyperkeratosis phenotype. We propose that in psoriatic lesion, KLK8 modulates hyperproliferation and prevents excessive hyperkeratosis by shedding the corneocytes.

DOI： 10.1074/jbc.M607998200

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

Kallikrein type serine proteases, KLK8/neuropsin, KLK6, and KLK7, have been implicated in the proliferation and differentiation of epidermal keratinocytes and in the pathogenesis of psoriasis. However, their mechanistic roles in these processes remain largely unknown. We applied 12-O-tetradecanoylphorbol-13-acetate on the wild type (WT) and the Klk8 gene-disrupted (Klk8(-/-)) mouse skin, inducing keratinocyte proliferation similar to the human psoriatic lesion. Klk8 mRNA as well as Klk6 and Klk7 mRNA were up-regulated after 12-O-tetradecanoylphorbol-13-acetate application in the WT mice. In contrast, Klk8(-/-) mice showed minimum increases of Klk6 and Klk7 transcripts, the proteins, and enzymatic activities. Relative to the WT, the Klk8(-/-) skin showed less proliferation and an increase in the number of cell layers in the stratum corneum. However, overexpression of Klk8 by adenovirus vector in knock-out keratinocytes did not result in an increase in Klk6 or Klk7 mRNA. The inefficient cleavage of adhesion molecules DSG1 and CDSN in Klk8(-/-) skin contributes to a delay in corneocyte shedding, resulting in the hyperkeratosis phenotype. We propose that in psoriatic lesion, KLK8 modulates hyperproliferation and prevents excessive hyperkeratosis by shedding the corneocytes.

DOI： 10.1074/jbc.M607998200

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER IRELAND LTD  

Protease M/neurosin is a serine protease expressed by oligodendrocytes (OLGs) in the central nervous system (CNS). To investigate the role of protease M/neurosin during experimental demyelination and remyelination, mice were fed cuprizone (bis-cyclohexanon oxaldihydrazone). Semi-quantitative RT-PCR analysis and immunohistochemistry revealed that the expressions of protease M/neurosin mRNA and protein were rapidly reduced in demyelination, whereas the expression of protease M/neurosin was increased in pi form of glutathione-S-transferases (GST-pi)-positive OLGs during remyelination. Cultured primary OLGs displayed a strong correlation between protease M/neurosin and myelin basic protein (MBP). After tumor necrosis factor-alpha (TNF-alpha) and IFN-gamma stimulation, these proteins showed colocalization in the oligodendroglial process. The suppression of protease M/neurosin using RNAi reduced the level of MBP mRNA in cultured OLGs. In contrast, the reduced level of protease M/neurosin was not associated with oligodendroglial cell death or differentiation in cultured OLGs. This study identifies that protease M/neurosin in OLGs is closely associated with the expression of the MBP and the PLP gene. Our data emphasize that the maintenance of myelination is an important function of protease M/neurosin in OLGs, suggesting its relation to the oligodendroglial response to myelin disorders. (c) 2006 Elsevier Ireland Ltd. All rights reserved.

DOI： 10.1016/j.neulet.2006.06.030

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER IRELAND LTD  

Protease M/neurosin is a serine protease expressed by oligodendrocytes (OLGs) in the central nervous system (CNS). To investigate the role of protease M/neurosin during experimental demyelination and remyelination, mice were fed cuprizone (bis-cyclohexanon oxaldihydrazone). Semi-quantitative RT-PCR analysis and immunohistochemistry revealed that the expressions of protease M/neurosin mRNA and protein were rapidly reduced in demyelination, whereas the expression of protease M/neurosin was increased in pi form of glutathione-S-transferases (GST-pi)-positive OLGs during remyelination. Cultured primary OLGs displayed a strong correlation between protease M/neurosin and myelin basic protein (MBP). After tumor necrosis factor-alpha (TNF-alpha) and IFN-gamma stimulation, these proteins showed colocalization in the oligodendroglial process. The suppression of protease M/neurosin using RNAi reduced the level of MBP mRNA in cultured OLGs. In contrast, the reduced level of protease M/neurosin was not associated with oligodendroglial cell death or differentiation in cultured OLGs. This study identifies that protease M/neurosin in OLGs is closely associated with the expression of the MBP and the PLP gene. Our data emphasize that the maintenance of myelination is an important function of protease M/neurosin in OLGs, suggesting its relation to the oligodendroglial response to myelin disorders. (c) 2006 Elsevier Ireland Ltd. All rights reserved.

DOI： 10.1016/j.neulet.2006.06.030

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER IRELAND LTD  

The abdominal sympathetic system is unique in that its postganglionic axons do not directly innervate gastrointestinal smooth muscle layers but exert their effects through the enteric nervous system. The purpose of the present study was to examine the ability of neurons in abdominal sympathetic ganglia to regenerate after axonal injury and to determine whether reinnervation occurs after the removal of ganglia. Axons from the celiac ganglion and superior mesenteric ganglion complex (CG/SMG) of adult female BALB/c mice were crushed or the ganglion complex was removed. Immunohistochemistry, western blotting and in situ hybridization were performed to examine the changes in tyrosine hydroxylase (TH) and growth-associated protein 43 (GAP-43) in the duodenum and the sympathetic ganglia. Terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling and injection of the tracer dye, fluorogold were also performed. After crushing the nerve, TH in the duodenum disappeared and reappeared within 90 days. In the CG/SMG, TH decreased and increased as in the duodenum, while the expression of GAP-43 changed in the opposite direction. Nerve crushing caused cell death to limited number of neurons in the CG/SMG. The removal of CG/SMG decreased TH in the duodenum and stomach, but 180 days later TH-positive innervation was recovered. Fluorogold injection revealed that the inferior mesenteric ganglion reinnervated the stomach. Therefore, postganglionic sympathetic nerves in the abdomen are able to regenerate and reinnervation occurs even after the removal of sympathetic ganglia. (c) 2005 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved.

DOI： 10.1016/j.neures.2005.12.007

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER IRELAND LTD  

The abdominal sympathetic system is unique in that its postganglionic axons do not directly innervate gastrointestinal smooth muscle layers but exert their effects through the enteric nervous system. The purpose of the present study was to examine the ability of neurons in abdominal sympathetic ganglia to regenerate after axonal injury and to determine whether reinnervation occurs after the removal of ganglia. Axons from the celiac ganglion and superior mesenteric ganglion complex (CG/SMG) of adult female BALB/c mice were crushed or the ganglion complex was removed. Immunohistochemistry, western blotting and in situ hybridization were performed to examine the changes in tyrosine hydroxylase (TH) and growth-associated protein 43 (GAP-43) in the duodenum and the sympathetic ganglia. Terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling and injection of the tracer dye, fluorogold were also performed. After crushing the nerve, TH in the duodenum disappeared and reappeared within 90 days. In the CG/SMG, TH decreased and increased as in the duodenum, while the expression of GAP-43 changed in the opposite direction. Nerve crushing caused cell death to limited number of neurons in the CG/SMG. The removal of CG/SMG decreased TH in the duodenum and stomach, but 180 days later TH-positive innervation was recovered. Fluorogold injection revealed that the inferior mesenteric ganglion reinnervated the stomach. Therefore, postganglionic sympathetic nerves in the abdomen are able to regenerate and reinnervation occurs even after the removal of sympathetic ganglia. (c) 2005 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved.

DOI： 10.1016/j.neures.2005.12.007

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：PERGAMON-ELSEVIER SCIENCE LTD  

Heparan sulfate proteoglycans, which bear long chains of heparan sulfate glycosaminoglycan, play significant roles during embryogenesis, including the formation of the CNS. However, their involvement in nerve regeneration has not yet been clarified. Here, we found that the mRNA expression of EXT2, one of the crucial enzymes for heparan sulfate-glycosaminoglycan synthesis, was markedly up-regulated in injured hypoglossal motor neurons after axotomy. In addition, immunohistochemical staining with an antibody specific for heparan sulfate-glycosaminoglycan chains demonstrated increased expression of heparan sulfate-glycosaminoglycan chains in the injured nucleus. Furthermore, the mRNA expressions of glypican-1 and syndecan-1, which are both well-known heparan sulfate proteoglycans, were prominently up-regulated in injured motor neurons. These results suggest that the biosynthesis of heparan sulfate chains promoted by EXT2 is activated in injured motor neurons, and that glypican-1 and syndecan-1 are potent candidates for heparan sulfate proteoglycans involved in peripheral nerve regeneration. (c) 2006 IBRO. Published by Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.neuroscience.2006.05.026

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：PERGAMON-ELSEVIER SCIENCE LTD  

Heparan sulfate proteoglycans, which bear long chains of heparan sulfate glycosaminoglycan, play significant roles during embryogenesis, including the formation of the CNS. However, their involvement in nerve regeneration has not yet been clarified. Here, we found that the mRNA expression of EXT2, one of the crucial enzymes for heparan sulfate-glycosaminoglycan synthesis, was markedly up-regulated in injured hypoglossal motor neurons after axotomy. In addition, immunohistochemical staining with an antibody specific for heparan sulfate-glycosaminoglycan chains demonstrated increased expression of heparan sulfate-glycosaminoglycan chains in the injured nucleus. Furthermore, the mRNA expressions of glypican-1 and syndecan-1, which are both well-known heparan sulfate proteoglycans, were prominently up-regulated in injured motor neurons. These results suggest that the biosynthesis of heparan sulfate chains promoted by EXT2 is activated in injured motor neurons, and that glypican-1 and syndecan-1 are potent candidates for heparan sulfate proteoglycans involved in peripheral nerve regeneration. (c) 2006 IBRO. Published by Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.neuroscience.2006.05.026

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-LISS  

Inflammation, demyelination, and axonal damage of the central nervous system (CNS) are major pathological features of multiple sclerosis (MS). Proteolytic digestion of the blood-brain barrier and myelin protein by serine proteases is known to contribute to the development and progression of MS. Neuropsin, a serine protease, has a role in neuronal plasticity, and its expression has been shown to be upregulated in response to injury to the CNS. To determine the possible involvement of neuropsin in demyelinating diseases of the CNS, we examined its expression in myelin oligodendrocyte glycoprotein (MOG)-induced experimental autoimmune encephalomyelitis (EAE), a recognized animal model for MS. Neuropsin mRNA expression was induced in the spinal cord white matter of mice with EAR Combined in situ hybridization and immunohistochemistry demonstrated that most of the cells expressing neuropsin mRNA showed immunoreactivity for CNPase, a cell-specific marker for oligodendrocytes. Mice lacking neuropsin (neuropsin(-/-)) exhibited an altered EAE progression characterized by delayed onset and progression of clinical symptoms as compared to wild-type mice. Neuropsin(-/-) mice also showed attenuated demyelination and delayed oligodendroglial death early during the course of EAE. These observations suggest that neuropsin is involved in the pathogenesis of EAE mediated by demyelination and oligodendroglial death. (c) 2005 Wiley-Liss, Inc.

DOI： 10.1002/glia.20226

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-LISS  

Inflammation, demyelination, and axonal damage of the central nervous system (CNS) are major pathological features of multiple sclerosis (MS). Proteolytic digestion of the blood-brain barrier and myelin protein by serine proteases is known to contribute to the development and progression of MS. Neuropsin, a serine protease, has a role in neuronal plasticity, and its expression has been shown to be upregulated in response to injury to the CNS. To determine the possible involvement of neuropsin in demyelinating diseases of the CNS, we examined its expression in myelin oligodendrocyte glycoprotein (MOG)-induced experimental autoimmune encephalomyelitis (EAE), a recognized animal model for MS. Neuropsin mRNA expression was induced in the spinal cord white matter of mice with EAR Combined in situ hybridization and immunohistochemistry demonstrated that most of the cells expressing neuropsin mRNA showed immunoreactivity for CNPase, a cell-specific marker for oligodendrocytes. Mice lacking neuropsin (neuropsin(-/-)) exhibited an altered EAE progression characterized by delayed onset and progression of clinical symptoms as compared to wild-type mice. Neuropsin(-/-) mice also showed attenuated demyelination and delayed oligodendroglial death early during the course of EAE. These observations suggest that neuropsin is involved in the pathogenesis of EAE mediated by demyelination and oligodendroglial death. (c) 2005 Wiley-Liss, Inc.

DOI： 10.1002/glia.20226

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER IRELAND LTD  

To determine the possible involvement of protease M/neurosin in demyelinating diseases of the CNS, we examined its expression in myelin oligodendrocyte glycoprotein (MOG)-induced experimental autoimmune encephalomyelitis (EAE), a recognized animal model of multiple sclerosis (NIS). In situ hybridization, immunohistochemistry and quantitative real-time polymerase chain reaction (PCR) demonstrated that EAE caused an increase in the expression of protease M/neurosin mRNA and its protein product throughout the white and gray matter surrounding demyelinating lesions. Combined in situ hybridization and immunohistochemistry demonstrated that most of the cells expressing protease M/neurosin mRNA within control spinal cord showed immunoreactivity for CNPase or NG2, cell-specific markers for oligodendrocytes and their progenitors, respectively. In the spinal cord from mice with EAE, the expression of protease M/neurosin mRNA in CNPase-positive cells appeared to be increased while double-labeled cells positive for protease M/neurosin mRNA and NG2 were rarely found in areas associated with demyelinating lesions. Although a prominent accumulation of inflammatory cells including T-cells was observed in the vicinity of demyelinated lesions, these cells were not associated with protease M/neurosin mRNA expression. The levels of protease M/neurosin mRNA expression were unchanged in the spleen and even decreased in the thymus during the course of EAE. These observations suggest that the differential expression of protease M/neurosin in mature oligodendrocytes and their progenitors is involved in the pathogenesis of NIS and EAE. (c) 2005 Elsevier Ireland Ltd. All rights reserved.

DOI： 10.1016/j.neulet.2005.03.022

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER IRELAND LTD  

To determine the possible involvement of protease M/neurosin in demyelinating diseases of the CNS, we examined its expression in myelin oligodendrocyte glycoprotein (MOG)-induced experimental autoimmune encephalomyelitis (EAE), a recognized animal model of multiple sclerosis (NIS). In situ hybridization, immunohistochemistry and quantitative real-time polymerase chain reaction (PCR) demonstrated that EAE caused an increase in the expression of protease M/neurosin mRNA and its protein product throughout the white and gray matter surrounding demyelinating lesions. Combined in situ hybridization and immunohistochemistry demonstrated that most of the cells expressing protease M/neurosin mRNA within control spinal cord showed immunoreactivity for CNPase or NG2, cell-specific markers for oligodendrocytes and their progenitors, respectively. In the spinal cord from mice with EAE, the expression of protease M/neurosin mRNA in CNPase-positive cells appeared to be increased while double-labeled cells positive for protease M/neurosin mRNA and NG2 were rarely found in areas associated with demyelinating lesions. Although a prominent accumulation of inflammatory cells including T-cells was observed in the vicinity of demyelinated lesions, these cells were not associated with protease M/neurosin mRNA expression. The levels of protease M/neurosin mRNA expression were unchanged in the spleen and even decreased in the thymus during the course of EAE. These observations suggest that the differential expression of protease M/neurosin in mature oligodendrocytes and their progenitors is involved in the pathogenesis of NIS and EAE. (c) 2005 Elsevier Ireland Ltd. All rights reserved.

DOI： 10.1016/j.neulet.2005.03.022

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SOC NEUROSCIENCE  

The subthalamic nucleus (STN) is a key component of basal ganglia circuitry that mediates a variety of motor functions. The STN neurons send glutamatergic projections to the output structures of basal ganglia, including the substantia nigra pars reticulata (SNr) and the entopeduncular nucleus, and also innervate the globus pallidus ( GP). However, the mechanism by which the STN regulates motor functions in the neural circuitry is not fully understood. Here we performed conditional ablation of the STN neurons by using immunotoxin-mediated cell targeting. We then analyzed dopamine (DA)-mediated motor behavior and firing activity of the SNr and GP neurons. Ablation of the STN neurons increased spontaneous movement and reduced hyperactivity in response to DA stimulation. Ablation of these neurons modulated the pattern and rate of spontaneous firing of the SNr neurons, although it did not substantially affect spontaneous firing of the GP neurons. The ablation attenuated DA-induced suppression of the firing rate of the SNr neurons and inhibited DA-induced elevation of the rate of the GP neurons. In addition, pharmacological blockade of GP activation in response to DA stimulation inhibited the suppression of SNr activity and the resultant motor activation. These results suggest that the STN neurons suppress spontaneous behavior through their direct projection to the output neurons and that, in response to DA, they contribute to expression of behavior by acting on the output neurons mainly through the GP-mediated pathways. We conclude that the STN coordinates motor behavior through differential neural pathways depending on the state of DA transmission.

DOI： 10.1523/JNEUROSCI.1904-05.2005

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Authorship：Lead author   Language：Japanese   Publishing type：Research paper (bulletin of university, research institution)  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-LISS  

Neuropsin and protease M/neurosin are serine proteases expressed by neurons and glial cells, and serve a variety of functions in the central nervous system (CNS). The current study demonstrates changes in the expression of these proteases following hemisection of the mouse spinal cord. Within unlesioned spinal cord, neuropsin mRNA expression was occasionally observed in the gray but not white matter, while the level of protease M/neurosin mRNA was higher in the white matter. After injury to the spinal cord, neuropsin mRNA expression was induced in the white matter in the area immediately adjacent to the lesion, peaking at 4 days post-injury and disappearing by 14 days. Enhanced expression of protease M/neurosin mRNA was observed throughout the white and gray matter surrounding the lesion, peaking at 4 days and persisting for 14 days. Neuropsin mRNA was expressed predominantly by CNPase-positive oligodendrocytes. Furthermore, most of these cells were also associated with immunoreactivity for protease M/neurosin protein. Within unlesioned spinal cord, most protease M/neurosin mRNA-expressing cells were CNPase-positive oligodendrocytes, and a substantial fraction of these cells also showed immunoreactivity for NG2, a marker for oligodendrocyte progenitors. After injury, protease M/neurosin mRNA expression within NG2-positive cells was significantly decreased, while the constitutive expression in CNPase-positive oligodendrocytes appeared to be preserved. These findings suggest that each subpopulation of oligodendrocytes based on the expression of neuropsin and protease M/neurosin has different roles in the response of the spinal cord to injury as well as in normal homeostasis. (C) 2004 Wiley-Liss, Inc.

DOI： 10.1002/glia.20058

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-LISS  

Neuropsin and protease M/neurosin are serine proteases expressed by neurons and glial cells, and serve a variety of functions in the central nervous system (CNS). The current study demonstrates changes in the expression of these proteases following hemisection of the mouse spinal cord. Within unlesioned spinal cord, neuropsin mRNA expression was occasionally observed in the gray but not white matter, while the level of protease M/neurosin mRNA was higher in the white matter. After injury to the spinal cord, neuropsin mRNA expression was induced in the white matter in the area immediately adjacent to the lesion, peaking at 4 days post-injury and disappearing by 14 days. Enhanced expression of protease M/neurosin mRNA was observed throughout the white and gray matter surrounding the lesion, peaking at 4 days and persisting for 14 days. Neuropsin mRNA was expressed predominantly by CNPase-positive oligodendrocytes. Furthermore, most of these cells were also associated with immunoreactivity for protease M/neurosin protein. Within unlesioned spinal cord, most protease M/neurosin mRNA-expressing cells were CNPase-positive oligodendrocytes, and a substantial fraction of these cells also showed immunoreactivity for NG2, a marker for oligodendrocyte progenitors. After injury, protease M/neurosin mRNA expression within NG2-positive cells was significantly decreased, while the constitutive expression in CNPase-positive oligodendrocytes appeared to be preserved. These findings suggest that each subpopulation of oligodendrocytes based on the expression of neuropsin and protease M/neurosin has different roles in the response of the spinal cord to injury as well as in normal homeostasis. (C) 2004 Wiley-Liss, Inc.

DOI： 10.1002/glia.20058

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：BLACKWELL PUBLISHING INC  

Lamellar granules (LG) of the epidermis appear as discrete round or oblong shaped granules in classical transmission electron micrographs, but a recent cryo-transmission electron microscopy study has claimed that LG are in fact branched tubular structures. LG contain various cargoes including lipids, hydrolytic enzymes, and several other proteins. It is not known whether there are any differences in the timing of expression among them and whether they are sorted into the granules individually or collectively. In order to address these questions, we studied the expression of glucosylceramides (GlcCer), cathepsin D (CatD), corneodesmosin (Cdsn), kallikrein (KLK)7, and KLK8 in normal human epidermis using confocal laser scanning microscopy and immunoelectron microscopy. The results were consistent with the model that LG are parts of a branched tubular structure. In this structure, all the components were shown to be distributed as separate aggregates. In the trans-Golgi network (TGN), bulbous protrusions containing GlcCer, Cdsn, KLK7 and KLK8, and small CatD-positive vesicles were observed. The molecules were shown to be delivered to the apical region of granular keratinocytes. This study provides strong evidence for the sequential synthesis and independent trafficking of various LG cargoes, including for the first time CatD and KLK8, from TGN.

DOI： 10.1111/j.0022-202X.2004.22515.x

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DOI： 10.1111/j.0022-202X.2004.22515.x

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：BLACKWELL PUBLISHING LTD  

Background Neuropsin (KLK8), a serine protease of the kallikrein family, is thought to be involved in the function of keratinocytes, i.e. migration, differentiation and desquamation. However, how neuropsin participates is still unknown.
Objective To observe the epidermal function of serine protease in neuropsin-deficient mice.
Methods We irradiated the skin of neuropsin-deficient mice with ultraviolet light to induce acute inflammation and compared the morphology with that of wild-type mice.
Results We observed a phenotypic change in the epidermis. An acute inflammatory dose of ultraviolet light induced a marked increase in neuropsin mRNA expression in the skin. The signal intensity of the mRNA expression was highest on day 2-3 after irradiation, when keratinocytes were aligned irregularly in the recovery period. Morphological comparison between neuropsin -/- and +/+ mice revealed that an irregular alignment of cells in the thickened epidermis was obvious on day 2 after irradiation in the wild-type mice, whereas it was prolonged for at least 2 days in the neuropsin-deficient mice. The stratum corneum of neuropsin-deficient mice was remarkably thicker than that of the wild-type mice at 5, 14 and 21 days after irradiation. The increase, as a response to this stimulus, in involucrin immunoreactivity, a marker for cell envelope assembly, was delayed in the mutant mice.
Conclusions Thus, neuropsin might be involved early in the process of differentiation, such as in the assembly of the cell envelope, but not in migration and desquamation.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SOC STUDY REPRODUCTION  

Uterine decidualization is accompanied by the remodeling of the cell-matrix and cell-cell interactions around the endometrial stromal cells to allow an appropriate invasion of trophoblasts. This remodeling is thought to require the proteolysis of extracellular matrix proteins or cell adhesion molecules; however, the molecular mechanism remains poorly understood. In this study, decidualization induced the expression and activation of an extracellular serine protease neuropsin in the mouse uterus. Although nonpregnant uteri contained little neuropsin, the protein content and enzymatic activity increased markedly and peaked at the midgestational period in pregnant uteri. Neuropsin expression and activity was also upregulated in artificially induced deciduomata but not in nondecidualized pseudopregnant uteri. Neuropsin is the first extracellular protease to show the evident induction of expression and activity by decidualization and might contribute to the remodeling of extracellular components after decidualization.

DOI： 10.1095/biolreprod.102.006080

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SOC STUDY REPRODUCTION  

Uterine decidualization is accompanied by the remodeling of the cell-matrix and cell-cell interactions around the endometrial stromal cells to allow an appropriate invasion of trophoblasts. This remodeling is thought to require the proteolysis of extracellular matrix proteins or cell adhesion molecules; however, the molecular mechanism remains poorly understood. In this study, decidualization induced the expression and activation of an extracellular serine protease neuropsin in the mouse uterus. Although nonpregnant uteri contained little neuropsin, the protein content and enzymatic activity increased markedly and peaked at the midgestational period in pregnant uteri. Neuropsin expression and activity was also upregulated in artificially induced deciduomata but not in nondecidualized pseudopregnant uteri. Neuropsin is the first extracellular protease to show the evident induction of expression and activity by decidualization and might contribute to the remodeling of extracellular components after decidualization.

DOI： 10.1095/biolreprod.102.006080

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：BRITISH MED JOURNAL PUBL GROUP  

Aim: The expression of human neuropsin (KLK8) mRNA in normal and pathological skin samples was analysed and the results compared with those for tissue plasminogen activator (tPA) mRNA.
Methods: Northern blot and in situ hybridisation analyses of KLK8 mRNA in normal and lesional skin of patients with cutaneous diseases were performed .
Results: A weak signal for KLK8 mRNA and no signal for tPA mRNA was seen in normal skin on northern blot analysis. Weak signals for KLK8 were localised to the superficial cells beneath the cornified layer in normal skin on in situ hybridisation. Psoriasis vulgaris, seborrheic keratosis, lichen planus, and squamous cell carcinoma skin samples, which show severe hyperkeratosis, displayed a high density of KLK8 mRNA on northern and in situ hybridisation analyses. The signals were localised in granular and spinous layers of lesional skin in all hyperkeratic samples, including the area surrounding the horn pearls of squamous cell carcinoma. To examine the relation between mRNA expression and terminal differentiation, the expression of KLK8 mRNA was analysed in cell cultures. When keratinisation proceeded in high calcium medium, a correlative increase in the expression of KLK8 mRNA was observed.
Conclusion: The results are consistent with a role for this protease in the terminal differentiation of keratinocytes.

DOI： 10.1136/mp.55.4.235

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Language：English   Publishing type：Research paper (scientific journal)  

Aim: The expression of human neuropsin (KLK8) mRNA in normal and pathological skin samples was analysed and the results compared with those for tissue plasminogen activator (tPA) mRNA. Methods: Northern blot and in situ hybridisation analyses of KLK8 mRNA in normal and lesional skin of patients with cutaneous diseases were performed. Results: A weak signal for KLK8 mRNA and no signal for tPA mRNA was seen in normal skin on northern blot analysis. Weak signals for KLK8 were localised to the superficial cells beneath the cornified layer in normal skin on in situ hybridisation. Psoriasis vulgaris, seborrheic keratosis, lichen planus, and squamous cell carcinoma skin samples, which show severe hyperkeratosis, displayed a high density of KLK8 mRNA on northern and in situ hybridisation analyses. The signals were localised in granular and spinous layers of lesional skin in all hyperkeratic samples, including the area surrounding the horn pearls of squamous cell carcinoma. To examine the relation between mRNA expression and terminal differentiation, the expression of KLK8 mRNA was analysed in cell cultures. When keratinisation proceeded in high calcium medium, a correlative increase in the expression of KLK8 mRNA was observed. Conclusion: The results are consistent with a role for this protease in the terminal differentiation of keratinocytes.

DOI： 10.1136/mp.55.4.235

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCI IRELAND LTD  

Myelin associated glycoprotein (MAG) has growth promoting effect on mouse cerebellar neurons. In the present study, we examined which isoform of MAG has the effect. cDNA for L-MAG and S-MAG was stably transfected into BALB/c 3T3 cells, on which cerebellar neurons were cultured. The neurons were stained with antibody against microtubule-associated protein-2. Neurites of the neurons cultured on cells expressing L-MAG extended significantly further than those cultured on cells expressing S-MAG or on control cells. Therefore, intracellular domain of MAG may have the potential to affect MAG-neurite interaction. (C) 2001 Elsevier Science Ireland Ltd. All rights reserved.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCI IRELAND LTD  

Myelin associated glycoprotein (MAG) has growth promoting effect on mouse cerebellar neurons. In the present study, we examined which isoform of MAG has the effect. cDNA for L-MAG and S-MAG was stably transfected into BALB/c 3T3 cells, on which cerebellar neurons were cultured. The neurons were stained with antibody against microtubule-associated protein-2. Neurites of the neurons cultured on cells expressing L-MAG extended significantly further than those cultured on cells expressing S-MAG or on control cells. Therefore, intracellular domain of MAG may have the potential to affect MAG-neurite interaction. (C) 2001 Elsevier Science Ireland Ltd. All rights reserved.

DOI： 10.1016/S0304-3940(01)02113-9

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：LIPPINCOTT WILLIAMS & WILKINS  

The human kallikrein gene family consists of 15 serine proteases. We examined the expression of the kallikrein genes in human cerebral cortex and hippocampus by RT-PCR and compared their expression between Alzheimer's disease (AD) and control tissue. KLKI, 4, 5, 6, 7, 8, 10, 11, 13 and 14 are expressed in both cerebral cortex and hippocampus. KLK9 is expressed in cortex but not hippocampus, whereas KLK2, 3, 12 and 15 are not expressed in either tissue. We demonstrate an I 1.5-fold increase in KLK8 mRNA levels in AD hippocampus compared to controls. The KLK8 gene product, neuropsin, processes extracellular matrix and is important for neuronal plasticity. Therefore, the increase in KLK8 could have detrimental effects on hippocampal function in AD. NeuroReport 12:2747-2751 (C) 2001 Lippincott Williams & Wilkins.

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Proteases are involved in a variety of processes including demyelination after injury to the central nervous systyem. Neuropsin is a serine protease. which is constitutively expressed in the neurons of the limbic system. In the present study. intrahippocampal kainate injection and enucleation were performed on adult mice. Neuropsin mRNA and protein expression was detected by in situ hybridization and immunohistochemistry. Double in situ hybridization confirmed that the mRNA expression was induced in oligodendrocytes. One day after kainate injection to the hippocampus, neuropsin mRNA was expressed. peaking 4-8 days postoperatively and disappearing at 14 days. Immunohistochemistry and immunoelectron microscopy revealed that neuropsin was expressed in the cell body of oligodendrocytes and myelin. To see if neuropsin degrades myelin protein, purified myelin was incubated with recombinant neuropsin. A decrease in the intensity of the bands of myelin basic protein was observed. These results indicate that neuropsin is involved in demyelination. (C) 2001 Elsevier Science Ireland Ltd and the Japan Neuroscience Society. All rights reserved.

DOI： 10.1016/S0168-0102(01)00200-0

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCI IRELAND LTD  

Proteases are involved in a variety of processes including demyelination after injury to the central nervous systyem. Neuropsin is a serine protease. which is constitutively expressed in the neurons of the limbic system. In the present study. intrahippocampal kainate injection and enucleation were performed on adult mice. Neuropsin mRNA and protein expression was detected by in situ hybridization and immunohistochemistry. Double in situ hybridization confirmed that the mRNA expression was induced in oligodendrocytes. One day after kainate injection to the hippocampus, neuropsin mRNA was expressed. peaking 4-8 days postoperatively and disappearing at 14 days. Immunohistochemistry and immunoelectron microscopy revealed that neuropsin was expressed in the cell body of oligodendrocytes and myelin. To see if neuropsin degrades myelin protein, purified myelin was incubated with recombinant neuropsin. A decrease in the intensity of the bands of myelin basic protein was observed. These results indicate that neuropsin is involved in demyelination. (C) 2001 Elsevier Science Ireland Ltd and the Japan Neuroscience Society. All rights reserved.

DOI： 10.1016/S0168-0102(01)00200-0

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC  

In the present study,we produced null-mutant mice of neuropsin, an extracellular matrix serine protease, to examine the neural functions of this protein particularly in the hippocampus. Golgi-Cox impregnation and NissI-staining revealed morphological change of cell soma in the mutant mice compared to wild-type mice. However, Golgi-Cox impregnation revealed no apparent change in the dendritic arborization and spine density. Quantitative electronmicroscopic analysis revealed that number of asymmetrical synapses were significantly decreased in the stratum radiatum, the major terminal field of Schaffer-collaterals, whereas free boutons still holding synaptic vesicles but with no synaptic specialization were increased in number in the same microscopic fields. An increased number of parvalbumin-immunoreactive cells (known as fast spiking cells) in mutant was also observed. These results strongly suggest that neuropsin is involved in connectivity of a group of CA1 synapses and consequently in the hippocampal networking.

DOI： 10.1006/mcne.2000.0945

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC  

In the present study,we produced null-mutant mice of neuropsin, an extracellular matrix serine protease, to examine the neural functions of this protein particularly in the hippocampus. Golgi-Cox impregnation and NissI-staining revealed morphological change of cell soma in the mutant mice compared to wild-type mice. However, Golgi-Cox impregnation revealed no apparent change in the dendritic arborization and spine density. Quantitative electronmicroscopic analysis revealed that number of asymmetrical synapses were significantly decreased in the stratum radiatum, the major terminal field of Schaffer-collaterals, whereas free boutons still holding synaptic vesicles but with no synaptic specialization were increased in number in the same microscopic fields. An increased number of parvalbumin-immunoreactive cells (known as fast spiking cells) in mutant was also observed. These results strongly suggest that neuropsin is involved in connectivity of a group of CA1 synapses and consequently in the hippocampal networking.

DOI： 10.1006/mcne.2000.0945

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCI IRELAND LTD  

Neuropsin is a serine protease cloned from the mouse hippocampus. Since neuropsin is a secreted protein which effectively cleaves fibronectin, it may affect cell adhesion or cell migration by modulating the content and/or chemical characteristics of fibronectin in extracellular matrix (ECM). In adhesion assays, alpha 5B2 cells expressing integrin alpha5 beta1 bound less effectively to fibronectin teated with neuropsin than intact fibronectin. In Boyden chamber chemotaxis assays, the fibronectin-induced migration of alpha 5B2 cells was not affected by neuropsin treatment. These findings suggest that neuropsin regulates the local microenvironment by modulating the interaction between cells acid fibronectin in ECM. (C) 2001 Elsevier Science Ireland Ltd and the Japan Neuroscience Society. All rights reserved.

DOI： 10.1016/S0168-0102(00)00213-3

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Neuropsin is a serine protease cloned from the mouse hippocampus. Since neuropsin is a secreted protein which effectively cleaves fibronectin, it may affect cell adhesion or cell migration by modulating the content and/or chemical characteristics of fibronectin in extracellular matrix (ECM). In adhesion assays, alpha 5B2 cells expressing integrin alpha5 beta1 bound less effectively to fibronectin teated with neuropsin than intact fibronectin. In Boyden chamber chemotaxis assays, the fibronectin-induced migration of alpha 5B2 cells was not affected by neuropsin treatment. These findings suggest that neuropsin regulates the local microenvironment by modulating the interaction between cells acid fibronectin in ECM. (C) 2001 Elsevier Science Ireland Ltd and the Japan Neuroscience Society. All rights reserved.

DOI： 10.1016/S0168-0102(00)00213-3

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER ASSOC CLINICAL CHEMISTRY  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER ASSOC CLINICAL CHEMISTRY  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：BLACKWELL SCIENCE LTD  

We found that neuropsin, an extracellular matrix serine protease, has a regulatory effect on Schaffer-collateral long-term potentiation (LTP) in the mouse hippocampus. Bath application of 1-170 nM recombinant neuropsin modulated early phase LTP in the Schaffer-collateral pathway with a 'bell-shape' dose-response curve. The maximum enhancing activity (134% of control LTP) was found at similar to 2.5 nM. Bath application of a neutralizing antibody against neuropsin in the hippocampal slice resulted in a marked inhibition of the tetanus-induced early phase of LTP. The in vivo continuous intraventricular infusion of an antisense oligonucleotide against neuropsin significantly reduced the amplitude of the tetanus-induced early phase of LTP in vitro. Neuropsin did not directly change the N-methyl D-aspartate (NMDA) current. Thus, neuropsin appears to act as a regulatory molecule in the early phase of LTP via its proteolytic function on extracellular matrix rather than affecting NMDA receptor-mediated calcium increase.

DOI： 10.1046/j.1460-9568.2000.00035.x

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：BLACKWELL SCIENCE LTD  

We found that neuropsin, an extracellular matrix serine protease, has a regulatory effect on Schaffer-collateral long-term potentiation (LTP) in the mouse hippocampus. Bath application of 1-170 nM recombinant neuropsin modulated early phase LTP in the Schaffer-collateral pathway with a 'bell-shape' dose-response curve. The maximum enhancing activity (134% of control LTP) was found at similar to 2.5 nM. Bath application of a neutralizing antibody against neuropsin in the hippocampal slice resulted in a marked inhibition of the tetanus-induced early phase of LTP. The in vivo continuous intraventricular infusion of an antisense oligonucleotide against neuropsin significantly reduced the amplitude of the tetanus-induced early phase of LTP in vitro. Neuropsin did not directly change the N-methyl D-aspartate (NMDA) current. Thus, neuropsin appears to act as a regulatory molecule in the early phase of LTP via its proteolytic function on extracellular matrix rather than affecting NMDA receptor-mediated calcium increase.

DOI： 10.1046/j.1460-9568.2000.00035.x

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：KARGER  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

No proteases have been identified to be oligodendrocyte-specific though there are phenomena in which proteases may be committed. We have cloned a cDNA for mouse homologue of protease M/neurosin, serine protease, and localized its mRNA in the mouse brain. The cDNA and the deduced amino acid sequences were 66% identical to those of human protease M/neurosin. In situ hybridization histochemistry revealed that the mRNA was localized in the mature oligodendrocytes which expressed proteolipid protein mRNA. Developmentally, protease M/neurosin mRNA was expressed by oligodendrocytes 2 to 7 days after the maturation. Thus, protease M/neurosin may be important to the processes occurring after the maturation of oligodendrocytes such as myelination or turnover of the proteins in the myelin. (C) 1999 Elsevier Science B.V. All rights reserved.

DOI： 10.1016/S0169-328X(99)00187-4

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

No proteases have been identified to be oligodendrocyte-specific though there are phenomena in which proteases may be committed. We have cloned a cDNA for mouse homologue of protease M/neurosin, serine protease, and localized its mRNA in the mouse brain. The cDNA and the deduced amino acid sequences were 66% identical to those of human protease M/neurosin. In situ hybridization histochemistry revealed that the mRNA was localized in the mature oligodendrocytes which expressed proteolipid protein mRNA. Developmentally, protease M/neurosin mRNA was expressed by oligodendrocytes 2 to 7 days after the maturation. Thus, protease M/neurosin may be important to the processes occurring after the maturation of oligodendrocytes such as myelination or turnover of the proteins in the myelin. (C) 1999 Elsevier Science B.V. All rights reserved.

DOI： 10.1016/S0169-328X(99)00187-4

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Language：English   Publishing type：Research paper (scientific journal)  

A cDNA for a novel serine protease, termed brain type granzyme K (B-GRK) was cloned from the mouse brain. The cDNA codes a protein similar to granzyme K (GRK) but completely different at the N-terminus. Genomic Southern and PCR analysis of the gene suggests B-GRK is the alternative transcription form of GRK. B-GRK and GRK have a different organ-specific expression pattern: B-GRK is expressed in the brain, while GRK is expressed in the spleen. The recombinant fusion protein was detected in the neuro2a cells transfected with a plasmid containing B-GRK sequence. The mRNA for B-GRK/GRK was detected in cerebral cortex, hippocampus and diencephalon of the mouse brain. In situ hybridization for B-GRK/GRK revealed that several regions in the forebrain and hypothalamus express the mRNA. Developmental analysis showed that in the prenatal stage, the mRNA was expressed also in pituitary and pineal body in addition to the brain. Copyright (C) 1999 Elsevier Science B.V.

DOI： 10.1016/S0169-328X(99)00166-7

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Language：English   Publishing type：Research paper (scientific journal)  

A cDNA for a novel serine protease, termed brain type granzyme K (B-GRK) was cloned from the mouse brain. The cDNA codes a protein similar to granzyme K (GRK) but completely different at the N-terminus. Genomic Southern and PCR analysis of the gene suggests B-GRK is the alternative transcription form of GRK. B-GRK and GRK have a different organ-specific expression pattern: B-GRK is expressed in the brain, while GRK is expressed in the spleen. The recombinant fusion protein was detected in the neuro2a cells transfected with a plasmid containing B-GRK sequence. The mRNA for B-GRK/GRK was detected in cerebral cortex, hippocampus and diencephalon of the mouse brain. In situ hybridization for B-GRK/GRK revealed that several regions in the forebrain and hypothalamus express the mRNA. Developmental analysis showed that in the prenatal stage, the mRNA was expressed also in pituitary and pineal body in addition to the brain. Copyright (C) 1999 Elsevier Science B.V.

DOI： 10.1016/S0169-328X(99)00166-7

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCI IRELAND LTD  

We have isolated cDNAs to two transcripts, granzyme M and alternative granzyme M mRNA from the mouse eye. Analysis of genomic DNA revealed these transcripts were derived from alternative transcription initiations. Northern blot analysis and reverse transcription-polymerase chain reactions revealed that both transcripts were expressed in the eye, though the alternative form was the major type. In situ hybridization studies demonstrated that alternative granzyme hi mRNA localized exclusively in the photoreceptor cells in the retina and expressed only after the Opening of the eye, suggesting that these transcripts are related to the maintenance of the retinal structure or functions of matured photoreceptor cells rather than the development or differentiation of retinal cells. (C) 1999 Elsevier Science Ireland Ltd. All rights reserved.

DOI： 10.1016/S0168-0102(99)00036-X

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCI IRELAND LTD  

We have isolated cDNAs to two transcripts, granzyme M and alternative granzyme M mRNA from the mouse eye. Analysis of genomic DNA revealed these transcripts were derived from alternative transcription initiations. Northern blot analysis and reverse transcription-polymerase chain reactions revealed that both transcripts were expressed in the eye, though the alternative form was the major type. In situ hybridization studies demonstrated that alternative granzyme hi mRNA localized exclusively in the photoreceptor cells in the retina and expressed only after the Opening of the eye, suggesting that these transcripts are related to the maintenance of the retinal structure or functions of matured photoreceptor cells rather than the development or differentiation of retinal cells. (C) 1999 Elsevier Science Ireland Ltd. All rights reserved.

DOI： 10.1016/S0168-0102(99)00036-X

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER VERLAG  

The expression of neuropsin mRNA in vivo in mouse skin was examined by in situ hybridization and Northern blotting under stimulated conditions. Two kinds of epidermal stimuli, a topical application of a chemical tumor promoter and incisional wounding, were used. A single topical application of 12-O-tetradecanoyl-phorbol 13-acetate induced epidermal hyperplasia and simultaneously induced an extensive increase in neuropsin mRNA in the suprabasal cells. A full-thickness skin incision also induced a profound increase in neuropsin mRNA in the suprabasal cells surrounding the mound but not in actively proliferating basal cells. The increases in neuropsin mRNA occurred rather late and were limited to the site of drug application or around the incision. Interestingly, neuropsin mRNA was not expressed in the epithelial tongue migrating toward the wound during re-epithelialization. Thus, neuropsin might participate in accelerated epidermal differentiation rather than in the proliferation or migration of keratinocytes in the wound.

DOI： 10.1007/s004030050418

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER VERLAG  

The expression of neuropsin mRNA in vivo in mouse skin was examined by in situ hybridization and Northern blotting under stimulated conditions. Two kinds of epidermal stimuli, a topical application of a chemical tumor promoter and incisional wounding, were used. A single topical application of 12-O-tetradecanoyl-phorbol 13-acetate induced epidermal hyperplasia and simultaneously induced an extensive increase in neuropsin mRNA in the suprabasal cells. A full-thickness skin incision also induced a profound increase in neuropsin mRNA in the suprabasal cells surrounding the mound but not in actively proliferating basal cells. The increases in neuropsin mRNA occurred rather late and were limited to the site of drug application or around the incision. Interestingly, neuropsin mRNA was not expressed in the epithelial tongue migrating toward the wound during re-epithelialization. Thus, neuropsin might participate in accelerated epidermal differentiation rather than in the proliferation or migration of keratinocytes in the wound.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

We have shown that neuropsin is expressed in the neurons of the limbic system in the adult mouse. After the central nervous system was injured by incision or intraperitoneal kainate injection, neuropsin mRNA was induced in the peri-lesioned region. The cells in which neuropsin mRNA was induced were localized mainly in axon fiber pathways and closely associated to proteolipid protein (PLP) mRNA expressing oligodendrocytes, (C) 1999 Elsevier Science B.V. All rights reserved.

DOI： 10.1016/S0006-8993(99)01238-X

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

We have shown that neuropsin is expressed in the neurons of the limbic system in the adult mouse. After the central nervous system was injured by incision or intraperitoneal kainate injection, neuropsin mRNA was induced in the peri-lesioned region. The cells in which neuropsin mRNA was induced were localized mainly in axon fiber pathways and closely associated to proteolipid protein (PLP) mRNA expressing oligodendrocytes, (C) 1999 Elsevier Science B.V. All rights reserved.

DOI： 10.1016/S0006-8993(99)01238-X

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

Neuropsin is a novel serine protease, the expression of which is highly localized in the limbic areas of the mouse brain and which is suggested to be involved in kindling epileptogenesis and hippocampal plasticity, The 2.1-Angstrom resolution crystal structure of neuropsin provides the first three-dimensional view of one of the serine proteases highly expressed in the nervous system, and reveals a serine protease fold that exhibits chimeric features between trypsin and nerve growth factor-gamma (NGF gamma), a member of the kallikrein family. Neuropsin possesses an N-glycosylated "kallikrein loop" but forms six disulfide bonds corresponding to those of trypsin, The ordered kallikrein loop projects proline toward the active site tee restrict smaller residues or proline at the P2 position of substrates. Loop F, which participates in forming the S3/S4 sites, is similar to trypsin rather than NGF gamma. The unique conformations of loops G and H form an S1 pocket specific for both arginine and lysine, These characteristic loop structures forming the substrate-binding site suggest the novel substrate specificity of neuropsin and give a clue to the design of its specific inhibitors.

DOI： 10.1074/jbc.274.7.4220

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

Neuropsin is a novel serine protease, the expression of which is highly localized in the limbic areas of the mouse brain and which is suggested to be involved in kindling epileptogenesis and hippocampal plasticity, The 2.1-Angstrom resolution crystal structure of neuropsin provides the first three-dimensional view of one of the serine proteases highly expressed in the nervous system, and reveals a serine protease fold that exhibits chimeric features between trypsin and nerve growth factor-gamma (NGF gamma), a member of the kallikrein family. Neuropsin possesses an N-glycosylated "kallikrein loop" but forms six disulfide bonds corresponding to those of trypsin, The ordered kallikrein loop projects proline toward the active site tee restrict smaller residues or proline at the P2 position of substrates. Loop F, which participates in forming the S3/S4 sites, is similar to trypsin rather than NGF gamma. The unique conformations of loops G and H form an S1 pocket specific for both arginine and lysine, These characteristic loop structures forming the substrate-binding site suggest the novel substrate specificity of neuropsin and give a clue to the design of its specific inhibitors.

DOI： 10.1074/jbc.274.7.4220

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

In the course of a study conducted to identify the mouse homologue of Drosophila eyes absent (eya), we isolated a novel mouse cDNA fragment which show little homology to eya but encodes a protein with Kruppel (C2H2)-type zinc finger motifs. By further screening using this cDNA fragment as a probe, we obtained the short and long forms of full-length cDNAs, which were apparently alternatively spliced products from one gene. Since both mRNAs encode proteins with widely-interspaced zinc finger motifs, we termed this gene wiz and refer to the short and long wiz transcripts as wizS and WizL, respectively. In situ hybridization studies using the probe against the region common to wizS and wizL showed that these mRNAs were expressed abundantly in the granule cell layers of the mouse cerebellum, the olfactory bulb, and the dentate gyrus, whereas the same technique using the probe against only wizL could not detect positive signals in the developing cerebellum, indicating that there is no expression of wizL mRNA there. Northern blot and in situ hybridization analyses demonstrated that the extracerebellar regions expressed both wizS and wizL mRNAs from the midgestational period to adulthood. The finding that two types of wiz transcripts (wizS and wizL) are expressed with different developmental patterns might indicate separate transcription functions in the cerebellar granule cells and the extracerebellar regions. (C) 1998 Elsevier Science B.V. All rights reserved.

DOI： 10.1016/S0169-328X(98)00216-2

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

In the course of a study conducted to identify the mouse homologue of Drosophila eyes absent (eya), we isolated a novel mouse cDNA fragment which show little homology to eya but encodes a protein with Kruppel (C2H2)-type zinc finger motifs. By further screening using this cDNA fragment as a probe, we obtained the short and long forms of full-length cDNAs, which were apparently alternatively spliced products from one gene. Since both mRNAs encode proteins with widely-interspaced zinc finger motifs, we termed this gene wiz and refer to the short and long wiz transcripts as wizS and WizL, respectively. In situ hybridization studies using the probe against the region common to wizS and wizL showed that these mRNAs were expressed abundantly in the granule cell layers of the mouse cerebellum, the olfactory bulb, and the dentate gyrus, whereas the same technique using the probe against only wizL could not detect positive signals in the developing cerebellum, indicating that there is no expression of wizL mRNA there. Northern blot and in situ hybridization analyses demonstrated that the extracerebellar regions expressed both wizS and wizL mRNAs from the midgestational period to adulthood. The finding that two types of wiz transcripts (wizS and wizL) are expressed with different developmental patterns might indicate separate transcription functions in the cerebellar granule cells and the extracerebellar regions. (C) 1998 Elsevier Science B.V. All rights reserved.

DOI： 10.1016/S0169-328X(98)00216-2

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

Neuropsin is a serine protease which is thought to function in a variety of tissues including the brain and skin. This protease has been shown to have important roles in neural plasticity in mice. Here we have cloned a cDNA and analyzed the gene for human neuropsin by polymerase chain reaction-based strategies. The cDNA. had 72% identity to mouse neuropsin. The deduced amino acid sequence showed 72% identity to mouse neuropsin. Key amino acid residues for the enzyme activity and all cysteine residues were conserved between human and mouse neuropsin. The gene for human neuropsin had six exons and five introns, and the gene organization is similar to trypsin-type serine proteases. The mRNA was expressed in primary cultures of keratinocytes. (C) 1998 Elsevier Science B.V. All rights reserved.

DOI： 10.1016/S0378-1119(98)00232-7

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

Neuropsin is a serine protease which is thought to function in a variety of tissues including the brain and skin. This protease has been shown to have important roles in neural plasticity in mice. Here we have cloned a cDNA and analyzed the gene for human neuropsin by polymerase chain reaction-based strategies. The cDNA. had 72% identity to mouse neuropsin. The deduced amino acid sequence showed 72% identity to mouse neuropsin. Key amino acid residues for the enzyme activity and all cysteine residues were conserved between human and mouse neuropsin. The gene for human neuropsin had six exons and five introns, and the gene organization is similar to trypsin-type serine proteases. The mRNA was expressed in primary cultures of keratinocytes. (C) 1998 Elsevier Science B.V. All rights reserved.

DOI： 10.1016/S0378-1119(98)00232-7

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：BLACKWELL SCIENCE INC  

Neuropsin serine protease that was first cloned from the mouse brain as a factor related to neural plasticity. Subsequent in situ hybridization histochemical analysis indicated a broad localization of its mRNA throughout the whole body, although the details remain obscure, In this study, we showed that neuropsin immunoreactivity is localized in the keratinized stratified epithelia of the mouse epidermis, hair, tongue, palate, nasal cavity, pharynges, esophagus, and forestomach. In the skin and mucous membranes, neuropsin immunoreactivity was found in the stratum spinosum and the stratum granulosum. The immunoreactivity in the former sublayer was mainly present in the cytoplasm, but that in the latter sublayer was exclusively present in the intercellular space or on the outer surface is of the cell membrane and thus exhibited a lamellar-like peripheral distribution. During development, the appearance of neuropsin immunoreactivity in the various epithelia was found at embryonic days 14.5-15.5, prior to formation of the stratum corneum, More extensive expression of neuropsin immunoreactivity was found in the nude mouse skin and mucous membranes than in wild-type mice. Because the nude mouse is characterized by genetic impairment of keratinization, such abnormal neuropsin expression might be caused or affected by this impairment. Therefore, neuropsin, an extracellular serine protease, is suggested to be involved in keratinization in the stratified epithelia.

DOI： 10.1046/j.1523-1747.1998.00212.x

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：BLACKWELL SCIENCE INC  

Neuropsin serine protease that was first cloned from the mouse brain as a factor related to neural plasticity. Subsequent in situ hybridization histochemical analysis indicated a broad localization of its mRNA throughout the whole body, although the details remain obscure, In this study, we showed that neuropsin immunoreactivity is localized in the keratinized stratified epithelia of the mouse epidermis, hair, tongue, palate, nasal cavity, pharynges, esophagus, and forestomach. In the skin and mucous membranes, neuropsin immunoreactivity was found in the stratum spinosum and the stratum granulosum. The immunoreactivity in the former sublayer was mainly present in the cytoplasm, but that in the latter sublayer was exclusively present in the intercellular space or on the outer surface is of the cell membrane and thus exhibited a lamellar-like peripheral distribution. During development, the appearance of neuropsin immunoreactivity in the various epithelia was found at embryonic days 14.5-15.5, prior to formation of the stratum corneum, More extensive expression of neuropsin immunoreactivity was found in the nude mouse skin and mucous membranes than in wild-type mice. Because the nude mouse is characterized by genetic impairment of keratinization, such abnormal neuropsin expression might be caused or affected by this impairment. Therefore, neuropsin, an extracellular serine protease, is suggested to be involved in keratinization in the stratified epithelia.

DOI： 10.1046/j.1523-1747.1998.00212.x

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

Activity-dependent changes in neuropsin gene expression in the hippocampus implies an involvement of neuropsin in neural plasticity. Since the deduced amino acid sequence of the gene contained the complete triplet (His-Asp-Ser) of the serine protease domain, the protein was postulated to have proteolytic activity. Recombinant full-length neuropsin produced in the baculovirus/insect cell system was enzymatically inactive but was readily converted to active enzyme by endoprotease processing. The activational processing of prototype neuropsin involved the specific cleavage of the Lys(32)-Ile(33) bond near its N terminus. Native neuropsin that was purified with a purity of 1,100-fold from mouse brain had enzymatic characteristics identical to those of active-type recombinant neuropsin. Both brain and recombinant neuropsin had amidolytic activities cleaving Arg-X and Lys-X bonds in the synthetic chromogenic substrates, and the highest specific activity was found against Boc-Val-Pro-Arg-4-methylcoumaryl-7-amide. The active-type recombinant neuropsin effectively cleaved fibronectin, an extracellular matrix protein. Taken together, these results indicate that this protease, which is enzymatically novel, has significant limbic effects by changing the extracellular matrix environment.

DOI： 10.1074/jbc.273.18.11189

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

Activity-dependent changes in neuropsin gene expression in the hippocampus implies an involvement of neuropsin in neural plasticity. Since the deduced amino acid sequence of the gene contained the complete triplet (His-Asp-Ser) of the serine protease domain, the protein was postulated to have proteolytic activity. Recombinant full-length neuropsin produced in the baculovirus/insect cell system was enzymatically inactive but was readily converted to active enzyme by endoprotease processing. The activational processing of prototype neuropsin involved the specific cleavage of the Lys(32)-Ile(33) bond near its N terminus. Native neuropsin that was purified with a purity of 1,100-fold from mouse brain had enzymatic characteristics identical to those of active-type recombinant neuropsin. Both brain and recombinant neuropsin had amidolytic activities cleaving Arg-X and Lys-X bonds in the synthetic chromogenic substrates, and the highest specific activity was found against Boc-Val-Pro-Arg-4-methylcoumaryl-7-amide. The active-type recombinant neuropsin effectively cleaved fibronectin, an extracellular matrix protein. Taken together, these results indicate that this protease, which is enzymatically novel, has significant limbic effects by changing the extracellular matrix environment.

DOI： 10.1074/jbc.273.18.11189

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：HISTOCHEMICAL SOC INC  

Neuropsin is a novel serine protease whose mRNA is expressed in the mouse central nervous system. We examined the expression of neuropsin mRNA during embryonic development using Northern and in situ hybridization in non-neural tissues. The pregnant uterus showed strong expression of neuropsin mRNA, whereas the nonpregnant uterus did not express this mRNA. Expression was first detected in the primary decidual zone at 5.5 days post coitum and was maximized at 10 days post coitum, decreasing remarkably thereafter. During mouse organogenesis, neuropsin expression was observed in the developing heart, lung, thymus, pituitary, choroid plexus, and epithelial linings of the skin, oral cavity, tongue, esophagus, and forestomach. In adult mouse organs, neuropsin mRNA was expressed in epithelial tissues covered by keratinocytes with moderate density, whereas low expression was observed in lung, thymus, and spleen. Neuropsin mRNA expression in developing organs and adult keratinocytes suggests that neuropsin is associated with extracellular matrix modifications and cell migrations.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：HISTOCHEMICAL SOC INC  

Neuropsin is a novel serine protease whose mRNA is expressed in the mouse central nervous system. We examined the expression of neuropsin mRNA during embryonic development using Northern and in situ hybridization in non-neural tissues. The pregnant uterus showed strong expression of neuropsin mRNA, whereas the nonpregnant uterus did not express this mRNA. Expression was first detected in the primary decidual zone at 5.5 days post coitum and was maximized at 10 days post coitum, decreasing remarkably thereafter. During mouse organogenesis, neuropsin expression was observed in the developing heart, lung, thymus, pituitary, choroid plexus, and epithelial linings of the skin, oral cavity, tongue, esophagus, and forestomach. In adult mouse organs, neuropsin mRNA was expressed in epithelial tissues covered by keratinocytes with moderate density, whereas low expression was observed in lung, thymus, and spleen. Neuropsin mRNA expression in developing organs and adult keratinocytes suggests that neuropsin is associated with extracellular matrix modifications and cell migrations.

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Publisher：Elsevier {BV}  

DOI： 10.1016/s0168-0102(98)82456-5

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DOI： 10.1016/S0167-4781(98)00116-X

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DOI： 10.1016/S0167-4781(98)00116-X

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

We evaluated the effects of oxidative stress in mouse brain induced by the intraperitoneal injection of diethyldithiocarbamate (DDC) on gene expression of the novel serine protease, neuropsin, and on shock-avoidance learning. The level of neuropsin mRNA in the hippocampal pyramidal neurons increased at 2 h after DDC treatment and decreased thereafter. At 7 days neuropsin mRNA significantly decreased to 60% of the pretreated control level and then returned to the control level at 30 days. Genes for tissue plasminogen activator, manganese superoxide dismutase, and heat shock protein did not differ in DDC-treated mice vs. the control group at 7 and 30 days. The shuttle-box avoidance learning was retarded at 7 days after DDC administration. However, it recovered to the control level at 30 days after DDC administration. The results suggest that generation of reactive oxygen species has an important role in neuropsin transcript in the Limbic areas which might be related to the disturbance in avoidance learning. (C) 1997 Elsevier Science B.V.

DOI： 10.1016/S0006-8993(97)00674-4

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

We evaluated the effects of oxidative stress in mouse brain induced by the intraperitoneal injection of diethyldithiocarbamate (DDC) on gene expression of the novel serine protease, neuropsin, and on shock-avoidance learning. The level of neuropsin mRNA in the hippocampal pyramidal neurons increased at 2 h after DDC treatment and decreased thereafter. At 7 days neuropsin mRNA significantly decreased to 60% of the pretreated control level and then returned to the control level at 30 days. Genes for tissue plasminogen activator, manganese superoxide dismutase, and heat shock protein did not differ in DDC-treated mice vs. the control group at 7 and 30 days. The shuttle-box avoidance learning was retarded at 7 days after DDC administration. However, it recovered to the control level at 30 days after DDC administration. The results suggest that generation of reactive oxygen species has an important role in neuropsin transcript in the Limbic areas which might be related to the disturbance in avoidance learning. (C) 1997 Elsevier Science B.V.

DOI： 10.1016/S0006-8993(97)00674-4

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

We evaluated the effects of oxidative stress in mouse brain induced by the intraperitoneal injection of diethyldithiocarbamate (DDC) on gene expression of the novel serine protease, neuropsin, and on shock-avoidance learning. The level of neuropsin mRNA in the hippocampal pyramidal neurons increased at 2 h after DDC treatment and decreased thereafter. At 7 days neuropsin mRNA significantly decreased to 60% of the pretreated control level and then returned to the control level at 30 days. Genes for tissue plasminogen activator, manganese superoxide dismutase, and heat shock protein did not differ in DDC-treated mice vs. the control group at 7 and 30 days. The shuttle-box avoidance learning was retarded at 7 days after DDC administration. However, it recovered to the control level at 30 days after DDC administration. The results suggest that generation of reactive oxygen species has an important role in neuropsin transcript in the Limbic areas which might be related to the disturbance in avoidance learning. (C) 1997 Elsevier Science B.V.

DOI： 10.1016/S0006-8993(97)00674-4

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC JNL-COMP SUBSCRIPTIONS  

Neuropsin (M-r 25 032) is a serine protease expressed in the limbic system of mouse brain. It has been implicated in various neurological processes including formation of memory and may be important as a drug target in the treatment of epilepsy. The recombinant protein was produced using a baculovirus expression system and was purified. Two crystal forms were obtained by a hanging-drop vapor-diffusion method with polyethylene glycol. Preliminary X-ray crystallographic analysis revealed that; crystal form I belongs to triclinic space group P1 with unit cell dimensions a = 97.16 Angstrom, b = 97.12 Angstrom, c = 46.75 Angstrom and alpha = 99.17 degrees, beta = 99.17 degrees, gamma = 117.35 degrees. Self-rotation function analysis of these data of form I indicates the position of a noncrystallographic threefold axis. There are six molecules in the crystallographic asymmetric unit. Crystal form II also belongs to triclinic space group P1 but has unit cell dimensions of a = 38.40 Angstrom, b = 55.16 Angstrom, c = 65.37; Angstrom and alpha = 95.38 degrees, beta = 89.98 degrees, gamma = 110.46 degrees with two molecules in the crystallographic asymmetric unit. Form II has a noncrystallographic twofold axis, Intensity data to 3.1 Angstrom resolution for form I and to 2.2 Angstrom resolution for form II have been collected. (C) 1997 Academic Press.

DOI： 10.1006/jsbi.1997.3862

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC JNL-COMP SUBSCRIPTIONS  

Neuropsin (M-r 25 032) is a serine protease expressed in the limbic system of mouse brain. It has been implicated in various neurological processes including formation of memory and may be important as a drug target in the treatment of epilepsy. The recombinant protein was produced using a baculovirus expression system and was purified. Two crystal forms were obtained by a hanging-drop vapor-diffusion method with polyethylene glycol. Preliminary X-ray crystallographic analysis revealed that; crystal form I belongs to triclinic space group P1 with unit cell dimensions a = 97.16 Angstrom, b = 97.12 Angstrom, c = 46.75 Angstrom and alpha = 99.17 degrees, beta = 99.17 degrees, gamma = 117.35 degrees. Self-rotation function analysis of these data of form I indicates the position of a noncrystallographic threefold axis. There are six molecules in the crystallographic asymmetric unit. Crystal form II also belongs to triclinic space group P1 but has unit cell dimensions of a = 38.40 Angstrom, b = 55.16 Angstrom, c = 65.37; Angstrom and alpha = 95.38 degrees, beta = 89.98 degrees, gamma = 110.46 degrees with two molecules in the crystallographic asymmetric unit. Form II has a noncrystallographic twofold axis, Intensity data to 3.1 Angstrom resolution for form I and to 2.2 Angstrom resolution for form II have been collected. (C) 1997 Academic Press.

DOI： 10.1006/jsbi.1997.3862

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DOI： 10.1016/S0027-5107(97)00053-5

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DOI： 10.1016/S0027-5107(97)00053-5

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

Neuropsin mRNA expression was analyzed and mapped in the mouse brains after kindling epileptogenesis by using in situ hybridization histochemistry. Dynamic increases of the neuropsin mRNA were observed in the layer II of prelimbic, somatosensory, auditory, perirhinal, entorhinal, and piriform cortices in an activity-dependent manner, though no neuropsin gene was expressed in these areas in control mice. In addition to the confirmation of our previous studies showing increases of mRNA in the hippocampus and amygdaloid complex, there were also remarkable increases of the neuropsin mRNA in the limbic areas, such as the accessory olfactory nucleus, the medial and lateral septal nucleus, the nucleus of diagonal band, the substantia innominata and the zona incerta. The dynamic activity-dependent changes of the gene expression and the site-specificity of neuropsin localization are suggesting that this molecule is implicated in cortical- and limbic-specific neuronal reorganization.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

Neuropsin mRNA expression was analyzed and mapped in the mouse brains after kindling epileptogenesis by using in situ hybridization histochemistry. Dynamic increases of the neuropsin mRNA were observed in the layer II of prelimbic, somatosensory, auditory, perirhinal, entorhinal, and piriform cortices in an activity-dependent manner, though no neuropsin gene was expressed in these areas in control mice. In addition to the confirmation of our previous studies showing increases of mRNA in the hippocampus and amygdaloid complex, there were also remarkable increases of the neuropsin mRNA in the limbic areas, such as the accessory olfactory nucleus, the medial and lateral septal nucleus, the nucleus of diagonal band, the substantia innominata and the zona incerta. The dynamic activity-dependent changes of the gene expression and the site-specificity of neuropsin localization are suggesting that this molecule is implicated in cortical- and limbic-specific neuronal reorganization.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCI PUBL IRELAND LTD  

In our previous study, we found a novel gene encoding a serine protease termed neuropsin (NP) which exhibited activity-dependent gene expression or repression in the mouse hippocampus (Chen et al., 1995). In the present study, we examined the ontogeny of NP mRNA expression by in situ hybridization in the brain. Weak hybridization signals were first observed in the developing pens, medulla oblongata and spinal cord of embryonic day-12 embryos. Hybridization signals were also observed in the choroid plexus at this stage in addition to neuronal labeling. The signals continued to show this localization pattern until postnatal day 12. After embryonic day 18, the number of hybridization signals localized in the neurons of the forebrain limbic area were more predominant than those in the lower brainstem. NP gene expression spread in the anterior olfactory nucleus, hippocampus, septal nuclei, diagonal band of Broca, amygdala and limbic cortex successively from early embryonic to adult stage, though signals in the other brain regions were gradually decreased after birth. Thus, the widespread localization and two types of expression pattern, constitutive or transient, suggest that NP is a multiple functional protein involved in development, neuronal plasticity and cerebrospinal fluid production.

DOI： 10.1016/0168-0102(95)00960-2

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SOC NEUROSCIENCE  

A novel murine cDNA which encodes a protein designated neuropsin was cloned, Northern and in situ hybridization analyses demonstrated that neuropsin mRNA is expressed specifically in the limbic system of mouse brain and is localized at highest concentration in pyramidal neurons of the hippocampal CA1-3 subfields. Direct hippocampal stimulation and kindling induced by amygdaloid stimulation caused a significant bilateral change in neuropsin mRNA level in the hippocampal pyramidal neurons, The activity-dependent changes and the specific localization indicate that neuropsin is involved in hippocampal plasticity.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SOC NEUROSCIENCE  

A novel murine cDNA which encodes a protein designated neuropsin was cloned, Northern and in situ hybridization analyses demonstrated that neuropsin mRNA is expressed specifically in the limbic system of mouse brain and is localized at highest concentration in pyramidal neurons of the hippocampal CA1-3 subfields. Direct hippocampal stimulation and kindling induced by amygdaloid stimulation caused a significant bilateral change in neuropsin mRNA level in the hippocampal pyramidal neurons, The activity-dependent changes and the specific localization indicate that neuropsin is involved in hippocampal plasticity.

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DOI： 10.1016/0168-0102(95)00960-2

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCI IRELAND LTD  

By in situ hybridization histochemistry, fibroblast growth factor receptor gene (flg)-expressing neurons were newly identified in the basal magnocellular nuclei (the vertical and horizontal limbs of the diagonal band, and Meynert's nucleus). The present study also confirmed flg localization in the laterodorsal tegmental nucleus and the pedunculopontine tegmental nucleus of the pens. Immuno- and in situ hybridization histochemistry on the same sections demonstrated that choline acetyltransferase and flg were colocalized in single neurons of the diagonal band, Meynert's nucleus and the pontine tegmental areas. The results suggest that a significant number of the basal magnocellular and a majority of the mesopontine cholinergic neurons are directly affected by fibroblast growth factors (FGF) via FGF receptor gene.

DOI： 10.1016/0168-0102(94)90020-5

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCI IRELAND LTD  

By in situ hybridization histochemistry, fibroblast growth factor receptor gene (flg)-expressing neurons were newly identified in the basal magnocellular nuclei (the vertical and horizontal limbs of the diagonal band, and Meynert's nucleus). The present study also confirmed flg localization in the laterodorsal tegmental nucleus and the pedunculopontine tegmental nucleus of the pens. Immuno- and in situ hybridization histochemistry on the same sections demonstrated that choline acetyltransferase and flg were colocalized in single neurons of the diagonal band, Meynert's nucleus and the pontine tegmental areas. The results suggest that a significant number of the basal magnocellular and a majority of the mesopontine cholinergic neurons are directly affected by fibroblast growth factors (FGF) via FGF receptor gene.

DOI： 10.1016/0168-0102(94)90020-5

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：JAPAN SOC HISTOCHEMISTRY & CYTOCHEMISTRY  

In situ hybridization histochemistry with the use of a digoxigenin-labeled ribonucleotide probe for basic fibroblast growth factor (bFGF) mRNA demonstrated bFGF transcripts in the masseter muscle of dystrophic (mdx) mouse and in vibrissae and small hair follicle of the rat peri-oral skin. A conspicuous hybridization signal was detected in the central part of the cytoplasm of the smallest myoblasts in the process of initial regeneration or differentiation. bFGF mRNA staining decreased in intensity as the myoblasts increased in size due to the production of myofilaments. Endomysial fibroblasts and extracellular matrix did not exhibit any detectable bFGF mRNA expression. In transverse sections of the hair follicles and vibrissae, a bFGF mRNA positive reaction was noted in the central portion of individual follicles, which were endowed with a non-hybridized core of variable diameter. The hybridization signal in longituidinal sections of hair follicles was localized mainly to keratinizing hair cortical cells; the matrix was scarcely labeled with the probe. The dermal papillae of hair follicles were devoid of bFGF mRNA staining. These findings suggest that early regenerating or differentiating myoblasts produce bFGF, rather than internalizing the growth factor originating in other tissues and that part of bFGF generated in the hair cortical cells is conveyed to the hair matrix and external root sheath which has been shown, by immunohistochemistry, to contain bFGF-like substances.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

The immunohistochemical localization of basic fibroblast growth factor (bFGF) was studied in the adult rat brain, using a specific antibody against a synthetic bFGF fragment (the N-terminal 12 residues). Widespread but uneven regional localization of bFGF-like immunoreactive neurons and fibers was observed. Ependymal cells were also stained. The immunoreactive neurons were found in the cerebral cortex, olfactory bulb, septum, basal magnocellular nuclei, thalamus, hypothalamus, globus pallidus, hippocampus, amygdala, red nucleus, central gray of the midbrain, cerebellum, dorsal tegmental area, reticular formation, cranial motor nuclei and spinal cord. Immunoreactive fiber bundles and nerve terminals were also detected. These results indicate that bFGF is produced by or present in a specific neuronal cell population of the central nervous system.

DOI： 10.1016/0006-8993(92)91427-G

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

The immunohistochemical localization of basic fibroblast growth factor (bFGF) was studied in the adult rat brain, using a specific antibody against a synthetic bFGF fragment (the N-terminal 12 residues). Widespread but uneven regional localization of bFGF-like immunoreactive neurons and fibers was observed. Ependymal cells were also stained. The immunoreactive neurons were found in the cerebral cortex, olfactory bulb, septum, basal magnocellular nuclei, thalamus, hypothalamus, globus pallidus, hippocampus, amygdala, red nucleus, central gray of the midbrain, cerebellum, dorsal tegmental area, reticular formation, cranial motor nuclei and spinal cord. Immunoreactive fiber bundles and nerve terminals were also detected. These results indicate that bFGF is produced by or present in a specific neuronal cell population of the central nervous system.

DOI： 10.1016/0006-8993(92)91427-G

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

The localization of the glycine cleavage system was examined in the rat brain by immunohistochemistry using an antibody to P-protein (a constituent of the system). In all sites studied, the enzyme was confined to the astrocytes. The intensity of astrocyte staining varied in different brain regions, with the strongest staining being noted in the hippocampus, the cerebellar cortex, the Bergmann glia in the cerebellum and the Muller cells in the retina. The weakest staining was seen in the brainstem and spinal cord. P-protein was found to be located in the mitochondria by an ultrastructural study.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

The localization of the glycine cleavage system was examined in the rat brain by immunohistochemistry using an antibody to P-protein (a constituent of the system). In all sites studied, the enzyme was confined to the astrocytes. The intensity of astrocyte staining varied in different brain regions, with the strongest staining being noted in the hippocampus, the cerebellar cortex, the Bergmann glia in the cerebellum and the Muller cells in the retina. The weakest staining was seen in the brainstem and spinal cord. P-protein was found to be located in the mitochondria by an ultrastructural study.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-LISS  

The postnatal ontogeny of cells expressing prepro-neurotensin/neuromedin N messenger RNA (prepro-NT/NN mRNA) in the rat forebrain and midbrain was investigated by in situ hybridization histochemistry. According to the pattern of expression during development, the cells which express prepro-NT/NN mRNA can be roughly divided into 2 groups. In type I cells, prepro-NT/NN mRNA expression reaches a maximum in terms of content during the postnatal period. After this early peak, cells of this type express the same or less prepro-NT/NN mRNA, reaching a plateau at an adult level that still contains a high level of expression. In type Il cells, prepro-NT/NN mRNA appears during the postnatal period, and the expression decreases dramatically after the first postnatal week, being almost undetectable by a few weeks after birth. Type I cells were observed in the following areas: the piriform cortex, field CA1 of Ammon's horn, subiculum, vertical, and horizontal limbs of the diagonal band of Broca, intermediate part of the lateral septal nucleus, bed nucleus of the stria terminalis, medial preoptic area, lateral hypothalamus, caudal part of the caudate putamen, medial, cortical, and central amygdaloid nuclei, ventral tegmental area, deep mesencephalic nucleus, cuneiform nucleus, dorsal raphe nucleus, laterodorsal tegmental nucleus, parabrachial nucleus, and oral part of the pontine reticular nucleus. Cells of type II were observed in the following areas: the mitral cell layer of the olfactory bulb, rostral part of the caudate putamen, (anterior) cingulate cortex, and retrosplenial cortex (posterior cingulate cortex).

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-LISS  

The postnatal ontogeny of cells expressing prepro-neurotensin/neuromedin N messenger RNA (prepro-NT/NN mRNA) in the rat forebrain and midbrain was investigated by in situ hybridization histochemistry. According to the pattern of expression during development, the cells which express prepro-NT/NN mRNA can be roughly divided into 2 groups. In type I cells, prepro-NT/NN mRNA expression reaches a maximum in terms of content during the postnatal period. After this early peak, cells of this type express the same or less prepro-NT/NN mRNA, reaching a plateau at an adult level that still contains a high level of expression. In type Il cells, prepro-NT/NN mRNA appears during the postnatal period, and the expression decreases dramatically after the first postnatal week, being almost undetectable by a few weeks after birth. Type I cells were observed in the following areas: the piriform cortex, field CA1 of Ammon's horn, subiculum, vertical, and horizontal limbs of the diagonal band of Broca, intermediate part of the lateral septal nucleus, bed nucleus of the stria terminalis, medial preoptic area, lateral hypothalamus, caudal part of the caudate putamen, medial, cortical, and central amygdaloid nuclei, ventral tegmental area, deep mesencephalic nucleus, cuneiform nucleus, dorsal raphe nucleus, laterodorsal tegmental nucleus, parabrachial nucleus, and oral part of the pontine reticular nucleus. Cells of type II were observed in the following areas: the mitral cell layer of the olfactory bulb, rostral part of the caudate putamen, (anterior) cingulate cortex, and retrosplenial cortex (posterior cingulate cortex).

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

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Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

DOI： 10.1016/0006-8993(91)91336-y

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

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DOI： 10.1016/0306-4522(90)90113-I

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

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J-GLOBAL

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Authorship：Corresponding author   Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (scientific journal)   Publisher：金芳堂  

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J-GLOBAL

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J-GLOBAL

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J-GLOBAL

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J-GLOBAL

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J-GLOBAL

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J-GLOBAL

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J-GLOBAL

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J-GLOBAL

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J-GLOBAL

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J-GLOBAL

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J-GLOBAL

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J-GLOBAL

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Language：Japanese   Publisher：(一社)日本解剖学会  

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Language：Japanese   Publisher：(一社)日本解剖学会  

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Language：English   Publisher：BLACKWELL SCIENCE LTD  

The behavioural and electrographical abnormalities associated with seizures in epileptic (kindled) mice correspond with those of human epilepsy. In kindled mice, neuropsin was markedly increased in the hippocampus and cerebral cortices. A single intraventricular injection of monoclonal antibodies specific to neuropsin reduced or eliminated the epileptic pattern noted on electroencephalograms and, as a result markedly inhibited the progression of kindling. Therefore, neuropsin appears to be a key protein controlling pathogenic events in the hippocampus, and thus neuropsin inhibitors might be useful for treatment of epilepsy.

DOI： 10.1046/j.1460-9568.1998.00068.x

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Language：Japanese   Publisher：The Biophysical Society of Japan General Incorporated Association  

CiNii Books

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Language：Japanese   Publisher：日本組織細胞化学会  

CiNii Books

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