Shigeru Yokoyama

Publications of Shigeru Yokoyama

• Expression and immunolocalization of Gpnmb, a glioma-associated glycoprotein, in normal and inflamed central nervous systems of adult rats.

  Authors: Jian-Jun Huang, Wen-Jie Ma, Shigeru Yokoyama

  Brain and behavior. 03/2012; 2(2):85-96.

  Glycoprotein nonmetastatic melanoma B (Gpnmb) is a type I transmembrane protein implicated in cell differentiation, inflammation, tissue regeneration, and tumor progression. Gpnmb, which is highlyGlycoprotein nonmetastatic melanoma B (Gpnmb) is a type I transmembrane protein implicated in cell differentiation, inflammation, tissue regeneration, and tumor progression. Gpnmb, which is highly expressed in glioblastoma cells, is a potential therapeutic target. However, little is known about its expression, cellular localization, and roles in non-tumorous neural tissues. In this study, we examined Gpnmb expression in the central nervous system of adult rats under both normal and inflammatory conditions. Reverse transcription-polymerase chain reaction analysis revealed that Gpnmb mRNA was expressed in the cerebrum, cerebellum, brain stem, and spinal cord of normal adult rats. Immunoperoxidase staining revealed that Gpnmb-immunoreactive cells were widely distributed in the parenchyma of all brain regions examined, with the cells being most prevalent in the hippocampal dentate gyrus, cerebellar cortex, spinal dorsal horn, choroid plexus, ependyma, periventricular regions, and in layers II and III of the cerebral cortex. Double immunofluorescence staining showed that these cells were co-stained most frequently with the microglia/macrophage marker OX42, and occasionally with the radial glia marker RC2 or the neuronal marker NeuN. Furthermore, an intraperitoneal injection of bacterial endotoxin lipopolysaccharide increased the number of Gpnmb and OX42 double-positive cells in the area postrema, which is one of the circumventricular organs, indicating infiltration of hematogenous macrophages. These results suggest that Gpnmb, which is expressed in microglia and macrophages in non-tumorous neural tissues, plays an important role in the regulation of immune/inflammatory responses.

• Social memory, amnesia, and autism: Brain oxytocin secretion is regulated by NAD(+) metabolites and single nucleotide polymorphisms of CD38.

  Authors: Haruhiro Higashida, Shigeru Yokoyama, Jian-Jun Huang, Li Liu, Wen-Jie Ma, Shirin Akther, Chiharu Higashida, Mitsuru Kikuchi, Yoshio Minabe, Toshio Munesue

  Neurochemistry international. 02/2012;

  Previously, we demonstrated that CD38, a transmembrane protein with ADP-ribosyl cyclase activity, plays a critical role in mouse social behavior by regulating the release of oxytocin (OXT), which isPreviously, we demonstrated that CD38, a transmembrane protein with ADP-ribosyl cyclase activity, plays a critical role in mouse social behavior by regulating the release of oxytocin (OXT), which is essential for mutual recognition. When CD38 was disrupted, social amnesia was observed in Cd38 knockout mice. The autism spectrum disorders (ASDs), characterized by defects in reciprocal social interaction and communication, occur either sporadically or in a familial pattern. However, the etiology of ASDs remains largely unknown. Therefore, the theoretical basis for pharmacological treatments has not been established. Hence, there is a rationale for investigating single nucleotide polymorphisms (SNPs) in the human CD38 gene in ASD subjects. We found several SNPs in this gene. The SNP rs3796863 (C>A) was associated with high-functioning autism (HFA) in American samples from the Autism Gene Resource Exchange. Although this finding was partially confirmed in low-functioning autism subjects in Israel, it has not been replicated in Japanese HFA subjects. The second SNP of interest, rs1800561 (4693C>T), leads to the substitution of an arginine (R) at codon 140 by tryptophan (W; R140W) in CD38. This mutation was found in four probands of ASD and in family members of three pedigrees with variable levels of ASD or ASD traits. The plasma levels of OXT in ASD subjects with the R140W allele were lower than those in ASD subjects lacking this allele. The OXT levels were unchanged in healthy subjects with or without this mutation. One proband with the R140W allele receiving intranasal OXT for approximately 3years showed improvement in areas of social approach, eye contact and communication behaviors, emotion, irritability, and aggression. Five other ASD subjects with mental deficits received nasal OXT for various periods; three subjects showed improved symptoms, while two showed little or no effect. These results suggest that SNPs in CD38 may be possible risk factors for ASD by abrogating OXT function and that some ASD subjects can be treated with OXT in preliminary clinical trials.

• Dopamine release via the vacuolar ATPase V0 sector c-subunit, confirmed in N18 neuroblastoma cells, results in behavioral recovery in hemiparkinsonian mice.

  Authors: Duo Jin, Shin-Ichi Muramatsu, Nobuaki Shimizu, Shigeru Yokoyama, Hirokazu Hirai, Kiyofumi Yamada, Hong-Xiang Liu, Chiharu Higashida, Minako Hashii, Akihiko Higashida, Masahide Asano, Shoji Ohkuma, Haruhiro Higashida

  Neurochemistry international. 01/2012;

  A 16-kDa proteolipid, mediatophore, in Torpedo electric organs mediates Ca(2+)-dependent acetylcholine release. Mediatophore is identical to the pore-forming stalk c-subunit of the V0 sector ofA 16-kDa proteolipid, mediatophore, in Torpedo electric organs mediates Ca(2+)-dependent acetylcholine release. Mediatophore is identical to the pore-forming stalk c-subunit of the V0 sector of vacuolar proton ATPase (ATP6V0C). The function of ATP6V0C in the mammalian central nervous system is not clear. Here, we report transfection of adeno-associated viral vectors harboring rat ATP6V0C into the mouse substantia nigra, in which high potassium stimulation increased overflow of endogenous dopamine (DA) measured in the striatum by in vivo microdialysis. Next, in the striatum of 6-hydroxydopamine-lesioned mice, a model of Parkinson's disease (PD), human tyrosine hydroxylase, aromatic l-amino-acid decarboxylase and guanosine triphosphate cyclohydrolase 1, together with or without ATP6V0C, were expressed in the caudoputamen for rescue. Motor performance on the accelerating rotarod test and amphetamine-induced ipsilateral rotation were improved in the rescued mice coexpressing ATP6V0C. [(3)H]DA, taken up into cultured N18 neuronal tumor cells transformed to express ATP6V0C, was released by potassium stimulation. These results indicated that ATP6V0C mediates DA release from nerve terminals in the striatum of DA neurons of normal mice and from gene-transferred striatal cells of parkinsonian mice. The results suggested that ATP6V0C may be useful as a rescue molecule in addition to DA-synthetic enzymes in the gene therapy of PD.

• CD38 and its role in oxytocin secretion and social behavior.

  Authors: Haruhiro Higashida, Shigeru Yokoyama, Mitsuru Kikuchi, Toshio Munesue

  Hormones and behavior. 12/2011;

  Here, we review the functional roles of cyclic ADP-ribose and CD38, a transmembrane protein with ADP-ribosyl cyclase activity, in mouse social behavior via the regulation of oxytocin (OXT) release,Here, we review the functional roles of cyclic ADP-ribose and CD38, a transmembrane protein with ADP-ribosyl cyclase activity, in mouse social behavior via the regulation of oxytocin (OXT) release, an essential component of social cognition. Herein we describe data detailing the molecular mechanism of CD38-dependent OXT secretion in CD38 knockout mice. We also review studies that used OXT, OXT receptor (OXTR), or CD38 knockout mice. Additionally, we compare the behavioral impairments that occur in these knockout mice in relation to the OXT system and CD38. This review also examines autism spectrum disorder (ASD), which is characterized by social and communication impairments, in relation to defects in the OXT system. Two single nucleotide polymorphisms (SNPs) in the human CD38 gene are possible risk factors for ASD via inhibition of OXT function. Further analysis of CD38 in relation to the OXT system may provide a better understanding of the neuroendocrinological roles of OXT and CD38 in the hypothalamus and of the pathophysiology of ASD.

• A Missense Mutation in CD38 Associated with Autism Spectrum Disorder in Three Pedigrees

  Authors: Haruhiro Higashida, Toshio Munesue, Shigeru Yokoyama, Minako Hashii, Keita Koizumi, Akihiro Matsushima

  08/2011;

  ISBN: 978-953-307-493-1

• CD38 gene knockout juvenile mice: a model of oxytocin signal defects in autism.

  Authors: Haruhiro Higashida, Shigeru Yokoyama, Toshio Munesue, Mitsuru Kikuchi, Yoshio Minabe, Olga Lopatina

  Biological & pharmaceutical bulletin. 01/2011; 34(9):1369-72.

  Oxytocin (OXT) in the hypothalamus is the biological basis of social recognition, trust, and bonding. We showed that CD38, a leukaemia cell marker, plays an important role in the hypothalamus in theOxytocin (OXT) in the hypothalamus is the biological basis of social recognition, trust, and bonding. We showed that CD38, a leukaemia cell marker, plays an important role in the hypothalamus in the process of OXT release in adult mice. Disruption of Cd38 (Cd38(-/-)) produced impairment of maternal behavior and male social recognition in mice, similar to the behavior observed in Oxt and OXT receptor (Oxtr) gene knockout (Oxt(-/-) and Oxtr(-/-), respectively) mice. Locomotor activity induced by separation from the dam was higher and the number of ultrasonic vocalization (USV) calls was lower in Cd38(-/-) than Cd38(+/+) pups. These phenotypes seemed to be caused by the high plasma OXT levels during development from neonates to 3-week-old juvenile mice. ADP-ribosyl cyclase activity was markedly lower in the knockout mice from birth, suggesting that weaning for mice is a critical time window of differentiating plasma OXT. Contribution by breastfeeding was an important exogenous source for regulating plasma OXT before weaning by the presence of OXT in milk and the dam's mammary glands. The dissimilarity of Cd38(-/-) infant behaviour to Oxt(-/-) or Oxtr(-/-) mice can be explained partly by this exogenous source of OXT. These results suggest that secretion of OXT into the brain in a CD38-dependent manner may play an important role in the development of social behavior, and mice with OXT signalling deficiency, including Cd38(-/-), Oxt(-/-) and Oxtr(-/-) mice are good animal models for developmental disorders, such as autism.

• Two genetic variants of CD38 in subjects with autism spectrum disorder and controls.

  Authors: Toshio Munesue, Shigeru Yokoyama, Kazuhiko Nakamura, Ayyappan Anitha, Kazuo Yamada, Kenshi Hayashi, Tomoya Asaka, Hong-Xiang Liu, Duo Jin, Keita Koizumi [......] Minako Hashii, Sarwat Amina, Fabio Malavasi, Eric J Huang, Jiasheng Zhang, Nobuaki Shimizu, Takeo Yoshikawa, Akihiro Matsushima, Yoshio Minabe, Haruhiro Higashida

  Neuroscience research. 06/2010; 67(2):181-91.

  The neurobiological basis of autism spectrum disorder (ASD) remains poorly understood. Given the role of CD38 in social recognition through oxytocin (OT) release, we hypothesized that CD38 may play aThe neurobiological basis of autism spectrum disorder (ASD) remains poorly understood. Given the role of CD38 in social recognition through oxytocin (OT) release, we hypothesized that CD38 may play a role in the etiology of ASD. Here, we first examined the immunohistochemical expression of CD38 in the hypothalamus of post-mortem brains of non-ASD subjects and found that CD38 was colocalized with OT in secretory neurons. In studies of the association between CD38 and autism, we analyzed 10 single nucleotide polymorphisms (SNPs) and mutations of CD38 by re-sequencing DNAs mainly from a case-control study in Japan, and Caucasian cases mainly recruited to the Autism Genetic Resource Exchange (AGRE). The SNPs of CD38, rs6449197 (p<0.040) and rs3796863 (p<0.005) showed significant associations with a subset of ASD (IQ>70; designated as high-functioning autism (HFA)) in the U.S. 104 AGRE family trios, but not with Japanese 188 HFA subjects. A mutation that caused tryptophan to replace arginine at amino acid residue 140 (R140W; (rs1800561, 4693C>T)) was found in 0.6-4.6% of the Japanese population and was associated with ASD in the smaller case-control study. The SNP was clustered in pedigrees in which the fathers and brothers of T-allele-carrier probands had ASD or ASD traits. In this cohort OT plasma levels were lower in subjects with the T allele than in those without. One proband with the T allele who was taking nasal OT spray showed relief of symptoms. The two variant CD38 poloymorphysms tested may be of interest with regard of the pathophysiology of ASD.

• Interleukin-6 upregulates the expression of PMP22 in cultured rat Schwann cells via a JAK2-dependent pathway.

  Authors: Takaaki Ito, Kazuo Ikeda, Katsuro Tomita, Shigeru Yokoyama

  Neuroscience letters. 03/2010; 472(2):104-8.

  The interleukin-6 (IL-6) family of cytokines is thought to be involved in the development and regeneration of peripheral nerves; however, their roles in myelination remain unclear. In this study, weThe interleukin-6 (IL-6) family of cytokines is thought to be involved in the development and regeneration of peripheral nerves; however, their roles in myelination remain unclear. In this study, we examined the effects of IL-6 on the expression of genes for compact myelin proteins using Schwann cell cultures prepared by multiple explantation of adult rat sciatic nerves. In semi-quantitative reverse transcription-polymerase chain reaction analysis, stimulation of Schwann cells with IL-6 significantly increased the mRNA level of peripheral myelin protein 22 (PMP22), but not those of myelin protein zero and myelin basic protein. The increase in PMP22 mRNA was markedly suppressed by AG490, a Janus kinase 2 (JAK2) inhibitor, but not significantly by PD098059, a mitogen-activated protein kinase inhibitor. Immunocytochemical staining revealed that IL-6 enhanced immunoreactivities for the phosphorylated forms of both JAK2 and signal transducer and activator of transcription 3 (STAT3), as well as that for PMP22. These results indicate that IL-6 can enhance PMP22 production in Schwann cells via a JAK2-dependent pathway by probably activating STAT3 and thus may contribute to myelination.

• Reply.

  Authors: Mikito Seto, Hisashi Bunko, Noriyuki Shuke, Keisuke Takahashi, Youji Sakaibori, Hitoshi Terada, Etsuko Imabayashi, Ichiei Kuji, Hiroshi Matsuda, Shigeru Yokoyama

  Nuclear medicine communications. 05/2009; 30(4):320-321.

• Quantitative regional blood flow measurements in exercising leg skeletal muscle based on 99mTc-pertechnetate clearance.

  Authors: Mikito Seto, Hisashi Bunko, Noriyuki Shuke, Keisuke Takahashi, Youji Sakaibori, Hitoshi Terada, Etsuko Imabayashi, Ichiei Kuji, Hiroshi Matsuda, Shigeru Yokoyama

  Nuclear medicine communications. 10/2008; 29(9):770-4.

  BACKGROUND AND AIM: Skeletal muscle blood flow (SMBF) is a crucial indicator of impaired peripheral circulation. Although 133Xe has long been used for estimation of regional SMBF, its lipophilic andBACKGROUND AND AIM: Skeletal muscle blood flow (SMBF) is a crucial indicator of impaired peripheral circulation. Although 133Xe has long been used for estimation of regional SMBF, its lipophilic and volatile nature hampers precise measurement. Therefore, we established an improved method based on 99mTc-pertechnetate ion (99mTcO-4) clearance. METHODS: Ten healthy male volunteers including five rugby players and five non-athletes (each group aged 25-35 years) received injection of 99mTcO-4 into the bilateral tibialis anterior muscles and gastrocnemius muscles (GCMs). The radioactivity of 99mTcO-4 before, during, and after toe-up or treadmill exercise was traced using a gamma-camera. Regional SMBF in absolute values (in ml/min/100 g muscle) was then calculated based on the half-time obtained from the time-activity curve. RESULTS: In both the groups, SMBF in tibialis anterior muscle changed similarly with values at the same levels. In contrast, SMBF in GCM showed marked difference between the two groups: in rugby players, SMBF in GCM returned to the baseline level (6.5+/-1.7, n=10) immediately after treadmill exercise, whereas that in non-athletes remained high (16.2+/-3.2, n=10). CONCLUSION: Regional SMBF measured by 99mTcO-4 clearance can indicate effectiveness of exercise training, and would be a diagnostic tool and prognostic indicator for use in patients with impaired peripheral circulation.

• Locomotor activity, ultrasonic vocalization and oxytocin levels in infant CD38 knockout mice.

  Authors: Hong-Xiang Liu, Olga Lopatina, Chiharu Higashida, Takahiro Tsuji, Ichiro Kato, Shin Takasawa, Hiroshi Okamoto, Shigeru Yokoyama, Haruhiro Higashida

  Neuroscience letters. 10/2008;

  Oxytocin (OT), a neurohormone involved in reproduction, plays a critical role in social behavior in a wide range of mammalian species from rodents to humans. The role of CD38 in regulating OTOxytocin (OT), a neurohormone involved in reproduction, plays a critical role in social behavior in a wide range of mammalian species from rodents to humans. The role of CD38 in regulating OT secretion for social behavior has been demonstrated in adult mice, but has not been examined in pups or during development. Separation from the dam induces stress in 7-day-old mouse pups. During such isolation, locomotor activity was higher in CD38 knockout (CD38(-/-)) pups than in wild-type (CD38(+/+)) or heterozygous (CD38(+/-)) controls. The number of ultrasonic vocalizations was lower in CD38(-/-) pups than in CD38(+/+) pups. However, the difference between the two genotypes was less severe than in OT knockout or OT receptor knockout mice. To explain this, we measured plasma OT levels. The level was not lower in CD38(-/-) pups during the period 1-3 weeks after birth, but was significantly reduced after weaning (</=3 weeks). ADP-ribosyl cyclase activities in the hypothalamus and pituitary were markedly lower from 1 week after birth in CD38(-/-) mice and were consistently lower thereafter to the adult stage (2 months old). These results showed that the reduced severity of behavioral abnormalities in CD38(-/-) pups was due to partial compensation by the high level of plasma OT.

• CD38 is critical for social behaviour by regulating oxytocin secretion.

  Authors: Duo Jin, Hong-Xiang Liu, Hirokazu Hirai, Takashi Torashima, Taku Nagai, Olga Lopatina, Natalia A Shnayder, Kiyofumi Yamada, Mami Noda, Toshihiro Seike [......] Naoya Noguchi, Ichiro Kato, Hiroshi Okamoto, Akihiro Matsushima, Alla Salmina, Toshio Munesue, Nobuaki Shimizu, Sumiko Mochida, Masahide Asano, Haruhiro Higashida

  Nature. 04/2007; 446(7131):41-5.

  CD38, a transmembrane glycoprotein with ADP-ribosyl cyclase activity, catalyses the formation of Ca2+ signalling molecules, but its role in the neuroendocrine system is unknown. Here we show thatCD38, a transmembrane glycoprotein with ADP-ribosyl cyclase activity, catalyses the formation of Ca2+ signalling molecules, but its role in the neuroendocrine system is unknown. Here we show that adult CD38 knockout (CD38-/-) female and male mice show marked defects in maternal nurturing and social behaviour, respectively, with higher locomotor activity. Consistently, the plasma level of oxytocin (OT), but not vasopressin, was strongly decreased in CD38-/- mice. Replacement of OT by subcutaneous injection or lentiviral-vector-mediated delivery of human CD38 in the hypothalamus rescued social memory and maternal care in CD38-/- mice. Depolarization-induced OT secretion and Ca2+ elevation in oxytocinergic neurohypophysial axon terminals were disrupted in CD38-/- mice; this was mimicked by CD38 metabolite antagonists in CD38+/+ mice. These results reveal that CD38 has a key role in neuropeptide release, thereby critically regulating maternal and social behaviours, and may be an element in neurodevelopmental disorders.

• Overexpression of human CD38/ADP-ribosyl cyclase enhances acetylcholine-induced Ca2+ signalling in rodent NG108-15 neuroblastoma cells.

  Authors: Haruhiro Higashida, Sarah E H Bowden, Shigeru Yokoyama, Alla Salmina, Minako Hashii, Naoto Hoshi, Jia-Sheng Zhang, Rimma Knijnik, Mami Noda, Zen-Guo Zhong, Duo Jin, Kazuhiro Higashida, Hisashi Takeda, Tenpei Akita, Kenji Kuba, Sayaka Yamagishi, Noriaki Shimizu, Shin Takasawa, Hiroshi Okamoto, Jon Robbins

  Neuroscience research. 04/2007; 57(3):339-46.

  The role of cyclic ADP-ribose (cADPR) and its synthetic enzyme, CD38, as a downstream signal of muscarinic acetylcholine receptors (mAChRs) was examined in neuroblastoma cells expressing M1 mAChRsThe role of cyclic ADP-ribose (cADPR) and its synthetic enzyme, CD38, as a downstream signal of muscarinic acetylcholine receptors (mAChRs) was examined in neuroblastoma cells expressing M1 mAChRs (NGM1). NGM1 cells were further transformed with both wild-type and mutant (C119K/C201E) human CD38. The dual transformed cells exhibited higher cADPR formation than ADPR production and elevated intracellular free Ca(2+) concentrations ([Ca(2+)](i)) in response to ACh. These phenotypes were analyzed in detail in a representative CD38 clone. The intracellular cADPR concentration by ACh application was significantly increased by CD38 overexpression. Digital image analysis by a confocal microscopy revealed that topographical distribution of the sites of Ca(2+) release was unchanged between control and overexpressed cells. These results indicate that cADPR is an intracellular messenger of Ca(2+) signalling, suggesting that CD38 can contribute to mAChR-cADPR signalling.

• Bradykinin activates ADP-ribosyl cyclase in neuroblastoma cells: intracellular concentration decrease in NAD and increase in cyclic ADP-ribose.

  Authors: Haruhiro Higashida, Alla Salmina, Minako Hashii, Shigeru Yokoyama, Jia-Sheng Zhang, Mami Noda, Zen-Guo Zhong, Duo Jin

  FEBS letters. 10/2006; 580(20):4857-60.

  ADP-ribosyl cyclase activity in the crude membrane fraction of neuroblastomaxglioma NGPM1-27 hybrid cells was measured by monitoring [(3)H] cyclic ADP-ribose (cADPR) formation from [(3)H] NAD(+).ADP-ribosyl cyclase activity in the crude membrane fraction of neuroblastomaxglioma NGPM1-27 hybrid cells was measured by monitoring [(3)H] cyclic ADP-ribose (cADPR) formation from [(3)H] NAD(+). Bradykinin (BK) at 100nM increased ADP-ribosyl cyclase activity by about 2.5-fold. Application of 300nM BK to living NGPM1-27 cells decreased NAD(+) to 78% of the prestimulation level at 30s. In contrast, intracellular cADPR concentrations were increased by 2-3-fold during the period from 30 to 120s after the same treatment. Our results suggest that cADPR is one of the second messengers downstream of B(2) BK receptors.

• Induction of tumor necrosis factor-alpha in Schwann cells after gradual elongation of rat sciatic nerve.

  Authors: Norio Hagiwara, Kazuo Ikeda, Haruhiro Higashida, Katsuro Tomita, Shigeru Yokoyama

  Journal of orthopaedic science : official journal of the Japanese Orthopaedic Association. 12/2005; 10(6):614-21.

  BACKGROUND: Although limb lengthening has become a common treatment, the biochemical responses underlying the adaptation of elongated nerves are unclear. The purpose of this study was to clarifyBACKGROUND: Although limb lengthening has become a common treatment, the biochemical responses underlying the adaptation of elongated nerves are unclear. The purpose of this study was to clarify whether expression of cytokines and neurotrophins is altered in gradually elongated peripheral nerves. METHODS: Left sciatic nerves of adult rats were elongated by lengthening the femur up to 20 mm at a rate of 1, 2, or 20 mm/day. The ipsilateral and contralateral sciatic nerves of each group were resected 1, 4, 8, and 16 days after 20 mm of lengthening. mRNAs for interleukin-1beta, interleukin-6, tumor necrosis factor-alpha (TNFalpha), nerve growth factor, brain-derived neurotrophic factor, neurotrophin-3, and neurotrophin-4/5 were semiquantified by reverse transcription-polymerase chain reaction. Histological changes were assessed by immunoperoxidase and immunofluorescence staining. RESULTS: Expression of TNFalpha mRNA was markedly induced in the ipsilateral sciatic nerves of the gradually elongated, 1 mm/day and 2 mm/day groups, although to a lesser extent than in the acutely elongated, 20 mm/day group. In contrast, mRNAs for other factors remained undetectable. The mRNA level for TNFalpha in each group was highest 1 day after 20 mm of lengthening. The highly up-regulated level in the acute group declined rapidly within 4 days and slowly thereafter; in contrast, the decrease in the gradual groups was always slow. Even 16 days later, the levels in all groups remained significantly elevated. Unexpectedly, TNFalpha mRNA expression was also induced in the contralateral side of all groups. Immunohistochemical staining showed that TNFalpha-immunoreactive cells in gradually elongated nerves were also positive for S-100 protein but negative for proliferating nuclear cell antigen, indicating that TNFalpha was produced by nonproliferating Schwann cells. CONCLUSIONS: Gradual nerve elongation by limb lengthening induces production of TNFalpha in Schwann cells. Presumably, TNFalpha plays a critical role in the adaptation of peripheral nerves to elongation.

• Induction of interleukin-6 in dorsal root ganglion neurons after gradual elongation of rat sciatic nerve.

  Authors: Naoki Osamura, Kazuo Ikeda, Takaaki Ito, Haruhiro Higashida, Katsuro Tomita, Shigeru Yokoyama

  Experimental neurology. 10/2005; 195(1):61-70.

  In the reconstruction of a segmental defect in injured peripheral nerves, gradual nerve elongation has become an important alternative to nerve grafting. To clarify biochemical responses inIn the reconstruction of a segmental defect in injured peripheral nerves, gradual nerve elongation has become an important alternative to nerve grafting. To clarify biochemical responses in peripheral sensory neurons after nerve elongation, we examined the expression of cytokines and neurotrophins related to nerve regeneration. We first established rat elongation models by lengthening left femurs up to 20.0 mm at the rate of 1.0, 2.0, or 20.0 mm/day. In toluidine blue staining, the acutely elongated, 20-mm/day group showed nuclear eccentricity in the nerve cell body in L5 dorsal root ganglion (DRG) and axonal degeneration in the sciatic nerves; in contrast, the gradually elongated, 1- and 2-mm/day groups remained intact, indicating adaptation. Reverse transcription-polymerase chain reaction analysis revealed that interleukin-6 (IL-6) mRNA was induced in ipsilateral L4-6 DRG in an elongation rate-dependent manner. In contrast, none of the elongated groups exhibited a significant change in mRNA levels for interleukin-1beta, tumor necrosis factor-alpha, nerve growth factor, brain-derived neurotrophic factor, neurotropnin-3, and neurotrophin-4/5. Levels of IL-6 mRNA in all the elongated groups reached the maximum level at day 4 after 20-mm lengthening, while the axotomized group showed a decrease from the maximum level at day 1. Induction of IL-6 mRNA was also detected in the contralateral L4-6 DRG of all the elongated groups, but not detected in the axotomized group. In histochemical analysis, IL-6-immunoreactivity was predominant in neurofilament-positive, medium to large DRG neurons. Application of IL-6 to cultured Schwann cells increased mRNA for peripheral myelin protein 22 (PMP22), a major myelin component. These results suggest that IL-6 plays a key role in biochemical responses in peripheral sensory neurons after gradual nerve elongation.

• Cyclin-dependent kinase 5 activators p35 and p39 facilitate formation of functional synapses.

  Authors: Jenny U Johansson, Lena Lilja, Xiao-Liang Chen, Haruhiro Higashida, Björn Meister, Mami Noda, Zhen-guo Zhong, Shigeru Yokoyama, Per-Olof Berggren, Christina Bark

  Brain research. Molecular brain research. 09/2005; 138(2):215-27.

  Cyclin-dependent kinase 5 (Cdk5) has emerged as a key coordinator of cell signaling in neurite outgrowth. Cdk5 needs to associate with one of the regulatory proteins p35 or p39 to be an activeCyclin-dependent kinase 5 (Cdk5) has emerged as a key coordinator of cell signaling in neurite outgrowth. Cdk5 needs to associate with one of the regulatory proteins p35 or p39 to be an active enzyme. To investigate if Cdk5 plays a role in the establishment of functional synapses, we have characterized the expression of Cdk5, p35, and p39 in the neuroblastoma-glioma cell line NG108-15, and recorded postsynaptic activity in myotubes in response to presynaptic overexpression of Cdk5, p35, and p39. Endogenous Cdk5 and p35 protein levels increased with cellular differentiation and preferentially distributed to soluble pools, whereas the level of p39 protein remained low and primarily was present in membrane and cytoskeletal fractions. Transient transfection of a dominant-negative mutant of Cdk5 in NG108-15 cells and subsequent culturing on differentiating muscle cells resulted in a significant reduction in synaptic activity, as measured by postsynaptic miniature endplate potentials (mEPPs). Overexpression of either Cdk5/p35 or Cdk5/p39 resulted in a substantial increase in synaptic structures that displayed postsynaptic activities, as well as mEPP frequency. These findings demonstrate that Cdk5, p35, and p39 are endogenously expressed in NG108-15 cells, exhibit distinct subcellular localizations, and that both Cdk5/p35 and Cdk5/p39 are central in formation of functional synapses.

• Protein kinase C bound with A-kinase anchoring protein is involved in muscarinic receptor-activated modulation of M-type KCNQ potassium channels.

  Authors: Haruhiro Higashida, Naoto Hoshi, Jia-Sheng Zhang, Shigeru Yokoyama, Minako Hashii, Duo Jin, Mami Noda, Jon Robbins

  Neuroscience research. 04/2005; 51(3):231-4.

  The second messenger for closure of M/KCNQ potassium channels in post-ganglionic neurons and central neurons had remained as a 'mystery in the neuroscience field' for over 25 years. However, recentlyThe second messenger for closure of M/KCNQ potassium channels in post-ganglionic neurons and central neurons had remained as a 'mystery in the neuroscience field' for over 25 years. However, recently the details of the pathway leading from muscarinic acetylcholine receptor (mAChR)-stimulation to suppression of the M/KCNQ-current were discovered. A key molecule is A-kinase anchoring protein (AKAP; AKAP79 in human, or its rat homolog, AKAP150) which forms a trimeric complex with protein kinase C (PKC) and KCNQ channels. AKAP79 or 150 serves as an adapter that brings the anchored C-kinase to the substrate KCNQ channel to permit the rapid and 'definitive' phosphorylation of serine residues, resulting in avoidance of signal dispersion. Thus, these findings suggest that mAChR-induced short-term modulation (or memory) does occur within the already well-integrated molecular complex, without accompanying Hebbian synapse plasticity. However, before this identity is confirmed, many other modulators which affect M-currents remain to be addressed as intriguing issues.

• AKAP150 signaling complex promotes suppression of the M-current by muscarinic agonists.

  Authors: Naoto Hoshi, Jia-Sheng Zhang, Miho Omaki, Takahiro Takeuchi, Shigeru Yokoyama, Nicolas Wanaverbecq, Lorene K Langeberg, Yukio Yoneda, John D Scott, David A Brown, Haruhiro Higashida

  Nature neuroscience. 07/2003; 6(6):564-71.

  M-type (KCNQ2/3) potassium channels are suppressed by activation of G(q/11)-coupled receptors, thereby increasing neuronal excitability. We show here that rat KCNQ2 can bind directly to theM-type (KCNQ2/3) potassium channels are suppressed by activation of G(q/11)-coupled receptors, thereby increasing neuronal excitability. We show here that rat KCNQ2 can bind directly to the multivalent A-kinase-anchoring protein AKAP150. Peptides that block AKAP150 binding to the KCNQ2 channel complex antagonize the muscarinic inhibition of the currents. A mutant form of AKAP150, AKAP(DeltaA), which is unable to bind protein kinase C (PKC), also attenuates the agonist-induced current suppression. Analysis of recombinant KCNQ2 channels suggests that targeting of PKC through association with AKAP150 is important for the inhibition. Phosphorylation of KCNQ2 channels was increased by muscarinic stimulation; this was prevented either by coexpression with AKAP(DeltaA) or pretreatment with PKC inhibitors that compete with diacylglycerol. These inhibitors also reduced muscarinic inhibition of M-current. Our data indicate that AKAP150-bound PKC participates in receptor-induced inhibition of the M-current.

• Subtype-specific coupling with ADP-ribosyl cyclase of metabotropic glutamate receptors in retina, cervical superior ganglion and NG108-15 cells.

  Authors: Haruhiro Higashida, Jia-Sheng Zhang, Sumiko Mochida, Xiao-Liang Chen, Yeonsook Shin, Mami Noda, Kazi Z Hossain, Naoto Hoshi, Minako Hashii, Ryuichi Shigemoto, Shigetada Nakanishi, Yutaka Fukuda, Shigeru Yokoyama

  Journal of neurochemistry. 07/2003; 85(5):1148-58.

  Cyclic ADP-ribose (cADP-ribose) is a putative second messenger or modulator. However, the role of cADP-ribose in the downstream signals of the metabotropic glutamate receptors (mGluRs) is unclear.Cyclic ADP-ribose (cADP-ribose) is a putative second messenger or modulator. However, the role of cADP-ribose in the downstream signals of the metabotropic glutamate receptors (mGluRs) is unclear. Here, we show that glutamate stimulates ADP-ribosyl cyclase activity in rat or mouse crude membranes of retina via group III mGluRs or in superior cervical ganglion via group I mGluRs. The retina of mGluR6-deficient mice showed no increase in the ADP-ribosyl cyclase level in response to glutamate. GTP enhanced the initial rate of basal and glutamate-stimulated cyclase activity. GTP-gamma-S also stimulated basal activity. To determine whether the coupling mode of mGluRs to ADP-ribosyl cyclase is a feature common to individual cloned mGluRs, we expressed each mGluR subtype in NG108-15 neuroblastoma x glioma hybrid cells. The glutamate-induced stimulation of the cyclase occurs preferentially in NG108-15 cells over-expressing mGluRs1, 3, 5, and 6. Cells expressing mGluR2 or mGluRs4 and 7 exhibit inhibition or no coupling, respectively. Glutamate-induced activation or inhibition of the cyclase activity was eliminated after pre-treatment with cholera or pertussis toxin, respectively. Thus, the subtype-specific coupling of mGluRs to ADP-ribosyl cyclase via G proteins suggests that some glutamate-evoked neuronal functions are mediated by cADP-ribose.