Hideyoshi Higashi

Glycosphingolipid synthesis in cerebellar Purkinje neurons: Roles in myelin formation and axonal homeostasis

Glycosphingolipids (GSLs) occur in all mammalian plasma membranes. They are most abundant in neuronal cells and have essential roles in brain development. Glucosylceramide (GlcCer) synthase, which is encoded by the Ugcg gene, is the key enzyme driving the synthesis of most neuronal GSLs. Experiments using conditional Nestin‐Cre Ugcg knockout mice have shown that GSL synthesis in vivo is essential, especially for brain maturation. However, the roles of GSL synthesis in mature neurons remain elusive, since Nestin‐Cre is expressed in neural precursors as well as in postmitotic neurons. To address this problem, we generated Purkinje cell‐specific Ugcg knockout mice using mice that express Cre under the control of the L7 promoter. In these mice, Purkinje cells survived for at least 10–18 weeks after Ugcg deletion. We observed apparent axonal degeneration characterized by the accumulation of axonal transport cargos and aberrant membrane structures. Dendrites, however, were not affected. In addition, loss of GSLs disrupted myelin sheaths, which were characterized by detached paranodal loops. Notably, we observed doubly myelinated axons enveloped by an additional concentric myelin sheath around the original sheath. Our data show that axonal GlcCer‐based GSLs are essential for axonal homeostasis and correct myelin sheath formation.

Comparative characterization of GPRC5B and GPRC5CLacZ knockin mice; behavioral abnormalities in GPRC5B-deficient mice.

Although GPRC5B and GPRC5C are categorized into the G protein-coupled receptor family C, including glutamate receptors, GABA receptors, and taste receptors, their physiological functions remain unknown. Since both receptors are expressed in the brain and evolutionarily conserved from fly to human, it is conceivable that they have significant biological roles particularly in the central nervous system (CNS). We generated GPRC5B- and GPRC5C-deficient mice to examine their roles in the CNS. Both homozygous mice were viable, fertile, and showed no apparent histological abnormalities, though GPRC5B-deficient mice resulted in partial perinatal lethality. We demonstrated that the expressions of GPRC5B and GPRC5C are developmentally regulated and differentially distributed in the brain. GPRC5B-deficient mice exhibited altered spontaneous activity pattern and decreased response to a new environment, while GPRC5C-deficient mice have no apparent behavioral deficits. Thus, GPRC5B has important roles for animal behavior controlled by the CNS. In contrast, GPRC5C does not affect behavior, though it has a high sequence similarity to GPRC5B. These findings suggest that family C, group 5 (GPRC5) receptors in mammals are functionally segregated from their common ancestor.

Loss of α1,6-Fucosyltransferase Decreases Hippocampal Long Term Potentiation IMPLICATIONS FOR CORE FUCOSYLATION IN THE REGULATION OF AMPA RECEPTOR HETEROMERIZATION AND CELLULAR SIGNALING

Background: High expression levels of core fucosylated N-glycans in brain tissues remain unexplained. Results: Loss of core fucosylation enhanced AMPA receptor heteromerization and decreased long term potentiation. Conclusion: Core fucosylation is required for hippocampal long term potentiation. Significance: Core fucosylation may be very important for the neuronal synaptic plasticity that is required for learning and memory. Core fucosylation is catalyzed by α1,6-fucosyltransferase (FUT8), which transfers a fucose residue to the innermost GlcNAc residue via α1,6-linkage on N-glycans in mammals. We previously reported that Fut8-knock-out (Fut8−/−) mice showed a schizophrenia-like phenotype and a decrease in working memory. To understand the underlying molecular mechanism, we analyzed early form long term potentiation (E-LTP), which is closely related to learning and memory in the hippocampus. The scale of E-LTP induced by high frequency stimulation was significantly decreased in Fut8−/− mice. Tetraethylammonium-induced LTP showed no significant differences, suggesting that the decline in E-LTP was caused by postsynaptic events. Unexpectedly, the phosphorylation levels of calcium/calmodulin-dependent protein kinase II (CaMKII), an important mediator of learning and memory in postsynapses, were greatly increased in Fut8−/− mice. The expression levels of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate receptors (AMPARs) in the postsynaptic density were enhanced in Fut8−/− mice, although there were no significant differences in the total expression levels, implicating that AMPARs without core fucosylation might exist in an active state. The activation of AMPARs was further confirmed by Fura-2 calcium imaging using primary cultured neurons. Taken together, loss of core fucosylation on AMPARs enhanced their heteromerization, which increase sensitivity for postsynaptic depolarization and persistently activate N-methyl-d-aspartate receptors as well as Ca2+ influx and CaMKII and then impair LTP.

Gangliosides and chondroitin sulfate desensitize and internalize B2 bradykinin receptors.

Prolonged or repeated agonist activation of G-protein-coupled receptors (GPCRs) initiates their desensitization and internalization, rendering them unresponsive to agonist activation. We analyzed how gangliosides and chondroitin sulfate affect B2 bradykinin (BK) receptors (B2Rs). Gangliosides and chondroitin sulfate did not stimulate intracellular Ca(2+) release from B2R-expressing CHO-K1 cells, but repeated exposure desensitized B2Rs to BK stimulation. Microscopic observation of DsRed-fused B2Rs revealed that several gangliosides and chondroitin sulfate C (CSC) effectively internalized B2Rs. Ganglioside-CSC treatment of B2R mutant-expressing cells failed to desensitize and internalize the mutant receptors. As this mutant lacks the first extracellular domain and cannot activate GPCR kinase (GRK), gangliosides and CSC likely initiate B2R desensitization and endocytosis through GRK-mediated B2R phosphorylation.

Intraplantar injection of gangliosides produces nociceptive behavior and hyperalgesia via a glutamate signaling mechanism

&NA; Gangliosides are abundant in neural tissue and play important roles in cell‐cell adhesion, signal transduction, and cell differentiation. Gangliosides are divided into 4 groups: asialo‐, a‐, b‐, and c‐series gangliosides, based on their biosynthetic pathway. St8sia1 knockout mice, which lack b‐ and c‐series gangliosides, exhibit altered nociceptive responses. The mechanism underlying this defect, however, remains unclear. To address this issue, we first investigated the possibility that gangliosides in peripheral nociceptor endings are involved in nociception. Intraplantar injection of the b‐series ganglioside GT1b, but not a‐series gangliosides such as GM1, produced nociceptive responses and enhanced low‐concentration formalin‐induced nociception. N‐methyl‐d‐aspartic acid receptor and type I metabotropic glutamate receptor antagonists inhibited GT1b‐induced hyperalgesia, suggesting the involvement of glutamate receptors. Furthermore, microdialysis analysis revealed elevated glutamate content in subdermal tissues due to intraplantar injection of GT1b. Co‐injection of glutamate dehydrogenase with GT1b attenuated GT1b‐induced hyperalgesia. These findings suggested that GT1b induced extracellular glutamate to accumulate in subdermal tissues, thereafter activating glutamate receptors, which in turn resulted in hyperalgesia and nociception. On the other hand, intraplantar injection of sialidase, which cleaves sialyl residues from glycoconjugates such as gangliosides, attenuated the late phase of 2% formalin‐induced nociception. Thus, the antinociceptive effects of sialidase and the nociceptive effects of GT1b indicated that endogenous gangliosides are involved in nociceptive responses. These results suggest that gangliosides play important roles in nociceptive responses originating in peripheral nociceptor endings. Ganglioside GT1b induced extracellular glutamate to accumulate in subdermal tissues, thereafter activating glutamate receptors, which in turn resulted in hyperalgesia and nociception.

Close association of B2 bradykinin receptors with P2Y2 ATP receptors

Two G-protein-coupled receptors (GPCRs) that couple with Gαq/11, B2 bradykinin (BK) receptor (B2R) and ATP/UTP receptor P2Y2 (P2Y2R), are ubiquitously expressed and responsible for vascular tone, inflammation, and pain. We analysed the cellular signalling of P2Y2Rs in cells that express B2Rs. B2R desensitization induced by BK or B2R internalization-inducing glycans cross-desensitized the P2Y2R response to ATP/UTP. Fluorescence resonance energy transfer from P2Y2R-AcGFP to B2R-DsRed was detected in the cells and on the cell surfaces, showing the close association of these GPCRs. BK- and ATP-induced cross-internalization of P2Y2R and B2R, respectively, was shown in a β-galactosidase complementation assay using P2Y2R or B2R fused to the H31R substituted α donor peptide of a β-galactosidase reporter enzyme (P2Y2R-α or B2R-α) with coexpression of the FYVE domain of endofin, an early endosome protein, fused to the M15 acceptor deletion mutant of β-galactosidase (the ω peptide, FYVE-ω). Arrestin recruitment to the GPCRs by cross-activation was also shown with the similar way. Coimmunoprecipitation showed that B2R and P2Y2R were closely associated in the cotransfected cells. These results indicate that B2R couples with P2Y2R and that these GPCRs act together to fine-tune cellular responsiveness. The collaboration between these receptors may permit rapid onset and turning off of biological events.

Involvement of ganglioside GT1b in glutamate release from neuroblastoma cells.

Since gangliosides play many important roles in neural systems, we investigated whether gangliosides are involved in glutamate release from neural cells. Differentiated neruro2a cells were treated with gangliosides, including GM3, GM1, GD1a, GD3, GD1b, or GT1b, for 30 min, and glutamate concentration in the culture media was measured using o-phthalaldehyde derivatization. Among the tested gangliosides, GT1b significantly increased the glutamate concentration when compared with untreated cells. Moreover, GT1b increased the glutamate concentration in the culture media of neuroblastoma × dorsal root ganglion neuron hybrid F11 cells. These results suggested that gangliosides are important in regulating extracellular glutamate concentration in the nervous system.

The roles of gangliosides in nociception

s / Neuroscience Research 68S (2010) e109–e222 e165 sciatic nerve ligation (PSL) in mice. MIP-1 mRNA and its protein were upregulated after PSL, and MIP-1 was localized on recruited macrophages and activated Schwann cells in the injured sciatic nerve (SCN). PSL-induced long-lasting tactile allodynia and thermal hyperalgesia were prevented by the perineural injection of neutralizing antibody for MIP-1 (anti-MIP-1 ). Intraneural and perineural injection of recombinant MIP-1 elicited tactile allodynia and thermal hyperalgesia in sham-operated mice. Receptors for MIP-1 (CCR1 and CCR5) mRNA and their proteins were also up-regulated in the SCN after PSL, and were localized on macrophages and Schwann cells. PSL-induced tactile allodynia was attenuated by perineural injection of siRNA against CCR1 and CCR5. On the other hand, PSL-induced thermal hyperalgesia was prevented by siRNA against CCR5, but not CCR1. Interleukin-1 (IL-1 ) mRNA and its precursor protein in macrophages and Schwann cells were also up-regulated in the SCN after PSL, and PSL-induced neuropathic pain was prevented by the perineural injection of neutralizing antibody for IL-1 . Up-regulation of IL-1 after PSL was suppressed by anti-MIP-1 and siRNA against CCR1 and CCR5. Perineural injection of nicotine, a macrophage suppressor, prevented PSL-induced neuropathic pain and suppressed the expressions of MIP-1 and IL-1 . In conclusion, we propose a novel critical molecule MIP-1 derived from macrophages and Schwann cells appears to play a crucial role in the development of neuropathic pain induced by PSL. doi:10.1016/j.neures.2010.07.2302 P1-j14 Role of estrogen in pain modulation in ovariectomized adult rats Li-Hong LI Institute of Neurobiology, Fudan University, Shanghai, China Many pain syndromes are prevalent in women and it is known that estrogen plays a key role in pain perception. This study was to measure the basal nociceptive responses to mechanical and thermal stimuli in naive, sham and ovariectomized (OVX) adult SD rats and to study if estrogen could modulate it. We used von Frey test (hindlimbs), Hargreavesquot test (hindlimbs) and tail flick test (proximal and distal tail) in different groups from pre-surgery till seven weeks after surgery one or two times per week. The body weight, uterus weight and vaginal smear were monitored to ensure the success of the surgery. We found that rats developed allodynia in von Frey test and hyperalgesia in tail flick test from 4-5 weeks after OVX till 7 weeks after surgery but not in Hargreavesquot test. The body weight increased and uterus weight decreased significantly in OVX group than in the other groups. And all of these phenomena could be reversed by the replacement of 17betaestradial. The estrogen plays an important role in stabilizing the basal pain threshold of female rats. doi:10.1016/j.neures.2010.07.2303 P1-j15 The roles of gangliosides in nociception Shun Watanabe 1 , Koichi Tan-No 2, Takeshi Tadano 2, Hideyoshi Higashi 1 1 Division of Glyco-signal Research, Institute of Molecular Biomembrane and Glycobiology, Tohoku Pharmaceutical University, sendai, Japan 2 Department of Pharmacology, Tohoku Pharmaceutical University, Sendai, Japan Gangliosides are abundant in neural tissue and play important roles in cell-cell adhesion and signal transduction. Based on their biosynthetic pathway, gangliosides are classified into four groups (asialo-, a-, b-, and c-series gangliosides). GD3 synthase (St8sia1) knockout mice, which lack band c-series gangliosides, showed altered nociceptive responses (Handa Y et al. 2005, Pain). However, the mechanism remains unclear. To address this issue, we investigated whether gangliosides in peripheral nociceptor endings are involved in nociception. Intraplantar injection of the b-series ganglioside GT1b, but not a-series gangliosides such as GM1, induced nociceptive responses and hyperalgesia. Furthermore, co-injection of glutamate receptor antagonists with GT1b reduced GT1b-induced hyperalgesia. Microdialysis analysis revealed elevated glutamate in the subdermal tissues by intraplantar GT1b injection. These findings suggested that intraplantar GT1b injection leads to accumulation of extracellular glutamate in subdermal tissues, thereafter activating glutamate receptors, which in turn resulted in hyperalgesia. These results suggest that gangliosides play important roles in nociceptive responses in peripheral nociceptor endings and that gangliosidemanipulating agents are useful for pain relief. doi:10.1016/j.neures.2010.07.2304 P1-j16 A local anesthetic, ropivacaine, suppresses the activated spinal glial cells in neuropathic pain Atsushi Sakai 1 , Shigeru Toda 2, Yumiko Ikeda 1, Sakamoto Atsuhiro 2, Hidenori Suzuki 1 1 Dept Pharmacol, Nippon Med Sch, Tokyo 2 Dept Anesthesiol, Nippon Med Sch, Tokyo Local anesthetics alleviate neuropathic pain in some cases in clinical practice, exhibiting longer durations of action than that predicted on the basis of the pharmacokinetics of their blocking effect on voltage-dependent sodium channels. Therefore, local anesthetics may contribute to additional mechanisms for reversal of the sensitization of nociceptive pathways which occurs in the neuropathic pain state. Here we examined the involvement of spinal glial cells in the analgesic effect of a local anesthetic, ropivacaine, in a rat model of neuropathic pain. The sciatic nerve was loosely ligated in rats to produce a chronic constrictive injury. The resulting thermal hyperalgesia was significantly reduced by the repetitive epidural administration of ropivacaine. Concomitantly with this analgesia, ropivacaine suppressed in the dorsal spinal cord the increase in immunoreactivities of OX-42 and GFAP, markers of activated microglia and astrocyte respectively, which followed the constrictive nerve injury. Nerve growth factor (NGF) was upregulated in the injured dorsal root ganglion following the ropivacaine administration. The epidural administration of TrkA-IgG fusion protein which blocks NGF action prevented the inhibitory effect of ropivacaine on microglia, but not astrocyte. The blockade of NGF action also abolished the analgesic effect of ropivacaine on neuropathic pain. These results suggest that ropivacaine suppresses the activated spinal glial cells in the dorsal spinal cord at least in part via NGF action, resulting in the prolonged analgesia. doi:10.1016/j.neures.2010.07.2305 P1-j17 Clinical mu-opioid receptor agonist remifentanil inhibits activities of inspiratory neurons in the medulla Yuki Kaku , Shun-ichi Kuwana Faculty of Health Sciences, Uekusa Gakuen University, Chiba, Japan While anesthetic drugs enable us to undergo invasive operations without pain and fears, they have potential risks of inducing serious side effects, e.g., disturbance of respiration. Remifentanil, a short-acting anesthetic agent, is recently used in clinical scene and known to induce respiratory depression with high-dose administration. However, the neural mechanisms of remifentanil-induced respiratory modulation remain unclear. We recently reported that remifentanil could reduced the rate of respiratory related burst activities of phrenic nerves in the isolated brain-spinal cord of newborn rats. The result suggests that remifentanil acts on the neural structures involved in respiratory rhythm generation. In the present study, therefore, we investigated the effects of remifentanil on activities of respiratory neurons in the ventrolateral medulla where the respiratory rhythm generator exists. The brainstem and cervical spinal cord of a newborn rat were isolated and maintained in the artificial cerebrospinal fluid. We recorded rhythmic bursts of C4 ventral root as an index of inspiratory activity. Activities of respiratory neurons in the ventrolateral medulla were recorded by a perforated whole cell patch-clamp technique. The patch-clamp recordings in the ventrolateral medulla revealed that remifentanil reduced the rate of burst firing (burst/min) of inspiratory neurons residing in the pre-Boetzinger complex. Thus, respiratory depression caused by remifentanil is due to inhibition of inspiratory neurons in the pre-Boetzinger complex. In contrast, there is little change in rhythm of burst firing of pre-inspiratory neurons, which may suggest that primary rhythm of respiration is generated by pre-inspiratory neurons. doi:10.1016/j.neures.2010.07.2306 P1-j18 CO2 compensates for dorsomedial medullary 5HT2 receptor activity on hypoxic ventilatory and airway responses Mitsuko Kanamaru , Ikuo Homma Department of Physiology, Showa University School of Medicine In the dorsomedial medulla oblongata (DMM), including the hypoglossal nucleus and the solitary tract nucleus, 5-HT acting on 5-HT2 receptors mediates the immediate onset of hypoxic hyperventilation and airway dilation. Hypercapnia compensates for airway narrowing and reduced ventilation elicited by 5-HT2 antagonism in the DMM. In this study, mice underwent

1021 Regional expression of an o-acetylated ganglioside epitope in subsets of neuroepithelial cells of chicken embryonic forebrain

Shinobu C. Fujita, Rika Kato, Hideyoshi Higashi Monoclonal antibody(MAb) 85A9 stains a certain subset of neurons and undifferentiated neuroepithelial cells of restricted regions of the chicken embryonic brain. Immunochemical results had indicated a glycolipid nature of the antigen. In this work MAb 85A9-immunoreactive bands were identified on TLC of extracts of embryonic brain. The stained bands were similar but not identical to those of MAbs 8A2 or 493D4 with reported specificity to certain 0-acetylated gangliosides. The 85A9 immunoreactivity was lost by treatment with sialidase or sodium methylate, a deacetylating reagent. The histochemical immunoreactivity was destroyed by sialidase but not by periodate treatments. Thus the antigen was considered to be 0-acetylated gangioside(s). A whole mount immunostaining procedure that does not use detergents was developed to visualize the glycolipid antigen in chicken embryos up to embryonic day 5, and the diencephalic staining patterns deduced from the sections were confirmed.

S3-1 Visualization of calcium/calmodulin-dependent protein kinase ii activity in cultured hippocampal neurons

A number of genes for hereditary neurodegenerative diseases have been identified by means of positional cloning. Among the causative genes, expansions of trinucleotide repeatshavebeenidentifiedas acommonmechanismofdominantlyinheritedneurodegenerative diseases. To elucidate the molecular mechanisms of neurodegeneration in these diseases two major questions come out; 1. What are the molecular mechanisms of meiotic as well as