Wakate Dojo

Wakate Dojo グリア、ミエリン、回路網形成

1DJ4-1 The soluble form of LOTUS suppresses Nogo receptor type 1-mediated signaling Yutaka Kawakami¹，Yuji Kurihara¹，Yu Saito¹，Ryota Nakagawa¹，Yuki Fujita²，Toshihide Yamashita²，Kohtaro Takei¹ ¹Department of Molecular Medical Bioscience, Graduate School of Medical Life Science, Yokohama City University, Yokohama, Japan，²Department of Molecular Neuroscience, Osaka University Graduate School of Medicine, Suita, Japan Nogo receptor type 1 (NgR1) is a main cause of neuroregenerative inhibition in the central nervous system (CNS) as a receptor of myelin-associated inhibitors (MAI: Nogo-A, MAG, OMgp), BLyS, and CSPGs. Binding of these molecules to NgR1 leads to signal transduction with its co-receptor p75NTR that results in growth cone collapse and neurite outgrowth inhibition through activation of low molecular GTPase Rho. We have previously identified lateral olfactory tract usher substance (LOTUS) as an endogenous antagonist of NgR1. LOTUS is a glycosylphosphatidylinositol-anchored membranous protein, and its binding to NgR1 suppresses MAI-induced growth cone collapse and neurite outgrowth inhibition. Although the soluble form of LOTUS (s-LOTUS) is expected to be a potent agent for promotion of neuronal regeneration after injury in the CNS, the function of s-LOTUS is not fully determined. In this study, we examined the functions of s-LOTUS on NgR1-mediated signaling. While s-LOTUS did not inhibit the binding of the ligands to NgR1, s-LOTUS bound to both p75NTR and NgR1 and suppressed the molecular interaction between NgR1 and p75NTR. Treatment with s-LOTUS inhibited MAI-induced RhoA activation in cultured cortical neurons. In functional analyses, s-LOTUS suppressed NgR1 ligand-induced growth cone collapse and neurite outgrowth inhibition in cultured chick dorsal root ganglia neurons. s-LOTUS rescued NgR1 ligand-induced growth cone collapse in cultured olfactory bulb neurons from LOTUS-knockout mice. Finally, treatment of s-LOTUS promoted axonal regeneration of retinal ganglion cells after optic nerve crush injury in mice. These findings suggest that s-LOTUS inhibits NgR1-mediated signaling and has therapeutic potential for neuronal regeneration in the damaged CNS.		1DJ4-2 The regulatory function of Wnt5a, secreted from periodontal ligament cells loaded with mechanical stimuli, in axonal elongation of peripheral nerves Kaori Takahashi，Takashi Yoshida，Minoru Wakamori Div. Mol. Pharmacol. Cell Biophys., Tohoku Univ. Grad Sch. Dent. The periodontal ligaments (PDL), located at the interface between tooth and alveolar bones, are responsible for tooth planting and pressure absorbing actions. Recent studies have shown that PDL cells produce neurotrophic factors, NGF and BDNF, and the axon guidance protein, Wnt5a⁽¹⁾, and that PC12 cells are differentiated by co-culture with human PDL cells⁽²⁾. Receptor organs in the interface, such as Ruffini endings and free nerve endings, play a pivotal role in pressure sensing. However, the regulatory mechanism of the nerve architecture by the PDL cells is not clear. To elucidate the mechanism, we measured the time-dependent changes of amounts of neurotrophic factors and axon guidance proteins produced by the PDL cells during mechanical stimuli.We established primary PDL cell lines derived from rat (rPDL). The rPDL cells were seeded on silicon chamber, and loaded with periodic mechanical stimulation (0.5 Hz, 15%, 72 hrs). The qPCR analysis revealed that the expression of Wnt5a in rPDL cells increased in a stimulation period-dependent manner, while the expression of neurotrophic factors was constant. To analyze biological function of released factors, the supernatant media of the PDL cells with or without mechanical stimulation were added in the primary mouse trigeminal ganglion (mTG) cells. The supernatant of the mechanical-stimulated PDL cells enhanced the neurite extension in the mTG cells.These results suggest that the mechanical stimulation regulates the neurite extension partly through the increase of Wnt5a secreted from PDL cells.(1)Zhang L. et al., Biochim. Biophys. Acta., 1860 (10), 2211-9, 2016 (2)Tomokiyo A. et al., J. Cell. Physiol., 227, 2040-2050, 2012

1DJ4-3 Specific activation of GABAergic interneurons in the cervical dorsal horn suppresses chronic itch Kensho Kanehisa，Miho Shiratori-Hayashi，Keisuke Koga，Yuta Kohro，Makoto Tsuda Dept Life Innov, Grad Sch Pharm Sci, Kyushu Univ Chronic itch is a debilitating symptom of inflammatory skin conditions, such as atopic and contact dermatitis and dry skin, for which existing treatments are largely ineffective. A recent study has shown that mice with a loss of a subpopulation of inhibitory interneurons in the spinal dorsal horn (SDH) display itch-related behaviors, including scratching. This behavioral phenotype reduces following transplantation of embryonic GABAergic precursor neurons into the SDH. Thus, SDH inhibitory interneurons may play a role in chronic itch. However, whether acute and specific stimulation of inhibitory interneurons that are intrinsically integrated into SDH neuronal circuits alleviates chronic itch remains to be determined. To acutely and specifically stimulate SDH inhibitory interneurons in vivo, we combined designer receptors exclusively activated by designer drugs (DREADD) technology with the flip-excision (FLEX) switch technology. To express the excitatory DREADD, hM3Dq, precisely in SDH inhibitory interneurons, an AAV vector carrying FLEX hM3Dq and mCherry was microinjected into the cervical SDH of Vgat-Cre mice (Vgat-Cre;AAV-hM3DqFLEX mice). Using immunostaining and electrophysiological manner, we confirmed that in Vgat-Cre;AAV-hM3DqFLEX mice, hM3Dq is expressed in the SDH neuron and inhibitory interneuron is activated by CNO, a specific agonist of hM3Dq receptor. We next examined the effect on chronic itch using models of contact dermatitis and dry skin and found that CNO attenuated scratching behavior in both chronic itch models. These results suggest that the activation of inhibitory interneuron suppresses scratching in chronic itch and proposed that enhancing the tone of GABAergic SDH neurons is an effective way to relieve chronic itch.		1DJ4-4 A novel mutation in the IP₃R1 suppressor domain causes spinocerebellar ataxia 29 with altered Ca²⁺ signal patterns Taisei Hirouchi^1,2，Jillian P Casey^3,4，Chihiro Hisatsune²，Akitoshi Miyamoto²，Katsuhiko Mikoshiba² ¹Laboratory of Tumor cell Biology, Department of Computational Biology and Medical Sciences, Graduate School of Frontier Sciences, The University of Tokyo，²Laboratory for Developmental Neurobiology, BrainScience Institute (BSI), RIKEN，³Clinical Genetics, Temple Street Children’s UniversityHospital，⁴UCD Academic Centre on Rare Diseases, School ofMedicine and Medical Sciences, University College Dublin Type 1 inositol 1,4,5-trisphosphate (IP₃) receptor (IP₃R1) is an IP₃-gated Ca²⁺ channel and is recently identified as a causative gene of spinocerebellar ataxia type 15/16 (SCA15/16), type 29 (SCA29) and Gillespie syndrome. While several studies have reported the effect of SCA15/16- or Gillespie syndrome-associated mutations on the IP₃R1 properties, how SCA29-associated mutations affect IP₃R1 function remains unknown. In this study, we identified a novel SCA29-associated missense mutation, p.Arg36Cys (R36C), in the IP₃R1 gene, which localizes within the most NH₂ -terminal region of IP₃R1, a suppressor domain (residues 1-225). Using the GST-fusion protein with the NH₂-terminal region of human IP₃R1 containing both the suppressor domain and the IP₃ binding core domain (589hIP₃R1: residues 1-589), we found that R36C variant significantly increased binding affinity for IP₃. Moreover, we also found that cells expressing R36C variant drastically changed the property of intracellular Ca²⁺ signal from a transient to a sigmoidal pattern. These results indicate that the R36C variant increases the IP₃ binding affinity and enhances the Ca²⁺ release activity of IP₃R1, suggesting a gain-of-function disease mechanism. This is the ﬁrst report of a gain-of-function disease mutation in IP₃R1 and provides novel insights into how enhanced Ca²⁺ release can also contribute to the pathogenesis of SCA29.

1DJ4-5 Identification of unique membrane lipid profile of human immortalized astrocyte Keita Kitamura¹，Hanae Morio^1,2，Kosuke Saito³，Ryo Ito¹，Yoshiro Saito³，Naohiko Anzai²，Hidetaka Akita¹，Tomomi Furihata^1,2 ¹Lab. Pharmacol & Toxicol., Grad. Sch. Pharmceut Sci., Chiba Univ，²Dept. Pharmacol., Grad. Sch. Med., Chiba Univ，³Div. Med. Safety Sci., NIHS 【Purpose】Astrocytes actively participate in central nervous system functions utilizing various membrane protein activities and membrane lipid-mediated signaling. It has been known that these membrane functions are dependent on membrane lipid composition, and, therefore, in order to understand molecular basis of astrocytic membrane functions, characterization of their membrane lipid profile is necessary. In this study, by taking advantage of human immortalized astrocyte cell line (HASTR/ci35), we aimed to identify membrane lipid composition profile of human astrocytes.【Methods】HASTR/ci35 cells were cultured in D-MEM containing N2 supplement. For comparison, HCT116, HepG2 and HEK293 cells were also cultured using the same condition. The lipidomic analysis was performed by LC-TOF/MS. In addition, fatty acid desaturation/elongation enzyme mRNA expression were examined by qPCR. 【Result】The results of lipidomic analysis showed that HASTR/ci35 cells contained significantly larger amount of poly-unsaturated fatty acid (PUFA) in the phosphatidylcholine fraction (4.2 ± 0.1 mol%) than other cell lines (1.4 ± 0.1 mol%, HCT116; 0.6 ± 0.03 mol%, HEK293; 1.3 ± 0.1 mol%, HepG2). Similar PUFA enrichment was also observed in the phosphatidylethanolamine fraction. More specifically, among the PUFA species, 18:2 acyl chains showed the highest content (1.9 ± 0.01 mol%), followed by 20:4 and 20:3 acyl chains. Consistently, fatty acid desaturation/elongation enzyme mRNAs were abundantly expressed in HASTR/ci35 cells. 【Conclusion】To summarize, the lipidomic analysis has revealed that HASTR/ci35 cells are equipped with PUFA-enriched cellular membrane. These results suggested that PUFA-abundant cellular membrane may be critically involved in various astrocytic-specific functions.		1DJ4-6 The neuroprotective effect of erythropoietin on microglial activation, including morphological changes, phagocytosis, and cytokine production Kohki Toriuchi¹，Tetsuya Tamura^2,3，Mineyoshi Aoyama¹，Hiroki Kakita⁴，Kazuya Sobue³，Kiyofumi Asai² ¹Department of Pathobiology, Nagoya City University Graduate School of Pharmaceutical Sciences, Nagoya, Japan，²Department of Molecular Neurobiology, Nagoya City University Graduate School of Medical Sciences, Nagoya, Japan，³Department of Anesthesiology and Intensive Care Medicine, Nagoya City University Graduate School of Medical Sciences, Nagoya, Japan，⁴Department of Perinatal and Neonatal Medicine, Aichi Medical University, Nagakute, Japan Erythropoietin (EPO), a hematopoietic hormonal cytokine induced in response to hypoxia, has neuroprotective effects. EPO receptor (EPOR) is expressed in microglia, resident immune cells in the brain. However, the effect of EPO on microglial activation is not clear. In the present study, we demonstrated that the EPOR is highly expressed in microglia, rather than in neurons or astrocytes, in in vitro experiments. Therefore, we investigated whether EPO could decrease lipopolysaccharide (LPS)-mediated activation of microglia in vitro. The BV-2 microglial cell line was treated with LPS in the absence or presence of EPO. In the presence of EPO, microglial expression of LPS-induced inflammatory cytokine genes was significantly decreased. In addition, EPO suppressed the LPS-induced phagocytic activity of BV-2 cells towards fluorescent beads, as well as induction of inducible nitric oxide synthase. In in vivo experiments, EPO significantly decreased the LPS-induced expression of inflammatory cytokine genes in mouse brains. Furthermore, morphological analysis of cortical microglia in the brains of mice stimulated with LPS revealed that combined treatment with EPO alleviated LPS-induced morphological changes in the microglia. These data indicate that EPO attenuates microglial activation, including morphological changes in vivo, phagocytosis in vitro, and the production of inflammatory cytokines in vivo and in vitro. Further investigation of EPO modulation of LPS-induced microglial activation may contribute to the development of novel neuroprotective therapy.