Oral Session

Oral Session 7 一般口演7

O7-1 Ezrin controls morphology and proliferation of neural stem cells in the postnatal ventricular-subventricular zone ERM蛋白質Ezrinによる脳室下帯神経幹細胞の形態・増殖制御 Kaneko Naoko（金子奈穂子）¹，石崎友崇^1,2，田村淳³，樋口慧⁴，月田早智子³，澤本和延^1,5 ¹Dept. Dev. Regen. Biol., Nagoya City Univ. Grad. Sch. Med. Sci., Nagoya, Japan ²Dept. Neurosurg., Nagoya Univ. Grad. Sch. Med., Nagoya, Japan ³Lab. Biol. Sci., Grad. Sch. Front. Biosci. and Grad. Sch. Med., Osaka Univ., Osaka, Japan ⁴Lab. Drug Dispos. & Pharmacokinet., Fac. Pharma-Sci., Teikyo Univ., Tokyo, Japan ⁵Div. Neural Dev. Regen., Natl. Inst. Physiol. Sci., Okazaki, Japan In the ventricular-subventricular zone (V-SVZ) located at the lateral wall of the lateral ventricles, neural stem cells (NSCs) continuously generate new neurons throughout life. Although the NSCs share many chemical properties with astrocytes, they possess distinct morphology with fewer processes that make direct contact with the lateral ventricle and parenchymal vasculature at their apical and basal ends, respectively. However, the mechanisms determining this unique morphology and its involvement in stem cell activity are largely unknown. Ezrin, an ezrin-radixin-moesin (ERM) family protein linking actin to the plasma membrane, is exclusively expressed in the V-SVZ astrocytes/NSCs, but not in other cell-types in this region or in parenchymal astrocytes. Here, using ezrin knockout (KO) mice, we investigated the involvement of ezrin in shape determination and neurogenic activity of the NSCs. In the ezrin KO mice, NSCs showed more branched and irregularly extended basal processes, and a significantly reduced proliferation rate compared with those in the age-matched control mice. In addition, proliferation of their progenies, rapidly dividing neuronal progenitors and migratory immature neurons, was reduced, although ezrin expression was not observed in these cells. Taken together, these results suggest that ezrin controls the morphology and proliferation activity of NSCs in the V-SVZ, which influence neurogenesis in the postnatal brain.		O7-2 Effect of mesenchymal stem/stromal cells (MSCs) on CCL5 expression after spinal cord injury 脊髄損傷後のCCL5の増加に対する骨髄間葉系幹/間質細胞の効果 Ohtaki Hirokazu（大滝博和）¹，矢倉一道^1,2，圓谷智海²，佐藤敦²，渡邊潤³，宮本和幸⁴，平泉裕⁵，本田一穂¹ ¹Dept. of Anatomy, Showa Univ. Sch. of Med. ²Dept. of Orthopedic Surg., Showa Univ. Fujigaoka Hosp, Yokohama, Japan ³Ctr for Biotech., Showa Univ., Tokyo, Japan ⁴Dept. of Emerg. & Clin. Care Med., Showa Univ. Fujigaoka Hosp., Yokohama, Japan ⁵Dept. of Orthopedic Surg., Showa Univ. Sch. of Med., Tokyo, Japan Spinal cord injury (SCI) is mainly caused by traffic and sports accidents, and is often subjected to permanent motor dysfunction. Mesenchymal stem/stromal cells (MSCs) have been attracted to be suppressed and/or resolved the SCI in animal and clinical experiments. Microglia/macrophages (MG/Mφ) mobilize to injury site and contribute to both deterioration and resolution of the inflammation. One of the mechanism of human MSCs (hMSCs) are considered to an induction of alternative activating MG/MΦ (M2-type MG/Mφ). However, it remains unclear the mechanism of hMSCs with MG/Mφ. The present study is to determine role of CCL5 after SCI with/without hMSCs transplantation and to determine the gene expression of M2-type MG/Mφ activating and axonal regenerating markers.Male C57/BL6 mice were subjected to SCI between TH9 and 10 intervertebral with thin razor. The hMSCs (5 x10⁵ cells) injected one caudal vertebral at day 1. The spinal cord (TH7 to12) was collected and carried out quantitative PCR and immunohistochemistry for CCL5, the receptors, MG/Mφ markers, and axonal regenerative markers.The expression ccl5 drastically increased 14 days after SCI. Although all receptors increased at day1, and ccr1 and ccr3 were returned to basal level by day 7, ccr5 kept higher level for 2 weeks. The CCL5 and CCR5 localized in neurons and microglia. hMSCs injection further increased ccl5 expression but not CCR5, and increased significantly M2-type MG/Mφ marker, arg1, chil3 and il4 and axonal regenerative marker, dpysl2 and gap43 gene expressions at day 14. Injection of mouse recombinant CCL5 also increased the expression of chil3 and gap43. These results suggest that CCL5 which increased the expression by hMSCs might enhance M2-type MG/Mφ activation and axonal regeneration.

O7-3 In vivo interaction between GTP cyclohydrolase 1 and its regulatory protein GFRP GTPシクロヒドロラーゼ1とその調節タンパク質GFRPのin vivo相互作用 Bellier Jean-Pierre¹，森村敏史¹，謝宇²，遠山育夫¹ ¹Mol. Neurosci. Res. Cen., Shiga Univ. Med. Sci. ²Life Sci. Res. Cen., Beihua Univ., Jilin, China The co-factor 6R-L-erythro-5,6,7,8-tetrahydrobiopterin (BH4)is required for the synthesis of tyrosine, nitric oxide, and the monoamines neurotransmitters (serotonin and catecholamines) by phenylalanine hydroxylase, nitric oxide synthase and the BH4-dependent amino acid hydroxylases: tyrosine hydroxylase, and tryptophan hydroxylase, respectively. De novo biosynthesis of BH4 involves a metabolic pathway with several enzymes, including the GTP cyclohydrolase 1 (GCH1) that is the first and rate-limiting enzyme. In vitro, GCH1 feedback regulation by BH4 is known to be mediated by GCH1 Feedback Regulatory Protein (GFRP) however it remains unclear whether such regulation may occur in vivo. Herein, we investigated the regulation of GCH1 activity by GFRP in vivo, using tissues bearing GCH1 enzymatic activity (i.e: liver, brainstem and dorsal root ganglia). The distribution and colocalization of GCH1 and its regulatory protein GFRP were studied on paraformaldehyde-fixed sections of rat using home-made antisera against each protein. Protein-protein interaction between GFRP and GCH1 was examined using native-PAGE and proximity ligation assay, and the activity of the BH4 metabolic pathway was monitored using HLPC-ECD in presence of a GFRP-dependent GCH1 inhibitor. GCH1 and GFRP protein were localized in hepatocytes, DRG neurons and some population of central monoaminergic neurons. We evidenced GFRP/GCH1 protein complex in liver and DRG, but not in brainstem of normal rat. These results suggest that feedback inhibition of GCH1 activity by GFRP takes place in vivo in liver and DRG neurons, but is unlikely to occur in central nervous system, at least in normal physiological condition.