| 幹細胞・細胞分化 |
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| P3-15
神経幹細胞およびグリオーマ幹細胞におけるCHD7の機能解析
大多 茂樹1，Chneiweis Herves2，河上 裕3，岡野 栄之1
慶應大・医・生理学1，ピエール・マリー・キュリー大・パリセーヌ神経科学研2，慶應大・医・先端研3
Macrophage migration inhibitory factor（MIF）plays an important role in supporting the proliferation and/or survival of murine neural stem/progenitor cells（NSPCs）, yet the downstream effectors of this factor remain unknown. Here, we show that MIF increases the expression of Pax6 and Chd7 in NSPCs in vitro. During neural development, Chd7 is expressed in the ventricular zone of the telencephalon of mouse brains at embryonic day 14.5, as well as in cultured NSPCs. Retroviral overexpression of Pax6 in NSPCs increased Chd7 gene expression. Lentivirally-expressed Chd7 shRNA suppressed cell proliferation and neurosphere formation, and inhibited neurogenesis in vitro, while decreasing gene expression of Hes5 and N-Myc. In addition, Chd7 overexpression increased cell proliferation in human ES cell-derived NSPCs. In Chd7 mutant fetal mouse brains, there were fewer Tbr2/Ki67 double-positive compared to wild type brains, indicating that Chd7 contributes to neurogenesis in the early developmental mouse brain. Furthermore, database analysis showed high expression of CHD7 in gliomas among the CHD family and the prognosis of patients with high CHD7 expression was worse than that of patients with low expression of the gene. We observed high levels of CHD7 expression in glioma stem cells compared to astrocytes, and CHD7 was shown to contribute to glioma stem cell proliferation. Collectively, our data demonstrate that CHD7 is an important factor in the proliferation and stemness maintenance of NSPCs and CHD7 is a promising therapeutic target for the treatment of gliomas. |
| P3-16
ヒストン脱アセチル化酵素阻害薬が神経幹細胞の増殖及び分化に与える影響
河内 琢也1，渡部 遼1，樽田 淳樹1，井上 亜耶1，吾郷 由希夫1，田熊 一敞1,2，松田 敏夫1,3
大阪大学大学院薬学研究科薬物治療学分野1，大阪大学大学院歯学研究科薬理学教室2，大阪大学・金沢大学・浜松医科大学・千葉大学・福井大学連合小児発達学研究科3
Valproic acid（VPA）is a multi-target drug and known to inhibit histone deacetylase（HDAC）activity. We have recently demonstrated that prenatal exposure to VPA at embryonic day 12.5（E12.5）causes autism-like behavioral abnormalities in male mice, and that the HDAC inhibition and neuronal loss by VPA may be involved in the autism-like pathology（Int. J. Neuropsychopharamacol., 2013）. On the other hand, we also found that chronic treatment with VPA in juvenile improved memory impairment and dendritic spine loss in the autism model mice（unpublished observation）. In view of the previous observation that HDAC inhibitors have complex effects on neuronal vulnerability and maturation, the present study examines the effects of HDAC inhibitors on proliferation and differentiation in neural stem cells（NSCs）, which are source of mature neurons. NSCs were prepared from the cerebral cortex at E16, and primary and secondary cultures were used to analyze cellular differentiation and proliferation, respectively. The proliferation study using secondary NSCs at 1 day in vitro（DIV）showed that treatment with VPA（0.1～0.3 mM）for 3 days decreased cellular proliferation and neurosphere formation. The other HDAC inhibitors sodium butyrate and trichostatin A also decreased proliferation in NSCs. To study the effect on differentiation, primary NSCs at 7 DIV were treated with HDAC inhibitors for 24 h, reseeded, cultivated under differentiating condition for 14 days, and then neuronal maturation was evaluated immunohistochemically. The neurons derived from HDAC inhibitors-treated NSCs exhibited higher total numbers and length of dendrites, compared with vehicle-treated control. These findings suggest that HDAC inhibitors suppress proliferation while they stimulate differentiation in NSCs. |
| P3-17
小脳のオリゴデンドロサイト分化におけるFGF-2の作用
成瀬 雅衣，柴崎 貢志，横山 就一，倉知 正，石崎 泰樹
群馬大学大学院医学系研究科分子細胞生物学
CD44 is a widely expressed transmembrane glycoprotein involved in the cell-cell and cell-matrix interactions, and is known as an astrocyte precursor marker. In this study, we examined the characteristics of CD44-expressing cells in developing mouse cerebellum. We also examined the effect of fibroblast growth factor-2（FGF-2）on these cells. In mouse cerebellum, CD44 expression was observed not only in GLAST-positive astrocyte precursor cells but also in Sox2-positive neural stem cells and Olig2-positive oligodendrocyte precursor cells at early postnatal stages. Neurospheres were obtained from CD44-positive cells purified from P3 cerebellum using fluorescence-activated cell sorting（FACS）, but not from CD44-negative cells. In addition, FGF-2 and heparin promoted oligodendrocytic differentiation of CD44-positive cells prepared from P3 cerebellum. On the other hand, CD44-negative cells did not differentiate into oligodendrocytes by FGF-2 and heparin. FGF-2 and heparin promoted proliferation of oligodendrocyte precursor cells, increased the number of oligodendrocytes, and delayed astrocyte differentiation in slice culture prepared from P3 cerebellum. These results suggest that FGF-2 has a significant effect on cerebellar oligodendrocyte differentiation, and CD44-positive cells in early postnatal cerebellum might differentiate into oligodendrocytes by FGF signaling. |
| P3-18
Hydrogen Sulfide is Essential for Schwann Cell Responses to Peripheral Nerve Injury.
Park Byung Sun
Department of Anatomy and Neurobioloy, School of Medicine, Biomedical Science Institution, Kyung Hee University
Hydrogen sulfide（H2S）functions as a physiological gas transmitter in both normal and pathophysiological cellular events. H2S is produced from substances by three enzymes：cystathionine β-synthase（CBS）, cystathionine γ-lyase（CSE）and 3-mercaptopyruvate sulfurtransferase（MST）. In human tissues, these enzymes are involved in tissue-specific biochemical pathways for H2S production. For example, CBS and CAT/MST are present in the brain, but CSE is not. Thus, we examined the expression of H2S production-related enzymes in peripheral nerves. Here, we found that CSE and MST/CAT, but not CBS, were present in normal peripheral nerves. In addition, injured sciatic nerves in vivo up-regulated CSE in Schwann cells during Wallerian degeneration（WD）；however, CSE was not up-regulated in peripheral axons. Using an ex vivo sciatic nerve explant culture, we found that the inhibition of H2S production broadly prevented the process of nerve degeneration, including myelin fragmentation, axonal degradation, Schwann cell dedifferentiation and Schwann cell proliferation in vitro and in vivo. Thus, these results indicate that H2S signaling is essential for Schwann cell responses to peripheral nerve injuryKeywords：Hydrogen sulfide, Schwann cells, cytstathionine-γ-lyase（CSE）, demyelination, Wallerian degeneration, axonal degradation. |
| P3-19
神経幹細胞遊走能活性化因子としてのp38MAPキナーゼ
浜之上 誠1,2，緒方 徹3，高松 研1,2
東邦大・医・細胞生理1，東邦大学慢性炎症性疾患の先進医療技術開発センター2，国立障害者リハビリテーションセンター研究所 運動機能系障害研究部3
Adult neural stem/progenitor cells（NPCs）in the subventricular zone（SVZ）migrate into olfactory bulb through rostral migratory stream, and also migrate into the injured sites through blood vessel as a source of regenerative tissue. In the previous study, we have shown that p38 MAP kinase（p38）was expressed in NPCs, but its function remains unknown. In this study, p38-overexpressing-NPCs showed the spheroid formation on culture dish compared to the process-bearing cells which express p38 dominant negative plasmid. To ascertain the roles of p38 on cell migraion, p38-overexpressing-NPCs were seeded onto upper chamber of Transwell, and the cell number in lower chamber was measured after 16h. The cell number of p38-overexpressing-NPCs in lower chamber was increased compared to that of control and p38-dominant negtive cells, suggesting the enhancement of NPC migration by p38-overexpression. In order to enhance NPCs migration more simply, we generated the recombinant p38 protein fused to cell-penetrating peptide, Tat, at N-terminus（Tat-p38）, and determined the roles of Tat-p38 on NPC migration in Transwell. Migrated cell number treated with Tat-p38 protein were increase, and such enhancement was inhibited by p38-specific chemical inhibitor, SB203580. Migrated cells number was not changed by thte treatment with Tat-p38-kinase-dead-protein and p38 protein without Tat-peptide. Furthermore, the number of migrating cells and the distance of migrated cells were enhanced in in adult brain slice cultre treated with Tat-p38 protein. These results indicated that p38 could be potent inducer of NPCs migration, and enhancement of NPCs by Tat-p38 protein might help to regenerate the damaged brain tissue. | | | |
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