若手道場口演（Wakate Dojo）

Wakate Dojo Development and Regeneration 1 若手道場口演発生・発達・再生1

7月25日（木）10:00～10:20　第10会場（万代島ビル　6F　会議室） 1WD10am2-1 新規遺伝子Nwd1は大脳皮質発生を制御する Seiya Yamada（山田晴也），Hiroki Akiyama（秋山博紀），Shin-ichi Sakakibara（榊原伸一）早稲田大学人間科学研究科　分子神経科学 During brain development, immature neurons differentiated from neural stem/progenitor cells (NSPCs) migrate to their destinations. Differentiation from NSPCs and neurite outgrowth and ensuing polarity formation are essential for proper migration, and aberrant neuronal migration cause various psychiatric disorders. However, despite the importance of neuronal migration in brain development, mechanisms controlling the migration of immature neurons differentiated from NSPCs remain largely unclear. Here, we identified NACHT and WD repeat domain containing protein 1 (Nwd1) as the novel gene expressed in NSPCs. First, we revealed the detailed Nwd1 expression pattern in the developing mouse brain. In situ hybridization and immunohistochemistry using a specific antibody to Nwd1 revealed that Nwd1 was localized near the mitochondria of NSPCs and immature neurons in the embryonic cerebral cortex. These observations suggest the involvement of Nwd1 in early brain development. To investigate the Nwd1 functions in developing cerebral cortex using in utero electroporation, nwd1 cDNA or nwd1 shRNA were electroporated into neocortex at E14.5, and analyzed at 2 or 4 days postelectroporation. Nwd1 overexpression in NSPCs suppressed neuronal migration in periventricular area. On the other hand, Nwd1 knockdown caused the accumulation of Tbr2- positive intermediate progenitor cells and doublecortin- positive immature neurons in the VZ, suggesting the induction of premature neuronal differentiation. To investigate the effect of Nwd1 knockdown in the neocortex formations, Nwd1 shRNA was electroporated into E14.5 cerebral cortex and analyzed at P7. Nwd1 knockdown caused periventricular heterotopias due to defects in NSPCs populations and accumulation of β-tubulinIII-positive neurons. Next, to examine the effect of nwd1 on the postmitotic immature neurons, cortical neurons prepared from E16.5 embryos were transfected with the expression vector for nwd1 cDNA or nwd1 shRNA, and cultured for three days. Overexpression and knockdown of nwd1 significantly decreased the number of neurites and axons, suggesting the role in neurite outgrowth and ensuring polarity formation of the newborn neurons. These results suggest that Nwd1 is essential for neocortex development through the controls of neuronal migration from NSPCs. Our findings will contribute to better understanding of brain development, and thus to therapeutic strategy for neurological diseases caused by migration defects.		7月25日（木）10:20～10:40　第10会場（万代島ビル　6F　会議室） 1WD10am2-2 生後小脳EGLにおけるNotch signalによる顆粒前駆細胞の未分化性制御機構について Toma Adachi（足立透真）^1,2，Satoshi Miyashita（宮下聡）¹，Mikio Hoshino（星野幹雄）¹ ¹国立研究開発法人　国立精神・神経医療研究センター ²早稲田大学生命医科学科 Cerebellar granule precursor cells (GCPs) are precursors of cerebellar granule cells which account for 80% of the central nerve cells in our brain. They develop from rhombic lip, constitute external granular layer (EGL) in the outermost layer of cerebellum. First, GCPs proliferate in the outer EGL (oEGL) then move to inner EGL (iEGL)and differentiate to granule neuron (GCs). Development of GCPs is a good model of analysis of molecular mechanism of development for several reasons such as occur in postnatal phase, simplicity to transfect gene and link between layer construction and differentiation state. The development of GCPs is controlled by various signaling systems such as BMP signal, wnt signal, SHH signal, and it is known that when the balance of the signals is lost, our cerebellum development become abnormal one like medulloblastoma. Notch signaling is a signal that is important to the proper development of both of vertebrate and invertebrate. It is also known that notch signal plays an important role in the development of appropriate neurons in the cerebral cortex of mouse. In cerebellar GCPs, it is already known that the notch signal-related proteins are expressed, but whether notch signal exists in GCPs or not is still elusive. Even if it exists, the cell type which GCP contact with via notch signaling is not known. We have overexpressed and downregulated notch signal related protein in GCPs by in vivo electroporation and found that notch signal related proteins play an important role in maintaining undifferentiated states of GCPs. We also found the fact that notch signal works in GCPs by using Hes1 promoter vectors and time lapse imaging. Finally, we got some data that shows the notch signal is done between two GCP cells, so we will present about it together.

7月25日（木）10:40～11:00　第10会場（万代島ビル　6F　会議室） 1WD10am2-3 非ヒト霊長類における神経幹細胞分化メカニズムの解析 Yuji Okano（岡野雄士）¹，Sato Tsukika（佐藤月花）¹，Kase Yoshitaka（加瀬義高）^1,2，Okano Hideyuki（岡野栄之）¹ ¹慶應大医生理 ²東京大学大学院医学研究科加齢医学講座 It has been known that γ-secretase inhibitor inhibits Notch signaling and suppresses the expression of genes such as Hes, a bHLH gene which is involved in the proliferation of neural stem cells (NSCs), and inhibits neuronal differentiation of NSCs. We analyzed how DAPT, a γ-secretase inhibitor, is involved in the differentiation fate of NSCs mainly focused on p38, Dual-specificity phosphatase (DUSP) family. From the developmental aspect, we also analyzed the temporal change in the differentiation potential of NSCs. In mouse-ES cells-derived NSCs, their differentiation ability is changed from neuronal to glial production over time, and we analyzed how DUSP, p38 is involved. We have also analyzed the differentiation ability of ES cells of common marmoset (Callithrix jacchus), a small non-human primate which has been considered genetically, physiologically, and anatomically close to humans. NSCs derived from marmoset ES cells were analyzed using the neurosphere method in the same manner as in experiments on NSCs derived from mouse ES cells. As a result, the proportion of differentiating into glia increased with the passage of marmoset ES cells-derived neurosoheres. This suggests that changes in differentiation ability is common in both rodents and non-human primate. It is under analysis whether this change in non-human primate occurs under the same mechanism as rodents, by focusing on DUSP and p38. In order to investigate neurodegenerative diseases using common marmosets accurately, it is extremely important to know how the differentiation of marmoset NSCs is regulated.