一般口演
神経発生とグリア発生 2
Neurogenesis and Gliogenesis 2
座長：田中 謙二（慶應義塾大学） |
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| 2022年7月2日 16:10～16:25 沖縄コンベンションセンター 会議場B3・4 第6会場
3O06e1-01
光操作技術を用いた神経幹細胞の制御メカニズムの解析
Analysis of neural stem cell regulatory mechanisms using optogenetics
*山田 真弓(1,2)、長崎 真治(1,2)、Seongchun Yang(1,2)、今吉 格(1,2,3)
1. 京都大学大学院生命科学研究科、2. 京都大学大学院生命科学研究科附属生命動態研究センター、3. 京都大学ウイルス再生医科学研究所
*Mayumi Yamada(1,2), Shinji C Nagasaki(1,2), Seongchun Yang(1,2), Itaru Imayoshi(1,2,3)
1. Grad Sch of Biostudies, Kyoto Univ, Kyoto, Japan, 2. Research Center for Dynamic Living Systems, Grad Sch of Biostudies, Kyoto Univ, Kyoto, Japan, 3. Institute for Frontier Life and Medical Sciences, Kyoto Univ, Kyoto, Japan
Keyword: Neural stem cell, optogenetics, neuronal differentiation, RNA-seq
Neural stem cells (NSCs) generate neurons, astrocytes and oligodendrocytes in mammalian brain. The production of these cells is precisely controlled by various regulatory mechanisms in NSCs during development and remodelling. Among such regulations, basic helix-loop-helix (bHLH) transcription factors function as key molecules in the cell proliferation, multipotency, fate determination, and differentiation of NSCs. Several bHLH transcription factors such as Ascl1, Hes1 and Olig2, are co-expressed in NSCs and change its expression patterns dynamically during cell proliferation and cell-fate determination. Previously, we found that these factors show oscillatory expression patterns during cell proliferation of NSCs (Imayoshi et al., 2013). In this study, we focus on the importance of the expression dynamics of Ascl1 during cell proliferation and neuronal differentiation. To analyze the functional roles of dynamic changes in Ascl1 expression, we induced oscillatory or sustained Ascl1 expressions in Ascl1 conditional knock-out cultured NSCs by our novel photoactivatable (PA)-Tet-ON system (Yamada et al., 2018). We found that oscillatory Ascl1 expression proceeded in cell proliferation and sustained Ascl1 expression promoted neuronal differentiation. Now, we are analyzing the downstream gene expression changes caused by these light-induced Ascl1 expressions by RNA sequencing. Some of the genes show similar oscillatory expression according to the oscillatory expression of Ascl1. In addition, some genes show increased expression according to the sustained expression of Ascl1. Many of the former genes are involved in cell proliferation and tissue development, while many of the latter genes are involved in neuronal differentiation. Furthermore, we are investigating the chromatin accessibility of Ascl1 target genes in cell proliferation and differentiation by Assay for Transposase-Accessible Chromatin (ATAC)-Sequencing analysis. In the process of cell proliferation and differentiation, chromatin accessibility of Ascl1 target genes seem to show several patterns for each gene. Some DNA regions are observed to gradually open according to cell proliferation or differentiation, while some remain open throughout. In addition, we are utilizing single cell RNA sequencing to analyze Ascl1 expression in individual cells and the behavior of its target gene in detail. We would like to comprehensively analyze these results to elucidate the regulatory mechanism of NSCs. | | 2022年7月2日 16:25～16:40 沖縄コンベンションセンター 会議場B3・4 第6会場
3O06e1-02
種特異的プロモーターノンコーディングRNA（pancRNA）によるヒト・マウス神経幹細胞制御
Differential regulation of human and mouse neural stem cells mediated by species-specific promoter-associated non-coding RNAs (pancRNAs)
*今村 拓也(1)、安 博洋(1)、安東 明莉(1)、槇村 有紗(1)、徳永 真結莉(1)、植木 龍也(1)、森下 文浩(1)
1. 広島大学
*Takuya Imamura(1), Boyang An(1), Akari Ando(1), Arisa Makimura(1), Mayuri Tokunaga(1), Tatsuya Ueki(1), Fumihiro Morishita(1)
1. Hiroshima University
Keyword: non-coding RNA, species-specific, neural stem cell
【Introduction】 Expansion and folding in the neocortex are associated with unique cognitive abilities that distinguish human from other mammalian species. At present, a set of human- or primate-specific genes have been proven that promote cortical expansion and folding, such as TMEM14B. Since cortical folding emerges progressively during evolution, multiple genes, not only specific genes, but also conserved genes, should be involved in this process by finely tuning their expression levels. We have previously reported that promoter-associated non-coding RNAs (pancRNAs) transcribed from bidirectional promoter act on cis-acting elements to differentiate transcription levels of neighboring genes. First, among such pancRNAs, we are focusing on pancCD63: it is expressed in the human, but not in the mouse neural stem cells (NSCs). CD63, known as an exosomal marker, is also expressed much higher in human NSCs. 【Materials and Methods】 human NSCs (AF22 cell line) and embryonic (E) 14.5 mouse NSCs were infected by using lentivirus in knockdown and overexpression experiments. Immunostaining detecting active caspase3 and cell cycle labeling assay using EdU were performed to analyze cell apoptosis and proliferation, respectively. As a gain-of-function experiment, in-utero electroporation was performed on E13.5 mouse embryos, and plasmid DNA was microinjected through the uterus into the lateral ventricle. Western blot analysis was performed to explore the downstream target about CD63. 【Results】 Knockdown pancCD63 reduced expression level of CD63, suggesting that pancCD63 can be as a regulatory molecule to affect CD63 expression. Knockdown of either CD63 or pancCD63 resulted in a dramatical decrease in EdU+ cells and an increase in active caspase3+ cells, suggesting that pancCD63-CD63 pair promote human NSC proliferation. Overexpression of CD63 in mouse brain increased number of basal progenitors (BPs) marked by Pax6 and Tbr2 at E15.5. Immunohistochemical and western blot analyses showed that CD63 might increase BPs proliferative ability through ITGB1-Akt pathway. It is to be noted that, at E18.5, CD63 overexpression generated a large number of upper layer neurons and showed a folding-like structure. 【Conclusion】 evolutionary acquired quantitative difference in the expression of pancCD63-CD63 pair can play a key role in cortical development and folding for developing brain individuality. We will also discuss the involvement of other species-specific pancRNA-partnered genes that include Ucp2, Nrsn2 and Bmi1. | | 2022年7月2日 16:40～16:55 沖縄コンベンションセンター 会議場B3・4 第6会場
3O06e1-03
加齢過程における成体海馬神経幹細胞のDNAメチロームの解読
Decoding the DNA methylome of adult hippocampal neural stem cells during the aging process
*松田 泰斗(1)、岩本 昌和(1)、中島 欽一(1)
1. 九州大学
*Taito Matsuda(1), Masakazu Iwamoto(1), Kinichi Nakashima(1)
1. Kyushu University
Keyword: Neural stem cell, Neurogenesis, aging, DNA methylation
Recent studies have demonstrated that reduction of neural stem cell (NSC) proliferation in the adult hippocampus with age and subsequent decrease in neurogenesis cause age-related impairment of learning and memory. DNA methylation, one of the epigenetic modifications, has been shown to control the differentiation of NSCs into neurons or glial cells in the developing brain. However, it is unclear whether DNA methylation changes with age lead to NSC dysfunctions in the adult hippocampus. Here, we examined how gene expression alterations associated with age-related changes in DNA methylation affect the NSC behavior by conducting whole-genome bisulfite sequencing (WGBS) and RNA sequencing (RNA-seq). We isolated EGFP-labeled NSCs from the hippocampus of 5 day-, 12 week-, and 24 week-old Nestin-EGFP transgenic mice that express EGFP under the control of the promotor of NSC marker gene Nestin and directly subjected them to WGBS and RNA-seq. In addition to transcriptomic alteration, we found the chronological alteration of the CpG and non-CpG DNA methylation profiles in hippocampal NSCs. We then identified differentially methylated regions with age, which consisted of many hypermethylated regions and a few hypomethylated regions. Interestingly, the non-CpG DMR and the CpG DMR were not located close to each other on the genome. Moreover, motif discovery analysis of DMR showed that CpG DMR but not non-CpG DMR contains motif sequences for NFI family members, known regulators for DNA methylation landscape. These results suggest that CpG methylation and non-CpG methylation, regulated by distinct factors, control the expression of different gene sets in NSC during the aging process. Thus, our data highlight the in vivo implications of DNA methylation dynamics that impair NSC functions with age. | | 2022年7月2日 16:55～17:10 沖縄コンベンションセンター 会議場B3・4 第6会場
3O06e1-04
Neurotrophin-3が成体海馬歯状回の神経新生と行動に与える影響
The role of neurotrophin-3 in the hippocampal adult neurogenesis and stress-related behaviors
*瀬木-西田 恵里(1)、笠倉 奈々美(1)
1. 東京理科大学 先進工 生命システム工
*Eri Segi-Nishida(1), Nanami Kasakura(1)
1. Tokyo Uni of Sci, Tokyo, Japan
Keyword: neurotrophin-3, hippocampus, neurogenesis, stress
The hippocampus is one of the brain regions which regulate stress-related emotional behaviors and where neurogenesis occurs throughout life. The adult hippocampal neurogenesis includes proliferation, survival, and neuronal differentiation in the sugranular zone of the dentate gyrus (DG). One of the neurotrophic factors: neurotrophin-3 (NT-3) regulates neuronal differentiation, survival, and synaptic formation, and is highly expressed in the DG in central nervous systems. Although stress generally decreases neurotrophic factor expression and neurogenesis, it has been reported that NT-3 expression in the hippocampus is increased by stress environment. To investigate the role of NT-3 in the hippocampal function, NT-3 was overexpressed in the hippocampal dentate gyrus by injecting NT-3 containing adeno-associated virus (AAV-NT-3). After 4 weeks of AAV-NT-3 injection, high NT-3 expression was observed in neurons in the hilus region, and the expression increased to more than 10 folds compared with the control group. We examined the influence of NT-3 overexpression on the neurogenic process in the DG. NT-3 overexpression decreased the number of cell proliferation and immature neurons in the subgranular zone of the DG. We also observed the mice with high expression of NT-3 in the hippocampus showed high spontaneous motor activity. These results suggest that high level of NT-3 in the hippocampus suppress the early phase of neurogenic process. Currently, we are identifying the mechanism by which NT3 controls neurogenesis and examining its effect on hippocampal-involved behavior. | |
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