S-Space College of Natural Sciences (자연과학대학) Dept. of Biological Sciences (생명과학부) Theses (Ph.D. / Sc.D._생명과학부)
Roles of Stress-related Factors in Embryonic Neurogenesis : Embryonic Neurogenesis에서 스트레스 관련 인자들의 역할
- 자연과학대학 생명과학부
- Issue Date
- 서울대학교 대학원
- Embryonic Neurogenesis ; NPC differentiation ; Prenatal Stress ; Nrf2 ; GSK3 ; IFN-γ ; SC4MOL
- 학위논문 (박사)-- 서울대학교 대학원 : 생명과학부, 2016. 2. 최석우.
- Stress exposure during development can lead to severe neurological diseases in infants and children. Many genes are known to be related to stress induction, but their roles in brain development have not been clearly elucidated. To rapidly screen for biologically meaningful factors involved in brain development, I first selected several genes that are known to be up-regulated in the fetal brain during prevalent stressed conditions such as prenatal infection and alcohol exposure, including nuclear factor (erythroid-derived 2)-like 2 (Nrf2), glycogen synthase kinase 3 (GSK3) α, GSK3β, interferon-gamma (IFN-γ), and sterol-C4-methyl oxidase-like gene (SC4MOL). These genes were overexpressed in primary embryonic day (E) 14.5 murine neural progenitor cells (NPCs) using retroviral vectors. The percentage of neuronal cells was measured by TuJ1 expression. Using this method, GSK3β and IFN-γ were found to have strong negative effects on neurogenesis, and they were further characterized in vitro and in vivo using various molecular techniques.
GSK3 is known as an important regulator during the proliferation and differentiation of NPCs, but the roles of the isoforms of this molecule (GSK3α and GSK3β) have not been clearly defined. Thus, the functions of GSK3α and GSK3β in the context of neuronal differentiation of NPCs were characterized. Treatment of primary NPCs with a GSK3 inhibitor (SB216763) resulted in an increase in the percentage of TuJ1-positive immature neurons, suggesting an inhibitory role of GSK3 in embryonic neurogenesis. Downregulation of GSK3β expression increased the percentage of TuJ1-positive cells, whereas the knock-down of GSK3α appeared to have no effect. Mutant GSK3β (Y216F) failed to suppress neuronal differentiation, indicating that the kinase activity of GSK3β is important for this regulatory function. Similar results were obtained in vivo when a retroviral vector expressing GSK3β was delivered to E9.5 mouse brains. In addition, SB216763 was found to block the rapamycin-mediated inhibition of neuronal differentiation of NPCs. Taken together, these data demonstrate that GSK3β, but not GSK3α, negatively controls the neuronal differentiation of NPCs and that GSK3β may act downstream of the mTORC1 signaling pathway.
IFN-γ is one of the critical cytokines released by host immune cells upon infection. Despite the important role(s) of IFN-γ in host immune responses, there have been no in vivo studies of the effects of IFN-γ on brain development, and the results obtained from many in vitro studies have been controversial. Treatment of E14.5 murine NPCs with IFN-γ resulted in a decrease in the percentage of TuJ1-positive immature neurons but an increase in the percentage of Nestin-positive NPCs. Similar results were obtained in vivo. Treatment of NPCs with a JAK inhibitor or the knock-down of STAT1 expression abrogated the IFN-γ-mediated inhibition of neurogenesis. Interestingly, the expression of one of proneural genes, Neurogenin2 (Neurog2) was inhibited dramatically upon IFN-γ treatment, and cells overexpressing Neurog2 did not respond to IFN-γ. Both IFN-γ treatment and overexpression of the constitutively active form of STAT1 reduced the Neurog2 promoter activity by nearly half. These results suggest that IFN-γ inhibits the neuronal differentiation of NPCs by negatively regulating the expression of Neurog2 partially at the promoter level via the JAK/STAT1 pathway.
In this thesis work, I identified GSK3β and IFN-γ as negative controllers of neuronal differentiation, which act downstream of the mTORC1 signaling pathway and upstream of the JAK/STAT1 pathway, respectively. This is the first study to clearly distinguish the roles of GSK3 isoforms in the context of neuronal differentiation and to investigate the effects of IFN-γ on embryonic neurogenesis in vivo. The findings from this thesis may provide insights into the mechanism of action of different stress-related factors during the early period of brain development, especially the initiation of neurogenesis and the possible consequences of congenital stress exposure.