Eom Hyeon Soo, Park Hae Ran, Jo Sung Kee, Kim Young Sang, Moon Changjong, Kim Sung-Ho, Jung Uhee
Radiation Biotechnology Research Division, Advanced Radiation Technology Institute, Korea Atomic Energy Research Institute, Jeongeup, Republic of Korea.
Department of Biochemistry, College of Natural Sciences, Chungnam National University, Daejeon, Republic of Korea.
PLoS One. 2016 Feb 1;11(2):e0147538. doi: 10.1371/journal.pone.0147538. eCollection 2016.
Most studies of IR effects on neural cells and tissues in the brain are still focused on loss of neural stem cells. On the other hand, the effects of IR on neuronal differentiation and its implication in IR-induced brain damage are not well defined. To investigate the effects of IR on C17.2 mouse neural stem-like cells and mouse primary neural stem cells, neurite outgrowth and expression of neuronal markers and neuronal function-related genes were examined. To understand this process, the signaling pathways including PI3K, STAT3, metabotrophic glutamate receptor 1 (mGluR1) and p53 were investigated. In C17.2 cells, irradiation significantly increased the neurite outgrowth, a morphological hallmark of neuronal differentiation, in a dose-dependent manner. Also, the expression levels of neuronal marker proteins, β-III tubulin were increased by IR. To investigate whether IR-induced differentiation is normal, the expression of neuronal function-related genes including synaptophysin, a synaptic vesicle forming proteins, synaptotagmin1, a calcium ion sensor, γ-aminobutyric acid (GABA) receptors, inhibitory neurotransmitter receptors and glutamate receptors, excitatory neurotransmitter receptors was examined and compared to that of neurotrophin-stimulated differentiation. IR increased the expression of synaptophysin, synaptotagmin1 and GABA receptors mRNA similarly to normal differentiation by stimulation of neurotrophin. Interestingly, the overall expression of glutamate receptors was significantly higher in irradiated group than normal differentiation group, suggesting that the IR-induced neuronal differentiation may cause altered neuronal function in C17.2 cells. Next, the molecular mechanism of the altered neuronal differentiation induced by IR was studied by investigating signaling pathways including p53, mGluR1, STAT3 and PI3K. Increases of neurite outgrowth, neuronal marker and neuronal function-related gene expressions by IR were abolished by inhibition of p53, mGluR-1, STAT3 or PI3K. The inhibition of PI3K blocked both p53 signaling and STAT3-mGluR1 signaling but inhibition of p53 did not affect STAT3-mGluR1 signaling in irradiated C17.2 cells. Finally, these results of the IR-induced altered differentiation in C17.2 cells were verified in ex vivo experiments using mouse primary neural stem cells. In conclusion, the results of this study demonstrated that IR is able to trigger the altered neuronal differentiation in undifferentiated neural stem-like cells through PI3K-STAT3-mGluR1 and PI3K-p53 signaling. It is suggested that the IR-induced altered neuronal differentiation may play a role in the brain dysfunction caused by IR.
大多数关于辐射对大脑神经细胞和组织影响的研究仍聚焦于神经干细胞的损失。另一方面，辐射对神经元分化的影响及其在辐射诱导的脑损伤中的意义尚未明确。为了研究辐射对C17.2小鼠神经干细胞样细胞和小鼠原代神经干细胞的影响，检测了神经突生长、神经元标志物的表达以及神经元功能相关基因。为了解这一过程，研究了包括PI3K、STAT3、代谢型谷氨酸受体1（mGluR1）和p53在内的信号通路。在C17.2细胞中，辐射以剂量依赖的方式显著增加了神经突生长，这是神经元分化的形态学标志。此外，辐射还增加了神经元标志物蛋白β-III微管蛋白的表达水平。为了研究辐射诱导的分化是否正常，检测了包括突触素（一种突触小泡形成蛋白）、突触结合蛋白1（一种钙离子传感器）、γ-氨基丁酸（GABA）受体（抑制性神经递质受体）和谷氨酸受体（兴奋性神经递质受体）等神经元功能相关基因的表达，并与神经营养因子刺激分化的情况进行比较。辐射增加突触素、突触结合蛋白1和GABA受体mRNA的表达，与神经营养因子刺激的正常分化情况相似。有趣的是，辐射组谷氨酸受体的总体表达明显高于正常分化组，这表明辐射诱导的神经元分化可能导致C17.2细胞中神经元功能改变。接下来，通过研究包括p53、mGluR1、STAT3和PI3K在内的信号通路，探讨了辐射诱导神经元分化改变的分子机制。抑制p53、mGluR-1、STAT3或PI3K可消除辐射引起的神经突生长增加、神经元标志物和神经元功能相关基因表达增加。在辐射的C17.2细胞中，抑制PI3K可阻断p53信号通路和STAT3-mGluR1信号通路，但抑制p53并不影响STAT3-mGluR1信号通路。最后，在使用小鼠原代神经干细胞的体外实验中验证了辐射诱导C17.2细胞分化改变的这些结果。总之，本研究结果表明，辐射能够通过PI3K-STAT3-mGluR1和PI3K-p53信号通路触发未分化神经干细胞样细胞中神经元分化的改变。提示辐射诱导的神经元分化改变可能在辐射引起的脑功能障碍中起作用。
