Tianjin Key Laboratory of Neurotrauma Repair, Pingjin Hospital Brain Center, Logistics University of Chinese People's Armed Police Forces, Tianjin, China.
Department of Cardiology, Yichang Second People's Hospital, Hubei, China.
J Alzheimers Dis. 2018;63(1):303-318. doi: 10.3233/JAD-180002.
Electrical excitability by membrane depolarization is crucial for survival and maturation of newborn cells in the dentate gyrus of the hippocampus. However, traditional technology for membrane depolarization lacks temporal and spatial precision. Optogenetics can be used to activate channelrhodopsin-2 (ChR2), allowing cationic current to depolarize genetically targeted cells. In this study, we used ChR2-EGFP driven by doublecortin (DCX) to promote survival and maturation of newborn cells in the dentate gyrus after traumatic brain injury (TBI). C57BL/6 mice underwent lateral fluid percussion TBI. TBI mice were transfected with a lentivirus carrying the DCX-ChR2-EGFP gene. We observed that not only immature neurons but also type-2b intermediate progenitor (IPs) and neuroblasts expressed DCX-EGFP, indicating that DCX-expressing newborn cells could provide a long time window for electrical activity regulation. Quantitative results showed that the number of EGFP-expressing cells began to rise at 3 days after TBI and peaked at 9 days after TBI. By optical depolarization of DCX-EGFP-expressing cells between 3 and 12 days, we observed significantly improved cognitive deficits after TBI with enhanced survival and maturation of newborn cells in the dentate gyrus. We also investigated the role of optical depolarization in neural stem cells transfected with a lentivirus carrying the ChR2-DCX-EGFP gene in vitro. By administrating verapamil to block L-type calcium channels, we verified that the up-regulation of MAP2, NeuN, Neurog2, NeuroD1 and GluR2 in newborn cells was mediated by ChR2-elicted depolarization. By using β-catenin inhibitor Dkk1, we demonstrated that optical depolarization of DCX-EGFP-expressing cells facilitated survival and maturation probably through the Wnt/β-catenin signaling cascade.
细胞膜去极化引起的电兴奋性对于海马齿状回新生细胞的存活和成熟至关重要。然而，传统的膜去极化技术缺乏时空精度。光遗传学可用于激活通道视紫红质-2（ChR2），允许阳离子电流去极化基因靶向细胞。在这项研究中，我们使用双皮质素（DCX）驱动的 ChR2-EGFP 促进创伤性脑损伤（TBI）后齿状回新生细胞的存活和成熟。C57BL/6 小鼠接受侧方液压冲击 TBI。TBI 小鼠用携带 DCX-ChR2-EGFP 基因的慢病毒转染。我们观察到不仅不成熟神经元，而且 2b 型中间祖细胞（IP）和神经母细胞表达 DCX-EGFP，表明表达 DCX 的新生细胞可以提供长时间的电活动调节窗口。定量结果显示，EGFP 表达细胞的数量在 TBI 后 3 天开始增加，并在 TBI 后 9 天达到峰值。通过在 3 至 12 天期间对 DCX-EGFP 表达细胞进行光学去极化，我们观察到 TBI 后认知缺陷明显改善，齿状回新生细胞的存活和成熟得到增强。我们还研究了在体外转染携带 ChR2-DCX-EGFP 基因的慢病毒的神经干细胞中光学去极化的作用。通过给予维拉帕米阻断 L 型钙通道，我们验证了 ChR2 诱导的去极化介导了新生细胞中 MAP2、NeuN、Neurog2、NeuroD1 和 GluR2 的上调。通过使用β-连环蛋白抑制剂 Dkk1，我们证明了 DCX-EGFP 表达细胞的光去极化可能通过 Wnt/β-连环蛋白信号级联促进存活和成熟。
