Institute of Neuroscience and Brain Research Center, National Yang-Ming University, Taiwan, Republic of China.
Cell Death Dis. 2013 Aug 1;4(8):e751. doi: 10.1038/cddis.2013.274.
Purinergic receptors have been shown to be involved in neuronal development, but the functions of specific subtypes of P2 receptors during neuronal development remain elusive. In this study we investigate the distribution of P2X7 receptors (P2X7Rs) in the embryonic rat brain using in situ hybridization. At E15.5, P2X7R mRNA was observed in the ventricular zone and subventricular zone, and colocalized with nestin, indicating that P2X7R might be expressed in neural progenitor cells (NPCs). P2X7R mRNA was also detected in the subgranular zone and dentate gyrus of the E18.5 and P4 brain. To investigate the roles of P2X7R and elucidate its mechanism, we established NPC cultures from the E15.5 rat brain. Stimulation of P2X7Rs induced Ca(2+) influx, inhibited proliferation, altered cell cycle progression and enhanced the expression of neuronal markers, such as TUJ1 and MAP2. Similarly, knockdown of P2X7R by shRNA nearly abolished the agonist-stimulated increases in intracellular Ca(2+) concentration and the expression of TUJ1 and NeuN. Furthermore, stimulation of P2X7R induced activation of ERK1/2, which was inhibited by the removal of extracellular Ca(2+) and treatment with blockers for P2X7R and PKC activity. Stimulation of P2X7R also induced translocation of PKCα and PKCγ, but not of PKCβ, whereas knockdown of either PKCα or PKCγ inhibited ERK1/2 activation. Inhibition of PKC or p-ERK1/2 also caused a decrease in the number of TUJ1-positive cells and a concomitant increase in the number of GFAP-positive cells. Taken together, the activation of P2X7R in NPCs induced neuronal differentiation through a PKC-ERK1/2 signaling pathway.
嘌呤能受体参与神经元发育，但特定亚型 P2 受体在神经元发育过程中的功能仍不清楚。在这项研究中，我们使用原位杂交技术研究了 P2X7 受体（P2X7R）在胚胎大鼠大脑中的分布。在 E15.5 时，P2X7R mRNA 观察到在脑室区和室下区，与巢蛋白共定位，表明 P2X7R 可能在神经祖细胞（NPCs）中表达。P2X7R mRNA 也在 E18.5 和 P4 大脑的颗粒下层和齿状回中检测到。为了研究 P2X7R 的作用并阐明其机制，我们从 E15.5 大鼠脑中建立了 NPC 培养物。P2X7R 的刺激诱导 Ca2+内流，抑制增殖，改变细胞周期进程，并增强神经元标志物如 TUJ1 和 MAP2 的表达。同样，通过 shRNA 敲低 P2X7R 几乎消除了激动剂刺激引起的细胞内 Ca2+浓度增加和 TUJ1 和 NeuN 的表达。此外，P2X7R 的刺激诱导 ERK1/2 的激活，该激活可通过去除细胞外 Ca2+和用 P2X7R 和 PKC 活性的抑制剂处理来抑制。P2X7R 的刺激还诱导 PKCα 和 PKCγ的易位，但不诱导 PKCβ的易位，而 PKCα 或 PKCγ 的敲低抑制 ERK1/2 的激活。PKC 或 p-ERK1/2 的抑制也导致 TUJ1 阳性细胞数量减少，GFAP 阳性细胞数量增加。总之，NPC 中 P2X7R 的激活通过 PKC-ERK1/2 信号通路诱导神经元分化。
