Department of Molecular Pharmacology and Biological Chemistry, Feinberg School of Medicine, Northwestern University, Chicago, Illinois 60611.
Department of Discovery Toxicology, Amgen, Inc., Thousand Oaks, California 91320.
J Neurosci. 2014 Jul 2;34(27):9107-23. doi: 10.1523/JNEUROSCI.0263-14.2014.
Calcium signals regulate many critical processes during vertebrate brain development including neurogenesis, neurotransmitter specification, and axonal outgrowth. However, the identity of the ion channels mediating Ca(2+) signaling in the developing nervous system is not well defined. Here, we report that embryonic and adult mouse neural stem/progenitor cells (NSCs/NPCs) exhibit store-operated Ca(2+) entry (SOCE) mediated by Ca(2+) release-activated Ca(2+) (CRAC) channels. SOCE in NPCs was blocked by the CRAC channel inhibitors La(3+), BTP2, and 2-APB and Western blots revealed the presence of the canonical CRAC channel proteins STIM1 and Orai1. Knock down of STIM1 or Orai1 significantly diminished SOCE in NPCs, and SOCE was lost in NPCs from transgenic mice lacking Orai1 or STIM1 and in knock-in mice expressing the loss-of-function Orai1 mutant, R93W. Therefore, STIM1 and Orai1 make essential contributions to SOCE in NPCs. SOCE in NPCs was activated by epidermal growth factor and acetylcholine, the latter occurring through muscarinic receptors. Activation of SOCE stimulated gene transcription through calcineurin/NFAT (nuclear factor of activated T cells) signaling through a mechanism consistent with local Ca(2+) signaling by Ca(2+) microdomains near CRAC channels. Importantly, suppression or deletion of STIM1 and Orai1 expression significantly attenuated proliferation of embryonic and adult NPCs cultured as neurospheres and, in vivo, in the subventricular zone of adult mice. These findings show that CRAC channels serve as a major route of Ca(2+) entry in NPCs and regulate key effector functions including gene expression and proliferation, indicating that CRAC channels are important regulators of mammalian neurogenesis.
钙信号调节脊椎动物大脑发育过程中的许多关键过程，包括神经发生、神经递质特化和轴突生长。然而，介导发育中神经系统钙信号的离子通道的身份尚未明确定义。在这里，我们报告说，胚胎和成年小鼠神经干细胞/祖细胞 (NSC/NPC) 表现出由钙释放激活钙 (CRAC) 通道介导的储存操纵钙 (SOCE)。NPC 中的 SOCE 被 CRAC 通道抑制剂 La(3+)、BTP2 和 2-APB 阻断，Western blot 显示存在典型的 CRAC 通道蛋白 STIM1 和 Orai1。STIM1 或 Orai1 的敲低显著减少 NPC 中的 SOCE，并且缺乏 Orai1 或 STIM1 的转基因小鼠和表达失活 Orai1 突变体 R93W 的敲入小鼠中的 NPC 中失去 SOCE。因此，STIM1 和 Orai1 对 NPC 中的 SOCE 做出了重要贡献。NPC 中的 SOCE 被表皮生长因子和乙酰胆碱激活，后者通过毒蕈碱受体发生。SOCE 的激活通过钙调神经磷酸酶/NFAT（激活 T 细胞的核因子）信号转导刺激基因转录，通过一种与 CRAC 通道附近的钙微区通过局部钙信号转导一致的机制。重要的是，STIM1 和 Orai1 表达的抑制或缺失显着减弱了作为神经球培养的胚胎和成年 NPC 的增殖，并且在体内，在成年小鼠的侧脑室下区。这些发现表明 CRAC 通道是 NPC 中钙内流的主要途径，并调节包括基因表达和增殖在内的关键效应功能，表明 CRAC 通道是哺乳动物神经发生的重要调节剂。
