Department of Pharmacology, School of Pharmacy, Nanjing Medical University, Nanjing, People's Republic of China.
Stem Cells. 2010 Nov;28(11):2041-52. doi: 10.1002/stem.522.
It has been demonstrated that neuronal nitric oxide synthase (nNOS) negatively regulates adult neurogenesis. However, the cellular and molecular mechanisms underlying are poorly understood. Here, we show that nNOS from neural stem cells (NSCs) and from neurons play opposite role in regulating neurogenesis. The NSCs treated with nNOS inhibitor N(5)-(1-imino-3-butenyl)-L- ornithine (L-VNIO) or nNOS gene deletion exhibited significantly decreased proliferation and neuronal differentiation, indicating that NSCs-derived nNOS is essential for neurogenesis. The NSCs cocultured with neurons displayed a significantly decreased proliferation, and deleting nNOS gene in neurons or scavenging extracellular nitric oxide (NO) abolished the effects of coculture, suggesting that neurons-derived nNOS, a source of exogenous NO for NSCs, exerts a negative control on neurogenesis. Indeed, the NSCs exposed to NO donor DETA/NONOate displayed decreased proliferation and neuronal differentiation. The bidirectional regulation of neurogenesis by NSCs- and neurons-derived nNOS is probably related to their distinct subcellular localizations, mainly in nuclei for NSCs and in cytoplasm for neurons. Both L-VNIO and DETA/NONOate inhibited telomerase activity and proliferation in wild-type (WT) but not in nNOS(-/-) NSCs, suggesting a nNOS-telomerase signaling in neurogenesis. The NSCs exposed to DETA/NONOate exhibited reduced cAMP response element binding protein (CREB) phosphorylation, nNOS expression, and proliferation. The effects of DETA/NONOate were reversed by forskolin, an activator of CREB signaling. Moreover, disrupting CREB phosphorylation by H-89 or LV-CREB133-GFP simulated the effects of DETA/NONOate, and inhibited telomerase activity. Thus, we conclude that NSCs-derived nNOS stimulates neurogenesis via activating telomerase, whereas neurons-derived nNOS represses neurogenesis by supplying exogenous NO that hinders CREB activation, in turn, reduces nNOS expression in NSCs.
已经证明神经元型一氧化氮合酶（nNOS）负向调节成年神经发生。然而，其背后的细胞和分子机制尚不清楚。在这里，我们表明神经干细胞（NSCs）和神经元中的 nNOS 发挥相反的作用来调节神经发生。用 nNOS 抑制剂 N(5)-(1-亚氨基-3-丁烯基)-L-鸟氨酸（L-VNIO）处理的 NSCs 或敲除 nNOS 基因后，增殖和神经元分化显著减少，表明 NSCs 来源的 nNOS 对神经发生至关重要。与神经元共培养的 NSCs 增殖显著减少，而敲除神经元中的 nNOS 基因或清除细胞外一氧化氮（NO）消除了共培养的影响，表明神经元来源的 nNOS 作为 NSCs 的外源性 NO 来源，对神经发生施加负向控制。事实上，暴露于 NO 供体 DETA/NONOate 的 NSCs 显示出增殖和神经元分化减少。NSCs 和神经元来源的 nNOS 对神经发生的双向调节可能与其不同的亚细胞定位有关，主要在细胞核中为 NSCs，在细胞质中为神经元。L-VNIO 和 DETA/NONOate 抑制野生型（WT）但不抑制 nNOS(-/-) NSCs 的端粒酶活性和增殖，表明神经发生中的 nNOS-端粒酶信号通路。暴露于 DETA/NONOate 的 NSCs 表现出 cAMP 反应元件结合蛋白（CREB）磷酸化、nNOS 表达和增殖减少。DETA/NONOate 的作用可被 CREB 信号的激活剂 forskolin 逆转。此外，通过 H-89 或 LV-CREB133-GFP 破坏 CREB 磷酸化模拟了 DETA/NONOate 的作用，并抑制了端粒酶活性。因此，我们得出结论，NSCs 来源的 nNOS 通过激活端粒酶刺激神经发生，而神经元来源的 nNOS 通过提供抑制 CREB 激活的外源性 NO 来抑制神经发生，从而减少 NSCs 中的 nNOS 表达。
