NADPH oxidases (Nox) are membrane‐bound multi‐subunit protein complexes producing reactive oxygen species (ROS) that regulate many cellular processes. Emerging evidence suggests that Nox‐derived ROS also control neuronal development and axonal outgrowth. However, whether Nox act downstream of receptors for axonal growth and guidance cues is presently unknown. To answer this question, we cultured retinal ganglion cells (RGCs) derived from zebrafish embryos and exposed these neurons to netrin‐1, slit2, and brain‐derived neurotrophic factor (BDNF). To test the role of Nox in cue‐mediated growth and guidance, we either pharmacologically inhibited Nox or investigated neurons from mutant fish that are deficient in Nox2. We found that slit2‐mediated growth cone collapse, and axonal retraction were eliminated by Nox inhibition. Though we did not see an effect of either BDNF or netrin‐1 on growth rates, growth in the presence of netrin‐1 was reduced by Nox inhibition. Furthermore, attractive and repulsive growth cone turning in response to gradients of BDNF, netrin‐1, and slit2, respectively, were eliminated when Nox was inhibited in vitro. ROS biosensor imaging showed that slit2 treatment increased growth cone hydrogen peroxide levels via mechanisms involving Nox2 activation. We also investigated the possible relationship between Nox2 and slit2/Robo2 signaling in vivo. astray/nox2 double heterozygote larvae exhibited decreased area of tectal innervation as compared to individual heterozygotes, suggesting both Nox2 and Robo2 are required for establishment of retinotectal connections. Our results provide evidence that Nox2 acts downstream of slit2/Robo2 by mediating growth and guidance of developing zebrafish RGC neurons.

中文翻译：

---

神经元 NADPH 氧化酶 2 调节 slot2/robo2 下游的生长锥导向

NADPH 氧化酶 (Nox) 是膜结合的多亚基蛋白复合物，可产生调节许多细胞过程的活性氧 (ROS)。新出现的证据表明，Nox 衍生的 ROS 也控制神经元发育和轴突生长。然而，目前尚不清楚 Nox 是否作用于轴突生长和引导线索的受体下游。为了回答这个问题，我们培养了源自斑马鱼胚胎的视网膜神经节细胞 (RGC)，并将这些神经元暴露于 netrin-1、slit2 和脑源性神经营养因子 (BDNF)。为了测试 Nox 在提示介导的生长和引导中的作用，我们要么通过药理学抑制 Nox，要么研究来自缺乏 Nox2 的突变鱼的神经元。我们发现，slit2 介导的生长锥塌陷和轴突回缩被 Nox 抑制消除。虽然我们没有看到 BDNF 或 netrin-1 对生长速率的影响，但在 netrin-1 存在下，Nox 抑制会降低生长。此外，当在体外抑制 Nox 时，分别响应 BDNF、netrin-1 和 slot2 梯度的吸引和排斥生长锥转动被消除。ROS 生物传感器成像显示，slit2 处理通过涉及 Nox2 激活的机制增加了生长锥的过氧化氢水平。我们还研究了体内 Nox2 和 slot2/Robo2 信号传导之间的可能关系。ROS 生物传感器成像显示，slit2 处理通过涉及 Nox2 激活的机制增加了生长锥的过氧化氢水平。我们还研究了体内 Nox2 和 slot2/Robo2 信号传导之间的可能关系。ROS 生物传感器成像显示，slit2 处理通过涉及 Nox2 激活的机制增加了生长锥的过氧化氢水平。我们还研究了体内 Nox2 和 slot2/Robo2 信号传导之间的可能关系。与单个杂合子相比， astray/nox2双杂合子幼虫的顶盖神经支配面积减少，这表明 Nox2 和 Robo2 都是建立视网膜顶盖连接所必需的。我们的研究结果提供了证据表明 Nox2 通过介导斑马鱼 RGC 神经元的生长和指导作用于 slot2/Robo2 的下游。