Department of Biological Sciences.
Purdue Institute of Inflammation, Immunology and Infectious Disease.
J Neurosci. 2018 Jun 27;38(26):5854-5871. doi: 10.1523/JNEUROSCI.1483-16.2018. Epub 2018 May 23.
NADPH oxidase (Nox)-derived reactive oxygen species (ROS) have been linked to neuronal polarity, axonal outgrowth, cerebellar development, regeneration of sensory axons, and neuroplasticity. However, the specific roles that individual Nox isoforms play during nervous system development remain unclear. To address this problem, we investigated the role of Nox activity in the development of retinotectal connections in zebrafish embryos. Zebrafish broadly express four genes (, , , and ) throughout the CNS during early development. Application of a pan-Nox inhibitor, celastrol, during the time of optic nerve (ON) outgrowth resulted in significant expansion of the ganglion cell layer (GCL), thinning of the ON, and a decrease in retinal axons reaching the optic tectum (OT). With the exception of GCL expansion, these effects were partially ameliorated by the addition of HO, a key ROS involved in Nox signaling. To address isoform-specific Nox functions, we used CRISPR/Cas9 to generate mutations in each zebrafish gene. We found that chimeric mutants displayed ON thinning and decreased OT innervation. Furthermore, homozygous mutants () showed significant GCL expansion and mistargeted retinal axons in the OT. Neurite outgrowth from cultured zebrafish retinal ganglion cells was reduced by Nox inhibitors, suggesting a cell-autonomous role for Nox in these neurons. Collectively, our results show that Nox2/Cybb is important for retinotectal development in zebrafish. Most isoforms of NADPH oxidase (Nox) only produce reactive oxygen species (ROS) when activated by an upstream signal, making them ideal candidates for ROS signaling. Nox enzymes are present in neurons and their activity has been shown to be important for neuronal development and function largely by studies. However, whether Nox is involved in the development of axons and formation of neuronal connections has remained unclear. Using mutant zebrafish embryos, this study shows that a specific Nox isoform, Nox2/Cybb, is important for the establishment of axonal connections between retinal ganglion cells and the optic tectum.
NADPH 氧化酶（Nox）衍生的活性氧（ROS）与神经元极性、轴突生长、小脑发育、感觉轴突再生和神经可塑性有关。然而，个别 Nox 同工型在神经系统发育过程中所起的具体作用尚不清楚。为了解决这个问题，我们研究了 Nox 活性在斑马鱼胚胎视网膜-视顶盖连接发育中的作用。在视神经（ON）生长期间，广谱表达于整个中枢神经系统的四个基因（、、、和）。应用泛 Nox 抑制剂白藜芦醇（celastrol）会导致神经节细胞层（GCL）显著扩张，视神经变薄，视网膜轴突到达视顶盖（OT）的数量减少。除了 GCL 扩张外，这些效应部分被关键的 Nox 信号 ROS 氢过氧化物（HO）的添加所缓解。为了研究同工型特异性的 Nox 功能，我们使用 CRISPR/Cas9 技术在每个斑马鱼基因中产生突变。我们发现，ON 变薄和 OT 神经支配减少的突变杂合子。此外，纯合突变体（）显示出显著的 GCL 扩张和视网膜轴突在 OT 中的错误靶向。体外培养的斑马鱼视网膜神经节细胞的突起生长被 Nox 抑制剂所抑制，这表明 Nox 在这些神经元中具有细胞自主性作用。总之，我们的结果表明 Nox2/Cybb 对斑马鱼的视网膜-视顶盖发育很重要。大多数 NADPH 氧化酶（Nox）同工型只有在被上游信号激活时才会产生活性氧（ROS），这使它们成为 ROS 信号的理想候选物。Nox 酶存在于神经元中，其活性对神经元的发育和功能很重要，这主要是通过研究来证明的。然而，Nox 是否参与轴突的发育和神经元连接的形成仍然不清楚。本研究使用突变的斑马鱼胚胎表明，一种特定的 Nox 同工型 Nox2/Cybb 对视网膜神经节细胞和视顶盖之间轴突连接的建立很重要。
