Phylogenetic conservation of the interdependent homeostatic relationship of sleep regulation and redox metabolism.

Sleep is an essential and evolutionarily conserved process that affects many biological functions that are also strongly regulated by cellular metabolism. The interdependence between sleep homeostasis and redox metabolism, in particular, is such that sleep deprivation causes redox metabolic imbalances in the form of over-production of ROS. Likewise (and vice versa), accumulation of ROS leads to greater sleep pressure. Thus, it is theorized that one of the functions of sleep is to act as the brain's "antioxidant" at night by clearing oxidation built up from daily stress of the active day phase. In this review, we will highlight evidence linking sleep homeostasis and regulation to redox metabolism by discussing (1) the bipartite role that sleep-wake neuropeptides and hormones have in redox metabolism through comparing cross-species cellular and molecular mechanisms, (2) the evolutionarily metabolic changes that accompanied the development of sleep loss in cavefish, and finally, (3) some of the challenges of uncovering the cellular mechanism underpinning how ROS accumulation builds sleep pressure and cellularly, how this pressure is cleared.

ROS Live Cell Imaging During Neuronal Development.

Reactive oxygen species (ROS) are well-established signaling molecules, which are important in normal development, homeostasis, and physiology. Among the different ROS, hydrogen peroxide (H2O2) is best characterized with respect to roles in cellular signaling. H2O2 has been implicated during the development in several species. For example, a transient increase in H2O2 has been detected in zebrafish embryos during the first days following fertilization. Furthermore, depleting an important cellular H2O2 source, NADPH oxidase (NOX), impairs nervous system development such as the differentiation, axonal growth, and guidance of retinal ganglion cells (RGCs) both in vivo and in vitro. Here, we describe a method for imaging intracellular H2O2 levels in cultured zebrafish neurons and whole larvae during development using the genetically encoded H2O2-specific biosensor, roGFP2-Orp1. This probe can be transiently or stably expressed in zebrafish larvae. Furthermore, the ratiometric readout diminishes the probability of detecting artifacts due to differential gene expression or volume effects. First, we demonstrate how to isolate and culture RGCs derived from zebrafish embryos that transiently express roGFP2-Orp1. Then, we use whole larvae to monitor H2O2 levels at the tissue level. The sensor has been validated by the addition of H2O2. Additionally, this methodology could be used to measure H2O2 levels in specific cell types and tissues by generating transgenic animals with tissue-specific biosensor expression. As zebrafish facilitate genetic and developmental manipulations, the approach demonstrated here could serve as a pipeline to test the role of H2O2 during neuronal and general embryonic development in vertebrates.

Neuronal NADPH oxidase 2 regulates growth cone guidance downstream of slit2/robo2.

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.

The role of NADPH oxidases in neuronal development.

Reactive oxygen species (ROS) are critical for maintaining cellular homeostasis and function when produced in physiological ranges. Important sources of cellular ROS include NADPH oxidases (Nox), which are evolutionary conserved multi-subunit transmembrane proteins. Nox-mediated ROS regulate variety of biological processes including hormone synthesis, calcium signaling, cell migration, and immunity. ROS participate in intracellular signaling by introducing post-translational modifications to proteins and thereby altering their functions. The central nervous system (CNS) expresses different Nox isoforms during both development and adulthood. Here, we review the role of Nox-mediated ROS during CNS development. Specifically, we focus on how individual Nox isoforms contribute to signaling in neural stem cell maintenance and neuronal differentiation, as well as neurite outgrowth and guidance. We also discuss how ROS regulates the organization and dynamics of the actin cytoskeleton in the neuronal growth cone. Finally, we review recent evidence that Nox-derived ROS modulate axonal regeneration upon nervous system injury.

nox2/cybb Deficiency Affects Zebrafish Retinotectal Connectivity.

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 in vivo 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 nox genes (nox1, nox2/cybb, nox5, and duox) 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 H2O2, a key ROS involved in Nox signaling. To address isoform-specific Nox functions, we used CRISPR/Cas9 to generate mutations in each zebrafish nox gene. We found that nox2/cybb chimeric mutants displayed ON thinning and decreased OT innervation. Furthermore, nox2/cybb homozygous mutants (nox2/cybb-/-) 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.SIGNIFICANCE STATEMENT 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 in vitro studies. However, whether Nox is involved in the development of axons and formation of neuronal connections in vivo 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.