المؤلفون: Haley Roeder, Aslihan Terzi, Daniel M. Suter, Cory J. Weaver

المصدر: Dev Neurobiol

مصطلحات موضوعية: Retinal Ganglion Cells, 0301 basic medicine, Growth Cones, Retinal ganglion, Article, 03 medical and health sciences, Cellular and Molecular Neuroscience, 0302 clinical medicine, Developmental Neuroscience, Neurotrophic factors, medicine, Animals, Receptors, Immunologic, Growth cone, Receptor, Zebrafish, chemistry.chemical_classification, Reactive oxygen species, NADPH oxidase, biology, Brain-Derived Neurotrophic Factor, Intracellular Signaling Peptides and Proteins, Netrin-1, Zebrafish Proteins, biology.organism_classification, Cell biology, 030104 developmental biology, medicine.anatomical_structure, nervous system, Retinal ganglion cell, chemistry, NADPH Oxidase 2, cardiovascular system, biology.protein, Reactive Oxygen Species, 030217 neurology & neurosurgery, circulatory and respiratory physiology

الوصف: 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.

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::733b2bf530a2d0a126ec91545c5c2668Test
https://doi.org/10.1002/dneu.22791Test

المؤلفون: Aslihan Terzi (9188978)

مصطلحات موضوعية: Neuroscience, NADPH oxidase, ROS, axon guidance, axon development, cellular neuroscience, development, zebrafish, hydrogen peroxide, slit/robo

الوصف: 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 evolutionarily conserved multi-subunit transmembrane proteins. Nox-mediated ROS regulate a variety of biological processes including stem cell proliferation and differentiation, 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. There is now emerging evidence that Nox-derived ROS also control neuronal development and pathfinding. Our lab has recently shown that retinal ganglion cells (RGCs) from nox2 mutant zebrafish exhibit pathfinding errors. However, whether Nox could act downstream of receptors for axonal growth and guidance cues is presently unknown. To investigate this question, we conducted a detailed characterization of the zebrafish nox2 mutants that were previously established in our group. Abnormal axon projections were found throughout the CNS of the nox2 mutant zebrafish. Anterior commissural axons failed proper fasciculation, and aberrant axon projections were detected in the dorsal longitudinal fascicle of the spinal cord. We showed that the major brain regions are intact and that the early development of CNS is not significantly altered in nox2 mutants. Hence, the axonal deficits in nox2 mutants are not due to general developmental problems, and Nox2 plays a role in axonal pathfinding and targeting. Next, we investigated whether Nox2 could act downstream of slit2/Robo2-mediated guidance during RGC pathfinding. We found that slit2-mediated RGC growth cone collapse was abolished in nox2 mutants in vitro. Further, ROS biosensor imaging showed that slit2 treatment increased growth cone hydrogen peroxide levels via mechanisms through Nox2 activation. Finally, we investigated the possible relationship between slit2/Robo2 and Nox2 signaling in vivo. Astray/nox2 double heterozygous mutant larvae exhibited decreased tectal area as opposed to individual heterozygous mutants, suggesting both Nox2 and Robo2 are required for the establishment of retinotectal connections. Our results suggest that Nox2 is part of a signal transduction pathway downstream of slit2/Robo2 interaction regulating axonal guidance cell-autonomously in developing zebrafish retinal neurons.

العلاقة: https://figshare.com/articles/thesis/THE_ROLE_OF_NADPH_OXIDASE_2_IN_AXON_GUIDANCE_DURING_ZEBRAFISH_VISUAL_SYSTEM_DEVELOPMENT/12743375Test

المؤلفون: Aslihan Terzi, Daniel M. Suter, SM Sabbir Alam

المصدر: J Vis Exp

مصطلحات موضوعية: Retinal Ganglion Cells, Cell signaling, animal structures, Neurogenesis, Transgene, General Chemical Engineering, Biosensing Techniques, Biology, Retinal ganglion, Article, General Biochemistry, Genetics and Molecular Biology, Live cell imaging, Animals, Zebrafish, Cells, Cultured, chemistry.chemical_classification, Reactive oxygen species, NADPH oxidase, General Immunology and Microbiology, General Neuroscience, Hydrogen Peroxide, biology.organism_classification, Molecular Imaging, Cell biology, chemistry, biology.protein, Reactive Oxygen Species, Oxidation-Reduction, Intracellular

الوصف: Reactive oxygen species (ROS) are well-established signaling molecules, which are important in normal development, homeostasis, and physiology. Among the different ROS, hydrogen peroxide (H(2)O(2)) is best characterized with respect to roles in cellular signaling. H(2)O(2) has been implicated during development in a number of species. For example, a transient increase in H(2)O(2) has been detected in zebrafish embryos during the first days following fertilization. Furthermore, depleting an important cellular H(2)O(2) 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 H(2)O(2) levels in cultured zebrafish neurons and whole larvae during development using the genetically encoded H(2)O(2)-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 H(2)O(2) levels at the tissue level. The sensor has been validated by the addition of H(2)O(2). Additionally, this methodology could be used to measure H(2)O(2) 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 H(2)O(2) during neuronal and general embryonic development in vertebrates.

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::82a77fa527241a4be57b0a32f4e094fbTest
https://doi.org/10.3791/62165-vTest

المؤلفون: Aslihan Terzi, Daniel M. Suter

المصدر: Free radical biologymedicine. 154

مصطلحات موضوعية: 0301 basic medicine, Central Nervous System, Neurons, Neurite, Chemistry, Neurogenesis, Cellular homeostasis, NADPH Oxidases, Cell migration, Actin cytoskeleton, Biochemistry, Neural stem cell, Cell biology, 03 medical and health sciences, 030104 developmental biology, 0302 clinical medicine, Physiology (medical), Growth cone, Reactive Oxygen Species, 030217 neurology & neurosurgery, Intracellular, Calcium signaling

الوصف: 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.

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::a9fbf41e1dcf619d48354b24da47c9b1Test
https://pubmed.ncbi.nlm.nih.gov/32370993Test

المؤلفون: Aslihan Terzi

مصطلحات موضوعية: cardiovascular system, circulatory and respiratory physiology, Neuroscience

الوصف: 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 evolutionarily conserved multi-subunit transmembrane proteins. Nox-mediated ROS regulate a variety of biological processes including stem cell proliferation and differentiation, 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. There is now emerging evidence that Nox-derived ROS also control neuronal development and pathfinding. Our lab has recently shown that retinal ganglion cells (RGCs) from nox2 mutant zebrafish exhibit pathfinding errors. However, whether Nox could act downstream of receptors for axonal growth and guidance cues is presently unknown. To investigate this question, we conducted a detailed characterization of the zebrafish nox2 mutants that were previously established in our group. Abnormal axon projections were found throughout the CNS of the nox2 mutant zebrafish. Anterior commissural axons failed proper fasciculation, and aberrant axon projections were detected in the dorsal longitudinal fascicle of the spinal cord. We showed that the major brain regions are intact and that the early development of CNS is not significantly altered in nox2 mutants. Hence, the axonal deficits in nox2 mutants are not due to general developmental problems, and Nox2 plays a role in axonal pathfinding and targeting. Next, we investigated whether Nox2 could act downstream of slit2/Robo2-mediated guidance during RGC pathfinding. We found that slit2-mediated RGC growth cone collapse was abolished in nox2 mutants in vitro. Further, ROS biosensor imaging showed that slit2 treatment increased growth cone hydrogen peroxide levels via mechanisms through Nox2 activation. Finally, we investigated the possible relationship between slit2/Robo2 and Nox2 signaling in vivo. Astray/nox2 double heterozygous mutant larvae exhibited decreased tectal area as opposed to individual heterozygous mutants, suggesting both Nox2 and Robo2 are required for the establishment of retinotectal connections. Our results suggest that Nox2 is part of a signal transduction pathway downstream of slit2/Robo2 interaction regulating axonal guidance cell-autonomously in developing zebrafish retinal neurons.

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::47c266a87e65845df8151ffa81090c8eTest

المؤلفون: Haley Roeder, Aslihan Terzi, Theodore Gurol, Daniel M. Suter, Qing Deng, Cory J. Weaver, Yuk Fai Leung

المصدر: The Journal of neuroscience : the official journal of the Society for Neuroscience. 38(26)

مصطلحات موضوعية: 0301 basic medicine, inorganic chemicals, Embryo, Nonmammalian, Neurite, Neurogenesis, Biology, Retinal ganglion, Retina, 03 medical and health sciences, medicine, Animals, Visual Pathways, CYBB, Ganglion cell layer, Zebrafish, Research Articles, NADPH oxidase, General Neuroscience, Optic Lobe, Nonmammalian, biology.organism_classification, Cell biology, 030104 developmental biology, medicine.anatomical_structure, Retinal ganglion cell, NOX1, NADPH Oxidase 2, biology.protein, cardiovascular system, circulatory and respiratory physiology

الوصف: 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.

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::2d0b86e6d56192471398f3985cdc4911Test
https://pubmed.ncbi.nlm.nih.gov/29793976Test