The Role of NADPH Oxidases in Neurite Outgrowth and Zebrafish Neurodevelopment

Cory J Weaver, Purdue University

Abstract

Nicotinamide adenine dinucleotide phosphate oxidases (NOX) are a family of enzymes that produce reactive oxygen species (ROS). The first NOX enzyme was discovered in leukocytes and associated with host defense in the immune system. Subsequent findings of ROS production in non-immune cells led to the identification of six additional NOX isoforms, and opened new avenues for research into NOX-mediated cellular functions. Since then, NOX-derived ROS have been found to be involved in a tremendous number of cell signaling pathways. Of particular interest is the well-established function of NOX-derived ROS in signaling pathways that drive cytoskeletal rearrangements and motility in several cell types. Our lab is interested in the highly motile neuronal growth cone that guides axonal growth during neurodevelopment and regeneration. Others have reported that inhibition of NOX enzymes during development causes a decrease in the size of some brain areas, and NOX deficiencies in humans are correlated with diminished cognitive function. Despite the fact that NOX activity is necessary for some cell motility in non-neuronal cells and a loss of NOX function during development has impacts on brain structure, it is still unclear what role NOX plays in axonal growth and guidance or the establishment of connections in the central nervous system. In this series of studies, I investigated the function of NOX enzymes in neurite outgrowth and neurodevelopment using both in vitro and in vivo approaches. I report that NOX activity is necessary for the extension of neurites in culture. Inhibition NOX activity leads to a disorganization of the actin cytoskeleton, a decrease in retrograde flow and a loss of neurite outgrowth. Using embryonic zebrafish, I showed that four separate nox genes are expressed broadly throughout the central nervous system during the first two days of development. Functional studies in zebrafish revealed that NOX inhibition leads to expansion of the ganglion cell layer in the retina and decrease in the size of the optic nerve. Subsequent studies using a site-specific, RNA-guided mutagenesis approach (CRISPR/Cas9) showed that optic nerve thinning resulted from developmental delays while ganglion cell expansion was a permanent defect caused by the mutation of a specific NOX isoform, NOX2/CYBB. The targeting of optic nerve axons to the optic tectum of the midbrain was also disrupted in NOX2/CYBB mutants. These results show that NOX activity is critical for neurite outgrowth in cultured cells in addition to retinal development and axon targeting in vivo.

Degree

Ph.D.

Advisors

Suter, Purdue University.

Subject Area

Neurosciences|Cellular biology|Developmental biology

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