A synergistic relationship between NADPH oxidases (NOXes) and F-actin in Aplysia californica neuronal growth cones
Abstract
Reactive oxygen species (ROS) are well known for their damaging effects due to oxidation of lipids, proteins and DNA which ultimately results in cell death, for example after spinal cord and brain injuries, in neurodegenerative diseases and glutamate excitotoxicity. Accumulating evidence indicates that ROS also have important signaling functions in cell proliferation, differentiation, cell motility and apoptosis. Here, I investigated the role of ROS in regulating F-actin structure and dynamics in neuronal growth cones. Lowering cytoplasmic levels of ROS by scavengers, NADPH oxidase inhibitors and lipoxygenase inhibitors decreased F-actin content in the peripheral domain of growth cones. NADPH oxidase inhibition resulted in actin assembly inhibition and reduced retrograde actin flow, while lipoxygenase inhibition increased arc contractility, possibly by activating the Rho pathway. When neurons were cultured overnight in the presence of these pharmacological agents, growth cone formation and neurite outgrowth was severely impaired. I also studied the sub-cellular localization of the NADPH oxidase (NOX2) homolog in Aplysia californica bag cell neuronal growth cones. NOX2 consists of plasma membrane and cytoplasmic bound subunits. I verified that the gp91 phox subunit has a membranous localization, while p40 phox has a cytoplasmic localization in growth cones. In control growth cones, a small percentage of gp91 phox and p40 phox subunits exhibit co-localization, which may be indicative of the low level or transient nature of NOX2 activity without stimuli. Interestingly, the p40 phox subunit exhibits distinctive localization along F-actin bundles. When the F-actin cytoskeleton was disrupted in neuronal growth cones with Cytochalasin B, p40 phox levels were decreased suggesting that NOX2 localization and activation depends on F-actin. I propose a tight interdependent relationship between NOX2 activity and the F-actin cytoskeleton in growth cones. Therefore, I conclude that NADPH oxidases redox-regulate F-actin structure and dynamics in neuronal growth cones and neurite outgrowth. Finally, identification of upstream extracellular signals and downstream intracellular signal transduction pathways will be essential for establishing ROS as physiological second messengers in growth cone motility and guidance. Due to the very short half-lives of several ROS, identifying the localization of relevant ROS sources will be an important next step in this project.
Degree
Ph.D.
Advisors
Suter, Purdue University.
Subject Area
Neurobiology
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