The proper function of the adult nervous system depends on a precise pattern of neuronal connections established during development. A myriad of molecular guidance providing both attractive or repulsive guidance information delineates the path of growth cones to their appropriate, often distant targets. Growth cones, upon contacting guidance cues, respond with changes in behavior and morphology resulting from the activation of numerous signaling pathways which ultimately induces a reorganization of distinct actin filament structures. Yet, the mechanisms how growth cones simultaneously receive, translate and integrate attractive and repulsive guidance informational into navigational decisions remains to be determined. Small GTPases including rac1 might play a key role in translating guidance information into a coordinated actin filament organization. Recently, a rac1-dependent plasma membrane oxidase has been identified in non-neuronal cells and in neurons, which produces superoxide, a potential signaling intermediate. This proposal will investigate the hypothesis that rac1- mediated superoxide production in the neuronal plasma membrane represents a novel mechanism for regulating actin filament dynamics underlying growth cone behavior in response to pro-inflammatory cytokines. This study will assess behavior, superoxide production and actin reorganization in motor neuron growth cone in vitro in response to a discrete exposure either to pro-inflammatory cytokine-coated beads. The role of rac1 and superoxide will be addressed using adenoviral expression of specific rac1 mutants and pharmacological modulation of superoxide levels, respectively. Ultra-localization of rac1 in growth cones with respect to its activity and the isolation and characterization of the neuronal oxidase will supplement this study. Key optical methods employed will include live video microscopy under DIC, phase and fluorescence illumination. Understanding superoxide-mediated actin dynamics and its regulation through rac1 could provide a novel approach for manipulating growth cone motility to enhance regeneration and/or ameliorate cytoskeletal alterations associated with neurodegenerative diseases.
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