The experiments in this proposal investigate how two guidance cues, nerve growth factor (NGF) and Semaphorin3A (Sema3A), regulate the migration of the growing tips (growth cones) of sensory (DRG) axons. NGF is produced in targets of sensory neurons, like skin. Immature axons navigate to the skin, then, invade and branch to form sensory endings. However, if nerve growth factor is absent in skin, sensory innervation does not occur. Similar innervation deficits occur if neurotrophins, NGF, BDNF or NT3, are absent from other target organs. Sema3A is expressed in central and peripheral tissues that sensory growth cones avoid, and animals with defective functions of Sema3A or its receptors have defasciculated and misrouted axons. Knowledge of how NGF and Sema3A regulate the motility of sensory axonal growth cones can be applied to understanding developmental deficits in innervation and neural circuits. In addition, neurotrophins can promote axonal regeneration in the central and peripheral nervous systems, and Sema3A contributes to the inhibitory environment that blocks axonal regeneration. Thus, our findings will be generally relevant in repairing diseased and damaged neural circuits. The long term goal of our project is to understand how guidance cues, like NGF and Sema3A regulate growth cone migration, as sensory axons innervate skin targets. At the tip of a growing axon, protrusive motility explores the environment, makes adhesive contacts to promote axonal growth and initiates responses that direct axonal pathfinding. Activation of trkA receptors by NGF triggers polymerization of actin filaments and protrusion of the growth cone margin. Sema3A activates receptors to inhibit protrusion and keep growing axons from straying on their way to peripheral and central target zones. We will test the hypothesis that NGF and Sema3A act through three actin-associated proteins to mediate the motility of growth cones.
The aims of this proposal are to investigate the roles and necessity of ADF/cofilin, the Arp2/3 complex, and ERM proteins in the regulation of growth cone behaviors. These proposed experiments involve in vitro and in vivo studies, using DRG sensory neurons from chicken embryos. Cellular and molecular methods, time lapse microscopy, immunocytochemistry, image analysis, and neuronal transfection will be used in three aims.
The first aim i nvestigates the roles of ADF/cofilin, Arp2/3 and ERM proteins in mediating the responses of sensory neuronal growth cones to NGF. ADF/cofilin sever actin filaments to generate new barbed ends for actin polymerization, the Arp2/3 complex may nucleate actin filaments and ERM proteins link actin filaments to the plasma membrane.
The second aim will investigate whether inhibition of these activities mediates the loss of protrusive motility and avoidance responses that Sema3A induces in growth cones.
The third aim i nvolves studies to investigate the roles of these proteins in mediating in vivo responses of sensory axons to NGF and Sema3A.

Public Health Relevance

Normal behavior requires correct wiring of neural connections during embryonic development, and neural repair and regeneration also require accurate regrowth of neural processes. This project investigates the mechanisms by which growing axons are guided by molecular attractants in their target tissues. This knowledge will be used to promote regeneration and repair of disabled, diseased and damaged nervous systems.

National Institute of Health (NIH)
Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD)
Research Project (R01)
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Special Emphasis Panel (ZRG1-MDCN-N (02))
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Henken, Deborah B
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University of Minnesota Twin Cities
Schools of Medicine
United States
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San Miguel-Ruiz, José E; Letourneau, Paul C (2014) The role of Arp2/3 in growth cone actin dynamics and guidance is substrate dependent. J Neurosci 34:5895-908
Gomez, Timothy M; Letourneau, Paul C (2014) Actin dynamics in growth cone motility and navigation. J Neurochem 129:221-34
Marsick, Bonnie M; Roche, Florence K; Letourneau, Paul C (2012) Repulsive axon guidance cues ephrin-A2 and slit3 stop protrusion of the growth cone leading margin concurrently with inhibition of ADF/cofilin and ERM proteins. Cytoskeleton (Hoboken) 69:496-505
Marsick, Bonnie M; San Miguel-Ruiz, Jose E; Letourneau, Paul C (2012) Activation of ezrin/radixin/moesin mediates attractive growth cone guidance through regulation of growth cone actin and adhesion receptors. J Neurosci 32:282-96
Marsick, Bonnie M; Letourneau, Paul C (2011) Labeling F-actin barbed ends with rhodamine-actin in permeabilized neuronal growth cones. J Vis Exp :
Marsick, Bonnie M; Flynn, Kevin C; Santiago-Medina, Miguel et al. (2010) Activation of ADF/cofilin mediates attractive growth cone turning toward nerve growth factor and netrin-1. Dev Neurobiol 70:565-88
Roche, Florence K; Marsick, Bonnie M; Letourneau, Paul C (2009) Protein synthesis in distal axons is not required for growth cone responses to guidance cues. J Neurosci 29:638-52
Letourneau, Paul C (2009) Actin in axons: stable scaffolds and dynamic filaments. Results Probl Cell Differ 48:65-90
Harder, Jennifer; Xu, Xiaohua; Letourneau, Paul et al. (2008) The actin cross-linking protein AFAP120 regulates axon elongation in a tyrosine phosphorylation-dependent manner. Neurosci Lett 444:132-6
Blackmore, Murray; Letourneau, Paul C (2007) Protein synthesis in distal axons is not required for axon growth in the embryonic spinal cord. Dev Neurobiol 67:976-86

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