Mechanisms involved in the regulation of the axonal cytoskeleton during NGF-induced axon branching

Mirela Spillane

doi:10.17918/00008589

The formation of a complex and functional neuronal circuitry depends on the ability of neuronal processes to extend over long distances and through complex environments to synapse with their targets. Further augmentation of the nervous system innervation is achieved by collateral branching of axonal shafts. Branching enables single axons to make connections with multiple neurons within the same or different nervous system regions. Insight into the signaling mechanisms underlying de novo formation of axon branches during embryogenesis may reveal therapeutic targets in promoting or preventing branching during regeneration in the context of nervous system injury. In vitro and in vivo, axon collateral branches arise through the maturation of transient finger-like protrusions, termed axonal filopodia. In turn, axonal filopodia emerge from dynamic axonal accumulations of actin filaments, termed F-actin patches. The mechanisms of neuronal filopodia formation are poorly understood. In this thesis, I show that the actin related protein (Arp) 2/3 complex plays a significant role in the formation of axonal actin patches, filopodia and branches formed in response to nerve growth factor (NGF). The Arp2/3 complex is shown to target to actin patches through immunocytochemistry, live-cell imaging analysis and platinum replica electron microscopy of actin filament organization in patches. Inhibition of Arp2/3 function impaired formation of axonal filopodia and branches in vitro and in vivo. Furthermore, branching induced by NGF requires intra-axonal synthesis of Arp2/3 complex subunits and the upstream regulators, cortactin and Wiskott-Aldrich syndrome protein-family verprolin homologous protein 1 (WAVE1). NGF, through phosphoinositide 3-kinase (PI3K) signaling, increases axonal levels of Arp2, cortactin, and WAVE1 in axons severed from their cell bodies in a manner sensitive to translation inhibition. Supporting these findings, using PCR-based methods in purified axonal preparations, we detected axonal mRNAs of these three proteins. Cortactin over-expression induces sensory axon collateral branch and filopodia formation in the living spinal cord of chicken embryos. Collectively, these data identify a cellular signaling mechanism for branch formation and link the intraaxonal protein synthesis of cytoskeletal proteins to defined cytoskeletal structures, providing a new mechanism of NGF effect on axonal morphogenesis.

Mechanisms involved in the regulation of the axonal cytoskeleton during NGF-induced axon branching

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Mechanisms involved in the regulation of the axonal cytoskeleton during NGF-induced axon branching

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