Nerve Growth Factor-Induced Formation of Axonal Filopodia and Collateral Branches Involves the Intra-Axonal Synthesis of Regulators of the Actin-Nucleating Arp2/3 Complex

Abstract

Nerve growth factor (NGF) induces collateral branching along sensory axons by promoting the formation of axonal filopodia dependent on the actin-nucleating Arp2/3 complex. This study shows that chicken embryonic sensory axons contain mRNAs for the actin-nucleating Arp2/3 complex activator WAVE1 and the complex stabilizer cortactin. NGF increases the axonal levels of WAVE1 and cortactin through localized protein synthesis even in axons isolated from the cell body. Inhibition of protein synthesis in severed axons impairs NGF-induced branching, the formation of axonal filopodia, and the initiation of Arp2/3-dependent axonal actin patches, which serve as precursors to the emergence of filopodia. Overexpression of WAVE1 or cortactin in axons not treated with NGF increased the rate of actin patch formation and the frequency of the emergence of filopodia from actin patches, respectively. Antisense inhibition of cortactin mRNA translation in isolated axons blocked NGF-induced filopodia. NGF also activated the Rac1 GTPase, which drives WAVE1 activity, in a protein synthesis-independent manner. Similarly, inhibition of protein synthesis did not impair the effects of NGF on the axonal microtubule cytoskeleton during branching. The effects of NGF on Rac1 activity and increases in axonal levels of WAVE1 and cortactin were both dependent on phosphoinositide 3-kinase (PI3K) signaling. Collectively, the data indicate that NGF promotes sensory axon branching through regulation of the actin cytoskeleton using both canonical signaling mechanisms and intra-axonal protein synthesis downstream of PI3K signaling. Finally, we present experimental evidence of axonal mRNA translation in sensory axons in the living embryonic spinal cord.