SUMMARY The branching of axons is a fundamental aspect of nervous system development and neuroplasticity. We report that branching of sensory axons in the presence of NGF occurs at sites populated by stalled mitochondria. Translational machinery targets to presumptive branching sites, followed by recruitment of mitochondria to these sites. The mitochondria promote branching through ATP generation and the determination of localized hotspots of active axonal mRNA translation, which contribute to actin-dependent aspects of branching. In contrast, mitochondria do not have a role in the regulation of the microtubule cytoskeleton during NGF-induced branching. Collectively, these observations indicate that sensory axons exhibit multiple potential sites of translation, defined by presence of translational machinery, but active translation occurs following the stalling and respiration of mitochondria at these potential sites of translation. This study reveals a local role for axonal mitochondria in the regulation of the actin cytoskeleton and axonal mRNA translation underlying branching.

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. Over-expression 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.

Dual leucine-zipper kinase (DLK) is critical for axon-to-soma retrograde signaling following nerve injury. However, it is unknown how DLK, a predicted soluble kinase, conveys long-distance signals and why homologous kinases cannot compensate for loss of DLK. Here, we report that DLK, but not homologous kinases, is palmitoylated at a conserved site adjacent to its kinase domain. Using short-hairpin RNA knockdown/rescue, we find that palmitoylation is critical for DLK-dependent retrograde signaling in sensory axons. This functional importance is because of three novel cellular and molecular roles of palmitoylation, which targets DLK to trafficking vesicles, is required to assemble DLK signaling complexes and, unexpectedly, is essential for DLK's kinase activity. By simultaneously controlling DLK localization, interactions, and activity, palmitoylation ensures that only vesiclebound DLK is active in neurons. These findings explain how DLK specifically mediates nerve injury responses and reveal a novel cellular mechanism that ensures the specificity of neuronal kinase signaling.

The initiation of axonal filopodia is the first step in the formation of collateral branches and synaptic structures. In sensory neurons, nerve growth factor (NGF) promotes the formation of axonal filopodia and branches. However, the signaling and cytoskeletal mechanisms of NGF-induced initiation of axonal filopodia are not clear. Axonal filopodia arise from precursor axonal cytoskeletal structures termed filamentous actin (F-actin) patches. Patches form spontaneously and are transient. Although filopodia emerge from patches, only a fraction of patches normally gives rise to filopodia. Using chicken sensory neurons and live imaging of enhanced yellow fluorescent protein (eYFP)-actin dynamics, we report that NGF promotes the formation of axonal filopodia by increasing the rate of F-actin patch formation but not the fraction of patches that give rise to filopodia. We also demonstrate that activation of the phosphatidylinositol 3-kinase (PI3K)-Akt pathway is sufficient and required for driving the formation of axonal F-actin patches, filopodia, and axon branches. Using the green fluorescent protein-plekstrin homology domain of Akt, which targets to PI3K-generated phosphatidylinositol-3,4,5-triphosphate (PIP 3 ), we report localized microdomains of PIP 3 accumulation that form in synchrony with F-actin patches and that NGF promotes the formation of microdomains of PIP 3 and patches. Finally, we find that, in NGF, F-actin patches form in association with axonal mitochondria and oxidative phosphorylation is required for patch formation. This investigation demonstrates that surprisingly NGF promotes formation of axonal filopodia by increasing the formation of cytoskeletal filopodial precursors (patches) through localized microdomains of PI3K signaling but not the emergence of filopodia from patches.