Andrea Ketschek

Mitochondria Coordinate Sites of Axon Branching through Localized Intra-axonal Protein Synthesis

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 nerve growth factor (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 hot spots 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.

The actin nucleating Arp2/3 complex contributes to the formation of axonal filopodia and branches through the regulation of actin patch precursors to filopodia

The emergence of axonal filopodia is the first step in the formation of axon collateral branches. In vitro, axonal filopodia emerge from precursor cytoskeletal structures termed actin patches. However, nothing is known about the cytoskeletal dynamics of the axon leading to the formation of filopodia in the relevant tissue environment. In this study we investigated the role of the actin nucleating Arp2/3 complex in the formation of sensory axon actin patches, filopodia, and branches. By combining in ovo chicken embryo electroporation mediated gene delivery with a novel acute ex vivo spinal cord preparation, we demonstrate that actin patches form along sensory axons and give rise to filopodia in situ. Inhibition of Arp2/3 complex function in vitro and in vivo decreases the number of axonal filopodia. In vitro, Arp2/3 complex subunits and upstream regulators localize to actin patches. Analysis of the organization of actin filaments in actin patches using platinum replica electron microscopy reveals that patches consist of networks of actin filaments, and filaments in axonal filopodia exhibit an organization consistent with the Arp2/3‐based convergent elongation mechanism. Nerve growth factor (NGF) promotes formation of axonal filopodia and branches through phosphoinositide 3‐kinase (PI3K). Inhibition of the Arp2/3 complex impairs NGF/PI3K‐induced formation of axonal actin patches, filopodia, and the formation of collateral branches. Collectively, these data reveal that the Arp2/3 complex contributes to the formation of axon collateral branches through its involvement in the formation of actin patches leading to the emergence of axonal filopodia.

Nerve Growth Factor Induces Axonal Filopodia through Localized Microdomains of Phosphoinositide 3-Kinase Activity That Drive the Formation of Cytoskeletal Precursors to Filopodia

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 (PIP3), we report localized microdomains of PIP3 accumulation that form in synchrony with F-actin patches and that NGF promotes the formation of microdomains of PIP3 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.

Kinesin-5 Is Essential for Growth-Cone Turning

Inhibition of kinesin-5, a mitotic motor protein also expressed in neurons, causes axons to grow faster as a result of alterations in the forces on microtubules (MTs) in the axonal shaft. Here, we investigate whether kinesin-5 plays a role in growth-cone guidance. Growth-cone turning requires that MTs in the central (C-) domain enter the peripheral (P-) domain in the direction of the turn. We found that inhibition of kinesin-5 in cultured neurons prevents MTs from polarizing within growth cones and causes them to grow past cues that would normally cause them to turn. We found that kinesin-5 is enriched in the transition (T-) zone of the growth cone and that kinesin-5 is preferentially phosphorylated on the side opposite the invasion of MTs. Moreover, when a growth cone encounters a turning cue, phospho-kinesin-5 polarizes even before the growth cone turns. Additional studies indicate that kinesin-5 works in part by antagonizing cytoplasmic dynein and that these motor-driven forces function together with the dynamic properties of the MTs to determine whether MTs can enter the P-domain. We propose that kinesin-5 permits MTs to selectively invade one side of the growth cone by opposing their entry into the other side.

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

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.

The roles of neuronal and glial precursors in overcoming chondroitin sulfate proteoglycan inhibition

The extension of axons through the major inhibitory component of the glial scar, chondroitin sulfate proteoglycans (CSPGs), remains a key obstacle for regeneration following spinal cord injury (SCI). We have previously shown that transplants composed of neuronal and glial restricted precursors (NRP and GRP respectively) promote regeneration and connectivity in the injured spinal cord (Bonner et al., 2010, 2011), however, little is known about the properties of these precursors at a cellular level. We now report that NRP-derived neurons, in contrast to dorsal root ganglion (DRG) neurons, have the ability to extend axons and cross over from a permissive substratum (laminin) onto inhibitory CSPG in vitro. Growth cones of neurons derived from NRP, compared to DRG, exhibit significantly lower levels of the CSPG receptors protein tyrosine phosphatase sigma (PTPσ) and leukocyte common antigen-related phosphatase (LAR). GRP-conditioned medium prepared from the same cell densities did not affect the response of primary sensory neurons to CSPG confirming that the ability of NRP-derived neurons to cross onto CSPG is determined intrinsically. However, GRP-conditioned medium collected from high density cultures increased the probability of DRG axons to cross from LN onto CSPG and increased the length of DRG axons extending on CSPG. Collectively, these results suggest that (1) neurons derived from NRPs are intrinsically insensitive to CSPGs due to low levels of receptor expression, and (2) high levels of factors secreted by GRP can reduce the inhibitory effects of CSPG and promote axonal growth. These observations provide mechanistic insights into the specific roles of NRPs and GRPs in promoting regeneration and repair following SCI.

Nerve growth factor promotes reorganization of the axonal microtubule array at sites of axon collateral branching.

The localized debundling of the axonal microtubule array and the entry of microtubules into axonal filopodia are two defining features of collateral branching. We report that nerve growth factor (NGF), a branch‐inducing signal, increases the frequency of microtubule debundling along the axon shaft of chicken embryonic sensory neurons. Sites of debundling correlate strongly with the localized targeting of microtubules into filopodia. Platinum replica electron microscopy suggests physical interactions between debundled microtubules and axonal actin filaments. However, as evidenced by depolymerization of actin filaments and inhibition of myosin II, actomyosin force generation does not promote debundling. In contrast, loss of actin filaments or inhibition of myosin II activity promotes debundling, indicating that axonal actomyosin forces suppress debundling. MAP1B is a microtubule associated protein that represses axon branching. Following treatment with NGF, microtubules penetrating filopodia during the early stages of branching exhibited lower levels of associated MAP1B. NGF increased and decreased the levels of MAP1B phosphorylated at a GSK‐3β site (pMAP1B) along the axon shaft and within axonal filopodia, respectively. The levels of MAP1B and pMAP1B were not altered at sites of debundling, relative to the rest of the axon. Unlike the previously determined effects of NGF on the axonal actin cytoskeleton, the effects of NGF on microtubule debundling were not affected by inhibition of protein synthesis. Collectively, these data indicate that NGF promotes localized axonal microtubule debundling, that actomyosin forces antagonize microtubule debundling, and that NGF regulates pMAP1B in axonal filopodia during the early stages of collateral branch formation.

Palmitoylation controls DLK localization, interactions and activity to ensure effective axonal injury signaling

Significance Dual leucine-zipper kinase (DLK) is essential for responses to nerve injury and subsequent neural regeneration by controlling transfer of signals from damaged distal axons to neuronal cell bodies. However, DLK is predicted to be freely diffusible, raising the question of how it conveys long-distance, directional signals. Here we report that direct modification of DLK with the lipid palmitate, a process called palmitoylation, is critical for retrograde injury signaling. At the molecular level, palmitoylation targets DLK to retrograde trafficking vesicles, assembles DLK-dependent signaling complexes, and is also essential for DLK kinase activity. This “multifunctional” palmitoylation explains how DLK mediates injury responses and may be a previously unappreciated mechanism that ensures the specificity of enzymatic signaling in diverse cell types. 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 vesicle-bound 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.

Chondroitinase activity can be transduced by a lentiviral vector in vitro and in vivo.

The bacterial enzyme chondroitinase ABC (ChABC), which cleaves chondroitin sulfate glycosaminoglycan chains, can degrade inhibitory scar tissue formed following spinal cord injury, thereby promoting axonal growth and regeneration. However, delivering the active enzyme for prolonged periods presents practical limitations. To overcome these problems, we prepared a lentiviral vector (LV) encoding chondroitinase AC (Chase) together with the green fluorescent protein (GFP) reporter (Chase/LV) and demonstrated its expression and enzymatic activity in vitro and in vivo. Neural precursor cells infected with Chase/LV expressed the GFP reporter at levels that increased dramatically with time in culture. Enzymatic activity from the supernatant of the infected cells was demonstrated by dot blot assay using an antibody that recognizes the digested form of CSPG and was compared with the bacterial ChABC enzyme. Chick DRG cultures plated adjacent to the CSPG border and incubated with supernatant from Chase/LV-infected cells showed neurites growing into the CSPG area, a response similar to that after treatment with ChABC. In contrast, in control cultures, the neurites turned to avoid the inhibitory CSPG interface. Degradation of CSPG in these cultures was confirmed by specific CSPG antibodies. A single injection of Chase/LV into the spinal cord resulted in sustained secretion of the enzyme, whose activity was detected for 8 weeks by expression of GFP and evidence of the digested form of CSPG. This study demonstrates the efficacy of the Chase/LV vector and its potential as a therapeutic tool to reduce scar inhibition and promote axonal growth and repair following central nervous system injury.

Drebrin coordinates the actin and microtubule cytoskeleton during the initiation of axon collateral branches

Drebrin is a cytoskeleton‐associated protein which can interact with both actin filaments and the tips of microtubules. Its roles have been studied mostly in dendrites, and the functions of drebrin in axons are less well understood. In this study, we analyzed the role of drebrin, through shRNA‐mediated depletion and overexpression, in the collateral branching of chicken embryonic sensory axons. We report that drebrin promotes the formation of axonal filopodia and collateral branches in vivo and in vitro. Live imaging of cytoskeletal dynamics revealed that drebrin promotes the formation of filopodia from precursor structures termed axonal actin patches. Endogenous drebrin localizes to actin patches and depletion studies indicate that drebrin contributes to the development of patches. In filopodia, endogenous drebrin localizes to the proximal portion of the filopodium. Drebrin was found to promote the stability of axonal filopodia and the entry of microtubule plus tips into axonal filopodia. The effects of drebrin on the stabilization of filopodia are independent of its effects on promoting microtubule targeting to filopodia. Inhibition of myosin II induces a redistribution of endogenous drebrin distally into filopodia, and further increases branching in drebrin overexpressing neurons. Finally, a 30 min treatment with the branch‐inducing signal nerve growth factor increases the levels of axonal drebrin. This study determines the specific roles of drebrin in the regulation of the axonal cytoskeleton, and provides evidence that drebrin contributes to the coordination of the actin and microtubule cytoskeleton during the initial stages of axon branching.