Bioenergetic Requirements and Spatiotemporal Profile of Nerve Growth Factor Induced PI3K-Akt Signaling Along Sensory Axons.

Nerve Growth Factor (NGF) promotes the elaboration of axonal filopodia and branches through PI3K-Akt. NGF activates the TrkA receptor resulting in an initial transient high amplitude burst of PI3K-Akt signaling followed by a maintained lower steady state, hereafter referred to as initiation and steady state phases. Akt initially undergoes phosphorylation at T308 followed by phosphorylation at S473, resulting in maximal kinase activation. We report that during the initiation phase the localization of PI3K signaling, reported by visualizing sites of PIP3 formation, and Akt signaling, reflected by Akt phosphorylation at T308, correlates with the positioning of axonal mitochondria. Mitochondrial oxidative phosphorylation but not glycolysis is required for Akt phosphorylation at T308. In contrast, the phosphorylation of Akt at S473 is not spatially associated with mitochondria and is dependent on both oxidative phosphorylation and glycolysis. Under NGF steady state conditions, maintenance of phosphorylation at T308 shows dual dependence on oxidative phosphorylation and glycolysis. Phosphorylation at S473 is more dependent on glycolysis but also requires oxidative phosphorylation for maintenance over longer time periods. The data indicate that NGF induced PI3K-Akt signaling along axons is preferentially initiated at sites containing mitochondria, in a manner dependent on oxidative phosphorylation. Steady state signaling is discussed in the context of combined contributions by mitochondria and the possibility of glycolysis occurring in association with endocytosed signalosomes.

The Axonal Glycolytic Pathway Contributes to Sensory Axon Extension and Growth Cone Dynamics.

Understanding the bioenergetics of axon extension and maintenance has wide ranging implications for neurodevelopment and disease states. Glycolysis is a pathway consisting of 10 enzymes and separated into preparatory and payoff phases, the latter producing ATP. Using embryonic chicken sensory neurons, we report that glycolytic enzymes are found through the axon and the growth cone. Pharmacological inhibition of glycolysis in the presence of NGF impairs axon extension and growth cone dynamics within minutes without affecting axon maintenance. Experiments using microfluidic chambers show that the effect of inhibiting glycolysis on axon extension is local along distal axons and can be reversed by promoting mitochondrial respiration. Knockdown of GAPDH simplifies growth cone morphology and is rescued by shRNA-resistant GAPDH expression. Rescue of GAPDH using KillerRed fused to GAPDH followed by localized chromophore-assisted light inactivation of KillerRed-GAPDH in distal axons halts growth cone dynamics. Considering filament polymerization requires ATP, inhibition of glycolysis results in a paradoxical increase in axonal actin filament levels. The effect on actin filaments is because of enzymes before GAPDH, the first enzyme in the payoff phase. In the absence of NGF, inhibition of glycolysis along distal axons results in axon degeneration independent of cell death. These data indicate that the glycolytic pathway is operative in distal axons and contributes to the rate of axon extension and growth cone dynamics in the presence of NGF and that, in the absence of NGF, the axonal glycolytic pathway is required for axon maintenance.SIGNIFICANCE STATEMENT Elucidation of the sources of ATP required for axon extension and maintenance has implications for understanding the mechanism of neuronal development and diseases of the nervous system. While recent work has emphasized the importance of mitochondrial oxidative phosphorylation, the role of the glycolytic pathway in axon morphogenesis and maintenance remains minimally understood. The data reveal that the glycolytic pathway is required for normal sensory axon extension in the presence of NGF, while in the absence of NGF the glycolytic pathway is required for axon maintenance. The results have implications for the understanding of the bioenergetics of axon morphogenesis and plasticity and indicate that NGF has protective effects on sensory axon maintenance in hypoglycemic states.

Neurotrophins induce fission of mitochondria along embryonic sensory axons.

Neurotrophins are growth factors that have a multitude of roles in the nervous system. We report that neurotrophins induce the fission of mitochondria along embryonic chick sensory axons driven by combined PI3K and Mek-Erk signaling. Following an initial burst of fission, a new steady state of neurotrophin-dependent mitochondria length is established. Mek-Erk controls the activity of the fission mediator Drp1 GTPase, while PI3K may contribute to the actin-dependent aspect of fission. Drp1-mediated fission is required for nerve growth factor (NGF)-induced collateral branching in vitro and expression of dominant negative Drp1 impairs the branching of axons in the developing spinal cord in vivo. Fission is also required for NGF-induced mitochondria-dependent intra-axonal translation of the actin regulatory protein cortactin, a previously determined component of NGF-induced branching. Collectively, these observations unveil a novel biological function of neurotrophins; the regulation of mitochondrial fission and steady state mitochondrial length and density in axons.

Chondroitin sulfate proteoglycans negatively regulate the positioning of mitochondria and endoplasmic reticulum to distal axons.

Chondroitin sulfate proteoglycans (CSPGs) are components of the extracellular matrix that inhibit the extension and regeneration of axons. However, the underlying mechanism of action remains poorly understood. Mitochondria and endoplasmic reticulum (ER) are functionally inter-linked organelles important to axon development and maintenance. We report that CSPGs impair the targeting of mitochondria and ER to the growth cones of chicken embryonic sensory axons. The effect of CSPGs on the targeting of mitochondria is blocked by inhibition of the LAR receptor for CSPGs. The regulation of the targeting of mitochondria and ER to the growth cone by CSPGs is due to attenuation of PI3K signaling, which is known to be downstream of LAR receptor activation. Dynactin is a required component of the dynein motor complex that drives the normally occurring retrograde evacuation of mitochondria from growth cones. CSPGs elevate the levels of p150Glu dynactin found in distal axons, and inhibition of the interaction of dynactin with dynein increased axon lengths on CSPGs. CSPGs decreased the membrane potential of mitochondria, and pharmacological inhibition of mitochondria respiration at the growth cone independent of manipulation of mitochondria positioning impaired axon extension. Combined inhibition of dynactin and potentiation of mitochondria respiration further increased axon lengths on CSPGs relative to inhibition of dynactin alone. These data reveal that the regulation of the localization of mitochondria and ER to growth cones is a previously unappreciated aspect of the effects of CSPGs on embryonic axons. © 2017 Wiley Periodicals, Inc. Develop Neurobiol 77: 1351-1370, 2017.

CSPGs inhibit axon branching by impairing mitochondria-dependent regulation of actin dynamics and axonal translation.

Chondroitin sulfate proteoglycans (CSPGs) inhibit the formation of axon collateral branches. The regulation of the axonal cytoskeleton and mitochondria are important components of the mechanism of branching. Actin-dependent axonal plasticity, reflected in the dynamics of axonal actin patches and filopodia, is greatest along segments of the axon populated by mitochondria. It is reported that CSPGs partially depolarize the membrane potential of axonal mitochondria, which impairs the dynamics of the axonal actin cytoskeleton and decreases the formation and duration of axonal filopodia, the first steps in the mechanism of branching. The effects of CSPGs on actin cytoskeletal dynamics are specific to axon segments populated by mitochondria. In contrast, CSPGs do not affect the microtubule content of axons, or the localization of microtubules into axonal filopodia, a required step in the mechanism of branch formation. It is also reported that CSPGs decrease the mitochondria-dependent axonal translation of cortactin, an actin associated protein involved in branching. Finally, the inhibitory effects of CSPGs on axon branching, actin cytoskeletal dynamics and the axonal translation of cortactin are reversed by culturing neurons with acetyl-l-carnitine, which promotes mitochondrial respiration. Collectively these data indicate that CSPGs impair mitochondrial function in axons, an effect which contributes to the inhibition of axon branching. © 2016 Wiley Periodicals, Inc. Develop Neurobiol 77: 419-437, 2017.

Cytoskeletal and signaling mechanisms of neurite formation.

The formation of a neurite, the basis for axons and dendrites, begins with the concerted accumulation and organization of actin and microtubules. Whereas much is known about the proteins that play a role in these processes, because they perform similar functions in axon branching and filopodia formation, much remains to be discovered concerning the interaction of these individual cytoskeletal regulators during neurite formation. Here, we review the literature regarding various models of filopodial formation and the way in which proteins that control actin organization and polymerization induce neurite formation. Although several different regulators of actin polymerization are involved in neurite initiation, redundancy occurs between these regulators, as the effects of the loss of a single regulator can be mitigated by the addition of neurite-promoting substrates and proteins. Similar to actin dynamics, both microtubule stabilizing and destabilizing proteins play a role in neurite initiation. Furthermore, interactions between the actin and microtubule cytoskeleton are required for neurite formation. Several lines of evidence indicate that the interactions between these two components of the cytoskeleton are needed for force generation and for the localization of microtubules at sites of nascent neurites. The general theme that emerges is the existence of several central regulatory pathways on which extracellular cues converge to control and organize both actin and microtubules to induce the formation of neurites.

The dynein inhibitor Ciliobrevin D inhibits the bidirectional transport of organelles along sensory axons and impairs NGF-mediated regulation of growth cones and axon branches.

The axonal transport of organelles is critical for the development, maintenance, and survival of neurons, and its dysfunction has been implicated in several neurodegenerative diseases. Retrograde axon transport is mediated by the motor protein dynein. In this study, using embryonic chicken dorsal root ganglion neurons, we investigate the effects of Ciliobrevin D, a pharmacological dynein inhibitor, on the transport of axonal organelles, axon extension, nerve growth factor (NGF)-induced branching and growth cone expansion, and axon thinning in response to actin filament depolymerization. Live imaging of mitochondria, lysosomes, and Golgi-derived vesicles in axons revealed that both the retrograde and anterograde transport of these organelles was inhibited by treatment with Ciliobrevin D. Treatment with Ciliobrevin D reversibly inhibits axon extension and transport, with effects detectable within the first 20 min of treatment. NGF induces growth cone expansion, axonal filopodia formation and branching. Ciliobrevin D prevented NGF-induced formation of axonal filopodia and branching but not growth cone expansion. Finally, we report that the retrograde reorganization of the axonal cytoplasm which occurs on actin filament depolymerization is inhibited by treatment with Ciliobrevin D, indicating a role for microtubule based transport in this process, as well as Ciliobrevin D accelerating Wallerian degeneration. This study identifies Ciliobrevin D as an inhibitor of the bidirectional transport of multiple axonal organelles, indicating this drug may be a valuable tool for both the study of dynein function and a first pass analysis of the role of axonal transport.

Plexin A3 and turnout regulate motor axonal branch morphogenesis in zebrafish.

During embryogenesis motor axons navigate to their target muscles, where individual motor axons develop complex branch morphologies. The mechanisms that control axonal branching morphogenesis have been studied intensively, yet it still remains unclear when branches begin to form or how branch locations are determined. Live cell imaging of individual zebrafish motor axons reveals that the first axonal branches are generated at the ventral extent of the myotome via bifurcation of the growth cone. Subsequent branches are generated by collateral branching restricted to their synaptic target field along the distal portion of the axon. This precisely timed and spatially restricted branching process is disrupted in turnout mutants we identified in a forward genetic screen. Molecular genetic mapping positioned the turnout mutation within a 300 kb region encompassing eight annotated genes, however sequence analysis of all eight open reading frames failed to unambiguously identify the turnout mutation. Chimeric analysis and single cell labeling reveal that turnout function is required cell non-autonomously for intraspinal motor axon guidance and peripheral branch formation. turnout mutant motor axons form the first branch on time via growth cone bifurcation, but unlike wild-type they form collateral branches precociously, when the growth cone is still navigating towards the ventral myotome. These precocious collateral branches emerge along the proximal region of the axon shaft typically devoid of branches, and they develop into stable, permanent branches. Furthermore, we find that null mutants of the guidance receptor plexin A3 display identical motor axon branching defects, and time lapse analysis reveals that precocious branch formation in turnout and plexin A3 mutants is due to increased stability of otherwise short-lived axonal protrusions. Thus, plexin A3 dependent intrinsic and turnout dependent extrinsic mechanisms suppress collateral branch morphogenesis by destabilizing membrane protrusions before the growth cone completes navigation into the synaptic target field.

Motion from occlusion.

Vision uses specific image features or cues to infer physical properties of the world. Here, we use a novel illusion to show that occlusion, traditionally thought of as a cue to depth, is also a powerful cue to motion. A display of stacking disks that contains only occlusion as a cue to depth generates a vivid sense of movement that is likely computed in early or middle levels of visual processing.