Formation of microtubule-based traps controls the sorting and concentration of vesicles to restricted sites of regenerating neurons after axotomy

Transformation of a transected axonal tip into a growth cone (GC) is a critical step in the cascade leading to neuronal regeneration. Critical to the regrowth is the supply and concentration of vesicles at restricted sites along the cut axon. The mechanisms underlying these processes are largely unknown. Using online confocal imaging of transected, cultured Aplysia californica neurons, we report that axotomy leads to reorientation of the microtubule (MT) polarities and formation of two distinct MT-based vesicle traps at the cut axonal end. Approximately 100 microm proximal to the cut end, a selective trap for anterogradely transported vesicles is formed, which is the plus end trap. Distally, a minus end trap is formed that exclusively captures retrogradely transported vesicles. The concentration of anterogradely transported vesicles in the former trap optimizes the formation of a GC after axotomy.     

Rapid changes in the distribution of GAP-43 correlate with the expression of neuronal polarity during normal development and under experimental conditions

Hippocampal neurons growing in culture initially extend several, short minor processes that have the potential to become either axons or dendrites. The first expression of polarity occurs when one of these minor processes begins to elongate rapidly, becoming the axon. Before axonal outgrowth, the growth-associated protein GAP-43 is distributed equally among the growth cones of the minor processes; it is preferentially concentrated in the axonal growth cone once polarity has been established (Goslin, K., D. Schreyer, J. Skene, and G. Banker. 1990. J. Neurosci. 10:588-602). To determine when the selective segregation of GAP-43 begins, we followed individual cells by video microscopy, fixed them as soon as the axon could be distinguished, and localized GAP-43 by immunofluorescence microscopy. Individual minor processes acquired axonal growth characteristics within a period of 30-60 min, and GAP-43 became selectively concentrated to the growth cones of these processes with an equally rapid time course. We also examined changes in the distribution of GAP-43 after transection of the axon. After an axonal transection that is distant from the soma, neuronal polarity is maintained, and the original axon begins to regrow almost immediately. In such cases, GAP-43 became selectively concentrated in the new axonal growth cone within 12-30 min. In contrast, when the axon is transected close to the soma, polarity is lost; the original axon rarely regrows, and there is a significant delay before a new axon emerges. Under these circumstances, GAP-43 accumulated in the new growth cone much more slowly, suggesting that its ongoing selective routing to the axon had been disrupted by the transection. These results demonstrate that the selective segregation of GAP-43 to the growth cone of a single process is closely correlated with the acquisition of axonal growth characteristics and, hence, with the expression of polarity.     

HDAC5 is a novel injury-regulated tubulin deacetylase controlling axon regeneration

Axon regeneration is an essential process to rebuild functional connections between injured neurons and their targets. Regenerative axonal growth requires alterations in axonal microtubule dynamics, but the signalling mechanisms involved remain incompletely understood. Our results reveal that axon injury induces a gradient of tubulin deacetylation, which is required for axon regeneration both in vitro and in vivo. This injury-induced tubulin deacetylation is specific to peripheral neurons and fails to occur in central neurons. We found that tubulin deacetylation is initiated by calcium influx at the site of injury, and requires protein kinase C-mediated activation of the histone deacetylase 5 (HDAC5). Our findings identify HDAC5 as a novel injury-regulated tubulin deacetylase that plays an essential role in growth cone dynamics and axon regeneration. In addition, our results suggest a mechanism for the spatial control of tubulin modifications that is required for axon regeneration.     

Initial stages of neural regeneration in Helisoma trivolvis are dependent upon PLA2 activity

Neuronal regeneration after damage to an axon tract requires the rapid sealing of the injured plasma membrane and the subsequent formation of growth cones that can lead regenerating processes to their appropriate target. Membrane sealing and growth cone formation are Ca(2+)-dependent processes, but the signaling pathways activated by Ca(2+) to bring about these effects remain poorly understood. An in vitro injury model was employed in which neurites from identified snail neurons (Helisoma trivolvis) were transected with a glass microknife, and the formation of new growth cones from the distal portions of transected neurites was recorded at defined times after transection. This study presents three main results. First, phospholipase A(2) (PLA(2)), a calcium-activated enzyme, is necessary for membrane sealing in vitro. Second, PLA(2) activity is also required for the formation of a new growth cone after the membrane has sealed successfully. Thus, PLA(2) plays a dual role by affecting both growth cone formation and membrane sealing. Third, the injury-induced activation of PLA(2) by Ca(2+) controls growth cone formation through the production of leukotrienes, secondary metabolites of PLA(2) activity. Taken together, these results suggest that the injury-induced Ca(2+) influx acts via PLA(2) and leukotriene production to assure growth cone formation. These findings indicate that events that cause an inhibition of PLA(2) or lipoxygenases, enzymes that produce leukotrienes, could result in the inability of neurites to regenerate.     

Developmental regulation of sensory axon regeneration in the absence of growth cones

The actin filament (F-actin) cytoskeleton is thought to be required for normal axon extension during embryonic development. Whether this is true of axon regeneration in the mature nervous system is not known, but a progressive simplification of growth cones during development has been described and where specifically investigated, mature spinal cord axons appear to regenerate without growth cones. We have studied the cytoskeletal mechanisms of axon regeneration in developmentally early and late chicken sensory neurons, at embryonic day (E) 7 and 14 respectively. Depletion of F-actin blocked the regeneration of E7 but not E14 sensory axons in vitro. The differential sensitivity of axon regeneration to the loss of F-actin and growth cones correlated with endogenous levels of F-actin and growth cone morphology. The growth cones of E7 axons contained more F-actin and were more elaborate than those of E14 axons. The ability of E14 axons to regenerate in the absence of F-actin and growth cones was dependent on microtubule tip polymerization. Importantly, while the regeneration of E7 axons was strictly dependent on F-actin, regeneration of E14 axons was more dependent on microtubule tip polymerization. Furthermore, E14 axons exhibited altered microtubule polymerization relative to E7, as determined by imaging of microtubule tip polymerization in living neurons. These data indicate that the mechanism of axon regeneration undergoes a developmental switch between E7 and E14 from strict dependence on F-actin to a greater dependence on microtubule polymerization. Collectively, these experiments indicate that microtubule polymerization may be a therapeutic target for promoting regeneration of mature neurons.     

The Yin and Yang of Wnt/Ryk axon guidance in development and regeneration

In the developing embryo,nascent axons navigate towards their specific targets to establish the intricate network of axonal connections linking neurons within the mature nervous system.Molecular navigational systems comprising repulsive and attractive guidance cues form chemotactic gradients along the pathway of the exploring growth cone.Axon-bound receptors detect these gradients and determine the trajectory of the migrating growth cone.In contrast to their benevolent role in the developing nervous system,repulsive guidance receptors are detrimental to the axon’s ability to regenerate after injury in the adult.In this review we explore the essential and beneficial role played by the chemorepulsive Wnt receptor,Ryk/Derailed in axon navigation in the embryonic nervous system(the Yin function).Specifically,we focus on the role of Wnt5a/Rykmediated guidance in the establishment of two major axon tracts in the mammalian central nervous system,the corticospinal tract and the corpus callosum.Recent studies have also identified Ryk as a major suppressor of axonal regeneration after spinal cord injury.Thus,we also discuss this opposing aspect of Ryk function in axonal regeneration where its activity is a major impediment to axon regrowth(the Yang function).     

RhoA-kinase and myosin II are required for the maintenance of growth cone polarity and guidance by nerve growth factor

Growth cones are highly polarized and dynamic structures confined to the tips of axons. The polarity of growth cones is in part maintained by suppression of protrusive activity from the distal axon shaft, a process termed axon consolidation. The mechanistic basis of axon consolidation that contributes to the maintenance of growth cone polarity is not clear. We report that inhibition of RhoA-kinase (ROCK) or myosin II resulted in unstable consolidation of the distal axon as evidenced by increased filopodial and lamellipodial extension. Furthermore, when ROCK or myosin II was inhibited lamellipodia formed at the growth cone migrated onto the axon shaft. Analysis of EYFP-actin dynamics in the distal axon revealed that ROCK negatively regulates actin polymerization and initiation of protrusive structures from spontaneously formed axonal F-actin patches, the latter being an effect attributable to ROCK-mediated regulation of myosin II. Inhibition of ROCK or myosin II blocked growth cone turning toward NGF by preventing suppression of protrusive activity away from the source of NGF, resulting in aborted turning responses. These data elucidate the mechanism of growth cone polarity, provide evidence that consolidation of the distal axon is a component of guidance, and identify ROCK as a negative regulator of F-actin polymerization underlying protrusive activity in the distal axon.     

Eph-dependent tyrosine phosphorylation of ephexin1 modulates growth cone collapse

Ephs regulate growth cone repulsion, a process controlled by the actin cytoskeleton. The guanine nucleotide exchange factor (GEF) ephexin1 interacts with EphA4 and has been suggested to mediate the effect of EphA on the activity of Rho GTPases, key regulators of the cytoskeleton and axon guidance. Using cultured ephexin1-/- mouse neurons and RNA interference in the chick, we report that ephexin1 is required for normal axon outgrowth and ephrin-dependent axon repulsion. Ephexin1 becomes tyrosine phosphorylated in response to EphA signaling in neurons, and this phosphorylation event is required for growth cone collapse. Tyrosine phosphorylation of ephexin1 enhances ephexin1's GEF activity toward RhoA while not altering its activity toward Rac1 or Cdc42, thus changing the balance of GTPase activities. These findings reveal that ephexin1 plays a role in axon guidance and is regulated by a switch mechanism that is specifically tailored to control Eph-mediated growth cone collapse.     

Ndel1 promotes axon regeneration via intermediate filaments

Failure of axons to regenerate following acute or chronic neuronal injury is attributed to both the inhibitory glial environment and deficient intrinsic ability to re-grow. However, the underlying mechanisms of the latter remain unclear. In this study, we have investigated the role of the mammalian homologue of aspergillus nidulans NudE, Ndel1, emergently viewed as an integrator of the cytoskeleton, in axon regeneration. Ndel1 was synthesized de novo and upregulated in crushed and transected sciatic nerve axons, and, upon injury, was strongly associated with neuronal form of the intermediate filament (IF) Vimentin while dissociating from the mature neuronal IF (Neurofilament) light chain NF-L. Consistent with a role for Ndel1 in the conditioning lesion-induced neurite outgrowth of Dorsal Root Ganglion (DRG) neurons, the long lasting in vivo formation of the neuronal Ndel1/Vimentin complex was associated with robust axon regeneration. Furthermore, local silencing of Ndel1 in transected axons by siRNA severely reduced the extent of regeneration in vivo. Thus, Ndel1 promotes axonal regeneration; activating this endogenous repair mechanism may enhance neuroregeneration during acute and chronic axonal degeneration.     

RhoA‐kinase and myosin II are required for the maintenance of growth cone polarity and guidance by nerve growth factor

Growth cones are highly polarized and dynamic structures confined to the tips of axons. The polarity of growth cones is in part maintained by suppression of protrusive activity from the distal axon shaft, a process termed axon consolidation. The mechanistic basis of axon consolidation that contributes to the maintenance of growth cone polarity is not clear. We report that inhibition of RhoA‐kinase (ROCK) or myosin II resulted in unstable consolidation of the distal axon as evidenced by increased filopodial and lamellipodial extension. Furthermore, when ROCK or myosin II was inhibited lamellipodia formed at the growth cone migrated onto the axon shaft. Analysis of EYFP‐actin dynamics in the distal axon revealed that ROCK negatively regulates actin polymerization and initiation of protrusive structures from spontaneously formed axonal F‐actin patches, the latter being an effect attributable to ROCK‐mediated regulation of myosin II. Inhibition of ROCK or myosin II blocked growth cone turning toward NGF by preventing suppression of protrusive activity away from the source of NGF, resulting in aborted turning responses. These data elucidate the mechanism of growth cone polarity, provide evidence that consolidation of the distal axon is a component of guidance, and identify ROCK as a negative regulator of F‐actin polymerization underlying protrusive activity in the distal axon. © 2006 Wiley Periodicals, Inc. J Neurobiol, 2006     

The role of microtubules in growth cone turning at substrate boundaries

To understand the role of microtubules in growth cone turning, we observed fluorescently labeled microtubules in neurons as they encountered a substrate boundary. Neurons growing on a laminin-rich substrate avoided growing onto collagen type IV. Turning growth cones assumed heterogeneous morphologies and behaviors that depended primarily in their extent of adhesion to the substrate. We grouped these behaviors into three categories-sidestepping, motility, and growth-mediated reorientation. In sidestepping and motility-mediated reorientation, the growth cone and parts of the axon were not well attached to the substrate so the acquisition of an adherent lamella caused the entire growth cone to move away from the border and consequently reoriented the axon. In these cases, since the motility of the growth cone dominates its reorientation, the microtubules were passive, and reorientation occurred without significant axon growth. In growth-mediated reorientation, the growth cone and axon were attached to the substrate. In this case, microtubules reoriented within the growth cone to stabilize a lamella. Bundling of the reoriented microtubules was followed by growth cone collapse to form new axon, and further, polarized lamellipodial extension. These observations indicate that when the growth cone remains adherent to the substrate during turning, the reorientation and bundling of microtubules is an important, early step in growth cone turning.     

Rho inhibition recruits DCC to the neuronal plasma membrane and enhances axon chemoattraction to netrin 1

Molecular cues, such as netrin 1, guide axons by influencing growth cone motility. Rho GTPases are a family of intracellular proteins that regulate the cytoskeleton, substrate adhesion and vesicle trafficking. Activation of the RhoA subfamily of Rho GTPases is essential for chemorepellent axon guidance; however, their role during axonal chemoattraction is unclear. Here, we show that netrin 1, through its receptor DCC, inhibits RhoA in embryonic spinal commissural neurons. To determine whether netrin 1-mediated chemoattraction requires Rho function, we inhibited Rho signaling and assayed axon outgrowth and turning towards netrin 1. Additionally, we examined two important mechanisms that influence the guidance of axons to netrin 1: substrate adhesion and transport of the netrin receptor DCC to the plasma membrane. We found that inhibiting Rho signaling increased plasma membrane DCC and adhesion to substrate-bound netrin 1, and also enhanced netrin 1-mediated axon outgrowth and chemoattractive axon turning. Conversely, overexpression of RhoA or constitutively active RhoA inhibited axonal responses to netrin 1. These findings provide evidence that Rho signaling reduces axonal chemoattraction to netrin 1 by limiting the amount of plasma membrane DCC at the growth cone, and suggest that netrin 1-mediated inhibition of RhoA activates a positive-feedback mechanism that facilitates chemoattraction to netrin 1. Notably, these findings also have relevance for CNS regeneration research. Inhibiting RhoA promotes axon regeneration by disrupting inhibitory responses to myelin and the glial scar. By contrast, we demonstrate that axon chemoattraction to netrin 1 is not only maintained but enhanced, suggesting that this might facilitate directing regenerating axons to appropriate targets.     

Pioneering studies on the mechanisms of axonal regeneration

While ultimately, focus must be placed on experimentation using adult systems, vastly important clues to regeneration can be found in the study of the embryonic nervous system. In embryonic systems, axonal regeneration is successful before a critical period, and numerous advances have resulted from the study of isolated cells and tissues in vitro. Studies over many decades from the laboratory of Paul C. Letourneau have probed the cellular and molecular phenomena involved in axon outgrowth and guidance in the embryonic central and peripheral nervous system and have laid the framework for many current advances in regeneration research. Letourneau’s pioneering work related to growth cone behavior, guidance, and regeneration has resulted in considerable contributions toward our understanding not only of cellular mechanisms that underlie axon growth, but also of the specific areas of study that require attention to accomplish future breakthroughs. The present article summarizes some of the major contributions from Paul Letourneau and his team in the area of axonal regeneration.     

The region-specific activities of lipid rafts during axon growth and guidance

The generation and control of cell polarity is a fundamental mechanism for directed migration of the cell. In developing neurons, the axonal growth cone recognizes environmental molecular cues and migrates toward its correct target, thereby forming neuronal networks. The spatial information provided by environmental cues directs axon growth and guidance through generating polarity of intracellular signals and cytoskeletal organization in the growth cone. This polarization process is dependent on lipid rafts, specialized microdomains in the cell membrane. Lipid rafts in specific regions of the growth cone are involved in axon growth and guidance. For example, forward migration of the growth cone requires raft membranes in its leading front. Recent experiments have suggested that lipid rafts function as a platform for localized signaling downstream of adhesion molecules and guidance receptors. The rafts assemble into an active membrane domain that captures and reorganizes the cytoskeletal machinery. In this way, the spatial control of signaling through raft membranes plays a critical role in translating extracellular information into polarized motility of the growth cone.     

Changes within maturing neurons limit axonal regeneration in the developing spinal cord

Embryonic birds and mammals display a remarkable ability to regenerate axons after spinal injury, but then lose this ability during a discrete developmental transition. To explain this transition, previous research has emphasized the emergence of myelin and other inhibitory factors in the environment of the spinal cord. However, research in other CNS tracts suggests an important role for neuron-intrinsic limitations to axon regeneration. Here we re-examine this issue quantitatively in the hindbrain-spinal projection of the embryonic chick. Using heterochronic cocultures we show that maturation of the spinal cord environment causes a 55% reduction in axon regeneration, while maturation of hindbrain neurons causes a 90% reduction. We further show that young neurons transplanted in vivo into older spinal cord can regenerate axons into myelinated white matter, while older axons regenerate poorly and have reduced growth cone motility on a variety of growth-permissive ligands in vitro, including laminin, L1, and N-cadherin. Finally, we use video analysis of living growth cones to directly document an age-dependent decline in the motility of brainstem axons. These data show that developmental changes in both the spinal cord environment and in brainstem neurons can reduce regeneration, but that the effect of the environment is only partial, while changes in neurons by themselves cause a nearly complete reduction in regeneration. We conclude that maturational events within neurons are a primary cause for the failure of axon regeneration in the spinal cord.     

Inhibition of PTEN activity promotes IB4‐positive sensory neuronal axon growth

Traumatic nerve injuries have become a common clinical problem, and axon regeneration is a critical process in the successful functional recovery of the injured nervous system. In this study, we found that peripheral axotomy reduces PTEN expression in adult sensory neurons; however, it did not alter the expression level of PTEN in IB4‐positive sensory neurons. Additionally, our results indicate that the artificial inhibition of PTEN markedly promotes adult sensory axon regeneration, including IB4‐positive neuronal axon growth. Thus, our results provide strong evidence that PTEN is a prominent repressor of adult sensory axon regeneration, especially in IB4‐positive neurons.     

Contact inhibition in the failure of mammalian CNS axonal regeneration

Anamniote animals, such as fish and amphibians, are able to regenerate damaged CNS nerves following injury, but regeneration in the mammalian CNS tracts, such as the optic nerve, does not occur. However, severed adult mammalian retinal axons can regenerate into peripheral nerve segments grafted into the brain and this finding has emphasized the importance of the environment in explaining regenerative failure in the adult mammalian CNS. Following lesions, regenerating axons encounter the glial cells, oligodendrocytes and astro-cytes, and their derivatives, respectively myelin and the astrocytic scar. Experiments to investigate the influence of these components on axon growth in culture have revealed cell-surface and extracellular matrix molecules that inhibit axon extension and growth cone motility. Structural and functional characterization of these ligands and their receptors is underway, and may solve the interesting neurobiological conundrum posed by the failure of mammalian CNS regeneration. Simultaneously, this might allow new possibilities for treatment of the severe clinical disabilities resulting from injury to the brain and spinal cord.     

Induction of axon growth arrest without growth cone collapse through the N-terminal region of four-transmembrane glycoprotein M6a

During development, axons elongate vigorously, carefully controlling their speed, to connect with their targets. In general, rapid axon growth is correlated with active growth cones driven by dynamic actin filaments. For example, when the actin-driven tip is collapsed by repulsive guidance molecules, axon growth is severely impaired. In this study, we report that axon growth can be suppressed, without destroying the actin-based structure or motility of the growth cones, when antibodies bind to the four-transmembrane glycoprotein M6a concentrated on the growth cone edge. Surprisingly, M6a-deficient axons grow actively but are not growth suppressed by the antibodies, arguing for an inductive action of the antibody. The binding of antibodies clusters and displaces M6a protein from the growth cone edge membrane, suggesting that the spatial rearrangement of this protein might underlie the unique growth cone behavior triggered by the antibodies. Molecular dissection of M6a suggested involvement for the N-terminal intracellular domain in this antibody-induced growth cone arrest.     

Common mechanisms underlying growth cone guidance and axon branching

During development, growth cones direct growing axons into appropriate targets. However, in some cortical pathways target innervation occurs through the development of collateral branches that extend interstitially from the axon shaft. How do such branches form? Direct observations of living cortical brain slices revealed that growth cones of callosal axons pause for many hours beneath their cortical targets prior to the development of interstitial branches. High resolution imaging of dissociated living cortical neurons for many hours revealed that the growth cone demarcates sites of future axon branching by lengthy pausing behaviors and enlargement of the growth cone. After a new growth cone forms and resumes forward advance, filopodial and lamellipodial remnants of the large paused growth cone are left behind on the axon shaft from which interstitial branches later emerge. To investigate how the cytoskeleton reorganizes at axon branch points, we fluorescently labeled microtubules in living cortical neurons and imaged the behaviors of microtubules during new growth from the axon shaft and the growth cone. In both regions microtubules reorganize into a more plastic form by splaying apart and fragmenting. These shorter microtubules then invade newly developing branches with anterograde and retrograde movements. Although axon branching of dissociated cortical neurons occurs in the absence of targets, application of a target-derived growth factor, FGF-2, greatly enhances branching. Taken together, these results demonstrate that growth cone pausing is closely related to axon branching and suggest that common mechanisms underlie directed axon growth from the terminal growth cone and the axon shaft.     

DSCR1 is required for both axonal growth cone extension and steering

Local information processing in the growth cone is essential for correct wiring of the nervous system. As an axon navigates through the developing nervous system, the growth cone responds to extrinsic guidance cues by coordinating axon outgrowth with growth cone steering. It has become increasingly clear that axon extension requires proper actin polymerization dynamics, whereas growth cone steering involves local protein synthesis. However, molecular components integrating these two processes have not been identified. Here, we show that Down syndrome critical region 1 protein (DSCR1) controls axon outgrowth by modulating growth cone actin dynamics through regulation of cofilin activity (phospho/dephospho-cofilin). Additionally, DSCR1 mediates brain-derived neurotrophic factor–induced local protein synthesis and growth cone turning. Our study identifies DSCR1 as a key protein that couples axon growth and pathfinding by dually regulating actin dynamics and local protein synthesis.