Axons in the CNS do not regrow after injury, whereas lesioned axons in the peripheral nervous system (PNS) regenerate. Lesioned CNS axons form characteristic swellings at their tips known as ...retraction bulbs, which are the nongrowing counterparts of growth cones. Although much progress has been made in identifying intracellular and molecular mechanisms that regulate growth cone locomotion and axonal elongation, a comprehensive understanding of how retraction bulbs form and why they are unable to grow is still elusive. Here we report the analysis of the morphological and intracellular responses of injured axons in the CNS compared with those in the PNS. We show that retraction bulbs of injured CNS axons increase in size over time, whereas growth cones of injured PNS axons remain constant. Retraction bulbs contain a disorganized microtubule network, whereas growth cones possess the typical bundling of microtubules. Using in vivo imaging, we find that pharmacological disruption of microtubules in growth cones transforms them into retraction bulb-like structures whose growth is inhibited. Correspondingly, microtubule destabilization of sensory neurons in cell culture induces retraction bulb formation. Conversely, microtubule stabilization prevents the formation of retraction bulbs and decreases axonal degeneration in vivo. Finally, microtubule stabilization enhances the growth capacity of CNS neurons cultured on myelin. Thus, the stability and organization of microtubules define the fate of lesioned axonal stumps to become either advancing growth cones or nongrowing retraction bulbs. Our data pinpoint microtubules as a key regulatory target for axonal regeneration.
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.
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.
Directed axonal growth depends on correct coordination of the actin and microtubule cytoskeleton in the growth cone. However, despite the relatively large number of proteins implicated in ...actin-microtubule crosstalk, the mechanisms whereby actin polymerization is coupled to microtubule stabilization and advancement in the peripheral growth cone remained largely unclear. Here, we identified the formin Dishevelled-associated activator of morphogenesis (DAAM) as a novel factor playing a role in concerted regulation of actin and microtubule remodeling in
primary neurons.
, DAAM binds to F-actin as well as to microtubules and has the ability to crosslink the two filament systems. Accordingly, DAAM associates with the neuronal cytoskeleton, and a significant fraction of DAAM accumulates at places where the actin filaments overlap with that of microtubules. Loss of
affects growth cone and microtubule morphology, and several aspects of microtubule dynamics; and biochemical and cellular assays revealed a microtubule stabilization activity and binding to the microtubule tip protein EB1. Together, these data suggest that, besides operating as an actin assembly factor, DAAM is involved in linking actin remodeling in filopodia to microtubule stabilization during axonal growth.
The development of precise connectivity patterns during the establishment of the nervous system depends on the regulated action of diverse, conserved families of guidance cues and their neuronal ...receptors. Determining how these signaling pathways function to regulate axon growth and guidance is fundamentally important to understanding wiring specificity in the nervous system and will undoubtedly shed light on many neural developmental disorders. Considerable progress has been made in defining the mechanisms that regulate the correct spatial and temporal distribution of guidance receptors and how these receptors in turn signal to the growth cone cytoskeleton to control steering decisions. This review focuses on recent advances in our understanding of the mechanisms mediating growth cone guidance with a particular emphasis on the control of guidance receptor regulation and signaling.
The cellular prion protein (PrP
C
), mainly known for its role in neurodegenerative diseases, is involved in several physiological processes including neuritogenesis. In addition, its ability to bind ...copper or zinc has been suggested for its role in metal homeostasis. Although PrP
C
has been known as a copper-binding molecule, little is known about how copper can affect PrP
C
physiological functions. By combining genomic approaches, cellular assays, and focal stimulation technique, we found that PrP
C
neuritogenesis function is directly influenced by N-terminal copper-binding amino acids. Several recombinant mouse PrP (recMoPrP) mutants at N-terminal copper-binding sites were produced, and primary hippocampal cultures were treated either in bulk or exposed near the hippocampal growth cones (GC) of single neurons in local stimulation manner. While focal stimulation of GC with wild-type recMoPrP induced neurite outgrowth and rapid GC turning toward the source, N-terminal mutants fail to support this effect. Indeed, disrupting all the copper-binding sites at the N-terminus of PrP
C
was toxic to neurons indicating that these regions are crucial for the protein function. Mutants at both octarepeat and non-octarepeat region abolished the neuritogenesis effect. Altogether, our findings indicate the crucial role of copper-binding sites in maintaining the neuritogenesis function in PrP, suggesting a potential link between loss-of-function of the protein and disease initiation.
Previous studies show that the main cannabinoid receptor in the brain—cannabinoid type 1 receptor (CB1R)—is required for establishment of axonal projections in developing neurons but questions remain ...regarding the cellular and molecular mechanisms, especially in neurons developing in their native environment. We assessed the effects of CB1R signalling on growth cone filopodia and axonal projections of retinal ganglion cells (RGCs) in whole mount brains from Xenopus laevis tadpoles. Our results indicate that growth cones of RGC axons in brains from tadpoles exposed to a CB1R agonist had fewer filopodial protrusions, whereas growth cones from tadpoles exposed to a CB1R inverse agonist had more filopodia than growth cones of RGC axons in whole brains from control tadpoles. However, application of both the CB1R agonist and inverse agonist resulted in RGC axons that were overly dispersed and undulatory in the optic tract in situ. In addition, expression of a mutant for cadherin adhesive factor, β‐catenin, that disrupts its binding to α‐catenin, and application of an inhibitor for actin regulator non‐muscle Myosin II, phenocopied the effects of the CB1R agonist and inverse agonist on growth cone filopodia, respectively. These findings suggest that both destablization and stabilization of growth cone filopodia are required for RGC axonal fasciculation/defasciculation in the optic tract and that CB1R regulates growth cone filopodia and axon dispersion of RGCs by oppositely modulating β‐catenin adhesive and Myosin II actin regulatory functions. This study extends and confirms our understanding of cannabinoid mechanisms in sculpting developing neuronal circuits in vivo.
Growth cones exposed to CB1R agonist had fewer filopodial protrusions, whereas those exposed to CB1R inverse agonist had more filopodial protrusions than controls. A β‐catenin mutant induced the same effects as CBR agonist, while an inhibitor of Myosin II phenocopied the inverse agonist on growth cone filopodia. These results imply that the activation of CB1R results in negative regulation of β‐catenin and positive regulation of Myosin II, which both ultimately leads to fewer filopodia protrusions. By oppositely modulating β‐catenin and Myosin II, CB1R regulates growth cone filopodia and the balance of destabilization/stabilization of the filopodia results in proper axonal defasciculation in the tract.
Injured axons of the adult CNS undergo lengthy retraction from the initial site of axotomy after spinal cord injury. Macrophage infiltration correlates spatiotemporally with this deleterious ...phenomenon, but the direct involvement of these inflammatory cells has not been demonstrated. In the present study, we examined the role of macrophages in axonal retraction within the dorsal columns after spinal cord injury in vivo and found that retraction occurred between days 2 and 28 after lesion and that the ends of injured axons were associated with ED-1+ cells. Clodronate liposome-mediated depletion of infiltrating macrophages resulted in a significant reduction in axonal retraction; however, we saw no evidence of regeneration. We used time-lapse imaging of adult dorsal root ganglion neurons in an in vitro model of the glial scar to examine macrophage-axon interactions and observed that adhesive contacts and considerable physical interplay between macrophages and dystrophic axons led to extensive axonal retraction. The induction of retraction was dependent on both the growth state of the axon and the activation state of the macrophage. Only dystrophic adult axons were susceptible to macrophage "attack." Unlike intrinsically active cell line macrophages, both primary macrophages and microglia required activation to induce axonal retraction. Contact with astrocytes had no deleterious effect on adult dystrophic axons, suggesting that the induction of extensive retraction was specific to phagocytic cells. Our data are the first to indicate a direct role of activated macrophages in axonal retraction by physical cell-cell interactions with injured axons.
Suppression of glycogen synthase kinase 3 (GSK3) activity in neurons yields pleiotropic outcomes, causing both axon growth promotion and inhibition. Previous studies have suggested that specific GSK3 ...substrates, such as adenomatous polyposis coli (APC) and collapsin response mediator protein 2 (CRMP2), support axon growth by regulating the stability of axonal microtubules (MTs), but the substrate(s) and mechanisms conveying axon growth inhibition remain elusive. Here we show that CLIP (cytoplasmic linker protein)-associated protein (CLASP), originally identified as a MT plus end-binding protein, displays both plus end-binding and lattice-binding activities in nerve growth cones, and reveal that the two MT-binding activities regulate axon growth in an opposing manner: The lattice-binding activity mediates axon growth inhibition induced by suppression of GSK3 activity via preventing MT protrusion into the growth cone periphery, whereas the plus end-binding property supports axon extension via stabilizing the growing ends of axonal MTs. We propose a model in which CLASP transduces GSK3 activity levels to differentially control axon growth by coordinating the stability and configuration of growth cone MTs.
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