Wallerian degeneration involves Rho/Rho-kinase signaling

Local axon degeneration is a common pathological feature of many neurodegenerative diseases, whereas the underlying molecular mechanisms are largely unknown. In this study, we used the degeneration of transected axons, termed "Wallerian degeneration," as a model to examine the possible involvement of Rho. Nogo-66, a myelin-derived inhibitor of axon regeneration, significantly accelerated axon degeneration of the dorsal root ganglion explant in vitro, whereas inhibiting Rho-kinase activity abolished the effect. Rho activation was observed in the distal part of the injured axons after spinal cord injury. We demonstrate that degeneration of the injured cortico-spinal axons was significantly retarded by a Rho-kinase inhibitor in vivo. Our findings suggest that inhibiting the signaling pathway may retard axon degeneration in pathological conditions.     

ZNRF1 promotes Wallerian degeneration by degrading AKT to induce GSK3B-dependent CRMP2 phosphorylation

Wallerian degeneration is observed in many neurological disorders, and it is therefore important to elucidate the axonal degeneration mechanism to prevent, and further develop treatment for, such diseases. The ubiquitin-proteasome system (UPS) has been implicated in Wallerian degeneration, but the underlying molecular mechanism remains unclear. Here we show that ZNRF1, an E3 ligase, promotes Wallerian degeneration by targeting AKT to degrade through the UPS. AKT phosphorylates glycogen synthase kinase-3β (GSK3B), and thereby inactivates it in axons. AKT overexpression significantly delays axonal degeneration. Overexpression of the active (non-phosphorylated) form of GSK3B induces CRMP2 phosphorylation, which is required for the microtubule reorganization observed in the degenerating axon. The inhibition of GSK3B and the overexpression of non-phosphorylated CRMP2 both protected axons from Wallerian degeneration. These findings indicate that the ZNRF1-AKT-GSK3B-CRMP2 pathway plays an important role in controlling Wallerian degeneration.     

NAD and axon degeneration: From the Wlds gene to neurochemistry

Neurodegenerative diseases have become a global issue due to the aging population. These disorders affect a vast patient population and represent a huge area of unmet therapeutic need. Axon degeneration is a common pathological character of those neurodegenerative diseases. It results in the loss of communication between neurons. Two decades ago, the Wallerian degeneration slow (Wlds) mouse strain was identified, in which the degeneration of transected axons is delayed. The phenotype is attributed to the overexpression of a chimeric protein Wlds which contains a short fragment of the ubiquitin assembly protein UFD2 and the full-length nicotinamide adenine dinucleotide (NAD) synthetic enzyme Nicotinamide mononucleotide adenylyl-transferase-1 (Nmnat-1). However, the underlying molecular mechanism remains largely unknown. Recently, it''s reported by independent researchers that the full length coding sequence of mouse Nmnat-1 could mimic the axonal protective effect of the Wlds gene when overexpressed in primary neural cultures. Together with a significant number of subsequential reports, this finding highlighted the substantial role of nicotinamide adenine dinucleotide (NAD) in the process of axon degeneration. Here we reviewed the history of axon degeneration research from a neurochemical standpoint and discuss the potential involvement of NAD synthesis, NAD consumption and NAD-dependent proteins and small molecules in axon degeneration.Key words: axon degeneration, Wallerian degeneration, Wlds, NAD, UPS, neurodegenerative diseases     

A local mechanism mediates NAD-dependent protection of axon degeneration

Axon degeneration occurs frequently in neurodegenerative diseases and peripheral neuropathies. Important insight into the mechanisms of axon degeneration arose from findings that the degeneration of transected axons is delayed in Wallerian degeneration slow (Wlds) mice with the overexpression of a fusion protein with the nicotinamide adenine dinucleotide (NAD) synthetic enzyme, nicotinamide mononucleotide adenylyltransferase (Nmnat1). Although both Wld(s) and Nmnat1 themselves are functional in preventing axon degeneration in neuronal cultures, the underlying mechanism for Nmnat1- and NAD-mediated axon protection remains largely unclear. We demonstrate that NAD levels decrease in degenerating axons and that preventing this axonal NAD decline efficiently protects axons from degeneration. In support of a local protective mechanism, we show that the degeneration of axonal segments that have been separated from their soma could be prevented by the exogenous application of NAD or its precursor nicotinamide. Furthermore, we provide evidence that such Nmnat1/NAD-mediated protection is primarily mediated by their effects on local bioenergetics. Together, our results suggest a novel molecular pathway for axon degeneration.     

The Drosophila cell corpse engulfment receptor Draper mediates glial clearance of severed axons

Neuron-glia communication is central to all nervous system responses to trauma, yet neural injury signaling pathways remain poorly understood. Here we explore cellular and molecular aspects of neural injury signaling in Drosophila. We show that transected Drosophila axons undergo injury-induced degeneration that is morphologically similar to Wallerian degeneration in mammals and can be suppressed by the neuroprotective mouse Wlds protein. Axonal injury elicits potent morphological and molecular responses from Drosophila glia: glia upregulate expression of the engulfment receptor Draper, undergo dramatic changes in morphology, and rapidly recruit cellular processes toward severed axons. In draper mutants, glia fail to respond morphologically to axon injury, and severed axons are not cleared from the CNS. Thus Draper appears to act as a glial receptor for severed axon-derived molecular cues that drive recruitment of glial processes to injured axons for engulfment.     

Soluble Axoplasm Enriched from Injured CNS Axons Reveals the Early Modulation of the Actin Cytoskeleton

Axon injury and degeneration is a common consequence of diverse neurological conditions including multiple sclerosis, traumatic brain injury and spinal cord injury. The molecular events underlying axon degeneration are poorly understood. We have developed a novel method to enrich for axoplasm from rodent optic nerve and characterised the early events in Wallerian degeneration using an unbiased proteomics screen. Our detergent-free method draws axoplasm into a dehydrated hydrogel of the polymer poly(2-hydroxyethyl methacrylate), which is then recovered using centrifugation. This technique is able to recover axonal proteins and significantly deplete glial contamination as confirmed by immunoblotting. We have used iTRAQ to compare axoplasm-enriched samples from naïve vs injured optic nerves, which has revealed a pronounced modulation of proteins associated with the actin cytoskeleton. To confirm the modulation of the actin cytoskeleton in injured axons we focused on the RhoA pathway. Western blotting revealed an augmentation of RhoA and phosphorylated cofilin in axoplasm-enriched samples from injured optic nerve. To investigate the localisation of these components of the RhoA pathway in injured axons we transected axons of primary hippocampal neurons in vitro. We observed an early modulation of filamentous actin with a concomitant redistribution of phosphorylated cofilin in injured axons. At later time-points, RhoA is found to accumulate in axonal swellings and also colocalises with filamentous actin. The actin cytoskeleton is a known sensor of cell viability across multiple eukaryotes, and our results suggest a similar role for the actin cytoskeleton following axon injury. In agreement with other reports, our data also highlights the role of the RhoA pathway in axon degeneration. These findings highlight a previously unexplored area of axon biology, which may open novel avenues to prevent axon degeneration. Our method for isolating CNS axoplasm also represents a new tool to study axon biology.     

MEK Inhibitor U0126 Reverses Protection of Axons from Wallerian Degeneration Independently of MEK–ERK Signaling

Wallerian degeneration is delayed when sufficient levels of proteins with NMNAT activity are maintained within axons after injury. This has been proposed to form the basis of ''slow Wallerian degeneration'' (Wld ^S), a neuroprotective phenotype conferred by an aberrant fusion protein, Wld^S. Proteasome inhibition also delays Wallerian degeneration, although much less robustly, with stabilization of NMNAT2 likely to play a key role in this mechanism. The pan-MEK inhibitor U0126 has previously been shown to reverse the axon-protective effects of proteasome inhibition, suggesting that MEK-ERK signaling plays a role in delayed Wallerian degeneration, in addition to its established role in promoting neuronal survival. Here we show that whilst U0126 can also reverse Wld^S-mediated axon protection, more specific inhibitors of MEK1/2 and MEK5, PD184352 and BIX02189, have no significant effect on the delay to Wallerian degeneration in either situation, whether used alone or in combination. This suggests that an off-target effect of U0126 is responsible for reversion of the axon protective effects of Wld^S expression or proteasome inhibition, rather than inhibition of MEK1/2-ERK1/2 or MEK5-ERK5 signaling. Importantly, this off-target effect does not appear to result in alterations in the stabilities of either Wld^S or NMNAT2.     

Calcium-Mediated Degeneration of the Axonal Cytoskeleton in the Ola Mouse

Abstract: The C57BL/Ola (Ola) mouse is a mutant substrain in which transected axons undergo very slow Wallerian degeneration. Because axonal degradation during Wallerian degeneration is calcium dependent, we tested whether Ola axons are susceptible to calcium-mediated axonal degeneration by comparing neuro-filament degradation between Ola and C57BL/6 mice in sciatic nerve explants. Using immunoblot analysis of neurofilament degradation and electron microscopy we found that as in normal axons, axonal degeneration in the Ola is calcium dependent. However, when compared with normal animals, higher levels of calcium were required for complete degradation of neurofilaments in Ola nerve, suggesting a relative insensitivity to calcium-mediated degeneration in the Ola. We conclude that calcium-activated proteases are present and active in Ola axons but that higher levels of calcium are required to accomplish complete axonal degradation. These results suggest a possible mechanism for prolonged survival of transected Ola axons and provide potential insight into the pathophysiology of axonal degeneration in injury and disease.     

NAD and axon degeneration: From the Wlds gene to neurochemistry

Neurodegenerative diseases have become a global issue due to the aging population. These disorders affect a vast patient population and represent a huge area of unmet therapeutic need. Axon degeneration is a common pathological character of those neurodegenerative diseases. It results in the loss of communication between neurons. Two decades ago, the Wallerian degeneration slow (Wlds) mouse strain was identified, in which the degeneration of transected axons is delayed. The phenotype is attributed to the over-expression of a chimeric protein Wlds which contains a short fragment of the ubiquitin assembly protein UFD2 and the full-length nicotinamide adenine dinucleotide (NAD) synthetic enzyme Nicotinamide mononucleotide adenylyl-transferase-1 (Nmnat-1). However, the underlying molecular mechanism remains largely unknown. Recently, it’s reported by independent researchers that the full length coding sequence of mouse Nmnat-1 could mimic the axonal protective effect of the Wlds gene when over-expressed in primary neural cultures. Together with a significant number of subsequential reports, this finding highlighted the substantial role of nicotinamide adenine dinucleotide (NAD) in the process of axon degeneration. Here we reviewed the history of axon degeneration research from a neurochemical standpoint and discuss the potential involvement of NAD synthesis, NAD consumption, and NAD-dependent proteins and small molecules in axon degeneration.     

Axon damage and repair in multiple sclerosis

It is well known that within long-standing multiple sclerosis (MS) lesions there is axonal loss but whether it is an early or late event has been more difficult to establish. The use of immunocytochemical methods that reveal axonal end-bulbs is a valuable approach to investigating acute axonal injury in human pathological material. The application of these techniques to multiple sclerosis tissue reveals evidence of axonal injury in acute lesions; the distribution of the end-bulbs in acute and active-chronic lesions is associated with regions of maximal density of infiltrating macrophages. Axon injury within the MS lesion will result in both Wallerian degeneration of the axon and also retrograde degeneration of the cell body. The functional consequences of the axon injury will depend upon numbers of axons injured and the topographical organization of the fibres coursing through the lesion. The molecular mechanisms by which the recruited leucocytes damage or transect the axons are not known. However, investigations in the Wld mutant mouse with very slow Wallerian degeneration demonstrate that axon degeneration is not simply a passive disintegration of the axon but has clear parallels with the active processes of programmed cell death. The presence of early axon injury and the consequences of an ever increasing load of neuronal damage has important implications not only for when therapy should be initiated in MS but also the therapeutic target.     

Endogenous Nmnat2 Is an Essential Survival Factor for Maintenance of Healthy Axons

The molecular triggers for axon degeneration remain unknown. We identify endogenous Nmnat2 as a labile axon survival factor whose constant replenishment by anterograde axonal transport is a limiting factor for axon survival. Specific depletion of Nmnat2 is sufficient to induce Wallerian-like degeneration of uninjured axons which endogenous Nmnat1 and Nmnat3 cannot prevent. Nmnat2 is by far the most labile Nmnat isoform and is depleted in distal stumps of injured neurites before Wallerian degeneration begins. Nmnat2 turnover is equally rapid in injured Wld ^S neurites, despite delayed neurite degeneration, showing it is not a consequence of degeneration and also that Wld^S does not stabilize Nmnat2. Depletion of Nmnat2 below a threshold level is necessary for axon degeneration since exogenous Nmnat2 can protect injured neurites when expressed at high enough levels to overcome its short half-life. Furthermore, proteasome inhibition slows both Nmnat2 turnover and neurite degeneration. We conclude that endogenous Nmnat2 prevents spontaneous degeneration of healthy axons and propose that, when present, the more long-lived, functionally related Wld^S protein substitutes for Nmnat2 loss after axon injury. Endogenous Nmnat2 represents an exciting new therapeutic target for axonal disorders.     

Wlds protection distinguishes axon degeneration following injury from naturally occurring developmental pruning

Axon pruning by degeneration remodels exuberant axonal connections and is widely required for the development of proper circuitry in the nervous system from insects to mammals. Developmental axon degeneration morphologically resembles injury-induced Wallerian degeneration, suggesting similar underlying mechanisms. As previously reported for mice, we show that Wlds protein substantially delays Wallerian degeneration in flies. Surprisingly, Wlds has no effect on naturally occurring developmental axon degeneration in flies or mice, although it protects against injury-induced degeneration of the same axons at the same developmental age. By contrast, the ubiquitin-proteasome system is intrinsically required for both developmental and injury-induced axon degeneration. We also show that the glial cell surface receptor Draper is required for efficient clearance of axon fragments during developmental axon degeneration, similar to its function in injury-induced degeneration. Thus, mechanistically, naturally occurring developmental axon pruning by degeneration and injury-induced axon degeneration differ significantly in early steps, but may converge onto a common execution pathway.     

NAD(+) and axon degeneration revisited: Nmnat1 cannot substitute for Wld(S) to delay Wallerian degeneration

The slow Wallerian degeneration protein (Wld(S)), a fusion protein incorporating full-length nicotinamide mononucleotide adenylyltransferase 1 (Nmnat1), delays axon degeneration caused by injury, toxins and genetic mutation. Nmnat1 overexpression is reported to protect axons in vitro, but its effect in vivo and its potency remain unclear. We generated Nmnat1-overexpressing transgenic mice whose Nmnat activities closely match that of Wld(S) mice. Nmnat1 overexpression in five lines of transgenic mice failed to delay Wallerian degeneration in transected sciatic nerves in contrast to Wld(S) mice where nearly all axons were protected. Transected neurites in Nmnat1 transgenic dorsal root ganglion explant cultures also degenerated rapidly. The delay in vincristine-induced neurite degeneration following lentiviral overexpression of Nmnat1 was significantly less potent than for Wld(S), and lentiviral overexpressed enzyme-dead Wld(S) still displayed residual neurite protection. Thus, Nmnat1 is significantly weaker than Wld(S) at protecting axons against traumatic or toxic injury in vitro, and has no detectable effect in vivo. The full protective effect of Wld(S) requires more N-terminal sequences of the protein.     

Wallerian degeneration of zebrafish trigeminal axons in the skin is required for regeneration and developmental pruning

Fragments of injured axons that detach from their cell body break down by the molecularly regulated process of Wallerian degeneration (WD). Although WD resembles local axon degeneration, a common mechanism for refining neuronal structure, several previously examined instances of developmental pruning were unaffected by WD pathways. We used laser axotomy and time-lapse confocal imaging to characterize and compare peripheral sensory axon WD and developmental pruning in live zebrafish larvae. Detached fragments of single injured axon arbors underwent three stereotyped phases of WD: a lag phase, a fragmentation phase and clearance. The lag phase was developmentally regulated, becoming shorter as embryos aged, while the length of the clearance phase increased with the amount of axon debris. Both cell-specific inhibition of ubiquitylation and overexpression of the Wallerian degeneration slow protein (Wld(S)) lengthened the lag phase dramatically, but neither affected fragmentation. Persistent Wld(S)-expressing axon fragments directly repelled regenerating axon branches of their parent arbor, similar to self-repulsion among sister branches of intact arbors. Expression of Wld(S) also disrupted naturally occurring local axon pruning and axon degeneration in spontaneously dying trigeminal neurons: although pieces of Wld(S)-expressing axons were pruned, and some Wld(S)-expressing cells still died during development, in both cases detached axon fragments failed to degenerate. We propose that spontaneously pruned fragments of peripheral sensory axons must be removed by a WD-like mechanism to permit efficient innervation of the epidermis.     

Engulfment of Axon Debris by Microglia Requires p38 MAPK Activity

The clearance of debris after injuries to the nervous system is a critical step for restoration of the injured neural network. Microglia are thought to be involved in elimination of degenerating neurons and axons in the central nervous system (CNS), presumably restoring a favorable environment after CNS injuries. However, the mechanism underlying debris clearance remains elusive. Here, we establish an in vitro assay system to estimate phagocytosis of axon debris. We employed a Wallerian degeneration model by cutting axons of the cortical explants. The cortical explants were co-cultured with primary microglia or the MG5 microglial cell line. The cortical neurites were then transected. MG5 cells efficiently phagocytosed the debris, whereas primary microglia showed phagocytic activity only when they were activated by lipopolysaccharide or interferon-β. When MG5 cells or primary microglia were co-cultured with degenerated axons, p38 mitogen-activated protein kinase (MAPK) was activated in these cells. Engulfment of axon debris was blocked by the p38 MAPK inhibitor SB203580, indicating that p38 MAPK is required for phagocytic activity. Receptors that recognize dying cells appeared not to be involved in the process of phagocytosis of the axon debris. In addition, the axons undergoing Wallerian degeneration did not release lactate dehydrogenase, suggesting that degeneration of the severed axons and apoptosis may represent two distinct self-destruction programs. We observed regrowth of the severed neurites after axon debris was removed. This finding suggests that axon debris, in addition to myelin debris, is an inhibitory factor for axon regeneration.Axon degeneration is an active, tightly controlled, and versatile process of axon segment self-destruction. The lesion-induced degeneration process was first described by Waller (1) and has since been known as Wallerian degeneration (2, 3). This degeneration involves rapid blebbing and fragmentation of an entire axonal stretch into short segments, which are then removed by locally activated phagocytic cells. Phagocytic removal of damaged axons and their myelin sheaths distal to the injury is important for creating a favorable environment for axonal regeneration in the nervous system. Although the debris of degenerated axons and myelin is cleared by phagocytes in the peripheral nervous system (PNS), the debris is removed very slowly in the central nervous system (CNS)³ (4, 5). This is considered to be one of the obstacles for regeneration of the injured axons in the CNS.Apoptotic neurons are also engulfed by activated phagocytic cells. Apoptosis is very well documented in the CNS where a significant proportion of neurons undergo programmed cell death (6). To prevent the diffusion of damaging degradation products into surrounding tissues, dying neurons are phagocytosed. In the brain, apoptotic cells are engulfed mainly by the resident population of phagocytes known as microglia. Microglia are generally considered to be immune cells of the CNS (7). They respond to any kind of pathology with a reaction termed “microglial activation.” After injuries to the CNS, microglia react within a few hours with a migratory response toward the lesion site.Although insight into the mechanism of phagocytosis of dying cells by microglia has improved, little is known about the mechanism of clearance of degenerated axons and myelin debris by microglia after axonal injury in the CNS. Interestingly, the axons undergoing Wallerian degeneration do not seem to possess detectable activation of the caspase family (8), suggesting that Wallerian degeneration and apoptosis may represent two distinct self-destruction programs. Thus, the mechanism of microglial phagocytosis of dying cells might be different from that of axon/myelin debris. We aimed to elucidate the mechanism of debris clearance by microglia after an axonal injury. We established an in vitro assay system to estimate phagocytosis of degenerated axon debris. We found that p38 mitogen-activated protein kinase (MAPK) was critical for the phagocytic activity of microglia. Treatment with lipopolysaccharide (LPS) or interferon-β (IFN-β) was necessary for the primary microglia to become phagocytic. In addition, clearance of degenerated axon debris allowed axonal growth from the severed neurites, suggesting that removal of the axon debris provides a favorable environment for axonal regeneration.     

Death of an axon: studying axon loss in development and disease

The loss of axon branches is a common feature of both the developing and the diseased nervous system. Despite its fundamental importance, a clear mechanistic understanding is lacking on how axonal loss occurs. However, the first molecular inroads into post-traumatic (Wallerian) axon degeneration have recently been made. In parallel, imaging techniques that allow visualizing single axons in vivo are providing a first glimpse at the cellular mechanisms of active dismantling of superfluous or diseased axons. This gives hope that soon a clearer mechanistic understanding of axon loss will emerge: comparing different forms of axon loss will reveal the spectrum of axon loss mechanisms; studies aimed at integrating the known molecular and cellular players during axon loss will provide mechanistic insight into axon dismantling; finally—by understanding how axons are normally lost—we will hopefully find ways to protect them during neurological disease or after trauma. Note: Robert Feulgen Prize Lecture 2005 presented at the Joint meeting of The Histochemical Society and The Society for Histochemistry in Noordwijkerhout, The Netherlands. After completion of this review, a comprehensive and highly informative overview of axon loss events in development and disease was published (Luo and O’Leary 2005).     

Early events of peripheral nerve regeneration

Early regeneration of injured peripheral nerves involves a series of events that are important in the success of eventual reconnection. In many nerve injuries, such as transections with gaps, axons and Schwann cells (SCs) penetrate into new microenvironments de novo, not involving zones of Wallerian degeneration. We studied unexplored axon-SC interactions by sampling of newly forming connections through a silicone conduit across transected rat sciatic peripheral nerve gaps. Axon and SC participation in bridge formation was addressed by light microscopy, electron microscopy and by double-labeling immunohistochemistry,including confocal imaging, and several, less appreciated aspects of early regrowth were identified. There are limitations to early and widespread regeneration of axons and SCs into bridges initially formed from connective tissue and blood vessels.Regrowth is 'staggered' such that only a small percentage of parent axons sampled the early bridge. There is an intimate, almost invariable relationship between SCs and extension of axons, which challenges the concept that axons lead and SCs follow.'Naked' axons were infrequent and limited in scope. Axons did not seek out and adhere to vascular laminin but intimately followed laminin deposits associated with apposed SCs. Growth cones identified by labeling of beta III tubulin, PGP(9 x 5) and GAP(43)/B(50) were complex, implying a pause in their regrowth, and were most prominent at the proximal stump-regenerative bridge interface. There is surprising and substantial hostility to local regrowth of axons into newly forming peripheral nerve bridges.Early axon outgrowth, associated with apposed Schwann cell processes, is highly constrained even when not exposed to adjacent myelin and products of Wallerian degeneration.     

In vivo imaging of axonal degeneration and regeneration in the injured spinal cord

The poor response of central axons to transection underlies the bleak prognosis following spinal cord injury. Here, we monitor individual fluorescent axons in the spinal cords of living transgenic mice over several days after spinal injury. We find that within 30 min after trauma, axons die back hundreds of micrometers. This acute form of axonal degeneration is similar in mechanism to the more delayed Wallerian degeneration of the disconnected distal axon, but acute degeneration affects the proximal and distal axon ends equally. In vivo imaging further shows that many axons attempt regeneration within 6-24 h after lesion. This growth response, although robust, seems to fail as a result of the inability of axons to navigate in the proper direction. These results suggest that time-lapse imaging of spinal cord injury may provide a powerful analytical tool for assessing the pathogenesis of spinal cord injury and for evaluating therapies that enhance regeneration.     

Wld S,Nmnats and Axon degeneration—progress in the past two decades

A chimeric protein called Wallerian degeneration slow (Wld^S) was first discovered in a spontaneous mutant strain of mice that exhibited delayed Wallerian degeneration. This provides a useful tool in elucidating the mechanisms of axon degeneration. Over-expression of Wld^S attenuates the axon degeneration that is associated with several neurodegenerative disease models, suggesting a new logic for developing a potential protective strategy. At molecular level, although Wld^S is a fusion protein, the nicotinamide mononucleotide adenylyl transferase 1 (Nmnat1) is required and sufficient for the protective effects of Wld^S, indicating a critical role of NAD biosynthesis and perhaps energy metabolism in axon degeneration. These findings challenge the proposed model in which axon degeneration is operated by an active programmed process and thus may have important implication in understanding the mechanisms of neurodegeneration. In this review, we will summarize these recent findings and discuss their relevance to the mechanisms of axon degeneration.     

Axon pruning during Drosophila metamorphosis: evidence for local degeneration and requirement of the ubiquitin-proteasome system

Axon pruning is widely used for the refinement of neural circuits in both vertebrates and invertebrates, and may also contribute to the pathogenesis of neurodegenerative diseases. However, little is known about the cellular and molecular mechanisms of axon pruning. We use the stereotyped pruning of gamma neurons of the Drosophila mushroom bodies (MB) during metamorphosis to investigate these mechanisms. Detailed time course analyses indicate that MB axon pruning is mediated by local degeneration rather than retraction and that the disruption of the microtubule cytoskeleton precedes axon pruning. In addition, multiple lines of genetic evidence demonstrate an intrinsic role of the ubiquitin-proteasome system in axon pruning; for example, loss-of-function mutations of the ubiquitin activating enzyme (E1) or proteasome subunits in MB neurons block axon pruning. Our findings suggest that some forms of axon pruning during development may share similarities with degeneration of axons in response to injury.