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A little nip and tuck: axon refinement during development and axonal injury
A little nip and tuck: axon refinement during development and axonal injury Lawrence K Low and Hwai-Jong Cheng While building the nervous system, regions of some developing axons are eliminated; this can also happen as a result of axonal injury. During development, many axon branches that are formed in excess of an organism’s needs are fated for removal in a process called axon pruning. By contrast, when axons are injured the axon segment distal to the injury site is compartmentalized and eliminated. In both cases, the end result is similar — a region of an axon is selected for removal. Recent evidence suggests that there are some similarities in the cellular and molecular mechanisms that regulate axon elimination in development and during axonal injury. Addresses Center for Neuroscience, University of California, Davis, 1544 Newton Court, Davis, CA 95616, USA Corresponding author: Cheng, Hwai-Jong ([email protected])
Current Opinion in Neurobiology 2005, 15:549–556 This review comes from a themed issue on Neuronal and glial cell biology Edited by Fred H Gage and A Kimberley McAlliser Available online 6th September 2005 0959-4388/$ – see front matter # 2005 Elsevier Ltd. All rights reserved. DOI 10.1016/j.conb.2005.08.007
Introduction Connectional specificity is a hallmark of properly functioning adult nervous systems. Neurons of developing nervous systems, however, do not conform to this notion of specificity but rather extend their projections excessively to areas where the axon branches must later be pruned away [1]. Recent advances in molecular biology and cellular imaging techniques have provided new clues on the processes that help govern how these excessive projections are removed. In this review, the primary focus is on important findings in the past year for developmental axon pruning; later, we also cover some aspects of axon removal during axonal injury in adult nervous systems and discuss how axon removal in each scenario might be related.
Removal of excess axon branches during development Anatomy of the removal of short terminal arbors at the neuromuscular junction and in the cerebellum
Much of what is known about the anatomy of axon branch elimination comes from studies of synapse elimination at www.sciencedirect.com
the neuromuscular junction (NMJ) in the peripheral nervous system [2–5]. In early postnatal development, muscle cells are polyinnervated by short axonal branches, or ‘terminal arbors’, that derive from the distal ends of axons of motor neurons competing for the same target (Figure 1a). Initially, competing motor neurons occupy similar synaptic areas and have comparable synaptic strengths [6,7]. However, later in development, activity-dependent competition at the NMJ leads to a winner-take-all scenario: the stronger synapses are preserved while weaker synapses and their short arbors are eliminated [7–10]. Several changes in axonal morphology have been observed as an axon arbor withdraws from the NMJ [4]. Previous studies have shown that these withdrawing axon branches are generally thinner in appearance than permanent axons and contain characteristic spherical structures, retraction bulbs, at their distal ends [11,12]. Retraction bulbs were originally reported by Cajal in his lesion studies of the nervous system [13]. They have also been observed in target-deprived studies of axon elimination [14–17]. Ultrastructural analysis of retraction bulbs from retracting branches of target-deprived dorsal column neurons revealed rounded structures filled with vesicles, disorganized neurofilaments and microtubules, and damaged mitochondria. This suggests an importance of postsynaptic targets in maintaining the structural integrity of the synapse and perhaps an intrinsic mechanism of self-destruction termed ‘autophagia’ [16]. Unfortunately, it was unclear at the time how the thinning axons and their retraction bulbs could be removed in vivo. Two cellular mechanisms have traditionally been thought to account for the disappearance of exuberant axon branches during developmental axon elimination (Figure 2a). Axon branches could just retract in a distal to proximal fashion as axonal contents are recycled to other parts of the axon [11]; however, there was no direct evidence for axon retraction in the vertebrate nervous system, as in most cases studied, retraction was only cited in the absence of degeneration. Alternatively, the axon branches might degenerate in a process resembling classic Wallerian degeneration (see glossary) as observed from earlier ultrastructural studies of axons following nerve transection [18,19]. In an elegant study by Bishop et al. [20], axon elimination at the NMJ was shown to occur by piecemeal removal of distal parts of the axon that were shed off as ‘axosomes’ (see glossary) from retraction bulbs [21]. Interestingly, Current Opinion in Neurobiology 2005, 15:549–556
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Glossary Acute axonal degeneration (AAD): An acute phase of axonal degeneration that occurs within minutes at the site of injury after an axon is cut. The proximal and distal portions of the axon undergo acute degeneration. Unlike Wallerian degeneration, which only affects the distal axon, AAD also affects the proximal axon which is connected to the cell soma. However, AAD shares many of the same qualities of Wallerian degeneration — the characteristic axonal blebbing and kinetics of axonal degeneration in Wallerian degeneration are also observed in AAD. Axosome: Membrane-bound components derived from retraction bulbs during terminal arbor pruning at the neuromuscular junction. Similar to retraction bulbs, they contain numerous clear vesicles and occasional mitochondria. Calpain inhibitor: An inhibitor of calpain proteases. Calpain proteases are considered to be important mediators of cytoskeletal degradation during Wallerian degeneration and acute axonal degeneration. Nicotinamide adenine dinucleotide (NAD): An important coenzyme that participates in many redox reactions in the cell. It also functions as a substrate for nuclear localization proteins such as Sir2 deacetylases and poly (ADP-ribose) polymerases, which have been linked to processes involved in yeast longevity and DNA repair, respectively. Nicotinamide mononucleotide adnenylyl transferase (NMNAT): A nuclear biosynthetic enzyme that is involved in NAD biosynthesis. Wallerian degeneration: A form of axonal degeneration that is usually seen within days after an axon is cut from the cell body. Wallerian degeneration only affects the distal axon fragment that is no longer connected to the cell body. The degenerative process, which proceeds in a proximal to distal fashion, is characterized by axonal blebbing, the disruption of the axonal membrane, disorganized neurofilaments and microtubules, and the swelling of mitochondria. Ufd2a: A ubiquitin assembly protein that belongs to the U-box family of ubiquitin ligases and functions presumably as both an E3 and an E4 ubiquitination factor. Polyubiquitination of proteins by Ufd2a targets them for protein degradation via the ubiquitin–proteosome pathway.
axon elimination at the NMJ did not involve Wallerian degeneration. Axosomes contained numerous clusters of intact vesicles and mitochondria that were not degenerating. However, axosome shedding appears to be a cellular mechanism that also differs from axon retraction, because neurofilaments and microtubules were quite disorganized within distal retraction bulbs and the more proximal axon segments of axons undergoing axosome shedding; this suggested that the machinery for retrograde transport that is necessary for axon retraction was altered. What was perhaps most striking during axosome shedding was the participation of Schwann cells in the phagocytosis of the axosomes of retreating axons [20,21]. Unfortunately, it was unclear whether Schwann cells were active mediators of distal axon fragmentation or just playing the role of passive scavengers that remove axonal debris. Schwann cells are required for proper maintenance of synaptic structure and function at the developing NMJ. Importantly, it has been shown that without Schwann cells motor axons will retract from the NMJ [22]. This suggests an intriguing scenario in which Schwann cells are actively involved in the removal of unwanted axons but in their absence an independent pathway can be switched Current Opinion in Neurobiology 2005, 15:549–556
on in neurons for axon branch elimination. It will be interesting to see in future studies if separate pathways for axon branch elimination can be activated through different signals from within or outside the cell. Unlike the NMJ, few studies have reported the cellular aspects of axon branch elimination in the developing vertebrate central nervous system (CNS) in detail. As an exception, the organization of connections in the immature cerebellum has served as a simple model for studying the elimination of short terminal arbors in the CNS. Similar to the situation in the NMJ, multiple climbing fibers extend short terminal arbors onto a single Purkinje cell at around postnatal day 3 in the cerebellum [23,24]. Several weeks later, all of those except the climbing fiber arbor that provides the strongest input onto a Purkinje cell are removed [25]. The prevalence of bulbous structures at the end of withdrawing arbors is reminiscent of retraction bulbs seen during synapse elimination at the NMJ. Another interesting finding from earlier ultrastructural studies was that many double-membrane bound structures resembling axosomes could be observed near areas once occupied by axon terminals during natural climbing fiber synapse elimination [26]. This finding is intriguing in that it suggests the cellular mechanisms for terminal arbor axon elimination as reported at the NMJ are also conserved throughout the CNS. Anatomy of the stereotyped removal of long axon collaterals in the CNS
Some evidence suggests that different cellular mechanisms regulate pruning in different contexts. During development in the CNS, the removal of axon collaterals can be stereotyped and occurs on a much grander scale than terminal arbor pruning [1,27–29]. Several aspects of stereotyped long axonal pruning distinguish it from terminal arbor pruning (Figure 1b). First, long axon collaterals project to different anatomical target regions, whereas short terminal arbors from a single axon target the same anatomical area. Thus, the removal of long collaterals involves different target regions, rather than the same target area as in terminal arbor pruning. Second, axon elimination is indeed stereotyped, because it can be predicted which axon collaterals are destined for elimination well in advance of their removal. Third, axon elimination occurs on a much larger scale, with axons as long as several millimeters in length being pruned. A classic example of stereotyped elimination of long axon collaterals occurs during the refinement of subcortical processes arising from layer V cortical neurons [28–31]. Layer V pyramidal neurons from the motor cortex and the visual cortex initially extend their axons to overlapping targets in the superior colliculus and spinal cord. The neurons from the visual cortex prune their branches from the spinal cord, whereas the neurons from the motor www.sciencedirect.com
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Figure 1
Variations in developmental axon pruning. (a) Early in development the neuron extends an axon towards a target region (target region 1). When the axon reaches target region 1, it extends short terminal arbors towards cells within the same target region. During terminal arbor pruning, the inappropriate terminal arbors are removed. (b) Early in development, a neuron extends an axon towards a target region (target region 1). This is followed by collateral branching to another target region (indicated as target 2). During stereotyped axon pruning, the inappropriate axon collaterals are predictably pruned away. In this scenario, target 1 has been designated as the inappropriate target. Stereotyped pruning is distinct from terminal arbor pruning in (a) in many respects. During stereotyped pruning, the axon is removed from a different target region, the axon collateral designated for removal can be predicted before it is pruned, and pruning can occur on a much larger scale in the order of many millimeters.
cortex prune their branches from the superior colliculus. Selective elimination appears to be transcriptionally regulated by the homeodomain transcription factor OTX1, because OTX1 translocates to the nucleus of layer V neurons just before pruning [32]. In addition, mice with loss-of-function mutations for OTX1 fail to prune away their projections from the spinal cord [32]. The cellular mechanisms that contribute to the stereotyped elimination of layer V axonal projections are unclear, but one report suggests that there is an increase in axon degeneration reminiscent of Wallerian degeneration during pruning of the axons [33]. Stereotyped axon removal also seems to play an important part in topographical mapping. Retinal ganglion cells (RGCs) located along the temporal–nasal axis of the retina organize their projections along the anterior–posterior (AP) axis of the chick tectum [34–36]. During early axonal pathfinding, RGCs often overextend their axons www.sciencedirect.com
beyond their targets and then branch collaterally off the main axon to corrected areas along the AP axis. Surprisingly, during refinement of the axon there is a characteristic blebbing of processes near areas where the axons had overextended [37]. Similar to the situation of the pruning of layer V axonal projections, this strongly implied the presence of a process involving large-scale degeneration. The stereotyped pruning of long collateral branches could be regulated by extrinsic signals that are received by the axonal branches [38,39]. In the hippocampus of young postnatal mice, mossy fibers of the dentate gyrus send a main bundle (MB) of projections that course adjacent to apical dendrites of CA3 pyramidal cells. A few days later collateral branching of infrapyramidal projections (IPB) occurs towards the basal dendrites of CA3 pyramidal cells. Axon branch elimination from the IPB was initiated by a Sema3F signal expressed locally within the stratum oriens of the hippocampus [38]. During the peak time for Current Opinion in Neurobiology 2005, 15:549–556
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Figure 2
Cellular mechanisms of segmental axon elimination. (a) During development axon branches are pruned away by axon degeneration, axon retraction, or axosome shedding. Axon degeneration results in compartmentalized degeneration throughout the collateral followed by fragmentation. Axon retraction is the distal to proximal retraction and recycling of components of the axon to other parts of the cell. Axosome shedding is the distal to proximal removal of the branches but with the continuous disposal of its distal components. (b) When an axon is injured in the peripheral nervous system two phases of degeneration are apparent. An acute axon degenerative process ensues in the proximal and distal axon segments adjacent to the injury site. This is followed by retraction (not shown) and then re-extension of the axon connected to the cell body while the detached axon segment degenerates by Wallerian degeneration. (c) During many chronic neurological diseases, axons experience a unique form of elimination called ‘dying back’ degeneration. Dying back is a retrograde form of degeneration in which a progressive distal to proximal degeneration of the axon occurs.
stereotyped axon pruning, the authors observed neither any signs of degenerating fibers nor increased signs of apoptosis, suggesting that branch elimination was mediated through axon branch retraction. In vitro cultures exposing hippocampal neurons to semaphorins strongly inferred that, in stereotyped pruning, branch retraction occurred in the absence of degeneration and axosome shedding [38]. Because mossy fiber length can be affected by increased activity [40], it will be interesting to see in future studies how semaphorin expression affects neurotransmission or vice-versa to possibly regulate the remodeling of axons during development. Insights into molecular mechanisms of axon branch elimination from studies in Drosophila
Extensive remodeling of nervous system structures and their connections is a characteristic feature of insect metamorphosis. Analyzing this fundamental event, which is required for normal insect development, might reveal more details of how axonal remodeling is regulated. The Current Opinion in Neurobiology 2005, 15:549–556
Drosophila mushroom body (MB), a brain structure implicated in learning and memory, is one area where considerable remodeling of axons occurs [41,42,43]. Two populations of neurons, g and a’/b’, populate the MB. The axonal arbors of g MB neurons, in particular, are altered during metamorphosis. Before the midthird instar stage, g MB neurons extend their processes along an axon peduncle, which is followed by a bifurcation of the axon to dorsal and medial MB lobes. During metamorphosis, the dorsal and medial branches are removed in a degenerative process that requires intact glial cell activity (see Luo and O’Leary [1] for a detailed review on Drosophila MB pruning). Studies on MB remodeling have provided important clues about the molecular pathways involved in pruning. Recent findings implicate the ubiquitin proteosome system (UPS) in regulating axon branch elimination. Polyubiquitination of protein substrates targets them for degradation by the UPS [44]. Mutations in genes required www.sciencedirect.com
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for polyubiquitination, such as the ubiquitin activating enzyme 1, Uba1, and in two subunits for the 19S proteosome regulatory particle, Mov34 and Rpn6, lead to defects in g MB pruning and, thus, provide a strong link between a protein degradation pathway and degenerative types of axon pruning [45]. Likewise, overexpression of a deubiquitinating enzyme, UBP2, in g MB neurons leads to a similar pruning defect. Importantly, activation of a cell death pathway is not involved, because mutations for apoptosis activators, grim, hid or rpr did not produce a pruning defect [45]. The UPS perhaps mediates axon branch elimination by targeting proteins that are necessary for branch stabilization or branch extension. One candidate, p190RhoGAP, is a GTPase activating protein that negatively regulates RhoA as a requirement for branch stabilization in a’/b’ MB neurons [46]. Activation of RhoA, its effector kinase Drok, and a key output for Drok, the regulatory phosphorylation of the myosin regulatory light chain, unveiled a pathway that induces branch elimination through regulation of myosin II activity [46]. The branch elimination pathway is probably dormant, because null mutants for RhoA and Drok lead to only subtle phenotypes, so the balance between activation and de-activation of the pathway depends largely on the activity of p190RhoGAP [46]. Because the pathway only affects branch stability in a’/b’ and not g MB neurons, it might be more important in other remodeling events in development. By contrast, g MB neurons are the only population to express Trio, a RacGEF, during peak stages of metamorphosis. Targeted DRacGAP loss-of-function in g MB neurons results in overextension of dorsal branches which is opposite to the phenotype observed by knocking out p190RhoGAP [47]. Thus, the balance between Rac and Rho GTPase activity might ultimately decide whether branch retraction or branch stabilization occurs. Not all remodeling events in Drosophila are associated with axon degeneration, however. In a genetic screen for molecules that affect synapse stability, Eaton et al. showed that disassembly of synapses and their axon branches at the NMJ of Drosophila proceeds by retraction [48]. Importantly, intact dynactin function was required for synapse stability. It will be interesting to see if future genetic screens on the same retraction pathway implicate similar molecules that have been shown to be required for degenerative g MB axon pruning.
Axon removal in pathology Wallerian degeneration and the Wlds mouse
In recent years, several lines of evidence have suggested that the molecular and cellular processes that regulate Wallerian degeneration are consistent with factors that are involved in degenerative modes of developmental axon pruning and a dying-back form of degeneration unique to chronic neuropathies. Wallerian degeneration www.sciencedirect.com
is a classical form of compartmental axon elimination that is initiated when an axon is cut in the CNS or PNS [49,50] (Figure 2b). The injured axon, which can conduct action potentials for a few days, is characterized by disassembly of the myelin sheath, disorganization of neurofilaments, swelling of mitochondria, and axonal thinning and membrane fragmentation that is accompanied by phagocytosis [50]. More recent studies have revealed, however, that transecting axons leads to a degenerative process that cannot be explained solely by classical Wallerian degeneration. An acute phase of degeneration occurs within minutes at the site of axonal injury [51]. During acute axonal degeneration (AAD; see glossary), injured portions of the cell-intact proximal axon and the detached distal axon segments break down into fragments in bi-directional fashion — an indication that perhaps some of the molecular mechanisms regulating AAD and Wallerian degeneration are shared. Indeed, a calpain inhibitor (see glossary) applied to transected axons effectively blocks cytoskeletal degradation usually seen in AAD and Wallerian degeneration [51]. After AAD, the proximal axon retracts but the cell body is left intact, whereas the isolated distal axon undergoes Wallerian degeneration. Why does axonal injury result in axon degeneration and not cell death? Some studies have suggested that axon degeneration and apoptosis are regulated by separate molecular pathways. Overexpression of human bcl-2, an inhibitor of apoptosis, in developing mouse retinal ganglion cells does not protect against axotomy-induced Wallerian degeneration but spares the neurons from cell death [52,53]. Additionally, the caspase family of cysteine proteases is normally activated during apoptosis but its activation is not observed in axotomized axons [54]. Likewise, blocking caspase activity does not affect the kinetics of Wallerian degeneration but apoptosis is markedly inhibited [54]. Apoptosis is also unaffected in a strain of mice in which Wallerian degeneration is slowed (Wlds). Interestingly, AAD is also slowed in Wlds mice but axon retraction occurs normally, indicating that perhaps axon retraction events are controlled by a separate process. The discovery of the Wlds mouse provided evidence that some forms of axon degeneration were regulated by an active process. Besides the protection from Wallerian degeneration, Wlds mice are phenotypically indistinguishable from their wild type counterparts. The Wlds gene was identified as an 85-kb tandem triplication that resulted in the overexpression of a chimeric protein: this protein included an intact nicotinamide adenine dinucleotide (NAD; see glossary) biosynthetic enzyme, a nicotinamide mononucleotide adnenylyl transferase (NMNAT; see glossary) fused to the amino-terminal 70 amino acids of Ufd2a, and an E4 ubiquitin ligase involved in ubiquitination [55–57]. Given that Wallerian degeneration and degenerative pruning in Drosophila could be slowed or inhibited by factors that disrupt UPS-mediated protein Current Opinion in Neurobiology 2005, 15:549–556
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degradation [58], it was not surprising that many studies focused on the potential neuroprotective effects of Ufd2a (see glossary). On the contrary, Araki et al. [59] discovered that it was not Ufd2a, but the activation of NMNAT and a downstream effector, SIRT1, a NAD-dependent histone deacetylase that has been shown to promote longevity in yeast and Caenorhabditis elegans, that mattered most. Interestingly, SIRT1 interacts with the tumor suppressor protein, p53, but other targets for SIRT1 need to be identified. When axons are locally deprived of nerve growth factor (NGF) from the targets they innervate, a degenerative process proceeds retrogradely from the axon terminal [60]. This ‘dying-back’ phenomenon is characteristic of many peripheral nerve diseases, and its progression is remarkably slowed in neurons that express the Wlds gene [61,62]. In Wlds neurons deprived of NGF, the cells die but the axons survive for up to a week [62]. Much like Wallerian degeneration, caspase activation is not seen in dying-back axons and the concurrent axon degeneration is not blocked by application of caspase inhibitors. The results suggest at the very least that ‘dying back’ degeneration and Wallerian degeneration share some of the same mechanisms. Given these results, in addition to the importance of the UPS in Wallerian degeneration [58], an attractive hypothesis is that SIRT1 might indirectly restrict the activity of molecules that promote axon degeneration. Future implications for studies on axon elimination in disease
Perhaps the most direct benefits of exploring mechanisms of axon elimination are its correlations with understanding axon pathology in neurological disease [63]. Dyingback axon degeneration (Figure 2c) is seen in many cases of chemical, metabolic and infectious insults on the nervous system. In the PNS, alcoholism, diabetes, AIDS and acrylamide poisoning all contribute to dying-back axon degeneration [63]. In Alzheimer’s, Parkinson’s and motor neuron diseases of the CNS, affected axons also degenerate accordingly (reviewed in [61,63]). Importantly, the WLDs gene protects axons in several mouse models in which axon degeneration is present, suggesting an important link between the processes that regulate Wallerian degeneration and the dying-back degeneration in chronic neuropathies [61,63].
Conclusions We have summarized some of the cellular and molecular processes associated with developmental axon elimination and experimentally induced axon degeneration. It seems that some molecular pathways are common in cases in which degeneration is the primary mode for axon elimination. In future studies, it will be important to address some of the following unresolved issues about axon elimination. First, are all forms of axon degeneration — Wallerian degeneration, degenerative axon pruning in Current Opinion in Neurobiology 2005, 15:549–556
development, NGF-deprived axon degeneration, and pathological degeneration in disease — regulated by the same molecular mechanism? Second, what molecular pathways activate axon retraction processes while suppressing degenerative ones? Third, and finally, is there a common link between pathways that regulate axon elimination through degeneration and those that regulate through retraction? The road ahead looks promising as more sophisticated techniques for visualizing cellular dynamics of axon elimination and for screening of related molecules that regulate the process become available.
Update
A recent study by Portera-Cailliau et al. [64] provides new insight into the development of axonal projections in the mouse cerebral cortex. By utilizing two-photon live imaging of GFP-expressing neurons that project axons to or within the cerebral cortex, the authors visualized the growth and pruning of long range axonal projections from thalamocortical (TC) neurons and much shorter axonal projections from the cortical interneurons of CajalRetzius (CR) cells in vivo. Importantly, it was found that developing axons can prune away their excess axons via either axonal degeneration or retraction. Axon retraction seems to be the preferred mechanism, because short range CR projections and long range TC projections prune most of their excess projections by retraction. Interestingly, axon pruning by axonal degeneration did occur, but it was only observed in the longer TC axons and occurred in only 5% of all pruning events. A recent report by Wang et al. [65] sheds new light on the axon-protective effects of NAD. Consistent with an earlier study by Araki et al. [59], the authors demonstrated that NAD levels are important for ensuring protection of the axon after axonal injury. NAD concentrations decrease after axonal injury, but the axon can be protected from axonal degeneration by applying exogenous NAD to prevent its levels from decreasing in the injured axons. Previously, Araki et al. [59] proposed that NAD levels were more important for regulating the activity of SIRT1 deacetylase in the nucleus. This was based on the evidence that exogenous NAD application did not offer any axonal protection when it was applied after axons were cut from the cell body; an axon protective signal from the nucleus was believed to be transduced from the nucleus to the axon. By contrast, Wang et al. [65] demonstrated that exogenous NAD applied at much higher concentrations than were used by Araki et al. [59] after an axon was cut could still protect the axon from degenerating — the result suggested that a local NAD protective effect was present. NAD levels might regulate SIRT1 activity in the cell nucleus as suggested by Araki et al. [59], but it is also an important coenzyme in ATP synthesis that might occur locally within the axon. In support of a local axon protective mechanism that was dependent on such bioenergetics, Wang et al. [65] www.sciencedirect.com
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showed that the application of exogenous pyruvate to prevent ATP depletion could protect cut axons as well.
18. Rosenthal JL, Taraskevich PS: Reduction in multiaxonal innervation at the neuromuscular junction of the rat during development. J Physiol 1977, 270:299-310.
Acknowledgements
19. Bixby JL: Ultrastructural observations on synapse elimination in neonatal rabbit skeletal muscle. J Neurocytol 1981, 10:81-100.
We thank X-B Liu, Z He, and members of the Cheng laboratory for helpful comments on the manuscript. This work was supported by grants from the National Institutes of Health (HD045757), University of California Davis MIND Institute, Whitehall Foundation, and fellowships from the Esther A. and Joseph Klingenstein Fund and the Alfred P. Sloan Foundation.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: of special interest of outstanding interest
20. Bishop DL, Misgeld T, Walsh MK, Gan WB, Lichtman JW: Axon branch removal at developing synapses by axosome shedding. Neuron 2004, 44:651-661. This study combines time-lapse imaging with electron microscopy of withdrawing terminal arbors at the NMJ. Interestingly, it was found that terminal arbors do not simply retract. Instead, while they are retracting, the distal ends of the axonal arbor are shed off as axosomes — nondegenerating membrane-bound structures that are sometimes filled with vesicles. The process is associated with glial cell activity, because it appears that glial cells are engulfing the axosomes. 21. Koirala S, Ko CP: Pruning an axon piece by piece: a new mode of synapse elimination. Neuron 2004, 44:578-580.
1.
Luo L, O’Leary DDM: Axon retraction and degeneration in development and disease. Annu Rev Neurosci 2005, 28:127-156.
22. Reddy LV, Koirala S, Sugiura Y, Herrera AA, Ko CP: Glial cells maintain synaptic structure and function and promote development of the neuromuscular junction in vivo. Neuron 2003, 40:563-580.
2.
Sanes JR, Lichtman JW: Development of the vertebrate neuromuscular junction. Annu Rev Neurosci 1999, 22:389-442.
23. Ito M: The modifiable neuronal network of the cerebellum. Jpn J Physiol 1984, 34:781-792.
3.
Nguyen QT, Lichtman JW: Mechanism of synapse disassembly at the developing neuromuscular junction. Curr Opin Neurobiol 1996, 6:104-112.
24. Mason CA, Gregory E: Postnatal maturation of cerebellar mossy and climbing fibers: transient expression of dual features on single axons. J Neurosci 1984, 4:1715-1735.
4.
Bernstein M, Lichtman JW: Axonal atrophy: the retraction reaction. Curr Opin Neurobiol 1999, 9:364-370.
5.
Lichtman JW, Colman H: Synapse elimination and indelible memory. Neuron 2000, 25:269-278.
25. Hashimoto K, Masanobu K: Functional differentiation of multiple climbing fiber inputs during synapse elimination in the developing cerebellum. Neuron 2003, 38:785-796.
6.
Balice-Gordon RJ, Lichtman JW: In vivo observations of preand postsynaptic changes during the transition from multiple to single innervation at developing neuromuscular junctions. J Neurosci 1993, 13:834-855.
7.
Colman H, Nabekura J, Lichtman JW: Alterations in synaptic strength preceding axon withdrawal. Science 1997, 275:356-361.
8.
Balice-Gordon RJ, Lichtman JW: Long-term synaptic loss induced by focal blockade of postsynaptic receptors. Nature 1994, 372:519-524.
9.
Gan WB, Lichtman JW: Synaptic segregation at the developing neuromuscular junction. Science 1998, 282:1508-1511.
10. Buffelli M, Burgess RW, Feng G, Lobe CG, Lichtman JW, Sanes JR: Genetic evidence that relative synaptic efficacy biases the outcome of synaptic competition. Nature 2003, 424:430-434. 11. Riley DA: Ultrastructural evidence for axon retraction during the spontaneous elimination of polyneuronal innervation of the rat soleus muscle. J Neurocytol 1981, 10:425-440. 12. Gan WB, Lichtman JW: Synaptic segregation at the developing neuromuscular junction. Science 1998, 282:1508-1511. 13. Ramon y Cajal S: Degeneration and regeneration of the nervous system. Translated and edited by May RM. Hafner Publishing; 1959. 14. Rossi F, Borsello T, Vaudano E, Strata P: Regressive modifications of climbing fibres following Purkinje cell degeneration in the cerebellar cortex of the adult rat. Neuroscience 1993, 53:759-778. 15. Marty S, Weinitz JM, Peschanski M: Target dependence of adult neurons: pattern of terminal arborizations. J Neurosci 1994, 14:5257-5266.
26. Eckenhoff MF, Pysh JJ: Double-walled coated vesicle formation: evidence for massive and transient conjugate internalization of plasma membranes during cerebellar development. J Neurocytol 1979, 8:623-638. 27. Innocenti GM: Growth and reshaping of axons in the establishment of visual callosal connections. Science 1981, 212:824-827. 28. O’Leary DDM, Bicknese AR, De Carlos JA, Heffner CD, Koester SE, Kutka LJ, Terashima T: Target selection by cortical axons: alternative mechanisms to establish axonal connections in the developing brain. Cold Spring Harb Symp Quant Biol 1990, 55:453-468. 29. O’Leary DDM: Remodelling of early axonal projections through the selective elimination of neurons and long axon collaterals. Ciba Found Symp 1987, 126:113-142. 30. Stanfield BB, O’Leary DDM, Fricks C: Selective collateral elimination in early postnatal development restricts cortical distribution of rat pyramidal tract neurones. Nature 1982, 298:371-373. 31. Stanfield BB, O’Leary DDM: The transient corticospinal projection from the occipital cortex during the postnatal development of the rat. J Comp Neurol 1985, 238:236-248. 32. Weimann JM, Zhang YA, Levin ME, Devine WP, Brulet P, McConnell SK: Cortical neurons require Otx1 for the refinement of exuberant axonal projections to subcortical targets. Neuron 1999, 24:819-831. 33. Reinoso BS, O’Leary DDM: Extension, branching and regression of visual corticospinal axons in rat. Soc Neurosci Abstr 1989, 15:1337. 34. McLaughlin T, Hindges R, O’Leary DDM: Regulation of axial patterning of the retina and its topographic mapping in the brain. Curr Opin Neurobiol 2003, 13:57-69.
16. Marty S, Peschanski M: Fine structural alteration in targetdeprived axonal terminals in the rat thalamus. Neuroscience 1994, 62:1121-1132.
35. McLaughlin T, Hindges R, Yates PA, O’Leary DDM: Bifunctional action of ephrin-B1 as a repellent and attractant to control bidirectional branch extension in dorsal-ventral retinotopic mapping. Development 2003, 130:2407-2418.
17. Pearson HE, Stoffler DJ, Sonstein WJ: Response of retinal terminals to loss of postsynaptic target neurons in the dorsal lateral geniculate nucleus of the adult cat. J Comp Neurol 1992, 315:333-343.
36. Yates PA, Roskies AL, McLaughlin T, O’Leary DDM: Topographic-specific axon branching controlled by ephrin-As is the critical event in retinotectal map development. J Neurosci 2001, 21:8548-8563.
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37. Nakamura H, O’Leary DDM: Inaccuracies in initial growth and arborization of chick retinotectal axons followed by course corrections and axon remodeling to develop topographic order. J Neurosci 1989, 9:3776-3795. 38. Bagri A, Cheng HJ, Yaron A, Pleasure SJ, Tessier-Lavigne M: Stereotyped pruning of long hippocampal axon branches triggered by retraction inducers of the semaphorin family. Cell 2003, 113:285-299.
52. Dubois-Dauphin M, Frankowski H, Tsujimoto Y, Huarte J, Martinou JC: Neonatal motoneurons overexpressing the bcl-2 protooncogene in transgenic mice are protected from axotomy-induced cell death. Proc Natl Acad Sci USA 1994, 91:3309-3313. 53. Burne JF, Staple JK, Raff MC: Glial cells are increased proportionally in transgenic optic nerves with increased numbers of axons. J Neurosci 1996, 16:2064-2073.
39. Sahay A, Molliver ME, Ginty DD, Kolodkin AL: Semaphorin-3F is critical for development of limbic system circuitry and is required in neurons for selective CNS axon guidance events. J Neurosci 2003, 23:6671-6680.
54. Finn JT, Weil M, Archer F, Simon R, Srinivasan A, Raff MC: Evidence that Wallerian degeneration and localized axon degeneration induced by local neurotrophin deprivation do not involve caspases. J Neurosci 2000, 20:1333-1341.
40. Adams B, Lee M, Fahnestock M, Racine RJ: Long-term potentiation trains induce mossy fiber sprouting. Brain Res 1997, 775:193-197.
55. Coleman MP, Conforti L, Buckmaster EA, Tarlton A, Ewing RM, Brown MC, Lyon MF, Perry VH: An 85-kb tandem triplication in the slow Wallerian degeneration (Wlds) mouse. Proc Natl Acad Sci USA 1998, 95:9985-9990.
41. Lee T, Marticke S, Sung C, Robinow S, Luo L: Cell-autonomous requirement of the USP/EcR-B ecdysone receptor for mushroom body neuronal remodeling in Drosophila. Neuron 2000, 28:807-818. 42. Watts RJ, Schuldiner O, Perrino J, Larsen C, Luo L: Glia engulf degenerating axons during developmental axon pruning. Curr Biol 2004, 14:678-684. This study provides evidence that stereotyped pruning of gamma mushroom body axons in Drosophila proceeds by axon degeneration. Axons destined for elimination were labeled using a clonal technique, mosaic analysis with a repressible cell marker (MARCM), and subsequently visualized under the electron microscope. It was found that axons degenerate during stereotyped pruning and the process is associated with glial cell activity. 43. Awasaki T, Ito K: Engulfing action of glial cells is required for programmed axon pruning during Drosophila metamorphosis. Curr Biol 2004, 14:668-677. This is a study published in parallel with the Watts et al. [42] work above. This study provides evidence that the engulfing activity of glial cells is required for mushroom body axon pruning. Furthermore, there is evidence suggesting an instructive role for pruning, because glial cell infiltration into areas occupied by mushroom body axons required intact ecdysone receptor signaling from gamma mushroom body neurons. 44. Voges D, Zwickl P, Baumeister W: The 26S proteosome: a molecular machine designed for controlled proteolysis. Annu Rev Biochem 1999, 68:1015-1068. 45. Watts RJ, Hoopfer ED, Luo L: Axon pruning during Drosophila metamorphosis: evidence for local degeneration and requirement of the ubiquitin-proteosome system. Neuron 2003, 38:871-885. 46. Billuart P, Winter CG, Maresh A, Zhao X, Luo L: Regulating axon branch stability: the role of p190 RhoGAP in repressing a retraction signaling pathway. Cell 2001, 107:195-207. 47. Awasaki T, Saito M, Sone M, Suzuki E, Sakai R, Ito K, Hama C: The Drosophila trio plays an essential role in patterning of axons by regulating their directional extension. Neuron 2000, 26:119-131. 48. Eaton BA, Fetter RD, Davis GW: Dynactin is necessary for synapse stabilization. Neuron 2002, 34:729-741. 49. Waller A: Experiments on the section of the glossopharyngeal and hypoglossal nerves of the frog, and observations on the alterations produced thereby in the structure of their primitive fibers. Philos Trans R Soc Lond B Biol Sci 1850, 140:423-429. 50. Gillingwater TH, Ribchester RR: Compartmental neurodegeneration and synaptic plasticity in the Wlds mutant mouse. J Physiol 2001, 534:627-639. 51. Kerschensteiner M, Schwab ME, Lichtman JW, Misgeld T: In vivo imaging of axonal degeneration and regeneration in the injured spinal cord. Nat Med 2005, 11:572-577. In this study, in vivo time-lapse imaging was performed on labeled dorsal root ganglion cell axons. When the axons were lesioned, time-lapse imaging revealed an acute form of axonal degeneration that affected areas proximal and distal to the lesion site. This acute axonal degeneration exhibited many of the same qualities as Wallerian degeneration.
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56. Conforti L, Tarlton A, Mack TGA, Mi W, Buckmaster EA, Wagner D, Perry VH, Coleman MP: A Ufd2/D4Cole1e chimeric protein and overexpression of Rbp7 in the slow Wallerian degeneration (Wlds) mouse. Proc Natl Acad Sci USA 2000, 97:11377-11382. 57. Mack TGA, Reiner M, Beirowski B, Mi W, Emanuelli M, Wagner D, Thomson D, Gillingwater T, Court F, Conforti L et al.: Wallerian degeneration of injured axons and synapses is delayed by a Ube4b/Nmnat chimeric gene. Nat Neurosci 2001, 4:1199-1206. 58. Zhai Q, Wang J, Kim A, Liu Q, Watts R, Hoopfer E, Mitchison T, Luo L, He Z: Involvement of the ubiquitin-proteasome system in the early stages of Wallerian degeneration. Neuron 2003, 39:217-225. 59. Araki T, Sasaki Y, Milbrandt J: Increased nuclear NAD biosynthesis and SIRT1 activation prevent axonal degeneration. Science 2004, 305:1010-1013. This study identifies a molecular pathway that helps to explain the axon protective effects imposed by the mutation in Wlds mice. The Wlds mutation results in the overexpression of a chimeric fusion protein. It was shown that the principal component of the fusion protein is NMNAT1, an enzyme involved in NAD biosynthesis. In addition, the authors demonstrated that the axon protective effects depend on activation of an NADdependent histone deacetylase in the nucleus, SIRT1, which acts as a downstream effector of NMNAT1. 60. Campenot RB: Development of sympathetic neurons in compartmentalized cultures. II. Local control of neurite survival by nerve growth factor. Dev Biol 1982, 93:13-21. 61. Coleman MP, Perry VH: Axon pathology in neurological disease: a neglected therapeutic target. Trends Neurosci 2002, 25:532-537. 62. Deckwerth TL, Johnson EM Jr: Neurites can remain viable after destruction of the neuronal soma by programmed cell death (apoptosis). Dev Biol 1994, 165:63-72. 63. Raff MC, Whitmore AV, Finn JT: Axonal self-destruction and neurodegeneration. Science 2002, 296:868-871. 64. Portera-Cailliau C, Weimer RM, De Paola V, Caroni P, Svoboda K: Diverse modes of axon elaboration in the developing neocortex. PLoS Biol 2005, 3:e272 [Epub ahead of print]. In an elegant study using in vivo two-photon imaging of GFP-expressing neurons in the mouse CNS, the authors demonstrate that developing neurons possess two conserved mechanisms for remodeling their axons. Cajal-Retzius interneurons that have shorter axonal arbors in the neocortex and thalamocortical (TC) neurons that project long axons to the neocortex both have a preference for pruning their axonal branches by axon retraction. However, in 5% of all pruning events, axonal degeneration was also observed in the TC projections during axonal remodeling. 65. Wang J, Zhai Q, Chen Y, Lin E, Gu W, McBurney MW, He Z: A local mechanism mediates NAD-dependent protection of axon degeneration. J Cell Biol 2005 Jul 25; [Epub ahead of print]. A second study demonstrating the axon protective effects of NAD from axonal degeneration. In contrast to an earlier study by Araki et al. [59], the authors propose that NAD acts locally on a bioenergetic pathway in axons after they have been cut from the cell body. Consistent with this hypothesis, the authors showed that exogenous addition of ATP or pyruvate could also protect axons from degenerating.
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Current Opinion in Neurobiology 2005, 15:549–556 This review comes from a themed issue on Neuronal and glial cell biology Edited by Fred H Gage and A Kimberley McAlliser Available online 6th September 2005 0959-4388/$ – see front matter # 2005 Elsevier Ltd. All rights reserved. DOI 10.1016/j.conb.2005.08.007
Introduction Connectional specificity is a hallmark of properly functioning adult nervous systems. Neurons of developing nervous systems, however, do not conform to this notion of specificity but rather extend their projections excessively to areas where the axon branches must later be pruned away [1]. Recent advances in molecular biology and cellular imaging techniques have provided new clues on the processes that help govern how these excessive projections are removed. In this review, the primary focus is on important findings in the past year for developmental axon pruning; later, we also cover some aspects of axon removal during axonal injury in adult nervous systems and discuss how axon removal in each scenario might be related.
Removal of excess axon branches during development Anatomy of the removal of short terminal arbors at the neuromuscular junction and in the cerebellum
Much of what is known about the anatomy of axon branch elimination comes from studies of synapse elimination at www.sciencedirect.com
the neuromuscular junction (NMJ) in the peripheral nervous system [2–5]. In early postnatal development, muscle cells are polyinnervated by short axonal branches, or ‘terminal arbors’, that derive from the distal ends of axons of motor neurons competing for the same target (Figure 1a). Initially, competing motor neurons occupy similar synaptic areas and have comparable synaptic strengths [6,7]. However, later in development, activity-dependent competition at the NMJ leads to a winner-take-all scenario: the stronger synapses are preserved while weaker synapses and their short arbors are eliminated [7–10]. Several changes in axonal morphology have been observed as an axon arbor withdraws from the NMJ [4]. Previous studies have shown that these withdrawing axon branches are generally thinner in appearance than permanent axons and contain characteristic spherical structures, retraction bulbs, at their distal ends [11,12]. Retraction bulbs were originally reported by Cajal in his lesion studies of the nervous system [13]. They have also been observed in target-deprived studies of axon elimination [14–17]. Ultrastructural analysis of retraction bulbs from retracting branches of target-deprived dorsal column neurons revealed rounded structures filled with vesicles, disorganized neurofilaments and microtubules, and damaged mitochondria. This suggests an importance of postsynaptic targets in maintaining the structural integrity of the synapse and perhaps an intrinsic mechanism of self-destruction termed ‘autophagia’ [16]. Unfortunately, it was unclear at the time how the thinning axons and their retraction bulbs could be removed in vivo. Two cellular mechanisms have traditionally been thought to account for the disappearance of exuberant axon branches during developmental axon elimination (Figure 2a). Axon branches could just retract in a distal to proximal fashion as axonal contents are recycled to other parts of the axon [11]; however, there was no direct evidence for axon retraction in the vertebrate nervous system, as in most cases studied, retraction was only cited in the absence of degeneration. Alternatively, the axon branches might degenerate in a process resembling classic Wallerian degeneration (see glossary) as observed from earlier ultrastructural studies of axons following nerve transection [18,19]. In an elegant study by Bishop et al. [20], axon elimination at the NMJ was shown to occur by piecemeal removal of distal parts of the axon that were shed off as ‘axosomes’ (see glossary) from retraction bulbs [21]. Interestingly, Current Opinion in Neurobiology 2005, 15:549–556
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Glossary Acute axonal degeneration (AAD): An acute phase of axonal degeneration that occurs within minutes at the site of injury after an axon is cut. The proximal and distal portions of the axon undergo acute degeneration. Unlike Wallerian degeneration, which only affects the distal axon, AAD also affects the proximal axon which is connected to the cell soma. However, AAD shares many of the same qualities of Wallerian degeneration — the characteristic axonal blebbing and kinetics of axonal degeneration in Wallerian degeneration are also observed in AAD. Axosome: Membrane-bound components derived from retraction bulbs during terminal arbor pruning at the neuromuscular junction. Similar to retraction bulbs, they contain numerous clear vesicles and occasional mitochondria. Calpain inhibitor: An inhibitor of calpain proteases. Calpain proteases are considered to be important mediators of cytoskeletal degradation during Wallerian degeneration and acute axonal degeneration. Nicotinamide adenine dinucleotide (NAD): An important coenzyme that participates in many redox reactions in the cell. It also functions as a substrate for nuclear localization proteins such as Sir2 deacetylases and poly (ADP-ribose) polymerases, which have been linked to processes involved in yeast longevity and DNA repair, respectively. Nicotinamide mononucleotide adnenylyl transferase (NMNAT): A nuclear biosynthetic enzyme that is involved in NAD biosynthesis. Wallerian degeneration: A form of axonal degeneration that is usually seen within days after an axon is cut from the cell body. Wallerian degeneration only affects the distal axon fragment that is no longer connected to the cell body. The degenerative process, which proceeds in a proximal to distal fashion, is characterized by axonal blebbing, the disruption of the axonal membrane, disorganized neurofilaments and microtubules, and the swelling of mitochondria. Ufd2a: A ubiquitin assembly protein that belongs to the U-box family of ubiquitin ligases and functions presumably as both an E3 and an E4 ubiquitination factor. Polyubiquitination of proteins by Ufd2a targets them for protein degradation via the ubiquitin–proteosome pathway.
axon elimination at the NMJ did not involve Wallerian degeneration. Axosomes contained numerous clusters of intact vesicles and mitochondria that were not degenerating. However, axosome shedding appears to be a cellular mechanism that also differs from axon retraction, because neurofilaments and microtubules were quite disorganized within distal retraction bulbs and the more proximal axon segments of axons undergoing axosome shedding; this suggested that the machinery for retrograde transport that is necessary for axon retraction was altered. What was perhaps most striking during axosome shedding was the participation of Schwann cells in the phagocytosis of the axosomes of retreating axons [20,21]. Unfortunately, it was unclear whether Schwann cells were active mediators of distal axon fragmentation or just playing the role of passive scavengers that remove axonal debris. Schwann cells are required for proper maintenance of synaptic structure and function at the developing NMJ. Importantly, it has been shown that without Schwann cells motor axons will retract from the NMJ [22]. This suggests an intriguing scenario in which Schwann cells are actively involved in the removal of unwanted axons but in their absence an independent pathway can be switched Current Opinion in Neurobiology 2005, 15:549–556
on in neurons for axon branch elimination. It will be interesting to see in future studies if separate pathways for axon branch elimination can be activated through different signals from within or outside the cell. Unlike the NMJ, few studies have reported the cellular aspects of axon branch elimination in the developing vertebrate central nervous system (CNS) in detail. As an exception, the organization of connections in the immature cerebellum has served as a simple model for studying the elimination of short terminal arbors in the CNS. Similar to the situation in the NMJ, multiple climbing fibers extend short terminal arbors onto a single Purkinje cell at around postnatal day 3 in the cerebellum [23,24]. Several weeks later, all of those except the climbing fiber arbor that provides the strongest input onto a Purkinje cell are removed [25]. The prevalence of bulbous structures at the end of withdrawing arbors is reminiscent of retraction bulbs seen during synapse elimination at the NMJ. Another interesting finding from earlier ultrastructural studies was that many double-membrane bound structures resembling axosomes could be observed near areas once occupied by axon terminals during natural climbing fiber synapse elimination [26]. This finding is intriguing in that it suggests the cellular mechanisms for terminal arbor axon elimination as reported at the NMJ are also conserved throughout the CNS. Anatomy of the stereotyped removal of long axon collaterals in the CNS
Some evidence suggests that different cellular mechanisms regulate pruning in different contexts. During development in the CNS, the removal of axon collaterals can be stereotyped and occurs on a much grander scale than terminal arbor pruning [1,27–29]. Several aspects of stereotyped long axonal pruning distinguish it from terminal arbor pruning (Figure 1b). First, long axon collaterals project to different anatomical target regions, whereas short terminal arbors from a single axon target the same anatomical area. Thus, the removal of long collaterals involves different target regions, rather than the same target area as in terminal arbor pruning. Second, axon elimination is indeed stereotyped, because it can be predicted which axon collaterals are destined for elimination well in advance of their removal. Third, axon elimination occurs on a much larger scale, with axons as long as several millimeters in length being pruned. A classic example of stereotyped elimination of long axon collaterals occurs during the refinement of subcortical processes arising from layer V cortical neurons [28–31]. Layer V pyramidal neurons from the motor cortex and the visual cortex initially extend their axons to overlapping targets in the superior colliculus and spinal cord. The neurons from the visual cortex prune their branches from the spinal cord, whereas the neurons from the motor www.sciencedirect.com
Axon refinement during development and axonal injury Low and Cheng 551
Figure 1
Variations in developmental axon pruning. (a) Early in development the neuron extends an axon towards a target region (target region 1). When the axon reaches target region 1, it extends short terminal arbors towards cells within the same target region. During terminal arbor pruning, the inappropriate terminal arbors are removed. (b) Early in development, a neuron extends an axon towards a target region (target region 1). This is followed by collateral branching to another target region (indicated as target 2). During stereotyped axon pruning, the inappropriate axon collaterals are predictably pruned away. In this scenario, target 1 has been designated as the inappropriate target. Stereotyped pruning is distinct from terminal arbor pruning in (a) in many respects. During stereotyped pruning, the axon is removed from a different target region, the axon collateral designated for removal can be predicted before it is pruned, and pruning can occur on a much larger scale in the order of many millimeters.
cortex prune their branches from the superior colliculus. Selective elimination appears to be transcriptionally regulated by the homeodomain transcription factor OTX1, because OTX1 translocates to the nucleus of layer V neurons just before pruning [32]. In addition, mice with loss-of-function mutations for OTX1 fail to prune away their projections from the spinal cord [32]. The cellular mechanisms that contribute to the stereotyped elimination of layer V axonal projections are unclear, but one report suggests that there is an increase in axon degeneration reminiscent of Wallerian degeneration during pruning of the axons [33]. Stereotyped axon removal also seems to play an important part in topographical mapping. Retinal ganglion cells (RGCs) located along the temporal–nasal axis of the retina organize their projections along the anterior–posterior (AP) axis of the chick tectum [34–36]. During early axonal pathfinding, RGCs often overextend their axons www.sciencedirect.com
beyond their targets and then branch collaterally off the main axon to corrected areas along the AP axis. Surprisingly, during refinement of the axon there is a characteristic blebbing of processes near areas where the axons had overextended [37]. Similar to the situation of the pruning of layer V axonal projections, this strongly implied the presence of a process involving large-scale degeneration. The stereotyped pruning of long collateral branches could be regulated by extrinsic signals that are received by the axonal branches [38,39]. In the hippocampus of young postnatal mice, mossy fibers of the dentate gyrus send a main bundle (MB) of projections that course adjacent to apical dendrites of CA3 pyramidal cells. A few days later collateral branching of infrapyramidal projections (IPB) occurs towards the basal dendrites of CA3 pyramidal cells. Axon branch elimination from the IPB was initiated by a Sema3F signal expressed locally within the stratum oriens of the hippocampus [38]. During the peak time for Current Opinion in Neurobiology 2005, 15:549–556
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Figure 2
Cellular mechanisms of segmental axon elimination. (a) During development axon branches are pruned away by axon degeneration, axon retraction, or axosome shedding. Axon degeneration results in compartmentalized degeneration throughout the collateral followed by fragmentation. Axon retraction is the distal to proximal retraction and recycling of components of the axon to other parts of the cell. Axosome shedding is the distal to proximal removal of the branches but with the continuous disposal of its distal components. (b) When an axon is injured in the peripheral nervous system two phases of degeneration are apparent. An acute axon degenerative process ensues in the proximal and distal axon segments adjacent to the injury site. This is followed by retraction (not shown) and then re-extension of the axon connected to the cell body while the detached axon segment degenerates by Wallerian degeneration. (c) During many chronic neurological diseases, axons experience a unique form of elimination called ‘dying back’ degeneration. Dying back is a retrograde form of degeneration in which a progressive distal to proximal degeneration of the axon occurs.
stereotyped axon pruning, the authors observed neither any signs of degenerating fibers nor increased signs of apoptosis, suggesting that branch elimination was mediated through axon branch retraction. In vitro cultures exposing hippocampal neurons to semaphorins strongly inferred that, in stereotyped pruning, branch retraction occurred in the absence of degeneration and axosome shedding [38]. Because mossy fiber length can be affected by increased activity [40], it will be interesting to see in future studies how semaphorin expression affects neurotransmission or vice-versa to possibly regulate the remodeling of axons during development. Insights into molecular mechanisms of axon branch elimination from studies in Drosophila
Extensive remodeling of nervous system structures and their connections is a characteristic feature of insect metamorphosis. Analyzing this fundamental event, which is required for normal insect development, might reveal more details of how axonal remodeling is regulated. The Current Opinion in Neurobiology 2005, 15:549–556
Drosophila mushroom body (MB), a brain structure implicated in learning and memory, is one area where considerable remodeling of axons occurs [41,42,43]. Two populations of neurons, g and a’/b’, populate the MB. The axonal arbors of g MB neurons, in particular, are altered during metamorphosis. Before the midthird instar stage, g MB neurons extend their processes along an axon peduncle, which is followed by a bifurcation of the axon to dorsal and medial MB lobes. During metamorphosis, the dorsal and medial branches are removed in a degenerative process that requires intact glial cell activity (see Luo and O’Leary [1] for a detailed review on Drosophila MB pruning). Studies on MB remodeling have provided important clues about the molecular pathways involved in pruning. Recent findings implicate the ubiquitin proteosome system (UPS) in regulating axon branch elimination. Polyubiquitination of protein substrates targets them for degradation by the UPS [44]. Mutations in genes required www.sciencedirect.com
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for polyubiquitination, such as the ubiquitin activating enzyme 1, Uba1, and in two subunits for the 19S proteosome regulatory particle, Mov34 and Rpn6, lead to defects in g MB pruning and, thus, provide a strong link between a protein degradation pathway and degenerative types of axon pruning [45]. Likewise, overexpression of a deubiquitinating enzyme, UBP2, in g MB neurons leads to a similar pruning defect. Importantly, activation of a cell death pathway is not involved, because mutations for apoptosis activators, grim, hid or rpr did not produce a pruning defect [45]. The UPS perhaps mediates axon branch elimination by targeting proteins that are necessary for branch stabilization or branch extension. One candidate, p190RhoGAP, is a GTPase activating protein that negatively regulates RhoA as a requirement for branch stabilization in a’/b’ MB neurons [46]. Activation of RhoA, its effector kinase Drok, and a key output for Drok, the regulatory phosphorylation of the myosin regulatory light chain, unveiled a pathway that induces branch elimination through regulation of myosin II activity [46]. The branch elimination pathway is probably dormant, because null mutants for RhoA and Drok lead to only subtle phenotypes, so the balance between activation and de-activation of the pathway depends largely on the activity of p190RhoGAP [46]. Because the pathway only affects branch stability in a’/b’ and not g MB neurons, it might be more important in other remodeling events in development. By contrast, g MB neurons are the only population to express Trio, a RacGEF, during peak stages of metamorphosis. Targeted DRacGAP loss-of-function in g MB neurons results in overextension of dorsal branches which is opposite to the phenotype observed by knocking out p190RhoGAP [47]. Thus, the balance between Rac and Rho GTPase activity might ultimately decide whether branch retraction or branch stabilization occurs. Not all remodeling events in Drosophila are associated with axon degeneration, however. In a genetic screen for molecules that affect synapse stability, Eaton et al. showed that disassembly of synapses and their axon branches at the NMJ of Drosophila proceeds by retraction [48]. Importantly, intact dynactin function was required for synapse stability. It will be interesting to see if future genetic screens on the same retraction pathway implicate similar molecules that have been shown to be required for degenerative g MB axon pruning.
Axon removal in pathology Wallerian degeneration and the Wlds mouse
In recent years, several lines of evidence have suggested that the molecular and cellular processes that regulate Wallerian degeneration are consistent with factors that are involved in degenerative modes of developmental axon pruning and a dying-back form of degeneration unique to chronic neuropathies. Wallerian degeneration www.sciencedirect.com
is a classical form of compartmental axon elimination that is initiated when an axon is cut in the CNS or PNS [49,50] (Figure 2b). The injured axon, which can conduct action potentials for a few days, is characterized by disassembly of the myelin sheath, disorganization of neurofilaments, swelling of mitochondria, and axonal thinning and membrane fragmentation that is accompanied by phagocytosis [50]. More recent studies have revealed, however, that transecting axons leads to a degenerative process that cannot be explained solely by classical Wallerian degeneration. An acute phase of degeneration occurs within minutes at the site of axonal injury [51]. During acute axonal degeneration (AAD; see glossary), injured portions of the cell-intact proximal axon and the detached distal axon segments break down into fragments in bi-directional fashion — an indication that perhaps some of the molecular mechanisms regulating AAD and Wallerian degeneration are shared. Indeed, a calpain inhibitor (see glossary) applied to transected axons effectively blocks cytoskeletal degradation usually seen in AAD and Wallerian degeneration [51]. After AAD, the proximal axon retracts but the cell body is left intact, whereas the isolated distal axon undergoes Wallerian degeneration. Why does axonal injury result in axon degeneration and not cell death? Some studies have suggested that axon degeneration and apoptosis are regulated by separate molecular pathways. Overexpression of human bcl-2, an inhibitor of apoptosis, in developing mouse retinal ganglion cells does not protect against axotomy-induced Wallerian degeneration but spares the neurons from cell death [52,53]. Additionally, the caspase family of cysteine proteases is normally activated during apoptosis but its activation is not observed in axotomized axons [54]. Likewise, blocking caspase activity does not affect the kinetics of Wallerian degeneration but apoptosis is markedly inhibited [54]. Apoptosis is also unaffected in a strain of mice in which Wallerian degeneration is slowed (Wlds). Interestingly, AAD is also slowed in Wlds mice but axon retraction occurs normally, indicating that perhaps axon retraction events are controlled by a separate process. The discovery of the Wlds mouse provided evidence that some forms of axon degeneration were regulated by an active process. Besides the protection from Wallerian degeneration, Wlds mice are phenotypically indistinguishable from their wild type counterparts. The Wlds gene was identified as an 85-kb tandem triplication that resulted in the overexpression of a chimeric protein: this protein included an intact nicotinamide adenine dinucleotide (NAD; see glossary) biosynthetic enzyme, a nicotinamide mononucleotide adnenylyl transferase (NMNAT; see glossary) fused to the amino-terminal 70 amino acids of Ufd2a, and an E4 ubiquitin ligase involved in ubiquitination [55–57]. Given that Wallerian degeneration and degenerative pruning in Drosophila could be slowed or inhibited by factors that disrupt UPS-mediated protein Current Opinion in Neurobiology 2005, 15:549–556
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degradation [58], it was not surprising that many studies focused on the potential neuroprotective effects of Ufd2a (see glossary). On the contrary, Araki et al. [59] discovered that it was not Ufd2a, but the activation of NMNAT and a downstream effector, SIRT1, a NAD-dependent histone deacetylase that has been shown to promote longevity in yeast and Caenorhabditis elegans, that mattered most. Interestingly, SIRT1 interacts with the tumor suppressor protein, p53, but other targets for SIRT1 need to be identified. When axons are locally deprived of nerve growth factor (NGF) from the targets they innervate, a degenerative process proceeds retrogradely from the axon terminal [60]. This ‘dying-back’ phenomenon is characteristic of many peripheral nerve diseases, and its progression is remarkably slowed in neurons that express the Wlds gene [61,62]. In Wlds neurons deprived of NGF, the cells die but the axons survive for up to a week [62]. Much like Wallerian degeneration, caspase activation is not seen in dying-back axons and the concurrent axon degeneration is not blocked by application of caspase inhibitors. The results suggest at the very least that ‘dying back’ degeneration and Wallerian degeneration share some of the same mechanisms. Given these results, in addition to the importance of the UPS in Wallerian degeneration [58], an attractive hypothesis is that SIRT1 might indirectly restrict the activity of molecules that promote axon degeneration. Future implications for studies on axon elimination in disease
Perhaps the most direct benefits of exploring mechanisms of axon elimination are its correlations with understanding axon pathology in neurological disease [63]. Dyingback axon degeneration (Figure 2c) is seen in many cases of chemical, metabolic and infectious insults on the nervous system. In the PNS, alcoholism, diabetes, AIDS and acrylamide poisoning all contribute to dying-back axon degeneration [63]. In Alzheimer’s, Parkinson’s and motor neuron diseases of the CNS, affected axons also degenerate accordingly (reviewed in [61,63]). Importantly, the WLDs gene protects axons in several mouse models in which axon degeneration is present, suggesting an important link between the processes that regulate Wallerian degeneration and the dying-back degeneration in chronic neuropathies [61,63].
Conclusions We have summarized some of the cellular and molecular processes associated with developmental axon elimination and experimentally induced axon degeneration. It seems that some molecular pathways are common in cases in which degeneration is the primary mode for axon elimination. In future studies, it will be important to address some of the following unresolved issues about axon elimination. First, are all forms of axon degeneration — Wallerian degeneration, degenerative axon pruning in Current Opinion in Neurobiology 2005, 15:549–556
development, NGF-deprived axon degeneration, and pathological degeneration in disease — regulated by the same molecular mechanism? Second, what molecular pathways activate axon retraction processes while suppressing degenerative ones? Third, and finally, is there a common link between pathways that regulate axon elimination through degeneration and those that regulate through retraction? The road ahead looks promising as more sophisticated techniques for visualizing cellular dynamics of axon elimination and for screening of related molecules that regulate the process become available.
Update
A recent study by Portera-Cailliau et al. [64] provides new insight into the development of axonal projections in the mouse cerebral cortex. By utilizing two-photon live imaging of GFP-expressing neurons that project axons to or within the cerebral cortex, the authors visualized the growth and pruning of long range axonal projections from thalamocortical (TC) neurons and much shorter axonal projections from the cortical interneurons of CajalRetzius (CR) cells in vivo. Importantly, it was found that developing axons can prune away their excess axons via either axonal degeneration or retraction. Axon retraction seems to be the preferred mechanism, because short range CR projections and long range TC projections prune most of their excess projections by retraction. Interestingly, axon pruning by axonal degeneration did occur, but it was only observed in the longer TC axons and occurred in only 5% of all pruning events. A recent report by Wang et al. [65] sheds new light on the axon-protective effects of NAD. Consistent with an earlier study by Araki et al. [59], the authors demonstrated that NAD levels are important for ensuring protection of the axon after axonal injury. NAD concentrations decrease after axonal injury, but the axon can be protected from axonal degeneration by applying exogenous NAD to prevent its levels from decreasing in the injured axons. Previously, Araki et al. [59] proposed that NAD levels were more important for regulating the activity of SIRT1 deacetylase in the nucleus. This was based on the evidence that exogenous NAD application did not offer any axonal protection when it was applied after axons were cut from the cell body; an axon protective signal from the nucleus was believed to be transduced from the nucleus to the axon. By contrast, Wang et al. [65] demonstrated that exogenous NAD applied at much higher concentrations than were used by Araki et al. [59] after an axon was cut could still protect the axon from degenerating — the result suggested that a local NAD protective effect was present. NAD levels might regulate SIRT1 activity in the cell nucleus as suggested by Araki et al. [59], but it is also an important coenzyme in ATP synthesis that might occur locally within the axon. In support of a local axon protective mechanism that was dependent on such bioenergetics, Wang et al. [65] www.sciencedirect.com
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showed that the application of exogenous pyruvate to prevent ATP depletion could protect cut axons as well.
18. Rosenthal JL, Taraskevich PS: Reduction in multiaxonal innervation at the neuromuscular junction of the rat during development. J Physiol 1977, 270:299-310.
Acknowledgements
19. Bixby JL: Ultrastructural observations on synapse elimination in neonatal rabbit skeletal muscle. J Neurocytol 1981, 10:81-100.
We thank X-B Liu, Z He, and members of the Cheng laboratory for helpful comments on the manuscript. This work was supported by grants from the National Institutes of Health (HD045757), University of California Davis MIND Institute, Whitehall Foundation, and fellowships from the Esther A. and Joseph Klingenstein Fund and the Alfred P. Sloan Foundation.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: of special interest of outstanding interest
20. Bishop DL, Misgeld T, Walsh MK, Gan WB, Lichtman JW: Axon branch removal at developing synapses by axosome shedding. Neuron 2004, 44:651-661. This study combines time-lapse imaging with electron microscopy of withdrawing terminal arbors at the NMJ. Interestingly, it was found that terminal arbors do not simply retract. Instead, while they are retracting, the distal ends of the axonal arbor are shed off as axosomes — nondegenerating membrane-bound structures that are sometimes filled with vesicles. The process is associated with glial cell activity, because it appears that glial cells are engulfing the axosomes. 21. Koirala S, Ko CP: Pruning an axon piece by piece: a new mode of synapse elimination. Neuron 2004, 44:578-580.
1.
Luo L, O’Leary DDM: Axon retraction and degeneration in development and disease. Annu Rev Neurosci 2005, 28:127-156.
22. Reddy LV, Koirala S, Sugiura Y, Herrera AA, Ko CP: Glial cells maintain synaptic structure and function and promote development of the neuromuscular junction in vivo. Neuron 2003, 40:563-580.
2.
Sanes JR, Lichtman JW: Development of the vertebrate neuromuscular junction. Annu Rev Neurosci 1999, 22:389-442.
23. Ito M: The modifiable neuronal network of the cerebellum. Jpn J Physiol 1984, 34:781-792.
3.
Nguyen QT, Lichtman JW: Mechanism of synapse disassembly at the developing neuromuscular junction. Curr Opin Neurobiol 1996, 6:104-112.
24. Mason CA, Gregory E: Postnatal maturation of cerebellar mossy and climbing fibers: transient expression of dual features on single axons. J Neurosci 1984, 4:1715-1735.
4.
Bernstein M, Lichtman JW: Axonal atrophy: the retraction reaction. Curr Opin Neurobiol 1999, 9:364-370.
5.
Lichtman JW, Colman H: Synapse elimination and indelible memory. Neuron 2000, 25:269-278.
25. Hashimoto K, Masanobu K: Functional differentiation of multiple climbing fiber inputs during synapse elimination in the developing cerebellum. Neuron 2003, 38:785-796.
6.
Balice-Gordon RJ, Lichtman JW: In vivo observations of preand postsynaptic changes during the transition from multiple to single innervation at developing neuromuscular junctions. J Neurosci 1993, 13:834-855.
7.
Colman H, Nabekura J, Lichtman JW: Alterations in synaptic strength preceding axon withdrawal. Science 1997, 275:356-361.
8.
Balice-Gordon RJ, Lichtman JW: Long-term synaptic loss induced by focal blockade of postsynaptic receptors. Nature 1994, 372:519-524.
9.
Gan WB, Lichtman JW: Synaptic segregation at the developing neuromuscular junction. Science 1998, 282:1508-1511.
10. Buffelli M, Burgess RW, Feng G, Lobe CG, Lichtman JW, Sanes JR: Genetic evidence that relative synaptic efficacy biases the outcome of synaptic competition. Nature 2003, 424:430-434. 11. Riley DA: Ultrastructural evidence for axon retraction during the spontaneous elimination of polyneuronal innervation of the rat soleus muscle. J Neurocytol 1981, 10:425-440. 12. Gan WB, Lichtman JW: Synaptic segregation at the developing neuromuscular junction. Science 1998, 282:1508-1511. 13. Ramon y Cajal S: Degeneration and regeneration of the nervous system. Translated and edited by May RM. Hafner Publishing; 1959. 14. Rossi F, Borsello T, Vaudano E, Strata P: Regressive modifications of climbing fibres following Purkinje cell degeneration in the cerebellar cortex of the adult rat. Neuroscience 1993, 53:759-778. 15. Marty S, Weinitz JM, Peschanski M: Target dependence of adult neurons: pattern of terminal arborizations. J Neurosci 1994, 14:5257-5266.
26. Eckenhoff MF, Pysh JJ: Double-walled coated vesicle formation: evidence for massive and transient conjugate internalization of plasma membranes during cerebellar development. J Neurocytol 1979, 8:623-638. 27. Innocenti GM: Growth and reshaping of axons in the establishment of visual callosal connections. Science 1981, 212:824-827. 28. O’Leary DDM, Bicknese AR, De Carlos JA, Heffner CD, Koester SE, Kutka LJ, Terashima T: Target selection by cortical axons: alternative mechanisms to establish axonal connections in the developing brain. Cold Spring Harb Symp Quant Biol 1990, 55:453-468. 29. O’Leary DDM: Remodelling of early axonal projections through the selective elimination of neurons and long axon collaterals. Ciba Found Symp 1987, 126:113-142. 30. Stanfield BB, O’Leary DDM, Fricks C: Selective collateral elimination in early postnatal development restricts cortical distribution of rat pyramidal tract neurones. Nature 1982, 298:371-373. 31. Stanfield BB, O’Leary DDM: The transient corticospinal projection from the occipital cortex during the postnatal development of the rat. J Comp Neurol 1985, 238:236-248. 32. Weimann JM, Zhang YA, Levin ME, Devine WP, Brulet P, McConnell SK: Cortical neurons require Otx1 for the refinement of exuberant axonal projections to subcortical targets. Neuron 1999, 24:819-831. 33. Reinoso BS, O’Leary DDM: Extension, branching and regression of visual corticospinal axons in rat. Soc Neurosci Abstr 1989, 15:1337. 34. McLaughlin T, Hindges R, O’Leary DDM: Regulation of axial patterning of the retina and its topographic mapping in the brain. Curr Opin Neurobiol 2003, 13:57-69.
16. Marty S, Peschanski M: Fine structural alteration in targetdeprived axonal terminals in the rat thalamus. Neuroscience 1994, 62:1121-1132.
35. McLaughlin T, Hindges R, Yates PA, O’Leary DDM: Bifunctional action of ephrin-B1 as a repellent and attractant to control bidirectional branch extension in dorsal-ventral retinotopic mapping. Development 2003, 130:2407-2418.
17. Pearson HE, Stoffler DJ, Sonstein WJ: Response of retinal terminals to loss of postsynaptic target neurons in the dorsal lateral geniculate nucleus of the adult cat. J Comp Neurol 1992, 315:333-343.
36. Yates PA, Roskies AL, McLaughlin T, O’Leary DDM: Topographic-specific axon branching controlled by ephrin-As is the critical event in retinotectal map development. J Neurosci 2001, 21:8548-8563.
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37. Nakamura H, O’Leary DDM: Inaccuracies in initial growth and arborization of chick retinotectal axons followed by course corrections and axon remodeling to develop topographic order. J Neurosci 1989, 9:3776-3795. 38. Bagri A, Cheng HJ, Yaron A, Pleasure SJ, Tessier-Lavigne M: Stereotyped pruning of long hippocampal axon branches triggered by retraction inducers of the semaphorin family. Cell 2003, 113:285-299.
52. Dubois-Dauphin M, Frankowski H, Tsujimoto Y, Huarte J, Martinou JC: Neonatal motoneurons overexpressing the bcl-2 protooncogene in transgenic mice are protected from axotomy-induced cell death. Proc Natl Acad Sci USA 1994, 91:3309-3313. 53. Burne JF, Staple JK, Raff MC: Glial cells are increased proportionally in transgenic optic nerves with increased numbers of axons. J Neurosci 1996, 16:2064-2073.
39. Sahay A, Molliver ME, Ginty DD, Kolodkin AL: Semaphorin-3F is critical for development of limbic system circuitry and is required in neurons for selective CNS axon guidance events. J Neurosci 2003, 23:6671-6680.
54. Finn JT, Weil M, Archer F, Simon R, Srinivasan A, Raff MC: Evidence that Wallerian degeneration and localized axon degeneration induced by local neurotrophin deprivation do not involve caspases. J Neurosci 2000, 20:1333-1341.
40. Adams B, Lee M, Fahnestock M, Racine RJ: Long-term potentiation trains induce mossy fiber sprouting. Brain Res 1997, 775:193-197.
55. Coleman MP, Conforti L, Buckmaster EA, Tarlton A, Ewing RM, Brown MC, Lyon MF, Perry VH: An 85-kb tandem triplication in the slow Wallerian degeneration (Wlds) mouse. Proc Natl Acad Sci USA 1998, 95:9985-9990.
41. Lee T, Marticke S, Sung C, Robinow S, Luo L: Cell-autonomous requirement of the USP/EcR-B ecdysone receptor for mushroom body neuronal remodeling in Drosophila. Neuron 2000, 28:807-818. 42. Watts RJ, Schuldiner O, Perrino J, Larsen C, Luo L: Glia engulf degenerating axons during developmental axon pruning. Curr Biol 2004, 14:678-684. This study provides evidence that stereotyped pruning of gamma mushroom body axons in Drosophila proceeds by axon degeneration. Axons destined for elimination were labeled using a clonal technique, mosaic analysis with a repressible cell marker (MARCM), and subsequently visualized under the electron microscope. It was found that axons degenerate during stereotyped pruning and the process is associated with glial cell activity. 43. Awasaki T, Ito K: Engulfing action of glial cells is required for programmed axon pruning during Drosophila metamorphosis. Curr Biol 2004, 14:668-677. This is a study published in parallel with the Watts et al. [42] work above. This study provides evidence that the engulfing activity of glial cells is required for mushroom body axon pruning. Furthermore, there is evidence suggesting an instructive role for pruning, because glial cell infiltration into areas occupied by mushroom body axons required intact ecdysone receptor signaling from gamma mushroom body neurons. 44. Voges D, Zwickl P, Baumeister W: The 26S proteosome: a molecular machine designed for controlled proteolysis. Annu Rev Biochem 1999, 68:1015-1068. 45. Watts RJ, Hoopfer ED, Luo L: Axon pruning during Drosophila metamorphosis: evidence for local degeneration and requirement of the ubiquitin-proteosome system. Neuron 2003, 38:871-885. 46. Billuart P, Winter CG, Maresh A, Zhao X, Luo L: Regulating axon branch stability: the role of p190 RhoGAP in repressing a retraction signaling pathway. Cell 2001, 107:195-207. 47. Awasaki T, Saito M, Sone M, Suzuki E, Sakai R, Ito K, Hama C: The Drosophila trio plays an essential role in patterning of axons by regulating their directional extension. Neuron 2000, 26:119-131. 48. Eaton BA, Fetter RD, Davis GW: Dynactin is necessary for synapse stabilization. Neuron 2002, 34:729-741. 49. Waller A: Experiments on the section of the glossopharyngeal and hypoglossal nerves of the frog, and observations on the alterations produced thereby in the structure of their primitive fibers. Philos Trans R Soc Lond B Biol Sci 1850, 140:423-429. 50. Gillingwater TH, Ribchester RR: Compartmental neurodegeneration and synaptic plasticity in the Wlds mutant mouse. J Physiol 2001, 534:627-639. 51. Kerschensteiner M, Schwab ME, Lichtman JW, Misgeld T: In vivo imaging of axonal degeneration and regeneration in the injured spinal cord. Nat Med 2005, 11:572-577. In this study, in vivo time-lapse imaging was performed on labeled dorsal root ganglion cell axons. When the axons were lesioned, time-lapse imaging revealed an acute form of axonal degeneration that affected areas proximal and distal to the lesion site. This acute axonal degeneration exhibited many of the same qualities as Wallerian degeneration.
Current Opinion in Neurobiology 2005, 15:549–556
56. Conforti L, Tarlton A, Mack TGA, Mi W, Buckmaster EA, Wagner D, Perry VH, Coleman MP: A Ufd2/D4Cole1e chimeric protein and overexpression of Rbp7 in the slow Wallerian degeneration (Wlds) mouse. Proc Natl Acad Sci USA 2000, 97:11377-11382. 57. Mack TGA, Reiner M, Beirowski B, Mi W, Emanuelli M, Wagner D, Thomson D, Gillingwater T, Court F, Conforti L et al.: Wallerian degeneration of injured axons and synapses is delayed by a Ube4b/Nmnat chimeric gene. Nat Neurosci 2001, 4:1199-1206. 58. Zhai Q, Wang J, Kim A, Liu Q, Watts R, Hoopfer E, Mitchison T, Luo L, He Z: Involvement of the ubiquitin-proteasome system in the early stages of Wallerian degeneration. Neuron 2003, 39:217-225. 59. Araki T, Sasaki Y, Milbrandt J: Increased nuclear NAD biosynthesis and SIRT1 activation prevent axonal degeneration. Science 2004, 305:1010-1013. This study identifies a molecular pathway that helps to explain the axon protective effects imposed by the mutation in Wlds mice. The Wlds mutation results in the overexpression of a chimeric fusion protein. It was shown that the principal component of the fusion protein is NMNAT1, an enzyme involved in NAD biosynthesis. In addition, the authors demonstrated that the axon protective effects depend on activation of an NADdependent histone deacetylase in the nucleus, SIRT1, which acts as a downstream effector of NMNAT1. 60. Campenot RB: Development of sympathetic neurons in compartmentalized cultures. II. Local control of neurite survival by nerve growth factor. Dev Biol 1982, 93:13-21. 61. Coleman MP, Perry VH: Axon pathology in neurological disease: a neglected therapeutic target. Trends Neurosci 2002, 25:532-537. 62. Deckwerth TL, Johnson EM Jr: Neurites can remain viable after destruction of the neuronal soma by programmed cell death (apoptosis). Dev Biol 1994, 165:63-72. 63. Raff MC, Whitmore AV, Finn JT: Axonal self-destruction and neurodegeneration. Science 2002, 296:868-871. 64. Portera-Cailliau C, Weimer RM, De Paola V, Caroni P, Svoboda K: Diverse modes of axon elaboration in the developing neocortex. PLoS Biol 2005, 3:e272 [Epub ahead of print]. In an elegant study using in vivo two-photon imaging of GFP-expressing neurons in the mouse CNS, the authors demonstrate that developing neurons possess two conserved mechanisms for remodeling their axons. Cajal-Retzius interneurons that have shorter axonal arbors in the neocortex and thalamocortical (TC) neurons that project long axons to the neocortex both have a preference for pruning their axonal branches by axon retraction. However, in 5% of all pruning events, axonal degeneration was also observed in the TC projections during axonal remodeling. 65. Wang J, Zhai Q, Chen Y, Lin E, Gu W, McBurney MW, He Z: A local mechanism mediates NAD-dependent protection of axon degeneration. J Cell Biol 2005 Jul 25; [Epub ahead of print]. A second study demonstrating the axon protective effects of NAD from axonal degeneration. In contrast to an earlier study by Araki et al. [59], the authors propose that NAD acts locally on a bioenergetic pathway in axons after they have been cut from the cell body. Consistent with this hypothesis, the authors showed that exogenous addition of ATP or pyruvate could also protect axons from degenerating.
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