- Email: [email protected]
Intrinsic neuronal regulation of axon and dendrite growth
Intrinsic neuronal regulation of axon and dendrite growth Jeffrey L Goldberg Neurons extend long axons and highly branched dendrites, and our understanding of the essential regulators of these processes has advanced in recent years. In the past year, investigators have shown that transcriptional control, posttranslational degradation and signaling cascades may be master regulators of axon and dendrite elongation and branching. Thus, evidence is mounting for the importance of the intrinsic growth state of a neuron as a crucial determinant of its ability to grow, or to regenerate, axons and dendrites. Addresses Department of Ophthalmology, McKnight Vision Research Center, Bascom Palmer Eye Institute, 1638 NW 10th Ave, Miami, Florida 33136, USA e-mail: [email protected]
Current Opinion in Neurobiology 2004, 14:551–557 This review comes from a themed issue on Neuronal and glial cell biology Edited by Silvia Arber and Rachel Wong Available online 12th September 2004 0959-4388/$ – see front matter # 2004 Elsevier Ltd. All rights reserved. DOI 10.1016/j.conb.2004.08.012
Abbreviations APC anaphase-promoting complex CaMK calmodulin kinase CNS central nervous system RGC retinal ganglion cell RNAi RNA interference
Introduction For over a century neurons have been defined by their morphology, yet we know little about how they extend their elaborate axons and dendrites. Investigating the regulation of axon growth is crucial to understanding why axons fail to regenerate in the central nervous system (CNS) after injury or in disease. How much is axon growth regulated intrinsically by the neuron versus extrinsically by the environment? Research has classically focused on identifying the extrinsic signals and receptors for neurite growth and guidance. Axon growth is not a default function of a surviving neuron, but must be specifically signaled [1–3]. Peptide trophic factors such as neurotrophins are typically the strongest extrinsic signals that are sufficient to induce axon and dendrite growth on their own, and extracellular matrix molecules such as laminin and heparin sulfate www.sciencedirect.com
proteoglycans, as well as cell adhesion molecules such as L1 and cadherins, promote axon growth in vitro and in vivo [4,5]. Furthermore, electrical activity initiated by presynaptic partners of a neuron has a significant role in shaping neurite and particularly dendrite outgrowth [6,7]. Thus, extrinsic signaling is clearly essential to the control of axon and dendrite outgrowth. Is the decision about whether to elongate axons or dendrites similarly determined extrinsically? A general hypothesis has been that, for a particular neuron, axon growth is supported by a one set of trophic factors and matrix molecules, and dendrite growth is supported by another set of signals (Figure 1a). For example, sympathetic, cortical and hippocampal neurons show dendritespecific growth in response to bone morphogenetic proteins [8–10], whereas rat sympathetic neurons that are supported only by nerve growth factor form axons but no dendrites [10]. Similarly, different matrix or adhesion molecules may preferentially support axon versus dendrite growth; for example, chick retinal ganglion cells (RGCs) cultured on top of glial endfeet grow mainly axons, whereas RGCs cultured on top of glial somata grow only dendrites [11]. Thus extrinsic signals can preferentially enhance axon or dendrite growth. What about the intrinsic state of the neuron? Studying the contribution of the intrinsic growth state of the neuron has been considerably harder, and has become confused in part because the word ‘intrinsic’ is often loosely substituted for other phrases such as ‘cell-autonomous’ or even ‘within the neuron’. Intrinsic must refer to a phenotype expressed independent of the environment, but because few model systems allow the study of neurons separated from glia and other cells, it has traditionally been difficult to investigate the intrinsic neurite growth ability of the neuron. Many intrinsic phenotypes are likely to be programmed in at the progenitor stages and maintained throughout part or all of the life of the neuron. At the most trivial level, the expression of a complement of receptors for survival, growth, and initial axon guidance is probably intrinsic to the neuron. For example, brainderived neurotrophic factor (BDNF) is able to elicit axon growth from retinal ganglion cells (RGCs). If BDNF is removed and then added back days later, RGCs remain responsive and begin to extend their axons again, demonstrating that responsiveness to BDNF is intrinsic to the RGCs [1]. Important differences between neighbouring neurons’ intrinsic responsiveness probably lead to the differences in their final patterning, as they are likely to experience very similar extrinsic environments. For example, brain-derived neurotrophic factor stimulates Current Opinion in Neurobiology 2004, 14:551–557
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Figure 1
Is axon versus dendrite growth a function of environment, a function of the intrinsic growth state of the neuron, or both? (a) In the extrinsic control hypothesis, axon-specific and dendrite-specific growth signals are presented to the neuron at different developmental windows or locations. (b) In the intrinsic control hypothesis, growth signals are available in general, and the neuron’s intrinsic growth state, either axonal or dendritic, determines which processes are preferentially responsive to extrinsic trophic signals. The ‘change in intrinsic state’ could come from an intrinsic, pre-programmed clock or aging mechanism, or could be elicited by an extrinsic signal, as for example amacrine cells are able to signal RGCs to decrease their intrinsic axon growth ability [26]. Note, however, that the amacrine signal does not have to be present in an ongoing fashion: once it signals RGCs to decrease their axon growth ability, they retain this new phenotype intrinsically. Thus, we hypothesize that trophic signals that are thought to be dendrite-specific growth promoters, that is, specifically stimulating the local outgrowth of dendritic but not axonal growth cones as in (a), could instead stimulate the neuron to change its intrinsic growth state as in (b).
cortical dendritic growth in layer 4 neurons but inhibits the growth of layer 6 neurons, whereas neurotrophin-3 has exactly the opposite effect [12]. Thus, the same trophic factors have different axon or dendrite promoting effects on different neurons, and these differences can be explained by the diverse intrinsic programs of trophic responsiveness of neurons.
Intrinsic regulation of axon and dendrite growth
For a given neuron, are such intrinsic programs static, or do they change with time? In vivo, for example, most CNS projection neurons first extend long axons and then greatly expand their dendritic arbors later in development, which suggests that axon growth and dendrite growth are separated at least partially in time. Must this be attributed to changes in extrinsic axon- or dendritepromoting signals, or could one neuron respond to the same extrinsic factors with either axon or dendrite growth, depending on changes in its own intrinsic growth state?
Work over recent years has clearly demonstrated that the intrinsic state of a neuron contributes to regulation of its axon growth ability. As mentioned above, neurons go through different periods of axon and dendrite growth in vivo. Further hints of intrinsic control have come from developmental studies in vivo demonstrating that embryonic spinal projection neurons can regenerate their axons after injury, whereas this ability is completely lost postnatally [13–15]. Similar data have been obtained in explant slice cultures, where young explants show better axon regeneration than do older explants [16,17]. Thus, it would appear that there is a developmental shift in the ability of neurons to regenerate their axons, but all of these studies are confounded by the simultaneous changes during development of the extrinsic glial environment, which can inhibit axon growth and regeneration [18,19].
In this review, I discuss recent evidence supporting the hypothesis that axon and dendrite growth is controlled intrinsically, and highlight exciting new data identifying potentially crucial intrinsic regulators of axon versus dendrite growth.
The lack of successful regeneration in experiments designed to overcome inhibitory CNS glial cues also hints at intrinsic axon growth regulation. Typically only a few per cent of axons regenerate and functional recovery proceeds very slowly [20–22]. For example, adult RGCs
Current Opinion in Neurobiology 2004, 14:551–557
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Intrinsic neuronal regulation of axon and dendrite growth Goldberg
take 2 months to regenerate through a peripheral nerve graft [23,24]. Similarly, eliminating oligodendrocytes and myelin from cerebellar slice cultures does not facilitate axon regeneration by Purkinje neurons [25]. As above, however, these experiments are confounded by the possible contribution of residual axon growth inhibitors, such as reactive astrocytes. Thus, it is difficult to address whether the developmental changes in axon growth ability both in vivo and in explant cultures are intrinsic to the neurons or are attributable to the changing environment. By separating CNS neurons away from CNS glia, however, recent data has definitively demonstrated that the intrinsic growth state of a neuron can dictate its axon and dendrite growth ability independently of the environmental milieu. Neonatal RGCs undergo a profound, irreversible loss in their intrinsic ability to extend axons rapidly [26]. In purified cultures without any CNS glia, at clonal density, and in defined growth media containing strongly growth promoting neurotrophic factors, embryonic RGCs extend axons up to ten times faster than do postnatal RGCs. This difference in axon growth ability is maintained in various trophic environments, including in response to neurotrophic factors or signals from visual pathway cells, and in vivo after transplantation into developing white matter pathways. These data suggest that the extrinsic growth-promoting environment is limited by an intrinsic maximum axon growth rate. Furthermore, purified embryonic RGCs that have been cultured for 10 days retain their fast growth rate and, similarly, purified postnatal RGCs continue to grow axons very slowly after 10 days in culture. Thus, RGC growth rates in vitro are not merely the result of an extrinsic signal to promote or inhibit axon growth that the RGCs were exposed to in vivo and carried into culture. Thus, RGCs exhibit a developmental change in their intrinsic axon growth ability that is independent of the environment and is exhibited in a variety of signaling milieus. These data also address whether this change in intrinsic growth ability of RGCs occurs by intrinsic aging, as if a clock ran out, or by a signaled event: the fact that embryonic RGCs do not lose their axon growth ability in purified cultures demonstrates that they are signaled by an extrinsic cue to decrease their axon growth ability. Interestingly, amacrine cells, which are presynaptic to RGCs in the retina, are sufficient to signal embryonic RGCs to decrease their axon growth ability [26]. Thus, RGCs can be induced by amacrine cells to decrease their intrinsic axon growth ability but, once they receive this signal, their poor axon growth ability is not dependent on ongoing signaling by neighboring cell types but rather is intrinsically maintained. Is the control of dendrite growth ability subject to similar intrinsic regulation? Remarkably, at about the same time www.sciencedirect.com
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that neonatal RGCs lose their ability to elongate axons, they gain the ability to generate dendrites rapidly [26]. Whether the increase in dendrite growth ability and the decrease in axon growth ability are mechanistically controlled by a single switch rather than by two concurrent phenotypic changes is not yet known, but the dendritic switch is also induced by a retinal cue [26]. These data from RGCs raise the hypothesis that, for other neurons, some of the signals that seem to stimulate dendrite but not axon growth may rather activate an intrinsic dendritic growth mode, after which the neurons extend dendrites in response to growth signals that are already present (Figure 1b). So far, axons and dendrites have been found to respond to many of the same growth and guidance signals [12,27,28]. Thus, the regulation of axon versus dendrite growth has a significant intrinsic component and is not determined solely by the presence of distinct extracellular cues.
Molecular mechanisms for regulating intrinsic growth state Transcriptional programming of intrinsic states
What molecular mechanisms could regulate the axonal or dendritic growth mode of a neuron? If properly regulated, almost any molecule in the cell could be crucial to determining axon versus dendrite growth [29]. It is attractive to discuss first transcription factors that could activate the expression of whole programs of axon or dendrite growth, and could also maintain an intrinsic phenotype in the absence of ongoing extrinsic signaling. In RGCs, the amacrine membrane-induced switch from fast to slower axon growth ability relies on new gene transcription, as it is blocked by actinomycin D (JL Goldberg, BA Barres, unpublished data). Genetic screens in Drosophila have been fruitful in yielding intrinsic, or at least cell-autonomous, determinants of neurite growth and branching [30,31]. In one set of experiments, for example, the homeodomain protein Cut has been shown to confer a dose-dependent increase in dendritic branching complexity and total length in a particular subset of peripheral sensory neurons [31]. Whether such molecules, which were discovered in Drosophila, will translate directly to the control of axon or dendrite growth in mammalian neurons remains to be shown, but the principle of cell-fate-determining transcription factors that control neurite outgrowth ability is certainly conserved. For example, the POU domain transcription factor Brn3b is essential for RGC development. In Brn3b / mice, RGCs fail to extend axons properly and 80% die before birth; in addition, Brn3b / RGCs in culture extend only rudimentary axons and instead elaborate abnormal, dendrite-like processes [32–36]. RGCs also express the related family members Brn3a and Brn3c: about 50% of RGCs express both Brn3b and Brn3c. The recently generated double knockout Brn3b / Brn3c / shows an almost total loss of axon outgrowth from explanted Current Opinion in Neurobiology 2004, 14:551–557
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retinas, suggesting that both Brn3b and Brn3c contribute to controlling RGC axon growth [37]. In these experiments, the only neurons that were found to elaborate neurites were those that did not express the alkaline phosphatase marker from the mutated Brn3c locus, which suggests that, in RGCs that do not normally express Brn3c, alternative transcription factors drive axon outgrowth [37]. Interestingly, in Brn3a knockout mice, sensory neurons demonstrate axon guidance and branching defects in vivo, and then undergo early cell death, analogous to the Brn3b effects in RGCs discussed above [38]. Thus, transcription factors involved in cell fate determination, which is often defined by cell morphology, may act directly via the control of axon and dendrite growth ability. What are the genes downstream of Brn3b that could effect changes in the intrinsic axon growth state of a neuron? Recently, Brn3b / retinas were compared with wild type retinas in microarray experiments to determine which genes depend on Brn3b for their expression [39]. Of interest, several genes linked to axon guidance were found to be downregulated in Brn3b / retinas. Two of the genes that were most strongly downregulated in the knockouts [39] were Sonic hedgehog, which has a role in axon guidance but not in axon elongation in the embryonic spinal cord [40], and persyn (also known as g-synuclein), a protein that is associated with neurofilaments [41]. a-Synuclein and b-synuclein seem to decrease neurite outgrowth in a rat neuronal cell line [42], however, raising the question of whether g-synuclein is indeed a pro-axon growth effector. Recently, the nerve growth factor (NGF) receptor trkA was identified as a transcriptional target of Brn3a, [43] providing a direct link from transcription factor expression to trophic responsiveness. In addition, these experiments point to the power of using focused microarray experiments in relation to established phenotypic models. For example, our research group is currently investigating which genes show altered expression during RGC development and thus may underlie the developmental loss of intrinsic axon growth ability in RGCs [5]. In some cases, it will be difficult to separate intrinsic neurite growth regulation from growth regulation directly induced by extrinsic signals. Recently, the Ca2+-dependent nuclear transactivator CREST has been cloned and found to regulate cortical and hippocampal dendrite development [44]. In CREST-deficient mice, dendritic arborization is severely compromised in vivo, and depolarization-dependent dendritic growth is similarly diminished in vitro. The downstream targets of CREST, as well as whether CREST deficiency causes an axonal phenotype, remain unknown. It is intriguing to propose that CREST regulation may be developmentally regulated by activity induced by specific presynaptic partners, and may switch the neuron into a dendritic growth mode to continue wiring presynaptically. Thus, CREST is a transcripCurrent Opinion in Neurobiology 2004, 14:551–557
tion factor whose activity is necessary for proper dendrite growth and development [44]. Is CREST expression regulated developmentally or in response to contact with neighboring cells? Determining how its expression is controlled will be essential to further elucidating any role in intrinsic dendrite growth ability. Posttranslational degradation mechanisms
Transcription factors are not the only master regulators of a cell. Levels of specific complements of proteins can be regulated posttranslationally by ubiquitination and degradation. Because there are hundreds of ubiquitin ligases in mammalian genomes, the potential for subcellular regulation of the levels of specific proteins is vast. The importance of ubiquitin-mediated degradation has been described for the normal axon degeneration that occurs during Drosophila development [45] or Wallerian degeneration after injury [46], as well as for axon guidance in primary neurons in vitro [47]; but what about its importance for axon elongation? Recently, Bonni and colleagues [48] have shown that the ubiquitin ligase anaphase-promoting complex (APC) has a significant role in regulating the axon growth rate of cerebellar granule neurons. APC was originally found to be essential for mitosis in dividing cells, but it is also expressed highly in postmitotic neurons. Using small hairpin constructs, Bonni and colleagues [48] inhibited the expression of Cdh1, which normally activates APC, in primary cultures of postnatal day 6 cerebellar granule neurons. RNA interference (RNAi)-mediated inhibition of Cdh1 led to a greater than twofold increase in axon length but not to a change in dendrite length, suggesting that Cdh1–APC complexes normally inhibit axon but not dendrite growth [48]. Similarly, an increase in axon growth was obtained when these researchers overexpressed a dominant-negative variant of APC or overexpressed the wild type form of an endogenous APC inhibitor, Emi1 [48]. Thus, Cdh1– APC is a potential candidate for a master regulator of a broader program that specifically controls the rate of axon, but not dendrite, growth. Cdh1–APC complexes were found in cerebellar granule neuron nuclei [48], but the substrates that are ubiquitinated by APC and regulate axon growth in these neurons remain to be identified. Second messengers and signaling cascades
How does the intrinsic state of neuron allow the same extrinsic cues to induce axon or dendrite growth preferentially (Figure 1b)? Cytoplasmic kinases or phosphatases may provide an axon- or dendrite-specific interpretation of extrinsic cues. For example, electrical activity seems to be crucial to potentiate both axon and dendrite growth in various neurons in response to trophic signals [6,29]. The influx of Ca2+ during depolarization activates a family of Ca2+-dependent signaling kinases, calmodulin www.sciencedirect.com
Intrinsic neuronal regulation of axon and dendrite growth Goldberg
kinases (CaMKs), which have now been implicated in both axon- and dendrite-specific growth. For axon growth, overexpression of a dominant-negative variant of CaMKI in embryonic hippocampal and postnatal cerebellar granule neurons decreases axon outgrowth [49], whereas expression of cytosolic dominant-negative CaMKII or nuclear dominant-negative CaMKIV constructs has no effect on axon growth in these cells. In other studies, CaMKII-b and nuclear CaMKIV have been found to stimulate dendrite but not axon growth in hippocampal or cortical cultures [50,51]. CaMKII is particularly interesting because its a-isoform has been long associated with the regulation of synaptic plasticity. By means of a b-isoform-specific actin-binding domain, however, CaMKII-b is able to increase dendrite motility and branching in hippocampal neurons [50]. Intriguingly, CaMKI is localized throughout the cytoplasm of all hippocampal neurites [49], raising issues of its role in dendrites and whether its localization or activation might become more axon-specific in vivo and in longer-lasting cultures. Thus, CaMK isoforms can differentially control axon- and dendrite-specific outgrowth in hippocampal neurons. Further experiments will hopefully point to mechanisms of either developmental or subcellular regulation, or both, for these kinases. How does the Ca2+-mediated activation of particular CaMKs lead to a program of specific axon or dendrite growth? Remarkably, CaMKII seems to activate another transcription factor involved in cell fate determination — the basic helix–loop–helix factor NeuroD [52]. Like APC, NeuroD is expressed in postmitotic neurons and has been recently shown to be phosphorylated by CaMKII in response to activity. Downregulation of NeuroD by RNAi in cerebellar granule neurons leads to a suppression of dendrite growth, but has no effect on axon growth either in dissociated cultures or in cerebellar slice explants [52]. Thus, the activation of dendrite-growthpromoting kinases leads to the activation of dendritegrowth-promoting transcription factors without activating the axon growth program of a neuron. Whether NeuroD interacts with CREST or activates an overlapping series of target genes will prove very interesting. The levels of second messengers such as cAMP and cGMP, if intrinsically maintained, could affect axon and dendrite growth differentially. Early indications of the importance of these second messengers came from studies on axon guidance molecules. For example, the attraction of growth cones towards a gradient of brainderived neurotrophic factor is dependent on cAMP [53]. Adequate levels of cAMP are crucial for RGCs to extend axons in response to neurotrophic factors [1], and the developmental decrease in cAMP levels has been proposed to underlie the developmental loss of axon regeneration in the presence of inhibitory myelin-associated www.sciencedirect.com
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cues [54]. In a strongly trophic, non-inhibitory environment, however, an increase in cAMP does not revert the slow axon growth of postnatal RGCs to the fast growth of their embryonic state [26]. Similarly, increases in cGMP convert the repulsion of growth cones away from point sources of semaphorin 3A or myelin-associated glycoprotein into attraction [55]. Could cGMP differentially affect axons and dendrites? There is already evidence for this effect in vivo: guanylate cyclase is asymmetrically localized to the apical dendrite in developing cortical pyramidal neurons, and apical sources of sema3A, which repel cortical axons basally, instead attract the dendrite apically [27]. Thus, the levels of cyclic nucleotides may provide another generalized mechanism for flipping the intrinsic growth state of a neuron, although further work that can dissociate the effects of these second messengers on the neuron’s intrinsic axon growth state from their effects on the axon’s response to extrinsic trophic or inhibitory signals will be crucial.
Conclusions: joining intrinsic and extrinsic control Thus, I propose here that the intrinsic growth state of the neuron is a crucial regulator of axon and dendrite elongation, and that extrinsic signals yet to be identified can stably change the intrinsic ability of a neuron to extend neurites during development. This change in intrinsic growth ability could be expressed at many different levels in the cell, but is likely to rely at least in part on altered gene expression. Further exploration of the intrinsic and extrinsic control of axon growth is essential, not only for our understanding of neuronal development but also for enhancing axon growth after injury or in disease. Attempts to address both issues simultaneously have proved more successful in enhancing regeneration in vivo. For example, macrophage-derived trophic factors can increase the regeneration of RGCs in the optic nerve, and this extrinsic enhancement of axon growth allows greater regeneration when combined with the expression of dominant-negative Nogo receptor constructs to decrease intrinsic responsiveness of the RGCs to the inhibitory Nogo protein [56]. Are these macrophagederived trophic factors switching the growth state of the RGC or simply providing a novel trophic support that is normally missing after injury in vivo? The identification of these factors and their further study may provide answers to this question. A stimulating final question is, can manipulating the intrinsic axon growth ability of neurons make their axons ignore extrinsic regulators? At first glance, the increase in axon growth seen after suppressing Cdh1–APC activity with RNAi was mirrored by an increase in axon growth on myelin substrates, which normally inhibit axon growth [48]. Neurons with suppressed Cdh1 elaborated axons Current Opinion in Neurobiology 2004, 14:551–557
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that were threefold longer on myelin substrates than those elaborated by control neurons on myelin; however, the axons still grew only half as fast as those of Cdh1suppressed neurons on control substrates [48]. These data suggest that activating a more rapid intrinsic axon growth program does not make neurons insensitive to extrinsic inhibitory signals, but rather they exemplify the point that both the intrinsic neuronal growth state and the extrinsic environment may have to be optimized for successful regeneration after injury.
Acknowledgements Thanks to members of the lab for helpful discussions throughout the writing process.
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
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36. Erkman L, McEvilly RJ, Luo L, Ryan AK, Hooshmand F, O’Connell SM, Keithley EM, Rapaport DH, Ryan AF, Rosenfeld MG: Role of transcription factors Brn-3.1 and Brn-3.2 in auditory and visual system development. Nature 1996, 381:603-606. 37. Wang SW, Mu X, Bowers WJ, Kim DS, Plas DJ, Crair MC, Federoff HJ, Gan L, Klein WH: Brn3b/Brn3c double knockout mice reveal an unsuspected role for Brn3c in retinal ganglion cell axon outgrowth. Development 2002, 129:467-477. 38. Eng SR, Gratwick K, Rhee JM, Fedtsova N, Gan L, Turner EE: Defects in sensory axon growth precede neuronal death in Brn3a-deficient mice. J Neurosci 2001, 21:541-549. 39. Mu X, Beremand PD, Zhao S, Pershad R, Sun H, Scarpa A, Liang S, Thomas TL, Klein WH: Discrete gene sets depend on POU domain transcription factor Brn3b/Brn-3.2/POU4f2 for their expression in the mouse embryonic retina. Development 2004, 131:1197-1210. The authors compare wild type and Brn3b knockout retinas by using microarrays to investigate which genes depend on Brn3b for expression, and confirm their results by using polymerase chain reaction with reverse transcription and in situ hybridization. Of 18 816 probes, only 87 genes differ in expression (being downregulated in general), but this short list contains interesting candidates for explaining the paucity of axon growth seen in the knockouts. 40. Charron F, Stein E, Jeong J, McMahon AP, Tessier-Lavigne M: The morphogen sonic hedgehog is an axonal chemoattractant that collaborates with netrin-1 in midline axon guidance. Cell 2003, 113:11-23. 41. Buchman VL, Adu J, Pinon LG, Ninkina NN, Davies AM: Persyn, a member of the synuclein family, influences neurofilament network integrity. Nat Neurosci 1998, 1:101-103. 42. Takenouchi T, Hashimoto M, Hsu LJ, Mackowski B, Rockenstein E, Mallory M, Masliah E: Reduced neuritic outgrowth and cell adhesion in neuronal cells transfected with human asynuclein. Mol Cell Neurosci 2001, 17:141-150. 43. Ma L, Lei L, Eng SR, Turner E, Parada LF: Brn3a regulation of TrkA/NGF receptor expression in developing sensory neurons. Development 2003, 130:3525-3534. 44. Aizawa H, Hu SC, Bobb K, Balakrishnan K, Ince G, Gurevich I, Cowan M, Ghosh A: Dendrite development regulated by CREST, a calcium-regulated transcriptional activator. Science 2004, 303:197-202. By using a variation on one-hybrid screens to search for transcription factors activated by depolarization, the authors identify a novel Ca2+activated transcription factor, CREST. They show both that CREST binds to cAMP response element-binding protein (CBP) and that CREST mutants extend fewer, shorter dendrites in vitro and in vivo, definitively linking this transcription factor to activity-dependent dendrite growth.
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47. Campbell DS, Holt CE: Chemotropic responses of retinal growth cones mediated by rapid local protein synthesis and degradation. Neuron 2001, 32:1013-1026. 48. Konishi Y, Stegmuller J, Matsuda T, Bonni S, Bonni A: Cdh1–APC controls axonal growth and patterning in the mammalian brain. Science 2004, 303:1026-1030. The authors demonstrate that the nuclear ubiquitin ligase anaphasepromoting complex specifically enhances the growth of axons but not dendrites of CNS neurons in both dissociated cultures and slice explants. 49. Wayman GA, Kaech S, Grant WF, Davare M, Impey S, Tokumitsu H, Nozaki N, Banker G, Soderling TR: Regulation of axonal extension and growth cone motility by calmodulindependent protein kinase I. J Neurosci 2004, 24:3786-3794. See annotation to [50]. 50. Fink CC, Bayer KU, Myers JW, Ferrell JE Jr, Schulman H, Meyer T: Selective regulation of neurite extension and synapse formation by the b but not the a isoform of CaMKII. Neuron 2003, 39:283-297. These two papers [49,50], together with [51], divide up axon- and dendrite-specific roles for the various members of the CaMK family. Specifically, by using various genetic and pharmacological inhibitors to modulate CaMK activities in primary hippocampal, cerebellar granule and cortical neurons in culture, the authors demonstrate that CaMKI promotes axon growth [49], cytosolic CaMKII-b promotes dendrite elongation and branching [50], and CaMKIV promotes dendrite growth via activation of specific transcription factors [51]. 51. Redmond L, Kashani AH, Ghosh A: Calcium regulation of dendritic growth via CaM kinase IV and CREB-mediated transcription. Neuron 2002, 34:999-1010. 52. Gaudilliere B, Konishi Y, de la Iglesia N, Yao G, Bonni A: A CaMKII–NeuroD signaling pathway specifies dendritic morphogenesis. Neuron 2004, 41:229-241. Bringing the story full circle from specific kinases to specific transcription factors, the authors show that the transcription factor NeuroD is necessary for normal dendrite growth in primary neurons, and that activation of NeuroD relies on CaMKII. 53. Song HJ, Ming GL, Poo MM: cAMP-induced switching in turning direction of nerve growth cones. Nature 1997, 388:275-279. 54. Cai D, Qiu J, Cao Z, McAtee M, Bregman BS, Filbin MT: Neuronal cyclic AMP controls the developmental loss in ability of axons to regenerate. J Neurosci 2001, 21:4731-4739. 55. Song H, Ming G, He Z, Lehmann M, McKerracher L, Tessier-Lavigne M, Poo M: Conversion of neuronal growth cone responses from repulsion to attraction by cyclic nucleotides. Science 1998, 281:1515-1518. 56. Fischer D, He Z, Benowitz LI: Counteracting the Nogo receptor enhances optic nerve regeneration if retinal ganglion cells are in an active growth state. J Neurosci 2004, 24:1646-1651.
Current Opinion in Neurobiology 2004, 14:551–557
Current Opinion in Neurobiology 2004, 14:551–557 This review comes from a themed issue on Neuronal and glial cell biology Edited by Silvia Arber and Rachel Wong Available online 12th September 2004 0959-4388/$ – see front matter # 2004 Elsevier Ltd. All rights reserved. DOI 10.1016/j.conb.2004.08.012
Abbreviations APC anaphase-promoting complex CaMK calmodulin kinase CNS central nervous system RGC retinal ganglion cell RNAi RNA interference
Introduction For over a century neurons have been defined by their morphology, yet we know little about how they extend their elaborate axons and dendrites. Investigating the regulation of axon growth is crucial to understanding why axons fail to regenerate in the central nervous system (CNS) after injury or in disease. How much is axon growth regulated intrinsically by the neuron versus extrinsically by the environment? Research has classically focused on identifying the extrinsic signals and receptors for neurite growth and guidance. Axon growth is not a default function of a surviving neuron, but must be specifically signaled [1–3]. Peptide trophic factors such as neurotrophins are typically the strongest extrinsic signals that are sufficient to induce axon and dendrite growth on their own, and extracellular matrix molecules such as laminin and heparin sulfate www.sciencedirect.com
proteoglycans, as well as cell adhesion molecules such as L1 and cadherins, promote axon growth in vitro and in vivo [4,5]. Furthermore, electrical activity initiated by presynaptic partners of a neuron has a significant role in shaping neurite and particularly dendrite outgrowth [6,7]. Thus, extrinsic signaling is clearly essential to the control of axon and dendrite outgrowth. Is the decision about whether to elongate axons or dendrites similarly determined extrinsically? A general hypothesis has been that, for a particular neuron, axon growth is supported by a one set of trophic factors and matrix molecules, and dendrite growth is supported by another set of signals (Figure 1a). For example, sympathetic, cortical and hippocampal neurons show dendritespecific growth in response to bone morphogenetic proteins [8–10], whereas rat sympathetic neurons that are supported only by nerve growth factor form axons but no dendrites [10]. Similarly, different matrix or adhesion molecules may preferentially support axon versus dendrite growth; for example, chick retinal ganglion cells (RGCs) cultured on top of glial endfeet grow mainly axons, whereas RGCs cultured on top of glial somata grow only dendrites [11]. Thus extrinsic signals can preferentially enhance axon or dendrite growth. What about the intrinsic state of the neuron? Studying the contribution of the intrinsic growth state of the neuron has been considerably harder, and has become confused in part because the word ‘intrinsic’ is often loosely substituted for other phrases such as ‘cell-autonomous’ or even ‘within the neuron’. Intrinsic must refer to a phenotype expressed independent of the environment, but because few model systems allow the study of neurons separated from glia and other cells, it has traditionally been difficult to investigate the intrinsic neurite growth ability of the neuron. Many intrinsic phenotypes are likely to be programmed in at the progenitor stages and maintained throughout part or all of the life of the neuron. At the most trivial level, the expression of a complement of receptors for survival, growth, and initial axon guidance is probably intrinsic to the neuron. For example, brainderived neurotrophic factor (BDNF) is able to elicit axon growth from retinal ganglion cells (RGCs). If BDNF is removed and then added back days later, RGCs remain responsive and begin to extend their axons again, demonstrating that responsiveness to BDNF is intrinsic to the RGCs [1]. Important differences between neighbouring neurons’ intrinsic responsiveness probably lead to the differences in their final patterning, as they are likely to experience very similar extrinsic environments. For example, brain-derived neurotrophic factor stimulates Current Opinion in Neurobiology 2004, 14:551–557
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Figure 1
Is axon versus dendrite growth a function of environment, a function of the intrinsic growth state of the neuron, or both? (a) In the extrinsic control hypothesis, axon-specific and dendrite-specific growth signals are presented to the neuron at different developmental windows or locations. (b) In the intrinsic control hypothesis, growth signals are available in general, and the neuron’s intrinsic growth state, either axonal or dendritic, determines which processes are preferentially responsive to extrinsic trophic signals. The ‘change in intrinsic state’ could come from an intrinsic, pre-programmed clock or aging mechanism, or could be elicited by an extrinsic signal, as for example amacrine cells are able to signal RGCs to decrease their intrinsic axon growth ability [26]. Note, however, that the amacrine signal does not have to be present in an ongoing fashion: once it signals RGCs to decrease their axon growth ability, they retain this new phenotype intrinsically. Thus, we hypothesize that trophic signals that are thought to be dendrite-specific growth promoters, that is, specifically stimulating the local outgrowth of dendritic but not axonal growth cones as in (a), could instead stimulate the neuron to change its intrinsic growth state as in (b).
cortical dendritic growth in layer 4 neurons but inhibits the growth of layer 6 neurons, whereas neurotrophin-3 has exactly the opposite effect [12]. Thus, the same trophic factors have different axon or dendrite promoting effects on different neurons, and these differences can be explained by the diverse intrinsic programs of trophic responsiveness of neurons.
Intrinsic regulation of axon and dendrite growth
For a given neuron, are such intrinsic programs static, or do they change with time? In vivo, for example, most CNS projection neurons first extend long axons and then greatly expand their dendritic arbors later in development, which suggests that axon growth and dendrite growth are separated at least partially in time. Must this be attributed to changes in extrinsic axon- or dendritepromoting signals, or could one neuron respond to the same extrinsic factors with either axon or dendrite growth, depending on changes in its own intrinsic growth state?
Work over recent years has clearly demonstrated that the intrinsic state of a neuron contributes to regulation of its axon growth ability. As mentioned above, neurons go through different periods of axon and dendrite growth in vivo. Further hints of intrinsic control have come from developmental studies in vivo demonstrating that embryonic spinal projection neurons can regenerate their axons after injury, whereas this ability is completely lost postnatally [13–15]. Similar data have been obtained in explant slice cultures, where young explants show better axon regeneration than do older explants [16,17]. Thus, it would appear that there is a developmental shift in the ability of neurons to regenerate their axons, but all of these studies are confounded by the simultaneous changes during development of the extrinsic glial environment, which can inhibit axon growth and regeneration [18,19].
In this review, I discuss recent evidence supporting the hypothesis that axon and dendrite growth is controlled intrinsically, and highlight exciting new data identifying potentially crucial intrinsic regulators of axon versus dendrite growth.
The lack of successful regeneration in experiments designed to overcome inhibitory CNS glial cues also hints at intrinsic axon growth regulation. Typically only a few per cent of axons regenerate and functional recovery proceeds very slowly [20–22]. For example, adult RGCs
Current Opinion in Neurobiology 2004, 14:551–557
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Intrinsic neuronal regulation of axon and dendrite growth Goldberg
take 2 months to regenerate through a peripheral nerve graft [23,24]. Similarly, eliminating oligodendrocytes and myelin from cerebellar slice cultures does not facilitate axon regeneration by Purkinje neurons [25]. As above, however, these experiments are confounded by the possible contribution of residual axon growth inhibitors, such as reactive astrocytes. Thus, it is difficult to address whether the developmental changes in axon growth ability both in vivo and in explant cultures are intrinsic to the neurons or are attributable to the changing environment. By separating CNS neurons away from CNS glia, however, recent data has definitively demonstrated that the intrinsic growth state of a neuron can dictate its axon and dendrite growth ability independently of the environmental milieu. Neonatal RGCs undergo a profound, irreversible loss in their intrinsic ability to extend axons rapidly [26]. In purified cultures without any CNS glia, at clonal density, and in defined growth media containing strongly growth promoting neurotrophic factors, embryonic RGCs extend axons up to ten times faster than do postnatal RGCs. This difference in axon growth ability is maintained in various trophic environments, including in response to neurotrophic factors or signals from visual pathway cells, and in vivo after transplantation into developing white matter pathways. These data suggest that the extrinsic growth-promoting environment is limited by an intrinsic maximum axon growth rate. Furthermore, purified embryonic RGCs that have been cultured for 10 days retain their fast growth rate and, similarly, purified postnatal RGCs continue to grow axons very slowly after 10 days in culture. Thus, RGC growth rates in vitro are not merely the result of an extrinsic signal to promote or inhibit axon growth that the RGCs were exposed to in vivo and carried into culture. Thus, RGCs exhibit a developmental change in their intrinsic axon growth ability that is independent of the environment and is exhibited in a variety of signaling milieus. These data also address whether this change in intrinsic growth ability of RGCs occurs by intrinsic aging, as if a clock ran out, or by a signaled event: the fact that embryonic RGCs do not lose their axon growth ability in purified cultures demonstrates that they are signaled by an extrinsic cue to decrease their axon growth ability. Interestingly, amacrine cells, which are presynaptic to RGCs in the retina, are sufficient to signal embryonic RGCs to decrease their axon growth ability [26]. Thus, RGCs can be induced by amacrine cells to decrease their intrinsic axon growth ability but, once they receive this signal, their poor axon growth ability is not dependent on ongoing signaling by neighboring cell types but rather is intrinsically maintained. Is the control of dendrite growth ability subject to similar intrinsic regulation? Remarkably, at about the same time www.sciencedirect.com
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that neonatal RGCs lose their ability to elongate axons, they gain the ability to generate dendrites rapidly [26]. Whether the increase in dendrite growth ability and the decrease in axon growth ability are mechanistically controlled by a single switch rather than by two concurrent phenotypic changes is not yet known, but the dendritic switch is also induced by a retinal cue [26]. These data from RGCs raise the hypothesis that, for other neurons, some of the signals that seem to stimulate dendrite but not axon growth may rather activate an intrinsic dendritic growth mode, after which the neurons extend dendrites in response to growth signals that are already present (Figure 1b). So far, axons and dendrites have been found to respond to many of the same growth and guidance signals [12,27,28]. Thus, the regulation of axon versus dendrite growth has a significant intrinsic component and is not determined solely by the presence of distinct extracellular cues.
Molecular mechanisms for regulating intrinsic growth state Transcriptional programming of intrinsic states
What molecular mechanisms could regulate the axonal or dendritic growth mode of a neuron? If properly regulated, almost any molecule in the cell could be crucial to determining axon versus dendrite growth [29]. It is attractive to discuss first transcription factors that could activate the expression of whole programs of axon or dendrite growth, and could also maintain an intrinsic phenotype in the absence of ongoing extrinsic signaling. In RGCs, the amacrine membrane-induced switch from fast to slower axon growth ability relies on new gene transcription, as it is blocked by actinomycin D (JL Goldberg, BA Barres, unpublished data). Genetic screens in Drosophila have been fruitful in yielding intrinsic, or at least cell-autonomous, determinants of neurite growth and branching [30,31]. In one set of experiments, for example, the homeodomain protein Cut has been shown to confer a dose-dependent increase in dendritic branching complexity and total length in a particular subset of peripheral sensory neurons [31]. Whether such molecules, which were discovered in Drosophila, will translate directly to the control of axon or dendrite growth in mammalian neurons remains to be shown, but the principle of cell-fate-determining transcription factors that control neurite outgrowth ability is certainly conserved. For example, the POU domain transcription factor Brn3b is essential for RGC development. In Brn3b / mice, RGCs fail to extend axons properly and 80% die before birth; in addition, Brn3b / RGCs in culture extend only rudimentary axons and instead elaborate abnormal, dendrite-like processes [32–36]. RGCs also express the related family members Brn3a and Brn3c: about 50% of RGCs express both Brn3b and Brn3c. The recently generated double knockout Brn3b / Brn3c / shows an almost total loss of axon outgrowth from explanted Current Opinion in Neurobiology 2004, 14:551–557
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retinas, suggesting that both Brn3b and Brn3c contribute to controlling RGC axon growth [37]. In these experiments, the only neurons that were found to elaborate neurites were those that did not express the alkaline phosphatase marker from the mutated Brn3c locus, which suggests that, in RGCs that do not normally express Brn3c, alternative transcription factors drive axon outgrowth [37]. Interestingly, in Brn3a knockout mice, sensory neurons demonstrate axon guidance and branching defects in vivo, and then undergo early cell death, analogous to the Brn3b effects in RGCs discussed above [38]. Thus, transcription factors involved in cell fate determination, which is often defined by cell morphology, may act directly via the control of axon and dendrite growth ability. What are the genes downstream of Brn3b that could effect changes in the intrinsic axon growth state of a neuron? Recently, Brn3b / retinas were compared with wild type retinas in microarray experiments to determine which genes depend on Brn3b for their expression [39]. Of interest, several genes linked to axon guidance were found to be downregulated in Brn3b / retinas. Two of the genes that were most strongly downregulated in the knockouts [39] were Sonic hedgehog, which has a role in axon guidance but not in axon elongation in the embryonic spinal cord [40], and persyn (also known as g-synuclein), a protein that is associated with neurofilaments [41]. a-Synuclein and b-synuclein seem to decrease neurite outgrowth in a rat neuronal cell line [42], however, raising the question of whether g-synuclein is indeed a pro-axon growth effector. Recently, the nerve growth factor (NGF) receptor trkA was identified as a transcriptional target of Brn3a, [43] providing a direct link from transcription factor expression to trophic responsiveness. In addition, these experiments point to the power of using focused microarray experiments in relation to established phenotypic models. For example, our research group is currently investigating which genes show altered expression during RGC development and thus may underlie the developmental loss of intrinsic axon growth ability in RGCs [5]. In some cases, it will be difficult to separate intrinsic neurite growth regulation from growth regulation directly induced by extrinsic signals. Recently, the Ca2+-dependent nuclear transactivator CREST has been cloned and found to regulate cortical and hippocampal dendrite development [44]. In CREST-deficient mice, dendritic arborization is severely compromised in vivo, and depolarization-dependent dendritic growth is similarly diminished in vitro. The downstream targets of CREST, as well as whether CREST deficiency causes an axonal phenotype, remain unknown. It is intriguing to propose that CREST regulation may be developmentally regulated by activity induced by specific presynaptic partners, and may switch the neuron into a dendritic growth mode to continue wiring presynaptically. Thus, CREST is a transcripCurrent Opinion in Neurobiology 2004, 14:551–557
tion factor whose activity is necessary for proper dendrite growth and development [44]. Is CREST expression regulated developmentally or in response to contact with neighboring cells? Determining how its expression is controlled will be essential to further elucidating any role in intrinsic dendrite growth ability. Posttranslational degradation mechanisms
Transcription factors are not the only master regulators of a cell. Levels of specific complements of proteins can be regulated posttranslationally by ubiquitination and degradation. Because there are hundreds of ubiquitin ligases in mammalian genomes, the potential for subcellular regulation of the levels of specific proteins is vast. The importance of ubiquitin-mediated degradation has been described for the normal axon degeneration that occurs during Drosophila development [45] or Wallerian degeneration after injury [46], as well as for axon guidance in primary neurons in vitro [47]; but what about its importance for axon elongation? Recently, Bonni and colleagues [48] have shown that the ubiquitin ligase anaphase-promoting complex (APC) has a significant role in regulating the axon growth rate of cerebellar granule neurons. APC was originally found to be essential for mitosis in dividing cells, but it is also expressed highly in postmitotic neurons. Using small hairpin constructs, Bonni and colleagues [48] inhibited the expression of Cdh1, which normally activates APC, in primary cultures of postnatal day 6 cerebellar granule neurons. RNA interference (RNAi)-mediated inhibition of Cdh1 led to a greater than twofold increase in axon length but not to a change in dendrite length, suggesting that Cdh1–APC complexes normally inhibit axon but not dendrite growth [48]. Similarly, an increase in axon growth was obtained when these researchers overexpressed a dominant-negative variant of APC or overexpressed the wild type form of an endogenous APC inhibitor, Emi1 [48]. Thus, Cdh1– APC is a potential candidate for a master regulator of a broader program that specifically controls the rate of axon, but not dendrite, growth. Cdh1–APC complexes were found in cerebellar granule neuron nuclei [48], but the substrates that are ubiquitinated by APC and regulate axon growth in these neurons remain to be identified. Second messengers and signaling cascades
How does the intrinsic state of neuron allow the same extrinsic cues to induce axon or dendrite growth preferentially (Figure 1b)? Cytoplasmic kinases or phosphatases may provide an axon- or dendrite-specific interpretation of extrinsic cues. For example, electrical activity seems to be crucial to potentiate both axon and dendrite growth in various neurons in response to trophic signals [6,29]. The influx of Ca2+ during depolarization activates a family of Ca2+-dependent signaling kinases, calmodulin www.sciencedirect.com
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kinases (CaMKs), which have now been implicated in both axon- and dendrite-specific growth. For axon growth, overexpression of a dominant-negative variant of CaMKI in embryonic hippocampal and postnatal cerebellar granule neurons decreases axon outgrowth [49], whereas expression of cytosolic dominant-negative CaMKII or nuclear dominant-negative CaMKIV constructs has no effect on axon growth in these cells. In other studies, CaMKII-b and nuclear CaMKIV have been found to stimulate dendrite but not axon growth in hippocampal or cortical cultures [50,51]. CaMKII is particularly interesting because its a-isoform has been long associated with the regulation of synaptic plasticity. By means of a b-isoform-specific actin-binding domain, however, CaMKII-b is able to increase dendrite motility and branching in hippocampal neurons [50]. Intriguingly, CaMKI is localized throughout the cytoplasm of all hippocampal neurites [49], raising issues of its role in dendrites and whether its localization or activation might become more axon-specific in vivo and in longer-lasting cultures. Thus, CaMK isoforms can differentially control axon- and dendrite-specific outgrowth in hippocampal neurons. Further experiments will hopefully point to mechanisms of either developmental or subcellular regulation, or both, for these kinases. How does the Ca2+-mediated activation of particular CaMKs lead to a program of specific axon or dendrite growth? Remarkably, CaMKII seems to activate another transcription factor involved in cell fate determination — the basic helix–loop–helix factor NeuroD [52]. Like APC, NeuroD is expressed in postmitotic neurons and has been recently shown to be phosphorylated by CaMKII in response to activity. Downregulation of NeuroD by RNAi in cerebellar granule neurons leads to a suppression of dendrite growth, but has no effect on axon growth either in dissociated cultures or in cerebellar slice explants [52]. Thus, the activation of dendrite-growthpromoting kinases leads to the activation of dendritegrowth-promoting transcription factors without activating the axon growth program of a neuron. Whether NeuroD interacts with CREST or activates an overlapping series of target genes will prove very interesting. The levels of second messengers such as cAMP and cGMP, if intrinsically maintained, could affect axon and dendrite growth differentially. Early indications of the importance of these second messengers came from studies on axon guidance molecules. For example, the attraction of growth cones towards a gradient of brainderived neurotrophic factor is dependent on cAMP [53]. Adequate levels of cAMP are crucial for RGCs to extend axons in response to neurotrophic factors [1], and the developmental decrease in cAMP levels has been proposed to underlie the developmental loss of axon regeneration in the presence of inhibitory myelin-associated www.sciencedirect.com
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cues [54]. In a strongly trophic, non-inhibitory environment, however, an increase in cAMP does not revert the slow axon growth of postnatal RGCs to the fast growth of their embryonic state [26]. Similarly, increases in cGMP convert the repulsion of growth cones away from point sources of semaphorin 3A or myelin-associated glycoprotein into attraction [55]. Could cGMP differentially affect axons and dendrites? There is already evidence for this effect in vivo: guanylate cyclase is asymmetrically localized to the apical dendrite in developing cortical pyramidal neurons, and apical sources of sema3A, which repel cortical axons basally, instead attract the dendrite apically [27]. Thus, the levels of cyclic nucleotides may provide another generalized mechanism for flipping the intrinsic growth state of a neuron, although further work that can dissociate the effects of these second messengers on the neuron’s intrinsic axon growth state from their effects on the axon’s response to extrinsic trophic or inhibitory signals will be crucial.
Conclusions: joining intrinsic and extrinsic control Thus, I propose here that the intrinsic growth state of the neuron is a crucial regulator of axon and dendrite elongation, and that extrinsic signals yet to be identified can stably change the intrinsic ability of a neuron to extend neurites during development. This change in intrinsic growth ability could be expressed at many different levels in the cell, but is likely to rely at least in part on altered gene expression. Further exploration of the intrinsic and extrinsic control of axon growth is essential, not only for our understanding of neuronal development but also for enhancing axon growth after injury or in disease. Attempts to address both issues simultaneously have proved more successful in enhancing regeneration in vivo. For example, macrophage-derived trophic factors can increase the regeneration of RGCs in the optic nerve, and this extrinsic enhancement of axon growth allows greater regeneration when combined with the expression of dominant-negative Nogo receptor constructs to decrease intrinsic responsiveness of the RGCs to the inhibitory Nogo protein [56]. Are these macrophagederived trophic factors switching the growth state of the RGC or simply providing a novel trophic support that is normally missing after injury in vivo? The identification of these factors and their further study may provide answers to this question. A stimulating final question is, can manipulating the intrinsic axon growth ability of neurons make their axons ignore extrinsic regulators? At first glance, the increase in axon growth seen after suppressing Cdh1–APC activity with RNAi was mirrored by an increase in axon growth on myelin substrates, which normally inhibit axon growth [48]. Neurons with suppressed Cdh1 elaborated axons Current Opinion in Neurobiology 2004, 14:551–557
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that were threefold longer on myelin substrates than those elaborated by control neurons on myelin; however, the axons still grew only half as fast as those of Cdh1suppressed neurons on control substrates [48]. These data suggest that activating a more rapid intrinsic axon growth program does not make neurons insensitive to extrinsic inhibitory signals, but rather they exemplify the point that both the intrinsic neuronal growth state and the extrinsic environment may have to be optimized for successful regeneration after injury.
Acknowledgements Thanks to members of the lab for helpful discussions throughout the writing process.
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
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