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A precise sequence of axon guidance events is required for the development of the ocular motor system. Three cranial nerves grow towards, and connect with, six extraocular muscles in a stereotyped pattern, in order to control eye movements. The signalling protein alpha2-chimaerin (α2-CHN) plays a pivotal role in the formation of the ocular motor system; mutations in CHN1, encoding α2-CHN, cause the human eye movement disorder Duane Retraction Syndrome (DRS). Our research has demonstrated that manipulation of α2-chn signalling in the zebrafish embryo leads to ocular motor axon wiring defects, although the signalling cascades regulated by α2-chn remain poorly understood. Here, we demonstrate that several cytoskeletal regulatory proteins - collapsin response mediator protein 2 (CRMP2), (encoded by the gene dpysl2), stathmin1 and stathmin 2 - bind to α2-CHN. dpysl2, stathmin1 and especially stathmin2 are expressed by ocular motor neurons. We find that manipulation of dpysl2 and of stathmins in zebrafish larvae leads to defects in both the axon wiring of the ocular motor system and the optokinetic reflex, impairing horizontal eye movements. Knockdowns of these molecules in zebrafish larvae of either sex caused axon guidance phenotypes that included defasciculation and ectopic branching; in some cases these phenotypes were reminiscent of DRS. chn1 knockdown phenotypes were rescued by overexpression of CRMP2 and STMN1, suggesting that these proteins act in the same signalling pathway. These findings suggest that CRMP2 and stathmins signal downstream of α2-CHN to orchestrate ocular motor axon guidance, and to control eye movements.Significance statementThe precise control of eye movement is crucial for the life of vertebrate animals, including humans. In humans, this control depends on the arrangement of nerve wiring of the ocular motor system, composed of three nerves and six muscles, a system that is conserved across vertebrate phyla. Mutations in the protein alpha2-chimaerin have previously been shown to cause eye movement disorders (squint) and axon wiring defects in humans. Our recent work has unravelled how alpha2-chimaerin co-ordinates axon guidance of the ocular motor system in animal models. In this paper, we demonstrate key roles for the proteins CRMP2 and stathmin 1/2 in the signalling pathway orchestrated by alpha2-chimaerin, potentially giving insight into the aetiology of eye movement disorders in humans.

We previously identified that ngr1 allele deletion limits the severity of experimental autoimmune encephalomyelitis (EAE) by preserving axonal integrity. However, whether this favorable outcome observed in EAE is a consequence of an abrogated neuronal-specific pathophysiological mechanism, is yet to be defined. Here we show that, Cre-loxP-mediated neuron-specific deletion of ngr1 preserved axonal integrity, whereas its re-expression in ngr1(−/−) female mice potentiated EAE-axonopathy. As a corollary, myelin integrity was preserved under Cre deletion in ngr1(flx/flx), retinal ganglion cell axons whereas, significant demyelination occurred in the ngr1(−/−) optic nerves following the re-introduction of NgR1. Moreover, Cre-loxP-mediated axon-specific deletion of ngr1 in ngr1(flx/flx) mice also demonstrated efficient anterograde transport of fluorescently-labeled ChTxβ in the optic nerves of EAE-induced mice. However, the anterograde transport of ChTxβ displayed accumulation in optic nerve degenerative axons of EAE-induced ngr1(−/−) mice, when NgR1 was reintroduced but was shown to be transported efficiently in the contralateral non- recombinant adeno-associated virus serotype 2-transduced optic nerves of these mutant mice. We further identified that the interaction between the axonal motor protein, Kinesin-1 and collapsin response mediator protein 2 (CRMP2) was unchanged upon Cre deletion of ngr1. Whereas, this Kinesin-1/CRMP2 association was reduced when NgR1 was re-expressed in the ngr1(−/−) optic nerves. Our data suggest that NgR1 governs axonal degeneration in the context of inflammatory-mediated demyelination through the phosphorylation of CRMP2 by stalling axonal vesicular transport. Moreover, axon-specific deletion of ngr1 preserves axonal transport mechanisms, blunting the induction of inflammatory demyelination and limiting the severity of EAE. SIGNIFICANCE STATEMENT Multiple sclerosis (MS) is commonly induced by aberrant immune-mediated destruction of the protective sheath of nerve fibers (known as myelin). However, it has been shown that MS lesions do not only consist of this disease pattern, exhibiting heterogeneity with continual destruction of axons. Here we investigate how neuronal NgR1 can drive inflammatory-mediated axonal degeneration and demyelination within the optic nerve by analyzing its downstream signaling events that govern axonal vesicular transport. We identify that abrogating the NgR1/pCRMP2 signaling cascade can maintain Kinesin-1-dependent anterograde axonal transport to limit inflammatory-mediated axonopathy and demyelination. The ability to differentiate between primary and secondary mechanisms of axonal degeneration may uncover therapeutic strategies to limit axonal damage and progressive MS.

Graded distribution of intracellular second messengers, such as Ca2+and cyclic nucleotides, mediates directional cell migration, including axon navigational responses to extracellular guidance cues, in the developing nervous system. Elevated concentrations of cAMP or cGMP on one side of the neuronal growth cone induce its attractive or repulsive turning, respectively. Although effector processes downstream of Ca2+have been extensively studied, very little is known about the mechanisms that enable cyclic nucleotides to steer migrating cells. Here, we show that asymmetric cyclic nucleotide signaling across the growth cone mediates axon guidance via modulating microtubule dynamics and membrane organelle transport. In embryonic chick dorsal root ganglion neurons in culture, contact of an extending microtubule with the growth cone leading edge induces localized membrane protrusion at the site of microtubule contact. Such a contact-induced protrusion requires exocytosis of vesicle-associated membrane protein 7 (VAMP7)-positive vesicles that have been transported centrifugally along the microtubule. We found that the two cyclic nucleotides counteractively regulate the frequency of microtubule contacts and targeted delivery of VAMP7 vesicles: cAMP stimulates and cGMP inhibits these events, thereby steering the growth cone in the opposite directions. By contrast, Ca2+signals elicit no detectable change in either microtubule contacts or VAMP7 vesicle delivery during Ca2+-induced growth cone turning. Our findings clearly demonstrate growth cone steering machinery downstream of cyclic nucleotide signaling and highlight a crucial role of dynamic microtubules in leading-edge protrusion for cell chemotaxis.SIGNIFICANCE STATEMENTDeveloping neurons can extend long axons toward their postsynaptic targets. The tip of each axon, called the growth cone, recognizes extracellular guidance cues and navigates the axon along the correct path. Here we show that asymmetric cyclic nucleotide signaling across the growth cone mediates axon guidance through localized regulation of microtubule dynamics and resulting recruitment of specific populations of membrane vesicles to the growth cone's leading edge. Remarkably, cAMP stimulates microtubule growth and membrane protrusion, whereas cGMP promotes microtubule retraction and membrane senescence, explaining the opposite directional polarities of growth cone turning induced by these cyclic nucleotides. This study reveals a novel microtubule-based mechanism through which cyclic nucleotides polarize the growth cone steering machinery for bidirectional axon guidance.

Functional neuronal connectivity requires proper neuronal morphogenesis and its dysregulation causes neurodevelopmental diseases. Transforming growth factor-β (TGF-β) family cytokines play pivotal roles in development, but little is known about their contribution to morphological development of neurons. Here we show that the Smad-dependent canonical signaling of TGF-β family cytokines negatively regulates neuronal morphogenesis during brain development. Mechanistically, activated Smads form a complex with transcriptional repressor TG-interacting factor (TGIF), and downregulate the expression of a neuronal polarity regulator, collapsin response mediator protein 2. We also demonstrate that TGF-β family signaling inhibits neurite elongation of human induced pluripotent stem cell-derived neurons. Furthermore, the expression of TGF-β receptor 1, Smad4, or TGIF, which have mutations found in patients with neurodevelopmental disorders, disrupted neuronal morphogenesis in both mouse (male and female) and human (female) neurons. Together, these findings suggest that the regulation of neuronal morphogenesis by an evolutionarily conserved function of TGF-β signaling is involved in the pathogenesis of neurodevelopmental diseases.SIGNIFICANCE STATEMENTCanonical transforming growth factor-β (TGF-β) signaling plays a crucial role in multiple organ development, including brain, and mutations in components of the signaling pathway associated with several human developmental disorders. In this study, we found that Smads/TG-interacting factor-dependent canonical TGF-β signaling regulates neuronal morphogenesis through the suppression of collapsin response mediator protein-2 (CRMP2) expression during brain development, and that function of this signaling is evolutionarily conserved in the mammalian brain. Mutations in canonical TGF-β signaling factors identified in patients with neurodevelopmental disorders disrupt the morphological development of neurons. Thus, our results suggest that proper control of TGF-β/Smads/CRMP2 signaling pathways is critical for the precise execution of neuronal morphogenesis, whose impairment eventually results in neurodevelopmental disorders.

The CRMP2 and CRMP4 proteins are strongly expressed in the developing nervous system, mediating neurite outgrowth, neuronal polarity, and axon guidance. In the present study, we demonstrate the interaction of the CRMP2 and CRMP4 proteins with the GluK5 subunit of the kainate (KA) receptor (KAR) and investigated the role of KARs in modulating the development of cultured mouse DRG neurons. We found that KARs modulate neuronal maturation and neurite outgrowth in a bidirectional manner. Accordingly, low concentrations of KA delayed maturation and enhanced neurite outgrowth, whereas maturation was promoted by higher concentrations of KA that attenuated neuritic elongation. The effects of weak KAR activation were prevented by blocking their noncanonical signaling and involved a differential regulation of CRMP2. Whereas the delay in maturation involves PKC-mediated phosphorylation of CRMP2 at T555 leading to a downregulation of membrane Cav2.2, the promotion of neurite outgrowth is achieved by dephosphorylation at T514 at the growth cones, the latter reflecting PKC-driven enhancement of GSK3β phosphorylation at S9. Together, these findings indicate that noncanonical KAR signaling influences neuronal development by modulating CRMP2 activity., This work was supported by grants to J.L. from the Spanish Ministerio de Ciencia e Innovación (BFU2006-07138 and BFU2011-24084), CONSOLIDER (CSD2007-00023), and Prometeo/2011/086. J.M.M. was supported by a doctoral fellowship from the Portuguese Fundação para a Ciência e a Tecnologia and R.J.R. was supported by a Juan de la Cierva fellowship. S.V. is an FPI fellow of the Ministry of Economy and Competitivity.

Axon regeneration after injury requires the extensive reconstruction, reorganization, and stabilization of the microtubule cytoskeleton in the growth cones. Here, we identify KIF3C as a key regulator of axonal growth and regeneration by controlling microtubule dynamics and organization in the growth cone. KIF3C is developmentally regulated. Rat embryonic sensory axons and growth cones contain undetectable levels of KIF3C protein that is locally translated immediately after injury. In adult neurons, KIF3C is axonally transported from the cell body and is enriched at the growth cone where it preferentially binds to tyrosinated microtubules. Functionally, the interaction of KIF3C with EB3 is necessary for its localization at the microtubule plus-ends in the growth cone. Depletion of KIF3C in adult neurons leads to an increase in stable, overgrown and looped microtubules because of a strong decrease in the microtubule frequency of catastrophes, suggesting that KIF3C functions as a microtubule-destabilizing factor. Adult axons lacking KIF3C, by RNA interference or KIF3C gene knock-out, display an impaired axonal outgrowth in vitro and a delayed regeneration after injury both in vitro and in vivo. Murine KIF3C knock-out embryonic axons grow normally but do not regenerate after injury because they are unable to locally translate KIF3C. These data show that KIF3C is an injury-specific kinesin that contributes to axon growth and regeneration by regulating and organizing the microtubule cytoskeleton in the growth cone.

Axon formation is a hallmark of initial neuronal polarization. This process is thought to be regulated by enhanced microtubule stability in the subsequent axon and changes in actin dynamics in the future axonal growth cone. Here, we show that the microtubule end-binding proteins cytoplasmic linker protein (CLIP)-115 and CLIP-170 were enriched in the axonal growth cone and extended into the actin-rich domain of the growth cone. CLIPs were necessary for axon formation and sufficient to induce an axon. The regulation of axonal microtubule stabilization by CLIPs enabled the protrusion of microtubules into the leading edge of the axonal growth cone. Moreover, CLIPs positively regulated growth cone dynamics and restrained actin arc formation, which was necessary for axon growth. In fact, in neurons without CLIP activity, axon formation was restored by actin destabilization or myosin II inhibition. Together, our data suggest that CLIPs enable neuronal polarization by controlling the stabilization of microtubules and growth cone dynamics.

International audience; Collapsin response mediator proteins (CRMPs) are involved in signaling of axon guidance and neurite outgrowth during neural development and regeneration. Among these, CRMP2 has been identified as an important actor in neuronal polarity and axon outgrowth, these activities being correlated with the reorganization of cytoskeletal proteins. In contrast, the function of CRMP5, expressed during brain development, remains obscure. Here, we find that, in contrast to CRMP2, CRMP5 inhibits tubulin polymerization and neurite outgrowth. Knockdown of CRMP5 expression by small interfering RNA confirms its inhibitory functions. CRMP5 forms a ternary complex with MAP2 and tubulin, the latter involving residues 475-522 of CRMP5, exposed at the molecule surface. Using different truncated CRMP5 constructs, we demonstrate that inhibition of neurite outgrowth by CRMP5 is mediated by tubulin binding. When both CRMP5 and CRMP2 are overexpressed, the inhibitory effect of CRMP5 abrogates neurite outgrowth promotion induced by CRMP2, suggesting that CRMP5 acts as a dominant signal. In cultured hippocampal neurons, CRMP5 shows no effect on axon growth, whereas it inhibits dendrite outgrowth and formation, at an early developmental stage, correlated with its strong expression in neurites. At later stages, when dendrites begin to extend, CRMP5 expression is absent. However, CRMP2 is constantly expressed. Overexpression of CRMP5 with CRMP2 inhibits CRMP2-induced outgrowth both on the axonal and dendritic levels. Deficiency of CRMP5 expression enhanced the CRMP2 effect. This antagonizing effect of CRMP5 is exerted through a tubulin-based mechanism. Thus, the CRMP5 binding to tubulin modulates CRMP2 regulation of neurite outgrowth and neuronal polarity during brain development.

The localization of specific mRNAs and their local translation in growth cones of developing axons has been shown to play an important mechanism to regulate growth cone turning responses to attractive or repulsive cues. However, the mechanism whereby local translation and growth cone turning may be controlled by specific mRNA-binding proteins is unknown. Here we demonstrate that brain-derived neurotrophic factor (BDNF) signals the Src-dependent phosphorylation of the beta-actin mRNA zipcode binding protein 1 (ZBP1), which is necessary for beta-actin synthesis and growth cone turning. We raised a phospho-specific ZBP1 antibody to Tyr396, which is a Src phosphorylation site, and immunofluorescence revealed BDNF-induced phosphorylation of ZBP1 within growth cones. The BDNF-induced increase in fluorescent signal of a green fluorescent protein translation reporter with the 3' untranslated region of beta-actin was attenuated with the Src family kinase-specific inhibitor PP2 [4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine]. Furthermore, a nonphosphorylatable mutant, ZBP1 Y396F, suppressed the BDNF-induced and protein synthesis-dependent increase in beta-actin localization in growth cones. Last, the ZBP1 Y396F mutant blocked BDNF-induced attractive growth cone turning. These results indicate that phosphorylation of ZBP1 at Tyr396 within growth cones has a critical role to regulate local protein synthesis and growth cone turning. Our findings provide new insight into how the regulated phosphorylation of mRNA-binding proteins influences local translation underlying growth cone motility and axon guidance.

Protein phosphatase 2A (PP2A) is indispensable in development, and deficits of PP2A and deterioration of neuronal axons have been observed in several neurodegenerative disorders, but the direct link between PP2A and the neuronal axon development is still missing. Here, we show that PP2A is essential for axon development in transfected rat brain and the dissociated hippocampal neurons. Upregulation of PP2A catalytic subunit (PP2Ac) not only promotes formation and elongation of the functional axons but also rescues axon retardation induced by PP2A inhibition. PP2A can dephosphorylate collapsin response mediator protein-2 (CRMP2) that implements the axon polarization, whereas constitutive expression of phosphomimic-CRMP2 abrogates the effect of PP2A upregulation. We also demonstrate that PP2Ac is enriched in the distal axon of the hippocampal neurons. Our results reveal a mechanistic link between PP2A and axonogenesis/axonopathy, suggesting that upregulation of PP2A may be a promising therapeutic for some neurodegenerative disorders.

Embryonic motoneurons from mutant SOD1 (mSOD1) mouse models of amyotrophic lateral sclerosis (ALS), but not wild-type motoneurons, can be triggered to die by exposure to nitric oxide (NO), leading to activation of a motoneuron-specific signaling pathway downstream of the death receptor Fas/CD95. To identify effectors of mSOD1-dependent cell death, we performed a proteomic analysis. Treatment of cultured mSOD1 motoneurons with NO led to a 2.5-fold increase in levels of collapsin response mediator protein 4a (CRMP4a). In vivo, the percentage of mSOD1 lumbar motoneurons expressing CRMP4 in mSOD1 mice increased progressively from presymptomatic to early-onset stages, reaching a maximum of 25%. Forced adeno-associated virus (AAV)-mediated expression of CRMP4a in wild-type motoneuronsin vitrotriggered a process of axonal degeneration and cell death affecting 60% of motoneurons, whereas silencing of CRMP4a in mSOD1 motoneurons protected them from NO-induced death.In vivo, AAV-mediated overexpression of CRMP4a but not CRMP2 led to the death of 30% of the lumbar motoneurons and an 18% increase in denervation of neuromuscular junctions in the gastrocnemius muscle. Our data identify CRMP4a as a potential early effector in the neurodegenerative process in ALS.

The transcription factor p53 suppresses tumorgenesis by regulating cell proliferation and migration. We investigated whether p53 could also control cell motility in postmitotic neurons. p53 isoforms recognized by phospho-p53-specific (at Ser-15) or “mutant” conformation-specific antibodies were highly and specifically expressed in axons and axonal growth cones in primary hippocampal neurons. Inhibition of p53 function by inhibitors, small interfering RNAs, or by dominant-negative forms, induced axonal growth cone collapse, whereas p53 overexpression led to larger growth cones. Furthermore, deletion of the p53 nuclear export signal blocked its axonal distribution and induced growth cone collapse. p53 inhibition-induced axonal growth cone collapse was significantly reduced by the Rho kinase (ROCK) inhibitor, Y27632 [(R)-(+)-trans-N-(4-pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide]. Our results reveal a new function for p53 as a critical regulator of axonal growth cone behavior by suppressing ROCK activity.

Collapsin response mediator protein 1 (CRMP1) is one of the CRMP family members that mediates signal transduction of axonal guidance and neuronal migration. We show here evidence that CRMP1 is involved in semaphorin3A (Sema3A)-induced spine development in the cerebral cortex. In the cultured cortical neurons fromcrmp1+/−mice, Sema3A increased the density of clusters of synapsin I and postsynaptic density-95, but this increase was markedly attenuated incrmp1−/−mice. This attenuation was also seen incyclin-dependent kinase 5(cdk5)−/−neurons. Furthermore, the introduction of wild-type CRMP1 but not CRMP1-T509A/S522A, (Thr 509 and Ser 522 were replaced by Ala), a mutant that cannot be phosphorylated by Cdk5, intocrmp1−/−neurons rescued the defect in Sema3A responsiveness. The Golgi-impregnation method showed that thecrmp1−/−layer V cortical neurons showed a lower density of synaptic bouton-like structures and that this phenotype had genetic interaction withsema3A. These findings suggest that Sema3A-induced spine development is regulated by phosphorylation of CRMP1 by Cdk5.

Collapsin response mediator protein 1 (CRMP1) is one of the CRMP family members that mediates signal transduction of axon guidance molecules. Here, we show evidence that CRMP1 is involved in Reelin (Reln) signaling to regulate neuronal migration in the cerebral cortex. Incrmp1−/−mice, radial migration of cortical neurons was retarded. This phenotype was not observed in thesema3A−/−andcrmp1+/−;sema3A+/−cortices. However, CRMP1 was colocalized with disabled-1 (Dab1), an adaptor protein in Reln signaling. In theRelnrl/rlcortex, CRMP1 and Dab1 were expressed at a higher level, yet tyrosine phosphorylated at a lower level. Loss ofcrmp1in adab1heterozygous background led to the disruption of hippocampal lamination, a Reeler-like phenotype. In addition to axon guidance, CRMP1 regulates neuronal migration by mediating Reln signaling.

Neurotrophins and semaphorin 3A are present along pathways and in targets of developing axons of dorsal root ganglion (DRG) sensory neurons. Growth cones of sensory axons are probably regulated by interaction of cytoplasmic signaling triggered coincidentally by both types of guidance molecules. We investigated the in vitro interactions of neurotrophins and semaphorin 3A (Sema3A) in modulating growth cone behaviors of axons extended from DRGs of embryonic day 7 chick embryos. Growth cones of DRGs raised in media containing 10(-9) m NGF or BDNF were more resistant to Sema3A-induced growth cone collapse than when DRGs were raised in 10(-11) m NGF. After overnight culture in 10(-11) m NGF, a 1 hr treatment with 10(-9) m NGF or BDNF was sufficient to increase growth cone resistance to Sema3A-induced collapse. This neurotrophin-mediated decrease in the collapse response of DRG growth cones was not associated with reduced expression on growth cones of the Sema3A-binding protein neuropilin-1. A series of pharmacological studies followed. Phosphatidylinositol 3 kinase activity is not required for these effects of NGF. The effects of inhibitors and activators of protein kinase A (PKA) indicate that PKA activity is involved in NGF modulation of Sema3A-induced growth cone collapse. The effects of inhibitors and activators of PKG indicate that PKG activity is involved in Sema3A-induced growth cone collapse. The effects of inhibitors also indicate that Rho-kinase activity is involved in Sema3A-induced growth cone collapse. These results are consistent with the idea that growth cone responses to an individual guidance cue depend on coincident signaling by other guidance cues and by other regulatory pathways.

Negative guidance molecules are important for guiding the growth of axons and ultimately for determining the wiring pattern in the developing nervous system. In tissue culture, growth cones at the tips of growing axons collapse in response to negative guidance molecules, such as ephrin-A2 and semaphorin 3A. The small GTPase Rac1 is involved in growth cone collapse, but the nature of its role is not clear. Rac1 activity assays showed that Rac1 is transiently inactivated after treatment with ephrin-A2. Ephrin-induced growth cone collapse, however, correlated with resumption of Rac1 activity. We demonstrate that Rac1 is required for endocytosis of the growth cone plasma membrane and reorganization of F-actin but not for the depolymerization of F-actin during growth cone collapse in response to ephrin-A2 and semaphorin 3A. Rac1, however, does not regulate constitutive endocytosis in growth cones. These findings show that in response to negative guidance molecules, the function of Rac1 changes from promoting actin polymerization associated with axon growth to driving endocytosis of the plasma membrane, resulting in growth cone collapse. Furthermore, Rac1 antisense injected into the embryonic chick eyein vivocaused the retinotectal projection to develop without normal topography in a manner consistent with Rac1 having an obligatory role in mediating ephrin signaling.

Precise growth cone guidance is the consequence of a continuous reorganization of actin filament structures within filopodia and lamellipodia in response to inhibitory and promoting cues. The small GTPases rac1, cdc42, and rhoA are critical for regulating distinct actin structures in non-neuronal cells and presumably in growth cones. Collapse, a retraction of filopodia and lamellipodia, is a typical growth cone behavior on contact with inhibitory cues and is associated with depolymerization and redistribution of actin filaments. We examined whether small GTPases mediate the inhibitory properties of CNS myelin or collapsin-1, a soluble semaphorin, in chick embryonic motor neuron cultures. As demonstrated for collapsin-1, CNS myelin-evoked growth cone collapse was accompanied by a reduction of rhodamine–phalloidin staining most prominent in the growth cone periphery, suggesting actin filament disassembly. Specific mutants of small GTPases were capable of desensitizing growth cones to CNS myelin or collapsin-1. Adenoviral-mediated expression of constitutively active rac1 or rhoA abolished CNS myelin-induced collapse and allowed remarkable neurite extension on a CNS myelin substrate. In contrast, expression of dominant negative rac1 or cdc42 negated collapsin-1-induced growth cone collapse and promoted neurite outgrowth on a collapsin-1 substrate. These findings suggest that small GTPases can modulate the signaling pathways of inhibitory stimuli and, consequently, allow the manipulation of growth cone behavior. However, the fact that opposite mutants of rac1 were effective against different inhibitory stimuli speaks against a universal signaling pathway underlying growth cone collapse.

Collapsin-1 or semaphorin III(D) inhibits axonal outgrowth by collapsing the lamellipodial and filopodial structures of the neuronal growth cones. Because growth cone collapse is associated with actin depolymerization, we considered whether small GTP-binding proteins of the rho subfamily might participate in collapsin-1 signal transduction. Recombinant rho, rac1, and cdc42 proteins were triturated into embryonic chick (DRG) neurons. Constitutively active rac1 increases the proportion of collapsed growth cones, and dominant negative rac1 inhibits collapsin-1-induced collapse of growth cones and collapsin-1 inhibition of neurite outgrowth. DRG neurons treated with dominant negative rac1 remain sensitive to myelin-induced growth cone collapse. Similar mutants of cdc42 do not alter growth cone structure, neurite elongation, or collapsin-1 sensitivity. Whereas the addition of activated rho has no effect, the inhibition of rho with Clostridium botulinum C3 transferase stimulates the outgrowth of DRG neurites. C3 transferase-treated growth cones exhibit little or no lamellipodial spreading and are minimally responsive to collapsin-1 and myelin. These data demonstrate a prominent role for rho and rac1 in modulating growth cone motility and indicate that rac1 may mediate collapsin-1 action.

Using two-dimensional gel electrophoresis we previously identified membrane-associated proteins that are upregulated over the course of neurogenesis. One of these, TOAD-64 (Turned On After Division, 64 kDa), is expressed immediately after neuronal birth and is dramatically downregulated in the adult. The gene encoding TOAD-64 has now been cloned, and its sequence shows homology to the unc-33 gene from C. elegans, mutations in which lead to aberrations in axon outgrowth. Northern and in situ hybridization show that TOAD-64 mRNA is enriched in the nervous system and is developmentally regulated in parallel with the protein. The expression of the TOAD-64 protein and gene coincident with initial neuronal differentiation and the downregulation when the majority of axon growth is complete suggests a role in axon elaboration. Three additional lines of evidence support this possibility: TOAD-64 is upregulated following neuronal induction of P19 and PC12 cells; the protein is found in lamellipodia and filopodia of growth cones; and axotomy of the sciatic nerve induces reexpression. While the sequence of TOAD-64 lacks a signal sequence and therefore is likely to encode a cytoplasmic protein, biochemical experiments demonstrate that the protein is tightly, but noncovalently, associated with membranes. The data presented here suggest that TOAD-64 could be a central element in the machinery underlying axonal outgrowth and pathfinding, perhaps playing a role in the signal transduction processes that permit growing axons to choose correct routes and targets.

Axonal growth cones are responsible for the correct guidance of developing axons and the establishment of functional neural networks. They are highly motile because of fast and continuous rearrangements of their actin-rich cytoskeleton. Here we have used live imaging of axonal growth cones of hippocampal neurons in culture and quantified their motility with a temporal resolution of 2 s. Using novel methods of analysis of growth cone dynamics, we show that transient activation of kainate receptors by bath-applied kainate induced a fast and reversible growth cone stalling. This effect depends on electrical activity and can be mimicked by the transient discharge of action potentials elicited in the neuron by intracellular current injections at the somatic level through a patch pipette. Growth cone stalling induced by electrical stimulation is mediated by calcium entry from the extracellular medium as well as by calcium release from intracellular stores that define spatially restricted microdomains directly affecting cytoskeletal dynamics. We propose that growth cone motility is dynamically controlled by transient bursts of spontaneous electrical activity, which constitutes a prominent feature of developing neural networksin vivo.

Neurite outgrowth is influenced by positive and negative signals that include the semaphorins, an important family of axonal outgrowth inhibitors. Here we report that the Rac GTPase activating protein (GAP)alpha2-chimaerin is involved in Semaphorin 3A (Sema 3A) signaling. In dorsal root ganglion neurons, Sema 3A-induced growth cone collapse was inhibited by alpha2-chimaerin mutated to eliminate GAP activity or interaction with phosphotyrosine. Activation of alpha2-chimaerin by phorbol ester caused growth cone collapse. Active alpha2-chimaerin interacts with collapsin response mediator protein-2 (CRMP-2) and cyclin-dependent kinase (Cdk) 5/p35 kinase through its SH2 and GAP domains, respectively. Cdk5 phosphorylates CRMP-2 at serine 522, possibly facilitating phosphorylation of serine 518 and threonine 514 by glycogen synthase kinase 3beta (GSK3beta), a kinase previously implicated in Sema 3A signaling. Phosphorylation of CRMP-2 serine 522 was essential for Sema 3A-induced growth cone collapse, which is dependent on Cdk5 but not Rho kinase activity. alpha2-chimaerin, like CRMP-2, can associate with the Sema 3A receptor. These results indicate that active alpha2-chimaerin Rac GAP, Cdk5/p35, and its substrate CRMP-2, are implicated in the dynamics of growth cone guidance initiated through Sema 3A signaling.

During development, EphB proteins serve as axon guidance molecules for retinal ganglion cell axon pathfinding toward the optic nerve head and in midbrain targets. To better understand the mechanisms by which EphB proteins influence retinal growth cone behavior, we investigated how axon responses to EphB were modulated by laminin and L1, two guidance molecules that retinal axons encounter duringin vivopathfinding. Unlike EphB stimulation in the presence of laminin, which triggers typical growth cone collapse, growth cones co-stimulated by L1 did not respond to EphB. Moreover, EphB exposure in the presence of both laminin and L1 resulted in a novel growth cone inhibition manifested as a pause in axon elongation with maintenance of normal growth cone morphology and filopodial activity. Pauses were not associated with loss of growth cone actin but were accompanied by a redistribution of the microtubule cytoskeleton with increased numbers of microtubules extending into filopodia and to the peripheral edge of the growth cone. This phenomenon was accompanied by reduced levels of the growth cone microtubule destabilizing protein SCG10. Antibody blockade of SCG10 function in growth cones resulted in both changes in microtubule distribution and pause responses mirroring those elicited by EphB in the presence of laminin and L1. These results demonstrate that retinal growth cone responsiveness to EphB is regulated by co-impinging signals from other axon guidance molecules. Furthermore, the results are consistent with EphB-mediated axon guidance mechanisms that involve the SCG10-mediated regulation of the growth cone microtubule cytoskeleton.

The Unc-33-like phosphoprotein/collapsin response mediator protein (Ulip/CRMP) family consists of four homologous phosphoproteins considered crucial for brain development. Autoantibodies produced against member(s) of this family by patients with paraneoplastic neurological diseases have made it possible to clone a fifth human Ulip/CRMP and characterize its cellular and anatomical distribution in developing brain. This protein, referred to as Ulip6/CRMP5, is highly expressed during rat brain development in postmitotic neural precursors and in the fasciculi of fibers, suggesting its involvement in neuronal migration/differentiation and axonal growth. In the adult, Ulip6/CRMP5 is still expressed in some neurons, namely in areas that retain neurogenesis and in oligodendrocytes in the midbrain, hindbrain, and spinal cord. Ulip2/CRMP2 and Ulip6/CRMP5 are coexpressed in postmitotic neural precursors at certain times during development and in oligodendrocytes in the adult. Because Ulip2/CRMP2 has been reported to mediate semaphorin-3A (Sema3A) signal in developing neurons, in studies to understand the function of Ulip6/CRMP5 and Ulip2/CRMP2 in the adult, purified adult rat brain oligodendrocytes were cultured in a Sema3A-conditioned medium. Oligodendrocytes were found to have Sema3A binding sites and to express neuropilin-1, the major Sema3A receptor component. In the presence of Sema3A, these oligodendrocytes displayed a dramatic reduction in process extension, which was reversed by removal of Sema3A and prevented by anti-neuropilin-1, anti-Ulip6/CRMP5, anti-Ulip2/CRMP2 antibodies, or VEGF-165, another neuropilin-1 ligand. These results indicate the existence in the adult brain of a Sema3A signaling pathway that modulates oligodendrocyte process extension mediated by neuropilin-1, Ulip6/CRMP5, and Ulip2/CRMP2, and they open new fields of investigation of neuron/oligodendrocyte interactions in the normal and pathological brain.

In cultured neurons, axon formation is preceded by the appearance in one of the multiple neurites of a large growth cone containing a labile actin network and abundant dynamic microtubules. The invasion-inducing T-lymphoma and metastasis 1 (Tiam1) protein that functions as a guanosine nucleotide exchange factor for Rac1 localizes to this neurite and its growth cone, where it associates with microtubules. Neurons overexpressing Tiam1 extend several axon-like neurites, whereas suppression of Tiam1 prevents axon formation, with most of the cells failing to undergo changes in growth cone size and in cytoskeletal organization typical of prospective axons. Cytochalasin D reverts this effect leading to multiple axon formation and penetration of microtubules within neuritic tips devoid of actin filaments. Taken together, these results suggest that by regulating growth cone actin organization and allowing microtubule invasion within selected growth cones, Tiam1 promotes axon formation and hence participates in neuronal polarization.

Members of the collapsin/semaphorin family play an important role in creating the complex pattern of neuronal connectivity. Inhibition of growth cone motility by chick collapsin is mediated by the intraneuronal protein CRMP-62. We have now isolated four rat sequences that are highly related to chick CRMP-62. All four genes are expressed exclusively in the nervous system and primarily during development. Rat CRMP-2/TOAD-64 is most closely related to chick CRMP-62 and is the most widely expressed CRMP within the nervous system. Rat CRMP-1 and CRMP-4/rUlip are expressed during discrete periods of neuronal development and are not found in the adult nervous system. Rat CRMP-3 has a distinct distribution, being expressed transiently in developing spinal cord and selectively in the postnatal cerebellum. The differential expression of these genes suggests that CRMPs may transduce signals from different semaphorins and that semaphorins may regulate the plasticity of the adult nervous system.