A molecular mechanism that regulates medially oriented axonal growth of upper layer neurons in the developing neocortex

During development, cortical neurons extend axons to their targets based on their laminar locations and cell types. Here we studied the molecular mechanism that regulates medially oriented axonal growth of upper layer neurons in the developing mouse cortex. Upper layer neurons were labeled with enhanced yellow fluorescent protein (EYFP) by in utero electroporation at E15.5. Cortical slices containing EYFP-labeled cells were dissected at E16, when axonal outgrowth from upper layer neurons is not initiated, and were cultured in an organotypic manner. After 3 days in culture, most labeled cells were found to extend axons medially in the same fashion as those observed in vivo. This oriented growth was disrupted when the lateral side of the cortical slice was removed, indicating that a laterally located repellent is involved in the medially oriented growth. Strikingly, the medially directed growth within the slices was reduced in the medium containing Semaphorin3A (Sema3A) or soluble form of Neuropilin-1 (Npn1), a receptor for Sema3A. Importantly, we found that Sema3A was expressed in a gradient from lateral-high to medial-low within the cortex, and callosal axons originating from upper layer neurons uniquely expressed Npn1. Consistent with these findings, ectopically expressed Sema3A repelled medially oriented elongation of upper layer cell axons in vivo. These results therefore suggest the operation of a repulsive mechanism for medially oriented axon growth of upper layer neurons, and further point to a role for a gradient expression of Sema3A in this directional axon growth along the mediolateral axis within the neocortex.     

Eye‐specific segregation and differential fasciculation of developing retinal ganglion cell axons in the mouse visual pathway

Prior to forming and refining synaptic connections, axons of projection neurons navigate long distances to their targets. While much is known about guidance cues for axon navigation through intermediate choice points, whether and how axons are organized within tracts is less clear. Here we analyze the organization of retinal ganglion cell (RGC) axons in the developing mouse retinogeniculate pathway. RGC axons are organized by both eye‐specificity and topography in the optic nerve and tract: ipsilateral RGC axons are segregated from contralateral axons and are offset laterally in the tract relative to contralateral axon topographic position. To identify potential cell‐autonomous factors contributing to the segregation of ipsilateral and contralateral RGC axons in the visual pathway, we assessed their fasciculation behavior in a retinal explant assay. Ipsilateral RGC neurites self‐fasciculate more than contralateral neurites in vitro and maintain this difference in the presence of extrinsic chiasm cues. To further probe the role of axon self‐association in circuit formation in vivo, we examined RGC axon organization and fasciculation in an EphB1^?/? mutant, in which a subset of ipsilateral RGC axons aberrantly crosses the midline but targets the ipsilateral zone in the dorsal lateral geniculate nucleus on the opposite side. Aberrantly crossing axons retain their association with ipsilateral axons in the contralateral tract, indicating that cohort‐specific axon affinity is maintained independently of guidance signals present at the midline. Our results provide a comprehensive assessment of RGC axon organization in the retinogeniculate pathway and suggest that axon self‐association contributes to pre‐target axon organization.     

Optogenetically enhanced axon regeneration: motor versus sensory neuron‐specific stimulation

Brief neuronal activation in injured peripheral nerves is both necessary and sufficient to enhance motor axon regeneration, and this effect is specific to the activated motoneurons. It is less clear whether sensory neurons respond in a similar manner to neuronal activation following peripheral axotomy. Further, it is unknown to what extent enhancement of axon regeneration with increased neuronal activity relies on a reflexive interaction within the spinal circuitry. We used mouse genetics and optical tools to evaluate the precision and selectivity of system‐specific neuronal activation to enhance axon regeneration in a mixed nerve. We evaluated sensory and motor axon regeneration in two different mouse models expressing the light‐sensitive cation channel, channelrhodopsin (ChR2). We selectively activated either sensory or motor axons using light stimulation combined with transection and repair of the sciatic nerve. Regardless of genotype, the number of ChR2‐positive neurons whose axons had regenerated successfully was greater following system‐specific optical treatment, with no effect on the number of ChR2‐negative neurons (whether motor or sensory neurons). We conclude that acute system‐specific neuronal activation is sufficient to enhance both motor and sensory axon regeneration. This regeneration‐enhancing effect is likely cell autonomous.     

Semaphorin 3A-mediated axon guidance regulates convergence and targeting of P2 odorant receptor axons

Semaphorins are known to play an important role in axon guidance of vertebrate olfactory sensory neurons to their targets in specific glomeruli of the olfactory bulb (OB). However, it is not clear how semaphorin-mediated guidance contributes to a systematic hierarchy of cues that govern the organization of this system. Because of the putative role that odorant receptor molecules such as P2 could play in establishing appropriate glomerular destinations for growing olfactory axons, we have also determined the spatial organization of P2 glomeruli in semaphorin 3A (Sema3A) mutant mice. First, in the postnatal OB of control and Sema3A(-/-) mice, we analysed the trajectories of olfactory axons that express the Sema3A receptor, neuropilin-1 (npn-1) and the positions of npn-1(+) glomeruli. Sema3A at the ventral OB midline guides npn-1(+) axons to targets in the lateral and medial OB. Absence of Sema3A permits many npn-1 axons to terminate aberrantly in the rostral and ventral OB. Second, in Sema3A(-/-) mice, many P2 axons are abnormally distributed throughout the ventral OB nerve layer and converge in atypical locations compared with littermate controls where P2 axons converge on stereotypically located lateral and medial glomeruli. In addition to their radically altered spatial distribution, P2 glomeruli in Sema3A(-/-) mice are significantly smaller and more numerous than in heterozygote littermates. These data show that Sema3A is an important repulsive olfactory guidance cue that establishes restricted npn-1(+) subcompartments in the olfactory bulb. Furthermore, Sema3A plays a key role in the convergence of axons expressing the odorant receptor P2 onto their appropriate targets.     

Ultrastructural observations on the expression of axonin-1: implications for the fasciculation of sensory axons during axonal outgrowth into the chick hindlimb.

To help understand how axons interact as they grow into the developing chick hindlimb, we used electron microscopy in conjunction with immunoperoxidase staining for the cell adhesion molecule axonin-1 to label sensory axons. The results showed that sensory axons travel together in bundles, tightly apposed to one another. In contrast, motoneuron axons are more widely spaced, although motoneuron axons situated at the perimeter of sensory axon bundles are found in close contact with neighboring sensory axons. Sensory growth cones and lamellipodia tend to be located centrally within the bundles, with several lamellipodia typically being found stacked together. Strikingly, regions of close axonal apposition are accompanied by axonin-1 expression, suggesting that such contacts are indeed adhesive. Taken together, these observations suggest that groups of sensory axons of a similar age grow together, with some of the older sensory axons fasciculating along motoneuron axons and younger sensory axons later fasciculating along older sensory axons. Axons situated at the periphery of sensory bundles are typically partly labelled, such that axonin-1 is expressed on membranes apposing other labelled axons but not on those facing unlabelled axons or unlabelled Schwann cells. Thus, axonin-1 appears to become redistributed within the membranes of axons growing into the limb, as it does on cultured neurons. In contrast, the neuron-glia cell adhesion molecule (NgCAM), which binds heterophilically to axonin-1, appears uniformly distributed on even those axons that would have an asymmetric distribution of axonin-1. Thus, the localization of axonin-1 strongly suggests that it plays an important role in sensory axon fasciculation, but the relative contributions of its interactions with various potential ligands are unclear. Finally, we found that some sensory growth cones have lamellipodia that are spread over considerable expanses. This suggests that although fasciculation is important in sensory axon guidance, sensory axons may also explore the local environment.     

Concentration-dependent requirement for local protein synthesis in motor neuron subtype-specific response to axon guidance cues

Formation of functional motor circuits relies on the ability of distinct spinal motor neuron subtypes to project their axons with high precision to appropriate muscle targets. While guidance cues contributing to motor axon pathfinding have been identified, the intracellular pathways underlying subtype-specific responses to these cues remain poorly understood. In particular, it remains controversial whether responses to axon guidance cues depend on axonal protein synthesis. Using a growth cone collapse assay, we demonstrate that mouse embryonic stem cell-derived spinal motor neurons (ES-MNs) respond to ephrin-A5, Sema3f, and Sema3a in a concentration-dependent manner. At low doses, ES-MNs exhibit segmental or subtype-specific responses, while this selectivity is lost at higher concentrations. Response to high doses of semaphorins and to all doses of ephrin-A5 is protein synthesis independent. In contrast, using microfluidic devices and stripe assays, we show that growth cone collapse and guidance at low concentrations of semaphorins rely on local protein synthesis in the axonal compartment. Similar bimodal response to low and high concentrations of guidance cues is observed in human ES-MNs, pointing to a general mechanism by which neurons increase their repertoire of responses to the limited set of guidance cues involved in neural circuit formation.     

The L1-type cell adhesion molecule Neuroglian is necessary for maintenance of sensory axon advance in the Drosophila embryo

Background

Cell adhesion molecules have long been implicated in the regulation of axon growth, but the precise cellular roles played by individual cell adhesion molecules and the molecular basis for their action are still not well understood. We have used the sensory system of the Drosophila embryo to shed light on the mechanism by which the L1-type cell adhesion molecule Neuroglian regulates axon growth.

Results

We have found a highly penetrant sensory axon stalling phenotype in neuroglian mutant embryos. Axons stalled at a variety of positions along their normal trajectory, but most commonly in the periphery some distance along the peripheral nerve. All lateral and dorsal cluster sensory neurons examined, except for the dorsal cluster neuron dbd, showed stalling. Sensory axons were never seen to project along inappropriate pathways in neuroglian mutants and stalled axons showed normal patterns of fasciculation within nerves. The growth cones of stalled axons possessed a simple morphology, similar to their appearance in wild-type embryos when advancing along nerves. Driving expression of the wild-type form of Neuroglian in sensory neurons alone rescued the neuroglian mutant phenotype of both pioneering and follower neurons. A partial rescue was achieved by expressing the Neuroglian extracellular domain. Over/mis-expression of Neuroglian in all neurons, oenocytes or trachea had no apparent effect on sensory axon growth.

Conclusion

We conclude that Neuroglian is necessary to maintain axon advance along axonal substrates, but is not required for initiation of axon outgrowth, axon fasciculation or recognition of correct growth substrates. Expression of Neuroglian in sensory neurons alone is sufficient to promote axon advance and the intracellular region of the molecule is largely dispensable for this function. It is unlikely, therefore, that Nrg acts as a molecular 'clutch' to couple adhesion of F-actin within the growth cone to the extracellular substrate. Rather, we suggest that Neuroglian mediates sensory axon advance by promoting adhesion of the surface of the growth cone to its substrate. Our finding that stalling of a pioneer sensory neuron is rescued by driving Neuroglian in sensory neurons alone may suggest that Neuroglian can act in a heterophilic fashion.     

Chemoattraction and chemorepulsion of olfactory bulb axons by different secreted semaphorins

During development, growth cones can be guided at a distance by diffusible factors, which are attractants and/or repellents. The semaphorins are the largest family of repulsive axon guidance molecules. Secreted semaphorins bind neuropilin receptors and repel sensory, sympathetic, motor, and forebrain axons. We found that in rat embryos, the olfactory epithelium releases a diffusible factor that repels olfactory bulb axons. In addition, Sema A and Sema IV, but not Sema III, Sema E, or Sema H, are able to orient in vitro the growth of olfactory bulb axons; Sema IV has a strong repulsive action, whereas Sema A appears to attract those axons. The expression patterns of sema A and sema IV in the developing olfactory system confirm that they may play a cooperative role in the formation of the lateral olfactory tract. This also represents a further evidence for a chemoattractive function of secreted semaphorins.     

Identification and characterization of a cell surface marker for embryonic rat spinal accessory motor neurons

The developing mammalian spinal cord contains distinct populations of motor neurons that can be distinguished by their cell body positions, by the expression of specific combinations of regulatory genes, and by the paths that their axons take to exit the central nervous system (CNS). Subclasses of spinal motor neurons are also thought to express specific cell surface proteins that function as receptors which control the guidance of their axons. We identified monoclonal antibody (mAb) SAC1 in a screen aimed at generating markers for specific subsets of neurons/axons in the developing rat spinal cord. During early embryogenesis, mAb SAC1 selectively labels a small subset of Isl1-positive motor neurons located exclusively within cervical segments of the spinal cord. Strikingly, these neurons extend mAb SAC1-positive axons along a dorsally directed trajectory toward the lateral exit points. Consistent with the finding that mAb SAC1 also labels spinal accessory nerves, these observations identify mAb SAC1 as a specific marker of spinal accessory motor neurons/axons. During later stages of embryogenesis, mAb SAC1 is transiently expressed on both dorsally and ventrally projecting spinal motor neurons/axons. Interestingly, mAb SAC1 also labels the notochord and floor plate during most stages of spinal cord development. The mAb SAC1 antigen is a 100-kD glycoprotein that is likely to be the rat homolog of SC1/BEN/DM-GRASP, a homophilic adhesion molecule that mediates axon outgrowth and fasciculation.     

Semaphorin III can induce death in sensory neurons

Semaphorin III has been described to function as a guidance molecule directing growing axons to their target. However, its effect on the neuron cell body has not been characterized. Semaphorin III has a highly dynamic expression pattern, which generally corroborates a chemorepellent guidance function, but also suggests additional functions, different from axon guidance. A number of studies show that some sensory neurons are eliminated, while their axons are still pathfinding. In this study we have investigated whether Semaphorin III also influences the survival of sensory neurons. We here present evidence that Semaphorin III can function in vitro as selective death factor for NGF-dependent sensory neurons. Semaphorin III induces a type of cell death that is characterized by slow onset, cell body shrinking, nuclear condensation, and TUNEL-positive staining of dying neurons. These are all hallmarks of neuronal apoptosis. We also show evidence that neurons can modulate the response to Semaphorin III. The novel function described here may also be relevant in vivo, contributing to active elimination of neurons during development or after injury.     

A semaphorin code defines subpopulations of spinal motor neurons during mouse development

Abstract In the spinal cord, motor neurons (MNs) with similar muscle targets and sensory inputs are grouped together into motor pools. To date, relatively little is known about the molecular mechanisms that control the establishment of pool-specific circuitry. Semaphorins, a large family of secreted and cell surface proteins, are important mediators of developmental processes such as axon guidance and cell migration. Here, we used mRNA in situ hybridization to study the expression patterns of semaphorins and their receptors, neuropilins and plexins, in the embryonic mouse spinal cord. Our data show that semaphorins and their receptors are differentially expressed in MNs that lie in distinct locations within the spinal cord. Furthermore, we report a combinatorial expression of class 3 (secreted) semaphorins and their receptors that characterizes distinct motor pools within the brachial and lumbar spinal cord. Finally, we found that a secreted semaphorin, Sema3A, elicits differential collapse responses in topologically distinct subpopulations of spinal MNs. These findings lead us to propose that semaphorins and their receptors might play important roles in the sorting of motor pools and the patterning of their afferent and efferent projections.     

Modulating Sema3A signal with a L1 mimetic peptide is not sufficient to promote motor recovery and axon regeneration after spinal cord injury

We examined whether Sema3A, which is upregulated at the site of spinal cord injury, exerts a direct effect on axons. We used ASNKL peptide that prevents specifically the inhibitory effect of Sema3A on L1/Neuropilin1 (Nrp1)-expressing axons. In the na?ve mouse spinal cord, L1 is located on a subset of corticospinal axons, whereas Nrp1 is barely detectable. After contusion injury, Nrp1 is found on L1-negative immune cells, whereas its expression does not increase on severed axons. L1-expressing axons sprout extensively into the lesion site but no difference in axon density could be detected in the lesion area of mice treated with ASNKL. In agreement, these mice did not recover a better motor function than controls. Similarly, culture of neurons sensitive to ASNKL on cryosections of lesioned spinal cords revealed no effect of Sema3A. Our data indicate a limited direct effect of Sema3A on axonal growth at the site of a contusion injury, and suggest that alternative mechanisms underlie positive effects of Sema3A inhibition on motor recovery.     

Secondary motoneuron axons localize DM-GRASP on their fasciculated segments

Cell surface adhesion molecules are thought to play a necessary role in axon guidance and fasciculation in the developing nervous system. We have studied a potential adhesion molecule using the zn-5 monoclonal antibody, which recognizes the surfaces of zebrafish spinal motoneurons. We show that zn-5 recognizes zebrafish DM-GRASP. DM-GRASP is a cell adhesion molecule of the immunoglobulin superfamily that mediates homophilic adhesion and neurite outgrowth in vitro. It is necessary for correct axon routing and fasciculation in the Drosophila visual system. In zebrafish, primary motoneurons pioneer the peripheral motor nerve pathways, and the axons of secondary motoneurons follow the routes established by the primary motoneuron axons. We show that, of the two classes of zebrafish spinal motoneurons, only the later growing secondary motoneurons express DM-GRASP. The secondary motoneurons restrict DM-GRASP protein to their cell bodies and fasciculated segments of their axons. Expression of DM-GRASP is transient: The protein is present during the period of axonal growth and disappears after axons have reached their muscle targets. Thus, homophilic adhesion mediated by DM-GRASP may play a role in fasciculation of secondary motoneuron axons but not in pathfinding by the pioneer axons of the primary motoneurons or in guidance of secondary motoneuron axons to their targets.     

Induction of neuronal apoptosis by Semaphorin3A-derived peptide

Collapsin-1/Semaphorin3A (Sema3A) belongs to the secreted type III semaphorins family of axon guidance molecules with chemorepulsive activity, and is suggested to play a major role in navigating axonal networks throughout development into their correct destinations. We have previously shown that semaphorins are mediators of neuronal apoptosis and can induce neuronal death in the absence of any other apoptotic trigger. We report here that exposure of neuronal cells to a small conserved peptide derived from Sema3A initiates an apoptotic death process. Administration of this peptide to cultured chick sympathetic and mouse cerebellar granule neurons caused a marked shrinkage of their axonal network and cell death, which was characterized as apoptotic, based on nuclear staining. Attenuation of neuronal cell death was obtained by treatment with antioxidants and by vascular endothelial growth factor. Survival of neurons exposed to this peptide increased by co-treatment with caspase inhibitors. Induction of apoptosis was specific to neuronal cells, similarly to that induced by the full-length Sema3A protein. Our findings therefore suggest active participation of this conserved Sema3A-derived peptide in semaphorin-induced neuronal death process.     

Semaphorins 3A, 3C, and 3F in mesencephalic dopaminergic axon pathfinding

By analyzing the mechanisms that govern dopaminergic axon pathfinding from the midbrain to the striatum in embryonic rat brains, we identified neuroepithelial regions that exert chemotropic effects on mesencephalic dopaminergic axons. Explants from the pretectum and the striatum showed an attractive effect, whereas those from the midhindbrain boundary, the dorsal thalamus, and the ventral thalamus had no effect. Expression of semaphorin (Sema) 3C and Sema3F in the pretectum and of Sema3A in the striatum suggested a role for these axon guidance molecules in dopaminergic axon pathfinding. When expressed in HEK293 cell aggregates, Sema3C had an attractive effect and enhanced axon growth, Sema3A enhanced axon growth, and Sema3F had a repulsive effect on dopaminergic axons. Antineuropilin-1 and antineuropilin-2 antibodies reduced attraction by the pretectum, whereas attraction by the striatum was not affected by the presence of antineuropilin-1 antibodies. Moreover, neuropilin-1- and neuropilin-2-soluble Fc chimeras reduced the attraction by the pretectum. These results suggest that semaphorins may help to establish the dopaminergic projection from the midbrain to the striatum during embryonic development.     

Upregulation of axon guidance molecules in the adult central nervous system of Nogo‐A knockout mice restricts neuronal growth and regeneration

Adult central nervous system axons show restricted growth and regeneration properties after injury. One of the underlying mechanisms is the activation of the Nogo‐A/Nogo receptor (NgR1) signaling pathway. Nogo‐A knockout (KO) mice show enhanced regenerative growth in vivo, even though it is less pronounced than after acute antibody‐mediated neutralization of Nogo‐A. Residual inhibition may involve a compensatory component. By mRNA expression profiling and immunoblots we show increased expression of several members of the Ephrin/Eph and Semaphorin/Plexin families of axon guidance molecules, e.g. EphrinA3 and EphA4, in the intact spinal cord of adult Nogo‐A KO vs. wild‐type (WT) mice. EphrinA3 inhibits neurite outgrowth of EphA4‐positive neurons in vitro. In addition, EphrinA3 KO myelin extracts are less growth‐inhibitory than WT but more than Nogo‐A KO myelin extracts. EphA4 KO cortical neurons show decreased growth inhibition on Nogo‐A KO myelin as compared with WT neurons, supporting increased EphA4‐mediated growth inhibition in Nogo‐A KO mice. Consistently, in vivo, Nogo‐A/EphA4 double KO mice show increased axonal sprouting and regeneration after spinal cord injury as compared with EphA4 KO mice. Our results reveal the upregulation of developmental axon guidance cues following constitutive Nogo‐A deletion, e.g. the EphrinA3/EphA4 ligand/receptor pair, and support their role in restricting neurite outgrowth in the absence of Nogo‐A.     

Two novel human NUMB isoforms provide a potential link between development and cancer

Background

In the developing hindbrain, cranial motor axon guidance depends on diffusible repellent factors produced by the floor plate. Our previous studies have suggested that candidate molecules for mediating this effect are Slits, Netrin-1 and Semaphorin3A (Sema3A). It is unknown to what extent these factors contribute to floor plate-derived chemorepulsion of motor axons, and the downstream signalling pathways are largely unclear.

Results

In this study, we have used a combination of in vitro and in vivo approaches to identify the components of floor plate chemorepulsion and their downstream signalling pathways. Using in vitro motor axon deflection assays, we demonstrate that Slits and Netrin-1, but not Sema3A, contribute to floor plate repulsion. We also find that the axon pathways of dorsally projecting branchiomotor neurons are disrupted in Netrin-1 mutant mice and in chick embryos expressing dominant-negative Unc5a receptors, indicating an in vivo role for Netrin-1. We further demonstrate that Slit and Netrin-1 signalling are mediated by Rho-kinase (ROCK) and myosin light chain kinase (MLCK), which regulate myosin II activity, controlling actin retrograde flow in the growth cone. We show that MLCK, ROCK and myosin II are required for Slit and Netrin-1-mediated growth cone collapse of cranial motor axons. Inhibition of these molecules in explant cultures, or genetic manipulation of RhoA or myosin II function in vivo causes characteristic cranial motor axon pathfinding errors, including the inability to exit the midline, and loss of turning towards exit points.

Conclusions

Our findings suggest that both Slits and Netrin-1 contribute to floor plate-derived chemorepulsion of cranial motor axons. They further indicate that RhoA/ROCK, MLCK and myosin II are components of Slit and Netrin-1 signalling pathways, and suggest that these pathways are of key importance in cranial motor axon navigation.     

Pontocerebellar axon guidance: neuropilin-1- and semaphorin 3A-sensitivity gradients across basilar pontine nuclei and semaphorin 3A variation across cerebellum

To assess the role of semaphorin 3A (Sema3A) and its receptor component neuropilin-1 (Npn-1) in pontocerebellar axon guidance, we compared the distributions of Sema3A, Npn-1, and DiI-labeled pontocerebellar axons in neonatal mouse cerebellum. Between embryonic day 18 and birth there was a large increase in Npn-1 expression in the basilar pontine nuclei (BPN), the major source of pontocerebellar axons. Sema3A expression in cerebellum also increased at this time. In the BPN, Npn-1 and the response of axons to Sema3A were graded with high Npn-1 and Sema3A responsiveness rostrally and lower levels caudally. The Npn-1 gradient was not smooth and cells with higher and lower expression were interspersed. Between birth and postnatal day 5, pontocerebellar axons projected to lobules of the hemispheres, including those with low to moderate levels of Sema3A, but did not enter regions with high levels of Sema3A, including the flocculus and much of the vermis. These results suggest that varying neuropilin levels on BPN axons, which correlated with their varying responses to Sema3A, combined with varying Sema3A levels across cerebellum, may contribute to guiding subsets of BPN axons to their distinct target regions within cerebellum.     

NGF enhances sensory axon growth induced by laminin but not by the L1 cell adhesion molecule

Neurotrophins and cell adhesion molecules regulate axon guidance, but their potential coordinate interactions are not well defined. In particular, it has been difficult to define the role of signaling from different surface molecules in neurotrophin-induced axon growth because of the strong dependence of embryonic neurons on this class of molecules for survival. We have addressed this issue using Bax deficient neurons, which do not require neurotrophins for survival. The L1 neural cell adhesion molecule and laminin each supported NGF-independent axon growth of cultured sensory neurons from dorsal root ganglia of embryonic Bax(-/-) mice. However, nerve growth factor (NGF) stimulated additional axon growth of sensory neurons on laminin but not on L1 substrates. Inhibition of the small GTPase RhoA by the dominant-negative mutant RhoA(T19N) restored NGF responsiveness of axon growth on L1 to Bax(-/-) neurons. Constitutively activated RhoA(Q63L) did not affect axon growth on L1 but inhibited NGF-stimulated axon growth on laminin. Consistent with the concept that RhoA was downregulated by NGF in neurons on laminin but not L1, the RhoA inhibitor C2IN-C3 toxin stimulated axon growth on L1 in wild-type DRG neurons in NGF. These results demonstrate a novel substrate-dependent regulation of NGF-induced growth of embryonic sensory axons mediated by RhoA GTPase.     

Modulation of semaphorin3A in perineuronal nets during structural plasticity in the adult cerebellum

In the adult central nervous system (CNS) subsets of neurons are enwrapped by densely organized extracellular matrix structures, called perineuronal nets (PNNs). PNNs are formed at the end of critical periods and contribute to synapse stabilization. Enzymatic degradation of PNNs or genetic deletion of specific PNN components leads to the prolongation of the plasticity period. PNNs consist of extracellular matrix molecules, including chondroitin sulfate proteoglycans, hyaluronan, tenascins and link proteins. It has been recently shown that the chemorepulsive axon guidance protein semaphorin3A (Sema3A) is also a constituent of PNNs, binding with high affinity to the sugar chains of chondroitin sulfate proteoglycans. To elucidate whether the expression of Sema3A is modified in parallel with structural plasticity in the adult CNS, we examined Sema3A expression in the deep cerebellar nuclei of the adult mouse in a number of conditions associated with structural reorganization of the local connectivity. We found that Sema3A in PNNs is reduced during enhanced neuritic remodeling, in both physiological and injury-induced conditions. Moreover, we provide evidence that Sema3A is tightly associated with Purkinje axons and their terminals and its amount in the PNNs is related to Purkinje cell innervation of DCN neurons, but not to glutamatergic inputs. On the whole these data suggest that Sema3A may contribute to the growth-inhibitory properties of PNNs and Purkinje neurons may directly control their specific connection pattern through the release and capture of this guidance cue in the specialized ECM that surrounds their terminals.