Distinct roles for secreted semaphorin signaling in spinal motor axon guidance

Neuropilins, secreted semaphorin coreceptors, are expressed in discrete populations of spinal motor neurons, suggesting they provide critical guidance information for the establishment of functional motor circuitry. We show here that motor axon growth and guidance are impaired in the absence of Sema3A-Npn-1 signaling. Motor axons enter the limb precociously, showing that Sema3A controls the timing of motor axon in-growth to the limb. Lateral motor column (LMC) motor axons within spinal nerves are defasciculated as they grow toward the limb and converge in the plexus region. Medial and lateral LMC motor axons show dorso-ventral guidance defects in the forelimb. In contrast, Sema3F-Npn-2 signaling guides the axons of a medial subset of LMC neurons to the ventral limb, but plays no major role in regulating their fasciculation. Thus, Sema3A-Npn-1 and Sema3F-Npn-2 signaling control distinct steps of motor axon growth and guidance during the formation of spinal motor connections.     

Semaphorin 3A Binds to the Perineuronal Nets via Chondroitin Sulfate Type E Motifs in Rodent Brains

Chondroitin sulfate (CS) and the CS-rich extracellular matrix structures called perineuronal nets (PNNs) restrict plasticity and regeneration in the CNS. Plasticity is enhanced by chondroitinase ABC treatment that removes CS from its core protein in the chondroitin sulfate proteoglycans or by preventing the formation of PNNs, suggesting that chondroitin sulfate proteoglycans in the PNNs control plasticity. Recently, we have shown that semaphorin3A (Sema3A), a repulsive axon guidance molecule, localizes to the PNNs and is removed by chondroitinase ABC treatment (Vo, T., Carulli, D., Ehlert, E. M., Kwok, J. C., Dick, G., Mecollari, V., Moloney, E. B., Neufeld, G., de Winter, F., Fawcett, J. W., and Verhaagen, J. (2013) Mol. Cell. Neurosci. 56C, 186–200). Sema3A is therefore a candidate for a PNN effector in controlling plasticity. Here, we characterize the interaction of Sema3A with CS of the PNNs. Recombinant Sema3A interacts with CS type E (CS-E), and this interaction is involved in the binding of Sema3A to rat brain-derived PNN glycosaminoglycans, as demonstrated by the use of CS-E blocking antibody GD3G7. In addition, we investigate the release of endogenous Sema3A from rat brain by biochemical and enzymatic extractions. Our results confirm the interaction of Sema3A with CS-E containing glycosaminoglycans in the dense extracellular matrix of rat brain. We also demonstrate that the combination of Sema3A and PNN GAGs is a potent inhibitor of axon growth, and this inhibition is reduced by the CS-E blocking antibody. In conclusion, Sema3A binding to CS-E in the PNNs may be a mechanism whereby PNNs restrict growth and plasticity and may represent a possible point of intervention to facilitate neuronal plasticity.     

Experience-dependent plasticity and modulation of growth regulatory molecules at central synapses

Structural remodeling or repair of neural circuits depends on the balance between intrinsic neuronal properties and regulatory cues present in the surrounding microenvironment. These processes are also influenced by experience, but it is still unclear how external stimuli modulate growth-regulatory mechanisms in the central nervous system. We asked whether environmental stimulation promotes neuronal plasticity by modifying the expression of growth-inhibitory molecules, specifically those of the extracellular matrix. We examined the effects of an enriched environment on neuritic remodeling and modulation of perineuronal nets in the deep cerebellar nuclei of adult mice. Perineuronal nets are meshworks of extracellular matrix that enwrap the neuronal perikaryon and restrict plasticity in the adult CNS. We found that exposure to an enriched environment induces significant morphological changes of Purkinje and precerebellar axon terminals in the cerebellar nuclei, accompanied by a conspicuous reduction of perineuronal nets. In the animals reared in an enriched environment, cerebellar nuclear neurons show decreased expression of mRNAs coding for key matrix components (as shown by real time PCR experiments), and enhanced activity of matrix degrading enzymes (matrix metalloproteinases 2 and 9), which was assessed by in situ zymography. Accordingly, we found that in mutant mice lacking a crucial perineuronal net component, cartilage link protein 1, perineuronal nets around cerebellar neurons are disrupted and plasticity of Purkinje cell terminal is enhanced. Moreover, all the effects of environmental stimulation are amplified if the afferent Purkinje axons are endowed with enhanced intrinsic growth capabilities, induced by overexpression of GAP-43. Our observations show that the maintenance and growth-inhibitory function of perineuronal nets are regulated by a dynamic interplay between pre- and postsynaptic neurons. External stimuli act on this interaction and shift the balance between synthesis and removal of matrix components in order to facilitate neuritic growth by locally dampening the activity of inhibitory cues.     

Type III neuregulin 1 regulates pathfinding of sensory axons in the developing spinal cord and periphery

Sensory axons must develop appropriate connections with both central and peripheral targets. Whereas the peripheral cues have provided a classic model for neuron survival and guidance, less is known about the central cues or the coordination of central and peripheral connectivity. Here we find that type III Nrg1, in addition to its known effect on neuron survival, regulates axon pathfinding. In type III Nrg1(-/-) mice, death of TrkA(+) nociceptive/thermoreceptive neurons was increased, and could be rescued by Bax elimination. In the Bax and type III Nrg1 double mutants, axon pathfinding abnormalities were seen for TrkA(+) neurons both in cutaneous peripheral targets and in spinal cord central targets. Axon guidance phenotypes in the spinal cord included penetration of axons into ventral regions from which they would normally be repelled by Sema3A. Accordingly, sensory neurons from type III Nrg1(-/-) mice were unresponsive to the repellent effects of Sema3A in vitro, which might account, at least in part, for the central projection phenotype, and demonstrates an effect of type III Nrg1 on guidance cue responsiveness in neurons. Moreover, stimulation of type III Nrg1 back-signaling in cultured sensory neurons was found to regulate axonal levels of the Sema3A receptor neuropilin 1. These results reveal a molecular mechanism whereby type III Nrg1 signaling can regulate the responsiveness of neurons to a guidance cue, and show that type III Nrg1 is required for normal sensory neuron survival and axon pathfinding in both central and peripheral targets.     

Ectopic myelinating oligodendrocytes in the dorsal spinal cord as a consequence of altered semaphorin 6D signaling inhibit synapse formation

Different types of sensory neurons in the dorsal root ganglia project axons to the spinal cord to convey peripheral information to the central nervous system. Whereas most proprioceptive axons enter the spinal cord medially, cutaneous axons typically do so laterally. Because heavily myelinated proprioceptive axons project to the ventral spinal cord, proprioceptive axons and their associated oligodendrocytes avoid the superficial dorsal horn. However, it remains unclear whether their exclusion from the superficial dorsal horn is an important aspect of neural circuitry. Here we show that a mouse null mutation of Sema6d results in ectopic placement of the shafts of proprioceptive axons and their associated oligodendrocytes in the superficial dorsal horn, disrupting its synaptic organization. Anatomical and electrophysiological analyses show that proper axon positioning does not seem to be required for sensory afferent connectivity with motor neurons. Furthermore, ablation of oligodendrocytes from Sema6d mutants reveals that ectopic oligodendrocytes, but not proprioceptive axons, inhibit synapse formation in Sema6d mutants. Our findings provide new insights into the relationship between oligodendrocytes and synapse formation in vivo, which might be an important element in controlling the development of neural wiring in the central nervous system.     

Motoneuronal Sema3C is essential for setting stereotyped motor tract positioning in limb-derived chemotropic semaphorins

The wiring of neuronal circuits requires complex mechanisms to guide axon subsets to their specific target with high precision. To overcome the limited number of guidance cues, modulation of axon responsiveness is crucial for specifying accurate trajectories. We report here a novel mechanism by which ligand/receptor co-expression in neurons modulates the integration of other guidance cues by the growth cone. Class 3 semaphorins (Sema3 semaphorins) are chemotropic guidance cues for various neuronal projections, among which are spinal motor axons navigating towards their peripheral target muscles. Intriguingly, Sema3 proteins are dynamically expressed, forming a code in motoneuron subpopulations, whereas their receptors, the neuropilins, are expressed in most of them. Targeted gain- and loss-of-function approaches in the chick neural tube were performed to enable selective manipulation of Sema3C expression in motoneurons. We show that motoneuronal Sema3C regulates the shared Sema3 neuropilin receptors Nrp1 and Nrp2 levels in opposite ways at the growth cone surface. This sets the respective responsiveness to exogenous Nrp1- and Nrp2-dependent Sema3A, Sema3F and Sema3C repellents. Moreover, in vivo analysis revealed a context where this modulation is essential. Motor axons innervating the forelimb muscles are exposed to combined expressions of semaphorins. We show first that the positioning of spinal nerves is highly stereotyped and second that it is compromised by alteration of motoneuronal Sema3C. Thus, the role of the motoneuronal Sema3 code could be to set population-specific axon sensitivity to limb-derived chemotropic Sema3 proteins, therefore specifying stereotyped motor nerve trajectories in their target field.     

Three-dimensional microanatomy of perineuronal proteoglycan nets enveloping motor neurons in the rat spinal cord

Summary. Spinal motor neurons possess reticular coats of extracellular matrix proteoglycans on their somata and proximal dendrites. In order to define the anatomical background of the network, spatial relationships of the perineuronal proteoglycans with synaptic boutons and astrocyte processes were analyzed in rat motor neurons by TEM after histochemical detection of the substances with cationic iron colloid, and by SEM after exposure of the cytoarchitecture with NaOH maceration. Narrow intercellular channels filled with proteoglycan were found to extend along the surface of the neurons to form a homogeneous network of a mesh size of about 1 µm. The system of perineuronal channels consisted of two parts: a primary intervaricose net which meandered among synaptic boutons on the surface of the motor neuron, and secondary subvaricose nets which irrigated interfaces between larger boutons and the neuron. No elements in the perineuronal cytoarchitecture coincided with the meshwork of proteoglycan, indicating the involvement of postsynaptic factors in the distribution of the substance. Thin astrocyte processes surrounding the neurons formed a distinct network with heterogeneous meshes corresponding to boutons of various sizes. The perineuronal glial nets extended their surface area in contact with the intervaricose nets of proteoglycan by complex cellular interdigitations. The subvaricose nets of proteoglycan compartmentalized multiple synapses on large boutons, suggesting an involvement in the division of the synapses during development.     

Semaphorin 5A is a bifunctional axon guidance cue for axial motoneurons in vivo

Semaphorins are a large class of proteins that function throughout the nervous system to guide axons. It had previously been shown that Semaphorin 5A (Sema5A) was a bifunctional axon guidance cue for mammalian midbrain neurons. We found that zebrafish sema5A was expressed in myotomes during the period of motor axon outgrowth. To determine whether Sema5A functioned in motor axon guidance, we knocked down Sema5A, which resulted in two phenotypes: a delay in motor axon extension into the ventral myotome and aberrant branching of these motor axons. Both phenotypes were rescued by injection of full-length rat Sema5A mRNA. However, adding back RNA encoding the sema domain alone significantly rescued the branching phenotype in sema5A morphants. Conversely, adding back RNA encoding the thrombospondin repeat (TSR) domain alone into sema5A morphants exclusively rescued delay in ventral motor axon extension. Together, these data show that Sema5A is a bifunctional axon guidance cue for vertebrate motor axons in vivo. The TSR domain promotes growth of developing motor axons into the ventral myotome whereas the sema domain mediates repulsion and keeps these motor axons from branching into surrounding myotome regions.     

Semaphorin 2a secreted by oenocytes signals through plexin B and plexin A to guide sensory axons in the Drosophila embryo

The semaphorin gene family has been shown to play important roles in axonal guidance in both vertebrates and invertebrates. Both transmembrane (Sema1a, Sema1b, Sema5c) and secreted (Sema2a, Sema2b) forms of semaphorins exist in Drosophila. Two Sema receptors, plexins (Plex) A and B, have also been identified. Many questions remain concerning the axon guidance functions of the secreted semaphorins, including the identity of their receptors. We have used the well-characterized sensory system of the Drosophila embryo to address these problems. We find novel sensory axon defects in sema2a loss-of-function mutants in which particular axons misproject and follow inappropriate pathways to the CNS. plexB loss-of-function mutants show similar phenotypes to sema2a mutants and sema2a interacts genetically with plexB, supporting the hypothesis that Sema2a signals through PlexB receptors. Sema2a protein is expressed by larval oenocytes, a cluster of secretory cells in the lateral region of the embryo and the sema2a mutant phenotype can be rescued by driving Sema2a in these cells. Ablation of oenocytes results in sensory axon defects similar to the sema2a mutant phenotype. These data support a model in which Sema2a, while being secreted from oenocytes, acts in a highly localized fashion: It represses axon extension from the sensory neuron cell body, but only in regions in direct contact with oenocytes.     

Cranial nerve fasciculation and Schwann cell migration are impaired after loss of Npn-1

Interaction of the axon guidance receptor Neuropilin-1 (Npn-1) with its repulsive ligand Semaphorin 3A (Sema3A) is crucial for guidance decisions, fasciculation, timing of growth and axon–axon interactions of sensory and motor projections in the embryonic limb. At cranial levels, Npn-1 is expressed in motor neurons and sensory ganglia and loss of Sema3A–Npn-1 signaling leads to defasciculation of the superficial projections to the head and neck. The molecular mechanisms that govern the initial fasciculation and growth of the purely motor projections of the hypoglossal and abducens nerves in general, and the role of Npn-1 during these events in particular are, however, not well understood. We show here that selective removal of Npn-1 from somatic motor neurons impairs initial fasciculation and assembly of hypoglossal rootlets and leads to reduced numbers of abducens and hypoglossal fibers. Ablation of Npn-1 specifically from cranial neural crest and placodally derived sensory tissues recapitulates the distal defasciculation of mixed sensory-motor nerves of trigeminal, facial, glossopharyngeal and vagal projections, which was observed in Npn-1^−/− and Npn-1^Sema− mutants. Surprisingly, the assembly and fasciculation of the purely motor hypoglossal nerve are also impaired and the number of Schwann cells migrating along the defasciculated axonal projections is reduced. These findings are corroborated by partial genetic elimination of cranial neural crest and embryonic placodes, where loss of Schwann cell precursors leads to aberrant growth patterns of the hypoglossal nerve. Interestingly, rostral turning of hypoglossal axons is not perturbed in any of the investigated genotypes. Thus, initial hypoglossal nerve assembly and fasciculation, but not later guidance decisions depend on Npn-1 expression and axon–Schwann cell interactions.     

Gating of Sema3E/PlexinD1 signaling by neuropilin-1 switches axonal repulsion to attraction during brain development

The establishment of functional neural circuits requires the guidance of axons in response to the actions of secreted and cell-surface molecules such as the semaphorins. Semaphorin 3E and its receptor PlexinD1 are expressed in the brain, but their functions are unknown. Here, we show that Sema3E/PlexinD1 signaling plays an important role in initial development of descending axon tracts in the forebrain. Early errors in axonal projections are reflected in behavioral deficits in Sema3E null mutant mice. Two distinct signaling mechanisms can be distinguished downstream of Sema3E. On corticofugal and striatonigral neurons expressing PlexinD1 but not Neuropilin-1, Sema3E acts as a repellent. In contrast, on subiculo-mammillary neurons coexpressing PlexinD1 and Neuropilin-1, Sema3E acts as an attractant. The extracellular domain of Neuropilin-1 is sufficient to convert repulsive signaling by PlexinD1 to attraction. Our data therefore reveal a "gating" function of neuropilins in semaphorin-plexin signaling during the assembly of forebrain neuronal circuits.     

Wiring the zebrafish: axon guidance and synaptogenesis

Many zebrafish mutants have specific defects in axon guidance or synaptogenesis, particularly in the retinotectal and motor systems. Several mutants have now been characterized in detail and/or cloned. A combination of genetic studies, in vivo imaging and new techniques for misexpressing genes or blocking their function promises to reveal the molecules and principles that govern wiring of the vertebrate nervous system.     

Characterization of a novel member of murine semaphorin family

Semaphorin gene family contains a large number of secreted and transmembrane proteins, and some of them are functioning as the repulsive and attractive cues of the axon guidance during development. Here we report murine orthologues of a novel member of class 6 semaphorin gene, semaphorin 6D (Sema6D), mapped on the chromosome 2. Sema6D is mainly expressed in the brain and lung, and the ubiquitous expression in the brain continues from embryonic late stage to adulthood, as determined by Northern blot and in situ hybridization. We also found that Sema6D has five different splicing variants, and the expression patterns of individual isoforms differ depending on the tissues. Thus, Sema6D may play important roles in various functions including the axon guidance during development and neuronal plasticity.     

Immunohistochemical mapping of perineuronal nets containing chondroitin unsulfate proteoglycan in the rat central nervous system

Subsets of neurons ensheathed by perineuronal nets containing chondroitin unsulfate proteoglycan have been immunohistochemically mapped throughout the rat central nervous system from the olfactory bulb to the spinal cord. A variable proportion of neurons were outlined by immunoreactivity for the monoclonal antibody (Mab 1B5), but only after chondroitinase ABC digestion. In forebrain cortical structures the only immunoreactive nets were around interneurons; in contrast, throughout the brainstem and spinal cord a large proportion of projection neurons were surrounded by intense immunoreactivity. Immunoreactivity was ordinarily found in the neuropil between neurons surrounded by an immunopositive net. By contrast, within the pyriform cortex the neuropil of the plexiform layer was intensely immunoreactive even though no perineuronal net could be found. The presence of perineuronal nets could not be correlated with any single class of neurons; however a few functionally related groups (e.g., motor and motor-related structures: motor neurons both in the spinal cord and in the efferent somatic nuclei of the brainstem, deep cerebellar nuclei, vestibular nuclei; red nucleus, reticular formation; central auditory pathway: ventral cochlear nucleus, trapezoid body, superior olive, nucleus of the lateral lemniscus, inferior colliculus, medial geniculate body) were the main components of the neuronal subpopulation displaying chondroitin unsulfate proteoglycans in the surrounding extracellular matrix. The immunodecorated neurons found in the present study and those shown by different monoclonal antibodies or by lectin cytochemisty, revealed consistent overlapping of their distribution patterns.     

Semaphorin3A regulates axon growth independently of growth cone repulsion via modulation of TrkA signaling

Regulation of axon growth is a critical event in neuronal development. Nerve growth factor (NGF) is a strong inducer of axon growth and survival in the dorsal root ganglia (DRG). Paradoxically, high concentrations of NGF are present in the target region where axon growth must slow down for axons to accurately identify their correct targets. Semaphorin3A (Sema3A), a powerful axonal repellent molecule for DRG neurons, is also situated in their target regions. NGF is a modulator of Sema3A-induced repulsion and death. We show that Sema3A is a regulator of NGF-induced neurite outgrowth via the TrkA receptor, independent of its growth cone repulsion activity. First, neurite outgrowth of DRG neurons is more sensitive to Sema3A than repulsion. Second, at concentrations sufficient to significantly inhibit Sema3A-induced repulsion, NGF has no effect on Sema3A-induced axon growth inhibition. Third, Sema3A-induced outgrowth inhibition, but not repulsion activity, is dependent on NGF stimulation. Fourth, Sema3A attenuates TrkA-mediated growth signaling, but not survival signaling, and over-expression of constitutively active TrkA blocks Sema3A-induced axon growth inhibition, suggesting that Sema3A activity is mediated via regulation of NGF/TrkA-induced growth. Finally, quantitative analysis of axon growth in vivo supports the possibility that Sema3A affects axon growth, in addition to its well-documented role in axon guidance. We suggest a model whereby NGF at high concentrations in the target region is important for survival, attraction and inhibition of Sema3A-induced repulsion, while Sema3A inhibits its growth-promoting activity. The combined and cross-modulatory effects of these two signaling molecules ensure the accuracy of the final stages in axon targeting.     

Axon response to guidance cues is stimulated by acetylcholine in Caenorhabditis elegans

Gradients of acetylcholine can stimulate growth cone turning when applied to neurons grown in culture, and it has been suggested that acetylcholine could act as a guidance cue. However, the role acetylcholine plays in directing axon migrations in vivo is not clear. Here, we show that acetylcholine positively regulates signaling pathways that mediate axon responses to guidance cues in Caenorhabditis elegans. Mutations that disrupt acetylcholine synthesis, transportation, and secretion affect circumferential axon guidance of the AVM neuron and in these mutants exogenously supplied acetylcholine improves AVM circumferential axon guidance. These effects are not observed for the circumferential guidance of the DD and VD motor neuron axons, which are neighbors of the AVM axon. Circumferential guidance is directed by the UNC-6 (netrin) and SLT-1 (slit) extracellular cues, and exogenously supplied acetylcholine can improve AVM axon guidance in mutants when either UNC-6- or SLT-1-induced signaling is disrupted, but not when both signaling pathways are perturbed. Not in any of the mutants does exogenously supplied acetylcholine improve DD and VD axon guidance. The ability of acetylcholine to enhance AVM axon guidance only in the presence of either UNC-6 or SLT-1 indicates that acetylcholine potentiates UNC-6 and SLT-1 guidance activity, rather than acting itself as a guidance cue. Together, our results show that for specific neurons acetylcholine plays an important role in vivo as a modulator of axon responses to guidance cues.     

Formation and remodeling of the brain extracellular matrix in neural plasticity: Roles of chondroitin sulfate and hyaluronan

BackgroundThe extracellular matrix (ECM) of the brain is rich in glycosaminoglycans such as chondroitin sulfate (CS) and hyaluronan. These glycosaminoglycans are organized into either diffuse or condensed ECM. Diffuse ECM is distributed throughout the brain and fills perisynaptic spaces, whereas condensed ECM selectively surrounds parvalbumin-expressing inhibitory neurons (PV cells) in mesh-like structures called perineuronal nets (PNNs). The brain ECM acts as a non-specific physical barrier that modulates neural plasticity and axon regeneration.Scope of reviewHere, we review recent progress in understanding of the molecular basis of organization and remodeling of the brain ECM, and the involvement of several types of experience-dependent neural plasticity, with a particular focus on the mechanism that regulates PV cell function through specific interactions between CS chains and their binding partners. We also discuss how the barrier function of the brain ECM restricts dendritic spine dynamics and limits axon regeneration after injury.Major conclusionsThe brain ECM not only forms physical barriers that modulate neural plasticity and axon regeneration, but also forms molecular brakes that actively controls maturation of PV cells and synapse plasticity in which sulfation patterns of CS chains play a key role. Structural remodeling of the brain ECM modulates neural function during development and pathogenesis.General significanceGenetic or enzymatic manipulation of the brain ECM may restore neural plasticity and enhance recovery from nerve injury.This article is part of a Special Issue entitled Neuro-glycoscience, edited by Kenji Kadomatsu and Hiroshi Kitagawa.     

Deletion of Sema3a or plexinA1/plexinA3 Causes Defects in Sensory Afferent Projections of Statoacoustic Ganglion Neurons

Statoacoustic ganglion (SAG) neurons project sensory afferents to appropriate targets in the inner ear to form functional vestibular and auditory circuits. Neuropilin1 (Npn1), a receptor for class 3 semaphorins, is required to generate appropriate afferent projections in SAG neurons; however, the ligands and coreceptors involved in Npn1 functioning remain unknown. Here we show that both plexinA1 and plexinA3 are expressed by SAG neurons, and plexinA1/plexinA3 double mutant mice show defects in afferent projections of SAG neurons in the inner ear. In control mice, sensory afferents of SAG neurons terminate at the vestibular sensory patches, whereas in plexinA1/plexinA3 double mutants, they extend more dorsally in the inner ear beyond normal vestibular target areas. Moreover, we find that semaphorin3a (Sema3a) is expressed in the dorsal otocyst, and Sema3a mutant mice show defects in afferent projections of SAG neurons similar to those observed in plexinA1/plexinA3 double mutants and in mice lacking a functional Npn1 receptor. Taken together, these genetic findings demonstrate that Sema3a repellent signaling plays a role in the establishment of proper afferent projections in SAG neurons, and this signaling likely occurs through a receptor complex involving Npn1 and either plexinA1 or plexinA3.