The role of repulsive guidance molecules in the embryonic and adult vertebrate central nervous system

During the development of the nervous system, outgrowing axons often have to travel long distances to reach their target neurons. In this process, outgrowing neurites tipped with motile growth cones rely on guidance cues present in their local environment. These cues are detected by specific receptors expressed on growth cones and neurites and influence the trajectory of the growing fibres. Neurite growth, guidance, target innervation and synapse formation and maturation are the processes that occur predominantly but not exclusively during embryonic or early post-natal development in vertebrates. As a result, a functional neural network is established, which is usually remarkably stable. However, the stability of the neural network in higher vertebrates comes at an expensive price, i.e. the loss of any significant ability to regenerate injured or damaged neuronal connections in their central nervous system (CNS). Most importantly, neurite growth inhibitors prevent any regenerative growth of injured nerve fibres. Some of these inhibitors are associated with CNS myelin, others are found at the lesion site and in the scar tissue. Traumatic injuries in brain and spinal cord of mammals induce upregulation of embryonic inhibitory or repulsive guidance cues and their receptors on the neurites. An example for embryonic repulsive directional cues re-expressed at lesion sites in both the rat and human CNS is provided with repulsive guidance molecules, a new family of directional guidance cues.     

Repulsive guidance molecule b inhibits neurite growth and is increased after spinal cord injury

Neuronal axons are guided by attractive and repulsive cues in their local environment. Since the identification of the repulsive guidance molecule (RGM) a (RGMa) as an axon repellent in the visual system, diverse functions, as part of the developing and adult central nervous system (CNS), have been ascribed to it. The binding of RGMa to its receptor neogenin has been shown to induce RhoA activation, leading to inhibitory/repulsive behavior and the collapse of the neuronal growth cone. In this paper, we provide evidence to suggest the involvement of RGMb, another member of the RGM family, in the rat CNS. RGMb inhibits neurite outgrowth in postnatal cerebellar granule neurons (CGNs) in vitro. RGMb is expressed by oligodendrocytes and neurons in the adult rat CNS, and the expression of this molecule is upregulated around the site of spinal cord injury. RGMb is present in myelin isolated from an adult rat brain. RGMb and neogenin are coexpressed in CGNs and entorhinal cortex neurons. These findings suggest that RGMb is a myelin-derived inhibitor of axon growth in the CNS. Inhibition of RGMb may provide an alternative approach for the treatment of spinal injuries.     

Antibody that neutralizes myelin-associated inhibitors of axon growth does not interfere with recognition of target-specific guidance information by rat retinal axons

During development, many CNS projection neurons establish topographically ordered maps in their target regions. Myelin-associated inhibitors of neurite growth contribute to the confinement of fiber tracts during development and limit plastic changes after CNS projections have been formed. Neutralization of myelin-associated growth inhibitors leads to an expansion of the retinal innervation of the superior colliculus (SC). In the lesioned adult mammalian CNS, these long projection neurons are usually unable to regrow axons over long distances after lesion due to myelin-associated inhibitors, which interfere with axonal growth in vivo and in vitro. Application of a specific antibody directed against myelin-inhibitors (IN-1) promotes regrowth of corticospinal tract or retinal ganglion cell axons. In the present study, we asked whether application of an antibody to myelin-associated growth inhibitors would lead to disturbances of target-specific axon guidance. To examine this issue, we used an in vitro model, the “stripe assay,” to examine the behavior of rat retinal ganglion cell axons on membranes from embryonic and deafferented adult rat SC. On membrane preparations from embryonic rat SC, retinal fibers avoid posterior tectal membranes, possibly due to the presence of a repulsive factor. Nasal retinal axons show a random growth pattern. On membranes prepared from the deafferented adult rat SC, temporal and nasal axons prefer to grow on membranes prepared from their specific target region, which suggests the involvement of target-derived attractive guidance components. The results of the present study show that retinal axons grow significantly faster in the presence of IN-1 antibody that neutralizes myelin-associated growth inhibitors present in the membrane preparations from the adult rat SC. IN-1 antibody, however, does not interfere with specific axonal guidance. This suggests that axonal guidance and specific target finding are independently regulated in retinal axons. © 1996 John Wiley & Sons, Inc.     

The function of leak and kuzbanian during growth cone and cell migration

Axonal growth cones require an evolutionary conserved repulsive guidance system to ensure proper crossing of the CNS midline. In Drosophila, the Slit protein is a repulsive signal secreted by the midline glial cells. It binds to the Roundabout receptors, which are expressed on CNS axons in the longitudinal tracts but not in the commissural tracts. Here we present an analysis of the genes leak and kuzbanian and show that both genes are involved in the repulsive guidance system operating at the CNS midline. Mutations in leak, which encodes the Roundabout-2 Slit receptor, were first recovered by Nüsslein-Volhard and co-workers based on defects in the larval cuticle. Analysis of the head phenotype suggests that slit may be able to act as an attractive guidance cue while directing the movements of the dorsal ectodermal cell sheath. kuzbanian also regulates midline crossing of CNS axons. It encodes a metalloprotease of the ADAM family and genetically interacts with slit. Expression of a dominant negative Kuzbanian protein in the CNS midline cells results in an abnormal midline crossing of axons and prevents the clearance of the Roundabout receptor from commissural axons. Our analyses support a model in which Kuzbanian mediates the proteolytic activation of the Slit/Roundabout receptor complex.     

Myosin IIA is required for neurite outgrowth inhibition produced by repulsive guidance molecule

Although myelin-associated neurite outgrowth inhibitors express their effects through RhoA/Rho-kinase, the downstream targets of Rho-kinase remain unknown. We examined the involvement of myosin II, which is one of the downstream targets of Rho-kinase, by using blebbistatin – a specific myosin II inhibitor – and small interfering RNA targeting two myosin II isoforms, namely, MIIA and MIIB. We found that neurite outgrowth inhibition by repulsive guidance molecule (RGMa) was mediated via myosin II, particularly MIIA, in cerebellar granule neurons. RGMa induced myosin light chain (MLC) phosphorylation by a Rho-kinase-dependent mechanism. After spinal cord injury in rats, phosphorylated MLC in axons around the lesion site was up-regulated, and this effect depends on Rho-kinase activity. Further, RGMa-induced F-actin reduction in growth cones and growth cone collapse were mediated by MIIA. We conclude that Rho-kinase-dependent activation of MIIA via MLC phosphorylation induces F-actin reduction and growth cone collapse and the subsequent neurite retraction/outgrowth inhibition triggered by RGMa.     

Outgrowth of neurites from NIE-115 neuroblastoma cells is prevented on repulsive substrates through the action of PAK

In the central nervous system (CNS), damaged axons are inhibited from regeneration by glial scars, where secreted chondroitin sulfate proteoglycan (CSPG) and tenascin repulse outgrowth of neurites, the forerunners of axons and dendrites. During differentiation, these molecules are thought to form boundaries for guiding neurons to their correct targets. In neuroblastoma NIE-115 cells, outgrowth of neurites on laminin could be induced by serum starvation or inhibition of RhoA by Clostridium botulinum C3 toxin. The outgrowing neurites avoided crossing onto the repulsive substrate CSPG or tenascin. This avoidance response was partially overcome on expression of membrane-targeted and kinase-inactive forms of PAK. In these cells, the endogenous PAK isoforms colocalized with actin in distinctive sites, alphaPAK in the cell center as small clusters and along the neurite shaft and betaPAK and gammaPAK in areas with membrane ruffles and filopodia, respectively. When isoform-specific N-terminal PAK sequences were introduced to interfere with PAK function, substantially more neurites crossed onto CSPG when cells contained a gammaPAK-derived peptide but not the corresponding alphaPAK- or betaPAK-derived peptide. Thus, while neurite outgrowth can be promoted by RhoA inhibition, overcoming the accompanying repulsive guidance response will require modulation of PAK activity. These results have therapeutic implications for CNS repair processes.     

Activated microglia inhibit axonal growth through RGMa

By causing damage to neural networks, spinal cord injuries (SCI) often result in severe motor and sensory dysfunction. Functional recovery requires axonal regrowth and regeneration of neural network, processes that are quite limited in the adult central nervous system (CNS). Previous work has shown that SCI lesions contain an accumulation of activated microglia, which can have multiple pathophysiological influences. Here, we show that activated microglia inhibit axonal growth via repulsive guidance molecule a (RGMa). We found that microglia activated by lipopolysaccharide (LPS) inhibited neurite outgrowth and induced growth cone collapse of cortical neurons in vitro--a pattern that was only observed when there was direct contact between microglia and neurons. After microglia were activated by LPS, they increased expression of RGMa; however, treatment with RGMa-neutralizing antibodies or transfection of RGMa siRNA attenuated the inhibitory effects of microglia on axonal outgrowth. Furthermore, minocycline, an inhibitor of microglial activation, attenuated the effects of microglia and RGMa expression. Finally, we examined whether these in vitro patterns could also be observed in vivo. Indeed, in a mouse SCI model, minocycline treatment reduced the accumulation of microglia and decreased RGMa expression after SCI, leading to reduced dieback in injured corticospinal tracts. These results suggest that activated microglia play a major role in inhibiting axon regeneration via RGMa in the injured CNS.     

The Involvement of the Myelin-Associated Inhibitors and Their Receptors in CNS Plasticity and Injury

The limited capacity for the central nervous system (CNS) to repair itself was first described over 100 years ago by Spanish neuroscientist Ramon Y. Cajal. However, the exact mechanisms underlying this failure in neuronal regeneration remain unclear and, as such, no effective therapeutics yet exist. Numerous studies have attempted to elucidate the biochemical and molecular mechanisms that inhibit neuronal repair with increasing evidence suggesting that several inhibitory factors and repulsive guidance cues active during development actually persist into adulthood and may be contributing to the inhibition of repair. For example, in the injured adult CNS, there are various inhibitory factors that impede the outgrowth of neurites from damaged neurons. One of the most potent of these neurite outgrowth inhibitors is the group of proteins known as the myelin-associated inhibitors (MAIs), present mainly on the membranes of oligodendroglia. Several studies have shown that interfering with these proteins can have positive outcomes in CNS injury models by promoting neurite outgrowth and improving functional recovery. As such, the MAIs, their receptors, and downstream effectors are valid drug targets for the treatment of CNS injury. This review will discuss the current literature on MAIs in the context of CNS development, plasticity, and injury. Molecules that interfere with the MAIs and their receptors as potential candidates for the treatment of CNS injury will additionally be introduced in the context of preclinical and clinical trials.     

R-Ras as a key player for signaling pathway of plexins

Axon guidance represents an important step in the formation of neuronal networks. Axons are guided by various guidance factors, such as semaphorins, slits, ephrins, and netrins. Plexins are cell surface receptors for the repulsive molecules of the semaphorin family. Cytoplasmic regions of plexins are responsible for initiating cellular signal transduction, resulting in axon repulsion. Recent advances have shed light on the signal transduction mechanism of plexins and the mechanisms by which it leads to a repulsive response. Plexin-B1 possesses an intrinsic guanine triphosphate (GTP)ase activating protein activity for R-Ras, a member of Ras family of small GTPases that has been implicated in promoting cell adhesion and neurite outgrowth through integrin activation. Stimulation of Plexin-B1 by Sema4D induces collapse of the growth cone through down-regulation of R-Ras activity. This article summarizes current understanding of the signaling mechanisms of plexins.     

Regulating actin dynamics in neuronal growth cones by ADF/cofilin and rho family GTPases

Growth cone motility and navigation in response to extracellular signals are regulated by actin dynamics. To better understand actin involvement in these processes we determined how and in what form actin reaches growth cones, and once there, how actin assembly is regulated. A continuous supply of actin is maintained at the axon tip by slow transport, the mobile component consisting of an unassembled form of actin. Actin is co-transported with actin-binding proteins, including ADF and cofilin, structurally related proteins essential for rapid turnover of actin filaments in vivo. ADF and cofilin activity is regulated through phosphorylation by LIM kinases, downstream effectors of the Rho family of GTPases, Cdc42, Rac and Rho. Attractive and repulsive extracellular guidance cues might locally alter actin dynamics by binding specific GTPase-linked receptors, activating LIM kinases, and subsequently modulating the activity of ADF/cofilin. ADF is enriched in growth cones and is required for neurite outgrowth. In addition, signals that influence growth cone behavior alter ADF/cofilin phosphorylation, and overexpression of ADF enhances neurite outgrowth. Growth promoting effects of laminin are mimicked by expression of constitutively active Cdc42 and blocked by expression of the dominant negative Cdc42. Repulsive effects of myelin and sema3D on growth cones are blocked by expression of constitutively active Rac1 and dominant negative Rac1, respectively. Thus a series of complex pathways must exist for regulating effectors of actin dynamics. The bifurcating nature of the ADF/cofilin phosphorylation pathway may provide the integration necessary for this complex regulation.     

Growth cone collapse through coincident loss of actin bundles and leading edge actin without actin depolymerization

Repulsive guidance cues can either collapse the whole growth cone to arrest neurite outgrowth or cause asymmetric collapse leading to growth cone turning. How signals from repulsive cues are translated by growth cones into this morphological change through rearranging the cytoskeleton is unclear. We examined three factors that are able to induce the collapse of extending Helisoma growth cones in conditioned medium, including serotonin, myosin light chain kinase inhibitor, and phorbol ester. To study the cytoskeletal events contributing to collapse, we cultured Helisoma growth cones on polylysine in which lamellipodial collapse was prevented by substrate adhesion. We found that all three factors that induced collapse of extending growth cones also caused actin bundle loss in polylysine-attached growth cones without loss of actin meshwork. In addition, actin bundle loss correlated with specific filamentous actin redistribution away from the leading edge that is characteristic of repulsive factors. Finally, we provide direct evidence using time-lapse studies of extending growth cones that actin bundle loss paralleled collapse. Taken together, these results suggest that actin bundles could be a common cytoskeletal target of various collapsing factors, which may use different signaling pathways that converge to induce growth cone collapse.     

Preferential outgrowth of central nervous system neurites on astrocytes and Schwann cells as compared with nonglial cells in vitro

I have compared central nervous system (CNS) neurite outgrowth on glial and nonglial cells. Monolayers of glial cells (astrocytes and Schwann cells) or nonglial cells (e.g., fibroblasts) were prepared and were shown to be greater than 95% pure as judged by cell type-specific markers. These monolayers were then tested for their ability to support neurite outgrowth from various CNS explants. While CNS neurites grew vigorously on the glial cells, most showed little growth on nonglial cell monolayers. Neurites grew singly or in fine fascicles on the glial cells at rates greater than 0.5 mm/d. The neurite outgrowth on astrocytes was investigated in detail. Scanning and transmission electron microscopy showed that the neurites were closely apposed to the astrocyte surface and that the growth cones were well spread with long filopodia. There was no evidence of significant numbers of explant- derived cells migrating onto the monolayers. Two types of experiments indicated that factors associated with the astrocyte surface were primarily responsible for the vigorous neurite outgrowth seen on these cells: (a) Conditioned media from either astrocytes or fibroblasts had no effect on the pattern of outgrowth on fibroblasts and astrocytes, and conditioned media factors from either cell type did not promote neurite outgrowth when bound to polylysine-coated dishes. (b) When growing CNS neurites encountered a boundary between astrocytes and fibroblasts, they stayed on the astrocytes and did not encroach onto the fibroblasts. These experiments strongly suggest that molecules specific to the surfaces of astrocytes make these cells particularly attractive substrates for CNS neurite outgrowth, and they raise the possibility that similar molecules on embryonic glial cells may play a role in guiding axonal growth during normal CNS development.     

Tenascin-C contains distinct adhesive, anti-adhesive, and neurite outgrowth promoting sites for neurons

The glia-derived extracellular matrix glycoprotein tenascin-C (TN-C) is transiently expressed in the developing CNS and may mediate neuron-glia interactions. Perturbation experiments with specific monoclonal antibodies suggested that TN-C functions for neural cells are encoded by distinct sites of the glycoprotein (Faissner, A., A. Scholze, and B. Gotz. 1994. Tenascin glycoproteins in developing neural tissues--only decoration? Persp. Dev. Neurobiol. 2:53-66). To characterize these further, bacterially expressed recombinant domains were generated and used for functional studies. Several short-term-binding sites for mouse CNS neurons could be assigned to the fibronectin type III (FNIII) domains. Of these, the alternatively spliced insert TNfnA1,2,4,B,D supported initial attachment for both embryonic day 18 (E18) rat and postnatal day 6 (P6) mouse neurons. Only TNfn1-3 supported binding and growth of P6 mouse cerebellar neurons after 24 h, whereas attachment to the other domains proved reversible and resulted in cell detachment or aggregation. In choice assays on patterned substrates, repulsive properties could be attributed to the EGF-type repeats TNegf, and to TNfnA1,2,4. Finally, neurite outgrowth promoting properties for E18 rat hippocampal neurons and P0 mouse DRG explants could be assigned to TNfnB,D, TNfnD,6, and TNfn6. The epitope of mAb J1/tn2 which abolishes the neurite outgrowth inducing effect of intact TN-C could be allocated to TNfnD. These observations suggest that TN-C harbors distinct cell- binding, repulsive, and neurite outgrowth promoting sites for neurons. Furthermore, the properties of isoform-specific TN-C domains suggest functional significance of the alternative splicing of TN-C glycoproteins.     

A neutralizing anti-Nogo66 receptor monoclonal antibody reverses inhibition of neurite outgrowth by central nervous system myelin

The Nogo66 receptor (NgR1) is a neuronal, leucine-rich repeat (LRR) protein that binds three central nervous system (CNS) myelin proteins, Nogo, myelin-associated glycoprotein, and oligodendrocyte myelin glycoprotein, and mediates their inhibitory effects on neurite growth. Although the LRR domains on NgR1 are necessary for binding to the myelin proteins, the exact epitope(s) involved in ligand binding is unclear. Here we report the generation and detailed characterization of an anti-NgR1 monoclonal antibody, 7E11. The 7E11 monoclonal antibody blocks Nogo, myelin-associated glycoprotein, and oligodendrocyte myelin glycoprotein binding to NgR1 with IC50 values of 120, 14, and 4.5 nm, respectively, and effectively promotes neurite outgrowth of P3 rat dorsal root ganglia neurons cultured on a CNS myelin substrate. Further, we have defined the molecular epitope of 7E11 to be DNAQLR located in the third LRR domain of rat NgR1. Our data demonstrate that anti-NgR1 antibodies recognizing this epitope, such as 7E11, can neutralize CNS myelin-dependent inhibition of neurite outgrowth. Thus, specific anti-NgR1 antibodies may represent a useful therapeutic approach for promoting CNS repair after injury.     

High-content neurite development study using optically patterned substrates

The study of neurite guidance in vitro relies on the ability to reproduce the distribution of attractive and repulsive guidance molecules normally expressed in vivo. The identification of subtle variations in the neurite response to changes in the spatial distribution of extracellular molecules can be achieved by monitoring the behavior of cells on protein gradients. To do this, automated high-content screening assays are needed to quantify the morphological changes resulting from growth on gradients of guidance molecules. Here, we present the use of laser-assisted protein adsorption by photobleaching (LAPAP) to allow the fabrication of large-scale substrate-bound laminin-1 gradients to study neurite extension. We produced thousands of gradients of different slopes and analyzed the variations in neurite attraction of neuron-like cells (RGC-5). An image analysis algorithm processed bright field microscopy images, detecting each cell and quantifying the soma centroid and the initiation, terminal and turning angles of the longest neurite.     

Th1 cells promote neurite outgrowth from cortical neurons via a mechanism dependent on semaphorins

The roles of T lymphocytes in the central nervous system (CNS) are diverse; their roles in the injured CNS have been reported to be both detrimental and advantageous. Hence, an investigation of the effects of specific subsets of T cells on neurons may provide an insight into the interaction between the nervous system and the immune system. In the present study, we demonstrate that a specific subset of T lymphocytes enhanced neurite outgrowth in vitro. When cultured T helper type 1 (Th1) cells were co-cultured with cortical neurons, neurite outgrowth from neurons was enhanced; however, the same was not observed when Th2 or naïve T cells were used. We observed that the promotion of neurite outgrowth by Th1 cells was completely inhibited by anti-interferon γ (IFN-γ) neutralizing antibody, but that IFN-γ did not directly promote neurite growth. Furthermore, experiments using knockout mice revealed that semaphorin 4A (Sema4A) but not Sema7A was required for the effect produced by Th1 cells. These results demonstrate that Sema4A and IFN-γ expressed in Th1 cells play a critical role in enhancing neurite outgrowth from cortical neurons.     

Regulation of growth cone actin dynamics by ADF/cofilin.

Nervous system development is reliant on neuronal pathfinding, the process in which axons are guided to their target cells by specific extracellular cues. The ability of neurons to extend over long distances in response to environmental guidance signals is made possible by the growth cone, a highly motile structure found at the end of neuronal processes. Growth cones detect directional cues and respond with either attractive or repulsive movements. The motility of growth cones is dependent on rapid reorganization of the actin cytoskeleton, presumably mediated by actin-associated proteins under the control of incoming guidance signals. This article reviews how one such family of proteins, the ADF/cofilins, are emerging as key regulators of growth cone actin dynamics. These proteins are essential for rapid actin turnover in a variety of different cell types. ADF/cofilins are heavily co-localized with actin in growth cones and are necessary for neurite outgrowth. ADF/cofilin activities are regulated through reversible phosphorylation by LIM kinases and slingshot phosphatases. LIM kinases are downstream effectors of the Rho GTPases Rho, Rac, and Cdc42. Growing evidence suggests that extracellular guidance cues may locally alter actin dynamics by regulating the activity of LIM kinase and ADF/cofilin phosphatases via the Rho GTPases. In this way, ADF/cofilins and their upstream effectors may be pivotal to our understanding of how guidance information is translated into physical alterations of the growth cone actin cytoskeleton.     

Functions of Nogo proteins and their receptors in the nervous system

The membrane protein Nogo-A was initially characterized as a CNS-specific inhibitor of axonal regeneration. Recent studies have uncovered regulatory roles of Nogo proteins and their receptors--in precursor migration, neurite growth and branching in the developing nervous system--as well as a growth-restricting function during CNS maturation. The function of Nogo in the adult CNS is now understood to be that of a negative regulator of neuronal growth, leading to stabilization of the CNS wiring at the expense of extensive plastic rearrangements and regeneration after injury. In addition, Nogo proteins interact with various intracellular components and may have roles in the regulation of endoplasmic reticulum (ER) structure, processing of amyloid precursor protein and cell survival.     

The role of Nogo-A in axonal plasticity, regrowth and repair

Axonal damage leads to permanent deficits in the adult central nervous system (CNS) not only because of the weak intrinsic ability of adult neurons to activate their growth program but importantly also because of the presence of specific growth inhibitors in the CNS tissue and the environment of the damaged axons. The well-studied myelin-derived protein Nogo-A is involved in various cellular and molecular events contributing to the failure of CNS axons to regrow and reconnect after transection. Recent studies have shown that, by acting in a negative way on the cytoskeleton and on the growth program of axotomized neurons, Nogo-A exerts fast and chronic inhibitory effects on neurite outgrowth. On the other hand, the blockade of Nogo-A results in a marked enhancement of compensatory and regenerative axonal extension in vivo; this enhancement is often paralleled by significant functional recovery, for example, of locomotion or skilled forelimb reaching after spinal cord or stroke lesions in rats and monkeys. Surprisingly, the blockade of Nogo-A or its receptor NgR in the hippocampus has recently been demonstrated to enhance long-term potentiation. A role of Nogo-A in synaptic plasticity/stability might therefore represent an additional, new and important aspect of CNS circuit remodeling. Function-blocking anti-Nogo-A antibodies are currently being tested in a clinical trial for improved outcome after spinal cord injury.     

Inhibitory effects of draxin on axonal outgrowth and migration of precerebellar neurons

The rhombic lip, a dorsal stripe of the neuroepithelium lining the edge of the fourth ventricle, is the site of origin of precerebellar neurons (PCN), which migrate tangentially towards the floor plate. After reaching the floor plate, they project their axons to the cerebellum. Although previous studies have shown that the guidance molecules Netrin/DCC and Slit/Robo have critical roles in PCN migration, the molecular mechanisms underlying this process remain poorly understood. Here, we report that draxin, a repulsive axon guidance protein, is involved in PCN development. We found that draxin is expressed in the rhombic lip and migratory stream of some PCN in the developing hindbrain of mice. In addition, draxin inhibited neurite outgrowth and nuclei migration from rhombic lip explants. These results suggest that draxin functions as a repulsive guidance cue for PCN migration. However, we observed no significant differences in PCN distribution between draxin^−/− and wild type embryos. Thus, draxin and other axon guidance cues may have redundant roles in PCN migration.