Multiple strategies for directed growth cone extension and navigation of peripheral neurons

Leeches have a diverse constellation of peripheral neural elements that are challenged to extend growth cones in highly specific ways in a constantly changing embryonic environment. Two major systems are reviewed here. In one, peripheral afferents extend growth cones toward the central nervous system (CNS), forming common pathways, and then segregate into particular tracts within the CNS. A majority of these afferents depend on CNS-derived guidance cues and projections from the CNS to guide their way. However, not all of the nerves are established this way and at least one of the peripheral nerves is likely to be pioneered by sensillar sensory afferents. The distribution of particular antigens (such as the lan3–2 antigen) suggests the identity of molecules involved in homophilic adhesion along common pathways, whereas others (such as the lan4–2 and 3–6 antigens) are candidates for mediating specific pathway choices. In the second system, the myo-organizing Comb cell (C cell) projects multiple growth cones simultaneously along oblique trajectories not influenced by segmental or midline boundaries. Its parallel growth cones exhibit space-filling as well as directional growth and are guided by local cues to extend in discrete phases that are coordinated with the development of the environment. Both systems exhibit highly directed outgrowth orchestrated by a hierarchy of cues, establish patterns of neurites used to direct later migrating cells, and seem to be regulated temporally and spatially by interactions with the embryonic environment. These systems illustrate the strengths of examining neural development in vivo across several levels of analysis. © 1995 John Wiley & Sons, Inc.     

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.     

Semaphorins in axon regeneration: developmental guidance molecules gone wrong?

Semaphorins are developmental axon guidance cues that continue to be expressed during adulthood and are regulated by neural injury. During the formation of the nervous system, repulsive semaphorins guide axons to their targets by restricting and channelling their growth. They affect the growth cone cytoskeleton through interactions with receptor complexes that are linked to a complicated intracellular signal transduction network. Following injury, regenerating axons stop growing when they reach the border of the glial-fibrotic scar, in part because they encounter a potent molecular barrier that inhibits growth cone extension. A number of secreted semaphorins are expressed in the glial-fibrotic scar and at least one transmembrane semaphorin is upregulated in oligodendrocytes surrounding the lesion site. Semaphorin receptors, and many of the signal transduction components required for semaphorin signalling, are present in injured central nervous system neurons. Here, we review evidence that supports a critical role for semaphorin signalling in axon regeneration, and highlight a number of challenges that lie ahead with respect to advancing our understanding of semaphorin function in the normal and injured adult nervous system.     

Receptor tyrosine phosphatases regulate axon guidance across the midline of the Drosophila embryo

Neural receptor-linked protein tyrosine phosphatases (RPTPs) are required for guidance of motoneuron and photoreceptor growth cones in Drosophila. These phosphatases have not been implicated in growth cone responses to specific guidance cues, however, so it is unknown which aspects of axonal pathfinding are controlled by their activities. Three RPTPs, known as DLAR, DPTP69D, and DPTP99A, have been genetically characterized thus far. Here we report the isolation of mutations in the fourth neural RPTP, DPTP10D. The analysis of double mutant phenotypes shows that DPTP10D and DPTP69D are necessary for repulsion of growth cones from the midline of the embryonic central nervous system. Repulsion is thought to be triggered by binding of the secreted protein Slit, which is expressed by midline glia, to Roundabout (Robo) receptors on growth cones. Robo repulsion is downregulated by the Commissureless (Comm) protein, allowing axons to cross the midline. Here we show that the Rptp mutations genetically interact with robo, slit and comm. The nature of these interactions suggests that DPTP10D and DPTP69D are positive regulators of Slit/Roundabout repulsive signaling. We also show that elimination of all four neural RPTPs converts most noncrossing longitudinal pathways into commissures that cross the midline, indicating that tyrosine phosphorylation controls the manner in which growth cones respond to midline signals.     

Central Nervous System Regeneration Inhibitors and their Intracellular Substrates

Injury to the central nervous system (CNS) initiates a cascade of responses that is inhibitory to the regeneration of neurons and full recovery. At the site of injury, glial cells conspire with an inhibitory biochemical milieu to construct both physical and chemical barriers that prevent the outgrowth of axons to or beyond the lesion site. These inhibitors include factors derived from myelin, repulsive guidance cues, and chondroitin sulfate proteoglycans. Each bind receptors on the axon surface to initiating intracellular signaling cascades that ultimately result in cytoskeletal reorganization and growth cone collapse. Here, we present an overview of the molecules, receptors, and signaling pathways that inhibit CNS regeneration, with a particular focus on the intracellular signaling machinery that may function as convergent targets for multiple inhibitory ligands.     

A requirement for filopodia extension toward Slit during Robo-mediated axon repulsion

Axons navigate long distances through complex 3D environments to interconnect the nervous system during development. Although the precise spatiotemporal effects of most axon guidance cues remain poorly characterized, a prevailing model posits that attractive guidance cues stimulate actin polymerization in neuronal growth cones whereas repulsive cues induce actin disassembly. Contrary to this model, we find that the repulsive guidance cue Slit stimulates the formation and elongation of actin-based filopodia from mouse dorsal root ganglion growth cones. Surprisingly, filopodia form and elongate toward sources of Slit, a response that we find is required for subsequent axonal repulsion away from Slit. Mechanistically, Slit evokes changes in filopodium dynamics by increasing direct binding of its receptor, Robo, to members of the actin-regulatory Ena/VASP family. Perturbing filopodium dynamics pharmacologically or genetically disrupts Slit-mediated repulsion and produces severe axon guidance defects in vivo. Thus, Slit locally stimulates directional filopodial extension, a process that is required for subsequent axonal repulsion downstream of the Robo receptor.     

Neogenin and repulsive guidance molecule signaling in the central nervous system

The repulsive guidance molecule (RGM) is a membrane-bound protein that was originally identified as an axon guidance molecule in the visual system. Functional studies have revealed that it has roles in axon guidance and laminar patterning in Xenopus and chick embryos, and in controlling cephalic neural tube closure in mouse embryos. The recent identification of neogenin as a receptor for RGM has provided evidence of the diverse functions of this ligand-receptor pair. Re-expression of RGM is observed after injury in the adult human and rat central nervous systems. Inhibition of RGM enhances growth of injured axons and promotes functional recovery after spinal cord injury in rats. Thus, re-expression of embryonic repulsive cues in adult tissues contributes to failure of axon regeneration in the central nervous system.     

Regulation of axonal outgrowth and pathfinding by integrin-ECM interactions

Developing neurons use a combination of guidance cues to assemble a functional neural network. A variety of proteins immobilized within the extracellular matrix (ECM) provide specific binding sites for integrin receptors on neurons. Integrin receptors on growth cones associate with a number of cytosolic adaptor and signaling proteins that regulate cytoskeletal dynamics and cell adhesion. Recent evidence suggests that soluble growth factors and classic axon guidance cues may direct axon pathfinding by controlling integrin-based adhesion. Moreover, because classic axon guidance cues themselves are immobilized within the ECM and integrins modulate cellular responses to many axon guidance cues, interactions between activated receptors modulate cell signals and adhesion. Ultimately, growth cones control axon outgrowth and pathfinding behaviors by integrating distinct biochemical signals to promote the proper assembly of the nervous system. In this review, we discuss our current understanding how ECM proteins and their associated integrin receptors control neural network formation.     

Netrin signaling leading to directed growth cone steering

In the developing nervous system, nerve cells and axons respond to various attractive and repulsive guidance cues while traveling to their final destination. Netrins are bifunctional guidance cues that attract several classes of axons but repel others. The response of an axon to netrins is dictated by the composition of netrin receptors on the cell surface and the internal state of the growth cone. Recent analyses have identified several signal transduction pathways that contribute to netrin-mediated guidance. A model emerges in which tyrosine phosphorylation, phosphatidylinositol signaling and regulation by Rho GTPases act in concert to trigger extension of axons and turning of growth cones in response to Netrin1.     

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.     

Growth and guidance cues for regenerating axons: where have they gone?

Both attractive and repellent cues are required to guide developing axons to their targets in the central nervous system. Critical guidance molecules in the developing brain include the semaphorins, netrins, slits, and ephrins. Current research indicates that many of these molecules and their receptors are expressed in the adult central nervous system (CNS), and that injury can alter the levels of these ligands/receptors. Recent studies have begun the process of elucidating the functions of these receptors in adult mammals, and the effects that they have on the regeneration of adult neurons. This review addresses our current knowledge with respect to the response of adult CNS neurons to axonal injury, interventions for enhancing the survival and regeneration of injured neurons, and the expression of developmental axon guidance cues in the injured mature CNS, with specific focus on the retino-tectal projection.     

Ena/VASP: proteins at the tip of the nervous system

The emergence of neurites from a symmetrical cell body is an essential feature of nervous system development. Neurites are the precursors of axons and dendrites and are tipped by growth cones, motile structures that guide elongating axons in the developing nervous system. Growth cones steer the axon along a defined path to its appropriate target in response to guidance cues. This navigation involves the dynamic extension and withdrawal of actin-filled finger-like protrusions called filopodia that continuously sample their environment. Ena/VASP proteins, a conserved family of actin-regulatory proteins, are crucial for filopodia formation and function downstream of several guidance cues. Here we review recent findings into Ena/VASP function in neurite initiation, axon outgrowth and guidance.     

A role for axon guidance receptors and ligands in blood vessel development and tumor angiogenesis

Nerves and blood vessels resemble each other in their ability to form branching networks. They are in close proximity suggesting possible molecular interactions. The patterning of nerves and blood vessels are not random but are regulated by attractive and repulsive cues. Four major neuronal guidance factors that are sensed by growth cones have been identified, Semaphorin, Ephrin, Slit and Netrin, and their cognate receptors, neuropilin, Eph, roundabouts (Robo) and uncoordinated-5 (UNC5). Unexpectedly, these ligand/receptor pairs also regulate developmental and tumor angiogenesis. Together, there is strong evidence that development of the nervous and vascular systems are regulated by common cues.     

Semaphorin-neuropilin-1 interactions in plasticity and regeneration of adult neurons

During development, axonal growth cones are guided to their appropriate targets by many attractive and repulsive cues. It has become increasingly clear over the last few years that how the growth cone responds to these cues depends both on the molecular nature of the cue and on the internal state of the neuron. The unexpected result is that the same molecule can act as an attractor or as a repellent. A number of guidance cues used by neurons during development are retained in the adult nervous system, where their function is often still unclear. Most of these molecules are implicated in plasticity in the adult nervous system and can play a role (sometimes maladaptive) in neuronal regeneration after injury. A group of axonal guidance cues that has been well studied in development is the semaphorin family of secreted and membrane-anchored proteins, which has been implicated in axon steering, fasciculation, branching and synapse formation. This review focuses on semaphorin-3A (probably the best-characterized semaphorin) and its receptors (in particular neuropilin-1) in the adult nervous system and argues that semaphorin-3A plays a role in the maintenance and regeneration of adult sensory neurons.     

Convergent and divergent signaling mechanisms of growth cone collapse by ephrinA5 and slit2

EphrinA5 and slit2 are important repulsive guidance cues in the developing retinotectal system. Both ephrinA5 and slit2 cause growth cone collapse of embryonic chick retinal ganglion growth cones cultured on EHS laminin. However, the signaling mechanism that these guidance cues initiate to cause collapse remains unclear. Here we provide evidence that while both ephrinA5 and slit2 cause collapse in morphologically similar ways, the intracellular signaling leading to the collapse involves shared as well as divergent paths. Pharmacological inhibition of either phosphatidylinositol 3-kinase (PI3K) or src family kinases prevented both ephrinA5-mediated and slit2-mediated growth cone collapse. In contrast, the inhibition of nonclassical protein kinase C (PKC) isoforms blocked ephrinA5-mediated collapse, but did not interfere with slit2-mediated collapse. PI3K was copurified by affinity chromatography with either the ephrinA5 receptors (ephAs) or the slit2 receptor (roundabout). Colocalization studies have also shown that src family kinase members are recruited to the ephA and roundabout receptors upon activation. In contrast, PKC members are recruited to the ephA receptors, but not to the roundabout receptors, upon activation. This demonstrates distinct points of convergence and divergence between the two signaling molecules, ephrinA5 and slit2, and their repulsive guidance in the chick retinotectal system.     

Molecules inhibiting neurite growth: A minireview

Molecules and activities which repulse growing neurites or induce growth cone collapse and long-lasting growth inhibition have been defined over the last 10 years. Recently, specific guidance roles for developing axons and pathways could be associated with such repulsive effects. A high molecular weight membrane protein located in CNS myelin acts as potent neurite growth inhibitor and may play a role as a negative control element for sprouting, neurite growth and regeneration, and for the plasticity of the adult CNS. Interestingly, some guidance molecules can have positive, growth-promoting as well as negative, repulsive effects for specific types of neurons. These results underline the complex mechanisms involved in neurite guidance which depends on the interpretation of combinations of incoming signals by particular growth cones. Special issue dedicated to Dr. Hans Thoenen.     

The Yin and Yang of Wnt/Ryk axon guidance in development and regeneration

In the developing embryo,nascent axons navigate towards their specific targets to establish the intricate network of axonal connections linking neurons within the mature nervous system.Molecular navigational systems comprising repulsive and attractive guidance cues form chemotactic gradients along the pathway of the exploring growth cone.Axon-bound receptors detect these gradients and determine the trajectory of the migrating growth cone.In contrast to their benevolent role in the developing nervous system,repulsive guidance receptors are detrimental to the axon’s ability to regenerate after injury in the adult.In this review we explore the essential and beneficial role played by the chemorepulsive Wnt receptor,Ryk/Derailed in axon navigation in the embryonic nervous system(the Yin function).Specifically,we focus on the role of Wnt5a/Rykmediated guidance in the establishment of two major axon tracts in the mammalian central nervous system,the corticospinal tract and the corpus callosum.Recent studies have also identified Ryk as a major suppressor of axonal regeneration after spinal cord injury.Thus,we also discuss this opposing aspect of Ryk function in axonal regeneration where its activity is a major impediment to axon regrowth(the Yang function).     

Distinct expression patterns of syndecans in the embryonic zebrafish brain

Axon pathfinding in the neuroepithelium of embryonic brain is dependent on a variety of short and long range guidance cues. Heparan sulfate proteoglycans such as syndecans act as modulators of these cues and their importance in neural development is highlighted by their phylogenetic conservation. In Drosophilia, a single syndecan is present on the surface of axon growth cones and is required for chemorepulsive signalling during midline crossing. Understanding the role of syndecans in the vertebrate nervous system is challenging given that there are four homologous genes, syndecans 1–4. We show here that syndecans 2–4 are expressed in the zebrafish embryonic brain during the major period of axon growth. These genes show differing expression patterns in the brain which provides putative insights into their functional specificity.     

Growth cones: the mechanism of neurite advance

Growth cones are the highly motile structures found at the tips of growing axons and dendrites (neurites), which extend from neurones, during the development of the nervous system. They function both as detectors and transducers of extrinsic guidance cues and as regions where the neurite assembly, advance cannot occur. Assembly of the neurite cytoskeleton in growing neurites chiefly involves microtubule assembly at the growth cone. Some of the factors that may influence microtubule assembly in growth cones are becoming apparent and include post-translational modification of tubulin itself and microtubule associated proteins, particularly tau and MAP1B.     

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.