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Axonal Pathfinding: Extracellular Matrix Role
Axonal Pathfinding: Extracellular Matrix Role
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Axonal Pathfinding: Extracellular Matrix Role P Letourneau, University of Minnesota, Minneapolis, MN, USA ã 2009 Elsevier Ltd. All rights reserved.
Introduction Normal behavior and other neural activities depend on the correct wiring of neural circuits during development. A critical step in forming neural circuits is the growth of axons from nerve cell bodies to the sites where synaptic connections are made. In spanning between neural somata and their synaptic targets, growing axons forge pathways that become the axonal tracts and peripheral nerves of the mature nervous system. The routes that axons take to reach their targets are determined by motile activities at their tips, called growth cones. Growth cones extend fine protrusions that adhere to nearby cells and surfaces. These adhesive contacts provide a toehold from which further protrusions are made. As growth cones crawl forward, they choose a path by detecting and responding to the spatial and temporal distributions of extracellular guidance molecules encountered in their local environments. Five major families of extracellular molecules – netrins, neurotrophins, semaphorins, slits, and ephrins – provide positive and negative cues that orient the migration of growth cones to their targets. These guidance molecules bind receptor proteins on growth cones and initiate cytoplasmic signals that regulate the motility and adhesive contacts that determine the advance, retreat, turning, branching, and stopping of growth cones. This article describes molecules that play a key role in axonal pathfinding by mediating the adhesive interactions necessary for growth cone migration.
Mechanism of Growth Cone Migration Cytoskeletal Dynamics
Growth cone migration and axonal elongation involve the cytoskeletal components, microtubules and actin filaments. Axon elongation requires the advance and polymerization of microtubules, which are bundled in the axon but which spread apart in the growth cone, where individual microtubules dynamically probe forward to the front of a growth cone via polymerization and movement involving microtubule motor molecules (Figure 1). Axonal growth occurs where the main microtubule bundle and associated organelles advance in the growth cone, as determined by the positions and stabilization of these forward
‘pioneering’ microtubules. These dynamic microtubules project forward into an actin filament network that fills flattened dynamic projections, called lamellipodia, and fingerlike filopodia. This extensive filament system is continually remodeled, as actin filaments initiate and polymerize at the front margin and are then moved back to be fragmented and depolymerized, recycling subunits to the front. Multiple actin-binding proteins regulate this dynamic organization of actin filaments. Growth cone migration is driven by forces produced within this actin filament domain. Actin polymerization creates protrusive forces that expand lamellipodia and elongate the tips of filopodia. Myosin motor molecules bind actin filaments and generate mechanical forces that move cargo bound to the myosin tail domains or pull on actin filaments to create tensions. Myosin II motor activity pulls actin filaments rearward, where they are depolymerized. Tensions generated by myosin II activity in the actin-rich leading margin can either direct or halt microtubule advance, depending on the situation. Myosin II-generated tensions produce the exploratory movements of lamellipodia and filopodia, whereas excessive levels of tension may sweep microtubules back in a contracting actin network that can collapse a growth cone. It is in the context of these dynamic cytoskeletal activities that adhesive interactions are critical to growth cone migration (Figure 2). Adhesive Contacts of Growth Cones
Growth cone plasma membranes contain adhesion receptors that bind noncovalently to adhesion molecules on other cells or surfaces. Lamellipodia and filopodia initiate adhesive interactions as they explore their environment, and if these bonds persist, receptors cluster to form discrete adhesive contacts, which include intracellular adhesion complexes. Adhesion complexes remain in place or shift rearward as a growth cone advances. These adhesive complexes play two roles in growth cone migration. First, they include proteins that anchor actin filaments at adhesive sites. These links constitute a ‘clutch’ that stops the retrograde movement of actin filaments and permits the advance of microtubules and axonal organelles (Figure 2). Without stabilization provided by adhesive contacts growth cone migration fails, and tensions within the axonal cytoskeleton cause axonal retraction. Second, these complexes include proteins of signaling cascades, protein kinases, protein phosphatases, and Rho GTPases, which act on proteins that regulate the organization of actin filaments and microtubules. Thus, adhesive contacts provide points
1140 Axonal Pathfinding: Extracellular Matrix Role
Figure 1 The distribution of microtubules and actin filaments in developing neurons and in axonal growth cones. Microtubules (green) are densely packed in the neuronal cell bodies (S) and are bundled in the axons and branches. Actin filaments are arrayed in filament networks and bundles in the peripheral domains (P) of the growth cones and along the shafts of the axons, where small areas of actin filament dynamics may give rise to collateral branches (B). In a growth cone, the microtubules from the central bundle of the central domain (C) splay apart and individual microtubules extend into the P domain and into filopodia (arrows).
Leading edge protrusion Myosin II
Advance of microtubules and organelles
Retrograde flow of actin
Actin polymerization
Myosin II
Clutch (stop retrograde flow)
Cell adhesion receptor
Attractive cue Repulsive cue Figure 2 A model of the mechanism of growth cone migration. Actin polymerization pushes the leading margin of the growth cone forward. Forces generated by myosin II pull actin filaments backwards, where filaments are disassembled. When growth cone receptors make adhesive contacts with a surface, a ‘clutch’ links the adhesive contact to actin filaments of the leading edge, and the retrograde flow of actin filaments stops. This permits the advance of microtubules and organelles and promotes axonal elongation. Intracellular signaling generated by attractive and repulsive axonal guidance cues interacts with the molecular mechanisms of actin polymerization, myosin II force generation, adhesive contacts, and microtubule advance to regulate the paths of growth cone migration.
of stability that are essential to growth cone migration, and they are signaling centers from which regulatory activities promote growth cone motility. The genetic regulation that determines neuronal phenotype also directs expression of receptors for adhesive ligands and guidance cues by the growth
cones of neurons of a particular type. Extracellular positive and negative axonal guidance cues, whether surface bound or soluble, signal through their receptors to modulate an interacting set of pathways that regulate cytoskeletal and membrane dynamics. Thus, growth cone behaviors reflect a complex integration
Axonal Pathfinding: Extracellular Matrix Role
of signaling events triggered at multiple receptors for guidance cues and adhesion molecules. By locally regulating the interplay of adhesive contacts and cytoskeletal dynamics within a growth cone, guidance cues determine the pathways of axonal growth (Figure 2). Three major types of adhesive interactions promote growth cone navigation. Growth cones migrate within extracellular spaces that contain a complex mixture of glycoproteins, organized into an extracellular matrix (ECM) of fibers, protein aggregates, and basal laminae, which are discrete ECM layers at tissue interfaces. One major adhesive interaction of growth cones involves binding of integrin receptors to adhesive ECM proteins, especially the laminins. Two other major adhesive interactions involve growth cone contacts with cells or other axons along their pathways. These interactions involve two groups of adhesive molecules, the cadherins and the immunoglobulin superfamily of cell adhesion molecules (IgCAMs). Cadherins are expressed on all tissue types, including neurons and axons. Cadherin adhesions involve homophilic binding between like cadherin molecules on two interacting cells. Weaker heterophilic interactions between different cadherins can also occur. IgCAMs are also expressed on all tissues, including neurons. Adhesive interactions of IgCAMs can involve homophilic interactions, similar to cadherins, but also heterophilic interactions in which an IgCAM on a growth cone binds a different IgCAM on adjacent cells. Even heterophilic interactions of IgCAMs with non-IgCAMs occur. Integrin adhesion receptors Integrin receptors are heterodimers of alpha and beta subunits. More than 20 integrin heterodimers have been identified in humans. The binding specificity for ECM components depends on the particular combination of alpha and beta subunits in a heterodimer. The 12 integrin dimers that are expressed in the mammalian nervous system include receptors for collagens, laminin-1 and laminin-5, fibronectin, tenascin, thrombospondin, vitronectin, and VCAM-1. A growth cone can express multiple integrins, allowing interactions with multiple ECM molecules. The cytoplasmic domains of integrins lack enzymatic activities, but when integrins bind adhesive ligands, conformational shifts in the cytoplasmic domains trigger formation of focal contacts that involve integrin clustering and creation of docking sites for proteins that initiate signaling and links to the cytoskeleton. When lamellipodia and filopodia of growth cones bind laminin-1, proteins that localize to the contact sites include paxillin, talin, vinculin, zyxin, and focal adhesion kinase (FAK). Vinculin and talin link actin filaments to the adhesive contacts, providing
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a clutch for growth cone migration (Figure 3). The presence of the adapter protein paxillin and activation of FAK initiates further protein interactions and signaling by Src family kinases, MAP kinases, and Rho GTPases. Activation of Rac1 and Cdc42 GTPases promotes actin polymerization by regulating actin-binding proteins and actin filament dynamics. Thus, when integrins on growth cones bind laminin-1, growth cone migration is stimulated by increased actin filament polymerization to protrude the leading margin and by the establishment of adhesions to stabilize these protrusions and promote the advance of microtubules. Cadherins and IgCAMs Stimulation of N-cadherin and IgCAMs, such as NCAM and L1, by ligand binding between cells leads to activation of the FGF receptor tyrosine kinase, which triggers signals involving PLC-gamma, DAG lipase, cytoplasmic [Ca2þ] elevation, and activation of MAPK. IgCAMs also signal via Src kinases to activate Rac1, PI3K, and MAPK. Cadherin signaling is also reported to activate Rac1. Thus, several pathways activated by cadherins and IgCAMs promote actin filament polymerization. Adhesive binding of cadherins and IgCAMs provides anchorage for actin filaments, creating the clutch necessary for growth cone migration. The cytoplasmic tails of many cadherins, such as N-cadherin, bind catenins, which bind actin filaments and link N-cadherin adhesive sites to the actin cytoskeleton in growth cones (Figure 4). The cytoplasmic domain of L1 binds the cytoskeletal linker ankyrin, but L1– ankyrin interactions are involved in stable adhesive junctions, such as at nodes of Ranvier, and not in growth cone migration. Members of the ezrin– moesin–radixin (ERM) proteins mediate actin filament binding to membranes, and interactions of L1 (and other IgCAMs) with ERM proteins may serve as a clutch in growth cone protrusions that bind via L1 or other IgCAMs. These adhesion receptors can be regulated in ways that are important to growth cone pathfinding. The expression levels of integrin receptors on growth cones are increased when laminin levels are low or when ECM proteins, such as proteoglycans, which interfere with laminin–integrin binding, are present. These responses would maintain growth cone adhesion and migration in environments when access to laminin is reduced. L1 is endocytosed from central regions and recycled to the leading margin of growth cones, increasing availability of L1 for adhesive contacts of lamellipodia and filopodia. The functions of adhesion receptors can also be modulated from the cytoplasm in an ‘inside-out’ manner or via cis interactions with other components of the plasma membrane. An important manner in which guidance
1142 Axonal Pathfinding: Extracellular Matrix Role
Collagen
PIP2
Integrin b
Arp2/3 complex
a
PIPKI g
Vinculin
Talin P
FAK
Actin
Figure 3 A model of integrin binding to ECM molecules and the formation of intracellular adhesive complexes. An alpha–beta integrin heterodimer is shown bound to a collagen fibril, and the intracellular adhesion complex is pictured, showing the proteins vinculin and talin, which are involved in linkage to actin filaments, and FAK kinase, which initiates signaling cascades. The Arp2/3 complex nucleates actin filament assembly. Reproduced from Brakebusch C and Fa¨ssler R (2003) The integrin–actin connection, an eternal love affair. EMBO Journal 22: 2324–2333, with permission from Nature Publishing Group.
molecules exert their positive and negative effects on growth cone pathfinding is by modulating the functions of adhesive receptors (Figure 2). For example, the negative cue semaphorin 3A may inhibit growth cone migration by blocking integrin-mediated cell adhesion. In addition, adhesion mediated by N-cadherin is inhibited by the negative guidance cue Slit protein via its receptor, Robo. Thus, the negative or repulsive effects of semaphorin 3A and Slit on growth cone pathfinding can involve these inhibitory effects on growth cone adhesion. On the other hand, the attractant netrin signals to activate the kinase FAK, which promotes integrin-mediated adhesion, suggesting that positive guidance cues activate adhesive interactions of growth cones.
Adhesion Molecules and Growth Cone Pathfinding What are the roles of these adhesion molecules in the pathfinding behaviors of growth cones? Major pathways that are followed by many growing axons offer multiple adhesive ligands for growth cone migration, such as laminins, fibronectin and collagens in the ECM, and cadherins and IgCAMs on adjacent cells and axons. These multiple options for adhesion may provide redundancy, ensuring growth cones form sufficient adhesive contacts for effective migration.
The first growth cones that ‘pioneer’ a pathway have limited options for binding to ECM or cell surface adhesion molecules on adjacent cells, whereas growth cones that enter an established pathway can track along previously extended axons by binding to cadherins and IgCAMs expressed on the surfaces of axonal shafts. Several in vivo examples of pathfinding roles of adhesion molecules are described in the following sections. Laminins
Laminins are large adhesive glycoproteins (MW 1 000 000 Da) that consist of heterotrimers of alpha, beta, and gamma chains. Ten laminin chains are known, forming 11 known heterotrimers with widely varied expression throughout different tissues. The laminins present several domains that mediate laminin binding to several cell surface receptors and to other ECM components. The most common laminins are typically present in basal laminae, an ultrastructural ECM layer associated with epithelia, muscle cells, Schwann cells, and glia. Laminin-1, which has been studied the most, promotes axonal growth in vitro from virtually every type of neuron, indicating that laminins have broad roles in promoting growth cone migration. Examples of growth cone migration along basal laminae include growth cones of Rohon–Beard
Axonal Pathfinding: Extracellular Matrix Role
b
Catenins a
Actin filament
Other actin-binding proteins
Actin monomer
Figure 4 A model of homophilic adhesion between cadherin adhesion molecules and the intracellular binding to actin filaments. Cadherin molecules bind between cells and become linked to actin filaments by way of interactions with alpha and beta catenins. From Weis WI and Nelson WJ (2006) Re-solving the cadherin–catenin–actin conundrum. Journal of Biological Chemistry 281: 35593–35597.
neurons in Xenopus, growth cones of retinal ganglion cells in the retina and optic nerve, and pioneer axons in the grasshopper limb bud. However, in addition to basal laminae, laminin is transiently expressed in the loose cellular environments of developing tissues, including the nervous system, on cell surfaces and associated with sparse ECM fibers. The growth cones that pioneer pathways, such as the corticofugal pathway of the neocortex or the medial longitudinal fasciculus from the brain into the spinal cord, migrate within loose extracellular spaces in the wall of the immature central nervous system (CNS), where the cells are labeled in a punctate manner by laminin antibodies. The expression of laminin on these cells is transient, and eventually laminin immunoreactivity is restricted to the basal lamina at the outer boundary of the CNS wall. In the developing peripheral nervous system (PNS), laminin is expressed in basal laminae and at early stages in the mesenchyme through which motor and sensory axons extend. Schwann cells express abundant laminin, forming the basal laminae that enclose axon–Schwann cell units. This punctate cellular
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expression of laminin diminishes during development, although laminin remains present in basal laminae. In view of the wide distribution of laminins and the ability of laminin-1 to promote robust axonal growth from many neuronal types, it is thought that laminins function permissively, providing adhesion that is required for growth cone migration, but not in an instructive manner to influence pathfinding decisions. Laminins and other ECM molecules may broadly promote growth cone migration along a pathway, whose boundaries are defined not by the absence of adhesive ECM molecules but, rather, by the expression in adjacent tissues of negative guidance cues, such as slits or semaphorins. This ‘surround repulsion’ occurs in both developing CNS and PNS. Several mutational studies have reported specific errors in pathfinding when a laminin is absent or blocked. Laminin function is essential for growth cone turning in the grasshopper limb bud, and zebra fish with mutations in the laminin-alpha-1 chain exhibit multiple axon guidance defects throughout the CNS, but not in every location. These results suggest that laminin-mediated adhesion is essential for growth cone navigation in at least some instances. Fibronectin
Fibronectin is a large adhesive glycoprotein (MW 250 000 Da) that is widely distributed in the ECM, including within ganglia and the endoneurium of the PNS. Like the laminins, the fibronectin molecule contains multiple domains that mediate binding to other ECM components and to multiple cellular receptors, including several integrin heterodimers. During development of the PNS and CNS, fibronectin is present in a punctate distribution in loose cellular spaces of immature nervous tissue, and eventually fibronectin expression diminishes as development ends, especially in the CNS. In tissue culture studies, fibronectin promotes axonal growth, but not as vigorously as does laminin. In addition, axonal growth by PNS neurons on fibronectin surfaces exceeds the responses of CNS neurons, probably because PNS neurons express higher levels of fibronectin receptors than CNS neurons. Evidence is lacking for a requirement for fibronectin in growth cone pathfinding. Integrins
Because neurons express multiple integrin subunits and because many ECM components, such as collagen, laminin, or fibronectin, can bind more than one integrin heterodimer, the essential roles of particular integrins in growth cone pathfinding are not clearly defined. Mouse knockouts of a1 and a6 integrins,
1144 Axonal Pathfinding: Extracellular Matrix Role
which are laminin-1 receptors, do not reveal clear defects, however, injections of anti-b1 integrin, part of several neuronal receptors for ECM proteins, into Xenopus embryos disrupts retinal axonal pathfinding. Similarly, conditional knockout of b1 integrin in sensory neurons results in deficits in innervation of skin, where sensory axons extend through the dermal ECM and along the epidermal basal lamina. The a4ß1 integrin heterodimer is specifically implicated in the growth and arborization of sympathetic axons within cardiac muscle. In Drosophila, mutations in the integrins a-PS1 and-PS2 lead to pathfinding errors. Cadherins
Cadherins are characterized as single-pass transmembrane proteins that contain an ectodomain of five cadherin repeats and a conserved cytoplasmic tail. Binding of calcium ion stabilizes an extended rodlike structure of the ectodomain, which is necessary for optimal adhesion by alignment of cadherin molecules on apposing cells. There are at least 100 cadherins, and most are expressed in the developing vertebrate brain on immature cells, neurons, and glia. Their functions are numerous, including cell sorting, boundary formation, target recognition, synaptogenesis, and synapse function. Regarding axonal pathfinding, the widely expressed N-cadherin stimulates in vitro axonal growth from a variety of CNS and PNS neurons. In vivo studies involving antibody injection or genetic mutation also implicate N-cadherin in axon growth and fasciculation. These results indicate that cadherins promote growth cone migration along axons in highly populated common pathways, but it is unclear whether cadherins play a role in the pathfinding of early pioneer growth cones. In some cases, a common pathway may be shared by several classes of elongating axons, which are distinguished by the expression of different cadherins. For example, the tectofugal projections of chickens express four different cadherins among different axon fascicles. These cadherins may mediate specific pathfinding, as the formation of homophilic adhesions of growth cones to axons expressing the same cadherin directs growth cones along specific axon fascicles toward their targets. Forced expression of specific cadherins causes growth cones to abnormally follow fascicles that express the same cadherin. Finally, growth cones often share expression of specific cadherins with neurons in their particular target. Thus, cadherins also have roles in target recognition and subsequent synaptogenesis. L1 and NCAM IgCAMs
Proteins that contain an immunoglobulin (Ig)-like domain constitute the Ig superfamily, which makes up more than 2% of human genes, constituting the largest gene family. The neuronal Ig superfamily
includes a large number of molecules, which have functions in axonal pathfinding not only as cell adhesion molecules but also as axonal guidance cues and as receptors of guidance cues. This discussion is restricted to two members of this large family, L1 and NCAM. The IgCAM L1 is widely expressed on axons in the developing CNS and PNS, and tissue culture studies show that substrates coated with L1 promote homophilic adhesion and axonal growth from many neuronal types. Spontaneous human mutations in the L1 gene and mouse L1 knockout studies both indicate important roles for L1 in brain development and function. Multiple anatomical and functional deficits result from human and mouse L1 mutations, including a failure of corticospinal axons to decussate in the hindbrain pyramids. Crossing defects were not found in other tracts or were not so extensive. L1 also interacts in cis with receptors for other guidance cues, including the semaphorin 3A receptor, suggesting that the defect in pyramidal decussation observed in L1 mutants could be due to disrupted pathfinding responses to semaphorin 3A and other guidance cues, as well as to reduced growth cone tracking along axons. Another prominent neuronal IgCAM is NCAM, the first neuronal IgCAM identified. NCAM is widely expressed on immature neurons and glia and also on other embryonic cells, such as myoblasts. In tissue culture studies, NCAM mediates neuronal adhesion and axon growth. In addition to homophilic adhesive interactions, NCAM also forms heterophilic adhesive interactions. Antibodies against NCAM can induce axon defasciculation in vitro and in vivo. Several isoforms of NCAM are expressed, and in some situations NCAM carries a carbohydrate polysialic acid (PSA) moiety that reduces NCAM adhesion. In NCAM-deficient mice defects in fasciculation of hippocampal axons were observed, but in general only minor defects in development or behavior were observed. Perhaps, in the absence of NCAM, other cell adhesion molecules serve the same functions.
Adhesion Molecules and Axonal Regeneration When the pathfinding phase of circuit construction ends, as growth cones reach their targets, the expression of neural cell adhesion molecules and their receptors is downregulated. However, injury or damage to nervous tissues can disconnect neural circuits, and axons must regenerate in order to reconnect neurons. When axons are injured in the PNS, axon regeneration is often robust, leading to varying degrees of functional recovery. Schwann cells, which ensheath all PNS axons, stimulate axonal regeneration by upregulating their expression of growth factors, and
Axonal Pathfinding: Extracellular Matrix Role
laminins, fibronectin, and cadherin, as substrates for growth cones. Axon regeneration in the PNS is also promoted by increased expression of integrin receptors by regenerating neurons. In the CNS of adult mammals, regeneration of injured axons is poor, and recent research has focused on inhibitory components of myelin and glial scars that block growth cone adhesion and trigger signals that inhibit growth cone motility. In lower vertebrates, CNS regeneration is often successful, and this involves the upregulation of expression of adhesive ligands, such as L1 and cadherins, as demonstrated in regenerating zebra fish optic nerves and spinal cords. Several strategies for improving axonal regeneration in mammalian model systems, and eventually humans, emphasize measures to improve the adhesive environment for growth cone migration. When stem cells that express L1 are transplanted into a mammalian CNS lesion, increased regeneration of corticospinal axons occurs. Purkinje cells transfected to express L1 and GAP43 show enhanced axonal regeneration. In vitro regeneration of axons by adult neurons on laminin and fibronectin is improved by transfection of neurons to express increased levels of the appropriate a integrin chains. Finally, many natural and synthetic bridges have been designed that include adhesion molecules to promote axonal regeneration across lesion sites. These studies demonstrate that strategies to increase the adhesive interactions of regenerating growth cones can stimulate axonal regeneration after injuries in adults. Probably, improved axonal regeneration in adults will also require an increase in the intrinsic ability of adult neurons to sprout and grow axons. This may involve upregulation of genes for adhesion receptors, for other guidance cue receptors, and for proteins that drive the dynamic cytoskeletal functions of immature neurons.
Summary Growth cone adhesion is integral to the mechanism of growth cone migration and pathfinding. Adhesive interactions of growth cones provide stability for lamellipodial and filopodial protrusions of growth cones and also act as signaling centers that regulate actin and microtubule dynamics and organization in a migrating growth cone. The adhesive interactions of growth cones are also a target of guidance cues that determine where growth cones turn, branch, and stop migrating. Migrating growth cones make three kinds of adhesive contacts with ECM and with other cells. These contacts involve integrin receptors, which recognize laminin and other ECM components; cadherins, which form homophilic adhesions; and IgCAMs, which can form homophilic and heterophilic adhesive interactions. Major pathways of
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growth cone migration during development contain one or, perhaps more typically, multiple adhesive ligands available to growth cones. The navigational decisions of growth cone pathfinding are based on local differences in adhesive stability for growth cone protrusions and in dynamic protrusive activity, as based on adhesive signaling and the integration of signaling triggered from other guidance cues. See also: Axon Guidance: Morphogens as Chemoattractants and Chemorepellants; Axon Guidance: Building Pathways with Molecular Cues in Vertebrate Sensory Systems; Axon Guidance: Guidance Cues and Guidepost Cells; Axonal Pathfinding: Netrins; Axonal Pathfinding: Guidance Activities of Sonic Hedgehog (Shh); Growth Cones; Semaphorins.
Further Reading Brakebusch C and Fa¨ssler R (2003) The integrin–actin connection, an eternal love affair. EMBO Journal 22: 2324–2333. Clegg DO, Wingerd KL, Hikita ST, and Tolhurst EC (2003) Integrins in the development, function and dysfunction of the nervous system. Frontiers in Bioscience 8: d723–d750. Colognato H, French-Constant C, and Feltri ML (2005) Human diseases reveal novel roles for neural laminins. Transactions in Neuroscience 28: 480–486. Dent EW and Gertler FB (2003) Cytoskeletal dynamics and transport in growth cone motility and axon guidance. Neuron 40: 209–227. Gordon-Weeks PR (2000) Neuronal Growth Cones. Cambridge, UK: Cambridge University Press. Hortsch M (2003) Neural cell adhesion molecules – brain glue and much more! Frontiers in Bioscience 8: d357–d359. Huber AB, Kolodkin AL, Ginty DD, and Cloutier JF (2003) Signaling at the growth cone: Ligand–receptor complexes and the control of axon growth and guidance. Annual Review of Neuroscience 28: 509–563. Hynes RO (2002) Integrins. Bidirectional, allosteric signaling machines. Cell 110: 673–687. Kamiguchi H (2003) The mechanism of axon growth: What we have learned from the cell adhesion molecule L1. Molecular Neurobiology 28: 219–228. Kiryusho D, Berezin V, and Bock E (2004) Regulators of neurite outgrowth: Role of cell adhesion molecules. Annals of the New York Academy of Sciences 1014: 140–154. Redies C, Treubert-Zimmermann U, and Luo J (2003) Cadherins as regulators for the emergence of neural nets from embryonic divisions. Journal of Physiology (Paris) 97: 5–15. Sakisaka T and Takai Y (2005) Cell adhesion molecules in the CNS. Journal of Cell Science 118: 5407–5410. Suter DM and Forscher P (2000) Substrate–cytoskeletal coupling as a mechanism for regulation of growth cone motility and guidance. Journal of Neurobiology 44: 97–113. Thiery JP (2003) Cell adhesion in development: A complex signaling network. Current Opinion in Genetics & Development 13: 365–371. Weis WI and Nelson WJ (2006) Re-solving the cadherin–catenin– actin conundrum. Journal of Biological Chemistry 281: 35593– 35597. Wen Z and Zheng JQ (2006) Directional guidance of nerve growth cones. Current Opinion in Neurobiology 16: 52–58.
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Axonal Pathfinding: Extracellular Matrix Role P Letourneau, University of Minnesota, Minneapolis, MN, USA ã 2009 Elsevier Ltd. All rights reserved.
Introduction Normal behavior and other neural activities depend on the correct wiring of neural circuits during development. A critical step in forming neural circuits is the growth of axons from nerve cell bodies to the sites where synaptic connections are made. In spanning between neural somata and their synaptic targets, growing axons forge pathways that become the axonal tracts and peripheral nerves of the mature nervous system. The routes that axons take to reach their targets are determined by motile activities at their tips, called growth cones. Growth cones extend fine protrusions that adhere to nearby cells and surfaces. These adhesive contacts provide a toehold from which further protrusions are made. As growth cones crawl forward, they choose a path by detecting and responding to the spatial and temporal distributions of extracellular guidance molecules encountered in their local environments. Five major families of extracellular molecules – netrins, neurotrophins, semaphorins, slits, and ephrins – provide positive and negative cues that orient the migration of growth cones to their targets. These guidance molecules bind receptor proteins on growth cones and initiate cytoplasmic signals that regulate the motility and adhesive contacts that determine the advance, retreat, turning, branching, and stopping of growth cones. This article describes molecules that play a key role in axonal pathfinding by mediating the adhesive interactions necessary for growth cone migration.
Mechanism of Growth Cone Migration Cytoskeletal Dynamics
Growth cone migration and axonal elongation involve the cytoskeletal components, microtubules and actin filaments. Axon elongation requires the advance and polymerization of microtubules, which are bundled in the axon but which spread apart in the growth cone, where individual microtubules dynamically probe forward to the front of a growth cone via polymerization and movement involving microtubule motor molecules (Figure 1). Axonal growth occurs where the main microtubule bundle and associated organelles advance in the growth cone, as determined by the positions and stabilization of these forward
‘pioneering’ microtubules. These dynamic microtubules project forward into an actin filament network that fills flattened dynamic projections, called lamellipodia, and fingerlike filopodia. This extensive filament system is continually remodeled, as actin filaments initiate and polymerize at the front margin and are then moved back to be fragmented and depolymerized, recycling subunits to the front. Multiple actin-binding proteins regulate this dynamic organization of actin filaments. Growth cone migration is driven by forces produced within this actin filament domain. Actin polymerization creates protrusive forces that expand lamellipodia and elongate the tips of filopodia. Myosin motor molecules bind actin filaments and generate mechanical forces that move cargo bound to the myosin tail domains or pull on actin filaments to create tensions. Myosin II motor activity pulls actin filaments rearward, where they are depolymerized. Tensions generated by myosin II activity in the actin-rich leading margin can either direct or halt microtubule advance, depending on the situation. Myosin II-generated tensions produce the exploratory movements of lamellipodia and filopodia, whereas excessive levels of tension may sweep microtubules back in a contracting actin network that can collapse a growth cone. It is in the context of these dynamic cytoskeletal activities that adhesive interactions are critical to growth cone migration (Figure 2). Adhesive Contacts of Growth Cones
Growth cone plasma membranes contain adhesion receptors that bind noncovalently to adhesion molecules on other cells or surfaces. Lamellipodia and filopodia initiate adhesive interactions as they explore their environment, and if these bonds persist, receptors cluster to form discrete adhesive contacts, which include intracellular adhesion complexes. Adhesion complexes remain in place or shift rearward as a growth cone advances. These adhesive complexes play two roles in growth cone migration. First, they include proteins that anchor actin filaments at adhesive sites. These links constitute a ‘clutch’ that stops the retrograde movement of actin filaments and permits the advance of microtubules and axonal organelles (Figure 2). Without stabilization provided by adhesive contacts growth cone migration fails, and tensions within the axonal cytoskeleton cause axonal retraction. Second, these complexes include proteins of signaling cascades, protein kinases, protein phosphatases, and Rho GTPases, which act on proteins that regulate the organization of actin filaments and microtubules. Thus, adhesive contacts provide points
1140 Axonal Pathfinding: Extracellular Matrix Role
Figure 1 The distribution of microtubules and actin filaments in developing neurons and in axonal growth cones. Microtubules (green) are densely packed in the neuronal cell bodies (S) and are bundled in the axons and branches. Actin filaments are arrayed in filament networks and bundles in the peripheral domains (P) of the growth cones and along the shafts of the axons, where small areas of actin filament dynamics may give rise to collateral branches (B). In a growth cone, the microtubules from the central bundle of the central domain (C) splay apart and individual microtubules extend into the P domain and into filopodia (arrows).
Leading edge protrusion Myosin II
Advance of microtubules and organelles
Retrograde flow of actin
Actin polymerization
Myosin II
Clutch (stop retrograde flow)
Cell adhesion receptor
Attractive cue Repulsive cue Figure 2 A model of the mechanism of growth cone migration. Actin polymerization pushes the leading margin of the growth cone forward. Forces generated by myosin II pull actin filaments backwards, where filaments are disassembled. When growth cone receptors make adhesive contacts with a surface, a ‘clutch’ links the adhesive contact to actin filaments of the leading edge, and the retrograde flow of actin filaments stops. This permits the advance of microtubules and organelles and promotes axonal elongation. Intracellular signaling generated by attractive and repulsive axonal guidance cues interacts with the molecular mechanisms of actin polymerization, myosin II force generation, adhesive contacts, and microtubule advance to regulate the paths of growth cone migration.
of stability that are essential to growth cone migration, and they are signaling centers from which regulatory activities promote growth cone motility. The genetic regulation that determines neuronal phenotype also directs expression of receptors for adhesive ligands and guidance cues by the growth
cones of neurons of a particular type. Extracellular positive and negative axonal guidance cues, whether surface bound or soluble, signal through their receptors to modulate an interacting set of pathways that regulate cytoskeletal and membrane dynamics. Thus, growth cone behaviors reflect a complex integration
Axonal Pathfinding: Extracellular Matrix Role
of signaling events triggered at multiple receptors for guidance cues and adhesion molecules. By locally regulating the interplay of adhesive contacts and cytoskeletal dynamics within a growth cone, guidance cues determine the pathways of axonal growth (Figure 2). Three major types of adhesive interactions promote growth cone navigation. Growth cones migrate within extracellular spaces that contain a complex mixture of glycoproteins, organized into an extracellular matrix (ECM) of fibers, protein aggregates, and basal laminae, which are discrete ECM layers at tissue interfaces. One major adhesive interaction of growth cones involves binding of integrin receptors to adhesive ECM proteins, especially the laminins. Two other major adhesive interactions involve growth cone contacts with cells or other axons along their pathways. These interactions involve two groups of adhesive molecules, the cadherins and the immunoglobulin superfamily of cell adhesion molecules (IgCAMs). Cadherins are expressed on all tissue types, including neurons and axons. Cadherin adhesions involve homophilic binding between like cadherin molecules on two interacting cells. Weaker heterophilic interactions between different cadherins can also occur. IgCAMs are also expressed on all tissues, including neurons. Adhesive interactions of IgCAMs can involve homophilic interactions, similar to cadherins, but also heterophilic interactions in which an IgCAM on a growth cone binds a different IgCAM on adjacent cells. Even heterophilic interactions of IgCAMs with non-IgCAMs occur. Integrin adhesion receptors Integrin receptors are heterodimers of alpha and beta subunits. More than 20 integrin heterodimers have been identified in humans. The binding specificity for ECM components depends on the particular combination of alpha and beta subunits in a heterodimer. The 12 integrin dimers that are expressed in the mammalian nervous system include receptors for collagens, laminin-1 and laminin-5, fibronectin, tenascin, thrombospondin, vitronectin, and VCAM-1. A growth cone can express multiple integrins, allowing interactions with multiple ECM molecules. The cytoplasmic domains of integrins lack enzymatic activities, but when integrins bind adhesive ligands, conformational shifts in the cytoplasmic domains trigger formation of focal contacts that involve integrin clustering and creation of docking sites for proteins that initiate signaling and links to the cytoskeleton. When lamellipodia and filopodia of growth cones bind laminin-1, proteins that localize to the contact sites include paxillin, talin, vinculin, zyxin, and focal adhesion kinase (FAK). Vinculin and talin link actin filaments to the adhesive contacts, providing
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a clutch for growth cone migration (Figure 3). The presence of the adapter protein paxillin and activation of FAK initiates further protein interactions and signaling by Src family kinases, MAP kinases, and Rho GTPases. Activation of Rac1 and Cdc42 GTPases promotes actin polymerization by regulating actin-binding proteins and actin filament dynamics. Thus, when integrins on growth cones bind laminin-1, growth cone migration is stimulated by increased actin filament polymerization to protrude the leading margin and by the establishment of adhesions to stabilize these protrusions and promote the advance of microtubules. Cadherins and IgCAMs Stimulation of N-cadherin and IgCAMs, such as NCAM and L1, by ligand binding between cells leads to activation of the FGF receptor tyrosine kinase, which triggers signals involving PLC-gamma, DAG lipase, cytoplasmic [Ca2þ] elevation, and activation of MAPK. IgCAMs also signal via Src kinases to activate Rac1, PI3K, and MAPK. Cadherin signaling is also reported to activate Rac1. Thus, several pathways activated by cadherins and IgCAMs promote actin filament polymerization. Adhesive binding of cadherins and IgCAMs provides anchorage for actin filaments, creating the clutch necessary for growth cone migration. The cytoplasmic tails of many cadherins, such as N-cadherin, bind catenins, which bind actin filaments and link N-cadherin adhesive sites to the actin cytoskeleton in growth cones (Figure 4). The cytoplasmic domain of L1 binds the cytoskeletal linker ankyrin, but L1– ankyrin interactions are involved in stable adhesive junctions, such as at nodes of Ranvier, and not in growth cone migration. Members of the ezrin– moesin–radixin (ERM) proteins mediate actin filament binding to membranes, and interactions of L1 (and other IgCAMs) with ERM proteins may serve as a clutch in growth cone protrusions that bind via L1 or other IgCAMs. These adhesion receptors can be regulated in ways that are important to growth cone pathfinding. The expression levels of integrin receptors on growth cones are increased when laminin levels are low or when ECM proteins, such as proteoglycans, which interfere with laminin–integrin binding, are present. These responses would maintain growth cone adhesion and migration in environments when access to laminin is reduced. L1 is endocytosed from central regions and recycled to the leading margin of growth cones, increasing availability of L1 for adhesive contacts of lamellipodia and filopodia. The functions of adhesion receptors can also be modulated from the cytoplasm in an ‘inside-out’ manner or via cis interactions with other components of the plasma membrane. An important manner in which guidance
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Collagen
PIP2
Integrin b
Arp2/3 complex
a
PIPKI g
Vinculin
Talin P
FAK
Actin
Figure 3 A model of integrin binding to ECM molecules and the formation of intracellular adhesive complexes. An alpha–beta integrin heterodimer is shown bound to a collagen fibril, and the intracellular adhesion complex is pictured, showing the proteins vinculin and talin, which are involved in linkage to actin filaments, and FAK kinase, which initiates signaling cascades. The Arp2/3 complex nucleates actin filament assembly. Reproduced from Brakebusch C and Fa¨ssler R (2003) The integrin–actin connection, an eternal love affair. EMBO Journal 22: 2324–2333, with permission from Nature Publishing Group.
molecules exert their positive and negative effects on growth cone pathfinding is by modulating the functions of adhesive receptors (Figure 2). For example, the negative cue semaphorin 3A may inhibit growth cone migration by blocking integrin-mediated cell adhesion. In addition, adhesion mediated by N-cadherin is inhibited by the negative guidance cue Slit protein via its receptor, Robo. Thus, the negative or repulsive effects of semaphorin 3A and Slit on growth cone pathfinding can involve these inhibitory effects on growth cone adhesion. On the other hand, the attractant netrin signals to activate the kinase FAK, which promotes integrin-mediated adhesion, suggesting that positive guidance cues activate adhesive interactions of growth cones.
Adhesion Molecules and Growth Cone Pathfinding What are the roles of these adhesion molecules in the pathfinding behaviors of growth cones? Major pathways that are followed by many growing axons offer multiple adhesive ligands for growth cone migration, such as laminins, fibronectin and collagens in the ECM, and cadherins and IgCAMs on adjacent cells and axons. These multiple options for adhesion may provide redundancy, ensuring growth cones form sufficient adhesive contacts for effective migration.
The first growth cones that ‘pioneer’ a pathway have limited options for binding to ECM or cell surface adhesion molecules on adjacent cells, whereas growth cones that enter an established pathway can track along previously extended axons by binding to cadherins and IgCAMs expressed on the surfaces of axonal shafts. Several in vivo examples of pathfinding roles of adhesion molecules are described in the following sections. Laminins
Laminins are large adhesive glycoproteins (MW 1 000 000 Da) that consist of heterotrimers of alpha, beta, and gamma chains. Ten laminin chains are known, forming 11 known heterotrimers with widely varied expression throughout different tissues. The laminins present several domains that mediate laminin binding to several cell surface receptors and to other ECM components. The most common laminins are typically present in basal laminae, an ultrastructural ECM layer associated with epithelia, muscle cells, Schwann cells, and glia. Laminin-1, which has been studied the most, promotes axonal growth in vitro from virtually every type of neuron, indicating that laminins have broad roles in promoting growth cone migration. Examples of growth cone migration along basal laminae include growth cones of Rohon–Beard
Axonal Pathfinding: Extracellular Matrix Role
b
Catenins a
Actin filament
Other actin-binding proteins
Actin monomer
Figure 4 A model of homophilic adhesion between cadherin adhesion molecules and the intracellular binding to actin filaments. Cadherin molecules bind between cells and become linked to actin filaments by way of interactions with alpha and beta catenins. From Weis WI and Nelson WJ (2006) Re-solving the cadherin–catenin–actin conundrum. Journal of Biological Chemistry 281: 35593–35597.
neurons in Xenopus, growth cones of retinal ganglion cells in the retina and optic nerve, and pioneer axons in the grasshopper limb bud. However, in addition to basal laminae, laminin is transiently expressed in the loose cellular environments of developing tissues, including the nervous system, on cell surfaces and associated with sparse ECM fibers. The growth cones that pioneer pathways, such as the corticofugal pathway of the neocortex or the medial longitudinal fasciculus from the brain into the spinal cord, migrate within loose extracellular spaces in the wall of the immature central nervous system (CNS), where the cells are labeled in a punctate manner by laminin antibodies. The expression of laminin on these cells is transient, and eventually laminin immunoreactivity is restricted to the basal lamina at the outer boundary of the CNS wall. In the developing peripheral nervous system (PNS), laminin is expressed in basal laminae and at early stages in the mesenchyme through which motor and sensory axons extend. Schwann cells express abundant laminin, forming the basal laminae that enclose axon–Schwann cell units. This punctate cellular
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expression of laminin diminishes during development, although laminin remains present in basal laminae. In view of the wide distribution of laminins and the ability of laminin-1 to promote robust axonal growth from many neuronal types, it is thought that laminins function permissively, providing adhesion that is required for growth cone migration, but not in an instructive manner to influence pathfinding decisions. Laminins and other ECM molecules may broadly promote growth cone migration along a pathway, whose boundaries are defined not by the absence of adhesive ECM molecules but, rather, by the expression in adjacent tissues of negative guidance cues, such as slits or semaphorins. This ‘surround repulsion’ occurs in both developing CNS and PNS. Several mutational studies have reported specific errors in pathfinding when a laminin is absent or blocked. Laminin function is essential for growth cone turning in the grasshopper limb bud, and zebra fish with mutations in the laminin-alpha-1 chain exhibit multiple axon guidance defects throughout the CNS, but not in every location. These results suggest that laminin-mediated adhesion is essential for growth cone navigation in at least some instances. Fibronectin
Fibronectin is a large adhesive glycoprotein (MW 250 000 Da) that is widely distributed in the ECM, including within ganglia and the endoneurium of the PNS. Like the laminins, the fibronectin molecule contains multiple domains that mediate binding to other ECM components and to multiple cellular receptors, including several integrin heterodimers. During development of the PNS and CNS, fibronectin is present in a punctate distribution in loose cellular spaces of immature nervous tissue, and eventually fibronectin expression diminishes as development ends, especially in the CNS. In tissue culture studies, fibronectin promotes axonal growth, but not as vigorously as does laminin. In addition, axonal growth by PNS neurons on fibronectin surfaces exceeds the responses of CNS neurons, probably because PNS neurons express higher levels of fibronectin receptors than CNS neurons. Evidence is lacking for a requirement for fibronectin in growth cone pathfinding. Integrins
Because neurons express multiple integrin subunits and because many ECM components, such as collagen, laminin, or fibronectin, can bind more than one integrin heterodimer, the essential roles of particular integrins in growth cone pathfinding are not clearly defined. Mouse knockouts of a1 and a6 integrins,
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which are laminin-1 receptors, do not reveal clear defects, however, injections of anti-b1 integrin, part of several neuronal receptors for ECM proteins, into Xenopus embryos disrupts retinal axonal pathfinding. Similarly, conditional knockout of b1 integrin in sensory neurons results in deficits in innervation of skin, where sensory axons extend through the dermal ECM and along the epidermal basal lamina. The a4ß1 integrin heterodimer is specifically implicated in the growth and arborization of sympathetic axons within cardiac muscle. In Drosophila, mutations in the integrins a-PS1 and-PS2 lead to pathfinding errors. Cadherins
Cadherins are characterized as single-pass transmembrane proteins that contain an ectodomain of five cadherin repeats and a conserved cytoplasmic tail. Binding of calcium ion stabilizes an extended rodlike structure of the ectodomain, which is necessary for optimal adhesion by alignment of cadherin molecules on apposing cells. There are at least 100 cadherins, and most are expressed in the developing vertebrate brain on immature cells, neurons, and glia. Their functions are numerous, including cell sorting, boundary formation, target recognition, synaptogenesis, and synapse function. Regarding axonal pathfinding, the widely expressed N-cadherin stimulates in vitro axonal growth from a variety of CNS and PNS neurons. In vivo studies involving antibody injection or genetic mutation also implicate N-cadherin in axon growth and fasciculation. These results indicate that cadherins promote growth cone migration along axons in highly populated common pathways, but it is unclear whether cadherins play a role in the pathfinding of early pioneer growth cones. In some cases, a common pathway may be shared by several classes of elongating axons, which are distinguished by the expression of different cadherins. For example, the tectofugal projections of chickens express four different cadherins among different axon fascicles. These cadherins may mediate specific pathfinding, as the formation of homophilic adhesions of growth cones to axons expressing the same cadherin directs growth cones along specific axon fascicles toward their targets. Forced expression of specific cadherins causes growth cones to abnormally follow fascicles that express the same cadherin. Finally, growth cones often share expression of specific cadherins with neurons in their particular target. Thus, cadherins also have roles in target recognition and subsequent synaptogenesis. L1 and NCAM IgCAMs
Proteins that contain an immunoglobulin (Ig)-like domain constitute the Ig superfamily, which makes up more than 2% of human genes, constituting the largest gene family. The neuronal Ig superfamily
includes a large number of molecules, which have functions in axonal pathfinding not only as cell adhesion molecules but also as axonal guidance cues and as receptors of guidance cues. This discussion is restricted to two members of this large family, L1 and NCAM. The IgCAM L1 is widely expressed on axons in the developing CNS and PNS, and tissue culture studies show that substrates coated with L1 promote homophilic adhesion and axonal growth from many neuronal types. Spontaneous human mutations in the L1 gene and mouse L1 knockout studies both indicate important roles for L1 in brain development and function. Multiple anatomical and functional deficits result from human and mouse L1 mutations, including a failure of corticospinal axons to decussate in the hindbrain pyramids. Crossing defects were not found in other tracts or were not so extensive. L1 also interacts in cis with receptors for other guidance cues, including the semaphorin 3A receptor, suggesting that the defect in pyramidal decussation observed in L1 mutants could be due to disrupted pathfinding responses to semaphorin 3A and other guidance cues, as well as to reduced growth cone tracking along axons. Another prominent neuronal IgCAM is NCAM, the first neuronal IgCAM identified. NCAM is widely expressed on immature neurons and glia and also on other embryonic cells, such as myoblasts. In tissue culture studies, NCAM mediates neuronal adhesion and axon growth. In addition to homophilic adhesive interactions, NCAM also forms heterophilic adhesive interactions. Antibodies against NCAM can induce axon defasciculation in vitro and in vivo. Several isoforms of NCAM are expressed, and in some situations NCAM carries a carbohydrate polysialic acid (PSA) moiety that reduces NCAM adhesion. In NCAM-deficient mice defects in fasciculation of hippocampal axons were observed, but in general only minor defects in development or behavior were observed. Perhaps, in the absence of NCAM, other cell adhesion molecules serve the same functions.
Adhesion Molecules and Axonal Regeneration When the pathfinding phase of circuit construction ends, as growth cones reach their targets, the expression of neural cell adhesion molecules and their receptors is downregulated. However, injury or damage to nervous tissues can disconnect neural circuits, and axons must regenerate in order to reconnect neurons. When axons are injured in the PNS, axon regeneration is often robust, leading to varying degrees of functional recovery. Schwann cells, which ensheath all PNS axons, stimulate axonal regeneration by upregulating their expression of growth factors, and
Axonal Pathfinding: Extracellular Matrix Role
laminins, fibronectin, and cadherin, as substrates for growth cones. Axon regeneration in the PNS is also promoted by increased expression of integrin receptors by regenerating neurons. In the CNS of adult mammals, regeneration of injured axons is poor, and recent research has focused on inhibitory components of myelin and glial scars that block growth cone adhesion and trigger signals that inhibit growth cone motility. In lower vertebrates, CNS regeneration is often successful, and this involves the upregulation of expression of adhesive ligands, such as L1 and cadherins, as demonstrated in regenerating zebra fish optic nerves and spinal cords. Several strategies for improving axonal regeneration in mammalian model systems, and eventually humans, emphasize measures to improve the adhesive environment for growth cone migration. When stem cells that express L1 are transplanted into a mammalian CNS lesion, increased regeneration of corticospinal axons occurs. Purkinje cells transfected to express L1 and GAP43 show enhanced axonal regeneration. In vitro regeneration of axons by adult neurons on laminin and fibronectin is improved by transfection of neurons to express increased levels of the appropriate a integrin chains. Finally, many natural and synthetic bridges have been designed that include adhesion molecules to promote axonal regeneration across lesion sites. These studies demonstrate that strategies to increase the adhesive interactions of regenerating growth cones can stimulate axonal regeneration after injuries in adults. Probably, improved axonal regeneration in adults will also require an increase in the intrinsic ability of adult neurons to sprout and grow axons. This may involve upregulation of genes for adhesion receptors, for other guidance cue receptors, and for proteins that drive the dynamic cytoskeletal functions of immature neurons.
Summary Growth cone adhesion is integral to the mechanism of growth cone migration and pathfinding. Adhesive interactions of growth cones provide stability for lamellipodial and filopodial protrusions of growth cones and also act as signaling centers that regulate actin and microtubule dynamics and organization in a migrating growth cone. The adhesive interactions of growth cones are also a target of guidance cues that determine where growth cones turn, branch, and stop migrating. Migrating growth cones make three kinds of adhesive contacts with ECM and with other cells. These contacts involve integrin receptors, which recognize laminin and other ECM components; cadherins, which form homophilic adhesions; and IgCAMs, which can form homophilic and heterophilic adhesive interactions. Major pathways of
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growth cone migration during development contain one or, perhaps more typically, multiple adhesive ligands available to growth cones. The navigational decisions of growth cone pathfinding are based on local differences in adhesive stability for growth cone protrusions and in dynamic protrusive activity, as based on adhesive signaling and the integration of signaling triggered from other guidance cues. See also: Axon Guidance: Morphogens as Chemoattractants and Chemorepellants; Axon Guidance: Building Pathways with Molecular Cues in Vertebrate Sensory Systems; Axon Guidance: Guidance Cues and Guidepost Cells; Axonal Pathfinding: Netrins; Axonal Pathfinding: Guidance Activities of Sonic Hedgehog (Shh); Growth Cones; Semaphorins.
Further Reading Brakebusch C and Fa¨ssler R (2003) The integrin–actin connection, an eternal love affair. EMBO Journal 22: 2324–2333. Clegg DO, Wingerd KL, Hikita ST, and Tolhurst EC (2003) Integrins in the development, function and dysfunction of the nervous system. Frontiers in Bioscience 8: d723–d750. Colognato H, French-Constant C, and Feltri ML (2005) Human diseases reveal novel roles for neural laminins. Transactions in Neuroscience 28: 480–486. Dent EW and Gertler FB (2003) Cytoskeletal dynamics and transport in growth cone motility and axon guidance. Neuron 40: 209–227. Gordon-Weeks PR (2000) Neuronal Growth Cones. Cambridge, UK: Cambridge University Press. Hortsch M (2003) Neural cell adhesion molecules – brain glue and much more! Frontiers in Bioscience 8: d357–d359. Huber AB, Kolodkin AL, Ginty DD, and Cloutier JF (2003) Signaling at the growth cone: Ligand–receptor complexes and the control of axon growth and guidance. Annual Review of Neuroscience 28: 509–563. Hynes RO (2002) Integrins. Bidirectional, allosteric signaling machines. Cell 110: 673–687. Kamiguchi H (2003) The mechanism of axon growth: What we have learned from the cell adhesion molecule L1. Molecular Neurobiology 28: 219–228. Kiryusho D, Berezin V, and Bock E (2004) Regulators of neurite outgrowth: Role of cell adhesion molecules. Annals of the New York Academy of Sciences 1014: 140–154. Redies C, Treubert-Zimmermann U, and Luo J (2003) Cadherins as regulators for the emergence of neural nets from embryonic divisions. Journal of Physiology (Paris) 97: 5–15. Sakisaka T and Takai Y (2005) Cell adhesion molecules in the CNS. Journal of Cell Science 118: 5407–5410. Suter DM and Forscher P (2000) Substrate–cytoskeletal coupling as a mechanism for regulation of growth cone motility and guidance. Journal of Neurobiology 44: 97–113. Thiery JP (2003) Cell adhesion in development: A complex signaling network. Current Opinion in Genetics & Development 13: 365–371. Weis WI and Nelson WJ (2006) Re-solving the cadherin–catenin– actin conundrum. Journal of Biological Chemistry 281: 35593– 35597. Wen Z and Zheng JQ (2006) Directional guidance of nerve growth cones. Current Opinion in Neurobiology 16: 52–58.