Guidance Cues and Growth Cone Motility
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Guidance Cues and Growth Cone Motility
As an axon grows, the growth cone at its advancing edge encounters specific ‘choice points’ at which guidance cues steer specific axons towards their appropriate destinations. Such cues may attract a subset of axons towards a given domain, repel axons from inappropriate target regions or simply provide a permissive substrate for axonal outgrowth. There are many different ways in which a guidance signal might intervene to steer the growth cone. For example, a guidance cue might promote the initiation, extension, stabilization, or retraction of individual filopodia, or the capture or stabilization of microtubules in specific regions of the growth cone. In each of these cases, guidance signals must be relayed through the growth cone to the actin cytoskeleton, a dynamic network of filaments and associated proteins that are largely responsible for motility and growth cone steering. Many diffusible and membrane-bound factors have been proposed as potential guidance cues in different systems. In some cases, it is netrins and semaphorin, and in other cases, it is ephrin, reelin or neurotrophin (Ref.1 & 2).

The targets for the signaling pathways downstream of guidance receptors are molecules such as ARP2/3 (to nucleate new actin filaments), EnaH (Enabled Homolog)/VASP proteins (to promote filament elongation), adhesion molecules (to couple actin filaments to the substrate), and myosins (to regulate the retrograde flow of actin filaments). Members of the Rho subfamily of small GTPases, including Rho, Rac and CDC42 play pivotal roles in conveying signals to the cytoskeleton. These molecules act as molecular switches that cycle between an active GTP-bound state and an inactive GDP-bound state brought on by intrinsic hydrolytic activity. EphA receptors directly activate RhoGTPase through the exchange factor Ephexin. These A-subclass ephrins are tethered to the membrane by a GPI (Glycosyl Phosphatidylinositol)-anchor and exchange factors activate GTPases by catalyzing the replacement of GDP with GTP. The EphR extracellular domain is composed of the ligand-binding Glob (Globular domain), a Cys (Cystein)-rich region and two FN (Fibronectin) Type-III repeats, and the cytoplasmic part of EphR is divided into four functional units; a classical protein tyrosine kinase domain (Eph Kinase), a SAM (Sterile Alpha Motif) and PBM (PDZ-domain Binding Motif) (Ref.4). Upon the formation of cell-cell contact, signaling through the EphR results in modulation of integrin activity and reorganization of the actin cytoskeleton. The activation of RhoA by Ephexin engages its downstream effector ROCK (Rho-Associated Coiled-Coil-Containing Protein Kinase), which in turn activates LIMK (LIM Kinase) or Myosin-II, thereby modulating actin dynamics. During synaptogenesis, the EphR help establish and modify the postsynaptic specialization by transmitting signals to the actin cytoskeleton through the Rho-family of GTPases. Intriguingly, EphA/EphrinA signals mediate a form of crosstalk between glial cells and neurons, which regulates the morphology of excitatory synapses in the mature hippocampus (Ref.3).

Netrins are secreted from the floor plate and ventral spinal cord and act as a chemoattractant for commissural axons. They are bifunctional and attract some axons and repel others. Netrin-induced attraction is mediated by the DCC (Deleted in Colorectal Cancer) family of receptors that include mammalian homologues DCC, Neogenin and UNC5H1, UNC5H2, UNC5H3 and UNC5H4. The intracellular domain of DCC has several motifs (P1, P2 and P3) that are conserved among C. elegans, Drosophila and mammals. Netrin engagement causes multimerization of DCC receptors, mediated by the association of P3 regions, and leads to attraction and stimulation of axonal growth (Ref.5). The intracellular domain of UNC5 consists of several recognizable domains, including a ZU5 domain, which is present in the GAP junction protein ZO1, and a DB (DCC-Binding) domain, and has an important role in mediating its interaction with DCC. Activation of DCC leads to activation of LCCs (L-Type Ca2+ Channels). In the absence of UNC5, DCC activation also triggers a cAMP (cyclic Adenosine 3, 5’-Monophosphate)-dependent signaling pathway and enhances LCC activity. In vertebrates, netrin attraction involves PLC (Phospholipase-C), Akt, PI3K(Phosphatidylinositol 3-Kinase), Nck, MAPK (Mitogen-Activated Protein Kinases), PKA (Protein Kinase-A) and the small GTPases CDC42, Rac and PAK (p21-Activated Kinase) (Ref.6 & 7). DSCAM (Down Syndrome Cell Adhesion Molecule), a member of the immunoglobulin superfamily represents a new class of neural cell adhesion molecules that binds directly to both the SH2 and SH3 domains of DOCK (Dedicator of Cytokinesis) and is involved in axon guidance. The Semaphorins are another family of guidance cues that can act as growth cone repellents. Both the Slit proteins and the semaphorin, particularly Sema3A, are expressed at high levels in the developing mammalian cortex and act as chemorepellants for cortical axons. Repulsive signals elicited by Semaphorin are exerted by two receptor families: Neuropilins (NRP1 and NRP2), and Plexins (subdivided into four subfamilies: PlxnA1-4, PlxnB1-3, PlxnC1, and PlxnD1). Many semaphorin bind directly to plexins and activate cytoplasmic signaling cascades. Sema3A (also known as Collapsin1) binds only to NRP1. At least two signaling pathways are involved in the response to Sema3A. One includes the small GTPase Rac1 and leads to the depolymerization of actin filaments. A second leads to the loss of integrin-mediated substrate adhesion. Activation of the NRP1/PlxnA1 receptor complex by Sema3A stimulates phosphorylation of Cofilin that promotes F-Actin turnover by severing and depolymerizing actin filaments. Phosphorylation of Cofilin by the serine/threonine kinase; LIMK downregulates cofilin’s ability to promote actin filament turnover (Ref.8). In addition to Rho-family GTPases, plexins directly and indirectly interact with several other molecules including the kinases Fes, Fyn, ROCK, CDK5 (Cyclin-Dependent Kinase-5), and the CRMP (Collapsin Response Mediator Protein)/CRAMS (CRMP-Associated Molecule) complex1, which has a role in the regulation of Actin polymerization and microtubule dynamics. Sema4D stimulates RhoA activation by a direct interaction between PlxnB and PDZ RhoGEF and LARG (Leukemia-Associated RhoGEF-12), which are PDZ domain-containing GEFs that are specific for Rho. The molecular target for Slit is a repulsive guidance transmembrane receptor known as the Robo (Roundabout). Robo is a cell surface receptor that contains five Ig (Immunoglobulin) domains and three FNIII (Fibronectin Type-III) repeats in its extracellular part. The cytoplasmic tails of human Robo1 contain four short blocks of conserved cytoplasmic motifs CC0, CC1, CC2 and CC3. All Robos are involved in multiple pathways of axon projection (including retinal and commissural axons) and neuronal migration (including the neocortex). The protein EnaH interacts with Robo to transduce part of Robo’s repulsive signal by binding to Robo’s CC2 motif. The non-receptor tyrosine kinase, Abl (Abelson), interacts with Robo through the CC3 region to antagonize Robo signaling-likely through a mechanism involving direct phosphorylation of the Robo receptor on the CC0 and CC1 motifs. The proline-rich CC3 motif in Robo binds directly to the SH3 domain in a subfamily of RhoGAPs-the srGAPs (Slit-Robo GTPase Activation Protein) and this interaction specifically inactivate CDC42 and RhoA, but not Rac1. Inactivation of CDC42 leads to a reduction in the activation of the N-WASP (Neuronal Wiskott -Aldrich syndrome Protein), thus decreasing the level of active ARP2/3 complex (Ref.8). Because active ARP2/3 promotes actin polymerization, the reduction of active CDC42 eventually decreases actin polymerization.

At the surface of target cells, Reelin binds to two lipoprotein receptors, VLDLR (Very-Low-Density Lipoprotein Receptor) and ApoER2 (Apolipoprotein-E Receptor-2), which relay the signal into the cell via the adapter Dab1 (Disabled-1). Binding requires calcium, and it is inhibited in the presence of ApoE (Apolipoprotein-E). Dab1 promotes an interaction with several nonreceptor tyrosine kinases, including Src, Fyn, and Abl through their SH2 domains, implying Dab1 functions in kinase signaling cascades during development. Growth factors have also long been implicated in axon guidance. Neurotrophins like NGF (Nerve Growth Factor) and BDNF (Brain-Derived Neurotrophic Factor) may function as chemorepellents. BDNF causes growth cone collapse, which can be prevented by elevating cAMP signaling. Neurotrophins bind two receptor types - a shared low-affinity receptor p75 and ligand-specific receptor tyrosine kinases of the TRK family (Ref.6). Myelin inhibitor NgR (Nogo Receptor) also requires p75(NTR) (p75 Neurotrophin Receptor) for transmembrane signaling. NgR is a GPI-linked LRR (Leucine-Rich Repeat) protein expressed in multiple types of neurons. It interacts with NgR to form a receptor complex that mediates signaling by Nogo66, MAG (Myelin-Associated Glycoprotein), and OMGP (Oligodendrocyte Myelin Glycoprotein)(Ref.10). MAG binds to neurons via a sialic acid linkage involving the gangliosides GT1b and GDAP1 (GD1a), a subclass of glycosphingolipids containing one or more sialic acid residues. Activation of TRK triggers multiple intracellular signaling pathways, including the Ras-MAP kinase pathway, Akt Signaling, PLC-Gamma (Phospholipase C-Gamma) and PI3K (Phosphoinositol-3 Kinase) pathway. In addition to neurotrophins, other growth factors may also act as attractants and repellents. HGF (Hepatocyte Growth Factor/ SF (Scatter Factor) acts as a survival factor for motor neurons and an attractant for their axons. cAMP and cGMP are key regulators for growth cone motility and axon guidance. Depending on the level of cyclic nucleotides within the neuron, the response of the growth cone to guidance cues can be attractive/ growth-promoting or repulsive/growth-inhibiting, with high levels favoring attraction and low levels favoring repulsion (Ref.9).

The regulation of guidance receptors and ligands allows a single guidance system to generate a variety of different responses. Some regulatory mechanisms, such as transcriptional regulation, are characteristic of all guidance systems. However, the functions of the axon guidance molecules are not limited to axon guidance, as Slit plays important roles in mesodermal cell migration, Ephrins are important in somitogenesis, vasculogenesis, and synaptic plasticity, and Semaphorin are crucial for heart and bone development. The identification of these regulatory pathways will facilitate the next step in understanding axon guidance and determining how guidance molecules function together in a biological context.