Neuroglian

EVOLUTIONARY HOMOLOGS

Neuroglian has extensive homology to vertebrate neural adhesion molecule L1 (Harper, 1991). Neuroglian is closer to L1 than it is to Drosophila Fasciclin II, indicating a more primitive common ancestor for the two (Bieber, 1989). Neuroglian intracellular domain resembles that of rat ankyrin binding glycoprotein, indicating that Neuroglian might signal through ankyrin to the cell cytoskeleton (Davis, 1993).

Members of the L1 family of neural cell adhesion molecules consist of multiple extracellular immunoglobulin and fibronectin type III domains that mediate the adhesive properties of this group of transmembrane proteins. In vertebrate genomes, these protein domains are separated by introns. It has been suggested that L1-type genes might have been subject to exon-shuffling events during evolution, however, comparison of the human L1-CAM and the chicken neurofascin gene with the genomic structure of their Drosophila homolog, neuroglian, indicates that no major rearrangement of protein domains has taken place subsequent to the split between the arthropod and chordate phyla. The Drosophila neuroglian gene appears to have lost most of the introns that have been conserved in the human L1-CAM and the chicken neurofascin gene. Nevertheless, exon shuffling or the generation of new exons by mutational changes might have been responsible for the generation of additional, alternatively spliced exons in L1-type genes (Zhao, 1998).

The L1-family of cell adhesion molecules is involved in many important aspects of nervous system development. Mutations in the human L1-CAM gene cause a complicated array of neurological phenotypes; however, the molecular basis of these effects cannot be explained by a simple loss of adhesive function. Human L1-CAM and its Drosophila homolog Neuroglian are rather divergent in sequence, with the highest degree of amino acid sequence conservation between segments of their cytoplasmic domains. In an attempt to elucidate the fundamental functions shared between these distantly related members of the L1-family, it has been demonstrated that the extracellular domains of mammalian L1-CAMs and Drosophila neuroglian are both able to induce the aggregation of transfected Drosophila S2 cells in vitro. To a limited degree they even interact with each other in cell adhesion and neurite outgrowth assays. The cytoplasmic domains of human L1-CAM and Neuroglian are both able to interact with the Drosophila homolog of the cytoskeletal linker protein ankyrin. Moreover, the recruitment of ankyrin to cell-cell contacts is completely dependent on L1-mediated cell adhesion. These findings support a model for L1 function in which the phenotypes of human L1-CAM mutations results from a disruption of the link between the extracellular environment and the neuronal cytoskeleton (Hortsch, 1998b).

Neuorfascin, a member of the L1 family of ankyrin-binding cell adhesion molecules, is a substrate for protein tyrosine kinase(s) and phosphatase(s); it identifies the highly conserved FIGQY tyrosine in the cytoplasmic domain as the principal site of phosphorylation, and demonstrates that phosphorylation of the FIGQY tyrosine abolishes ankyrin-binding activity. Neurofascin expressed in neuroblastoma cells is subject to tyrosine phosphorylation after activation of tyrosine kinases by NGF or bFGF or inactivation of tyrosine phosphatases with vanadate or dephostatin. Furthermore, both neurofascin and the related molecule Nr-CAM are tyrosine phosphorylated in a developmentally regulated pattern in rat brain. The FIGQY sequence is present in the cytoplasmic domains of all members of the L1 family of neural cell adhesion molecules. Phosphorylation of the FIGQY tyrosine abolishes ankyrin binding, as determined by coimmunoprecipitation of endogenous ankyrin and in vitro ankyrin-binding assays. Measurements of fluorescence recovery after photobleaching demonstrate that phosphorylation of the FIGQY tyrosine also increases lateral mobility of neurofascin expressed in neuroblastoma cells to the same extent as removal of the cytoplasmic domain. Ankyrin binding, therefore, appears to regulate the dynamic behavior of neurofascin and is the target for regulation by tyrosine phosphorylation in response to external signals. These findings suggest that tyrosine phosphorylation at the FIGQY site represents a highly conserved mechanism, used by the entire class of L1-related cell adhesion molecules, for regulation of ankyrin-dependent connections to the spectrin skeleton (Garver, 1997).

L1-type molecules are predominatly, although not exclusively, expressed in developing peripheral and central nervous systems. In vertebrates, L1-CAM, Nr-CAM, and neurofascin exhibit a differential, dynamic expression pattern during nervous system development. At various stages during embryonic and postembryonic deevelopment, many types of neurons and glial cells express one or several of these L1-type molecules on their surface. Experimental evidence indicates that these molecules are involved in diverse cellular processes, such as myelination, neurite outgrowth, growth cone morphology, axon fasciculation and pathfinding, neuronal cell migration, and long-term potentiation in the hippocampus (Hortsch, 1996 and references).

Manduca Neuroglian shares with Drosophila NRG a 58% identity. They share six successive and homologous Ig-like domains, the first domain beginning near the N-terminus. Manduca NRG is differentially expressed in the embryonic labial segment as two circular monolayers of epithelial cells. These cells serve as precursors of the prothoracic glands. Like all cells of tissues derived from ectoderm, the precursors of cells in the prothoracic glands initially occupy positions on the surface of the embryo. Movement of these cells from exterior positions to internal positions in embryos involves stereotypic morphological changes that are characteristic for all animal embryos. Cuboidal cells in the epithelial region, destined to invaginate, elongate to form a thickened, circular placode of columnar epithelial cells. The cells of the placode then broaden basally and constrict apically as all the pyramidal-shaped cells fold inward. Such constriction is known to be controlled by non-muscle myosin, an element of the cytoskeleton. The initiation of these morphological changes in the monolayer of ectodermal epithelial cells coincides with the initiation of NRG expression. A disparity in NRG expression occurs at the interface between the two circular placodes of cells in the labial segment and their surrounding cells. In Drosophila the prothoracic gland is part of a composite structure known as the ring gland that originates from three invaginations of the foregut ectoderm. In both moths and flies, cells of the presumptive prothoracic gland segregate from ectodermal monolayers in the embryonic head, even though these cells in the two insects arise as invaginations from different regions of the head ectoderm. It is suggested that adhesive disparities between precursor cells and surrounding cells lie at the basis of cell segregation (Chen, 1997).

L1-dependent neurite outgrowth can be triggered by a heterophilic interaction with the DM-GRASP cell adhesion molecule, the GPI-anchored membrane proteins F2/F11, and axonin-1/TAG-1, all of which are also members of the immunoglobulin domain superfamily. Other ligands that interact with vertebrate L1 family members include the extracellular matrix molecule laminin and the two chondroitin sulfate proteoglycans, neurocan and phosphacan. The phosphacan proteglycan represents the extracellular domain of a larger membrane protein that has an intracellular protein-tyrosine phosphatase domain. This protein is found in many glial and other cell types, which suggests the possibility that interactions with L1 family members may influence intracellular phosphotyrosine levels. In support of this, there is an L1-dependent stimulation of protein phosphatase activity in growth cone-enriched membranes. Phosphacan as well as neurocan inhibit Ng-CAM- and rat L1-CAM-mediated neuronal adhesion and neurite outgrowth (Hortsch, 1996 and references).

Cell adhesion molecules (CAMs) are good candidates for the positive cues that promote and/or guide axons out of the developing mammalian retina. The activation of a tyrosine kinase-phospholipase C gamma cascade is important for the neurite outgrowth responses stimulated by NCAM, N-cadherin (see Drosophila Cadherin-N) and L1 (Drosophila homolog: Neuroglian). It is thought that the neurite growth response stimulated by these CAMs is mediated by activation of the fibroblast growth factor receptor FGFR (Drosophila homolog: FGFR) in neurons. For example, fibroblast growth factor receptor function is required for the orderly projection of ganglion cells to the optic fissure. FGFR intracellular domain recruits and activates phospholipase C gamma via interactions of PLC gamma SH2 domain with the activated receptor. The key events downstream from activation of PLC gamma are the generation of diacylglycerol and the conversion of diacylglycerol to arachidonic acid via DAG lipase activity. Subsequently AA, interacting with calcium channels, induces an increased influx of calcium into neurons. The CAMs are able to interact with the FGFR extracellular domains, in cis (adjacently on the same cell membrane), via conserved interaction motifs, thus recruiting FGFR to sites of homophilic CAD interaction to engender FGFR activation, and thus promoting axonal growth (Doherty, 1996 and references).

Nr-CAM, most closely related to Drosophila Neuroglian, is a neuronal cell adhesion molecule (CAM) belonging to the immunoglobulin superfamily. Nr-CAM has been implicated as a ligand for another CAM, axonin-1, in guidance of commissural axons across the floor plate in the spinal cord. Nr-CAM also serves as a neuronal receptor for several other cell surface molecules, but its role as a ligand in neurite outgrowth is poorly understood. This problem has been studied using a chimeric Fc-fusion protein of the extracellular region of Nr-CAM (Nr-Fc) and potential neuronal receptors in the developing peripheral nervous system have been investigated. A recombinant Nr-CAM-Fc fusion protein, containing all six Ig domains and the first two fibronectin type III repeats of the extracellular region of Nr-CAM, retains cellular and molecular binding activities of the native protein. Injection in ovo of Nr-Fc into the central canal of the developing chick spinal cord results in guidance errors for commissural axons in the vicinity of the floor plate. This effect is similar to that resulting from treatment with antibodies against axonin-1, confirming that axonin-1/Nr-CAM interactions are important for the guidance of commissural axons through a spatially and temporally restricted Nr-CAM positive domain in the ventral spinal cord. When tested as a substrate, Nr-Fc induces robust neurite outgrowth from dorsal root ganglion and sympathetic ganglion neurons, but it is not effective for tectal and forebrain neurons. The peripheral but not the central neurons express high levels of axonin-1 both in vitro and in vivo. Moreover, antibodies against axonin-1 inhibit Nr-Fc-induced neurite outgrowth, indicating that axonin-1 is a neuronal receptor for Nr-CAM on these peripheral ganglion neurons. The results demonstrate a role for Nr-CAM as a ligand in axon growth by a mechanism involving axonin-1 as a neuronal receptor and suggest that dynamic changes in Nr-CAM expression can modulate axonal growth and guidance during development (Lustig, 1999).

The gene product termed TAG-1 in the rat and axonin-1 in the chicken belongs to a group of nervous tissue glycoproteins of the immunoglobulin (Ig) superfamily. It is present predominantly on the axons of specific nerve fiber tracts that have a restricted distribution pattern during neural development; TAG-1/axonin-1 can serve as a substrate for neurite outgrowth. The Ig superfamily of cell adhesion molecules can be subdivided into several subgroups, one of which comprises proteins having six Ig-like domains and four fibronectin type III repeats, but these proteins lack a transmembrane region and may be attached to the membrane by a glycosylphosphatidylinositol anchor. In terms of sequence homology, this group can be further divided into several subclasses. The TAG-1/axonin-1 subclass, whose members have been shown to occur in both soluble and membrane-bound forms, includes chicken axonin-1, rat TAG-1, and human TAX-1. Axonin-1 and TAG-1 have 75% amino acid sequence identity, and TAX-1 has 91% identity to TAG-1 and 75% to axonin-1. The protein most closely resembles CG1084 (e-115) of Drosophila and its sequence also resembles Neuroglian (BLAST search e-value = e-90) (Fitzli, 2000 and references therein).

An interaction of growth cone axonin-1 with the floor-plate NgCAM-related cell adhesion molecule (NrCAM) plays a crucial role in commissural axon guidance across the midline of the spinal cord. Axonin-1 mediates a guidance signal without promoting axon elongation. In an in vitro assay, commissural axons grow preferentially on stripes coated with a mixture of NrCAM and NgCAM. This preference is abolished in the presence of anti-axonin-1 antibodies without a decrease in neurite length. Consistent with these findings, commissural axons in vivo only fail to extend along the longitudinal axis when both NrCAM and NgCAM interactions are perturbed, but not when axonin-1 and NrCAM or axonin-1 and NgCAM interactions, are perturbed. Thus, it is concluded that axonin-1 is involved in guidance of commissural axons without promoting their growth (Fitzli, 2000).

In myelinated fibers of the vertebrate nervous system, glial-ensheathing cells interact with axons at specialized adhesive junctions, the paranodal septate-like junctions. The axonal proteins paranodin/Caspr and contactin (see Drosophila Contactin) form a cis complex in the axolemma at the axoglial adhesion zone, and both are required to stabilize the junction. There has been intense speculation that an oligodendroglial isoform of the cell adhesion molecule neurofascin, NF155, expressed at the paranodal loop might be the glial receptor for the paranodin/Caspr-contactin complex, particularly since paranodin/Caspr and NF155 colocalize to ectopic sites in the CNS of the dysmyelinated mouse Shiverer mutant. The extracellular domain of NF155 binds specifically to transfected cells expressing the paranodin/Caspr-contactin complex at the cell surface. This region of NF155 also binds the paranodin/Caspr-contactin complex from brain lysates in vitro. In support of the functional significance of this interaction, NF155 antibodies and the extracellular domain of NF155 inhibit myelination in myelinating cocultures, presumably by blocking the adhesive relationship between the axon and glial cell. These results demonstrate that the paranodin/Caspr-contactin complex interacts biochemically with NF155 and that this interaction is likely to be biologically relevant at the axoglial junction (Charles, 2002).

The myelin and lymphocyte protein (MAL) is a tetraspan raft-associated proteolipid predominantly expressed by oligodendrocytes and Schwann cells. Genetic ablation of mal resulted in cytoplasmic inclusions within compact myelin, paranodal loops that are everted away from the axon, and disorganized transverse bands at the paranode-axon interface in the adult central nervous system. These structural changes were accompanied by a marked reduction of contactin-associated protein/paranodin, neurofascin 155 (NF155), and the potassium channel Kv1.2, whereas nodal clusters of sodium channels are unaltered. Initial formation of paranodal regions appeared normal, but abnormalities become detectable when MAL starts to be expressed. Biochemical analysis revealed reduced myelin-associated glycoprotein, myelin basic protein, and NF155 protein levels in myelin and myelin-derived rafts. These results demonstrate a critical role for MAL in the maintenance of central nervous system paranodes, likely by controlling the trafficking and/or sorting of NF155 and other membrane components in oligodendrocytes (Schaeren-Wiemers, 2004).

Voltage-dependent Na⁺ channels are highly concentrated at nodes of Ranvier in myelinated axons and play a key role in promoting rapid and efficient conduction of action potentials by saltatory conduction. The molecular mechanisms that direct their localization to the node are not well understood but are believed to involve contact-dependent signals from myelinating Schwann cells and interactions of Na⁺ channels with the cytoskeletal protein, ankyrin G. Two cell adhesion molecules (CAMs) expressed at the axon surface, Nr-CAM and neurofascin, are also linked to ankyrin G and accumulate at early stages of node formation, suggesting that they mediate contact-dependent Schwann cell signals to initiate node development. To examine the potential role of Nr-CAM in this process, myelinating cocultures of DRG (dorsal root ganglion) neurons and Schwann cells were treated with an Nr-CAM-Fc (Nr-Fc) fusion protein. Nr-Fc has no effect on initial axon-Schwann cell interactions, including Schwann cell proliferation, or on the extent of myelination, but it strikingly and specifically inhibits Na⁺ channel and ankyrin G accumulation at the node. Nr-Fc binds directly to neurons and clusters and coprecipitates neurofascin expressed on axons. These results provide the first evidence that neurofascin plays a major role in the formation of nodes, possibly via interactions with Nr-CAM (Lustig, 2001).

Paranodal axoglial junctions in myelinated nerve fibers are essential for efficient action potential conduction and ion channel clustering. In the mature CNS, a fraction of the oligodendroglial 155 kDa isoform of neurofascin (NF-155), a major constituent of paranodal junctions, has key biochemical characteristics of a lipid raft-associated protein. However, despite its robust expression, NF-155 is detergent soluble before paranodes form and in purified oligodendrocyte cell cultures. Only during its progressive localization to paranodes is NF-155 (1) associated with detergent-insoluble complexes that float at increasingly lower densities of sucrose and (2) retained in situ after detergent treatment. Finally, mutant animals with disrupted paranodal junctions, including those lacking specific myelin lipids, have significantly reduced levels of raft-associated NF-155. Together, these results suggest that trans interactions between oligodendroglial NF-155 and axonal ligands result in cross-linking, stabilization, and formation of paranodal lipid raft assemblies (Schafer, 2004).

Distinct classes of GABAergic synapses are segregated into subcellular domains (i.e., dendrite, soma, and axon initial segment-AIS), thereby differentially regulating the input, integration, and output of principal neurons. In cerebellum, for example, basket interneurons make exquisitely precise 'pinceau synapses' on AIS of Purkinje neurons, but the underlying mechanism is unknown. Using BAC transgenic reporter mice, it was found that basket axons always contact Purkinje soma before innervating AIS. This synapse targeting process follows the establishment of a subcellular gradient of neurofascin186 (NF186), an L1 family immunoglobulin cell adhesion molecule (L1CAM), along the Purkinje AIS-soma axis. This gradient is dependent on ankyrinG, an AIS-restricted membrane adaptor protein that recruits NF186. In the absence of neurofascin gradient, basket axons lost directional growth along Purkinje neurons and precisely followed NF186 to ectopic locations. Disruption of NF186-ankyrinG interactions at AIS reduced pinceau synapse formation. These results implicate ankyrin-based localization of L1CAMs in subcellular organization of GABAergic synapses (Ango, 2004).

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