Intracellular signaling is changed after clustering of the neural cell adhesion molecules axonin-1 and NgCAM during neurite fasciculation.

Neural cell adhesion molecules of the immunoglobulin/fibronectin type III family on axons have been implicated in promotion of neurite outgrowth, fasciculation, and the mediation of specific cell adhesion. The present study demonstrates that two of these molecules on dorsal root ganglion neurons are associated with distinct protein kinases, axonin-1 with the src-related nonreceptor tyrosine kinase fyn and NgCAM with a casein kinase II-related activity and a serine/ threonine kinase related to S6 kinase. When neurites grew without contacts involving axonin-1 and NgCAM, strong fyn kinase activity was associated with axonin-1, whereas the NgCAM-associated kinase activities were low. Clustering of axonin-1 with NgCAM induced by the formation of cell-cell contacts correlated with a reduction of the axonin-1-associated fyn activity and an increased phosphorylation of NgCAM by the associated casein kinase II-related activity. Thus, axonin-1 and NgCAM trigger distinctive intracellular signals during in vitro differentiation depending on their state of association.

The axonally secreted protein axonin-1 is a potent substratum for neurite growth.

Axonin-1 is a neuronal glycoprotein occurring both as a membrane-bound and a secreted form. Membrane-bound axonin-1 is predominantly located in membranes of developing nerve fiber tracts and has recently been characterized as a cell adhesion molecule; the soluble form is secreted from axons and accumulates in the cerebrospinal fluid and the vitreous fluid of the eye. In the present study, we addressed the question as to whether secreted axonin-1 was released in a functionally competent form and we found that it strongly promotes neurite outgrowth when presented to neurons as an immobilized substratum. Neurite lengths elaborated by embryonic dorsal root ganglia neurons on axonin-1 were similar to those on the established neurite-promoting substrata L1 and laminin. Fab fragments of axonin-1 antibodies completely inhibited neurite growth on axonin-1, but not on other substrata. In soluble form, axonin-1 had an anti-adhesive effect, as revealed by perturbation of neurite fasciculation. In view of their structural similarity, we conclude that secreted and membrane-bound axonin-1 interact with the same growth-promoting neuritic receptor. The fact that secreted axonin-1 is functionally active, together with our previous findings that it is secreted from an internal cellular pool, suggests a functional dualism between membrane-bound and secreted axonin-1 at the site of secretion, which is most likely the growth cone. The secretion of adhesion molecules could represent a powerful and rapidly acting regulatory element of growth cone-neurite interactions in the control of neurite elongation, pathway selection, and possibly target recognition.

Neurite outgrowth on immobilized axonin-1 is mediated by a heterophilic interaction with L1(G4).

Axonin-1 is an axon-associated cell adhesion molecule with dualistic expression, one form being glycophosphatidylinositol-anchored to the axonal membrane, the other secreted from axons in a soluble form. When presented as a substratum for neuronal cultures it strongly promotes neurite outgrowth from chicken embryonic dorsal root ganglia neurons. In this study, the axon-associated cell adhesion molecule G4, which is identical with Ng-CAM and 8D9, and homologous or closely related to L1 of the mouse and NILE of the rat, was investigated with respect to a receptor function for axonin-1. Using fluorescent microspheres with covalently coupled axonin-1 or L1(G4) at their surface we showed that these proteins bind to each other. Within the sensitivity of this microsphere assay, no interaction of axonin-1 with itself could be detected. Axonin-1-coated microspheres also bound to the neurites of cultured dorsal root ganglia neurons. This interaction was exclusively mediated by L1(G4), as indicated by complete binding suppression by monovalent anti-L1(G4) antibodies. The interaction between neuritic L1(G4) and immobilized axonin-1 was found to mediate the promotion of neurite growth on axonin-1, as evidenced by the virtually complete arrest of neurite outgrowth in the presence of anti-L1(G4) antibodies. Convincing evidence has recently been presented that neurite growth on L1(8D9) is mediated by the homophilic binding of neuritic L1(G4) (1989. Neuron. 2: 1597-1603). Thus, both L1(G4)- and axonin-1-expressing axons may serve as "substrate pathways" for the guidance of following axons expressing L1(G4) into their target area. Conceivably, differences in the concentration of axonin-1 and L1(G4), and/or modulatory influences on their specific binding parameters in leading pathways and following axons could represent elements in the control of axonal pathway selection.

A homologue of the axonally secreted protein axonin-1 is an integral membrane protein of nerve fiber tracts involved in neurite fasciculation.

Axonin-1 is a glycoprotein that is released from axons of cultured neurons (Stoeckli, E. T., P. F. Lemkin, T. B. Kuhn, M. A. Ruegg, M. Heller, and P. Sonderegger. 1989. Eur. J. Biochem. 180:249-258). It has recently been purified from the ocular vitreous fluid of the chicken embryo (Ruegg, M. A., E. T. Stoeckli, T. B. Kuhn, M. Heller, R. Zuellig, and P. Sonderegger. 1989. EMBO (Eur. Mol. Biol. Organ.) J. 8:55-63). Immunohistochemistry localized axonin-1 prevalently in developing nerve fiber tracts. The presence of anti-axonin-1 Fab fragments during axon growth in vitro resulted in antibody binding to the axonal surfaces and in a marked perturbation of the fasciculation pattern. Hence, a fraction of axonin-1 is associated with axonal membranes and, by operational criteria, qualifies as a cell adhesion molecule. The major proportion of membrane-associated axonin-1 co-solubilized with the integral membrane proteins. By physico-chemical, immunological, and protein-chemical criteria, the integral membrane form was found to be highly similar to soluble axonin-1. In common with a number of other cell adhesion molecules, both soluble and membrane-bound axonin-1 express the L2/HNK-1 and the L3 epitopes. Radioactive pulse-chase and double-labeling experiments revealed that the released form was not derived from the membrane-bound form by shedding from the membrane surface, but directly secreted from an intracellular pool. Due to its high degree of similarity to the membrane-associated form and the presence of the L2/HNK-1 and L3 epitopes, reported to be ligands in adhesive cell interactions, adhesive properties are postulated for secreted axonin-1. As a soluble adhesive protein, it may function as a regulator of cell adhesion around its most likely site of secretion, the growth cone.

Cell adhesion molecules NgCAM and axonin-1 form heterodimers in the neuronal membrane and cooperate in neurite outgrowth promotion.

The axonal surface glycoproteins neuronglia cell adhesion molecule (NgCAM) and axonin-1 promote cell-cell adhesion, neurite outgrowth and fasciculation, and are involved in growth cone guidance. A direct binding between NgCAM and axonin-1 has been demonstrated using isolated molecules conjugated to the surface of fluorescent microspheres. By expressing NgCAM and axonin-1 in myeloma cells and performing cell aggregation assays, we found that NgCAM and axonin-1 cannot bind when present on the surface of different cells. In contrast, the cocapping of axonin-1 upon antibody-induced capping of NgCAM on the surface of CV-1 cells coexpressing NgCAM and axonin-1 and the selective chemical cross-linking of the two molecules in low density cultures of dorsal root ganglia neurons indicated a specific and direct binding of axonin-1 and Ng-CAM in the plane of the same membrane. Suppression of the axonin-1 translation by antisense oligonucleotides prevented neurite outgrowth in dissociated dorsal root ganglia neurons cultured on an NgCAM substratum, indicating that neurite outgrowth on NgCAM substratum requires axonin-1. Based on these and previous results, which implicated NgCAM as the neuronal receptor involved in neurite outgrowth on NgCAM substratum, we concluded that neurite outgrowth on an NgCAM substratum depends on two essential interactions of growth cone NgCAM: a trans-interaction with substratum NgCAM and a cis-interaction with axonin-1 residing in the same growth cone membrane.

Neurite fasciculation mediated by complexes of axonin-1 and Ng cell adhesion molecule.

Neural cell adhesion molecules composed of immunoglobulin and fibronectin type III-like domains have been implicated in cell adhesion, neurite outgrowth, and fasciculation. Axonin-1 and Ng cell adhesion molecule (NgCAM), two molecules with predominantly axonal expression exhibit homophilic interactions across the extracellular space (axonin- 1/axonin-1 and NgCAM/NgCAM) and a heterophilic interaction (axonin-1-NgCAM) that occurs exclusively in the plane of the same membrane (cis-interaction). Using domain deletion mutants we localized the NgCAM homophilic binding in the Ig domains 1-4 whereas heterophilic binding to axonin-1 was localized in the Ig domains 2-4 and the third FnIII domain. The NgCAM-NgCAM interaction could be established simultaneously with the axonin-1-NgCAM interaction. In contrast, the axonin-1-NgCAM interaction excluded axonin-1/axonin-1 binding. These results and the examination of the coclustering of axonin-1 and NgCAM at cell contacts, suggest that intercellular contact is mediated by a symmetric axonin-12/NgCAM2 tetramer, in which homophilic NgCAM binding across the extracellular space occurs simultaneously with a cis-heterophilic interaction of axonin-1 and NgCAM. The enhanced neurite fasciculation after overexpression of NgCAM by adenoviral vectors indicates that NgCAM is the limiting component for the formation of the axonin-12/NgCAM2 complexes and, thus, neurite fasciculation in DRG neurons.

A few axonal proteins distinguish ventral spinal cord neurons from dorsal root ganglion neurons.

A series of proteins putatively involved in the generation of axonal diversity was identified. Neurons from ventral spinal cord and dorsal root ganglia were grown in a compartmented cell-culture system which offers separate access to cell somas and axons. The proteins synthesized in the neuronal cell somas and subsequently transported into the axons were selectively analyzed by 2-dimensional gel electrophoresis. The patterns of axonal proteins were substantially less complex than those derived from the proteins of neuronal cell bodies. The structural and functional similarity of axons from different neurons was reflected in a high degree of similarity of the gel pattern of the axonal proteins from sensory ganglia and spinal cord neurons. Each axonal type, however, had several proteins that were markedly less abundant or absent in the other. These neuron-population enriched proteins may be involved in the implementation of neuronal diversity. One of the proteins enriched in dorsal root ganglia axons had previously been found to be expressed with decreased abundance when dorsal root ganglia axons were co-cultured with ventral spinal cord cells under conditions in which synapse formation occurs (P. Sonderegger, M. C. Fishman, M. Bokoum, H. C. Bauer, and P.G. Nelson, 1983, Science [Wash. DC], 221:1294-1297). This protein may be a candidate for a role in growth cone functions, specific for neuronal subsets, such as pathfinding and selective axon fasciculation or the initiation of specific synapses. The methodology presented is thus capable of demonstrating patterns of protein synthesis that distinguish different neuronal subsets. The accessibility of these proteins for structural and functional studies may contribute to the elucidation of neuron-specific functions at the molecular level.

Binding between the neural cell adhesion molecules axonin-1 and Nr-CAM/Bravo is involved in neuron-glia interaction.

Neural cell adhesion molecules of the immunoglobulin superfamily mediate cellular interactions via homophilic binding to identical molecules and heterophilic binding to other family members or structurally unrelated cell-surface glycoproteins. Here we report on an interaction between axonin-1 and Nr-CAM/Bravo. In search for novel ligands of axonin-1, fluorescent polystyrene microspheres conjugated with axonin-1 were found to bind to peripheral glial cells from dorsal root ganglia. By antibody blockage experiments an axonin-1 receptor on the glial cells was identified as Nr-CAM. The specificity of the interaction was confirmed with binding studies using purified axonin-1 and Nr-CAM. In cultures of dissociated dorsal root ganglia antibodies against axonin-1 and Nr-CAM perturbed the formation of contacts between neurites and peripheral glial cells. Together, these results implicate a binding between axonin-1 of the neuritic and Nr-CAM of the glial cell membrane in the early phase of axon ensheathment in the peripheral nervous system.

A direct interaction of axonin-1 with NgCAM-related cell adhesion molecule (NrCAM) results in guidance, but not growth of commissural axons.

An interaction of growth cone axonin-1 with the floor-plate NgCAM-related cell adhesion molecule (NrCAM) was shown to play a crucial role in commissural axon guidance across the midline of the spinal cord. We now provide evidence that axonin-1 mediates a guidance signal without promoting axon elongation. In an in vitro assay, commissural axons grew preferentially on stripes coated with a mixture of NrCAM and NgCAM. This preference was 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, but not when axonin-1 and NrCAM or axonin-1 and NgCAM interactions, are perturbed. Thus, we conclude that axonin-1 is involved in guidance of commissural axons without promoting their growth.

Axonal proteins of presynaptic neurons during synaptogenesis.

Changes occur in the synthesis and axonal transport of neuronal proteins in dorsal-root ganglia axons as a result of contact with cells from the spinal cord during synapse formation. Dorsal-root ganglia cells were cultured in a compartmental cel culture system that allows separate access to neuronal cell bodies and their axons. When cells from the ventral spinal cord were cultured with the dorsal-root ganglia axons, synapses were established within a few days. Metabolic labeling and two-dimensional electrophoresis revealed that four of more than 300 axonal proteins had changed in their expression by the time synapses were established. The highly selective nature of these changes suggests that the proteins involved may be important in the processes of axon growth and synapse formation and their regulation by the regional environment.

Interference with axonin-1 and NrCAM interactions unmasks a floor-plate activity inhibitory for commissural axons.

Axonin-1 and NrCAM were previously shown to be involved in the in vivo guidance of commissural growth cones across the floor plate of the embryonic chicken spinal cord. To further characterize their role in axon pathfinding, we developed a two-dimensional coculture system of commissural and floor-plate explants in which it was possible to study the behavior of growth cones upon floor-plate contact. Although commissural axons readily entered the floor plate under control conditions, perturbations of either axonin-1 or NrCAM interactions prevented the growth cones from entering the floor-plate explants. The presence of antiaxonin-1 resulted in the collapse of commissural growth cones upon contact with the floor plate. The perturbation of NrCAM interactions also resulted in an avoidance of the floor plate, but without inducing growth-cone collapse. Therefore, axonin-1 and NrCAM are crucial for the contact-mediated interaction between commissural growth cones and the floor plate, which in turn is required for the proper guidance of the axons across the ventral midline and their subsequent rostral turn into the longitudinal axis.

Continuous renewal of the axonal pathway sensor apparatus by insertion of new sensor molecules into the growth cone membrane.

BACKGROUND: Growth cones at the tips of growing axons move along predetermined pathways to establish synaptic connections between neurons and their distant targets. To establish their orientation, growth cones continuously sample for, and respond to, guidance information provided by cell surfaces and the extracellular matrix. To identify specific guidance cues, growth cones have sensor molecules on their surface, which are expressed differentially during the temporospatial progress of axon outgrowth, at levels that depend on the pattern of neural activity. However, it has not been elucidated whether a change in gene expression can indeed change the molecular composition and, hence, the function of the sensor apparatus of growth cones. RESULTS: We have constructed adenoviral gene transfer vectors of the chicken growth cone sensor molecules axonin-1 and Ng-CAM. Using these vectors, we initiated the expression of axonin-1 and Ng-CAM in rat dorsal root ganglia explants during ongoing neurite outgrowth. Using specific surface immunodetection at varying time points after infection, we found that axonin-1 and Ng-CAM are transported directly to the growth cone and inserted exclusively in the growth cone membrane and not in the axolemma of the axon shaft. Furthermore, we found that axonin-1 and Ng-CAM do not diffuse retrogradely, suggesting that the sensor molecules are integrated into multimolecular complexes in the growth cone. CONCLUSIONS: During axon outgrowth, the pathway sensor apparatus of the growth cone is continuously updated by newly synthesized sensor molecules that originate directly from the transcription/translation machinery. Changes in the expression of sensor molecules may have a direct impact, therefore, on the exploratory function of the growth cone.

Affinity capture of proteins from solution and their dissociation by contact printing.

Biological experiments at the solid/liquid interface, in general, require surfaces with a thin layer of purified molecules, which often represent precious material. Here, we have devised a method to extract proteins with high selectivity from crude biological sample solutions and place them on a surface in a functional, arbitrary pattern. This method, called affinity-contact printing (alphaCP), uses a structured elastomer derivatized with ligands against the target molecules. After the target molecules have been captured, they are printed from the elastomer onto a variety of surfaces. The ligand remains on the stamp for reuse. In contrast with conventional affinity chromatography, here dissociation and release of captured molecules to the substrate are achieved mechanically. We demonstrate this technique by extracting the cell adhesion molecule neuron-glia cell adhesion molecule (NgCAM) from tissue homogenates and cell culture lysates and patterning affinity-purified NgCAM on polystyrene to stimulate the attachment of neuronal cells and guide axon outgrowth.

Cloning and sequencing of the cDNA encoding human neurotrypsin.

cDNA clones encoding human neurotrypsin have been isolated from a human fetal brain cDNA library using a PCR-amplified probe. The assembled cDNA sequence contains a 2625 bp open reading frame encoding a multidomain serine protease with an overall sequence identity of 82.5% to murine neurotrypsin. Surprisingly, the human neurotrypsin exhibits an additional scavenger receptor cysteine-rich repeat.

Structure of the mouse gene for the serine protease inhibitor neuroserpin (PI12).

Neuroserpin (PI12), initially identified as an axonally secreted protein in cultured chicken dorsal root ganglion neurons, belongs to the serpin family of the serine protease inhibitors and is mainly expressed by neurons of both the developing and the adult nervous system. Here we report on the cloning and structural characterization of the neuroserpin gene of the mouse. The murine neuroserpin gene spans over more than 55kb and consists of nine exons. The positions and phases of the exonintron borders are completely conserved between neuroserpin and its nearest homologues, protease nexin-1 and plasminogen activator inhibitor-1. A single transcription initiation site, which is colocalized with a potential initiation (Inr) sequence, has been determined by primer extension and RNase protection. Sequence analysis revealed a TATA-less promoter with a CAAT box and several sites for the general transcription factor Sp1 and the neuron-specific transcription factor AP-2.

Crystal structure of neuroserpin: a neuronal serpin involved in a conformational disease.

The protease inhibitor neuroserpin regulates the development of the nervous system and its plasticity in the adult. Neuroserpins carrying the Ser53Pro or Ser56Arg mutation form polymers in neuronal cells. We describe here the structure of wild-type neuroserpin in a cleaved form. The structure provides a basis to understand the role of the mutations in the polymerization process. We propose that these mutations could delay the insertion of the reactive center loop into the central beta-sheet A, an essential step in the inhibition and possibly in the polymerization of neuroserpin.

Expression of the axonal cell adhesion molecules axonin-1 and Ng-CAM during the development of the chick retinotectal system.

Cell surface glycoproteins expressed on growth cones and axons during brain development have been postulated to be involved in the cell-cell interactions that guide axons into their target area. Nevertheless, an unequivocal description of the mechanism by which such molecules exert control over the pathway of a growing axon has not been done. As a crucial requirement in support of a relevant involvement of an axonal surface molecule in growth cone guidance, this molecule should be expressed in the growth cone. The developing retinotectal system provides an excellent opportunity to test whether a particular neuronal surface molecule fulfills the requirement of the spatiotemporal coincidence between its appearance and the emergence of growth cones because its setup follows the rule of chronotopy, i.e., the position of axons in a certain site is determined by the time of their arrival. We have analyzed axonin-1 and the neuron-glia cell adhesion molecule (Ng-CAM), two axonal surface molecules that promote neurite growth in vitro, for their expression in the retina and in the retinotectal system of the chick throughout its development. At stage 18, both axonin-like (A-LI) and Ng-CAM-like immunoreactivity (Ng-CAM-LI) are clearly present in the area where first retinal ganglion cells (RGCs) are generated. The immunoreactivity spreads synchronously with the formation of RGCs over the developing retina. From stage 32 on, the inner plexiform layer is also stained according to its temporospatial gradient of maturation. In later stages, the outer plexiform layer and the inner segments of photoreceptors also show immunoreactivity. The development of A-LI and Ng-CAM-LI along the optic nerve, chiasm, optic tract, and in the superficial layers of the optic tectum follows the chronotopic pattern of axons, as was found by earlier morphological investigations. Older axons loose their A-LI. This allows to localize the position of newly formed axons. The fact that A-LI and Ng-CAM-LI parallel the formation and maturation of axons suggests that axonin-1 and Ng-CAM may play an important role in the organization of the retinotectal system.

Distribution of TAG-1/axonin-1 in fibre tracts and migratory streams of the developing mouse nervous system.

The axonal cell adhesion molecule, TAG-1/axonin-1, stimulates axonal growth and supports neurite fasciculation in vitro. Using a polyclonal antiserum raised against chick axonin-1, which shares 75% of its sequence with TAG-1 of the rat, we have mapped the distribution of TAG-1/axonin-1 throughout the developing nervous system of the mouse. Although absent from proliferating neuroepithelia and from non-neuronal cells, immunoreactivity for TAG-1/axonin-1 is expressed by stage-specific subpopulations of differentiating neurons from embryonic day 10 to postnatal day 15. It stains their axons and the surface of their parent somata during the early phases of axogenesis. In agreement with a putative role of TAG-1/axonin-1 as an axon-bound growth substrate, immunoreactivity is found in developing spinal and cranial nerves, in corticothalamic projections, as well as in subsets of fasciculating long projecting tracts of the central nervous system, such as the dorsal funiculi of the spinal cord, the lateral olfactory and optic tracts, the fasciculus retroflexus, and the predorsal bundle. High levels of immunoreactivity characterise the development of the cerebellar molecular layer, the corpus callosum, anterior and hippocampal commissure, and of crossed projections in the spinal cord and at several levels of the brainstem. Intense immunoreactivity in fine collaterals of cutaneous afferents, including their growth cones that are in contact with the embryonic skin, suggests a role of TAG-1/axonin-1 in target recognition. While staining is weak on the somata of radially migrating neurons such as cortical neurons and cerebellar granule cells, strong immunoreactivity is associated with neural somata and processes of the three tangential migrations that form the precerebellar nuclei, indicating a possible involvement of TAG-1/axonin-1 in contacts between these neurons and the processes they migrate upon.

High frequency of mutations in four different disease genes in early-onset dementia.

Heterozygous mutations in the genes for amyloid precursor protein (APP), the presenilins (PS1, PS2), prion protein (PrP), neuroserpin, and tau are associated with early-onset dementia (EOD) with or without neurological signs in the early disease stage. To investigate the proportion of EOD without early neurological signs attributable to known genes we prospectively (i.e., ante mortem) screened these six genes for mutations in 36 patients with EOD before age 60. Family history for dementia was positive (PFH) in 16, negative (NFH) in 17, and unknown (UFH) in 3 patients. In 12 patients, we found 5 novel mutations (PS1: F105L; PS2: T122P, M239I; PrP: Q160X, T188K) and 5 previously reported mutations (APP: in three most likely unrelated patients V717I; PS1: A79V, M139V; PrP: P102L, T183A) that all are considered disease causing. Of these 12 patients, 9 had PFH. This indicates a detection rate of 56% (9/16) in patients with PFH. We found 2 mutations (APP V717I) in 2 of the 3 the UFH-patients, and only 1 mutation (PrP T188K) in 1 of the 17 patients with NFH. No mutation was found in tau and neuroserpin genes. To date, three patients died and FAD, predicted by PS mutations in two patients, and prion disease, predicted by a PrP mutation in the third one, were histopathologically confirmed at autopsy. Up to now, mutation findings may be the most specific biomarkers for an ante mortem diagnosis of FAD or hereditary prion disease.