The role of cell adhesion molecules for navigating axons: density matters.

Cell adhesion molecules of the immunoglobulin-super-family (IgSF-CAMs) do not only have a physical effect, mediating merely attachment between cell surfaces. For navigating axons, IgSF-CAMs also exert an instructive impact: Upon activation, they elicit intracellular signalling cascades in the tip of the axon, the growth cone, which regulate in a spatio-temporally concerted action both speed and direction of the axon. Density and distribution of IgSF-CAMs in the growth cone plasma membrane play important roles for the activation of IgSF-CAMs, their clustering, and the adhesive forces they acquire, as well as for the local restriction and effective propagation of their intracellular signals.

Role of cyclin-dependent kinase 5 and its activator P35 in local axon and growth cone stabilization.

Axons elongate and perform steering reactions with their growth cones constantly undergoing local collapse and stabilization. Our previous studies have shown that a type-1 phosphorylated form of microtubule-associated protein 1B, recognized by monoclonal antibody 1E11 (mab1E11), is present in stable regions and absent from unstable regions of turning growth cones of retinal ganglion cells. In contrast, the total population of microtubule-associated protein 1B is present in the entire growth cone. Here we demonstrate that inhibition of cyclin-dependent kinase 5 (Cdk5) results in loss of mab1E11 binding whereas inhibition of glycogen synthase kinase 3 has no such effect, revealing that mab1E11 recognizes a Cdk5 phosphorylation site on type-1 phosphorylated form of microtubule-associated protein 1B. We moreover show that kinase Cdk5 as well as its activator P35 is present in retinal ganglion cells in the early developing chick embryo retina and enriched in their extending axons. Cdk5 and P35 are concentrated in the youngest, distal axon region and the growth cone as also seen for Cdk5-phosphorylated type-1 phosphorylated form of microtubule-associated protein 1B. Inhibition of Cdk5 by antibodies or inhibitor Roscovitine results in growth cone collapse and axon retraction and prevents substantial axon outgrowth. In contrast, glycogen synthase kinase 3 inhibition causes only a transient axon retraction which is soon recovered and allows for axon formation. In growth cones induced to turn at substrate borders, where stable and instable parts of the growth cone are clearly defined, Cdk5 is present in the entire growth cone. P35, in contrast, is restricted to the stable parts of the growth cone, which do not collapse but instead transform into new distal axon. The local presence of Cdk5-phosphorylated type-1 phosphorylated form of microtubule-associated protein 1B in stabilized growth cone areas can be therefore attributed to the local activation of Cdk5 by P35 in these regions. Together our data demonstrate a crucial role of Cdk5 and its activator P35 in elongation and maintenance of axons as well as for stability and steering of their growth cones.

Role of cell adhesion molecule DM-GRASP in growth and orientation of retinal ganglion cell axons.

The cell adhesion molecule (CAM) DM-GRASP was investigated with respect to a role for axonal growth and navigation in the developing visual system. Expression analysis reveals that DM-GRASP's presence is highly spatiotemporally regulated in the chick embryo retina. It is restricted to the optic fiber layer (OFL) and shows an expression maximum in a phase when the highest number of retinal ganglion cell (RGC) axons extend. In the developing retina, axons grow between the DM-GRASP-displaying OFL and the Laminin-rich basal lamina. We show that DM-GRASP enhances RGC axon extension and growth cone size on Laminin substrate in vitro. Preference assays reveal that DM-GRASP-containing lanes guide RGC axons, partially depending on NgCAM in the axonal membrane. Inhibition of DM-GRASP in organ-cultured eyes perturbs orientation of RGC axons at the optic fissure. Instead of leaving the retina, RGC axons cross the optic fissure and grow onto the opposite side of the retina. RGC axon extension per se and navigation from the peripheral retina towards the optic fissure, however, is not affected. Our results demonstrate a role of DM-GRASP for axonal pathfinding in an early phase of the formation of the higher vertebrate central nervous system.

The microtubule-associated protein MAP1B is involved in local stabilization of turning growth cones.

For the development of the nervous system it is crucial that growth cones detect environmental information and react by altering their growth direction. The latter process is thought to depend on local stabilization of growth cone microtubules. We have obtained evidence of a role for the microtubule-associated protein MAP1B, in particular a mode 1 phosphoisoform of the molecule, P1-MAP1B, in this process. P1-MAP1B is tightly associated with the cytoskeleton and is present at highest concentrations in the distal axon and the growth cone of chick retinal ganglion cells. In growth cones turning at nonpermissive substrate borders, P1-MAP1B is restricted to regions which are stabilized. Unilateral neutralization of P1-MAP1B in one-half the growth cone by microscale chromophore-assisted laser inactivation changes growth cone motility, morphology, and growth direction. The results indicate a functional role for P1-MAP1B in local growth cone stabilization and thus growth cone steering.

Regulatory interrelations among topographic molecules CBF1, CBF2 and EphA3 in the developing chick retina.

It has been shown that topographic expression of two winged-helix transcription factors, CBF1/c-qin and CBF2, and a receptor tyrosine kinase EphA3 (Mek4/Cek4) play important roles in establishing the topographic retinotectal projection map along the rostrocaudal axis. The interrelationship among these topographic molecules in the chick retina was studied during development. The topographic expression of CBF1 and CBF2 preluded the graded expression of EphA3, but their precise expression profiles did not exactly fit together. However, interestingly, CBF1 and CBF2 were properly expressed, together with EphA3, in immortalized cell lines derived from the quail retina, which maintained position-specific characteristics. The expression of another topographic molecule SOHo-1, the sensory organ homeobox-1 transcription factor, was separate from EphA3 expression. Ectopic expression of CBF1 using in ovo electroporation repressed the expression of CBF2, and misexpression of CBF2 influenced the graded localization of EphA3 in the retina, albeit imperfectly. Taken together, it is suggested that retinal cells first begin to express CBF1 or CBF2 according to their topographic positions, generate cellular descendants in which the expression of CBF1 and CBF2 is maintained cell-autonomously, and then establish the nasotemporal gradient of EphA3 under the control of CBF2, although indirect.

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.

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.

Target preference of embryonic retina cells and retinal cell lines is cell-autonomous, position-specific, early determined and heritable.

Retinal ganglion cells (RGCs) form the topographic connection between retina and optic tectum in the developing avian embryo. In vitro, neurons with the morphological traits and marker expression of RGCs were found both in single-cell cultures from embryonic day (E) 6 chick retina and in retinal cell lines derived from E3.5 quail retina. Rapid and substantial differentiation of RGC-like cells could be induced in the lines by addition of fibroblast growth factor aFGF or bFGF. RGC-like cells were examined with respect to their target discrimination properties as single cells in the stripe carpet assay. In this assay system, alternating stripes of membrane vesicles prepared from the anterior and posterior tectum are offered to growing axonal processes as a substrate. Temporal RGC-like cells, both primary cells prepared from the temporal retina and immortalized cells of those retinal lines derived from the temporal retina, avoid stripes of membrane vesicles from posterior tectum; they prefer to grow on membrane vesicles from the anterior tectum, which is their in vivo target. Nasal RGC-like cells did not exhibit a target preference, in accordance with previous findings. Together the experiments show that target preference of RGCs is a cell-autonomous and heritable mechanism that is determined early and is position-dependent.

Two strategies to prepare neural cortical cytoskeleton components for the generation of monoclonal antibodies.

Like most other cells, neurons possess a spectrin/actin based network closely associated with the inner side of the cell membrane, the cortical cytoskeleton. This structure serves many diverse functions during axonal outgrowth. In the growth cone, the cortical cytoskeleton is involved in surface shaping, modulation of integral membrane proteins, and signal transduction. We developed two strategies to prepare material enriched for neural cortical cytoskeleton. The first strategy combined the isolation of a membrane/cortical cytoskeleton fraction by density gradient centrifugation with an enzymatic degradation of cell surface proteins. The second strategy is based on the attachment and crosslinking of single cells to beads, allowing for the removal of the cell contents by cell disruption; only membrane/cortical cytoskeleton patches are retained on the beads. Both strategies made use of the intimate association of the cortical cytoskeleton with the cell membrane, permitting the removal of cytoplasm, organelles and cytoplasmic cytoskeleton while retaining the cortical cytoskeleton. Monoclonal antibodies generated using both preparations as immunization material were screened for recognition of intracellular structures in axons and growth cones of retinal ganglion cells in culture. A quantitative specification of the antibodies is presented and six antibodies are characterized in immunolabelings and Western blot analysis.

Generation of cell lines from embryonic quail retina capable of mature neuronal differentiation.

The avian embryonic retina is widely used as a model system for cellular and molecular studies on central nervous system neurons. We aimed at the generation of cell lines from the early embryonic quail retina by retroviral oncogene transduction. For this, we made use of the retina organ culture system which exhibits both proliferation, necessary for stable oncogene transduction, and initial neuronal differentiation, a prerequisite for the generation of cell lines with mature neuronal properties. The oncogene myc was chosen as it is both proliferation-inducing and differentiation-compatible. A chimeric gene, mycER, containing v-myc and the hormone-binding domain of the estrogen receptor, was used for transduction in order to allow for hormone regulation of myc activity. Transduced organ-cultured cells from temporal and nasal retina were passaged into sparse single cell cultures. From these, colonies of rapidly dividing cells were isolated and the progeny expanded as cell lines. The lines contained cells with features of neuroepithelial cells, showing vimentin and A2B5. They also contained spontaneously differentiated neuronal cells showing neurofilament L and N-CAM180. A subpopulation of the neuronal cells exhibited the morphological characteristics of retinal ganglion cells, i.e., large pear-shaped somata each emitting one long process with a distinct growth cone. In addition, they showed the marker profile of retinal ganglion cells, i.e., expression of Thy-1, G4, DM-GRASP, Nr-CAM, neurofilament H, and tau. Neuronal differentiation could be induced by the addition of db cAMP and retinoic acid. The mature neuronal features of the lines open new possibilities to study properties of retinal neurons, including ganglion cells, in a defined and manipulable experimental system.

Cell adhesion molecule SC1/DMGRASP is expressed on growing axons of retina ganglion cells and is involved in mediating their extension on axons.

We determined expression and function of a cell membrane protein in the developing chick retinotectal system identified by a monoclonal antibody (mAb 4H5) and the corresponding antiserum. Our data revealed that the protein shares a series of properties, including the N-terminal amino acid sequence, with a cell adhesion molecule termed DM-GRASP, SC1, BEN, and JC7. It can therefore be considered identical with this molecule and is referred to as SC1/DMGRASP. In early development of the retinotectal system, SC1/DMGRASP is exclusively expressed on growing, far-projecting, tract-forming axons. Expression begins at the onset of retina ganglion cell axogenesis and its maximum overlaps with the phase of maximal axon extension. Later in development, SC1/DMGRASP appears on distinct laminae within plexiform layers in spatiotemporal correlation with synaptogenesis. In an in vitro assay system designed to study the elongation of RGC axonal processes on preexisting RGC axons, addition of SC1/DMGRASP antiserum specifically reduces lengths of axonal processes. In contrast, axonal growth on laminin or basal lamina preparations is not SC1/DMGRASP-dependent. Taken together, the data provide evidence for a role of SC1/DMGRASP in axonal elongation of SC1/DMGRASP-positive axons on such axons, thereby possibly contributing to the pathway and target finding mechanisms of far-projecting, tract-forming central nervous system neurons.

A functional role for the middle extracellular region of the neural cell adhesion molecule (NCAM) in axonal fasciculation and orientation.

A new monoclonal antibody (mAb HR1) was used to study the function of a previously neglected region in the extracellular domain of the neural cell adhesion molecule (NCAM). Application of mAb HR1 in retina organ culture interferes with several axonal functions in the developing eye. The antibody disturbs the orientation of axons growing in the peripheral retina and the tracking of axons in the middle retina. In the central retina, fasciculation is disturbed and a proportion of the ganglion cell axons do not leave the eye at the optic fissure but are misrouted to the contralateral side of the retina. Analysis of peptide fragments of NCAM indicates that the epitope of mAb HR1 resides in the region C-terminal to the fifth immunoglobulin (Ig) domain. Moreover, mAb HR1 binds to the oligopeptide comprising the 15 amino acids immediately C-terminal to the fifth Ig domain of NCAM. In addition, binding of mAb HR1 to NCAM is increased by removal of the large polysialic acid chains of the fifth Ig domain. Taken together, the data show that this region of NCAM--which has been previously reported to represent the flexible hinge region of the molecule--is crucial for the function of this molecule, in particular on cell surfaces in motion, e.g., those of growing axons in the developing nervous system.

Immunoelectron-microscopic localization of the 180 kD component of the neural cell adhesion molecule N-CAM in postsynaptic membranes.

In order to investigate the expression of cell adhesion molecules in synapses, we have studied the localization of the neural cell adhesion molecule N-CAM in the cerebellum and hippocampus of adult mice by immunocytological and immunochemical methods. Of the three molecular components of N-CAM with relative molecular masses (Mr) of 120, 140, and 180 kD, N-CAM 120 is not detectable in synaptosomal membranes, whereas N-CAM 140 is expressed on both pre- and postsynaptic membranes and N-CAM 180 is restricted to postsynaptic sites, with localization of the N-CAM 180-specific epitope in postsynaptic densities. Specificity of immunoreactivity is indicated by the observation that antibodies to the neural cell adhesion molecule L1 do not label synaptic membranes, whereas antibodies to two major components of postsynaptic densities, actin and erythrocyte spectrin, react with synaptic structures. Interestingly, N-CAM 180 is only detectable in subpopulations of synapses in the intact tissue. Isolated synaptosomes, opened for unimpeded accessibility of antibody by hypoosmotic treatment, also reveal a partial expression of N-CAM 180 in that 67% are labeled by antibodies to N-CAM 180, while antibodies to actin and erythrocyte spectrin react with 95% and 88% of all synaptosomes, respectively. N-CAM 180 does not appear to be differentially expressed in synapses of a particular morphological type, but is detectable in all types of synapses in the cerebellum and hippocampus, except for mossy fiber synapses and synapses between basket and Purkinje cells, which are generally N-CAM 180-negative. Since N-CAM 180 has been shown to be characteristic of stabilized or stabilizing cell contacts, possibly by its association with the cytoskeleton-membrane linker protein spectrin (Pollerberg et al.: J. Cell Biol. 101:1921-1929, '85; Nature 324:462-465, '86; Cell Tissue Res. 250:227-236, '87), we would like to suggest N-CAM 180 plays an important role in determining the stability of contacts between pre- and postsynaptic membranes and state of synaptic activity.

The 180-kD component of the neural cell adhesion molecule N-CAM is involved in cell-cell contacts and cytoskeleton-membrane interactions.

N-CAM180, the molecular form of the three neural cell adhesion molecules (N-CAM) with the largest cytoplasmic domain, is accumulated at sites of cell-cell contact (cell bodies, neurites, growth cones) in cultures of neuroblastoma and cerebellum. At these sites the cytoskeleton-membrane linker protein brain spectrin and actin are also accumulated. Brain spectrin copurifies with N-CAM180 by immunoaffinity chromatography and binds specifically to N-CAM180 but not to N-CAM140 or N-CAM120 in a solid-phase binding test. These observations indicate an association of N-CAM180 with the cytoskeleton in vivo. This association may underlie the reduced lateral mobility of N-CAM180 in the surface membrane compared to N-CAM140 (Pollerberg et al. 1986). Together with the fact that N-CAM180 is only expressed after termination of neuron migration in vivo (Persohn and Schachner, unpublished) these results suggest a role for N-CAM180 in stabilization of cell contacts.

Differentiation state-dependent surface mobilities of two forms of the neural cell adhesion molecule.

The neural cell adhesion molecule (N-CAM) has been implicated in morphogenetic events during formation of the nervous system. Three forms of N-CAM exist, all glycoprotein chains, of relative molecular masses 180,000 (180K), 140K and 120K (N-CAM180, N-CAM140 and N-CAM120) which are differentially expressed on neural cell types and during development. The three chains are thought to carry similar if not identical amino-acid sequences on their extracellular amino-terminal domains, but differ in the length of their carboxy-terminal cytoplasmic region. They occur in highly sialylated embryonic and less sialylated adult forms. N-CAM180 is selectively expressed in more differentiated neural cells and may play a role in the stabilization of cell contacts. To investigate this, we have studied in the surface membrane of a mouse neuroblastoma cell line N2A the lateral mobility of the two predominant forms of N-CAM, N-CAM180 and N-CAM140, as a function of differentiation. Here we report that as judged by fringe pattern photobleaching, the surface mobility of N-CAM140 is higher than that of N-CAM180, suggesting an association of N-CAM180 with the cytoskeleton or other stabilizing factors. We also show that brain spectrin, a membrane-cytoskeleton linker protein, binds only to N-CAM180. The immobilization of N-CAM in differentiated N2A cells is achieved by a shift in expression from N-CAM140 to N-CAM180.

Opioid antinociception and positive reinforcement are mediated by different types of opioid receptors.

Fentanyl (FEN) and diprenorphine's (DIPR) potentials for analgesia and reinforcement were assayed using rats. Analgesia was measured by the classic tail-flick test. The test germane to opioid reinforcement involved measuring pressing rates for direct electrical stimulation of the lateral hypothalamus and ventral tegmental area. FEN, as does morphine and heroin, produced strong analgesia and enhanced pressing rates for brain stimulation. DIPR produced no analgesia and antagonized FEN's analgesia. DIPR, at doses antagonizing FEN's analgesia, enhanced pressing for brain stimulation. DIPR's enhancement of pressing was antagonized by naloxone (100 micrograms/kg). When FEN and DIPR were given concurrently, pressing for brain stimulation was not reduced and was greater than after FEN alone was given. These data support a conclusion that different types of receptors are associated with opioid analgesia and reinforcement.