NgCAM and VAMP2 reveal that direct delivery and dendritic degradation maintain axonal polarity.

Neurons are polarized cells of extreme scale and compartmentalization. To fulfill their role in electrochemical signaling, axons must maintain a specific complement of membrane proteins. Despite being the subject of considerable attention, the trafficking pathway of axonal membrane proteins is not well understood. Two pathways, direct delivery and transcytosis, have been proposed. Previous studies reached contradictory conclusions about which of these mediates delivery of axonal membrane proteins to their destination, in part because they evaluated long-term distribution changes and not vesicle transport. We developed a novel strategy to selectively label vesicles in different trafficking pathways and determined the trafficking of two canonical axonal membrane proteins, neuron-glia cell adhesion molecule and vesicle-associated membrane protein-2. Results from detailed quantitative analyses of transporting vesicles differed substantially from previous studies and found that axonal membrane proteins overwhelmingly undergo direct delivery. Transcytosis plays only a minor role in axonal delivery of these proteins. In addition, we identified a novel pathway by which wayward axonal proteins that reach the dendritic plasma membrane are targeted to lysosomes. These results redefine how axonal proteins achieve their polarized distribution, a crucial requirement for elucidating the underlying molecular mechanisms.

Increased expression of the close homolog of the adhesion molecule L1 in different cell types over time after rat spinal cord contusion.

The close homolog of the adhesion molecule L1 (CHL1) is important during CNS development, but a study with CHL1 knockout mice showed greater functional recovery after spinal cord injury (SCI) in its absence. We investigated CHL1 expression from 1 to 28 days after clinically relevant contusive SCI in Sprague-Dawley rats. Western blot analysis showed that CHL1 expression was significantly up-regulated at day 1 and further increased over 4 weeks after SCI. Immunohistochemistry of tissue sections showed that CHL1 in the intact spinal cord was expressed at low levels. By 1 day and through 4 weeks after SCI, CHL1 became highly expressed in NG2(+) cells. Hypertrophic GFAP(+) astrocytes also expressed CHL1 by 1 week after injury. The increase in CHL1 protein paralleled that of NG2 in the first week and GFAP between 1 and 4 weeks after injury. At 4 weeks, NG2(+) /CHL1(+) cells and GFAP(+) /CHL1(+) astrocytes were concentrated at the boundary between residual spinal cord tissue and the central lesion. NF200(+) spinal cord axons approached but did not penetrate this boundary. In contrast, CHL1(+) cells in the central lesion at 1 week and later colabeled with p75 and NG2 and were chronically associated with many NF200(+) axons, presumably axons that had sprouted in association with CHL1(+) Schwann cells infiltrating the cord after contusion. Thus, our study demonstrates up-regulation of CHL1 in multiple cell types and locations in a rat model of contusion injury and suggests that this molecule may be involved both in inhibition of axonal regeneration and in recovery processes after SCI.

Células gliales y actividad sináptica: control traduccional del acople metabólico / Glial cells and synaptic activity: translational control of metabolic coupling

Introducción. Consideradas tradicionalmente como células de soporte, las células gliales constituyen la inmensa mayoría de las células cerebrales al superar en número a las neuronas por un factor de diez. Poco se conoce de su participación en la fisiología cerebral, a pesar de su ubicación privilegiada, envolviendo las sinapsis. Las células de la estirpe glial participan en la formación de la denominada barrera hematoencefálica y representan una conexión entre la concentración de metabolitos en el compartimiento sistémico y el líquido cefalorraquídeo. Desarrollo. En este artículo analizamos los fenómenos moleculares desencadenados por el ácido glutámico en las células gliales y su participación en el acople metabólico establecido entre estas células y las neuronas. Conclusiones. El control de la traducción selectiva de ARN mensajero constituye la base molecular del acople entre la liberación sostenida de glutamato, la captura de este neurotransmisor y la producción y liberación de glutamina por las células gliales (AU)

Introduction. Traditionally regarded as supportive cells, glial cells have been barely studied in the context of brain physiology. No attention has been paid to the fact that these cells outcome neurons by an estimated factor of ten, and more importantly that they surround synapses. Moreover, cells of glial linage influence the formation of the so-called brain blood barrier representing a link between the concentration of metabolites in the systemic compartment and thecerebrospinal fluid. Development. Using as a model system the cerebellar glutamatergic synapses, in this contribution, we analyze the molecular transactions triggered by glutamate within glial cells that are involved neuronal-glia metabolic coupling. Conclusions. A tight coupling between sustained neuronal glutamate release, glial glutamate uptake, glial glutamine production and release is based on the control of the translation of selective mRNAs (AU)

Ankyrin-dependent and -independent mechanisms orchestrate axonal compartmentalization of L1 family members neurofascin and L1/neuron-glia cell adhesion molecule.

Axonal initial segments (IS) and nodes of Ranvier are functionally important membrane subdomains in which the clustering of electrogenic channels enables action potential initiation and propagation. In addition, the initial segment contributes to neuronal polarity by serving as a diffusion barrier. To study the mechanisms of axonal compartmentalization, we focused on two L1 family of cell adhesion molecules (L1-CAMs) [L1/neuron-glia cell adhesion molecule (L1/NgCAM) and neurofascin (NF)] and two neuronal ankyrins (ankB and ankG). NF and ankG accumulate specifically at the initial segment, whereas L1/NgCAM and ankB are expressed along the entire lengths of axons. We find that L1/NgCAM and NF show distinct modes of steady-state accumulation during axon outgrowth in cultured hippocampal neurons. Despite their different steady-state localizations, both L1/NgCAM and NF show slow diffusion and low detergent extractability specifically in the initial segment but fast diffusion and high detergent extractability in the distal axon. We propose that L1-CAMs do not strongly bind ankB in the distal axon because of spatial regulation of ankyrin affinity by phosphorylation. NF, conversely, is initially enriched in an ankyrin-independent manner in the axon generally and accumulates progressively in the initial segment attributable to preferential binding to ankG. Our results suggest that NF and L1/NgCAM accumulate in the axon by an ankyrin-independent pathway, but retention at the IS requires ankyrin binding.

Neurine, an acetylcholine autolysis product, elevates secreted amyloid-beta protein precursor and amyloid-beta peptide levels, and lowers neuronal cell viability in culture: a role in Alzheimer's disease?

Classical hallmarks of Alzheimer's disease (AD) are a synaptic loss, cholinergic neuron death, and abnormal protein deposition, particularly of toxic amyloid-beta peptide (Abeta) that is derived from amyloid-beta protein precursor (AbetaPP) by the action of beta- and gamma-secretases. The trigger(s) initiating the biochemical cascades that underpin these hallmarks have yet to be fully elucidated. The typical forebrain cholinergic cell demise associated with AD brain results in a loss of presynaptic cholinergic markers and acetylcholine (ACh). Neurine (vinyl-trimethyl-ammonium hydroxide) is a breakdown product of ACh, consequent to autolysis and is an organic poison found in cadavre brain. The time- and concentration-dependent actions of neurine were assessed in human neuroblastoma (NB, SK-N-SH) cells in culture by quantifying cell viability by lactate dehydrogenase (LDH) and MTS assay, and AbetaPP and Abeta levels by Western blot and ELISA. NB cells displayed evidence of toxicity to neurine at > or = 3 mg/ml, as demonstrated by elevated LDH levels in the culture media and a reduced cell viability shown by the MTS assay. Using subtoxic concentrations of neurine, elevations in AbetaPP and Abeta1-40 peptide levels were detected in conditioned media samples.

Neuron-glia interaction in the insect nervous system.

In all complex organisms, glial cells are pivotal for neuronal development and function. Insects are characterized by having only a small number of these cells, which nevertheless display a remarkable molecular diversity. An intricate relationship between neurons and glia is initially required for glial migration and during axonal patterning. Recent data suggest that in organisms such as Drosophila, a prime role of glial cells lies in setting boundaries to guide and constrain axonal growth.

Adhesion proteins for a tight neuron-electrode contact.

The neural cell adhesion molecules axonin-1 and NgCAM have been genetically engineered and covalently immobilized on glass and silicon oxide surfaces in their correct orientation. Surfaces treated with these adhesion molecules were used as substrates for culturing dorsal root ganglion neurons. The cleft between the neuron cell membrane and the surface was determined using fluorescence interference contrast (FLIC) microscopy. For comparison, cell--material distances on laminin, RGDC, polylysine and amino-terminated surfaces were measured. When the neurons grow on axonin-1 the cell--surface distance is at a minimum (37 nm) probably because the glycocalyx hinders a closer contact. A selective treatment of extracellular electrodes with axonin-1 could be used to improve the cell-material contact and thus increase extracellularly recorded signals.

Developmental and reactive growth of dentate gyrus afferents: cellular and molecular interactions.

The lamination of dentate gyrus afferents established during development is maintained following lesion-induced reactive growth in the adult. After partial deafferentation sprouts from undamaged afferents restore most synapses, while respecting the laminae relative boundaries. No evidence of trans-laminar sprouting has been found. Here, we review the information gathered during the last decade on the cellular and molecular bases of dentate synaptogenesis, with special attention to the role of glia during development and that of reactive glia after deafferentation. The interactions of neurons with astroglia and astroglial macromolecules, particularly proteoglycans, influence synapse segregation in the dentate gyrus, providing us with a reasonable explanation for afferent lamination.

Radial glia phenotype: origin, regulation, and transdifferentiation.

Radial glial cells play a major guidance role for migrating neurons during central nervous system (CNS) histogenesis but also play many other crucial roles in early brain development. Being among the earliest cells to differentiate in the early CNS, they provide support for neuronal migration during embryonic brain development; provide instructive and neurotrophic signals required for the survival, proliferation, and differentiation of neurons; and may be multipotential progenitor cells that give rise to various cell types, including neurons. Radial glial cells constitute a major cell type of the developing brain in numerous nonmammalian and mammalian vertebrates, increasing in complexity in parallel with the organization of the nervous tissue they help to build. In mammalian species, these cells transdifferentiate into astrocytes when neuronal migration is completed, whereas, in nonmammalian species, they persist into adulthood as a radial component of astroglia. Thus, our perception of radial glia may have to change from that of path-defining cells to that of specialized precursor cells transiently fulfilling a guidance role during brain histogenesis. In that respect, their apparent change of phenotype from radial fiber to astrocyte probably constitutes one of the most common transdifferentiation events in mammalian development.

Estrogen promotes the initial migration and inception of NgCAM-dependent calcium-signaling by new neurons of the adult songbird brain.

The adult avian forebrain continues to generate neurons from ventricular zone (VZ) precursor cells, whose neuronal progeny then migrate into the brain parenchyma. Migrating neurons respond to the Ig-family adhesion molecule NgCAM with increments in cytosolic calcium, and migration is disrupted by anti-NgCAM Ig. The calcium response to NgCAM is developmentally restricted to bipolar migrants during a period spanning 6 to 9 DIV. This period corresponds to the postmitotic age at which new neurons leave the adult VZ to traverse a subjacent layer of estrogen-receptive "gatekeeper" neurons. Since neuronal passage through this layer occurs concurrently with the onset of NgCAM-dependent calcium signaling, we asked whether acquisition of the calcium response to NgCAM required estrogen exposure. Among neurons arising from explants of the adult finch neostriatal VZ, only those supplemented with estrogen developed calcium responses to NgCAM; neither explants raised in the absence of estrogen, nor those supplemented with testosterone, did so. Neurons in all three groups expressed NgCAM, had equivalent baseline calcium levels, and responded identically to K+-depolarization. Nonetheless, many more neurons migrated from explants of both finch and canary VZ raised in estrogen-supplemented media than from their estrogen-deprived counterparts, even though no effect of estrogen on neuronal survival per se was noted. These findings suggest that estrogen encourages the initial departure and assumption of signal competence by neurons arising from the adult avian VZ, thereby promoting their parenchymal recruitment and migration success.

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.

Neurolin, the goldfish homolog of DM-GRASP, is involved in retinal axon pathfinding to the optic disk.

Young axons of new retinal ganglion cells (RGCs) in the continuously growing goldfish retina fasciculate with one another and their immediate forerunners on their path toward the optic disk and along the optic nerve. They express the immunoglobulin superfamily cell adhesion molecules (CAMs) neurolin (DM-GRASP) and the L1-like E587 antigen. Repeated injections of Fab fragments from polyclonal antisera against neurolin (neurolin Fabs) into the eye of 3. 4-cm-long and rapidly growing goldfish caused highly aberrant pathways of young RGC axon subfascicles in the dorsal retina. Many axons grew in circles and failed to reach the optic disk. In contrast, E587 Fabs, used in parallel experiments, disrupted the fascicles but did not interfere with the disk-directed growth. Neurolin Fabs also disturbed axonal fasciculation in vivo as well as in vitro but less severely than E587 Fabs. Coinjections of both Fabs increased defasciculation of the dorsal axons in both aberrant and disk-directed routes. They also disrupted the order of young RGC axons in the optic nerve more severely than E587 Fabs alone. This demonstrates that the development of tight and orderly fascicles in the dorsal retina and in the optic nerve requires both E587 antigen and neurolin. More importantly, our results suggest an involvement of neurolin in RGC axonal guidance from the retinal periphery to the optic disk. Because disrupted fascicles and errant axon routes were found only in the dorsal retinal half, a cooperation with so-called positional markers may be conceived.

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.

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.

Axonal growth-related cell surface molecule, neurin-1, involved in neuron-glia interaction.

We purified and characterized a novel axonal growth-related molecule, neurin-1, which is anchored to the surface membrane via a phosphatidylinositol (PI) linkage. This molecule was detected by a combination of phosphatidylinositol-specific phospholipase C (PI-PLC) treatment from detergent-soluble mouse brain membranes and subsequent Western blot analysis with monoclonal antibody (MAb 2A). Neurin-1 is immunologically distinct from other known axonal growth associated surface glycoproteins. In immunoblots of embryonic mouse brain membrane, the MAb 2A recognized a single band at approximately 68 kDa, and showed that neurin-1 is mainly associated with fiber-containing regions of developing embryonic mouse brain. Expression is immunohistochemically similar to that of cell adhesion molecule L1, but in comparison, neurin-1 appears somewhat later. Late in embryonic development, neurin-1 appeared to be more stage- and region-specific. Its precise localization at the neural cell surface membranes was confirmed by immuno-electron microscopy using labeled and cultured live nerve cells. Neurin-1 was found only on the surface of the axon and growth cone. Neurin-1, otherwise termed PI anchor protein, corresponds closely in function to the other PI-anchored cell adhesion molecules. Anti-neurin-1 antibody (MAb 2A), however, perturbs the axonal growth and neural cell migration from the astrocyte feeder layer cultures. These results suggest that neurin-1 is one of the important cell surface molecules mediated in the neuron and glial cell interaction.

Clustering and functional cooperation of Ng-CAM and axonin-1 in the substratum-contact area of growth cones.

Growth cones and neurites of chicken dorsal root ganglia neurons cultured on laminin, Ng-CAM, or axonin-1 exhibit substratum-dependent morphology and growth patterns which are accompanied by distinctive distributions of axonin-1 and Ng-CAM in the growth cone membrane. On either Ng-CAM or axonin-1 substratum, both Ng-CAM and axonin-1 were depleted from some areas of the apical growth cone membrane. In contrast, on laminin, both axonin-1 and Ng-CAM remained randomly distributed. Removal of axonin-1 from growth cones resulted in a blockage of neurite outgrowth on both Ng-CAM and axonin-1 substrata, indicating that in these neurons axonin-1 cooperates with Ng-CAM in the activation of axon growth. Based on these results possible molecular models for cooperation between axonin-1 and Ng-CAM on the growth cone are discussed.