Enzymatic removal of polysialic acid from neural cell adhesion molecule interrupts gonadotropin releasing hormone (GnRH) neuron-glial remodeling.

There is abundant evidence to prove that the astrocytes are highly dynamic cell type in CNS and under physiological conditions such as reproduction, these cells display a remarkable structural plasticity especially at the level of their distal processes ensheathing the gonadotropin releasing hormone (GnRH) axon terminals. The morphology of GnRH axon terminals and astrocytes in the median eminence region of hypothalamus show activity dependent structural plasticity during different phases of estrous cycle. In the current study, we have assessed the functional contribution of ∞-2,8-linked polysialic acid (PSA) on neural cell adhesion molecule (PSA-NCAM) in this neuronal-glial plasticity using both in vitro and in vivo model systems. In vivo experiments were carried out after stereotaxic injection of endoneuraminidase enzyme (endo-N) near median eminence region of hypothalamus to specifically remove PSA residues on NCAM followed by localization of GnRH, PSA-NCAM and glial fibrillary acidic protein (GFAP) by immunostaining. Using in vitro model, structural remodeling of GnV-3 cells, (a conditionally immortalized GnRH cell line) co-cultured with primary astrocytes was studied after treating the cells with endo-N. Marked morphological changes were observed in GnRH axon terminals in proestrous phase rats and control GnV-3 cells as compared to endo-N treatment i.e. after removal of PSA. The specificity of endo-N treatment was also confirmed by studying the expression of PSA-NCAM by Western blotting in cultures treated with and without endo-N. Removal of PSA from surfaces with endo-N prevented stimulation associated remodeling of GnRH axon terminals as well as their associated glial cells under both in vivo and in vitro conditions. The current data confirms the permissive role of PSA to promote dynamic remodeling of GnRH axon terminals and their associated glia during reproductive cycle in rats.

Neural cell-cell and cell-substrate adhesion through N-cadherin, N-CAM and L1.

In this study neural (N)-cadherin, neural cell adhesion molecule (N-CAM) and L1 proteins and their antibody equivalents were covalently immobilized on a polyethylene-imine (PEI)-coated glass surface to form neuron-adhesive coatings. Impedance sensing and (supplementary) image analysis were used to monitor the effects of these CAMs. Immobilization of high concentrations of both N-cadherin protein and antibody led to good adhesion of neurons to the modified surface, better than surfaces treated with 30.0 and 100.0 µg ml(-1) N-CAM protein and antibody. L1 antibody and protein coating revealed no significant effect on neuronal cell-substrate adhesion. In a second series of combinatorial experiments, we used the same antibodies and proteins as medium-additives to inhibit cell-cell adhesion between neurons. Adhesion of neurons cultured on N-cadherin protein or antibody-modified surfaces was lowered by the addition of a soluble N-cadherin protein and antibody to the culturing medium, accelerating neuronal aggregation. The presence of a soluble N-CAM antibody or protein had no effect on the adhesion of neuronal cells on a N-cadherin protein-modified surface. On a N-cadherin antibody-coated surface, the addition of a soluble N-CAM protein led to cell death of neurons after 48 h, while a N-CAM antibody had no effect. In the presence of a soluble N-cadherin protein and antibody the aggregation of neurons was inhibited, both on N-CAM protein and N-CAM antibody-modified surfaces. Neurons cultured on immobilized antibodies were less affected by the addition of soluble CAM blockers than neurons cultured on immobilized proteins, indicating that antibody-protein bonds are more stable compared to protein-protein bonds.

¿Cómo se conecta la olfacción? Mecanismos celulares y moleculares que dirigen el desarrollo de las conexiones sinápticas desde la nariz a la corteza (I) / How is the sense of smell connected? Cellular and molecular mechanisms guiding the development

Resumen. Dentro del conjunto del sistema nervioso central, el sistema olfativo resulta fascinante por sus particularidades fisiológicas durante el desarrollo, siendo uno de los modelos más estudiados para entender los mecanismos relacionados con la guía y el crecimiento axonal hacia sus objetivos apropiados. Se conoce una constelación de mecanismos, unos mediados por contacto (lamininas, moléculas de adhesión celular, efrinas, etc.) y otros secretables (semaforinas, slits, factores de crecimiento, etc.), por desempeñar diversas funciones en el establecimiento de las interacciones sinápticas entre el epitelio olfativo, el bulbo olfativo y la corteza olfativa. También se han propuesto al respecto otros mecanismos específicos de este sistema, incluida la increíble familia de cerca de 1.000 receptores olfativos distintos. En los últimos años, diferentes revisiones se han concentrado en los elementos parciales de este sistema, sobre todo en los mecanismos implicados en la formación del nervio olfativo, echándose en falta una revisión detallada de aquellos relacionados con el desarrollo de las conexiones entre las distintas estructuras olfativas (epitelio, bulbo y corteza). En esta primera parte de la revisión, abordamos este tema desde el siguiente enfoque: los diversos mecanismos celulares y moleculares que dirigen la formación del nervio olfativo y el tracto olfativo lateral (AU)

Summary. The physiological particularities that occur during the development of the olfactory system make it one of the most fascinating parts of the central nervous system and one of models that has been most widely studied in order to understand the mechanisms related with axonal growth and guidance towards the right targets. A variety of mechanisms are known, some mediated by contact (laminins, cell adhesion molecules, ephrins, etc.) and others that are secreted (semaphorins, slits, growth factors, etc.), to play diverse roles in establishing the synaptic interactions among the olfactory epithelium, the olfactory bulb and the olfactory cortex. In relation to this, other specific mechanisms for this system have also been proposed, including the incredible family of close to 1000 different olfactory receptors. In recent years, different reviews have focused on the partial elements of this system, especially on the mechanisms involved in the formation of the olfactory nerve. However, no detailed review of those related with the development of the connections between the different olfactory structures (epithelium, bulb and cortex) has been put forward to date. In this first part of the review, we address this topic from the following perspective: the different cellular and molecular mechanisms that guide the formation of the olfactory nerve and the lateral olfactory tract (AU)

Structural basis for synaptic adhesion mediated by neuroligin-neurexin interactions.

The heterophilic synaptic adhesion molecules neuroligins and neurexins are essential for establishing and maintaining neuronal circuits by modulating the formation and maturation of synapses. The neuroligin-neurexin adhesion is Ca2+-dependent and regulated by alternative splicing. We report a structure of the complex at a resolution of 2.4 A between the mouse neuroligin-1 (NL1) cholinesterase-like domain and the mouse neurexin-1beta (NX1beta) LNS (laminin, neurexin and sex hormone-binding globulin-like) domain. The structure revealed a delicate neuroligin-neurexin assembly mediated by a hydrophilic, Ca2+-mediated and solvent-supplemented interface, rendering it capable of being modulated by alternative splicing and other regulatory factors. Thermodynamic data supported a mechanism wherein splicing site B of NL1 acts by modulating a salt bridge at the edge of the NL1-NX1beta interface. Mapping neuroligin mutations implicated in autism indicated that most such mutations are structurally destabilizing, supporting deficient neuroligin biosynthesis and processing as a common cause for this brain disorder.

Involvement of cell adhesion molecules in polydatin protection of brain tissues from ischemia-reperfusion injury.

Previous studies have demonstrated that polydatin, a crystal component extracted from the root stem of the perennial herbage Polygonum Cuspidatum Sieb.et Zucc, exerts a neuroprotective effect on cerebral injury induced by ischemia/reperfusion. To investigate the possible mechanism of this action, we determined the effects of polydatin on the expression of cell adhesion molecules (CAMs) after ischemia-induced cerebral injury. Rats were treated with polydatin (i.v.) immediately after the operation of middle cerebral artery occlusion (MCAO) for 1 h. It was found that polydatin improved neurological deficits and reduced the volume of brain infarction. In addition, polydatin decreased the levels of CAMs relative to the control (MCAO alone); these included intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), E-selectin, L-selectin and Integrins. These results suggest that polydatin exerts protective effects likely via inhibition of the expression of various CAMs; polydatin may be a potential agent for treatment of brain injury associated with stroke.

Neural cell adhesion molecule (NCAM-/-) null mice show impaired sensitization of the startle response.

The neural cell adhesion molecule (NCAM) plays important roles in development of the nervous system and in synaptic plasticity and memory formation in the adult. The present study sought to further investigate the role of NCAM in learning by testing habituation and footshock sensitization learning of the startle response (SR) in NCAM null mutant (NCAM-/-) and wildtype littermate (NCAM+/+) mice. Whereas habituation is a form of non-associative learning, footshock sensitization is induced by rapid contextual fear conditioning. Habituation was tested by repetitive presentation of acoustic and tactile startle stimuli. Although NCAM-/- mice showed differences in sensitivity in both stimulus modalities, habituation learning was intact in NCAM-/- mice, suggesting that NCAM does not play a role in the mechanisms underlying synaptic plasticity in the startle pathway. Footshock sensitization was elicited by presentation of electric footshocks between two series of acoustic stimuli. In contrast to habituation, footshock sensitization learning was attenuated in NCAM-/- mice: the acoustic SR increase after the footshocks was lower in the mutant than in wildtype mice, indicating that NCAM plays an important role in the relevant brain areas, such as amygdala and/or the hippocampus.

Neural cell adhesion molecule-null mice are not deficient in prepulse inhibition of the startle response.

Mice constitutively deficient in the neural cell adhesion molecule have morphological changes in the brain, which are hallmarks of schizophrenia. Schizophrenic patients are impaired in sensorimotor processing indicated by a deficit in prepulse inhibition of the acoustic startle response. Here we tested whether prepulse inhibition and prepulse facilitation are changed in neural cell adhesion molecule-deficient mice compared with their wild-type littermates. Neither prepulse inhibition nor prepulse facilitation (which occurred only at the lowest prepulse intensity used and was weak) was altered. This result is discussed in the light of the 'two-hit' hypothesis of schizophrenia, suggesting that in neural cell adhesion molecule-deficient mice, a prepulse inhibition deficit may become apparent only after treatment with a 'second hit' (such as increased stress).

A role of adhesion molecules in neuroglial plasticity.

Glial cells are exquisitely sensitive to changes in neuronal activity, and their capacity for structural plasticity including migration is critical for remodeling an repair of nervous tissue. Our in vitro studies suggest that isoforms of the neural cell adhesion molecule (NCAM) carrying an unconventional carbohydrate polymer, polysialic acid (PSA), are involved in these events. We have demonstrated that neurohypophyseal explants from newborn rats generate cellular outgrowth of immature astrocytes displaying the characteristics of oligodendrocyte-type 2 astrocyte (O-2A) progenitor cells previously identified in the optic nerve. Treatment of O-2A cells with the enzyme Endo N, which specifically removes PSA from the cells surface, produced a complete blockade of the dispersion of the O-2A cell population from the explant. Identical effects of Endo N were observed in migration assays using cortical O-2A cells. Neurohypophyseal O-2A cells express functional NMDA class of glutamate receptors and the pharmacological blockade of these receptors inhibit PSA-NCAM biosynthesis and dramatically diminish O-2A cell migration from neurohypophyseal explants. This suggests a potential mechanism through which neuronal activity via glutamate release may regulate PSA-NCAM expression on immature glial cells, which in turn is critical for their migration.

NCAM requires a cytoplasmic domain to function as a neurite outgrowth-promoting neuronal receptor.

The neural cell adhesion molecule (NCAM) promotes axonal growth via a homophilic binding mechanism by acting both as a neuronal receptor and a substratum ligand. We have previously shown that the GPI-linked 120-kDa isoform of NCAM, which lacks a cytoplasmic domain, is effective at promoting neurite outgrowth as a cellular ligand. To test its ability to function as a neuronal receptor, we have transfected PC12 cells with a cDNA encoding human GPI-linked NCAM and tested clones displaying stable cell surface expression of this isoform for their ability to respond to NCAM in a cellular substratum. Although they continued to express endogenous transmembrane rat isoforms of NCAM (140 and 180 kDa), PC12 cells expressing the GPI-linked NCAM lost their ability to extend neurites in response to substratum associated NCAM. However, their outgrowth response to N-cadherin and other activators of axonal growth was undiminished. Removal of GPI-linked NCAM from the surface of these clones using phosphatidylinositol-specific phospholipase C (PIPLC) fully restored their responsiveness to NCAM, indicating that the inhibition was a direct consequence of cell surface expression of this "dominant negative" isoform of NCAM. We have previously shown that expression of transfected 140- and 180-kDa isoforms of human NCAM in PC12 cells does not result in a loss of the neurite outgrowth response to NCAM. However, we show that deletion of the cytoplasmic domain of the 140-kDa isoform has the same effect as expression of GPI-linked NCAM. We conclude that the cytoplasmic domain of NCAM is required for an appropriate neurite outgrowth response.

The cytoplasmic domain of the cell adhesion molecule L1 is not required for homophilic adhesion.

L1 is a highly conserved cell adhesion molecule with complete homology of the cytoplasmic domain between the known mammalian protein sequences. Since the cytoplasmic domains of other adhesion molecules have been shown to influence adhesion, we have investigated the effects of deletion of the cytoplasmic domain on the ability of L1 to mediate homophilic adhesion. Full length L1 and a truncated L1, lacking 95% of the cytoplasmic domain, were expressed in myeloma cells. Independent stable transfectants were assayed for the ability to form aggregates. Myelomas expressing L1 lacking the cytoplasmic domain were able to form cell aggregates as well as the myelomas expressing full length L1. Cell aggregate formation was correlated with the level of L1 expression, and the aggregation could be blocked by anti-L1 Fabs. Similar results were obtained in adhesion assays of the myeloma cells to substrate-bound L1. These results indicate that the cytoplasmic domain of L1 is not required for homophilic interactions.