Role of the NR2A/2B subunits of the N-methyl-D-aspartate receptor in glutamate-induced glutamic acid decarboxylase alteration in cortical GABAergic neurons in vitro.

The vulnerability of brain neuronal cell subpopulations to neurologic insults varies greatly. Among cells that survive a pathological insult, for example ischemia or brain trauma, some may undergo morphological and/or biochemical changes that may compromise brain function. The present study is a follow-up of our previous studies that investigated the effect of glutamate-induced excitotoxicity on the GABA synthesizing enzyme glutamic acid decarboxylase (GAD65/67)'s expression in surviving DIV 11 cortical GABAergic neurons in vitro [Monnerie and Le Roux, (2007) Exp Neurol 205:367-382, (2008) Exp Neurol 213:145-153]. An N-methyl-D-aspartate receptor (NMDAR)-mediated decrease in GAD expression was found following glutamate exposure. Here we examined which NMDAR subtype(s) mediated the glutamate-induced change in GAD protein levels. Western blotting techniques on cortical neuron cultures showed that glutamate's effect on GAD proteins was not altered by NR2B-containing diheteromeric (NR1/NR2B) receptor blockade. By contrast, blockade of triheteromeric (NR1/NR2A/NR2B) receptors fully protected against a decrease in GAD protein levels following glutamate exposure. When receptor location on the postsynaptic membrane was examined, extrasynaptic NMDAR stimulation was observed to be sufficient to decrease GAD protein levels similar to that observed after glutamate bath application. Blocking diheteromeric receptors prevented glutamate's effect on GAD proteins after extrasynaptic NMDAR stimulation. Finally, NR2B subunit examination with site-specific antibodies demonstrated a glutamate-induced, calpain-mediated alteration in NR2B expression. These results suggest that glutamate-induced excitotoxic NMDAR stimulation in cultured GABAergic cortical neurons depends upon subunit composition and receptor location (synaptic vs. extrasynaptic) on the neuronal membrane. Biochemical alterations in surviving cortical GABAergic neurons in various disease states may contribute to the altered balance between excitation and inhibition that is often observed after injury.

The core protein of the chondroitin sulfate proteoglycan phosphacan is a high-affinity ligand of fibroblast growth factor-2 and potentiates its mitogenic activity.

Using a radioligand binding assay we have demonstrated that phosphacan, a chondroitin sulfate proteoglycan of nervous tissue that also represents the extracellular domain of a receptor-type protein tyrosine phosphatase, shows saturable, reversible, high-affinity binding (Kd approximately 6 nM) to fibroblast growth factor-2 (FGF-2). Binding was reduced by only approximately 35% following chondroitinase treatment of the proteoglycan, indicating that the interaction is mediated primarily through the core protein rather than the glycosaminoglycan chains. Immunocytochemical studies also showed an overlapping localization of FGF-2 and phosphacan in the developing central nervous system. At concentrations of 10 microg protein/ml, both native phosphacan and the core protein obtained by chondroitinase treatment potentiated the mitogenic effect of FGF-2 (5 ng/ml) on NIH/3T3 cells by 75-90%, which is nearly the same potentiation as that produced by heparin at an equivalent concentration. Although studies on the role of proteoglycans in mediating the binding and mitogenic effects of FGF-2 have previously focused on cell surface heparan sulfate, our results indicate that the core protein of a chondroitin sulfate proteoglycan may also regulate the access of FGF-2 to cell surface signaling receptors in nervous tissue.

Effect of synthetic peptides derived from SCO-spondin conserved domains on chick cortical and spinal-cord neurons in cell cultures.

SCO-spondin is a newly identified protein, strongly expressed in the subcommissural organ (SCO), an ependymal differentiation of the brain. When secreted into the cerebrospinal fluid at the entrance to the Sylvian aqueduct, it condenses and forms Reissner's fiber. Several conserved domains have previously been characterizedin SCO-spondin, e.g., thrombospondin type 1 repeats (TSRs), low-density lipoprotein receptor (LDLr) type A repeats, and epidermal-growth-factor-like domains, which are potent sites of protein-protein interaction. To clarify the role of this protein on neuronal development, we have tested the effect of oligopeptides, the sequences of which include highly conserved amino acids of TSRs, LDLr type A repeats and a potent site of attachment to proteoglycan, on cortical and spinal-cord neurons in primary cell cultures. One of these peptides (WSGWSSCSRSCG), corresponding to a SCO-spondin TSR sequence, markedly increases adhesivity and neuritic outgrowth of cortical neurons and induces an opposite effect on cortical and spinal-cord neuronal aggregation. These effects are specific, as no response is observed with the scrambled sequence of this peptide. Another peptide (WGPCSVSCG) is only slightly active on adhesivity and neuritic outgrowth of cortical neurons and has no effect on spinal-cord neurons. Peptides derived from other conserved domains of SCO-spondin are not effective under our experimental conditions. Thus, SCO-spondin may be responsible for at least a part of the effects previously observed on neuronal cells cultured in the presence of Reissner's fiber. In addition, SCO-spondin seems to interfere with neuronal development and/or axonal guidance during ontogenesis of the central nervous system in modulating side-to-side interactions and neuritic outgrowth.

An in vitro cell culture system for the aggregation of embryonic chick central nervous system neurons.

The use of dissociated neuronal cell cultures is a very widespread technique. It is useful to study specific interactions between cells and resolve molecular mechanisms underlying neural development and function. For instance, the extended family of neurotrophic factors was identified and further studied especially using such techniques. Several growth factors have also been studied in this way as well as other developmental molecules. In this paper we describe a method of culturing chick cortical cells, in the complete absence of serum, which results in an enhanced aggregation of neurons by few days in culture. This cell culture system is particularly convenient to perform functional analyses of various molecules involved in neuronal cell adhesion mechanisms, such as extracellular matrix proteins or cell adhesion molecules, that require the establishment of in vitro paradigms in order to analyze their influence on cell-substratum and cell-cell interactions, as previously reported. We have successfully studied the effect of specific glycoproteins from the subcommissural organ on neuronal cell adhesion using this cell culture system.

In vitro differentiation of chick spinal cord neurons in the presence of Reissner's fibre, an ependymal brain secretion.

The subcommissural organ (SCO), which belongs to the circumventricular organs, is a specialized ependymal structure of the brain that secretes glycoproteins into the cerebrospinal fluid (CSF) which condense to form a thread-like structure, the Reissner's fibre (RF). Regarding the presence of this ependymal brain secretion all along the central canal of the developing spinal cord, we analysed a putative developmental activity of RF on neuronal spinal cord cells. The effects of RF proper and soluble RF-material were examined in primary cultures of dissociated spinal cord cells from day 6 chicken embryos. In serum-containing mixed glial/neuronal cell cultures, both RF and soluble RF-material promoted neuronal survival. This effect was blocked by addition of specific antibodies raised against bovine RF into the culture medium. In serum-free neuron-enriched cultures, no neuronal survival activity was observed; however, under these conditions RF proper induced neuronal aggregation and neuritic outgrowth of spinal cord cells. Interestingly, neurites extending from the aggregates appeared mainly unfasciculated. Our results suggest a direct modulation of cell-cell interactions by SCO/RF glycoproteins and an indirect survival effect on neurons. These data strengthen the hypothesis of the involvement of SCO/RF complex in the development of the central nervous system (CNS) and are discussed regarding molecular features of SCO-spondin, a novel glycoprotein recently identified in this complex.

Reissner's fibre promotes neuronal aggregation and influences neuritic outgrowth in vitro.

Reissner's fibre is the condensed form of glycoproteins secreted by the subcommissural organ; it extends through the central canal to the caudal end of the spinal cord. The effect of Reissner's fibre was assessed on dissociated embryonic chick cortical neuronal cells grown in chemically defined medium, by using two cell culture systems: (1) low-density cultures, in which neuronal cells remained evenly distributed; (2) high-density cultures, in which neuronal cells aggregated, displaying prominent neuritic outgrowth. Reissner's fibre, when added to low-density cultures, induced neuronal aggregation and neuritic outgrowth but this effect was restricted to an area centred around Reissner's fibre. Reissner's fibre, when added to high-density cultures, potentiated cell aggregation. Antibodies inhibiting the neural cell adhesion molecule or N-cadherin, and soluble Reissner's fibre material (reported previously to have anti-aggregative activity), did not prevent the aggregative activity induced by Reissner's fibre. Neuronal cells showed a similar reaction pattern when heparin or Reissner's fibre was added to the culture. These results suggest that the subcommissural organ/Reissner's fibre complex has multifunctional activities and may modulate cell-cell interactions during the development of the central nervous system.

Soluble material from Reissner's fiber displays anti-aggregative activity in primary cultures of chick cortical neurons.

The subcommissural organ (SCO), which belongs to the circumventricular organs, is a specialized ependymal structure of the brain that secretes glycoproteins into the cerebrospinal fluid (CSF) which condense to form a thread-like structure, Reissner's fiber (RF). The effects of soluble material released by RF were examined in primary cultures of dissociated cortical cells from embryonic (day 8) chick brain. Under serum-free conditions, the presence in the cultures of soluble RF material markedly impaired neuronal cell aggregation. This effect was completely blocked by addition into the culture medium of specific antibodies raised against bovine RF. The anti-aggregative effect of soluble RF material is observed on poly-L-lysine as well as on different extracellular matrix proteins including collagen and laminin, but was less effective on fibronectin. The continuous exposure of the cultures to soluble RF material for 7 days significantly decreased choline acetyltransferase activity. On the other hand, soluble RF material did not appear to have mitogenic activity on neuronal cultures. Modulation of cell-cell interactions by SCO/RF glycoproteins strengthens the hypothesis of the involvement of RF in developmental events of the central nervous system.

SCO-spondin: a new member of the thrombospondin family secreted by the subcommissural organ is a candidate in the modulation of neuronal aggregation.

A number of cues are known to influence neuronal development including growth factors, cell-adhesion molecules, components of the extracellular matrix and guidance molecules. In this study, we present molecular and functional evidence that SCO-spondin, a novel relative of the thrombospondin family, could also be involved in neuronal development by modulating cell aggregative mechanisms. SCO-spondin corresponds to glycoproteins secreted by the subcommissural organ (SCO), an ependymal differentiation of the vertebrate brain located at the entrance to the Sylvian aqueduct. A cDNA clone of 2.6 kb, isolated from a bovine SCO cDNA library, was shown to be specifically and highly expressed in the bovine SCO by in situ hybridization and was subsequently sequenced. Analysis of the deduced amino acid sequence reveals the presence of four conserved domains known as thrombospondin (TSP) type I repeats. To account for the homology with thrombospondins and F-spondin, this secreted glycoprotein was called SCO-spondin. Two potent binding sites to glycosaminoglycan (BBXB) and to cytokine (TXWSXWS) are also found in the TSP type I repeats. The deduced amino acid sequence exhibits three other conserved domains called low density lipoprotein (LDL) receptor type A repeats. The possibility of SCO-spondin involvement in neuronal development as a component of the extracellular matrix is discussed regarding these molecular features. The idea of a modulation of cell-cell and/or cell-matrix interaction is further supported by the anti-aggregative effect observed on cultured neuronal cells of material solubilized from Reissner's fiber. That Reissner's fiber, the condensed secretory product of the SCO present along the whole spinal cord can be a potent morphogenetical structure is an important concept for the analysis of the molecular mechanisms leading to spinal cord differentiation.

Reissner's fibre supports the survival of chick cortical neurons in primary mixed cultures.

Reissner's fibre, a thread-like structure present in the central canal of the spinal cord, is a product of the condensation of specific glycoproteins that are released by specialized ependymal cells into the cerebrospinal fluid. These secretory ependymocytes constitute the subcommissural organ, a circumventricular organ that lines the roof of the third ventricle of the brain. The subcommissural organ/Reissner's fibre complex is a permanent structure in the vertebrate central nervous system. The addition of bovine Reissner's fibre itself or of soluble material released by Reissner's fibre to primary mixed cultures of chick cerebral cortical cells markedly enhances neuronal survival. The responsive cells have been identified as neurons by labelling them with antibodies to neurofilament proteins. This neuronal survival effect is dose-dependent and does not require the presence of serum in the culture medium. Affinity-purified polyclonal antibodies raised against bovine Reissner's fibre partially block the effect of Reissner's fibre on neuronal survival. These results suggest that Reissner's fibre is involved in developmental processes of the central nervous system.