Effects of SCO-spondin thrombospondin type 1 repeats (TSR) in comparison to Reissner's fiber material on the differentiation of the B104 neuroblastoma cell line.

SCO-spondin is a newly identified protein that is strongly expressed in the subcommissural organ (SCO), an ependymal differentiation of the brain. When released into the cerebrospinal fluid at the entrance to the Sylvian aqueduct, the glycoproteins condense and form a thread-like structure, Reissner's fiber (RF). To analyze the role of SCO-spondin on neuronal development, we studied the effects induced by an oligopeptide derived from a thrombospondin type 1 repeat (TSR) of SCO-spondin on neuroblastoma B104 cells and compared them with the effects of soluble RF material containing complete SCO-spondin proteins. In low density cell culture, the TSR peptide first induced a notable flattening of cells accompanied by increased neurite outgrowth. Grouping of these differentiated B104 cells, which later formed dense aggregates, was then observed with increasing time in culture. Soluble RF material induced similar morphological changes and neurite-promoting effects on B104 cells, although the cells remained evenly distributed throughout the culture time and no aggregates were visible. In high-density cell culture, both TSR peptide and RF material induced prominent neurite outgrowth and subsequent rapid cell aggregation. Whereas soluble RF material inhibited cell proliferation, no respective effect was observed in the presence of the TSR peptide. A direct interaction of TSR peptide and soluble RF material with a B104 cell binding site was revealed by increased B104 cell metabolic activity by flow cytometry.

Subcommissural organ/Reissner's fiber complex: characterization of SCO-spondin, a glycoprotein with potent activity on neurite outgrowth.

In the developing vertebrate nervous system, several proteins of the thrombospondin superfamily act on axonal pathfinding. By successive screening of a SCO-cDNA library, we have characterized a new member of this superfamily, which we call SCO-spondin. This extracellular matrix glycoprotein of 4,560 amino acids is expressed and secreted early in development by the subcommissural organ (SCO), an ependymal differentiation located in the roof of the Sylvian aqueduct. Furthermore, SCO-spondin makes part of Reissner's fiber (RF), a thread-like structure present in the central canal of the spinal cord. This novel protein shows a unique arrangement of several conserved domains, including 26 thrombospondin type 1 repeats (TSR), nine low-density lipoprotein receptor (LDLr) type A domains, two epidermal growth factor (EGF)-like domains, and N- and C-terminal von Willebrand factor (vWF) cysteine-rich domains, all of which are potent sites of protein-protein interaction. Regarding the huge number of TSR, the putative function of SCO-spondin on axonal guidance is discussed in comparison with other developmental molecules of the CNS exhibiting TSR. To correlate SCO-spondin molecular feature and function, we tested the effect of oligopeptides, whose sequences include highly conserved amino acids of the consensus domains on a neuroblastoma cell line B 104. One of these peptides (WSGWSSCSRSCG) markedly increased neurite outgrowth of B 104 cells and this effect was dose dependent. Thus, SCO-spondin is a favorable substrate for neurite outgrowth and may participate in the posterior commissure formation and spinal cord differentiation during ontogenesis of the central nervous system.

Neuroblastoma B104 cell line as a model for analysis of neurite outgrowth and neuronal aggregation induced by Reissner's fiber material.

The subcommissural organ (SCO) 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 on neuroblastoma B104 cells grown in serum-free medium, using "low-density" and "high-density" culture systems. In the presence of soluble RF material, low-density cultures were suitable for analysis of the enhanced neurite outgrowth of B104 cells, while high-density cultures allowed the increased B104 cell aggregation to be examined. RF-induced neuronal aggregation and neuritic outgrowth were restricted to a perimeter around the RF. This standardized cell culture system reproduced in part the effects observed previously with primary cortical and spinal cord cell cultures and may serve the analysis of the mechanisms leading to aggregation and neurite outgrowth. In the present study, we analyzed variations in the rate of neural cell adhesion molecules, such as N-CAM and N-cadherin, induced by soluble RF material in high-density cultures.