Stéphane Gobron

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 Reissners 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. GLIA 32:177–191, 2000.

SCO-spondin is evolutionarily conserved in the central nervous system of the chordate phylum.

Bovine SCO-spondin was shown to be a brain-secreted glycoprotein specifically expressed in the subcommissural organ, an ependymal differentiation located in the roof of the Sylvian aqueduct. Also, SCO-spondin makes part of Reissners fiber, a phylogenetically and ontogenetically conserved structure present in the central canal of the spinal cord of chordates. This secretion is a large multidomain protein probably involved in axonal growth and/or guidance. As Reissners fiber is highly conserved in the chordate central nervous system, we sought genes orthologous to the bovine SCO-spondin gene by Southern blot analysis in several members of the chordate phylum: urochordates, cephalochordates, cyclostomes, and lower and higher vertebrates, including humans. In addition, conserved glycoproteins present in the subcommissural organ and Reissners fiber were revealed by immunohistochemistry using antibodies raised against bovine Reissners fiber. Variation in the sites of Reissners fiber production according to chordate subphylum, presence of this structure in the spinal cord, and conservation of the SCO-spondin gene are discussed in the context of chordate central nervous system development. These results indicate that SCO-spondin is an ancient ependymal secretion, making part of Reissners fiber, that may have had an important function during the evolution of the central nervous system in chordates, including that of the spinal cord.

Complex expression pattern of the SCO-spondin gene in the bovine subcommissural organ: toward an explanation for Reissner's fiber complexity?

Bovine SCO-spondin is a glycoprotein secreted by the subcommissural organ (SCO), an ependymal derivative located in the roof of the third ventricle. It shows homology with developmental molecules involved in directional axonal growth. Using SCO-spondin cDNAs as probes, we analysed the specific expression of the corresponding gene in the bovine SCO by Northern blot and in situ hybridization (ISH). A strong expression was detected in the secretory ependymal and hypendymal cells of the SCO and the main transcripts showed a large size 14 kb. A single copy gene was revealed by Southern blot analysis of bovine genomic DNA. The presence of additional transcripts suggested a transcriptional regulation of the SCO-spondin gene. A comparative analysis of the results obtained by molecular and immunological techniques (immunoblotting and immunopurification) pointed to the presence of several SCO-spondin related proteins in the SCO encoded by the same gene. The presence in the cerebral hemispheres (CH) of a 54-kDa glycoprotein with a common epitope is discussed as a putative cleaved SCO-spondin product carried by the cerebrospinal fluid, that may act on neuronal development.

SCO-Spondin Derived Peptide NX210 Induces Neuroprotection In Vitro and Promotes Fiber Regrowth and Functional Recovery after Spinal Cord Injury

In mammals, the limited regenerating potential of the central nervous system (CNS) in adults contrasts with the plasticity of the embryonic and perinatal periods. SCO (subcommissural organ)-spondin is a protein secreted early by the developing central nervous system, potentially involved in the development of commissural fibers. SCO-spondin stimulates neuronal differentiation and neurite growth in vitro. NX210 oligopeptide was designed from SCO-spondins specific thrombospondin type 1 repeat (TSR) sequences that support the main neurogenic properties of the molecule. The objective of this work was to assess the neuroprotective and neuroregenerative properties of NX210 in vitro and in vivo for the treatment of spinal cord injury (SCI). In vitro studies were carried out on the B104 neuroblastoma cell line demonstrating neuroprotection by the resistance to oxidative damage using hydrogen peroxide and the measure of cell viability by metabolic activity. In vivo studies were performed in two rat models of SCI: (1) a model of aspiration of dorsal funiculi followed by the insertion of a collagen tube in situ to limit collateral sprouting; white matter regeneration was assessed using neurofilament immunostaining; (2) a rat spinal cord contusion model to assess functional recovery using BBB scale and reflex testing. We demonstrate for the first time that NX210 (a) provides neuroprotection to oxidative stress in the B104 neuroblastoma cells, (b) stimulates axonal regrowth in longitudinally oriented neofibers in the aspiration model of SCI and (c) significantly improves functional recovery in the contusive model of SCI.

Assessment of citalopram and escitalopram on neuroblastoma cell lines: Cell toxicity and gene modulation

Selective serotonin reuptake inhibitors (SSRI) are common antidepressants which cytotoxicity has been assessed in cancers notably colorectal carcinomas and glioma cell lines. We assessed and compared the cytotoxicity of 2 SSRI, citalopram and escitalopram, on neuroblastoma cell lines. The study was performed on 2 non-MYCN amplified cell lines (rat B104 and human SH-SY5Y) and 2 human MYCN amplified cell lines (IMR32 and Kelly). Citalopram and escitalopram showed concentration-dependent cytotoxicity on all cell lines. Citalopram was more cytotoxic than escitalopram. IMR32 was the most sensitive cell line. The absence of toxicity on human primary Schwann cells demonstrated the safety of both molecules for myelin. The mechanisms of cytotoxicity were explored using gene-expression profiles and quantitative real-time PCR (qPCR). Citalopram modulated 1 502 genes and escitalopram 1 164 genes with a fold change ≥ 2. 1 021 genes were modulated by both citalopram and escitalopram; 481 genes were regulated only by citalopram while 143 genes were regulated only by escitalopram. Citalopram modulated 69 pathways (KEGG) and escitalopram 42. Ten pathways were differently modulated by citalopram and escitalopram. Citalopram drastically decreased the expression of MYBL2, BIRC5 and BARD1 poor prognosis factors of neuroblastoma with fold-changes of -107 (p<2.26 10−7), -24.1 (p<5.6 10−9) and -17.7 (p<1.2 10−7). CCNE1, AURKA, IGF2, MYCN and ERBB2 were more moderately down-regulated by both molecules. Glioma markers E2F1, DAPK1 and CCND1 were down-regulated. Citalopram displayed more powerful action with broader and distinct spectrum of action than escitalopram.

SCO-spondin oligopeptide inhibits angiogenesis in glioblastoma

Angiogenesis plays a critical role in glioblastoma growth and progression. We therefore aimed at evaluating the anti-angiogenic properties of an oligopeptide originating from SCO-spondin (NX) on a model of human glioblastoma. To this end, we studied the impact of NX treatment on human brain endothelial cells (HBMECs) alone or co-cultured with glioblastoma cells (U87-MG) on apoptosis, proliferation, migration and release of angiogenic factors. We further investigated the anti-angiogenic potential of NX on human glioblastoma cells grown on chorio-allantoic membrane (CAM) or in glioblastoma xenografts. The results of our experiments showed that NX treatment impaired the microvascular network and induced a decrease in cell proliferation, vascularization and tumor growth in the CAM model as well as in xenotransplants. Interestingly, our in vitro experiments showed that NX impairs HBMECs migration but also regulates the release of angiogenic factors from U87-MG. These results are confirmed by the profiling of NX-treated U87-MG grown on CAM that highlighted modifications of several genes involved in angiogenesis. In conclusion, NX inhibits tumorigenesis by impairing the ability of glioblastoma cells to induce angiogenesis and by inhibiting endothelial cell migration. This molecule might therefore be an interesting candidate for future cancer therapies.