Adolescent idiopathic scoliosis (AIS) affects 3% to 4% of children between the ages of 11 and 18 [1, 2]. This disorder, characterized by abnormal three-dimensional spinal curvatures that typically develop during periods of rapid growth, occurs in the absence of congenital vertebral malformations or neuromuscular defects [1]. Genetic heterogeneity [3] and a historical lack of appropriate animal models [4] have confounded basic understanding of AIS biology; thus, treatment options remain limited [5, 6]. Recently, genetic studies using zebrafish have linked idiopathic-like scoliosis to irregularities in motile cilia-mediated cerebrospinal fluid flow [7-9]. However, because loss of cilia motility in human primary ciliary dyskinesia patients is not fully associated with scoliosis [10, 11], other pathogenic mechanisms remain to be determined. Here, we demonstrate that zebrafish scospondin (sspo) mutants develop late-onset idiopathic-like spinal curvatures in the absence of obvious cilia motility defects. Sspo is a large secreted glycoprotein functionally associated with the subcommissural organ and Reissner's fiber [12]-ancient and enigmatic organs of the brain ventricular system reported to govern cerebrospinal fluid homeostasis [13, 14], neurogenesis [12, 15-18], and embryonic morphogenesis [19]. We demonstrate that irregular deposition of Sspo within brain ventricles is associated with idiopathic-like scoliosis across diverse genetic models. Furthermore, Sspo defects are sufficient to induce oxidative stress and neuroinflammatory responses implicated in AIS pathogenesis [9]. Through screening for chemical suppressors of sspo mutant phenotypes, we also identify potent agents capable of blocking severe juvenile spine deformity. Our work thus defines a new preclinical model of AIS and provides tools to realize novel therapeutic strategies.

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.

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.

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-spondin's 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.

Hereditary hemochromatosis is an autosomal recessive disease highly prevalent in Northern Europe. Here we describe the performance of a genetic test for two mutations of the HFE gene (C282Y and H63D). It is based on a solid-phase PCR coupled with an α-phosphorothioate-mediated primer extension, conferring resistance to hydrolysis by ExoIII. Next, Elisa-like detection allows a colorimetric reading of the genetic test. We performed 322 tests (212 on the C282Y mutation, 110 on the H63D mutation) and compared the results with the RFLP method. Using OD ranges giving the minimum of uncertainty, the tests lead to high specificity and sensitivity, and they address the detection of mutated or normal bases in the HFE gene or the deduced phenotype (safe or ill), with positive predictive values or negative ones greater than 0.96. This method is therefore proposed as a primary test or as a confirming test.

The increase in the incidence of neurodegenerative diseases linked to aging or injury needs to be addressed in research into neuroprotective or neuroregenerative therapies, and requires the development of specific biological models. To achieve this goal we propose (1) the use of the mouse olfactory epithelium as a biological support which specifically exhibits a regenerative or a self-renewing capacity and during the lifetime necessitates the presence of neural stem cells, and (2) the use of an intraperitoneal injection of 2,6-dichlorobenzonitrile (diclobenil) as a chemical inducer of neurodegeneration in olfactory epithelium by selectively killing mature cells. We developed a biological model to follow the processes of neurodegeneration (chemically induced) and neuroregeneration (self-renewal of olfactory epithelium). The purpose of this study was to develop a method to monitor quickly neurodegeneration/neuroregeneration processes in order to further screen protective and regenerative therapies. For this purpose, we used the sedimentation field flow fractionation elution of olfactory epithelium. We obtained specific elution profiles and retention parameters allowing the monitoring of the induction and kinetics of biological processes. The use of insulin-like growth factor 1α as a neuroprotective agent in an innovative nebulization protocol showed sedimentation field flow fractionation to be a simple, fast and low-cost method to monitor such a biological event on the scale of an entire organism.

Detection of single nucleotide polymorphisms (SNPs) and of mutations is of importance in the field of genetics, biomedical research and in vitro diagnosis. We report here a genotyping procedure that can be virtually applied to any locus within a genome: it uses α-phosphorothioate deoxynucleotides in a primer-extension step followed by an ExoIII treatment. Non-extended primers are hydrolyzed whereas extended primers resist this treatment, indicating which nucleotide has been incorporated, i.e. the genotype of the locus. A 3-bp deletion in the CFTR gene (F508del, the most prevalent mutation involved in cystic fibrosis) was used as a model, in a single-tube procedure for each nucleotide to be tested. Human genomic DNA samples were correctly genotyped in less than 3 h by a solid-phase PCR followed by primer extension, ExoIII treatment and an ELISA-like detection method. The same principle (primer extension with α-phosphorothioate deoxynucleotide, ExoIII treatment) should also be combined with other detection systems such as gel or capillary electrophoresis, mass spectrometry or DNA chips.

The beneficial effects of Omega n - 3 polyunsaturated fatty acids (n - 3 PUFA) in situations of cognitive impairment may be associated with enhanced neuronal growth. Since neuronal growth is impaired in n - 3 PUFA deficiency, and enhanced by certain vitamins and trace elements, the effects of n - 3 PUFA, vitamin and mineral cell complex (VMC) and their association on neuronal growth were investigated in cultured rat neuroblastoma cells. Treatment of cells for 3 days with n - 3 PUFA significantly enhanced neurite length without affecting the number of neurites or cells. VMC significantly increased cell number without affecting neurite length or their number. Combined n - 3 PUFA and VMC significantly enhanced all three growth parameters. The data confirm the growth promoting effects of n - 3 PUFA and VMC in cultured neurons over a relatively short time frame.

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.

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 Reissner's 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 Reissner's 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 Reissner's fiber were revealed by immunohistochemistry using antibodies raised against bovine Reissner's fiber. Variation in the sites of Reissner's 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 Reissner's fiber, that may have had an important function during the evolution of the central nervous system in chordates, including that of the spinal cord. (C) 1998 IBRO. Published by Elsevier Science Ltd.

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.

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.

PARTIE II. - L'organe sous-commissural est une structure du diencephale dorsal d'origine ependymaire. Il est specialise dans la synthese de glycoproteines qui sont secretees dans le liquide cephalo-rachidien ou elles forment la Fibre de Reissner. L'analyse immunohistochimique a montre la grande specificite de cette secretion qui serait relarguee dans le 3 e ventricule mais egalement en direction des espaces lep-tomeninges. Cependant, le nombre des composants entrant dans cette secretion glyco-proteique reste encore sujet a discussion. Les premiers resultats obtenus grâce aux techniques de biologie moleculaire montre l'existence d'au moins un gene, specifiquement exprime dans l'OSC et de sequence originale. Le role de cet organe et de sa secretion dans le developpement du systeme nerveux central reste hypothetique, bien que certaines experiences laissent supposer une intervention lors de la morphogenese axiale ainsi que sur la survie neuronale.