Pascale Durbec

Control of planar divisions by the G-protein regulator LGN maintains progenitors in the chick neuroepithelium

The spatio-temporal regulation of symmetrical as opposed to asymmetric cell divisions directs the fate and location of cells in the developing CNS. In invertebrates, G-protein regulators control spindle orientation in asymmetric divisions, which generate progeny with different identities. We investigated the role of the G-protein regulator LGN (also called Gpsm2) in spindle orientation and cell-fate determination in the spinal cord neuroepithelium of the developing chick embryo. We show that LGN is located at the cell cortex and spindle poles of neural progenitors, and that it regulates spindle movements and orientation. LGN promotes planar divisions in the early spinal cord. Interfering with LGN function randomizes the plane of division. Notably, this does not affect cell fate, but frequently leads one daughter of proliferative symmetric divisions to exit the neuroepithelium prematurely and to proliferate aberrantly in the mantle zone. Hence, tight control of planar spindle orientation maintains neural progenitors in the neuroepithelium, and regulates the proper development of the nervous system.

PSA‐NCAM modulates BDNF‐dependent survival and differentiation of cortical neurons

We show that the loss or inactivation of the polysialic acid (PSA) tail of neural cell adhesion molecule (NCAM) on rat cortical neurons in culture leads to reduced differentiation and survival. The mechanism by which this negative effect is mediated appears to involve the neuronal response to brain‐derived neurotrophic factor (BDNF): (i) in the absence of PSA or in the presence of excess free PSA added to the culture medium, BDNF‐induced cell signalling is reduced; (ii) the addition of exogenous BDNF to the medium reverses the effect of PSA loss or inactivation. These data suggest that PSA‐NCAM, previously shown to modulate cell migration and plasticity, is needed for an adequate sensitivity of neurons to BDNF.

Transfected F3/F11 neuronal cell surface protein mediates intercellular adhesion and promotes neurite outgrowth

The mouse neuronal F3 glycoprotein and its chicken homolog F11 belong to a subclass of proteins of the immunoglobulin superfamily with preferential localization on axons and neurites. We have transfected F3 cDNA into CHO cells. Biochemical analysis establishes that the cDNA we have cloned codes for a 130 kd phosphatidylinositol-anchored polypeptide. F3-expressing transfectants exhibited enhanced self-adhesive properties, aggregating with faster kinetics and forming larger aggregates than F3-negative control cells. When used as a culture substrate for sensory neurons, F3-transfected cells showed a markedly enhanced ability to promote neurite outgrowth compared with nontransfected cells. The results support the idea that F3/F11 and other closely similar proteins function as cell adhesion molecules that play a role in axonal growth and guidance.

Revisiting the function of PSA-NCAM in the nervous system.

Since its first description the polysialylated form of NCAM (PSA-NCAM) is thought to be a major regulator of cell-cell interactions in the nervous system. Over the past few years many crucial questions have been answered concerning PSA biosynthesis and function. Among these are the identification and cloning of the key enzymes that are responsible for its synthesis and the fact that expression of PSA is not restricted to developmental stages but maintained in the adult nervous system. In the adult, PSA has been shown to be not only a marker of structural plasticity but seems to be a major player in these processes. Originally suggested to be a purely anti-adhesive factor, modulating cell-cell interactions in general and by this allowing plasticity, there is now increasing evidence that this might not be the whole story. Instead, it appears possible that PSA-NCAM interacts with secreted signaling molecules and by this fulfills a more instructive function in brain plasticity.

Oligodendrocyte precursor migration and differentiation: combined effects of PSA residues, growth factors, and substrates.

Using the oligosphere strategy (V. Avellana-Adalid et al., 1996, J. Neurosci. Res. 45, 558-570), we compared the migratory behavior of oligodendrocyte preprogenitors (OPP) that expressed the polysialylated form of the neural cell adhesion molecule (PSA-NCAM) and of GD3-positive oligodendrocyte progenitors (OP). To study the role of PSA in OPP migration, we used endoneuraminidase-N, which specifically cleaves PSA from NCAM. Kinetic data showed that (i) migration velocity decreased with time and was favored on polyornithine compared to Matrigel; (ii) cells emerging from spheres enriched in PSA-NCAM+ OPP migrated farther than those from spheres enriched in GD3+ OP, their migration being enhanced by the addition of growth factors; (iii) removal of PSA from NCAM moderately reduced OPP migration and induced their differentiation in GD3+ OP and GFAP+ astrocytes; (iv) blocking integrins reduced their migration, suggesting an alternative mechanism of migration. Altogether these data illustrate that motility and differentiation of OPP involve the combinatorial action of PSA-NCAM, molecules of the ECM and their receptors, and growth factors.

A lateral belt of cortical LGN and NuMA guides mitotic spindle movements and planar division in neuroepithelial cells

Knockdown or mislocalization of LGN complex components disrupts the stereotypic biphasic spindle movements regulating planar cell division and neuroepithelial structure in chick embryos.

A nude mice model of human rhabdomyosarcoma lung metastases for evaluating the role of polysialic acids in the metastatic process.

PSA is an oncodevelopmental antigen usually expressed in human tumors with high metastatic potential. Here we set up a metastatic model in nude mice by using TE671 cells, which strongly express PSA-NCAM. We observed the formation of lung metastases when TE671 cells were injected intravenously, intramuscularly, and intraperitoneously, but not subcutaneously. Intraperitoneous injections also induced peritoneal carcinosis, ascites, and liver metastases. To evaluate the putative role of PSA in the metastatic process we used a specific cleavage of PSA on NCAM by endoneuraminidase-N on intraperitoneous primary tumors. Mice with primary intramuscular tumors were taken as control. Repeated injections of endoneuraminidase-N led to a decrease in PSA expression in primary intraperitoneous nodules and ascites but not in intramuscular primary tumors. Endoneuraminidase-N also increased the delay in ascitic formation and decreased the number of lung or liver metastases in the case of intraperitoneous tumors but not in the case of intramuscular tumors. When metastases occurred in endoneuraminidase-N injected animals, they strongly expressed PSA-NCAM. Therefore, we established a relationship between PSA expression on the surface of primary tumor cells and the metastatic process.

Oligodendrogenesis in the normal and pathological central nervous system

Oligodendrocytes (OLGs) are generated late in development and myelination is thus a tardive event in the brain developmental process. It is however maintained whole life long at lower rate, and myelin sheath is crucial for proper signal transmission and neuronal survival. Unfortunately, OLGs present a high susceptibility to oxidative stress, thus demyelination often takes place secondary to diverse brain lesions or pathologies. OLGs can also be the target of immune attacks, leading to primary demyelination lesions. Following oligodendrocytic death, spontaneous remyelination may occur to a certain extent. In this review, we will mainly focus on the adult brain and on the two main sources of progenitor cells that contribute to oligodendrogenesis: parenchymal oligodendrocyte precursor cells (OPCs) and subventricular zone (SVZ)-derived progenitors. We will shortly come back on the main steps of oligodendrogenesis in the postnatal and adult brain, and summarize the key factors involved in the determination of oligodendrocytic fate. We will then shed light on the main causes of demyelination in the adult brain and present the animal models that have been developed to get insight on the demyelination/remyelination process. Finally, we will synthetize the results of studies searching for factors able to modulate spontaneous myelin repair.

Sonic Hedgehog Signaling Is a Positive Oligodendrocyte Regulator during Demyelination

The morphogen Sonic Hedgehog (Shh) controls the generation of oligodendrocyte (OLs) during embryonic development and regulates OL production in adulthood in the cortex and corpus callosum. The roles of Shh in CNS repair following lesions associated with demyelinating diseases are still unresolved. Here, we address this issue by using a model of focal demyelination induced by lysolecithin in the corpus callosum of adult mice. Shh transcripts and protein were not detected in control animals but were upregulated in a time-dependent manner in the oligodendroglial lineage within the lesion. We report an increased transcription of Shh target genes suggesting a broad reactivation of the Shh pathway. We show that the adenovirus-mediated transfer of Shh into the lesioned brain results in the attenuation of the lesion extent with the increase of OL progenitor cells (OPCs) and mature myelinating OL numbers due to survival, proliferation, and differentiation activities as well as the decrease of astrogliosis and macrophage infiltration. Furthermore, the blocking of Shh signaling during the lesion, using its physiological antagonist, Hedgehog interacting protein, results in a decrease of OPC proliferation and differentiation, preventing repair. Together, our findings identify Shh as a necessary factor playing a positive role during demyelination and indicate that its signaling activation stands as a potential therapeutic approach for myelin diseases.

Migrating and myelinating potential of neural precursors engineered to overexpress PSA-NCAM

Polysialic acid (PSA) on NCAM is an important modulator of cell-cell interactions during development and regeneration. Here we investigated whether PSA overexpression influences neural cell migration and myelination. We stably expressed a GFP-tagged polysialytransferase, PSTGFP, in mouse neurospheres and induced prolonged PSA synthesis. Using a chick xenograft assay for migration, we show that PSA can instruct precursor migration along the ventral pathway. PSA persistence did not change neural precursor multipotentiality in vitro but induced a delay in oligodendrocyte differentiation. PSTGFP+ precursors showed widespread engraftment in shiverer brain, closely similar to that observed with control precursors expressing a fluorescent protein. Initially, myelination by oligodendrocytes was delayed but, eventually, down-regulation of PSTGFP occurred, allowing myelination to proceed. Thus down-regulation of polysialyltransferases takes place even in cells where its RNA is under the control of a heterologous promoter and engineering PSA overexpression in neural precursors does not cause irreversible unphysiological effects.