Characterization of a new mouse line triggering transient oligodendrocyte progenitor depletion.

Oligodendrocyte progenitor cells (OPC) are the main proliferative cells in the healthy adult brain. They produce new myelinating oligodendrocytes to ensure physiological myelin remodeling and regeneration after various pathological insults. Growing evidence suggests that OPC have other functions. Here, we aimed to develop an experimental model that allows the specific ablation of OPC at the adult stage to unravel possible new functions. We generated a transgenic mouse expressing a floxed human diphtheria toxin receptor under the control of the PDGFRa promoter, crossed with an Olig2Cre mouse to limit the recombination to the oligodendrocyte lineage in the central nervous system. We determined a diphtheria toxin dose to substantially decrease OPC density in the cortex and the corpus callosum without triggering side toxicity after a few daily injections. OPC density was normalized 7 days post-treatment, showing high repopulation capacity from few surviving OPC. We took advantage of this strong but transient depletion to show that OPC loss was associated with behavioral impairment, which was restored by OPC recovery, as well as disruption of the excitation/inhibition balance in the sensorimotor cortex, reinforcing the hypothesis of a neuromodulatory role of OPC in the adult brain.

Validating the sensitivity of inhomogeneous magnetization transfer (ihMT) MRI to myelin with fluorescence microscopy.

Inhomogeneous Magnetization Transfer (ihMT) is a development from the MT MRI technique. IhMT can be considered as a dipolar order relaxation time (T1D) weighted imaging modality whose signal has shown an enhanced selectivity for myelin-rich structures. However, a formal validation of the ihMT sensitivity relative to a gold standard myelin density measurement has not yet been reported. To address this need, we compared ihMT MRI with green fluorescence protein (GFP) microscopy, in a study performed on genetically-modified plp-GFP mice, considered as a reference technique for myelin-content assessment. Various ihMT protocols consisting of variable T1D-filtering and radiofrequency power temporal distributions, were used for comparison with fluorescence microscopy. Strong and significant linear relationships (r2 (0.87-0.96), p < 0.0001) were found between GFP and ihMT ratio signals across brain regions for all tested protocol variants. Conventional MT ratios showed weaker correlations (r2 (0.24-0.78), p ≤ 0.02) and a much larger signal fraction unrelated to myelin, hence corresponding to a much lower specificity for myelin. T1D-filtering reduced the ihMT signal fraction not attributed to myelin by almost twofold relative to zero filtering suggesting that at least half of the unrelated signal has a substantially shorter T1D than myelin. Overall, these results strongly support the sensitivity of ihMT to myelin content.

Olesoxime favors oligodendrocyte differentiation through a functional interplay between mitochondria and microtubules.

Multiple sclerosis (MS) is a neurodegenerative disease characterized by episodes of immune attacks and oligodendrocyte death leading to demyelination and progressive functional deficits. New therapeutic strategies are needed to stimulate the spontaneous regenerative process observed in some patients. Spontaneous myelin repair relies on the mobilization and differentiation of endogenous oligodendrocyte progenitors at the lesion site. Olesoxime, a cholesterol-like compound, has been shown to favor oligodendrocyte maturation in culture and promote myelin regeneration in rodents. Here, we study the mode of action of this compound and show that it binds to oligodendrocyte mitochondria, leading to their hyperfilamentation. This is accompanied by a reduction of basal superoxide levels, and accumulation of End Binding Protein 1 (EB1) at growing ends of microtubules. In parallel, we demonstrate that Reactive Oxygen Species (ROS) scavengers also promote oligodendrocyte differentiation, together with increasing mitochondrial filamentation and EB1-dependent microtubule polymerization. Altogether, our data uncover the mechanisms by which olesoxime promotes oligodendrocyte maturation. They also reveal that a bidirectional relationship between mitochondria hyperfilamentation and ROS level modulation controls oligodendrocyte maturation. This study identifies new cellular mechanisms to target for the development of regenerative treatments for MS.

Characterization of heterogeneous glial cell populations involved in dehydration-induced proliferation in the adult rat neurohypophysis.

The adult neurohypophysis (NH) is a well-established site of CNS plasticity: its glial cells, the pituicytes, reorganize their structure and undergo increased proliferation in response to stimulations such as dehydration. However, it remains to be clarified whether the newly-formed cells derive from pituicytes re-entering the cell cycle or from glial precursors or stem cells. Here, we first analyze the expression of several glial markers in the adult rat NH and demonstrate that the pituicytes constitute a heterogeneous population. In particular, we identify a distinct subtype of glial cells expressing the oligodendrocyte precursor marker platelet-derived growth factor receptor alpha (pdgfralpha). In addition, adult NH explants can give rise to migratory precursors able to differentiate into mature oligodendrocytes, unlike NH cells in vivo. This led us to hypothesize that the adult NH could contain immature cells, therefore we used a neurosphere-forming assay to test for the presence of stem or progenitor cells. Adult NH cells can generate bipotent primary neurospheres but not secondary ones, suggesting that the structure contains glial progenitors but probably not stem cells. Finally, when the NH is stimulated by dehydration, we observe an increase in cell proliferation associated with an increase in cell death. By identifying the cells incorporating bromodeoxyuridine (BrdU) or positive for Ki67, we demonstrate that this increased proliferation concerns all glial cell types in the adult NH, including the pdgfralpha+ cells. Our study shows that the NH is a complex structure composed of multiple glial subtypes, which all participate in the physiological response to dehydration.

Relevance of combinatorial profiles of intermediate filaments and transcription factors for glioma histogenesis.

In order to define specific markers for histogenesis of three well-characterized subgroups of human gliomas (pilocytic astrocytomas, glioblastoma multiforme and oligodendrogliomas), we studied the expression of relevant markers that characterize gliomagenesis, by immunohistochemistry and in situ hybridization. They include the intermediate filament proteins glial fibrillary acidic protein (GFAP), vimentin and nestin, the transcription factors Olig2, Nkx2.2 and Sox10, and the proteolipid protein transcripts plp/dm20. We show that the three major categories of human gliomas express a combinatorial profile of markers that gives new insights to their histogenesis and may help diagnosis. Pilocytic astrocytomas strongly express GFAP, vimentin, Olig2, Nkx2.2 and Sox10 but not nestin. In contrast, glioblastomas strongly express GFAP, vimentin and nestin but these tumours are heterogeneous regarding the expression of the transcription factors studied. Finally, in oligodendrogliomas, intermediate filament proteins are generally not observed whereas Olig2 was found in almost all tumour cells nuclei while only a subpopulation of tumour cells expressed Nkx2.2 and Sox10.

In vitro identification and functional characterization of glial precursor cells in human gliomas.

Human gliomas including astrocytomas and oligodendrogliomas are defined as being composed of neoplastic astrocytes and oligodendrocytes respectively. Here, on the basis of in vitro functional assays, we show that gliomas contain a mixture of glial progenitor cells and their progeny. We have set up explant cultures from pilocytic astrocytomas, glioblastomas and oligodendrogliomas and studied antigens that characterize glial lineage, from the precursor cells (glial restricted precursors and oligodendrocyte-type2-astrocyte/oligodendrocyte precursor cells expressing the A2B5 ganglioside) to the differentiated cells (oligodendrocyte and type-1 and type-2 astrocytes). All tumoral explants contain A2B5+ cells and can generate migrating cells with distinctive functional properties according to glioma subtypes. In pilocytic astrocytomas, very few migrating cells are dividing and can differentiate in type-2 astrocytes or towards the oligodendrocyte lineage. In glioblastomas, most migrating cells are dividing, express A2B5 or glial fibrillary acid protein (GFAP) and can generate oligodendrocytes and type-1 and type-2 astrocytes in appropriate medium. Oligodendroglioma explants are made by actively dividing glial precursor cells expressing A2B5 or PSA-NCAM. Only few cells can migrate and differentiation towards oligodendrocyte lineage does not occur. Isolated A2B5+ cells from both glioblastomas and oligodendrogliomas showed similar genetic alterations as the whole tumour. Therefore, pilocytic astrocytomas contain slowly dividing oligodendrocyte-type2-astrocyte/oligodendrocyte precursor cells in keeping with their benign behaviour whereas both glioblastomas and oligodendrogliomas contain neoplastic glial restricted precursor cells. In oligodendrogliomas, these cells are trapped in undifferentiated and proliferating state. The precursor cells properties present in gliomas give new insight into their histogenesis and open up new avenues for research in the field of gliomagenesis.

A role for the polysialic acid-neural cell adhesion molecule in PDGF-induced chemotaxis of oligodendrocyte precursor cells.

Directed migration of oligodendrocyte precursor cells (OPCs) is important for myelin formation and repair but the mechanisms of directional control are poorly understood. Here we have tested the role of polysialic acid-neural cell adhesion molecule (PSA-NCAM) in the directional migration of OPCs towards platelet-derived growth factor (PDGF). Using a Boyden microchemotaxis chamber and the Dunn direct viewing chamber, we show that in concentration gradients of PDGF, PSA-positive OPCs polarize and efficiently migrate towards the source of PDGF (chemotaxis). The loss or inactivation of the polysialic tail of NCAM leads to an altered pattern of OPC migration in response to PDGF gradients. Cells under these conditions, while being polarized and migrating, show no bias of displacement towards the source of PDGF and make random turns. By contrast, directed migration of OPCs towards basic fibroblast growth factor was not affected by the removal of PSA. Moreover, inactivation of PSA does not interfere with the random migration pattern of cells in uniform concentrations of PDGF (chemokinesis). These results suggest that PSA-NCAM is specifically involved in establishing the directionality of OPC migration in response to the concentration gradient of PDGF, but it is not essential for cell motility per se.

Oligodendrocyte progenitor migration in response to injury of glial monolayers requires the polysialic neural cell-adhesion molecule.

Injury to the nervous system results in reactive astrogliosis that is a critical determinant of neuronal regeneration. To analyze glial responses to mechanical injury and the role of the polysialic neural cell adhesion molecule (PSA-NCAM) in this process, we established primary glia cultures from newborn rat cerebral cortex. Scratching a confluent monolayer of primary glial cells resulted in two major events: rapid migration of oligodendrocyte progenitor-like (O-2A) cells into the wounded area and development of polarized morphology of type 1 astrocytes at the wound edge. Migrating O-2A progenitors had a bipolar morphology and exhibited A2B5 and O4 immunolabeling. Once these cells were established inside the wounded area, they lost A2B5 immunoreactivity and differentiated into glial fibrillary acidic protein-positive astrocytes. Migrating O-2A cells expressed PSA-NCAM, but type 1 astrocytes at the wound edge did not. Treatment of wounded cultures with Endo-N, which specifically removes PSA from the surface of cells, resulted in a significant decrease in O-2A cell migration into the wounded area and completely blocked the wound closure. Video time-lapse analysis showed that, in the presence of Endo-N, O-2A cells remained motile and migrated short distances but did not move away from the monolayer. These results demonstrate that O-2A progenitors contribute to reactive astrogliosis in culture and that PSA-NCAM is involved in this process by regulating cell migration.

Activity and cross-reactivity of antibodies induced in mice by immunization with a group B meningococcal conjugate.

The capsular polysaccharide of group B Neisseria meningitidis is composed of a linear homopolymer of alpha(2-8) N-acetyl neuraminic acid or polysialic acid (PSA) that is also carried by isoforms of the mammalian neural cell adhesion molecule (NCAM), which is especially expressed on brain cells during development. Here we analyzed the ability of antibodies induced by the candidate vaccine N-propionyl polysaccharide tetanus toxoid conjugate to recognize PSA-NCAM. We hyperimmunized mice to produce a pool of antisera and a series of immunoglobulin G monoclonal antibodies and evaluated their self-reactivity profile by using a battery of tests (immunoprecipitation, immunoblotting, and immunofluorescence detection on live cells and human tissue sections) chosen for their sensitivity and specificity to detect PSA-NCAM in various environments. We also searched for the effects of the vaccine-induced antibodies in two functional assays involving cell lysis or cell migration. Although they were highly bactericidal, all the antibodies tested showed very low or no recognition of PSA-NCAM, in contrast to PSA-specific monoclonal antibodies used as controls. Different patterns of cross-reactions were revealed by the tests used, likely due to affinity and specificity differences among the populations of induced antibodies. Furthermore, neither cell lysis nor perturbation of migration was observed in the presence of the tested antibodies. Importantly, we showed that whereas enzymatic removal of PSA groups from the surfaces of live cells perturbed their migration, blocking them with PSA-specific antibodies was not functionally detrimental. Taken together, our data indicated that this candidate vaccine induced antibodies that could not demonstrate an immunopathologic effect.

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.

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 intraperitoneally, but not subcutaneously. Intraperitoneal 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 intraperitoneal 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 intraperitoneal 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 intraperitoneal 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.

Transplantation of mammalian olfactory progenitors into chick hosts reveals migration and differentiation potentials dependent on cell commitment.

In vertebrates, interneurons of the olfactory bulb are continuously generated postnatally and throughout life at the subventricular zone of the forebrain. From there, the neuronal progenitors migrate tangentially in a typical chain-like structure to the olfactory bulb in which they differentiate as interneurons. We have used a mouse/chick xenograft strategy to explore the migration and differentiation potential of the mouse olfactory progenitors in a heterochronic and heterotypic environment. We compared the migration of primary cells derived from the subventricular zone of adult or newborn lateral ventricule with the behavior of in vitro amplified cells derived from the same structures. We show that in the chick environment, olfactory bulb progenitors from newborn brain tissue perform chain migration along the neural crest cell routes, whereas grafted neurosphere-derived-cells migrate as isolated cells. These results, together with in vitro observations, allow us to propose that neuronal chain migration is a community effect independent of environmental cues but which is closely regulated by the differentiation program of the cells. We established that the progenitor cells performing chain migration are already committed, while neurosphere-derived-cells are able to integrate and differentiate as components of the peripheral nervous system.

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.

Brain-derived neurotrophic factor restores long-term potentiation in polysialic acid-neural cell adhesion molecule-deficient hippocampus.

The neural cell adhesion molecule (NCAM) and its polysialylated form (PSA-NCAM) contribute to long-term potentiation (LTP) in the CA1 hippocampus. Here we report that the deficient LTP found in slices prepared from NCAM knockout mice and in organotypic slice cultures treated with Endo-N, an enzyme that cleaves the PSA moiety of NCAM, can be rescued by brain-derived neurotrophic factor (BDNF). This effect is not reproduced by nerve growth factor, but can be obtained with high concentrations of NT4/5. The effect of BDNF cannot be accounted for by modifications of N-methyl-D-aspartate receptor-dependent responses or of high-frequency bursts. PSA-NCAM, however, could directly interact with BDNF. Exogenous application of PSA residues or recombinant PSA-NCAM also prevents LTP. Furthermore trkB phosphorylation, and thus BDNF signaling, is reduced in both NCAM knockout mice and Endo-N-treated slice cultures. These results suggest that one action of PSA-NCAM could be to sensitize pyramidal neurons to BDNF, thereby modulating activity-dependent synaptic plasticity.

Consequences of neural cell adhesion molecule deficiency on cell migration in the rostral migratory stream of the mouse.

In vertebrates, interneurons of the olfactory bulb (OB) are generated postnatally and throughout life at the subventricular zone of the forebrain. The neuronal precursors migrate tangentially through the forebrain using a well defined pathway, the rostral migratory stream (RMS), and a particular mode of migration in a chain-like organization. A severe size reduction of the OB represents the most striking morphological phenotype in neural cell adhesion molecule (NCAM)-deficient mice. This defect has been traced back to a migration deficit of the precursors in the RMS and linked to the lack of the polysialylated form of NCAM. In this study we investigate the morphological alterations and functional properties of the RMS in mice totally devoid of all isoforms of NCAM and polysialic acid (PSA). We show that a morphologically altered, but defined and continuous pathway exists in mutants, and we present in vivo and in vitro evidence that PSA-NCAM in the RMS is not essential for the formation and migration of chains. Instead, we find a massive gliosis associated with the formation of membrane specializations in a heterotypic manner, linking precursors to astrocytes. This finding and the over-representation and defasciculation of axons in the pathway suggest that important interactions between migrating cells and their stationary environment are perturbed in the mutants. Finally, we used transplantation experiments to demonstrate that lack of PSA-NCAM leads to a decrease but not a total blockade of migration and demonstrate that the mutant RMS is functional in transporting normal neuronal precursors to the OB.

Signalling by the RET receptor tyrosine kinase and its role in the development of the mammalian enteric nervous system.

RET is a member of the receptor tyrosine kinase (RTK) superfamily, which can transduce signalling by glial cell line-derived neurotrophic factor (GDNF) and neurturin (NTN) in cultured cells. In order to determine whether in addition to being sufficient, RET is also necessary for signalling by these growth factors, we studied the response to GDNF and NTN of primary neuronal cultures (peripheral sensory and central dopaminergic neurons) derived from wild-type and RET-deficient mice. Our experiments show that absence of a functional RET receptor abrogates the biological responses of neuronal cells to both GDNF and NTN. Despite the established role of the RET signal transduction pathway in the development of the mammalian enteric nervous system (ENS), very little is known regarding its cellular mechanism(s) of action. Here, we have studied the effects of GDNF and NTN on cultures of neural crest (NC)-derived cells isolated from the gut of rat embryos. Our findings suggest that GDNF and NTN promote the survival of enteric neurons as well as the survival, proliferation and differentiation of multipotential ENS progenitors present in the gut of E12.5-13.5 rat embryos. However, the effects of these growth factors are stage-specific, since similar ENS cultures established from later stage embryos (E14. 5-15.5), show markedly diminished response to GDNF and NTN. To examine whether the in vitro effects of RET activation reflect the in vivo function(s) of this receptor, the extent of programmed cell death was examined in the gut of wild-type and RET-deficient mouse embryos by TUNEL histochemistry. Our experiments show that a subpopulation of enteric NC undergoes apoptotic cell death specifically in the foregut of embryos lacking the RET receptor. We suggest that normal function of the RET RTK is required in vivo during early stages of ENS histogenesis for the survival of undifferentiated enteric NC and their derivatives.

III. Role Of the RET signal transduction pathway in development of the mammalian enteric nervous system.

The enteric nervous system (ENS) in vertebrates is derived from the neural crest and constitutes the most complex part of the peripheral nervous system. Natural and induced mutagenesis in mammals has shown that the tyrosine kinase receptor RET and its functional ligand glial cell line-derived neurotrophic factor (GDNF) play key roles in the development of the ENS in humans and mice. We have developed and briefly describe here a number of assays that analyze the specific function of the RET receptor and its ligand. Our data suggest that the RET signal transduction pathway has multiple roles in the development of the mammalian ENS.

GDNF signalling through the Ret receptor tyrosine kinase.

Mutational analysis in humans and mice has demonstrated that the Ret, the product of the c-ret proto-oncogene, a member of the receptor tyrosine kinase (RTK) superfamily, is essential for development of the enteric nervous system and kidney. Despite the established role of Ret in mammalian embryogenesis, its cognate ligand(s) is currently unknown. Here we demonstrate, by using a Xenopus embryo bioassay, that glial-cell-line-derived neurotrophic factor (GDNF), a distant member of the transforming growth factor (TGF)-beta superfamily, signals through the Ret RTK. Furthermore, using explant cultures from wild-type and Ret-deficient mouse embryos, we show that normal c-ret function is necessary for GDNF signalling in the peripheral nervous system. Our data strongly suggest that Ret is a functional receptor for GDNF, and that GDNF, in addition to its potential role in the differentiation and survival of central nervous system neurons, has profound effects on kidney organogenesis and the development of the peripheral nervous system.

The GPI-anchored adhesion molecule F3 induces tyrosine phosphorylation: involvement of the FNIII repeats.

The glycosyl-phosphatidylinositol (GPI)-anchored F3 molecule, a member of the Ig superfamily made up of Ig and FNIII-like domains, is involved in cell-cell adhesion, neuronal pathfinding and fasciculation. Little is known about the mechanism(s) that governs the F3-mediated cell-cell recognition. In particular, it is not known whether F3 transduces signals across the membrane. Here we show that in F3-transfected CHO cells (1A cells) an increase in tyrosine phosphorylation occurs during F3-mediated aggregation. Moreover, under aggregation conditions F3 immunoprecipitated from 32P-metabolically labeled 1A cells associated with three major phosphorylated proteins. Interestingly, genistein inhibited the F3-mediated aggregation. Increased tyrosine phosphorylation was also observed using antibody-mediated F3-cross-linking. Furthermore, F3 expressed both in 1A cells and in post-natal mouse cerebellum forms non-covalent soluble complexes with protein tyrosine kinase(s). In cerebellum the F3-associated kinase was identified as fyn. By contrast, a truncated F3 protein, expressed in CHO cells, from which all the FN type III repeats have been deleted, does not associate with a kinase. Cross-linking of the F3-truncated form does not induce modulation of tyrosine phosphorylation. Taken together these data demonstrate that F3 is a molecule that transduces signals through both association with protein tyrosine kinase and modulation of protein tyrosine phosphorylation. The presence of FN type III domains is essential for the activation of the intracellular signaling pathway.