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  Director : DUBOIS-DALCQ Monique (mdalcq@pasteur.fr)


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In the field of neurovirology, we study how Poliovirus establishes persistent infections in vitro and in vivo following the onset of paralytic poliomyelitis. Mutant viruses are able to persistently infect non-neural cells and show alterations in entry of the virus into cells. We analyse how poliovirus induces apoptosis in CNS motoneurons of paralytic mice.

In the field of neuroscience, we study the development and regeneration of oligodendrocytes, the myelin-forming cells of the CNS. Two different types of signals (neuregulins and sonic hedgehog) control the generation of oligodendrocytes from multipotential neural stem cells. How alpha chemokines and adhesion molecules regulate neural precursor cell migration is also investigated with the goal to design strategies to enhance myelin repair in demyelinating diseases.

Other research focuses on the molecular and cellular biology of connexins. This multigene family of proteins forms the intercellular channels clustered at gap junctions and allows cells to share ions, small metabolites and second messengers, thus coordinating a wide range of behaviors. One of the goals is to understand the molecular and cellular defects of human genetic diseases associated with connexin mutations. We are also pursuing the functional characterization of the connexin subgroup preferentially expressed in retinal neurons. Finally we study the diversity of function of connexins in neural stem cells during their differentiation.



  report

cale

Persistent poliovirus infection

(F. Colbère-Garapin, I. Pelletier, K. Labadie)

Poliovirus (PV) is the etiologic agent of paralytic poliomyelitis. It is among the best characterized enteroviruses, and the structure of the capsid, the viral genome and the multiplication cycle have been extensively studied. It is considered to be the prototype enterovirus. Recent studies have shown that some immunodeficient individuals can harbor and excrete poliovirus (PV) for more than 10 years. We have shown that PV can establish persistent infections in human cells of neuronal origin in culture, and have also developed several models to elucidate the molecular mechanisms of persistent PV infections. During persistent PV infection of neuroblastoma IMR-32 cells, mutant viruses (PVpi) are selected. Some PVpi mutations affect capsid residues located in regions involved in the interactions with the virus receptor (poliovirus receptor: PVR, or CD155), a protein belonging to the immunoglobulin superfamily; it has three extracellular domains, a transmembrane region and a cytoplasmic region. Only the first extracellular domain directly interacts with PV. PVpi selected in human neuronal cells are able to establish persistent infections in nonneural cells, whereas their parental viral strains are not. We have identified several viral determinants involved in this phenotype. A single determinant is in some cases sufficient to confer to a lytic virus the capacity to establish persistent infections in epidermoid cells, but the association of several determinants has a synergistic effect. These determinants profoundly affect the early steps of the virus cycle, including cell binding and the receptor-mediated conformational changes of the capsid. After adsorption onto PVR at 0°C, some persistent PV mutants have a lower sedimentation coefficient in sucrose gradient than virions (147S instead of 160S). We are currently studying the early steps of infection with purified soluble PVR molecules in order to further characterize 147S particles (collaboration with A. Nomoto & M. Arita (Tokyo)). Our results suggest that 147S particles may be an uncoating intermediate.

J. Martin (N.I.B.S.C., United Kingdom) has characterized PV strains isolated from a hypogammaglobulinemic individual, who remained asymptomatic while excreting PV for almost two years (J. Martin, et coll. J. Virol. 2000, 74, 3001-3010). PV persistence has never been studied in an in vitro model of human intestinal cells. To elucidate how PV persists in humans, we are developing a model in collaboration with Javier Martin, using the isolates from the hypogammaglobulinemic patient and human intestinal cells. This allows us to evaluate the respective importance of viral and cellular determinants in the molecular mechanisms of this persistent infection.

Poliovirus transcytosis in a model of the intestinal barrier (F. Colbère-Garapin, L. Ouzilou)

A model of the intestinal barrier, with human polarized enterocytes and cells having morphological and biochemical characteristics of M cells from Peyer's patches has been developed by S. Kerneis et al. (Science 1997, 277, 949-952) in E. Pringault's lab (ILE Laboratory, IP). The differentiation of M cells is induced upon coculture of polarized Caco-2 cells with lymphocytes isolated from murine Peyer's patches. In collaboration with the ILE lab, we are studying PV transcytosis through these M-like cells. The efficiency of PV transcytosis is 4 to 20-fold higher in cocultures containing M-like cells than in polarized enterocytes. This is consistent with M cells being involved in PV entry into the organism.

Poliovirus as a model for studying virus-nerve cell interactions : apoptosis and persistence (B. Blondel and T. Couderc).

Neurotropic viruses can persist in the central nervous system following the acute phase of infection and induce new pathologies several years after the initial infection. Poliovirus is currently one of the best-characterized neurotropic viruses. Patients having recovered from acute poliomyelitis developed after several decades of clinical stability a new disease, called post-polio syndrome, characterized notably by slowly progressive muscle weakness and atrophy. One hypothesis to explain this syndrome could be poliovirus persistence in the central nervous system, possibly associated with an immunopathological process.

We have previously developed a mouse model susceptible to poliovirus infection and we have shown that poliovirus can persist in the central nervous system after the onset of paralysis throughout the life of animals. We have also shown that the poliovirus persistence could be due, at least in part, to an inhibition of viral genome synthesis in the central nervous system. During the acute phase of poliomyelitis, we have demonstrated that poliovirus kills motoneurons by an apoptotic process (Fig. 1).

We have recently developed a model of mixed mouse primary nerve cell cultures to study the molecular mechanisms of poliovirus-induced apoptosis in nerve cells. We are currently investigating the role of interactions of poliovirus with its cellular receptor (CD155) and that of transduction signal in the induction of apoptosis versus persistence. In particular, we are analyzing caspases activation and mitochondrial dysfunctions as well as NF-k B activation following poliovirus/CD155 interaction.

Finally, mice surviving paralytic poliomyelitis represent a relevant animal model to study processes leading to regeneration of paralyzed muscle following virus-induced motoneuron death.

 

Myelin-forming cells development from neural stem cells (Dubois-Dalcq et al).

A Two major signaling pathways control oligodendrocyte development

(1) Neuregulin 1 (Nrg-1) isoforms have been shown to influence the emergence and growth of oligodendrocytes, the CNS myelin-forming cells. We have investigated how Nrg-1 signaling of ErbB receptors specifically controls the early stages of oligodendrocyte generation from multipotential neural precursors (NP). We show here that embryonic striatal NP express multiple Nrg-1 transcripts and proteins, as well as their specific receptors, ErbB2 and ErbB4, but not ErbB3. The major isoform synthesized by striatal NP is a transmembrane Type III isoform called Cystein-rich domain Nrg-1. To examine the biological effect of Nrg-1, we added soluble ErbB3 (sErbB3) to growing neurospheres. This inhibitor of Nrg-1 bioactivity decreased NP mitosis and increased their apoptosis, resulting in a significant reduction in neurosphere size and number. When NP were induced to migrate and differentiate by adhesion of neurospheres to the substratum, the level of type III isoforms detected by RT/PCR and Western blot decreased in parallel with a reduction in Nrg-1 fluorescence intensity in differentiating astrocytes, neurons and oligodendrocytes. Pretreatment of growing neurospheres with sErbB3 induced a three fold increase in the proportion of oligodendrocytes generated from NP migrating out of the neurosphere. This effect was not observed with an unrelated soluble receptor. Addition of sErbB3 during NP growth and differentiation enhanced oligodendrocyte maturation as shown by expression of galactocerebroside and myelin basic protein. We propose that both Type III Nrg-1 signaling and soluble ErbB receptors modulate oligodendrocyte development from NP.

(2)Sonic Hedgehog (Shh) induces oligodendrocyte development in the ventral neural tube and telencephalon but its role in oligodendrocyte generation in dorsal telencephalon is debated. Transcripts for Shh and its receptor complex were detected in subventricular zone and neocortex from E17 to birth. As Shh is not yet expressed in E15 neocortex, we grew E15 cortical precursors (CP) into neurospheres in the presence of recombinant Octyl-Shh (O-Shh). After sphere adhesion and removal of O-Shh, enhanced neurite outgrowth and cell migration were already observed at 3 hours. Three days after O-Shh treatment, oligodendrocyte progenitors (OP) emerged and continued to increase in number for 7 days while the ratio of neuronal cells decreased compared to control. Shh selectively triggered mitosis of OP but not neuronal progenitors and enhanced growth of neonatal OP. Thus Shh in E15-17 embryonic neocortex can signal CP to adopt an oligodendrocyte fate and favors expansion of this lineage.

B "Stem cells for myelin repair" is a research program carried on together with a network of european laboratories (Rougon et al, CNRS, LGPD, Marseille, France; Brüstle et al, Univ of Bonn, Germany; ffrench-Constant et al, Univ of Cambridge, England; Pena Rossi et al, Serono International, Geneva; Matsas et al, Pasteur Institute, Athens, Greece. The goal is to generate cells or therapeutic compounds that will repair myelin and restore normal nerve conduction in rodent animal models for demyelination and, on the long term, in Multiple Sclerosis.

To achieve this aim we are pursuing differents sets of experiments to (1) Generate neural precursor cells with enhanced migration properties that can reach and repair sites of demyelination in the central nervous system (CNS) following transplantation in animal models for the disease; (2) Develop techniques to manipulate neural precursor migration in a transient manner if differentiation of these exogenous cells is hampered in some way ; (3) Characterize the cell surface signalling that stimulate precursor cell migration in order to design compounds enhancing the recruitment of endogenous myelin-forming cells at sites of demyelination. Our strategy will be based on manipulating molecules known to be permissive for migration during development of the nervous system such as the polysialylated form of NCAM and chemokines or important for signalling during migration such as integrins. Our final assay will be remyelination and restoration of function in demyelinating animal model.

Connexins and cell communication (Roberto Bruzzone, bruzzone@pasteur.fr)In coll with Nathalie Duval, Danielle Gomès & Georgia Mitropoulou

Most cells communicate with their immediate neighbors through the exchange of cytosolic molecules such as ions, second messengers and small metabolites. This activity is made possible by clusters of intercellular channels called gap junctions, that connect adjacent cells. The molecular architecture of intercellular channels consists of two channels, called connexons, which interact to span the plasma membrane of two adjacent cells and directly join the cytoplasm of one cell to another. Connexons are made of structural proteins named connexins, that compose a multigene family whose members are distinguished according to their predicted molecular mass in kDa. Connexin channels participate in the regulation of signaling between developing and differentiated cell types. The recent discovery of human genetic diseases associated with mutations in six connexin genes and the study of knockout mice have validated the view that this form of intercellular signaling fulfills a crucial role in coordinating several aspects of tissue homeostasis. Understanding in detail how these channels gate offers the potential to develop specific drugs to deal with connexin-based disorders. To address this issue, we propose to take advantage of the naturally occurring connexin mutations to gain insight into the regulatory mechanisms of channel gating and further develop a related project on the specific role of connexins in the central nervous system (CNS), a largely unexplored area. Three questions are being specifically investigated: (1) What are the cellular and molecular basis of connexin diseases? (2) What are the functional properties of the novel subgroup of neuronal connexins? (3) What is the role of connexins in CNS development and differentiation?

Connexins and human diseases

Mutations in six connexin genes have been linked with five different pathologies. Mutations in Cx32 cause X-linked Charcot-Marie-Tooth disease (CMTX), the second most common inherited demyelinating neuropathy of the peripheral nervous system, while mutations in either Cx46 or Cx50 cause autosomal dominant cataracts. Three connexins, Cx26, Cx30, Cx31, have been linked to different forms of dominant and recessive nonsyndromic deafness, one of the most prevalent inherited sensory disorders. Cx26 mutations can also underlie syndromic forms of hearing loss that are associated with palmoplantar keratoderma, whereas Cx31 has been linked to erythrokeratodermia variabilis.

Connexins in the retina

Gap junctions play a key role in the functional organization of the vertebrate retina, as virtually every cell type is coupled to its neighbors by these intercellular channels. Moreover, a broad range of experimental studies has shown that the gap junctions connecting different types of retinal cells are selectively permeable to small tracers, exhibit unique physiological and pharmacological properties, and are differentially gated. Curiously, none of the distributions or functional properties of the known connexins studied in vitro is in good agreement with the distinct pharmacological properties of different gap junctional pathways in the vertebrate retina. The recent identification and cloning of members of the neuronal connexin subgroup make available, for the first time, participants in gap junction formation between various retinal neurons for study and reconstitution in experimental systems.

Connexins in the developing nervous system

It has been proposed that connexin channels are needed for the electrical and biochemical coordination of many processes, including cell migration, differentiation and formation of synaptic circuits. As development proceeds, connexins are expressed in complex and overlapping patterns whose significance is still unclear. One potential consequence of this program is the establishment of boundaries or gradients of communication between contacting cells, as formation of gap junction channels is a selective process that is also dependent on the presence of compatible connexins in adjacent cells. Gradients or boundaries of communication would result in distinct signals being sensed by neighboring cells, leading to the activation of different gene expression programs within a group of cells. Thus far, it is not known whether connexin-based communication pathways plays are implicated during specific steps of neurogenesis and gliogenesis. The regulation of the spatial and temporal expression of connexins during development makes it attractive to elucidate their role in the series of events leading to the emergence of differentiated cells from a pool of precursors.

Fig. 1 : Poliovirus-induced apoptosis in mouse neurones in culture. Neurons (green) and viral (red) antigens were identified by immunocytochemistry. Apoptosis was revealed by TUNEL (blue) labeling.



  publications

puce Publications of the unit on Pasteur's references database


  personnel

  Office staff Researchers Scientific trainees Other personnel
 

BARAN Corinne (cbaran@pasteur.fr)

DEMOND Anne (admond@pasteur.fr) a quitté l'unité en septembre 2001

DUBOIS-DALCQ Monique Professeur, Chef d'Unité mdalcq@pasteur.fr

BLONDEL Bruno Chargé de Recherche bblondel@pasteur.fr

BRUZZONE Roberto Chef de Laboratoire bruzzone@pasteur.fr

COLBERE-GARAPIN Florence Chef de Laboratoire fcolbere@pasteur.fr

COUDERC Thérèse Chargée de Recherche tcouderc@pasteur.fr

DUVAL Nathalie Assistante IP natoche@pasteur.fr

HARROCH Sheila Chargée de Recherche sharroch@pasteur.fr

BAULAC Stéphanie, en thèse

FRANCESCHINI Isabelle, en CDD

GOSSELIN Anne-Sophie, en thèse

LABADIE Karine, en thèse

LAZARINI Françoise, en CDD

MITROPOULOU Georgia, en thèse

OUZILOU Laurent, en thèse

SAULNIER Aure, en DEA

SIMONIN Yannick, en thèse

VACARESSE Nathalie, en CDD

VITRY Sandrine, postdoctorante

THAM to Nam , Ingénieur, ttham@pasteur.fr

GABELLEC Marie-Madeleine, Ingénieur, (a quitté en septembre 2001), mmg@pasteur.fr

PELLETIER Isabelle, Ingénieur, ipelleti@pasteur.fr

CASANOVA Philippe, technicien, pcasanov@pasteur.fr

GOMES Danielle, technicienne, dgomes@pasteur.fr

GUIVEL-BENHASSINE Florence, technicienne, fguivel@pasteur.fr

MURRAY Kerren, technicienne, kmurray@pasteur.fr

BELLANCE Edmond, aide de laboratoire


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