Unit: Neurovirology and Nervous System Regeneration

Director: Monique DUBOIS-DALCQ

The focus of our research is on

IThe Infections of the nervous system in particular the entry of neurotropic enteroviruses in the organism and poliovirus persistent infection in the mouse spinal cord. A new project concerns the mechanisms of propagation of prion infectivity from the periphery to the central nervous system.

IIDevelopment, communication/signalling and regeneration of the nervous system

We focus on the the development and regeneration of myelin-forming cells from neural stem cells, in particular on the molecular controls of migration and differenciation of oligodendrocytes as well as the role of Récepteur tyrosine phosphatases in the function of neural cells. Finally we investigate cell communication mediated by connexins in gap junctions in neural progenitors and neuronal networks as well as the molecular mechanisms underlying diseases caused by connexin mutations.

Neurotropic enteroviruses.
Persistence of vaccine-derived polioviruses (F. Colbère-Garapin et al.).

Immunodeficient patients whose gut is chronically infected by vaccine-derived poliovirus (VDPV) may excrete large amounts of virulent, mutated virus for years. To investigate how poliovirus (PV) establishes chronic infections in the gut, we tested whether it is possible to establish persistent VDPV infections in human intestinal cells. Four type 3 VDPV mutants, representative of the viral evolution in the gut of a hypogammaglobulinemic patient over almost two years (J. Martin et al., J. Virol., 2000, 74, 3001-10), were used to infect both undifferentiated, dividing cells, and differentiated, polarized enterocytes. A VDPV mutant excreted 36 days post-vaccination by the patient was lytic in both types of intestinal cell cultures, like the parental Sabin 3 strain. In contrast, three VDPVs, excreted 136, 442 and 637 days post-vaccination, established persistent infections both in undifferentiated cells and in enterocytes. Thus, viral determinants selected between day 36 and 136 conferred on VDPV mutants the capacity to infect intestinal cells persistently. The percentage of persistently VDPV-infected cultures was higher in enterocytes than in undifferentiated cells, implicating cellular determinants involved in the differentiation of enterocytes in persistent VDPV infections. The establishment of persistent infections in enterocytes was not due to poor replication of VDPVs in these cells, but was associated with reduced viral adsorption to the cellular receptor CD155.

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. 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 have shown that poliovirus-induced apoptosis involved both activation of initiator caspases and mitochondrial dysfunctions. Moreover, the interactions of poliovirus with its cellular receptor (CD155) could modulate apoptosis and this modulation could play a role in poliovirus persistence. We are currently studying the molecular mechanisms involved in mitochondrial dysfunctions following poliovirus binding to CD155.

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

Prion diseases The group of Françoise Lazarini focuses on the mechanisms of prion disease propagation from the periphery to the CNS such as occurred in Creutzfeldt-Jakob cases surging after the spongiform bovine encephalopathy epidemic and those resulting from growth hormone contamination. Using the murine model, we study among the immune cells the targets of this infectious agent, in particular dendritic cells and macrophages. We are also exploring whether repeated peripheral infection with sub-infectious prion doses can have a cumulative effect and eventually induce this deadly disease.

II Development, communication/signalling and regeneration of the nervous system

A Stem cell plasticity, PSA-NCAM and myelination (Monique Dubois-Dalcq et al).

Finding ways to enhance remyelination is a major challenge in treating demyelinating diseases. Recent studies have suggested that circulating bone marrow cells can home in brain and transdifferentiate into neural cells. To ask whether hematopoietic precursors can form myelinating cells, we investigated the neuropoietic potential of embryonic precursors sorted from the mouse Aorta-Gonads-Mesonephros (AGM) region. This cell fraction is capable of long term hematopoietic reconstitution and generates colonies containing multipotential precursors and lymphoid or erythro-myeloid progenies. When cultured in hematopoietic growth conditions, a fraction of CD45 positive AGM cells coexpress neural markers such as nestin, the polysialylated form of NCAM, the beta III tubulin isoform and glial fibrillary acidic protein. Yet, when hematopoietic precursors containing green fluorescent protein were co-cultured with embryonic striatal precursors into neurospheres, they maintained their hematopoietic phenotype without undergoing differentiation into neurons, astrocytes or oligodendrocytes. After intraventricular grafting, hematopoietic precursors integrated into the brain of wild-type or hypomyelinated newborn shiverer mice and gave rise to microglia but not neurons or glia. In contrast, when wild type embryonic striatal neurospheres were grafted in shiverer, they formed numerous myelin internode patches. Even when neural and hematopoietic precursors were grafted together into shiverer mice, only neural precursors generated myelin-forming cells and synthesized myelin. Thus embryonic neurospheres have myelin repair properties not shown by embryonic hematopoietic precursors. This suggests that the use of multipotential neural precursors to generate myelin-forming cells remains one of the most promising avenue toward remyelination therapies.

The polysialic acid (PSA) on NCAM plays a critical role in development of neural cells. We have investigated whether enhanced PSA expression influences oligodendrocyte development and myelination. We stably expressed a GFP-tagged polysialytransferase, PSTGFP, in mouse embryonic striatal neurospheres under the control of a viral LTR. The chimeric enzyme induced prolonged PSA synthesis in neural precursors without changing the cells'multipotentiality. When grafted intraventricularly into dysmyelinating shiverer mice, PSTGFP expressing precursors, as well as control precursors expressing a fluorescent protein, showed widespread engraftment in the postnatal brain and generated myelinating oligodendrocytes. Compared to control cells, however, PSTGFP expressing cells took longer to myelinate in vivo and fewer of the cells expressed myelin basic protein in vitro. While this delay in myelination was correlated with prolonged maintenance of PSA in PSTGFP expressing cells, differentiating oligodendrocytes consistently downregulated PSTGFP protein and PSA. Thus downregulation of polysialyltransferase is a prerequisite for oligodendrocyte differentiation.

B Role of CXCR4 signaling in neural cell migration ( Monique Dubois-Dalcq et al)

During forebrain development, precursors migrate tangentially from the subpallial telencephalum to the developing cortex. We show that CXCR4 signaling by SDF-1, an alpha chemokine conserved from fish to man, induces mouse striatal precursor migration. SDF-1 and CXCR4 are synthesized by E14 striatum as well as neurospheres grown from this region. Freshly dissociated striatal precursors show a chemotactic response toward SDF-1 which can be inhibited by SDF-1 neutralizing antibodies and this response is not triggered by a inactive truncated SDF-1. After adhesion, precursors coming out of neurospheres respond to SDF-1 in a dose-dependent manner by enhanced radial migration from the sphere. These bipolar cells express CXCR4 in the perinuclear area, at the membrane and along processes. Some of these cells coexpress the O4 antigen characteristic of oligodendrocyte precursors. When the latter mature into multiprocessed oligodendrocytes synthesizing membrane sheaths, expression of CXCR4 is down regulated. The data suggest that SDF-1 is a mediator of migration of striatal and early oligodendrocyte precursors during development.

The CXCR4 chemokine receptor is also expressed by interneuron precursors migrating from the basal forebrain to the neocortex. In addition, CXCR4 is present in early Cajal-Retzius cells of the cortical marginal zone. In mice with a null mutation in CXCR4 or SDF-1, interneurons are severely underrepresented in the superficial layers and ectopically placed in the deep layers of the neocortex. In contrast, the submeningeal positioning of Cajal-Retzius cells is unaffected. We propose that SDF-1, which is highly expressed in the embryonic leptomeninges, selectively regulates migration and layer-specific integration of CXCR4-expressing interneurons during neocortical development.


C Pathologies of the nervous system, signaling and repair (S. Harroch et al).

Research in our laboratory is focused on the development of mice model of patholologies of the nervous system in order to develop pharmacological and/or cellular therapies.

In view of this, we have generated mice deficient in receptor tyrosine phosphatase (RPTP), a family of molecule that have transmembrane and soluble isoforms, all strongly expressed in the central and peripheral nervous system. In particular, our interest focus on RPTPβ/PTPζ and RPTPγ, two RPTPs structurally homologous but expressed in different cell types. Indeed, while RPTPß is primarily expressed in glial cells, we detected RPTPγ in neurons .

These mutated animals allow us already to validate two pathological systems: the epilepsy and the multiple sclerosis. Indeed, RPTPß-deficient mice develop seizures while aging. And RPTPß-deficient mice remain paralyzed after inflammatory lesions and are unable to regenerate oligodendrocytes, necessary for remyelination.

Therefore, taking advantage of a new group of molecules with myelinating properties, the RPTPs, we aim to generate modified neural precursors with high potential of survival, and remyelination capabilities to repair myelin, in the perspective of an alternative cell therapy to demyelinating diseases.

Finally, we are also studying signaling pathways regulated by RPTPs, in particular, specific substrates of RPTPβ and RPTPγ to aim developing future pharmacological therapeutic drugs such as activator or inhibitor of these specific signaling pathways. .

D Functional role of gap junction channels (electrical synapses) in the brain (R Bruzzone)

Gap junctions are collections of intercellular channels that, in vertebrates, are formed by connexins, a multi-gene family of which 20 members have been identified in humans. Besides the undisputed role of chemical transmission in network oscillations, both computer simulations and electrophysiological recordings have recently emphasized a key role for electrical synapses in the generation of synchronous activity in the hippocampus and neocortex. However, the molecular identity of the connexins in pyramidal neurons has remained unknown thus far. One possibility is that a different class of molecules expressed in the mammalian brain forms electrical junctions between pyramidal cells. In collaboration with Hannah Monyer (University of Heidelberg, Germany), we have identified a small group of genes, the pannexins that share structural features with gap junction proteins of invertebrates and vertebrates. We have found that two of these genes, pannexin1 (Px1) and Px2, are abundantly expressed in the central nervous system. In many neuronal cell populations, including hippocampal pyramidal cells, there is co-expression of both pannexins. More importantly, in paired oocytes, Px1 alone and in combination with Px2 induced the formation of intercellular channels. We speculate that pannexins form cell type-specific gap junctions with distinct properties that may subserve different functions and we are currently generating knockout animals to test these hypotheses.

Photo 1 : Neuronal expression of RPTPg :

Identification of LacZ expressing cells in brain sections of RPTPg+/- mice.

A. Saggital section of adult mouse brain stained with X-gal.

B. Immunocytochemistry of saggital section labelled with Hoechst, LacZ, NeuN and GFAP in the hippocampus area.

Keywords: Neurotropic enterovirus, persistent infection

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