Fabrice Chareyre

Abstract: Neurons are characterized by extremely long axons. This exceptional cell shape is likely to depend on multiple factors including interactions between the cytoskeleton and membrane proteins. In many cell types, members of the protein 4.1 family play an important role in tethering the cortical actin-spectrin cytoskeleton to the plasma membrane. Protein 4.1B is localized in myelinated axons, enriched in paranodal and juxtaparanodal regions, and also all along the internodes, but not at nodes of Ranvier where are localized the voltage-dependent sodium channels responsible for action potential propagation. To shed light on the role of protein 4.1B in the general organization of myelinated peripheral axons, we studied 4.1B knockout mice. These mice displayed a mildly impaired gait and motility. Whereas nodes were unaffected, the distribution of Caspr/paranodin, which anchors 4.1B to the membrane, was disorganized in paranodal regions and its levels were decreased. In juxtaparanodes, the enrichment of Caspr2, which also interacts with 4.1B, and of the associated TAG-1 and Kv1.1, was absent in mutant mice, whereas their levels were unaltered. Ultrastructural abnormalities were observed both at paranodes and juxtaparanodes. Axon calibers were slightly diminished in phrenic nerves and preterminal motor axons were dysmorphic in skeletal muscle. βII spectrin enrichment was decreased along the axolemma. Electrophysiological recordings at 3 post-natal weeks showed the occurrence of spontaneous and evoked repetitive activity indicating neuronal hyperexcitability, without change in conduction velocity. Thus, our results show that in myelinated axons 4.1B contributes to the stabilization of membrane proteins at paranodes, to the clustering of juxtaparanodal proteins, and to the regulation of the internodal axon caliber.

Abstract: Meningiomas are among the most common primary central nervous system tumours in adults. Studies focused on the molecular basis for meningioma development are hampered by a lack of information with regard to the cell of origin for these brain tumours. Herein, we identify a prostaglandin D synthase-positive meningeal precursor as the cell of origin for murine meningioma, and show that neurofibromatosis type 2 (Nf2) inactivation in prostaglandin D2 synthase (PGDS) (+) primordial meningeal cells, before the formation of the three meningeal layers, accounts for the heterogeneity of meningioma histological subtypes. Using a unique PGDSCre strain, we define a critical embryonic and early postnatal developmental window in which biallelic Nf2 inactivation in PGDS (+) progenitor cells results in meningioma formation. Moreover, we identify differentially expressed markers that characterize the two major histological meningioma subtypes both in human and mouse tumours. Collectively, these findings establish the cell of origin for these common brain tumours as well as a susceptible developmental period in which signature genetic mutations culminate in meningioma formation.

Abstract: The axon initial segment (AIS) plays a crucial role: it is the site where neurons initiate their electrical outputs. Its composition in terms of voltage-gated sodium (Nav) and voltage-gated potassium (Kv) channels, as well as its length and localization determine the neuron's spiking properties. Some neurons are able to modulate their AIS length or distance from the soma in order to adapt their excitability properties to their activity level. It is therefore crucial to characterize all these parameters and determine where the myelin sheath begins in order to assess a neuron's excitability properties and ability to display such plasticity mechanisms. If the myelin sheath starts immediately after the AIS, another question then arises as to how would the axon be organized at its first myelin attachment site; since AISs are different from nodes of Ranvier, would this particular axonal region resemble a hemi-node of Ranvier?

Abstract: The NF2 gene product, merlin/schwannomin, is a cytoskeleton organizer with unique growth-inhibiting activity in specific cell types. A narrow spectrum of tumors is associated with NF2 deficiency, mainly schwannomas and meningiomas, suggesting cell-specific mechanisms of growth control. We have investigated merlin function in mouse Schwann cells (SCs). We found that merlin regulates contact inhibition of proliferation by limiting the delivery of several growth factor receptors at the plasma membrane of primary SCs. Notably, upon cell-to-cell contact, merlin downregulates the membrane levels of ErbB2 and ErbB3, thus inhibiting the activity of the downstream mitogenic signaling pathways protein kinase B and mitogen-activated protein kinase. Consequently, loss of merlin activity is associated with elevated levels of ErbB receptors in primary SCs. We also observed accumulation of growth factor receptors such as ErbB2 and 3, insulin-like growth factor 1 receptor and platelet-derived growth factor receptor in peripheral nerves of Nf2-mutant mice and in human NF2 schwannomas, suggesting that this mechanism could play an important role in tumorigenesis.

Abstract: Cerebral cavernous malformations (CCM) are vascular malformations of the brain that lead to cerebral hemorrhages. In 20% of CCM patients, this results from an autosomal dominant condition caused by loss-of-function mutations in one of the three CCM genes. High expression levels of the CCM genes in the neuroepithelium indicate that CCM lesions might be caused by a loss of function of these genes in neural cells rather than in vascular cells. However, their in vivo function, particularly during cerebral angiogenesis, is totally unknown. We developed mice with constitutive and tissue-specific CCM2 deletions to investigate CCM2 function in vivo. Constitutive deletion of CCM2 leads to early embryonic death. Deletion of CCM2 from neuroglial precursor cells does not lead to cerebrovascular defects, whereas CCM2 is required in endothelial cells for proper vascular development. Deletion of CCM2 from endothelial cells severely affects angiogenesis, leading to morphogenic defects in the major arterial and venous blood vessels and in the heart, and results in embryonic lethality at mid-gestation. These findings establish the essential role of endothelial CCM2 for proper vascular development and strongly suggest that the endothelial cell is the primary target in the cascade of events leading from CCM2 mutations to CCM cerebrovascular lesions.

Abstract: Pregnancy results in the transfer of stem cells from the fetus to the maternal circulation. These cells are able to migrate and differentiate within various damaged maternal tissues. We recently showed the presence of fetal-derived cells in human breast carcinomas during pregnancy. In this study, we aimed to reproduce these results in a murine model of "pregnancy-associated" breast carcinoma. We bred virgin MMTV-H-Ras transgenic female mice with male mice transgenic for luciferase under the control of the VEGFR2 promoter. Tumors that developed during or following gestation were analyzed and their nuclear grade classified. Fetal cells were detected by Y chromosome Fluorescence in situ hybridization FISH in 9/9 of breast carcinomas but only in 2 liver controls from the same animals. The number of fetal cells was 20 and 4.9 per million maternal cells in these tissues, respectively (p < 0.05). High grade tumors had significantly more fetal cells (p < 0.05). In vivo imaging of the luciferase signal under control of the VEGFR2 promoter as well as von Willebrand staining did not reveal an endothelial phenotype of fetal cells. Sixty two percent of the fetal cells expressed cytokeratins but were not tumoral. In conclusion, fetal cells-expressing cytokeratin-are always present in murine breast carcinomas associated with gestation. Interestingly, high-grade tumors contain more fetal cells.

Abstract: The integrin alpha(v)beta(3) is involved in multiple aspects of malignant cancer, including tumor angiogenesis and metastasis, which makes the receptor a key target for the development of anti-cancer therapies. We report here on the production, the characterization and the in vivo anti-angiogenic and anti-metastatic properties of a novel alpha(v)beta(3)-binding disintegrin, DisBa-01, isolated from a cDNA library made with RNAs from the venom gland of Bothrops alternatus. The 11,637 Da-recombinant monomeric form of DisBa-01 displayed an RGD motif and interacted with purified alpha(v)beta(3) integrin in surface plasmon resonance studies, in a dose-dependent and cation sensitive manner. A three-dimensional molecular model of DisBa-01 in complex with alpha(v)beta(3) predicted a large surface of contacts with the beta(3) subunit. DisBa-01 inhibited the adhesion of alpha(v)beta(3)-expressing human microvascular endothelial cell line-1 (HMEC-1) and murine melanoma cell line B16F10 to vitronectin (IC(50) = 555 nM and 225 nM, respectively), and transiently inhibited their proliferation without direct cell toxicity, but did not affect the binding nor the proliferation of a human breast cancer-derived cell line (MDA-MB-231) not expressing alpha(v)beta(3). In vivo, DisBa-01 dose-dependently decreased bFGF-induced angiogenesis in a matrigel plug assay in athymic nude mice (IC(50) = 83 nM). When injected intravenously to C57BL/6 mice together with B16F10 melanoma cells, DisBa-01 time- and dose-dependently inhibited lung metastasis monitored by bioluminescent imaging. We conclude that DisBa-01 is a potent new inhibitor of alpha(v)beta(3)-dependent adherence mechanisms involved in neo-vascularization and tumor metastasis processes.

Abstract: Meningiomas account for approximately 30% of all primary central nervous system tumors and are found in half of neurofibromatosis type 2 patients often causing significant morbidity. Although most meningiomas are benign, 10% are classified as atypical or anaplastic, displaying aggressive clinical behavior. Biallelic inactivation of the neurofibromatosis 2 (NF2) tumor suppressor is associated with meningioma formation in all NF2 patients and 60% of sporadic meningiomas. Deletion of the p16(INK4a)/p14(ARF) locus is found in both benign and malignant meningiomas, while mutation of the p53 tumor suppressor gene is uncommon. Previously, we inactivated Nf2 in homozygous conditional knockout mice by adenoviral Cre delivery and showed that Nf2 loss in arachnoid cells is rate-limiting for meningioma formation. Here, we report that additional nullizygosity for p16(Ink4a) increases the frequency of meningioma and meningothelial proliferation in these mice without modifying the tumor grade. In addition, by using magnetic resonance imaging (MRI) to screen a large cohort of mutant mice, we were able to detect meningothelial proliferation and meningioma development opening the way to future studies in which therapeutic interventions can be tested as preclinical assessment of their potential clinical application.

Abstract: The recent discovery of bacterial receptors such as NOD2 that contribute to crosstalk between innate and adaptive immune systems in the digestive tract constitutes an important challenge in our understanding of liver injury mechanisms. The present study focuses on NOD2 functions during liver injury. NOD2, TNF-alpha and IFN-gamma mRNA were quantified using real-time PCR in liver samples from patients and mice with liver injury. We evaluated the susceptibility of concanavalin A (ConA) challenge in NOD2-deficient mice (Nod2-/-) compared to wild-type littermates. We tested the effect of muramyl dipeptide (MDP), the specific activator of NOD2, on ConA-induced liver injury in C57BL/6 mice. We studied the cellular distribution and the role of NOD2 in immune cells and hepatocytes. We demonstrated that NOD2, TNF-alpha and IFN-gamma were upregulated during liver injury in mice and humans. Nod2-/- mice were resistant to ConA-induced hepatitis compared to their wild-type littermates, through reduced IFN-gamma production by immune cells. Conversely, administration of MDP exacerbated ConA-induced liver injury. MDP was a strong inducer of IFN-gamma in freshly isolated human PBMC, splenocytes and hepatocytes. Our study supports the hypothesis that NOD2 contributes to liver injury via a regulatory mechanism affecting immune cells infiltrating the liver and hepatocytes. Taken together, our results indicate that NOD2 may represent a new therapeutic target in liver diseases.

Abstract: Schwannomin/merlin is the product of a tumor suppressor gene mutated in neurofibromatosis type 2 (NF2). Although the consequences of NF2 mutations on Schwann cell proliferation are well established, the physiological role of schwannomin in differentiated cells is not known. To unravel this role, we studied peripheral nerves in mice overexpressing in Schwann cells schwannomin with a deletion occurring in NF2 patients (P0-SCH-Delta39-121) or a C-terminal deletion. The myelin sheath and nodes of Ranvier were essentially preserved in both lines. In contrast, the ultrastructural and molecular organization of contacts between Schwann cells and axons in paranodal and juxtaparanodal regions were altered, with irregular juxtaposition of normal and abnormal areas of contact. Similar but more severe alterations were observed in mice with conditional deletion of the Nf2 gene in Schwann cells. The number of Schmidt-Lanterman incisures, which are cytoplasmic channels interrupting the compact myelin and characterized by distinct autotypic contacts, was increased in the three mutant lines. P0-SCH-Delta39-121 and conditionally deleted mice displayed exuberant wrapping of nonmyelinated fibers and short internodes, an abnormality possibly related to altered control of Schwann cell proliferation. In support of this hypothesis, Schwann cell number was increased along fibers before myelination in P0-SCH-Delta39-121 mice but not in those with C-terminal deletion. Schwann cell numbers were also more numerous in mice with conditional deletion. Thus, schwannomin plays an important role in the control of Schwann cell number and is necessary for the correct organization and regulation of axoglial heterotypic and glio-glial autotypic contacts.

Abstract: Nucleotide oligomerisation domain 2 (NOD2) is a component of the innate immunity known to be involved in the homeostasis of Peyer patches (PPs) in mice. However, little is known about its role during gut infection in vivo. Yersinia pseudotuberculosis is an enteropathogen causing gastroenteritis, adenolymphitis and septicaemia which is able to invade its host through PPs. We investigated the role of Nod2 during Y. pseudotuberculosis infection. Death was delayed in Nod2 deleted and Crohn's disease associated Nod2 mutated mice orogastrically inoculated with Y. pseudotuberculosis. In PPs, the local immune response was characterized by a higher KC level and a more intense infiltration by neutrophils and macrophages. The apoptotic and bacterial cell counts were decreased. Finally, Nod2 deleted mice had a lower systemic bacterial dissemination and less damage of the haematopoeitic organs. This resistance phenotype was lost in case of intraperitoneal infection. We concluded that Nod2 contributes to the susceptibility to Y. pseudotuberculosis in mice.

Abstract: Fetal progenitor cells enter the maternal circulation during pregnancy and can persist for decades. We aimed to determine the role of these cells in tissue inflammation during pregnancy. WT female mice were mated to males transgenic for the EGFP (ubiquitous) or the luciferase gene controlled by the VEGF receptor 2 (VEGFR2; V-Luc) promoter. A contact hypersensitivity reaction was triggered during such pregnancies. Fetal cells were tracked by using real-time quantitative amplification of the transgene (real-time PCR), Y chromosome in situ hybridization (FISH), immunofluorescence or in vivo bioluminescence imaging. Real-time PCR disclosed fetal cells in the inflamed areas in all tested mice (17/17) with higher frequency and numbers in the inflamed compared with the control areas (P = 0.01). Double labeling demonstrated CD31+ EGFP+ fetal cells organized as blood vessels. In WT pregnant mice bearing V-Luc fetuses, a specific luciferase activity signal could be detected at the hypersensitivity site only, demonstrating the elective presence of VEGFR2-expressing fetal cells. In conclusion, using various techniques, we found the presence of fetal endothelial cells lining blood vessels in maternal sites of inflammation. These results imply that fetal endothelial progenitor cells are acquired by the mother and participate in maternal angiogenesis during pregnancy.

Abstract: Disruption of the intestinal homeostasis and tolerance towards the resident microbiota is a major mechanism involved in the development of inflammatory bowel disease. While some bacteria are inducers of disease, others, known as probiotics, are able to reduce inflammation. Because dendritic cells (DCs) play a central role in regulating immune responses and in inducing tolerance, we investigated their role in the anti-inflammatory potential of probiotic lactic acid bacteria.

Abstract: CARD15/NOD2 mutations are associated with susceptibility to Crohn's Disease (CD) and Graft Versus Host Disease (GVHD). CD and GVHD are suspected to be related with the dysfunction of Peyer's patches (PP) and isolated lymphoid follicles (LFs). Using a new mouse model invalidated for Card15/Nod2 (KO), we thus analysed the impact of the gene in these lymphoid formations together with the development of experimental colitis.

Abstract: Animal models allowing more sensitive and early detection of tumorigenesis and metastasis are instrumental in the fight for developing effective therapies against aggressive forms of cancer. In the present chapter, the advantages and limitations of the bioluminescent imaging (BLI) approach are discussed. Although BLI provides rapid, highly sensitive, noninvasive and quantitative detection of small tumors and micrometastases, several issues like the low anatomic resolution or the attenuation of the luminescent signal with tissue depth must be considered when using this technology.

Abstract: CARD15 is a major susceptibility gene for a frequent multifactorial chronic inflammatory bowel disorder, Crohn disease (CD). By using NF-kappaB activation assays, the cytosolic CARD15 was shown to efficiently detect bacterial peptidoglycan (PGN), reminiscent of the PGN recognition protein surveillance mechanism in Drosophila. The 3 CD-associated variants and 13 additional variants carried by CD patients demonstrated impaired PGN-dependent response revealing null, hypomorphic, or dominant-negative properties. Quantitative parametrization of this response, computed from the patients' CARD15 genotypes, was predictive of several variable CD manifestations. In contrast, CARD15 alleles associated with Blau's syndrome promoted PGN-independent NF-kappaB activation, an observation that accounts for the minimal microbial input in the etiology of this dominant, monogenic inflammatory disorder affecting solely aseptic sites.

Abstract: Crohn's disease is one of the principal human chronic inflammatory bowel diseases. Although its aetiology is still unknown, its complex pathogenesis has environmental, immunological, and genetic determinants. CARD15 is the first susceptibility gene implicated in the predisposition to Crohn's disease and is known to be expressed only in monocytes. However, its expression in situ has not yet been studied.