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Collapsin response mediator proteins (CRMPs) are highly expressed in the brain during early postnatal development and continue to be present in specific regions into adulthood, especially in areas with extensive neuronal plasticity including the hippocampus. They are found in the axons and dendrites of neurons wherein they contribute to specific signaling mechanisms involved in the regulation of axonal and dendritic development/maintenance. We previously identified CRMP3's role on the morphology of hippocampal CA1 pyramidal dendrites and hippocampus-dependent functions. Our focus here was to further analyze its role in the dentate gyrus where it is highly expressed during development and in adults. On the basis of our new findings, it appears that CRMP3 has critical roles both in axonal and dendritic morphogenesis of dentate granular neurons. In CRMP3-deficient mice, the dendrites become dystrophic while the infrapyramidal bundle of the mossy fiber shows aberrant extension into the stratum oriens of CA3. This axonal misguided projection of granular neurons suggests that the mossy fiber-CA3 synaptic transmission, important for the evoked propagation of the activity of the hippocampal trisynaptic circuitry, may be altered, whereas the dystrophic dendrites may impair the dynamic interactions with the entorhinal cortex, both expected to affect hippocampal function.

Although hippocampal neurons are well-distinguished by the morphological characteristics of their dendrites and their structural plasticity, the mechanisms involved in regulating their neurite initiation, dendrite growth, network formation and remodeling are still largely unknown, in part because the key molecules involved remain elusive. Identifying new dendrite-active cues could uncover unknown molecular mechanisms that would add significant understanding to the field and possibly lead to the development of novel neuroprotective therapy because these neurons are impaired in many neuropsychiatric disorders. In our previous studies, we deleted the gene encoding CRMP3 in mice and identified the protein as a new endogenous signaling molecule that shapes diverse features of the hippocampal pyramidal dendrites without affecting axon morphology. We also found that CRMP3 protects dendrites against dystrophy induced by prion peptide PrP(106-126). Here, we report that CRMP3 has a profound influence on neurite initiation and dendrite growth of hippocampal neurons in vitro. Our deletional mapping revealed that the C-terminus of CRMP3 probably harbors its dendritogenic capacity and supports an active transport mechanism. By contrast, overexpression of the C-terminal truncated CRMP3 phenocopied the effect of CRMP3 gene deletion with inhibition of neurite initiation or decrease in dendrite complexity, depending on the stage of cell development. In addition, this mutant inhibited the activity of CRMP3, in a similar manner to siRNA. Voltage-gated calcium channel inhibitors prevented CRMP3-induced dendritic growth and somatic Ca(2+) influx in CRMP3-overexpressing neurons was augmented largely via L-type channels. These results support a link between CRMP3-mediated Ca(2+) influx and CRMP3-mediated dendritic growth in hippocampal neurons.

Vascular endothelial growth factor (VEGF) regulates angiogenesis, but also has important, yet poorly characterized roles in neuronal wiring. Using several genetic and in vitro approaches, we discovered a novel role for VEGF in the control of cerebellar granule cell (GC) migration from the external granule cell layer (EGL) toward the Purkinje cell layer (PCL). GCs express the VEGF receptor Flk1, and are chemoattracted by VEGF, whose levels are higher in the PCL than EGL. Lowering VEGF levels in mice in vivo or ectopic VEGF expression in the EGL ex vivo perturbs GC migration. Using GC-specific Flk1 knock-out mice, we provide for the first time in vivo evidence for a direct chemoattractive effect of VEGF on neurons via Flk1 signaling. Finally, using knock-in mice expressing single VEGF isoforms, we show that pericellular deposition of matrix-bound VEGF isoforms around PC dendrites is necessary for proper GC migration in vivo. These findings identify a previously unknown role for VEGF in neuronal migration.

Background: In many neuroinflammatory diseases, dendritic cells (DCs) accumulate in several compartments of the central nervous system (CNS), including the cerebrospinal fluid (CSF). Myeloid DCs invading the inflamed CNS are thus thought to play a major role in the initiation and perpetuation of CNS-targeted autoimmune responses. We previously reported that, in normal rats, DCs injected intra-CSF migrated outside the CNS and reached the B-cell zone of cervical lymph nodes. However, there is yet no information on the migratory behavior of CSF-circulating DCs under neuroinflammatory conditions., Methodology/principal Findings: To address this issue, we performed in vivo transfer experiments in rats suffering from experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis. EAE or control rats were injected intra-CSF with bone marrow-derived myeloid DCs labeled with the fluorescent marker carboxyfluorescein diacetate succinimidyl ester (CFSE). In parallel experiments, fluorescent microspheres were injected intra-CSF to EAE rats in order to track endogenous antigen-presenting cells (APCs). Animals were then sacrificed on day 1 or 8 post-injection and their brain and peripheral lymph nodes were assessed for the presence of microspheres(+) APCs or CFSE(+) DCs by immunohistology and/or FACS analysis. Data showed that in EAE rats, DCs injected intra-CSF substantially infiltrated several compartments of the inflamed CNS, including the periventricular demyelinating lesions. We also found that in EAE rats, as compared to controls, a larger number of intra-CSF injected DCs reached the cervical lymph nodes. This migratory behavior was accompanied by an accentuation of EAE clinical signs and an increased systemic antibody response against myelin oligodendrocyte glycoprotein, a major immunogenic myelin antigen., Conclusions/significance: Altogether, these results indicate that CSF-circulating DCs are able to both survey the inflamed brain and to reach the cervical lymph nodes. In EAE and maybe multiple sclerosis, CSF-circulating DCs may thus support the immune responses that develop within and outside the inflamed CNS.

Collapsin response mediator proteins (CRMPs) are believed to play a crucial role in neuronal differentiation and axonal outgrowth. Among them, CRMP2 mediates axonal guidance by collapsing growth cones during development. This activity is correlated with the reorganization of cytoskeletal proteins. CRMP2 is implicated in the regulation of several intracellular signaling pathways. Two subtypes, A and B, and multiple cytosolic isoforms of CRMP2B with apparent masses between 62 and 66 kDa have previously been reported. Here, we show a new short isoform of 58 kDa, expressed during brain development, derived from C-terminal processing of the CRMP2B subtype. Although full-length CRMP2 is restricted to the cytoplasm, using transfection experiments, we demonstrate that a part of the short isoform is found in the nucleus. Interestingly, at the tissue level, this short CRMP2 is also found in a nuclear fraction of brain extract. By mutational analysis, we demonstrate, for the first time, that nuclear translocation occurs via nuclear localization signal (NLS) within residues Arg(471)-Lys(472) in CRMP2 sequence. The NLS may be unmasked after C-terminal processing; thereby, this motif may be surface-exposed. This short CRMP2 induces neurite outgrowth inhibition in neuroblastoma cells and suppressed axonal growth in cultured cortical neurons, whereas full-length CRMP2 promotes neurite elongation. The NLS-mutated short isoform, restricted to the cytoplasm, abrogates both neurite outgrowth and axon growth inhibition, indicating that short nuclear CRMP2 acts as a dominant signal. Therefore, post-transcriptional processing of CRMP2 together with its nuclear localization may be an important key in the regulation of neurite outgrowth in brain development.

Mucopolysaccharidosis IIIB is a lysosomal disease characterized by a severe neurological deterioration, the pathophysiological mechanisms of which are poorly understood. Recently FGF pathway was shown to be altered leading us to explore a downstream target involved in brain development: the collapsin response mediator protein-1 (CRMP-1). CRMP-1 transcript level was normal but a cleavage of CRMP-1 was observed with an abnormal expression of the truncated form until adult age. This truncated CRMP-1 protein could play a role in post-natal cortex maturation and be involved in neuronal alterations occurring in lysosomal diseases.

In vitro studies have pointed to the collapsin response mediator proteins (CRMPs) as key regulators of neurite outgrowth and axonal differentiation. CRMP3 is expressed mostly in the nervous system during development but remains at high levels in the hippocampus of adults. To explore CRMP3 function in vivo, we generated mice with targeted disruption of the CRMP3 gene. Immunohistochemistry and Golgi staining of CA1 showed abnormal dendrite and spine morphogenesis in the hippocampus of CRMP3-deficient mice. Apical dendrites displayed an increase in undulation and a reduction in length and branching points. Basal dendrites also exhibited a reduction in length with an alteration in soma stem distribution and an increased number of thick dendrites localized in stratum oriens (SO). Long-term potentiation (LTP) was impaired in this area. These data indicate an important role for CRMP3 in dendrite arborization, guide-posts navigation, and neuronal plasticity.

Although most pituitary tumors are benign, some are invasive or aggressive. In the absence of specific markers of malignancy, only tumors with metastases are considered malignant. To identify markers of invasion and aggressiveness, we focused on prolactin (PRL) tumors in the human and rat. Using radiology and histological methods, we classified 25 human PRL tumors into three groups (non-invasive, invasive, and aggressive-invasive) and compared them with a model of transplantable rat PRL tumors with benign and malignant lineages. Combining histological(mitoses and labeling for Ki-67, P53, pituitary transforming tumor gene (PTTG), and polysialic acid neural cell adhesion molecule) and transcriptomic (microarrays and q-RTPCR) methods with clinical data (post-surgical outcome with case-control statistical analysis), we found nine genes implicated in invasion (ADAMTS6, CRMP1, and DCAMKL3) proliferation (PTTG, ASK, CCNB1, AURKB, and CENPE), or pituitary differentiation (PITX1) showing differential expression in the three groups of tumors (P = 0.015 to 0.0001). A case-control analysis, comparing patients in remission (9 controls) and patients with persistent or recurrent tumors (14 cases) revealed that eight out of the nine genes were differentially up- or downregulated (P = 0.05 to 0.002), with only PTTG showing no correlation with clinical course (P = 0.258). These combined histological and transcriptomic analyses improve the pathological diagnosis of PRL tumors, indicating a reliable procedure for predicting tumor aggressiveness and recurrence potential. The similar gene profiles found between non-invasive human and benign rat tumors, as well as between aggressive-invasive human and malignant rat tumors provide new insights into malignancy in human pituitary tumors.

Background: In absence epilepsy, the neuronal hyper-excitation and hyper-synchronization, which induce spike and wave discharges in a cortico-thalamic loop are suspected to be due to an imbalance between GABA and glutamate (GLU) neurotransmission. In order to elucidate the role played by GLU in disease outcome, we measured cortical and thalamic extracellular levels of GLU and GABA. We used an in vivo quantitative microdialysis approach (no-net-flux method) in an animal model of absence epilepsy (GAERS). In addition, by infusing labelled glutamate through the microdialysis probe, we studied in vivo glutamate uptake in the cortex and thalamus in GAERS and non-epileptic control (NEC) rats. Expression of the vesicular glutamate transporters VGLUT1 and VGLUT2 and a synaptic component, synaptophysin, was also measured., Results: Although extracellular concentrations of GABA and GLU in the cortex and thalamus were not significantly different between GAERS and NEC rats, cortical GLU uptake was significantly decreased in unrestrained awake GAERS. Expression of VGLUT2 and synaptophysin was increased in the cortex of GAERS compared to NEC rats, but no changes were observed in the thalamus., Conclusion: The specific decrease in GLU uptake in the cortex of GAERS linked to synaptic changes suggests impairment of the glutamatergic terminal network. These data support the idea that a change in glutamatergic neurotransmission in the cortex could contribute to hyperexcitability in absence epilepsy.

Obesity results from disturbances of tightly regulated interactions between the nervous, endocrine, and metabolic systems that can be caused by external factors, such as viral infections. A mouse model of obesity induced by brain infection with a morbillivirus, canine distemper virus, allowed us to identify obesity-related genes. Using a subtractive library for the hypothalamus, the main brain structure regulating energy homeostasis, we identified a new gene on mouse chromosome 19 which we named upregulated obese product (Urop) 11 and, which has no homology with any known mRNA. A step-by-step molecular approach allowed us to isolate the full-length mRNA, predict the protein sequence, and identify consensus sites. Urop11 was mainly detected in the hypothalamus and adipocytes, and was dramatically upregulated in these central and peripheral structures in obese mice. Urop11 was also expressed in human neural and lymphoid samples and its expression seemed to be regulated by the state of lymphocyte activation. Interestingly, Urop11 expression was strongly upregulated both in vivo in mouse hypothalamus and in vitro in mouse neural cell lines, after leptin treatment. Taken together, our data show that Urop11 is a target of leptin, the satiety factor produced by adipocytes, in physiological and pathological conditions, including obesity. This new gene can be considered a key molecule in the hypothalamic integration pathway and demonstrates the importance of Urop11 as a target of leptin action.

Immunotherapy has been explored for several decades to try to improve the prognosis of gliomas, but until recently no therapeutic benefit has been achieved. The discovery of dendritic cells, the most potent professional antigen presenting cells to initiate specific immune response, and the possibility of producing them ex vivo gave rise to new protocols of active immunotherapy. In oncology, promising experimental and clinical therapeutic results were obtained using these dendritic cells loaded with tumor antigen. Patients bearing gliomas have deficit antigen presentation making this approach rational. In several experimental glioma models, independent research teams have showed specific antitumor responses using these dendritic cells. Phase I/II clinical trials have demonstrated the feasibility and the tolerance of this immunotherapeutic approach. In neuro-oncology, the efficiency of such an approach remains to be established, similarly in oncology where positive phase III studies are missing. Nevertheless, dendritic cells comprise a complex network which is only partially understood and capable of generating either immunotolerance or immune response. Numerous parameters remain to be explored before any definitive conclusion about their utility as an anticancer weapon can be drawn. It seems however logical that immunotherapy with dendritic cells could prevent or delay tumor recurrence in patients with minor active disease. A review on glioma and dendritic cells is presented.

Collapsin response mediator proteins (CRMPs) consist of five homologous cytosolic proteins that participate in signal transduction involved in a variety of physiological events. CRMP1 is highly expressed during brain development; however, its functions remains unclear. To gain insight into its function, we generated CRMP1(-/-) mice with a knock-in LacZ gene. No gross anatomical changes or behavioral alterations were observed. Expression of CRMP1 was examined by the expression of the knocked-in LacZ gene, in situ hybridization with riboprobes and by imunohistochemistry. CRMP1 was found to be highly expressed in the developing the cerebellum, olfactory bulbs, hypothalamus and retina. In adults, expression level was high in the olfactory bulbs and hippocampus but very low in the retina and cerebellum and undetectable in hypothalamus. To study potential roles of CRMP1, we focused on cerebellum development. CRMP1(-/-) mice showed a decrease in the number of granule cells migrating out of explants of developing cerebellum, as did treatment of the explants from normal mice with anti-CRMP1 specific antibodies. CRMP1(-/-) mice showed a decrease in granule cell proliferation and apoptosis in external granule cell layers in vivo. Adult cerebellum of CRMP1(-/-) did not show any abnormalities.

Recent evidence indicates that microglial cells may not derive from blood circulating mature monocytes as they express features of myeloid progenitors. Here, we observed that a subpopulation of microglial cells expressed CD34 and B220 antigens during brain development. We thus hypothesized that microglia, or a subset of microglial cells, originate from blood circulating CD34+/B220+ myeloid progenitors, which could target the brain under developmental or neuroinflammatory conditions. Using experimental allergic encephalomyelitis (EAE) as a model of chronic neuroinflammation, we found that a discrete population of CD34+/B220+ cells expands in both blood and brain of diseased animals. In EAE mice, intravenous transfer experiments showed that macrophage-colony stimulating factor (M-CSF) -expanded CD34+ myeloid progenitors target the inflamed central nervous system (CNS) while keeping their immature phenotype. Based on these results, we then assessed whether CD34+/B220+ cells display in vitro differentiation potential toward microglia. For this purpose, CD34+/B220+ cells were sorted from M-CSF-stimulated bone marrow (BM) cultures and exposed to a glial cell conditioned medium. Under these experimental conditions, CD34+/B220+ cells were able to differentiate into microglial-like cells showing the morphological and phenotypic features of native microglia. Overall, our data suggest that under developmental or neuroinflammatory conditions, a subpopulation of microglial cells derive from CNS-invading CD34+/B220+ myeloid progenitors.

The accurate identification and thorough characterization of tumorigenic cells in glioblastomas are essential to enhance our understanding of their malignant behavior and for the design of strategies that target this important cell population. We report here that, in rat brain, the scaffolding protein IQGAP1 is a marker of brain nestin+ amplifying neural progenitor cells. In a rat model of glioma, IQGAP1 also characterizes a subpopulation of nestin+ amplifying tumor cells in glioblastoma-like tumors but not in tumors with oligodendroglioma features. We next confirmed that IQGAP1 represents a new marker that may help to discriminate human glioblastoma from oligodendrogliomas. In human glioblastoma exclusively, IQGAP1 specifies a subpopulation of amplifying nestin+ cancer cells. Neoplastic IQGAP1+ cells from glioblastoma can be expanded in culture and possess all the characteristics of cancer stem-like progenitors. The similarities between amplifying neural progenitors and glioblastoma amplifying cancer cells may have significant implications for understanding the biology of glioblastoma.

Malignant rhabdoid tumours (MRT) are highly aggressive cancers of early childhood that arise in different organs or tissues. The unifying criterion for these tumours is the presence of inactivating mutations of the hSNF5/INI1 tumour suppressor gene which encodes a core subunit of the chromatin remodelling SWI/SNF complex. Using a variety of markers we analysed the phenotypic traits of MON and DEV cell lines derived respectively from an undifferentiated abdominal MRT and from a brain MRT. DEV cells express spontaneously a wide range of neural and glial markers. It can be induced to differentiate into the neural lineage following hSNF5/INI1 expression with appearance of neurite processes, strong increase of neural markers and decrease of glial markers. A less pronounced neural differentiation is also observed with MON cells, which possess more primitive polyphenotypic features with positivity for markers from the three embryonic layers. Finally, we show that the neural differentiation of rat PC12 cells in the presence of nerve growth factor (NGF) is strongly impaired when hSNF5/INI1 expression is inhibited by RNA interference. Altogether these results indicate that hSNF5/INI1 is an essential subunit for SWI/SNF-dependant induction of neural differentiation programs. Further experiments should enable documentation of whether it provides instructive or permissive signals for differentiation.

The choroid plexuses (CPs) form a protective interface between the blood and the ventricular cerebrospinal fluid (CSF). To probe into the pathways by which CPs provide brain protection, we sought to evaluate the efficiency of glutathione conjugation in this barrier as a mechanism to prevent the entry of blood-borne electrophilic, potentially toxic compounds into the CSF, and we investigated the fate of the resulting metabolites. Rat CPs, as well as human CPs from both fetal and adult brains, displayed high glutathione-S-transferase activities. Using an in vitro model of the blood-CSF barrier consisting of choroidal epithelial cells cultured in a two-chambered device, we showed that glutathione conjugation can efficiently prevent the entry of 1-chloro-2,4-dinitrobenzene (CDNB) into the CSF, a model for electrophilic compounds. The duration of this enzymatic protection was set by the concentration of CDNB to which the epithelium was exposed, and this barrier effect was impaired only on severe epithelial intracellular glutathione and cysteine depletion. The conjugate was excreted from the choroidal cells in a polarized manner, mostly at the blood-facing membrane, via a high-capacity transport process, which is not a rate-limiting step in this detoxification pathway, and which may involve transporters of the ATP-binding cassette c(Abcc) and/or solute carrier 21 (Slc21) families. Supplying the choroidal epithelium at the blood-facing membrane with a therapeutically relevant concentration of N-acetylcysteine sustained this neuroprotective effect. Thus, glutathione conjugation at the CP epithelium coupled with the basolateral efflux of the resulting metabolites form an efficient blood-CSF enzymatic barrier, which can be enhanced by pharmacologically increasing glutathione synthesis within the epithelial cells.

The interface between the blood and the cerebrospinal fluid (CSF) is formed by the choroid plexuses (CPs), which are specialized structures located within the brain ventricles. They are composed of a vascularized stroma surrounded by a tight epithelium that controls molecular and cellular traffic between the blood and the CSF. Cells expressing myeloid markers are present within the choroidal stroma. However, the exact identity, maturation state, and functions of these CP-associated myeloid cells are not fully clarified. We show here that this cell population contains immature myeloid progenitors displaying a high proliferative potential. Thus, in neonate rats and, to a lesser extent, in adult rats, cultured CP stroma cells form large colonies of macrophages, in response to M-CSF or GM-CSF, while, under the same conditions, peripheral blood monocytes do not. In addition, under GM-CSF treatment, free-floating colonies of CD11c(+) monocytic cells are generated which, when restimulated with GM-CSF and IL-4, differentiate into OX62(+)/MHC class II(+) dendritic cells. Interestingly, in CP stroma cultures, myeloid cells are found in close association with fibroblastic-like cells expressing the neural stem-cell marker nestin. Similarly, in the developing brain, macrophages and nestin(+) fibroblastic cells accumulate in vivo within the choroidal stroma. Taken together, these results suggest that the CP stroma represents a niche for myeloid progenitors and may serve as a reservoir for brain macrophages.

Measles virus (MV) infection causes an acute childhood disease, associated in certain cases with infection of the central nervous system and development of a severe neurological disease. We have generated transgenic mice ubiquitously expressing the human protein SLAM (signaling lymphocytic activation molecule), or CD150, recently identified as an MV receptor. In contrast to all other MV receptor transgenic models described so far, in these mice infection with wild-type MV strains is highly pathogenic. Intranasal infection of SLAM transgenic suckling mice leads to MV spread to different organs and the development of an acute neurological syndrome, characterized by lethargy, seizures, ataxia, weight loss, and death within 3 weeks. In addition, in this model, vaccine and wild-type MV strains can be distinguished by virulence. Furthermore, intracranial MV infection of adult transgenic mice generates a subclinical infection associated with a high titer of MV-specific antibodies in the serum. Finally, to analyze new antimeasles therapeutic approaches, we created a recombinant soluble form of SLAM and demonstrated its important antiviral activity both in vitro and in vivo. Taken together, our results show the high susceptibility of SLAM transgenic mice to MV-induced neurological disease and open new perspectives for the analysis of the implication of SLAM in the neuropathogenicity of other morbilliviruses, which also use this molecule as a receptor. Moreover, this transgenic model, in allowing a simple readout of the efficacy of an antiviral treatment, provides unique experimental means to test novel anti-MV preventive and therapeutic strategies.

The prognosis of malignant gliomas remains dismal and alternative therapeutic strategies are required. Immunotherapy with dendritic cells (DCs) pulsed with tumour antigens emerges as a promising approach. Many parameters influence the efficacy of DC-based vaccines and need to be optimised in preclinical models. The present study compares different vaccine schedules using DCs loaded with tumour cell lysate (DC-Lysate) for increasing long-term survival in the GL26 orthotopic murine glioma model, focusing on the number of injections and an optimal way to recall antitumour immune response. Double vaccination with DC-Lysate strongly prolonged median survival compared to unvaccinated animals (mean survival 87.5 days vs. 25 days; p < 0.0001). In vitro data showed specific cytotoxic activity against GL26. However, late tumour relapses frequently occurred after 3 months and only 20% of mice were finally cured at 7 months. While one, two or three DC injections gave identical survival, a boost using only tumour lysate after initial DC-Lysate priming dramatically improved long-term survival in vaccinated mice, compared to the double DC-Lysate group, with 67.5% of animals cured at 7 months (p < 0.0001). In vitro data showed better specific CTL response and also the induction of specific anti-GL26 antibodies in the DC-Lysate/Lysate group, which mediated Complement Dependent Cytotoxicity. These experimental data may be of importance for the design of clinical trials that currently use multiple DC injections.

The lack of draining lymphatic vessels in the central nervous system (CNS) contributes to the so-called "CNS immune privilege." However, despite such a unique anatomic feature, dendritic cells (DCs) are able to migrate from the CNS to cervical lymph nodes through a yet unknown pathway. In this report, labeled bone marrow-derived myeloid DCs were injected stereotaxically into the cerebrospinal fluid (CSF) or brain parenchyma of normal rats. We found that DCs injected within brain parenchyma migrate little from their site of injection and do not reach cervical lymph nodes. In contrast, intra-CSF-injected DCs either reach cervical lymph nodes or, for a minority of them, infiltrate the subventricular zone, where neural stem cells reside. Surprisingly, DCs that reach cervical lymph nodes preferentially target B-cell follicles rather than T-cell-rich areas. This report sheds a new light on the specific role exerted by CSF-infiltrating DCs in the control of CNS-targeted immune responses.

The semaphorin-signaling transducer collapsin response mediator protein 2 (CRMP2) has been identified in the nervous system where it mediates Sema3A-induced growth cone navigation. In the present study, we provide first evidence that CRMP2 is present in the immune system and plays a critical role in T lymphocyte function. CRMP2 redistribution at the uropod in polarized T cells, a structural support of lymphocyte motility, suggests that it may regulate T cell migration. This was evidenced in primary T cells by small-interfering RNA-mediated CRMP2 gene silencing and blocking Ab, as well as CRMP2 overexpression in Jurkat T cells tested in a chemokine- and semaphorin-mediated transmigration assay. Expression analysis in PBMC from healthy donors showed that CRMP2 is enhanced in cell subsets bearing the activation markers CD69+ and HLA-DR+. Heightened expression in T lymphocytes of patients suffering from neuroinflammatory disease with enhanced T cell-transmigrating activity points to a role for CRMP2 in pathogenesis. The elucidation of the signals and mechanisms that control this pathway will lead to a better understanding of T cell trafficking in physiological and pathological situations.

The choroid plexus epithelium forms the interface between the blood and the CSF. In conjunction with the tight junctions restricting the paracellular pathway, polarized specific transport systems in the choroidal epithelium allow a fine regulation of CSF-borne biologically active mediators. The highly vascularized stroma delimited by the choroidal epithelium can be a reservoir for retrovirus-infected or activated immune cells. In this work, new insight in the implication of the blood-CSF barrier in neuroinfectious and inflammatory diseases is provided by using a differentiated cellular model of the choroidal epithelium, exposed to infected T lymphocytes. We demonstrate that T cells activated by a retroviral infection, but not non-infected cells, reduce the transporter-mediated CSF-to-blood efflux of organic anions, in particular that of the potent pro-inflammatory prostaglandin PGE2, via the release of soluble factors. A moderate alteration of the paracellular permeability also occurs. We identified the viral protein Tax, oxygenated free radicals, matrix-metalloproteinases and pro-inflammatory cytokines as active molecules released during the exposure of the epithelium to infected T cells. Among them, tumour necrosis factor and interleukin 1 are directly involved in the mechanism underlying the decrease in some choroidal organic anion efflux. Given the strong involvement of CSF-borne PGE2 in sickness behaviour syndrome, these data suggest that the blood-CSF barrier plays an important role in the pathophysiology of neuroinflammation and neuroinfection, via changes in the transport processes controlling the CSF biodisposition of PGE2.

Human polycystic lipomembraneous osteodysplasia with sclerosing leukoencephalopathy, also known as Nasu-Hakola disease, has been described to be associated with mutations affecting the immunoreceptor tyrosine-based activation motif-bearing KARAP/DAP12 immunoreceptor gene. Patients present bone fragilities and severe neurological alterations leading to presenile dementia. Here we investigated whether the absence of KARAP/DAP12-mediated signals in loss-of-function (KDelta75) mice also leads to bone and central nervous system pathological features. Histological analysis of adult KDelta75 mice brains revealed a diffuse hypomyelination predominating in anterior brain regions. As this was not accompanied by oligodendrocyte degeneration or microglial cell activation it suggests a developmental defect of myelin formation. Interestingly, in postnatal KDelta75 mice, we observed a dramatic reduction in microglial cell numbers similar to in vitro microglial cell differentiation impairment. Our results raise the intriguing possibility that defective microglial cell differentiation might be responsible for abnormal myelin development. Histomorphometry revealed that bone remodeling is also altered, because of a resorption defect, associated with a severe block of in vitro osteoclast differentiation. In addition, we show that, among monocytic lineages, KARAP/DAP12 specifically controls microglial and osteoclast differentiation. Our results confirm that KARAP/DAP12-mediated signals play an important role in the regulation of both brain and bone homeostasis. Yet, important differences exist between the symptoms observed in Nasu-Hakola patients and KDelta75 mice.

Neurotropic viruses are involved in pathologies of the central nervous system, triggering transient or irreversible disorders, such as neurological diseases or homeostasis imbalance. In experimental animals, viruses have been shown to cause obesity, a complex disease depending on multiple factors, including genetic susceptibility and environmental components. Using a mouse model of virally induced obesity following brain infection by the Canine Distemper Virus (CDV), a morbillivirus closely related to the human measles virus, we investigated the modulation of expression of several hypothalamic neuropeptides known to intervene in the regulation of body weight and energy expenditure, both during the acute and late stages of infection. During the acute stage, while viral replication occurs, we found a dramatic decrease of expressions of neuropeptides, in particular neuropeptide Y, melanin-concentrating hormone (MCH), hypocretin, vasopressin and tachykinins, the magnitude of which seemed to be linked to the viral burden and the individual susceptibility. The effect of the virus, however, varied with the hypothalamic nucleus and neuropeptide involved, suggesting that certain circuits were affected while others remained intact. During the late stage of infection, marked recovery to the initial hypothalamic levels of peptide expression was seen in a number of lean animals, suggesting recovery of homeostasis equilibrium. Interestingly, some neuropeptidergic systems remained disturbed in mice exhibiting obese phenotype, arguing for their involvement in triggering/maintaining obesity. Even though our data could not fully explain the viral-induced obesity, they may be helpful in understanding the molecular events associated with obesity and in investigating therapeutic alternatives.

The cerebrospinal fluid (CSF) is in direct contact with the extracellular space of the CNS, thus biochemical processes in the CNS could potentially be reflected in the CSF. Changes in extracellular matrix (ECM) proteins can be studied through their analysis in the CSF. ECM plays an essential role in CNS homeostasis and several proteins such as laminin (LN), fibronectin (FN), thrombospondin (TS) and heparan sulphate proteoglycan (HS, perlecan) form part of its structure. Possible changes in the levels of these proteins were investigated in two different pathologies--tropical spastic paraparesis/HTLV-I-associated myelopathy (TSP/HAM) (n=25) and Creutzfeldt-Jakob disease (CJD) (n=19)--and compared with those in a control group with or without neurological disease (n=25). CSF analyses were carried out using monoclonal or monospecific polyclonal antibodies. In comparison with the control group, it was found that TSP/HAM patients presented significantly higher levels of LN, TS and HS, while in CJD patients the levels of FN, TS and HS were increased. In CJD patients the HS level was almost double that of the TSP/HAM patients. These results suggest a distinct pattern of ECM proteins in CSF in relation to the type of neurological disease. TSP/HAM is a chronic motor disease that affects the white matter of the spinal cord, while CJD is a subacute dementia that affects cerebral neurons and their synapsis.

Semaphorins are multifunctional factors implicated in various developmental processes. Little is known about the intracellular pathways ensuring appropriate signal transduction that encode the diverse functions observed. In this study, we investigated whether mitogen-activated protein kinases (MAPK), which are key elements of signal transduction in eukaryotic cells, were activated during semaphorin 3A (Sema3A)-induced repulsion or apoptosis of neural progenitor cells. We found that selective recruitment of the ERK1/2 pathway occurred during Sema3A-induced neural progenitor cell repulsion, whereas p38 MAPK activation was necessary for induction of apoptosis. Moreover, we provide evidence for the involvement of vascular endothelial growth factor receptor 1 (VEGFR1) in the activation of ERK1/2. Additional experiments performed with native cerebellar progenitors confirmed such a selective recruitment of MAPK during Sema3A-dependent migration or apoptosis. Altogether, our results suggest a model to explain how a single factor can exert different functions for a given cell type by the selective recruitment of intracellular pathways.

The pattern of sensory neuron extensions and connections is established during embryonic development through complex and varied guidance cues that control motility of growth cones and neurite morphogenesis. Semaphorins and neurotrophins are molecules that act as such cues. Collapsin response mediator proteins (CRMPs) are thought to be part of the semaphorin signal transduction pathway implicated in semaphorin-induced growth cone collapse. In this report, we present evidence that CRMPs are also involved in the neurite extension controlled by neurotrophins. We found that specific antibodies and the dominant-negative mutant protein for CRMP2 both potentiated the neurite extension induced by NGF, while specific antibodies and the corresponding mutant protein for CRMP1 both abolished the neurite extension induced by NT3. Our data suggest that CRMP2 has a negative effect on neurite extension induced by NGF and CRMP1 participates in the neurite formation/extension induced by NT3. These results point to a function for CRMPs in the regulation of neurite outgrowth induced by neurotrophins in sensory neurons.

An inappropriate cross talk between activated T lymphocytes infiltrating the CNS and neural cells can sustain the onset and progression of demyelination and axonal degeneration in neuroinflammatory diseases. To mimic this deleterious cross talk, we designed an experimental paradigm consisting of transient cocultures of T lymphocytes chronically activated by retrovirus infection (not virus productive) with human multipotent neural precursors or primary oligodendrocytes from rat brain. We showed that activated T lymphocytes induced apoptotic death of multipotent neural progenitors and immature oligodendrocytes after a progressive collapse of their process extensions. These effects were reminiscent of those induced by brain semaphorin on neural cells. Blockade by specific Abs of soluble CD100 (sCD100)/semaphorin 4D released by activated T cells, or treatment with rsCD100, demonstrated that this immune semaphorin has the ability to collapse oligodendrocyte process extensions and to trigger neural cell apoptosis, most likely through receptors of the plexin family. The specific presence of sCD100 in the cerebrospinal fluid and of CD100-expressing T lymphocytes in the spinal cord of patients suffering with neuroinflammatory demyelination pointed to the potential pathological effect of sCD100 in the CNS. Thus, our results show that CD100 is a new important element in the deleterious T cell-neural cell cross talk during neuroinflammation and suggest its role in demyelination or absence of remyelination in neuroinflammatory diseases including multiple sclerosis and human T lymphotropic virus type 1-associated myelopathy.

Neuroinflammation and neuroinfection trigger cytokine-mediated responses that include an increase in the cerebrospinal fluid (CSF) levels of pro-inflammatory matrix metalloproteinases (MMPs) and organic anions such as leukotrienes and prostaglandins. The choroid plexus (CP) epithelium forming the interface between the blood and the CSF regulates the CSF concentration of bioactive organic anions and is involved in neuro-immune regulation. We demonstrated that both fourth and lateral ventricle CPs are a source of pro- and active MMP-2 and MMP-9 in the brain. Using a cellular model of the blood-CSF barrier, we showed that a pro-inflammatory cytokine treatment leads to an increase in the choroidal MMP secretion at either the apical or the basolateral membrane, depending on the ventricular origin of the choroidal cells. This effect was not concomitant with an alteration in the structural blood-CSF barrier. Neither was the pool of antioxidant sulfhydryls in the choroidal cells challenged. In contrast, the efficiency of the choroidal epithelium to clear the CSF from organic anions was highly reduced. Thus, during inflammation, the CPs could be one source of MMPs found in the CSF facilitate leucocyte migration by secreting MMPs into the choroidal stroma, and promote the inflammatory process by failing in its ability to clear deleterious compounds from the brain.

Matrix metalloproteinases (MMPs) are responsible for the extensive extracellular proteolysis that plays a central role in regulating the pericellular environment, contributing to morphogenesis and developmental remodeling. In the CNS, there is increasing in vitro evidence for the involvement of MMPs in neurite elongation and axonal guidance. Here, we show that expression of MMP-9 is spatiotemporally related to cerebellar granule cell migration during postnatal development. Using cerebellar explant cultures, we demonstrated that a specific MMP-9-blocking antibody affects granular cell axonal outgrowth and migration in a dose-dependent manner. In addition, the in vivo analysis of MMP-9-deficient mice revealed abnormal accumulation of granular precursors (GPs) in the external granular layer (EGL) at a time when migration is normally extensive. Furthermore, GP migration was delayed and their programmed cell death was reduced in MMP-9-deficient mice, suggesting that MMP-9 is involved in the control of granule cell migration and apoptosis. These results provide direct evidence for a physiological role of MMP-9 in neuronal precursor migration and apoptosis in the developing cerebellum, and emphasize the importance of MMP-9 in the temporal regulation of the cerebellar microenvironment.

Objective: In AIDS, early suppression of the viral load in the central nervous system is critical for the efficacy of antiretroviral therapy, in order to prevent the emergence of a reservoir of resistant strains of virus, and brain impairment in late stages of the infection. The blood-cerebrospinal fluid (CSF) interface (i.e. the choroidal epithelium) constitutes the most direct route to reach the ventricular meningeal and perivascular infected macrophages, and may modulate the cerebral biodisposition of antiretroviral drugs through various transport systems. Our aim was to address nucleoside drug transfer specifically across the blood-CSF interface, and identify the possible mechanisms involved in their transport., Methods: Drug influx and efflux were measured using an in vitro cellular model that reproduces the barrier and transport properties of the blood-CSF interface in vivo. Transport mechanisms were investigated by competition studies., Results: The CSF influx rate of zidovudine was the highest, although moderate, followed by that of stavudine. The permeability coefficients of the other drugs tested were low. Zidovudine influx into the CSF is independent of thymidine transport systems, and more importantly is limited by an efflux mechanism. This efflux involves an apical (CSF-facing) carrier belonging to the solute carrier (Slc) 22 family of organic anion transporters, and can be inhibited by a therapeutic concentration of benzbromarone., Conclusions: The demonstration and characterization of this efflux mechanism is the basis for the development of specific inhibitory agents in view to increase the delivery of antiretroviral nucleoside analogs to the brain.

Primary papillary tumors of the central nervous system are rare. We have encountered a series of six papillary tumors of the pineal region with distinctive features that appear to represent a clinicopathologic entity. The tumors occurred in four women and two men, ranging in age from 19 to 53 years. Imaging studies showed a large well-circumscribed mass in the pineal region. The tumors were characterized by an epithelial-like growth pattern, in which the vessels were covered by a layer of tumoral cells. In papillary areas, the neoplastic cells were large, columnar or cuboidal, with a clear cytoplasm. Nuclei, round or infolded, were found generally at the basal pole of tumoral cells. Immunohistochemically, the tumor cells showed strong staining for cytokeratin, S-100 protein, neuron-specific enolase, and vimentin but only weak or no staining for epithelial membrane antigen and glial fibrillary acid protein. Ultrastructural examination of two cases revealed abundant rough endoplasmic reticulum with distended cisternae filled with secretory product, microvilli, and perinuclear intermediate filaments. The morphofunctional features of these papillary tumors of the pineal region, remarkably uniform within this series, are similar to those described for ependymal cells of the subcommissural organ, and the papillary tumors of the pineal region may be derived from these specialized ependymocytes.

The astrocyte, the major glial cell in the central nervous system, may influence many aspects of inflammation and immune reactivity within the brain. We have established a model of chronically activated T lymphocytes, interacting with neural cells of diverse origin to study the complex immune regulatory system suspected to lead to neuroinflammatory diseases. We show that human astrocytes became reactive following T cell contact, secreting proinflammatory cytokines, matrix metalloproteinases (MMP) and tissue inhibitors of metalloproteinase (TIMP). The altered MMP/TIMP system was shown to be involved in deleterious effects displayed by activated T cells towards human multipotent neural precursers by controlling their sensitivity to T cell-induced Fas-mediated apoptosis. MMP/TIMP was suspected to stabilize Fas at the cell membrane. In a model of mixed rat glial cells in primary culture (astrocytes, oligodendrocytes), activated T lymphocytes induced the collapse of processes and the death of immature oligodendrocytes. These effects were associated with upregulation of Fas at the cell surface of oligodendrocytes and secretion of MMP and TIMP by astrocytes. By amplifying the expression of inflammatory molecules including the MMP/TIMP system, astrocytes appear to be a crucial relay in the deleterious molecular cascade triggered by activated T lymphocytes. Detection of altered MMP/TIMP in patients suffering from myelopathy associated with retroviral infection (HTLV-1) strongly suggests its involvement in the physiopathological process of the disease.

Background: Macrophages, osteoclasts, dendritic cells, and microglia are highly specialized cells that belong to the mononuclear phagocyte system. Functional and phenotypic heterogeneity within the mononuclear phagocyte system may reveal differentiation plasticity of a common progenitor, but developmental pathways leading to such diversity are still unclear., Results: Mouse bone marrow cells were expanded in vitro in the presence of Flt3-ligand (FL), yielding high numbers of non-adherent cells exhibiting immature monocyte characteristics. Cells expanded for 6 days, 8 days, or 11 days (day 6-FL, day 8-FL, and day 11-FL cells, respectively) exhibited constitutive potential towards macrophage differentiation. In contrast, they showed time-dependent potential towards osteoclast, dendritic, and microglia differentiation that was detected in day 6-, day 8-, and day 11-FL cells, in response to M-CSF and receptor activator of NFkappaB ligand (RANKL), granulocyte-macrophage colony stimulating-factor (GM-CSF) and tumor necrosis factor-alpha (TNFalpha), and glial cell-conditioned medium (GCCM), respectively. Analysis of cell proliferation using the vital dye CFSE revealed homogenous growth in FL-stimulated cultures of bone marrow cells, demonstrating that changes in differential potential did not result from sequential outgrowth of specific precursors., Conclusions: We propose that macrophages, osteoclasts, dendritic cells, and microglia may arise from expansion of common progenitors undergoing sequential differentiation commitment. This study also emphasizes differentiation plasticity within the mononuclear phagocyte system. Furthermore, selective massive cell production, as shown here, would greatly facilitate investigation of the clinical potential of dendritic cells and microglia.

Ependymomas, rare neoplasms of the central nervous system, occur predominantly in children. They are highly vascularized, and histological findings show many perivascular rosettes of tumoral cells radially organized around capillaries. Treatment of ependymomas relies on surgery combined with radio- or chemotherapy, but the efficiency of chemotherapy is limited, probably because of their multidrug resistance (MDR) phenotype. Progress in the therapy of these neoplasms is dramatically limited by the absence of cell line models. We established conditions for the long-term culture of human tumoral ependymocytes and their 3D coculture in Matrigel with endothelial cells. Histological, immunological, and ultrastructural studies showed that the morphological features (microvilli, cilia, and caveolae) of these cultured cells were similar to those of the tumor in vivo. The cells expressed potential oncological markers related to the immature state of tumoral cells (nestin and Notch-1), their tumorigenicity [caveolae and epidermal growth factor-receptor (EGF-R)], or the MDR phenotype [P-glycoprotein (P-gp)]. The expression of P-gp, EGF-R, and caveolin-1 by these tumoral ependymocytes could be useful in studies on new drugs. This coculture model might represent a new powerful tool to study new therapeutic delivery strategies in tumoral cells.

Calretinin (CR) is an EF-hand calcium binding protein expressed at high level in neurons. To identify regulatory elements in CR gene promoter, cultured rat cortical cells were transfected with constructs containing its 5'-end deletion mutants and the luciferase reporter gene. A fragment ending at -115 bp upstream of the transcription start site had high promoter activity and was able to induce expression of luciferase specifically in neuronal cells of cortical cultures. The wild type sequence of -115/+54 CR promoter fragment preferentially drove the expression of green fluorescent protein analog in cells of neuronal phenotype differentiated from multipotent human cell line DEV. Electrophoretic mobility shift assays (EMSA) revealed that the -115/-71 CR gene promoter region contains a binding site for a factor present in brain nuclear extract. Among oligonucleotides containing consensus binding sites for transcription factors within this region, the one representing AP2 binding site was able to compete formation of a protein complex. Mutations of this site prevented the binding between brain protein(s) and the -115/+54 CR gene promoter region and abolished the preferential expression of reporter gene in neuronal cells of DEV line. Thus, the AP2-like element seems to be essential for the neuron-specific activity of the CR gene promoter.

In absence epilepsy, epileptogenic processes are suspected of involving an imbalance between GABAergic inhibition and glutamatergic excitation. Here, we describe alteration of the expression of glutamate transporters in rats with genetic absence (the Genetic Absence Epilepsy Rats from Strasbourg: GAERS). In these rats, epileptic discharges, recorded in the thalamo-cortical network, appear around 40 days after birth. In adult rats no alteration of the protein expression of the glutamate transporters was observed. In 30-day-old GAERS protein levels (quantified by western blot) were lower in the cortex by 21% and 35% for the glial transporters GLT1 and GLAST, respectively, and by 32% for the neuronal transporter EAAC1 in the thalamus compared to control rats. In addition, the expression and activity of GLAST were decreased by 50% in newborn GAERS cortical astrocytes grown in primary culture. The lack of modification of the protein levels of glutamatergic transporters in adult epileptic GAERS, in spite of mRNA variations (quantified by RT-PCR), suggests that they are not involved in the pathogeny of spike-and-wave discharges. In contrast, the alteration of glutamate transporter expression, observed before the establishment of epileptic discharges, could reflect an abnormal maturation of the glutamatergic neurone-glia circuitry.

The human T-lymphotropic virus type-1 (HTLV-1) is the causative agent of a chronic progressive myelopathy (TSP/HAM) in which lesions of the central nervous system (CNS) are associated with infiltration of HTLV-1-infected T-cells. In a model that mimics the interaction between glial and T-cells, we show that transient contact with T-lymphocytes chronically infected with HTLV-1 induce profound metabolic alterations in astrocytes. Within the first week post-contact, an overall activation of astrocyte metabolism was observed as assessed by enhanced uptake of glutamate and glucose, and lactate release. In contrast, longer examination showed a reduced astrocytic accumulation of glutamate. The time course of the change in glutamate uptake was in fact biphasic. Previous observations indicated that HTLV-1 protein Tax-1 was involved in this delayed decrease, via the induction of TNF-alpha. The expression of the glial glutamate transporters, GLAST and GLT-1 decreased in parallel. These decreases in glutamate uptake and transporters' expression were associated with an imbalance in the expression of the catabolic enzymes of glutamate, GS and GDH, presumably due to Tax-1. Given the fact that impairment of glutamate management in astrocytes is able to compromise the functional integrity of neurons and oligodendrocytes, our results altogether give new insights into the physiopathology of TSP/HAM.

The Unc-33-like phosphoprotein/collapsin response mediator protein (Ulip/CRMP) family consists of four homologous phosphoproteins considered crucial for brain development. Autoantibodies produced against member(s) of this family by patients with paraneoplastic neurological diseases have made it possible to clone a fifth human Ulip/CRMP and characterize its cellular and anatomical distribution in developing brain. This protein, referred to as Ulip6/CRMP5, is highly expressed during rat brain development in postmitotic neural precursors and in the fasciculi of fibers, suggesting its involvement in neuronal migration/differentiation and axonal growth. In the adult, Ulip6/CRMP5 is still expressed in some neurons, namely in areas that retain neurogenesis and in oligodendrocytes in the midbrain, hindbrain, and spinal cord. Ulip2/CRMP2 and Ulip6/CRMP5 are coexpressed in postmitotic neural precursors at certain times during development and in oligodendrocytes in the adult. Because Ulip2/CRMP2 has been reported to mediate semaphorin-3A (Sema3A) signal in developing neurons, in studies to understand the function of Ulip6/CRMP5 and Ulip2/CRMP2 in the adult, purified adult rat brain oligodendrocytes were cultured in a Sema3A-conditioned medium. Oligodendrocytes were found to have Sema3A binding sites and to express neuropilin-1, the major Sema3A receptor component. In the presence of Sema3A, these oligodendrocytes displayed a dramatic reduction in process extension, which was reversed by removal of Sema3A and prevented by anti-neuropilin-1, anti-Ulip6/CRMP5, anti-Ulip2/CRMP2 antibodies, or VEGF-165, another neuropilin-1 ligand. These results indicate the existence in the adult brain of a Sema3A signaling pathway that modulates oligodendrocyte process extension mediated by neuropilin-1, Ulip6/CRMP5, and Ulip2/CRMP2, and they open new fields of investigation of neuron/oligodendrocyte interactions in the normal and pathological brain.

Viral infection of the central nervous system (CNS) can result in perturbation of cell-to-cell communication involving the extracellular matrix (ECM). ECM integrity is maintained by a dynamic balance between the synthesis and proteolysis of its components, mainly as a result of the action of matrix metalloproteinases (MMPs) and the tissue inhibitors of metalloproteinases (TIMPs). An MMP/TIMP imbalance may be critical in triggering neurological disorders, in particular in virally induced neural disorders. In the present study, a mouse model of brain infection using a neurotropic strain of canine distemper virus (CDV) was used to study the effect of CNS infection on the MMP/TIMP balance and cytokine expression. CDV replicates almost exclusively in neurons and has a unique pattern of expression (cortex, hypothalamus, monoaminergic nuclei, hippocampus, and spinal cord). Here we show that although several mouse brain structures were infected, they exhibited a differential pattern in terms of MMP, TIMP, and cytokine expression, exemplified by (i) a large increase in pro-MMP9 levels, in particular in the hippocampus, which occurred mainly in neurons and was associated with in situ gelatinolytic activity, (ii) specific and significant upregulation of MT1-MMP mRNA expression in the cortex and hypothalamus, (iii) an MMP/TIMP imbalance, suggested by the upregulation of TIMP-1 mRNA in the cortex, hippocampus, and hypothalamus and of TIMP-3 mRNA in the cortex, and (iv) a concomitant region-specific large increase in expression of Th1-like cytokines, such as gamma interferon, tumor necrosis factor alpha, and interleukin 6 (IL-6), contrasting with weaker induction of Th2-like cytokines, such as IL-4 and IL-10. These data indicate that an MMP/TIMP imbalance in specific brain structures, which is tightly associated with a local inflammatory process as shown by the presence of immune infiltrating cells, differentially impairs CNS integrity and may contribute to the multiplicity of late neurological disorders observed in this viral mouse model.

Obesity is a complex disease involving genetic components and environmental factors and probably associated with the dysregulation of central homeostasis normally maintained by the hypothalamic neuroendocrine/neurotransmitter network. We previously reported that canine distemper virus (CDV), which is closely related to human measles virus, can target hypothalamic nuclei, and lead to obesity syndrome in the late stages of infection. Here, using differential display PCR, we demonstrate specific down-regulation of melanin-concentrating hormone precursor mRNA (ppMCH) in infected-obese mice. Although ppMCH was down-regulated in all infected mice during the acute stage of infection, this was only seen during the late stage of infection in infected-obese mice. In addition, ppMCH mRNA and protein expression in the lateral hypothalamus was decreased in the absence of neuronal death. These results show the importance of ppMCH in the establishment and maintenance of obesity and the involvement of a virus as an environmental factor.

The aim of our study was to investigate whether a human neural cell line could be used as a reliable screening tool to examine the functional conservation, in humans, of transcription factors involved in neuronal or glial specification in other species. Gain-of-function experiments were performed on DEV cells, a cell line derived from a human medulloblastoma. Genes encoding nine different transcription factors were tested for their influence on the process of specification of human DEV cells towards a neuronal or glial fate. In a first series of experiments, DEV cells were transfected with murine genes encoding transcription factors known to be involved in the neuronal differentiation cascade. Neurogenins-1, -2, and -3; Mash-1; and NeuroD increased the differentiation of DEV cells towards a neuronal phenotype by a factor of 2-3.5. In a second series of experiments, we tested transcription factors involved in invertebrate glial specification. In the embryonic Drosophila CNS, the development of most glial cells depends on the master regulatory gene glial cell missing (gcm). Expression of gcm in DEV cells induced a twofold increase of astrocytic and a sixfold increase of oligodendroglial cell types. Interestingly, expression of tramtrack69, which is required in all Drosophila glial cells, resulted in a fourfold increase of only the oligodendrocyte phenotype. Expression of the related tramtrack88 protein, which is not expressed in the fly glia, or the C. elegans lin26 protein showed no effect. These results show that the Drosophila transcription factor genes tested can conserve their function upon transfection into the human DEV cells, qualifying this cell line as a screening tool to analyze the mechanisms of neuronal and glial specification., (Copyright 2001 Wiley-Liss, Inc.)

The dynamic and coordinated interaction between cells and their microenvironment controls cell migration, proliferation, and apoptosis, mediated by different cell surface molecules. We have studied the response of a neuroectodermal progenitor cell line, Dev, to a guidance molecule, semaphorin 3A (Sema3A), described previously as a repellent-collapsing signal for axons, and we have shown that Sema3A acts as a repellent guidance cue for migrating progenitor cells and, on prolonged application, induces apoptosis. Both repulsion and induction of cell death are mediated by neuropilin-1, the ligand-binding component of the Sema3A receptor. The vascular endothelial growth factor, VEGF165, antagonizes Sema3A-induced apoptosis and promotes cell survival, migration, and proliferation. Surprisingly, repulsion by Sema3A also depends on expression of VEGFR1, a VEGF165 receptor, expressed in Dev cells. Moreover, we found that these repulsive effects of Sema3A require tyrosine kinase activity, which can be attributed to VEGFR1. These results indicate that the balance between guidance molecules and angiogenic factors can modulate the migration, apoptosis (or survival), and proliferation of neural progenitor cells through shared receptors.

SCO-ependymocytes have a secretory activity and a neural innervation relating them to neurosecretory nerve cells. To elucidate the cell lineage of the SCO-ependymocytes and emphasize the role of the neural innervation in their differentiation, in particular 5-HT innervation, we analyzed the developmental pattern of expression of several glial and neuronal markers: (1) in the SCO of mammals possessing (rat, cat) or devoid (mouse, rabbit) of 5-HT innervation, (2) in rat 5-HT deafferented SCO, and (3) in rat SCO transplanted in a foreign environment, the fourth ventricle. The ability of SCO-ependymocytes to transiently express GFAP during development and express the glial alpha alpha-enolase confirms the glial lineage of the SCO-ependymocytes. Synthesis of vimentin by SCO-ependymocytes relates them to the classical ependymocytes. The ability of mature SCO-ependymocytes to take up GABA only when they are innervated by 5-HT terminal underlines the role of the neural environment on the differentiation of these ependymocytes and suggests that differential maturation of the SCO according to its innervation, may lead to specific functional specialization of this organ in different species., (Copyright 2001 Wiley-Liss, Inc.)

Object: Somatostatin receptors have been found on a variety of tumours like neuroendocrine breast or brain tumours. Their detection opens new diagnostic and therapeutic paths. The aim of this work was to investigate their expression in medulloblastomas., Methods: Using both techniques, reverse transcriptase-polymerase chain reaction and immunohistochemistry, we analysed mRNA of different subtypes of somatostatin receptors in 15 medulloblastomas and the localisation of the subtype SSTR2 receptor at the cellular level in 13 medulloblastomas. All five subtypes mRNA were variably expressed in each medulloblastoma. The signal obtained after Southern blotting for SSTR2 receptor amplification was the highest as compared to the signal obtained for the other receptor subtypes. Immunostaining for SSTR2A receptor was present in every tumour specimen and was specifically located to the cellular membrane of neoplastic cells. No staining was identified at the level of peritumoral veins., Conclusion: The evidence of predominant expression of SSTR2 receptors in medulloblastomas opens interesting prospects for their diagnosis and therapy.