Régulation du métabolisme du glycogène cérébral sous l'effet de la méthionine sulfoximine

La raison pour laquelle les neurones synchronisent leurs activités pour générer l épilepsie est toujours inconnue. Au cours de l épilepsie expérimentale et humaine, le métabolisme énergétique cérébral subit de profondes altérations. Nous recherchons un lien éventuel entre ces altérations et l épileptogenèse. Pour cela, nous avons utilisé le modèle d épilepsie induit par la méthionine sulfoximine. Sous l effet de ce convulsivant, le taux de glycogène augmente dans l encéphale d une série de lignées de souris, mais, la chronologie et l amplitude de cette augmentation varient d une ligne e à l autre. Un mécanisme pouvant expliquer cette augmentation pourrait être la baisse du taux de sérotonine observée dans l encéphale. En stimulant les récepteurs 5-HT1A par un dérivé pyridinergique présentant une forte affinité pour ces récepteurs, le taux de sérotonine augmente. Ce dérivé pourrait alors antagoniser les effets de la méthionine sulfoximine. Étant donné que la sérotonine peut cibler un certain nombre de protéines régulant le taux de glycogène, les transcrits de ces protéines ont été étudiés. Le taux de transcrits de la principale protéine régulatrice, la Protein Targeting to Glycogen n augmente pas. Il en est de même des transcrits de la glycogénine, de la glycogène synthase et de la phosphorylase. Il ressort de l ensemble de nos résultats que la méthionine sulfoximine augmente le taux de glycogène en agissant directement sur les astrocytes et en modulant le taux de sérotonine. Il reste à montrer si ce sont les altérations du métabolisme du glycogène et/ou les altérations de la neurotransmission sérotoninergique qui jouent un rôle prépondérant dans l épileptogenèse.ORLEANS-BU Sciences (452342104) / SudocSudocFranceF

Early, middle, and late stages of neural cells from ovine embryo in primary cultures

The utilization of neural cells in culture has importantly increased the knowledge of the nervous system biology. In most studies, the investigations are performed on biological materials coming from common laboratory animals and the extrapolation of the results to other animals is not easy. For some studies, such as developmental biology of the nervous system, prion disease investigations, or agronomical production, the utilization of ovine neural cell cultures presents many advantages. Unfortunately, there are few data on the conditions of culture of such cells. In the present work, we investigated simple ways to obtain neurons and astrocytes from sheep brain. Viable neuronal cell cultures were obtained from 40 to 50 day old fetuses. Their morphologies were quite similar to that of neurons from rodent or chick brain and they were labeled by antineurofilament antibodies. Stages older than 50 days of pregnancy were unable to give viable culture of neurons. The stages of 40 day old fetus to newborn lamb were able to give viable astrocyte cultures. The common protoplasmic astrocytes were obtained and they were labeled by antiglial fibrillary acidic protein antibodies. The astrocytes contained glycogen, thus looking like the common astrocytes from rodents. Neuronal or astroglial cultures can be derived from 26 day old embryos, but the cultures contained contaminating cells. Among the latter cells, there were undifferentiated cells which were flat and epitheloid and which were grouped as islets. These cells could be maintained in culture for a time duration over 7 months, even after two passages. They differentiated principally in astrocytes with a radial configuration. This work shows how some neural cells can be simply and easily cultured from sheep brain. For the first time, neurons were cultured from the sheep embryonic brain. Moreover, stem cells were cultured for more than 7 months and, finally, glycogen accumulation in sheep astrocytes was shown to be the same as that in rodent astrocytes. The oligodendrocyte culture was already documented. Thus, sheep can easily be used as well as other models for neural cell studies

Biocompatibilité et trafic intracellulaire de nanoparticules de silice mésoporeuses

De part leurs propriétés physiques et chimiques, les nanoparticules de silice mésoporeuses (MSNs) sont de bonnes candidates pour la délivrance de principes actifs. Cependant, leurs toxicités et leurs devenirs intracellulaires sont largement méconnus. Au cours de ces travaux, nous avons étudié la cytotoxicité et l endocytose de MSNs. Nous avons montré que les MSNs peuvent être endocytées par une variété de lignées cellulaires et par des astrocytes de rat en culture sans signe apparent de cytotoxicité importante. Ces nanoparticules ne présentent pas une toxicité observable in vivo chez des souris. Après avoir montré que l endocytose des MSNs s effectue par la voie des puits de clathrines, nous avons procédé à la délivrance intracellulaire d une protéine. Nous avons montré un échappement des lysosomes de cette protéine grâce aux MSNs. En couplant l acide folique aux MSNs, les cellules tumorales ont été ciblées. Lors de ces études, nous avons également montré que l un des tests les plus utilisés en toxicologie surestime la cytotoxicité des MSNs. Cette surestimation est due à une modification du trafic intracellulaire. Nos travaux ont montré que les MSNs sont endocytés sans nuire à la viabilité cellulaire, ce qui nous a permis de réaliser les premiers essais de délivrances de principes actifs avec nos nanoparticules.Due to their physical and chemical properties, the mesoporous silica nanoparticles (MSNs) are good candidates for drug delivery applications. However, their toxicity and their intracellular trafficking remain unclear. During these works, we studied the cytotoxicity and the endocytosis of MSNs. We showed that the MSNs can be internalised by a variety of cell lines and rat astrocytes in culture without visible sign of important cytotoxicity. These nanoparticles did not present an observable in vivo toxicity in mice. Then we showed that the endocytosis of the MSNs was made by the clathrines coated pits and we proceeded to the intracellular delivery of a protein. We showed an escape of the lysosomes of this protein due to MSNs. Such an internalised protein escaped from lysosomes under the effect of MSNs. After linking folic acid to MSNs, we are able to target tumoral cells with these nanoparticles. During the preceding studies we observed that one of the most used tests in toxicology overestimated the cytotoxicity of MSNs because the latter nanoparticles modified intracellular traffic. Our works showed that the MSNs are internalized without damaging the cellular viability and we made the first experiments of drug delivery using our nanoparticles.ORLEANS-SCD-Bib. electronique (452349901) / SudocSudocFranceF

Mesoporous silica nanoparticles enhance MTT formazan exocytosis in HeLa cells and astrocytes

International audienceWe report on the observation that mesoporous silica nanoparticles (MSNs), after being endocytosed, interfere with the MTT test in HeLa cells and astrocytes by accelerating the exocytosis of formazan crystals. The stimulation of MTT formazan exocytosis is probably related to perturbation of intracellular vesicle trafficking by MSN uptake as revealed by experiments in presence of chloroquine and genistein. Similar effect has been previously observed with a number of chemicals, especially with neurotoxic beta amyloid peptides, but not with nanoparticles. We showed also that MTT reduction test gives an overestimation of the cytotoxicity of mesoporous silica nanoparticles compared to other tests such as LDH activity, WST-1 test and flow cytometry. These findings show that MTT assay should not be used for the study of MSN toxicity, and that perturbation of intracellular trafficking has to be taken into account in evaluating biocompatibility of MSNs

A fraction of neurofibromin interacts with PML bodies in the nucleus of the CCF astrocytoma cell line.

International audienceNeurofibromatosis type 1 is a common genetic disease that causes nervous system tumors, and cognitive deficits. It is due to mutations within the NF1 gene, which encodes the Nf1 protein. Nf1 has been shown to be involved in the regulation of Ras, cAMP and actin cytoskeleton dynamics. In this study, using immunofluorescence experiments, we have shown a partial nuclear localization of Nf1 in the astrocytoma cell line: CCF and we have demonstrated that Nf1 partially colocalizes with PML (promyelocytic leukemia) nuclear bodies. A direct interaction between Nf1 and the multiprotein complex has further been demonstrated using "in situ" proximity ligation assay (PLA)

Uptake of Functionalized Mesoporous Silica Nanoparticles by Human Cancer Cells

International audienceMesoporous silica nanoparticles (MSN) were functionalised by aminofluorescein (AMF) with diethylenetriaminepentaacetic acid spacer molecules which provide free carboxylic groups for binding cell-specific ligands such as folate. AMF allowed the exploration of cellular uptake by HeLa cells using confocal microscopy and flow cytometry. The functionalized nanoparticles (MSN-AMF) penetrated efficiently into HeLa cell cytoplasm through a clathrin dependent endocytosis mechanism. The number of endocytosed MSN-AMF was enhanced when using folate as a targeting molecule. Uptake kinetics revealed that most of MSN-AMF were internalized within 4 h of incubation. Moreover, we found that MSN-AMF were capable of escaping the acidic endolysosomal vesicles of HeLa cells. Cytotoxicity studies suggested that these nanoparticles are non-toxic to HeLa cells up to a dose level of 50 mu g/ml