Régulation du transport du glutamate par les récepteurs métabotropiques du glutamate dans des cultures primaires d'astrocytes activés : implication dans le déséquilibre glutamatergique caractéristique de la sclérose latérale amyotrophique

More than to provide a structural and metabolic support to the neurones, astrocytes actively participate in the neurotransmission. Indeed, involved in the control of the synaptic glutamate concentration, the astrocytes take up this neurotransmitter through specific transporters. Thus, they avoid the triggering of excitotoxicity process involved in the development and the progression of neurodegenerative pathologies including the Parkinsons, the Alzheimers diseases or the amyotrophic lateral sclerosis (ALS). Thus, the control of the glutamate uptake by the astrocytes constitutes an aspect of importance in the therapy of progressive neuropathological diseases. In this context, this work was aimed to evaluate the role of the metabotropic glutamate receptors of group I (mGluRI) in the regulation of the glutamate uptake in primary culture of astrocytes. A protocol of in vitro activation of astrocytes was determined to mimick pathological conditions. As documented in native nervous tissue, the cultured astrocytes take up glutamate through specific transporters and express functional mGluRI. The in vitro activation of astrocytes induces an increase in the expression and the functionality of the both partners. In the same model, a positive cross-talk is observed: a brief stimulation of mGluRI activates the glutamate uptake by the astrocytes. The mGluRI could be considered as a sensor of the extracellular glutamate concentration controlling the clearance of glutamate by astrocytes. It would play a role in the neuroprotection avoiding the triggering of glutamate-mediated excitotoxicity process. The last part of this work concerns the study of this regulation in the context of ALS. The in vitro activated astrocytes are obtained from hSOD1G93A transgenic rats and mimick the astrocytes involved in the development of gliose in ALS patients. In this model, the glutamate uptake parameters are not altered. On the other hand, while the expression and functionality of mGluRI are increased, these receptors doe not exert the acute control on the glutamate uptake in the in vitro activated astrocytes derived from transgenic rat. These results demonstrate a potential acute homologous regulation of glutamate transporters by mGluRI in cultured astrocytes reflecting physiological and pathological conditions and could open new tracks for the development of neuroprotective drugs.Non seulement les astrocytes constituent un support structurel et métabolique pour les neurones mais ils participent également à la neurotransmission. En effet, impliqués dans le contrôle de la concentration synaptique du glutamate, les astrocytes captent ce neurotransmetteur excitateur au travers de transporteurs spécifiques. Ils évitent ainsi le déclenchement du phénomène d'excitotoxicité impliqué dans le développement et la progression de maladies neurodégénératives telles la maladie de Parkinson, la maladie d'Alzheimer ou la sclérose latérale amyotrophique (SLA). La notion de contrôle du transport du glutamate par les astrocytes constitue donc un aspect important dans la thérapie de pathologies neurodégénératives progressives. Dans ce contexte, ce travail avait pour objectif d'évaluer le rôle des récepteurs métabotropiques du glutamate du groupe I (mGluRI) dans la régulation du transport du glutamate au sein de cultures primaires d'astrocytes. Un protocole d'activation in vitro de ce type cellulaire fut établi afin d'imiter les astrocytes impliqués in vivo dans la gliose, phénomène généralement observé dans ces mêmes neuropathologies. Comme décrit pour le tissu nerveux in vivo, les astrocytes cultivés captent le glutamate au travers de transporteurs spécifiques et expriment des mGluRI fonctionnels. L'activation in vitro de ces astrocytes induit une augmentation de l'expression et de l'activité de ces deux partenaires. Dans ce même modèle, s'établit une interaction positive où le mGluRI activé brièvement favorise le transport du glutamate par les astrocytes. Le mGluRI serait alors considéré comme un senseur de la concentration du glutamate extracellulaire et jouerait un rôle dans la neuroprotection en évitant le déclenchement de processus excitotoxiques lié au glutamate. La dernière partie de ce travail concerne l'étude de cette régulation dans le contexte précis de la SLA. Les astrocytes activés in vitro sont obtenus au départ de rats transgéniques hSOD1G93A, modèle pour la SLA, mimant les astrocytes impliqués dans le développement de gliose chez des patients atteints de cette pathologie. Dans ce modèle, les caractéristiques de transport du glutamate ne sont pas altérées. Cependant, alors que l'expression et la fonctionnalité de mGluRI sont augmentées, ceux-ci n'exercent pas de contrôle aigu de l'activité des transporteurs du glutamate dans les astrocytes activés in vitro. Ce travail met en exergue la régulation homologue des transporteurs par les récepteurs du glutamate dans les astrocytes en conditions physiologiques ou pathologiques et offre de nouvelles pistes au développement thérapeutique.Thèse de doctorat en sciences biomédicales (neuropharmacologie)(SBIM 3) -- UCL, 200

IS231-MIC231 elements from Bacillus cereus sensu lato are modular.

Summary IS231A was originally discovered in Bacillus thuringiensis as a typical 1.6 kb insertion sequence (IS) displaying 20 bp inverted repeats (IR) flanking a transposase gene. A first major variation of this canonical organization was found in MIC231A1. This mobile insertion cassette (MIC), delineated by IS231A-related extremities, contained an active d-stereospecific endopeptidase (adp) gene instead of a transposase. Interestingly, it was shown that MIC231A1 can be mobilized in trans by the IS231A transposase. In this paper, we show that this family of IS231-MIC231 elements can be extended to a broad range of related entities displaying higher levels of structural complexity. Several IS231A-like elements contained, upstream of their transposase gene, passenger genes coding for putative antibiotic resistances or regulatory factors. Furthermore, the diversity of the MIC231 elements ranged from empty cassettes to structures carrying up to three passenger genes. Among these, MIC231V carried, in addition to an adp gene, an active fosfomycin resistance determinant. In vivo transposition assays showed that MIC231V is also trans-activated by the IS231A transposase. These results lend further support to the potential contribution of these modular mobile elements to the genome plasticity of the Bacillus cereus/B. thuringiensis group

Molecular and functional characterisation of glutamate transporters in rat cortical astrocytes exposed to a defined combination of growth factors during in vitro differentiation.

In vitro culture of astroglial progenitors can be obtained from early post-natal brain tissues and several methods have been reported for promoting their maturation into differentiated astrocytes. Hence, a combination of several nutriments/growth factors -- the G5 supplement (insulin, transferrin, selenite, biotin, hydrocortisone, fibroblast growth factor and epidermal growth factor) -- is widely used as a culture additive favouring the growth, differentiation and maturation of primary cultured astrocytes. Considering the key role played by glial cells in the clearance of glutamate in the synapses, cultured astrocytes are frequently used as a model for the study of glutamate transporters. Indeed, it has been shown that when tested separately, growth factors influence the expression and activity of the GLAST and GLT-1. The present study aimed at characterising the functional expression of these transporters during the time course of differentiation of cultured cortical astrocytes exposed to the supplement G5. After a few days, the vast majority of cells exposed to this supplement adopted a typical stellate morphology (fibrous or type II astrocytes) and showed intense expression of the glial fibrillary acidic protein. Both RT-PCR and immunoblotting studies revealed that the expression of both GLAST and GLT-1 rapidly increased in these cells. While this was correlated with a significant increase in specific uptake of radiolabelled aspartate, fluorescence monitoring of the Na+ influx associated with glutamate transporters activity revealed that the exposure to the G5 supplement considerably increased the percentage of cells participating in the uptake. Biochemical and pharmacological studies revealed that this activity did not involve GLT-1 but most likely reflected an increase in GLAST-mediated uptake. Together, these data indicate that the addition of this classical combination of growth factors and nutriments drives the rapid differentiation toward a homogenous culture of fibrous astrocytes expressing functional glutamate transporters

Induction of glial glutamate transporters in adult mesenchymal stem cells.

Adult bone marrow mesenchymal stem cells are multipotent cells that can differentiate into a variety of mesodermal tissues. Recent studies have reported on their ability to also evolve into non-mesodermal cells, especially neural cells. While most of these studies revealed that manipulating these cells triggers the expression of typical neurone markers, less is known about the induction of neuronal- or glial-related physiological properties. The present study focused on the characterisation of glutamate transporters expression and activity in rat mesenchymal stem cells grown in culture conditions favouring their differentiation into astroglial cells. Ten days exposure of the cells to the culture supplement G5 was found to increase the expression of nestin (neuro-epithelial stem cell intermediate filament), an intermediate filament protein expressed by neural stem cells. Simultaneously, a robust induction of the high-affinity glutamate transporter GLT-1 (and GLAST) expression was detected by RT-PCR and immunocytochemistry. This expression was correlated with a highly significant increase in the Na+-dependent [3H]D-aspartate uptake. Finally, while glial fibrillary acidic protein immunoreactivity could not be detected, the induced expression of the astrocytic enzyme glutamine synthetase was demonstrated. These results indicate that in vitro differentiation of adult mesenchymal stem cells in neural precursors coincides with the induction of functional glutamate transport systems. Although the astrocytic nature of these cells remains to be confirmed, this observation gives support to the study of mesenchymal stem cells as a promising tool for the treatment of neurological diseases involving glutamate excitoxicity

In vitro differentiated neural stem cells express functional glial glutamate transporters.

The possibility to isolate stem cells from the adult central nervous system and to maintain and propagate these cells in vitro has raised a general interest with regards to their use in cell replacement therapy for degenerative brain diseases. Considering the critical role played by astrocytes in the control of glutamate homeostasis, we have characterised the expression of functional glutamate transporters in neural stem cells exposed to selected culture conditions favouring their differentiation into astrocytes. Commonly, neural stem cells proliferate in suspension as neurospheres in serum-free medium. The addition of serum or a supplement of growth factors (G5) to the culture medium was found to trigger cell adhesion on coated surfaces and to favour their differentiation. Indeed, after 7 days in these conditions, the vast majority of the cells adopted markedly distinct morphologies corresponding to protoplasmic (with serum) or fibrous (with G5 supplement) astrocytes and approximately 35-40% acquired the expression of the glial fibrillary acidic protein (GFAP). Immunocytochemical analysis also revealed that the treatments with serum or with the G5 supplement triggered the expression of the glial glutamate transporters GLT-1 (35 and 21%, respectively) and GLAST (29 and 69%, respectively). This effect was correlated with a robust increase in the Na+ -dependent [3H]-d-aspartate uptake, which was partially inhibited by dihydrokainate, a selective blocker of GLT-1. Together, these results indicate that in vitro differentiation of cultured neural stem cells can give rise to distinct populations of astrocytes expressing functional glutamate transporters

Pour une analyse macro-syntaxique et sémantique de n'importe quoi /

Verhandeling voorgelegd tot het behalen van de graad van Licentiaat in de Taal- en Letterkunde: Romaanse tale

A New Method for the Measurement of International Normalized Ratio in Hemodialysis Patients with Heparin-Locked Tunneled Dialysis Catheters

Background. To measure International Normalized Ratio (INR) in hemodialysis patients with tunneled dialysis catheters (TDCs), blood sampling is frequently obtained via the catheter at the start of the session. INR measurements via finger-prick point of care testing (POCT) and via blood sampling taken from the dialysis circuit are evaluated as alternatives. Methods. In 14 hemodialysis patients with TDCs, treated with vitamin K antagonists (VKA), INR measurements via POCT were compared with plasma INR samples taken via the catheter at the start of dialysis and via the dialysis circuit after 30 and 60 minutes during 3 nonconsecutive dialysis sessions. Results. Blood samples taken at the start of dialysis at the catheter site were frequently contaminated with heparin originating from the locking solution (unfractionated heparin concentration (UFH) >1.0 IU/ml in 13.2%). POCT INR at the start of dialysis was not different from plasma INR after 30 and 60 minutes (Wilcoxon test p=0.113, n = 37, and p=0.631, n = 36, respectively). Moreover, there was no difference between POCT INR at the start of dialysis and POCT INR after 30 and 60 minutes (Wilcoxon test p=0.797 and p = 0.801, respectively; n = 36). Passing and Bablok regression equation was used, y = 0.460 + 0.733x; n = 105. Treatment decisions based on these 2 methods showed a very good overall agreement (kappa = 0.810; 95% CI: 0.732–0.889; n = 105). Conclusions. Measuring plasma INR via the TDC at the start of dialysis should be abandoned. Measuring POCT INR via a finger prick at the start or even after 30 to 60 minutes is an alternative. The most elegant alternative is to take plasma INR samples via the dialysis circuit 30 minutes or later after the start of the dialysis

Acute up-regulation of glutamate uptake mediated by mGluR5a in reactive astrocytes.

Excitatory transmission in the CNS necessitates the existence of dynamic controls of the glutamate uptake achieved by astrocytes, both in physiological conditions and under pathological circumstances characterized by gliosis. In this context, this study was aimed at evaluating the involvement of group I metabotropic glutamate receptors (mGluR) in the regulation of glutamate transport in a model of rat astrocytes undergoing in vitro activation using a cocktail of growth factors (G5 supplement). The vast majority of the cells were found to take up aspartate, mainly through the glutamate/aspartate transporter (GLAST), and at least 60% expressed functional mGluR5a. When exposed for 15 s to the selective group I mGluR agonist (S)-3,5-dihydroxyphenylglycine, reactive astrocytes showed a significant increase in their capacity to take up aspartate. This effect was confirmed at the single-cell level, since activation of mGluRs significantly increased the initial slope of aspartate-dependent Na+ entry associated with the activity of glutamate transporters. This up-regulation was inhibited by an antagonist of mGluR5 and, more importantly, was sensitive to a specific glutamate transporter 1 (GLT-1) blocker. The acute influence of mGluR5 on aspartate uptake was phospholipase C- and protein kinase C-dependent, and was mimicked by phorbol esters. We conclude that mGluR5a contributes to a dynamic control of GLT-1 function in activated astrocytes, acting as a glial sensor of the extracellular glutamate concentration in order to acutely regulate the excitatory transmission