Influence of microglia-related neuroinflammation on the modulation of glutamatergic transmission by astrocytes

Glial cells have been shadowed by neurons for a century but are now regarded as forming an intricate network participating in the regulation of neurotransmission and to maintain an equilibrated neuronal environment. Microglia ensure the first line of brain defence by assuming a permanent surveillance throughout the central nervous system. Indeed, they respond to changes in the microenvironment by a phenotypic switch supporting migration, phagocytosis and elimination of disturbing elements through the release of inflammatory mediators. The excessive release of these mediators leads to neuroinflammation, which is a typical hallmark of several neurological disorders such as chronic neurodegenerative diseases and acute brain injuries. Activated microglia is considered as a detrimental element in these pathologies but exact mechanisms are not really understood. Besides, astrocytes play various functions which go from a structural support to the control of brain functionality, and are considered as crucial elements of the glutamatergic neurotransmission being nearly as much essential as neurons. Glutamate, the major excitatory neurotransmitter, must be cleared out of the synaptic cleft to avoid hyperactivation of post-synaptic receptors and the occurrence of excitotoxicity, which leads to neuronal death. This elementary function for ensuring the maintenance of the physiological concentration of glutamate in the synaptic cleft is essentially performed by the two glial glutamate transporters (GLAST and GLT-1). However, these glutamate transporters are dynamically regulated according to the surrounding environment, their location in the brain and the pathological state. Whereas modulation of glutamatergic receptors has been a major research topic for neuroscientists during decades, the regulation of glutamate transporters is now deeply investigated as disturbed systems of glutamate clearance has been frequently observed in several pathological circumstances. Since inflammatory mediators secreted by microglia exert various effects on neighbour astroglial cells, such molecules are potential regulators of glutamate handling by astrocytes. While the scientific community is arguing the putative neurotoxicity of microglia and its involvement in various brain pathologies, this thesis shed new light on the crosstalk between microglia and astrocytes. Its main focus was on the influence of the activated-microglia on the modulation of the glutamatergic transmission by astrocytes, which was examined by exposing rat primary cultured astrocytes to the conditioned medium from the LPS-activated microglia. The first observations were focused on the modifications of the astrocyte phenotype with a typical morphology of reactive astrocytes, which highlighted the ability of activated microglia to trigger a pathological reactive state in astrocytes. This astrogliosis process was associated with a down-regulation of the metabotropic glutamate receptor 5 (mGluR5), suggesting an alteration of the glutamatergic transmission in astrocytes. The main issue was to assess the influence of the LPS-activated microglia conditioned medium on glial glutamate transporters expression and activity. Our results indicated a drastic decrease in the glutamate uptake activity in astrocytes, resulting of a down-regulation of the expression of the GLAST, the predominant astrocytic glutamate transporter, while GLT-1 was up-modulated. This duality of regulation induced by the activated microglia was mimicked by the tumor necrosis factor alpha, suggesting a major role for this cytokine in such regulation. The putatively detrimental regulation of GLAST in response to inflammation was also studied in cells exposed to dibutyryl cAMP, recognized as a model of astrocytes exhibiting a typical mature/differentiated phenotype. In this model, the conditioned culture medium from activated microglia, as well as tumor necrosis factor alpha, were found to increase glutamate uptake capacity, resulting in increased activity of GLAST and GLT-1. Consistently, both of these treatments caused only modest induction of an inflammatory response in matured astrocytes compared to undifferentiated astrocytes, emphasizing on the ability of these differentiated astrocytes to maintain a finely tuned balance between pro- and anti-inflammatory mediators. In conclusion, these data indicate that astrocytes are very sensitive to the microglia-generated environment as demonstrated by the astrogliotic phenotype and the dual regulation of glutamate transporters, and may thus be involved in the neurotoxicity associated with neuroinflammation. Besides, our data give rise to the original concept that priming of astrocytes with dibutyryl cAMP to promote neuroprotection may be an attractive approach to confront microglia-mediated neuroinflammation without evolving toward a neurotoxic astrocyte phenotype.Thèse de doctorat en sciences pharmaceutiques (neuropharmacologie) (FARM 3) -- UCL, 200