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This study investigated the role of NMDARs in the differentiation of MEG-01 cells and in the activation of human platelets. This investigation demonstrated that the NR1, NR2D and NR3 subunit proteins are expressed in human platelets, with the NR1 subunit also expressed in MEG-01 cells. The NR2A subunit protein was not detectable in either MEG-01 cells or human platelets. PMA-induced differentiation of MEG-01 cells did not appear to stimulate changes in expression of any of the subunit proteins tested. Using assays to measure the changes in [Ca2+]i and ATP secretion, it was determined that donors could be separated into those who responded to the agonists applied and those who did not; responses also decreased over time in both assays. Human platelets from responding donors demonstrate an increase in [Ca2+]i in response to extracellular glutamate, and that increases in ATP secretion are detected at a 10-fold lower concentration. The same is also true with extracellular glycine. Increases in [Ca2+]i were elicited on the addition of extracellular NMDA; extracellular D-serine had no effect. NMDAR inhibitors, MK-801 and D-AP5, inhibited ATP secretion evoked by either glutamate alone or in combination with glycine. D-serine inhibited responses elicited by extracellular glycine. NMDARs play a role in MK differentiation, with the adhesion of MEG-01 cells cultured on a fibrinogen-surface and differentiated with PMA reduced by both inhibitors. PMA-treated MEG-01 cells increased both in size and irregularity, with the addition of NMDAR-specific inhibitors having no effect. S-nitrosylation also inhibits activation of NMDAR, and a new molecule has been developed which can detect S-nitrosylated proteins through a single step process in live cells. Overall, this study has shown that both human platelets and MEG-01 cells express NMDAR subunits, which have been demonstrated to form functional receptors in human platelets.

Tendon injury is painful and often debilitating, and is a one of the most prevalent soft tissue injuries encountered in the clinic. While common, the underlying molecular and genetic processes of tendon damage and repair remain poorly understood. The work described herein used genome-wide expression analyses to investigate tendon injury and healing from three perspectives. The first identified novel gene expression in tendon fibroblasts following their stimulation with nitric oxide (NO). Of particular relevance to tendon healing was the observation that stimulated fibroblasts express a number of extracellular matrix (ECM) genes in response to NO in a dose-dependent manner, and that NO significantly affects cellular adhesion, a critical process during tendon repair. These findings will be of use when optimising dosages of NO delivery in future work investigating NO as potential treatment for tendon injuries. The second study examined gene expression in an acute tendon injury model in the rat at 1, 7, and 21 days post injury, roughly representing the inflammation, proliferation, and remodelling phase of wound repair. Several novel genes and pathways were found to be differentially expressed at each stage of healing. Of particular interest were the presence of a significant number of genes related to glutamate signaling, a method of cellular communication that has not previously been shown to exist in tendon. Also upregulated were a number of genes which have previously only been associated with embryonic development. Finally, gene expression in a supraspinatus tendinopathy model in the rat was investigated. Several genetic pathways were identified in tendinopathic tendons which have not previously been associated with the disease, and, analogous to the acute injury model study, glutamate signaling gene overexpression was also prevalent. Further in vitro studies showed that the expression of these genes in tendon fibroblasts were stimulated by glutamate treatment, which in turn upregulated pro-apoptotic pathways causing cell death. This may prove to be an important causative factor in the tendon degeneration seen in tendinopathy, in which apoptosis has been identified as playing a significant role. The results of these studies contribute to a better understanding of the aetiology of several extremely common pathologies of this soft tissue, and may help to develop more targeted therapies for increasing the efficacy of tendon healing in future.

In rodents, stress and corticosteroids rapidly increase excitatory neurotransmission. During excitatory neurotransmission, glutamate concentrations are maintained by conversion of glutamine to glutamate. The hypothesis was that cortisol would alter human prefrontal glutamine or glutamate concentrations. Glutamine and glutamate were measured in prefrontal cortex (n = 12) using 3.0 Tesla proton magnetic resonance spectroscopy (1H-MRS) before and after intravenous cortisol (hydrocortisone 35mg), in a randomized, double blind, placebo-controlled, within-subjects design. Glutamine decreased following cortisol compared with placebo (session by time, F(2,22) = 5.51; p = 0.012), whereas glutamate did not change (F(4,44) = 0.71; p = 0.59). Glutamine may be utilized to maintain glutamate concentrations during increased excitatory neurotransmission following cortisol. A limitation is that 1H-MRS does not measure metabolic flux rates directly. The effects of cortisol on glutamine could be a useful measure of altered central glucocorticoid responses in psychiatric disorders.

Traumatic brain injury (TBI) is a devastating public health problem for patients and their families. The neurodegeneration that follows TBI is complex, but can be broadly subdivided into primary and secondary damage. Though primary damage is irreversible and therefore unsalvageable, extensive literature aimed at understanding the tissue, cellular, inflammatory and subcellular processes following the injury have proven unequivocally that secondary pathophysiological events are delayed and progressive in nature. Understanding these secondary events at the cellular levels is critical in the eventual establishment of targeted therapeutics aimed at limiting progressive injury after an initial trauma. One such secondary event is referred to in the literature as excitotoxicity; a sustained and de-regulated activation of glutamate receptors that leads to rapid cytotoxic edema and calcium overload. Our understanding of excitotoxicity has evolved to include not only a role for elevated extracellular glutamate in mediating neuronal damage, but also post-synaptic receptor modifications that render glutamate profoundly more toxic to injured neurons than healthy tissue. In this thesis, we explored the hypothesis that glutamate excitotoxicity can be perpetuated by trauma-induced post-synaptic modification of the AMPA receptor. Specifically, we used a cortical culture model of TBI as well as the fluid percussion injury device to test the hypothesis that TBI confers a reduction of surface GluR2 protein, an AMPA receptor subunit that limits neuronal calcium permeability. We conjectured that this decrease in the expression of surface GluR2 would increase the expression of calcium-permeable AMPA receptors, thereby rendering neurons vulnerable to secondary excitotoxic injury. We further investigated the subcellular mechanisms responsible for the internalization of surface GluR2, and the phenotypic consequences of GluR2 endocytosis in both models. Our data revealed that both models of TBI resulted in a regulated signaling cascade leading to the phosphorylation and internalization of GluR2. By exogenously interrupting the trafficking of GluR2 protein with an inhibitory peptide, we further observed that GluR2 internalization was mediated by a protein interaction involving protein interacting with C kinase 1 (PICK1) and protein kinase C alpha (PKCα), two PDZ domain-containing proteins that mediate GluR2 trafficking during constitutive synaptic plasticity. We observed that GluR2 endocytosis was NMDA receptor dependent, and resulted in increased neuronal calcium permeability, augmented AMPA receptor-mediated electrophysiological activity and increased susceptibility to delayed cell death. Finally, we demonstrated that the interruption of GluR2 trafficking is cytoprotective, suggesting that sustaining surface GluR2 protein protects neurons against secondary injury. Overall, our findings suggest that experimental TBI promotes the expression of injurious GluR2-lacking AMPA receptors, thereby enhancing cellular vulnerability to secondary excitotoxicity.

Traumatic brain injury (TBI) is a devastating public health problem for patients and their families. The neurodegeneration that follows TBI is complex, but can be broadly subdivided into primary and secondary damage. Though primary damage is irreversible and therefore unsalvageable, extensive literature aimed at understanding the tissue, cellular, inflammatory and subcellular processes following the injury have proven unequivocally that secondary pathophysiological events are delayed and progressive in nature. Understanding these secondary events at the cellular levels is critical in the eventual establishment of targeted therapeutics aimed at limiting progressive injury after an initial trauma. One such secondary event is referred to in the literature as excitotoxicity; a sustained and de-regulated activation of glutamate receptors that leads to rapid cytotoxic edema and calcium overload. Our understanding of excitotoxicity has evolved to include not only a role for elevated extracellular glutamate in mediating neuronal damage, but also post-synaptic receptor modifications that render glutamate profoundly more toxic to injured neurons than healthy tissue. In this thesis, we explored the hypothesis that glutamate excitotoxicity can be perpetuated by trauma-induced post-synaptic modification of the AMPA receptor. Specifically, we used a cortical culture model of TBI as well as the fluid percussion injury device to test the hypothesis that TBI confers a reduction of surface GluR2 protein, an AMPA receptor subunit that limits neuronal calcium permeability. We conjectured that this decrease in the expression of surface GluR2 would increase the expression of calcium-permeable AMPA receptors, thereby rendering neurons vulnerable to secondary excitotoxic injury. We further investigated the subcellular mechanisms responsible for the internalization of surface GluR2, and the phenotypic consequences of GluR2 endocytosis in both models. Our data revealed that both models of TBI resulted in a regulated signaling cascade leading to the phosphorylation and internalization of GluR2. By exogenously interrupting the trafficking of GluR2 protein with an inhibitory peptide, we further observed that GluR2 internalization was mediated by a protein interaction involving protein interacting with C kinase 1 (PICK1) and protein kinase C alpha (PKCα), two PDZ domain-containing proteins that mediate GluR2 trafficking during constitutive synaptic plasticity. We observed that GluR2 endocytosis was NMDA receptor dependent, and resulted in increased neuronal calcium permeability, augmented AMPA receptor-mediated electrophysiological activity and increased susceptibility to delayed cell death. Finally, we demonstrated that the interruption of GluR2 trafficking is cytoprotective, suggesting that sustaining surface GluR2 protein protects neurons against secondary injury. Overall, our findings suggest that experimental TBI promotes the expression of injurious GluR2-lacking AMPA receptors, thereby enhancing cellular vulnerability to secondary excitotoxicity.

Thèse de doctorat : Neurosciences : Paris 12 : 2006. / Titre provenant de l'écran-titre. Bibliogr. : 200 réf.

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