Equine protozoal myeloencephalitis (EPM), predominantly caused by the protozoa Saracocystis neurona, is a common neurologic disease in horses from North America. Equine exposure to the parasite occurs frequently as the protozoa is excreted in opossum (Didelphis virginiana) feces and contaminates the horse's environment. However, clinical neurologic disease only emerges in a small fraction of exposed horses. The seemingly protective immune response that develops in some exposed horses but not all is not fully defined. Previous reports utilizing horse EPM models and immune compromised mouse models, which develop disease simulating EPM after infection with S. neurona, have reported a role of T-lymphocytes and the cytokine interferon gamma, in disease protection. As part of this dissertation, the role of T-lymphocytes and IFNγ was further elucidated. It was determined that IFNγ production is essential for T-lymphocytes to offer protection against S. neurona induced encephalitis, in immune compromised mice. Another factor hindering prognosis of EPM affected horses is treatment failure. The efficacy of the antiprotozoal decoquinate, was tested and found to be ineffective at preventing S. neurona encephalitis, in immune compromised mice. However, the antiprotozoal, diclazuril, was found to be effective at preventing S. neurona encephalitis in immunocompromised mice but once treatment was terminated, infection persisted, and neurologic disease developed. In-situ methods were employed to extensively evaluate the immunopathology of spinal cord tissue samples collected from EPM affected horses. A novel in-situ hybridization technique was successfully utilized to identify S. neurona in tissue samples collected from horses with EPM. This technique will create new opportunities for investigating the immunopathology of EPM. Overall results from the studies conducted in this dissertation suggest that IFNγ production from T lymphocytes is essential for them to offer protection against S. neurona encephalitis. Additionally, further insight on FDA approved and non-FDA approved treatment options for S. neurona infection was gained through the use of the B6Ifnγ -/- mouse model. Collectively, these studies expanded on the knowledge of an understudied equine neurologic disease. / Doctor of Philosophy / Horses are susceptible to the neurologic disease Equine Protozoal Myeloencephalitis, more commonly referred to as EPM by equine enthusiasts. The disease results from ingestion of the parasite, Saracocystis neurona, which contaminates the horse's natural environment; therefore, horses are likely to come in contact with the parasite while eating or drinking. Not all horses that encounter S. neurona develop neurologic disease, some will be protected by their immune system with the only evidence of exposure being serological antibodies. In efforts to not experimentally induce EPM in horses, an immunocompromised mouse model is often used instead. Through the use of the immunocompromised mouse model, researchers have discovered that the immune cell, T lymphocytes, and signaling molecule, interferon gamma, are important for protection against S. neurona infection. In one study conducted for this dissertation it was found that T lymphocytes need to be able to produce interferon gamma in order to provide protection. Another issue that the immunocompromised mouse model has helped address, is EPM treatment efficacy. The inability of antiprotozoal drugs that are utilized for EPM treatment to fully eliminate the parasite from the horse's body is thought to cause reoccurring disease in some horses. One non-FDA approved treatment was evaluated here and determined not to be effective in the immunocompromised mouse model. One FDA approved treatment option, which is commonly used to treat EPM, was evaluated as well. This drug was proven to be effective at preventing disease while mice were being treated but termination of treatment led to development of neurologic disease, exemplifying treatment failure. One final study was conducted to examine the different types of immune cells and signaling molecules in spinal cord tissue samples collected, from horses which had to be euthanized due to poor prognosis related to EPM. In this study a novel experimental technique was successfully used which will help progress EPM research. Overall results of these studies offered more explanation on the immune response that protects against neurologic disease from S. neurona infection and demonstrated that not all treatments are effective and reoccurring disease may be a result of treatment failure.

L’adénosine est un neuromodulateur ubiquitaire impliqué dans différents processus physiologiques et neuroprotecteurs du système nerveux central. Elle agit via quatre sous-types de récepteurs couplés à protéine G (A1, A2a, A2b et A3). Le récepteur A2a (A2aR) est fortement exprimé dans des régions riches en dopamine, avec de fortes concentrations dans la zone du caudate-putamen du cerveau, où elle a un rôle important dans la transmission neuronale et dans le processus dégénératif d’origine extrapyramidale. Ainsi, le blocage du récepteur A2a s’est révélé être une cible prometteuse pour le traitement des maladies neurodégénératives tels la Maladie de Parkinson (MP) et la Maladie d’Alzheimer (MA). A ce jour, seulement trois composés sont en phase clinique dans le cadre du traitement de la MP. Toutefois, bien que très affins, ils ne possèdent pas des propriétés ADME ou une sélectivité optimales.Au début de ce projet, la sous-structure Tic-hydantoïne a été identifiée comme présentant une bonne affinité pour A2aR. Après étude des fonctions manquantes de cette molécule et en respectant les éléments pharmacophoriques nécessaires pour une bonne activité, nous avons imaginé et évalué dans la structure co-cristallisé du A2aR et du ligand antagoniste de référence ZM241385 différentes modulations autour de l’hétérocycle. Ceci a permis de proposer la famille des Tic-guanidines, qui présente une fonction donneur de liaison hydrogène avec le résidu Asn253 du site et qui a été synthétisé après optimisation d’un chemin synthétique original. De plus, 1700 molécules ont été conçues de novo et évaluées in silico. Parmi les familles potentiellement intéressantes, deux d’entre elles, les quinolizidinones et les amino-imidazopyridines ont été synthétisées et évaluées in vitro afin de déterminer leur affinité vis-à-vis le récepteur A2a ainsi que leur cytotoxicité vis-à-vis les cellules neuronales. / Adenosine is a ubiquitous neuromodulator able to regulate many physiological processes and plays an important neuroprotective role in the central nervous system. Its effects are transmitted by four distinct G protein receptor subtypes designated A1, A2a, A2b, and A3. A2a receptors (A2aR) show a restricted distribution, being characteristic of the dopamine enriched areas, the highest concentration being in the caudate-putamen in brain, where it has an important role in neuronal signaling with this region and potential involvement in neurologic disease of extrapyramidal origin.A2a antagonism was shown to be a promising pharmacological target for neurodegenerative diseases such as Parkinson’s disease (PD) and Alzheimer disease (AD). Currently, only three compounds are still in clinical phase for PD treatment. Even if they show good affinities for the receptor, there is still a need for improving their ADME properties by keeping their selectivity towards other adenosine receptors.At the beginning of this project, a Tic-hydantoin derivative was identified as a new ligand with a good affinity for the A2a receptor. Based on the recently published crystalline structure of the A2A receptor complexed with the selective and high-affinity antagonist ZM241385 and a pharmacophoric model, we identified the missing features needed for a good affinity in our molecule. We designed and evaluated in silico many pharmacomodulations around the heterocyclic ring and Tic-guanidin substructure was proposed to present favorable hydrogen bound with Asn253 of the A2a binding site. This structure was obtained after optimization of a new synthetic pathway. Moreover, 1700 molecules were originally designed and evaluated in silico. Among potential interesting families, two of them, quinolizidinones and amino-imidazopyridines were synthesized and evaluated in vitro toward their affinity for A2a receptor and their cytotoxicity towards neuronal cells.

No description available.