<p>Transition metals, such as iron, manganese, and copper are essential in the development and function of biological systems. However, disrupted levels of transition metals are highly cytotoxic, and metal homeostasis is strictly maintained in cells under normal conditions. The neuropathology of several brain disorders, such as Alzheimer’s disease and Parkinson’s disease has been linked to altered metal levels. This work focused on the measurement of iron, manganese, and copper, with the aim of better elucidating their role in brain disease.</p> <p>Two experiments were carried out in C57Bl/6 mice looking at metal homeostasis: <em>1.</em> following manganese injections typically administered in manganese-enhanced MRI animal studies, and <em>2.</em> following copper deficiency in a cuprizone model of demyelination. Metal measurements were made in the brain and visceral organs using X-ray fluorescence to measure iron and copper concentrations, and neutron activation analysis to measure manganese concentrations.</p> <p>In the MEMRI study in this work, in addition to the expected manganese concentration increases in brain regions, a statistically significant decrease in iron concentration in the thalamus was found. This change in iron levels in the thalamus following manganese injections should serve as a caution that care should be taken when interpreting signal changes in brain regions.</p> <p>The cuprizone study in this thesis confirmed that copper levels are reduced following cuprizone administration. Surprisingly, manganese concentrations were significantly higher in several brain regions that have demyelination in this model, but not iron or copper. The mechanism of cuprizone toxicity was related to manganese neurotoxicity that may contribute to demyelination.</p> / Master of Science (MSc)

CnrX est un senseur périplasmique, ancré à la membrane, appartenant au complexe CnrYXH qui contribue à réguler l'expression des gènes impliqués dans la résistance au nickel et au cobalt chez Cupriavidus metallidurans CH34. La résistance est induite par la libération de CnrH, un facteur sigma de type ECF (Extracytoplasmic Function), par le complexe CnrYX en réponse à Ni et Co. Nous avons cherché à comprendre la manière dont CnrXs, le domaine senseur de CnrX, détecte les ions métalliques, les stratégies utilisées pour sélectionner spécifiquement Ni ou Co ainsi que la nature du signal engendré par cette interaction. Les techniques spectroscopiques et biophysiques telles que l'UV-visible, la RPE, le XAS et l'ITC ont permis d'étudier les sites métalliques en solution. Le dimère de CnrXs possède quatre sites de liaison au cobalt. Deux des sites (sites F) sont retrouvés dans la protéine entière dont nous avons maintenant un excellent modèle avec le mutant CnrXs-H32A. Les deux autres sites (sites E) ont un signal spectroscopique atypique probablement dû à la formation d'un complexe binucléaire de cobalt. Nous présentons également des structures à haute résolution de CnrXs dans ses formes apo et métallées par le nickel, cobalt ou zinc. Nous avons établi que la forme zinc est la forme inactive de la protéine et que le mécanisme de détection est engendrée par la substitution du zinc par le nickel et le cobalt dans le site F, conduisant à une modification majeure du site de liaison au métal. Tandis que le zinc est pentacoordiné dans une sphère 3N2O, Ni et Co recrutent le soufre de la seule méthionine (Met123) comme sixième ligand pour former un site octaédrique. Nous suggérons que Met123 soit l'interrupteur moléculaire dont la liaison avec le métal fait évoluer la structure de la protéine vers une conformation active. A notre connaissance, ces résultats constituent la première étude structurale et spectroscopique d'un senseur de métal périplasmique impliqué dans un système de transduction du signal dépendant d'un facteur sigma de type ECF. / CnrX is the membrane-anchored periplasmic sensor of the CnrYXH complex that contributes to regulate the expression of the genes involved in cobalt and nickel resistance in Cupriavidus metallidurans CH34. This resistance is induced by the release of the ExtraCytoplasmic Function (ECF) sigma factor CnrH from the CnrYX complex upon sensing of Ni or Co. We addressed the metal sensing mechanisms of CnrXs, the strategies used to select Ni or Co and the nature of the signal onset. Biophysical and spectroscopic techniques allowed us to study the metal binding sites in solution. The CnrXs dimer contains four cobalt binding sites. Two (F sites) are present in the full-length protein which H32A-CnrXs mutant is an excellent model of. The two other sites have an unusual spectroscopic signal that might be due to the formation of a binuclear cobalt complex. We present also high-resolution structures of CnrXs in the apo, Ni-, Co-, and Zn-bound forms. We propose that Zn-bound CnrX typifies the resting state of the complex and that the sensing mechanism is triggered by the substitution of Zn for Ni or Co in the F site. This substitution leads to dramatic changes in the metal-binding site. While the Zn ion is pentacoordinated in a 3N2O sphere, Ni or Co ions recruit the thioether sulfur of the only methionine (Met123) residue as a sixth ligand to form an octahedral site. We propose that the Met123 side chain recruitment is the qualitative change that switches on the sensing mechanism by remodeling the four-helix bundle that accommodates the metal-binding site. To our knowledge these results represent the first structural and spectroscopic study of a periplasmic metal sensor involved in transmembrane signal transduction for the activation of an ECF-type sigma factor.