Cachexia or muscle atrophy is a condition that is associated with a variety of diseases such as chronic heart failure and cancer. In North America, Europe and Japan, more than 8 million patients suffer from cachexia, and it is estimated that cachexia is the cause of death in 30% of cancer patients. Unfortunately, there is no available treatment for cachexia. Bexarotene, a retinoid X receptor (RXR) agonist, is a FDA approved drug used to treat cancer and is able to induce myogenic differentiation in embryonic stem cells. In this study, we investigated the mechanism by which bexarotene enhances myogenic differentiation. The Akt signaling pathway is required for myogenesis and thus we examined its involvement in bexarotene-enhanced myogenic differentiation. We showed that bexarotene, through the activation of RXR signaling, regulates Akt2 expression to enhance myoblast differentiation and fusion. Additionally, we showed that Akt2, but neither Akt1 nor Akt3, is required for bexarotene-enhanced differentiation. Furthermore, we showed that the activation of RXR signaling by bexarotene correlates with a specific histone acetylation mark at the Akt2 locus. More importantly, we demonstrated that bexarotene is able to rescue myoblast differentiation in an in vitro cachexia system. Taken together, our data revealed the significance of Akt2 in bexarotene-enhanced myogenic differentiation and the potential of using bexarotene as a treatment for cachexia.

Remyelination is a spontaneous regenerative process whereby myelin sheaths are restored to demyelinated axons. Key players in this process are oligodendrocyte progenitor cells (OPCs), a widespread population of CNS progenitor cells which persist into adulthood. Remyelination is impaired in patients with chronic demyelinating conditions such as Multiple Sclerosis, and as with other regenerative processes, its efficiency declines with increasing age. Hence, there is a need for the development of therapeutic interventions that will aid in promoting endogenous remyelination when the endogenous regenerative potential is compromised. The nuclear receptor RXR$\gamma$ is an important positive regulator of OPC differentiation and an accelerator of endogenous remyelination in aged rats. RXR$\gamma$ functions as a ligand-induced transcription factor and is able to regulate gene transcription. It does so by heterodimerising with other nuclear receptors and recruiting co-regulators involved in chromatin remodelling. However, we lack understanding on the specific mechanism by which RXR$\gamma$ promotes OPC differentiation. With the work presented in this thesis I demonstrate that RXR$\gamma$ function is regulated at multiple signalling levels. Proximity ligation assays revealed that RXR$\gamma$ remains consistently bound to its partners throughout the oligodendrocyte lineage, and the biological relevance of each heterodimer is determined by the dynamic association of co-regulators. This is in turn influenced by ligand presence and subcellular receptor localisation. To identify the genes controlled by RXR$\gamma$ in OPCs I carried out ChIP sequencing, which revealed genes involved in proliferation and cell cycle control. Further functional assessments aided me in the development of a hypothesis whereby RXR$\gamma$ activation does not directly influence oligodendrocyte formation, but rather promotes cell cycle exit thereby accelerating and facilitating OPC differentiation. Altered nuclear receptor expression and ligand presence in ageing OPCs may consequently impair this process. My thesis provides an alternative hypothesis to how RXR$\gamma$ regulates lineage cell progression, highlighting a new avenue in the development of therapeutic interventions targeting generic stem cell functions for which drugs are already FDA approved, rather than oligodendrocyte-specific pathways.

La dépression est un trouble neurologique grave et la deuxième cause d’invalidité dans le monde. Récemment, la signalisation du récepteur X aux rétinoïdes et l’inflammation chronique ont été identifiées comme facteurs génétiques et environnementaux. Au cours de ma thèse, j'ai étudié la façon dont le récepteur X gamma aux rétinoïdes (RXRg) contrôle la neuroinflammation dans deux modèles précliniques de dépression. J'ai montré que RXRg contrôle un signal spécifique lié à l'âge, entrainant la sénescence et l'hypoactivité des cellules microgliales. Ce signal impacte également la phagocytose microgliale et contribue à l'hypertrophie neuronale dans le striatum, elle-même associée à des symptômes dépressifs. De plus, j'ai pu mettre en évidence une activité antidépressive des rexinoïdes et des fibrates dans le stress de défaite sociale chronique, ainsi que leur mécanisme cellulaire. Les données obtenues pourraient permettre le développement de nouveaux traitements antidépresseurs. / Depression is severe mental disorder that is a second leading contributor to diseases burden. Recently, retinoid X receptor signaling and chronic inflammation have been identified as genetic and environmental factors. However, a cross-talk between these two factors was poorly understood. During my PhD I have studied how retinoid X receptor gamma (RXRg) controls neuroinflammation in two pre-clinical models of depression. I have shown that RXRg controls age-specific signal that drives microglial senescence and hypoactivity. The latter, impacts also microglial phagocytosis and contribute to neuronal hypertrophy in the striatum that previously was associated with depressive symptoms. Additionally, I showed antidepressant activity and cellular mechanism of rexinoids and fibrates in chronic social defeat stress. Obtained data have strong therapeutic potential that may allow for development of new antidepressant therapies.

Os receptores nucleares são de suma importância para os processos de sinalização intercelular nos eucariotos, uma vez que possuem a capacidade de convergir diferentes sinais internos e externos na regulação de programas genéticos. Estas proteínas funcionam, na sua maioria, como fatores de transcrição ativados por ligantes, sendo a via de comunicação direta entre as moléculas de sinalização e a resposta transcricional eliciada pelas mesmas. A programação genética, estabilizada ou modificada pelos receptores, afeta virtualmente todos os aspectos da vida dos organismos multicelulares, tais como a embriogênese, a homeostase, a reprodução, o crescimento e a morte celular. A regulação transcricional e a seletividade promovida por estas proteínas têm fomentado intensas pesquisas, as quais estão decifrando a complexa rede de eventos moleculares que relatam sua forma de ação. Será um desafio para o futuro o conhecimento completo das regras moleculares que definem sua maneira de promover o controle espacial e temporal da expressão gênica. Estas informações prometem trazer detalhes cruciais para o desenvolvimento de drogas mais eficientes e de grande valor terapêutico. Neste contexto, o principal objetivo dos estudos aqui apresentados foi o de aumentar o conhecimento sobre o comportamento e estrutura do receptor nuclear humano dos hormônios tireoidianos, isoforma β1 (hTRβ1), e do receptor nuclear humano do ácido retinóico 9-cis, isoforma ? (hRXRα). Para tal, aplicou-se a técnica de espalhamento de raios X a baixos ângulos para determinar-se, em solução, o envelope destes receptores contendo os domínios de ligação ao DNA e ao ligante. Paralelamente, investiu-se em diversas tentativas de cristalização dos mesmos. Os resultados obtidos permitiram a determinação da localização espacial dos diferentes domínios e as organizações quaternárias dos homodímeros e homotetrâmeros. Conseqüentemente, foram propostos os primeiros modelos estruturais de receptores nucleares contendo os domínios de ligação ao DNA e ao ligante. O comportamento oligomérico, em solução, do hTRβ1 também foi analisado qualitativamente. Verificou-se que a formação do homodímero e do homotetrâmero é influenciada pela presença do hormônio T3, pela concentração protéica, pelos domínios presentes e por mutações específicas. Estes estudos geraram a hipótese de que o receptor nuclear hTRβ1 é capaz de se autoreprimir. Até então, dentro da superfamília dos receptores nucleares, esta capacidade de autorepressão somente havia sido descrita para o receptor hRXRα. Por fim, cristalizou-se o domínio LBD do receptor hTRβ1 com os ligantes T3, Triac e GC-1. O objetivo foi o de determinar estruturas cristalográficas importantes para o futuro desenvolvimento de tiromiméticos de ação isoforma-seletiva. / In eukaryotes, nuclear receptors are of major importance for intercellular signaling because they join different intra and extracellular signals during regulation of genetic programs. The great majority of these proteins function as ligand activated transcription factors providing a direct link between signaling molecules and the transcriptional responses elicited by them. The genetic programs that these receptors establish or modify affect virtually all aspects of the multicellular organisms? life, such as embryogenesis, homeostasis, reproduction, cell growth, and death. Their gene-regulatory power and selectivity has prompted intense research which is now starting to decipher the complex network of molecular events involved in transcription regulation. The future challenge will be to uncover the molecular rules that define spatial and temporal control of gene expression. Such knowledge would be essential to the development of more efficient drugs with better therapeutic values. Therefore, the main purpose in this study was to extend the understanding on the behavior and the structure of human thyroid receptor, isoform ?1 (hTRβ1), and human retinoic acid X receptor, isoform ? (hRXRα). It was applied the small angle X-ray scattering technique to determine, in solution, the envelop of both receptors containing DNA and ligand binding domains. Beside this, several crystallization conditions were tried for both receptors. The results made possible to define the spatial localization of the domains and the quaternary structure of the homodimers and homotetramers. Consequently, we were able to propose the first structural models for nuclear receptors containing the DNA and ligand binding domains. The oligomeric behavior of the hTRβ1, in solution, was also analyzed qualitatively. We verified that it was influenced by the presence of T3 hormone, the protein concentration, the presence of both DNA and ligand binding domains, and by specific mutations. Based on these results, we were able to hypothesize that the hTRβ1 has the capacity of autorepression. Up to now, only the hRXRα, in the whole nuclear receptor superfamily, had been described to behave similarly. Finally, we crystallized the ligand binding domain of the hTRβ1 in the presence of the ligands T3, Triac, and GC-1. The objective was to solve crystallographic structures essential for the future development of tiromimetics with isoform-selective action.

Os receptores nucleares são de suma importância para os processos de sinalização intercelular nos eucariotos, uma vez que possuem a capacidade de convergir diferentes sinais internos e externos na regulação de programas genéticos. Estas proteínas funcionam, na sua maioria, como fatores de transcrição ativados por ligantes, sendo a via de comunicação direta entre as moléculas de sinalização e a resposta transcricional eliciada pelas mesmas. A programação genética, estabilizada ou modificada pelos receptores, afeta virtualmente todos os aspectos da vida dos organismos multicelulares, tais como a embriogênese, a homeostase, a reprodução, o crescimento e a morte celular. A regulação transcricional e a seletividade promovida por estas proteínas têm fomentado intensas pesquisas, as quais estão decifrando a complexa rede de eventos moleculares que relatam sua forma de ação. Será um desafio para o futuro o conhecimento completo das regras moleculares que definem sua maneira de promover o controle espacial e temporal da expressão gênica. Estas informações prometem trazer detalhes cruciais para o desenvolvimento de drogas mais eficientes e de grande valor terapêutico. Neste contexto, o principal objetivo dos estudos aqui apresentados foi o de aumentar o conhecimento sobre o comportamento e estrutura do receptor nuclear humano dos hormônios tireoidianos, isoforma β1 (hTRβ1), e do receptor nuclear humano do ácido retinóico 9-cis, isoforma ? (hRXRα). Para tal, aplicou-se a técnica de espalhamento de raios X a baixos ângulos para determinar-se, em solução, o envelope destes receptores contendo os domínios de ligação ao DNA e ao ligante. Paralelamente, investiu-se em diversas tentativas de cristalização dos mesmos. Os resultados obtidos permitiram a determinação da localização espacial dos diferentes domínios e as organizações quaternárias dos homodímeros e homotetrâmeros. Conseqüentemente, foram propostos os primeiros modelos estruturais de receptores nucleares contendo os domínios de ligação ao DNA e ao ligante. O comportamento oligomérico, em solução, do hTRβ1 também foi analisado qualitativamente. Verificou-se que a formação do homodímero e do homotetrâmero é influenciada pela presença do hormônio T3, pela concentração protéica, pelos domínios presentes e por mutações específicas. Estes estudos geraram a hipótese de que o receptor nuclear hTRβ1 é capaz de se autoreprimir. Até então, dentro da superfamília dos receptores nucleares, esta capacidade de autorepressão somente havia sido descrita para o receptor hRXRα. Por fim, cristalizou-se o domínio LBD do receptor hTRβ1 com os ligantes T3, Triac e GC-1. O objetivo foi o de determinar estruturas cristalográficas importantes para o futuro desenvolvimento de tiromiméticos de ação isoforma-seletiva. / In eukaryotes, nuclear receptors are of major importance for intercellular signaling because they join different intra and extracellular signals during regulation of genetic programs. The great majority of these proteins function as ligand activated transcription factors providing a direct link between signaling molecules and the transcriptional responses elicited by them. The genetic programs that these receptors establish or modify affect virtually all aspects of the multicellular organisms? life, such as embryogenesis, homeostasis, reproduction, cell growth, and death. Their gene-regulatory power and selectivity has prompted intense research which is now starting to decipher the complex network of molecular events involved in transcription regulation. The future challenge will be to uncover the molecular rules that define spatial and temporal control of gene expression. Such knowledge would be essential to the development of more efficient drugs with better therapeutic values. Therefore, the main purpose in this study was to extend the understanding on the behavior and the structure of human thyroid receptor, isoform ?1 (hTRβ1), and human retinoic acid X receptor, isoform ? (hRXRα). It was applied the small angle X-ray scattering technique to determine, in solution, the envelop of both receptors containing DNA and ligand binding domains. Beside this, several crystallization conditions were tried for both receptors. The results made possible to define the spatial localization of the domains and the quaternary structure of the homodimers and homotetramers. Consequently, we were able to propose the first structural models for nuclear receptors containing the DNA and ligand binding domains. The oligomeric behavior of the hTRβ1, in solution, was also analyzed qualitatively. We verified that it was influenced by the presence of T3 hormone, the protein concentration, the presence of both DNA and ligand binding domains, and by specific mutations. Based on these results, we were able to hypothesize that the hTRβ1 has the capacity of autorepression. Up to now, only the hRXRα, in the whole nuclear receptor superfamily, had been described to behave similarly. Finally, we crystallized the ligand binding domain of the hTRβ1 in the presence of the ligands T3, Triac, and GC-1. The objective was to solve crystallographic structures essential for the future development of tiromimetics with isoform-selective action.

7 Abstract With the first appearance of life on Earth, organisms had to adapt to an ever-changing surrounding environment in order to survive. Since the emergence of metazoan multi- cellularity, subsets of cells could adapt to perform specific biological tasks beneficial to the whole organism, necessitating not only spatiotemporal regulation of gene expression during development, but also integration of tissue specific needs with overall organis- mal status. Within the set of evolutionary conserved regulatory systems, the family of nuclear receptor (NR) transcription factors stands out due to its high degree of evolu- tionary conservation, plasticity and uniqueness to the metazoan kingdom, regulating gene expression in response to, or in the absence of a ligand by genomic and non- genomic actions. With an increasing number of different compounds being recognized as ligands to NRs, it is now thought that ancient NRs were probably characterized by low ligand binding specificity, eventually serving as environmental sensors, integrating nutrient availability and gene expression at the base of metazoan evolution. Characteri- zation of the NR network in one of the simplest metazoan organisms, Trichoplax ad- haerens, revealed not only a functional network and sub-specialization of NR dependent gene regulation, but...

Abstract The hormonally active form of vitamin D, 1,25(OH)2D3, is involved in many biological functions throughout the body, such as regulation of calcium and phosphate homeostasis, bone remodeling and controlling cell proliferation and differentiation. Vitamin D receptor (VDR), a member of the nuclear hormone receptor (NHR) super family, mediates those genomic actions of 1,25 (OH)2D3 by actively repressing or activating its target genes. In the present study recombinant human nuclear VDR and its ligand binding domain (LBD) were expressed in Spodoptera frugiperda (Sf9) insect cells and in E.coli. Recombinant proteins were purified and their biochemical and biophysical properties were characterized. Recombinant VDR was shown to bind to the vitamin D response element (VDRE) of osteopontin and osteocalcin genes as a homodimer or as a heterodimer with the retinoid X receptor (RXR)-αΔAB. Full-length VDR and its LBD were demonstrated to bind natural ligand 1,25 (OH)2D3 with high affinity. The binding affinities of several vitamin D analogs were also determined. Ligand binding induced conformational change within the receptor was studied using several methods such as partial proteolytic digestion, small angle neutron scattering (SANS), native gel electrophoresis and circular dichroism (CD) spectroscopy. Results indicate that ligand binding induces conformational change within VDR and different 1,25(OH)2D3 analogs might induce a somewhat different conformation within the receptor. This is seen as an unequal capacity of analogs to stabilize receptor against proteases or heat and as differences in the promotion of receptor homodimerization. Compared to other nuclear hormone receptors, VDR presents a large insertion region at the N-terminal part of the LBD between helices H1 and H3, encoded by an additional exon. In the present study this additional exon was deleted and the properties of mutated LBD were compared to the wild type LBD. Biochemical analyses indicated that the mutant protein exhibits the same ligand binding, dimerization with RXR and transactivation properties as the wild-type VDR, suggesting that the insertion region does not affect these main functions. Furthermore, solution studies by small angle X-ray scattering indicated that the insertion region in the VDR locates on the surface of molecule and it is not structurally well ordered.

No description available.

Dans ce travail de thèse, j'ai cherché à comprendre les mécanismes de contrôle des fonctions mnésiques par Rxrγ ainsi que l’implication potentielle de la signalisation dopaminergique dans ces mécanismes. Dans ce cadre, j'ai focalisé mon étude sur les fonctions hippocampiques à différent niveaux. J'ai cherché (1) à identifier les populations cellulaires de l'hippocampe exprimant Rxrγ en utilisant des techniques histologiques(immunohistochimie, hybridation in situ) afin de pouvoir (2) étudier les fonctions électrophysiologiques de ces cellules en utilisant la technique du patch-clamp. Pour comprendre le rôle de Rxrγ dans le contrôle des fonctions autonomes de ces cellules et les conséquences sur le réseau neuronal environnant, j’ai étudié les effets de la perte de fonction chez les souris Rxrγ/.Étant donné que différentes sous régions de l'hippocampe sont impliquées dans différents processus mnésiques, et que notamment le gyrus denté a été associé au processus de pattern separation (Leutgeb et al., 2007), j'ai également (3) cherché à préciser les fonctions mnésiques qui dépendent de l'activité de Rxrγ en réalisant des analyses comportementales chez les souris Rxrγ/. Etant donné que Rxrγ possède une activité transcriptionnelle, entre autre sur le gène du récepteur D2 à la dopamine (Drd2) (Samad et al., 1997), j'ai également (4) étudié la signalisation dopaminergique dans l’hippocampe chez les souris sauvages et les mutants nuls Rxrγ/. Enfin, afin de démontrer la spécificité neuroanatomique et homéostatique du contrôle exercé par Rxrγ sur la mémoire,j’ai procédé à (5) l’inactivation spécifique de Rxrγ dans les hippocampes de souris floxées pour ce gène, au moyen d’un virus AAV exprimant la recombinase Cre. / The present thesis work is an attempt to understand the mechanisms of Rxrγ control of memory functions, as well as the potential involvement of dopaminergic signaling in these mecanisms. In this context, I focused my research on hippocampal functions at several distinct levels. The first part of my work (1) aimed at defining the hippocampal cell populations expressing Rxrγ using various histological techniques (immunohistochemistry, in situhybridization) in order to (2) study the electrophysiological functions of these cells using invitro patch-clamp.To identify the role of Rxrγ in the control of cell autonomous functions, as well as the consequences on the surrounding network, I have studied the effects of its loss of function in Rxrγ/mice.As the different subregions of the hippocampus are implicated indistinct aspects of learning and memory, and in particular the dentate gyrus being associated with pattern separation (Leutgeb et al., 2007), I have also tried to dissect the mnemonic processes that rely on Rxrγ activity by performing behavioral analyses of Rxrγ/mice. Considering the transcriptional activities of Rxrγ on Drd2, I have also (4) studied dopaminergic signaling in the hippocampus of wild type and Rxrγ null mutant mice. Finally, to demonstrate the neuroanatomical and homeostatic specificity of Rxrγ control on memory, I performed (5) specific inactivations of Rxrγ in hippocampi of conditional mutant mice that possessed floxed Rxrγ, using AAV vectors expressing recombinase Cre.

Cystic Fibrosis (CF) is an autosomal recessive disease that arises from mutations in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene. The deletion of phenylalanine-508 (ΔF508-CFTR) is the most prevalent CF mutation and results in a misfolded protein that fails to exit the endoplasmic reticulum (ER). Previous studies demonstrated that mutation of a di-arginine based ER retention motif (R553AR555) in the first nucleotide binding domain (NBD1) rescues the trafficking defect of ΔF508-CFTR. We hypothesized that if the R553AR555 motif mediates retention of the ΔF508-CFTR protein, peptides that mimic this motif should antagonize mistrafficking mediated by aberrant exposure of the endogenous R553AR555 motif. We generated a peptide bearing the R553AR555 motif (CF-RXR) and conjugated it to the cell penetrating peptide Tat (CPP-CF-RXR) to facilitate intracellular delivery and investigated its efficacy in rescuing the mistrafficking and function of ΔF508-CFTR. Using a variety of biochemical and functional assays we demonstrate that the CPP-CF-RXR peptide is effective at increasing surface expression of ΔF508-CFTR in baby hamster kidney (BHK) and human embryonic kidney (HEK) cell lines. Furthermore, the increased surface expression is accompanied by an increase in its functional expression as a chloride channel. Using Ussing chamber assays, we demonstrate that the CPP-CF-RXR peptide improved ΔF508-CFTR channel function in respiratory epithelial tissues obtained from CF patients. Additionally, we investigated the effects of small molecules on mediating biosynthetic rescue of a ΔF508-CFTR construct bearing the additional mutations R553K and R555K (ΔFRK-CFTR) to inactivate the R553AR555 motif. Interestingly, mutation of the R553AR555 motif exerts an additive effect with correctors VRT-325 and Corrector 4a. Taken together, our data suggests that abnormal accessibility of the RXR motif present in NBD1 is a key determinant of the mistrafficking of the major CF causing mutant.