No description available.

Cocaine abuse poses a substantial health and economic burden for which no effective treatment currently exists. Exposure to cocaine results in altered signaling in a number of central nervous system (CNS) pathways. Previous studies have primarily focused on neurotransmitter systems, such as the dopaminergic and glutamatergic systems, as well as on drug-induced neuroplasticity within the mesolimbic system, which is believed to contribute to reward, addiction, and relapse following withdrawal. Furthermore, cocaine exerts a number of effects on gene regulation that contribute to many pathological conditions commonly afflicting users such as mood disturbances, psychotic symptoms, and long-term cognitive dysfunction. While some mechanisms by which cocaine regulates gene expression have been well-characterized, a large gap in our understanding regarding its downstream actions still exists and must be elucidated in order to develop effective treatment strategies. One pathway we have discovered to be disrupted in an animal model of chronic cocaine abuse is the retinoid X receptor (RXR) signaling pathway. Retinoid X receptors serve as obligate heterodimer partners for a number of nuclear receptor transcription factors, including the thyroid hormone receptor (TR), retinoic acid receptor, vitamin D receptor, and peroxisome proliferator activated receptor. Heterodimeric complexes bind to specific recognition sequences in or around the promoter of target genes to activate, or in some cases, repress, transcriptional activity. Therefore, alterations in the levels and function of RXRs can potentially disrupt numerous signaling cascades. In this context, we observed a significant down-regulation in mRNA and protein levels of RXR-y, an isoform predominantly expressed in the CNS that is involved in dopaminergic signaling, in brains of cocaine-administered mice. Additionally, we observed significantly decreased levels of the neuroplasticity protein, neurogranin, which is regulated transcriptionally by TR/RXR heterodimers. Mechanisms underlying regulation of RXR levels in cells of the CNS are vastly unexplored. Studies in other organ systems, including liver and cardiac systems, demonstrate pro-inflammatory cytokines and cellular stress pathways exert repressive effects on RXR signaling, although these studies solely investigated regulation of the RXR-a isoform. Recently, studies have highlighted the role of the immune system during chronic drug abuse, and demonstrate that significant amounts of proinflammatory factors are produced in the brains of chronic cocaine abusers. Therefore, we hypothesized that cocaine-mediated induction of inflammatory cytokines, such as tumor necrosis factor (TNF)-a may contribute to decreased RXR-y expression within the CNS. Utilizing in vitro neuronal systems, we have demonstrated that cocaine exposure induces neuronal expression of TNF-a and that this contributes to decreased levels of RXR-y, as inhibition of TNF-a or its downstream effector c-Jun-NH2-terminal kinase (JNK) prevents cocaine-mediated reductions in RXR-y protein levels. Furthermore, treatment of neurons with TNF-a alone mimics the effects on RXR-y levels observed in cocaine-treated cells. Additionally, we show that proteasome-dependent protein degradation likely plays a role, as inhibition of the 26 S proteasome with Bortezomib during cocaine or TNF-a exposure blocks the down-regulation of RXR-y levels. Degradation of RXR-y in response to cocaine and TNF-a may involve nuclear export, as our results show an increased level of RXR-y in the cytoplasmic compartment shortly after treatment, and inhibiting nuclear export during treatment with Leptomycin B prevents decreases in whole cell protein levels of RXR-y. In addition to the effects of chronic cocaine abuse on neurons, other CNS cell types such as oligodendrocytes may be negatively impacted by exposure to cocaine. Imaging studies and post-mortem microarray data from human cocaine abuse patients reveal loss of myelin and down-regulated expression of myelin-related genes in the nucleus accumbens and frontal cortex. Altered myelin integrity likely contributes to cognitive deficits that present in many chronic cocaine abuse patients and may also exacerbate damage to neurons. However, limited investigation has been performed to evaluate the effects of cocaine on oligodendrocyte health and function. We have employed an in vivo murine model of chronic cocaine administration to evaluate the impact of cocaine on white matter protein levels. Our data reveal that cocaine induces a significant decrease in white matter protein levels, even following an extended period of withdrawal, in the nucleus accumbens. One potential mechanism for cocaine-mediated white matter damage involves perturbations of glutamate homeostasis, as glutamatergic signaling can induce excitotoxicity in CNS cells, including oligodendrocytes. In this context, we found that administration of the B-lactam antibiotic, ceftriaxone, during cocaine withdrawal ameliorates loss of white matter proteins. Ceftriaxone has previously been shown to upregulate expression and activity of the glial glutamate transporter GLT-1, lending support to the theory that cocaine-mediated myelin loss may be due, in part, to disruption of glutamatergic signaling. Ceftriaxone treatment also decreased expression of cleaved caspase-3, a pro-apoptotic signaling molecule activated during excitotoxic cell death, in cocaine-administered mice. Taken together, our studies characterize two novel consequences of cocaine exposure: (1) decreased neuronal RXR-y expression and down-regulation of RXR-target genes, such as neurogranin, and (2) loss of myelin proteins in the nucleus accumbens which can be attenuated by administration of ceftriaxone. These findings yield insight into mechanisms underlying cocaine-mediated CNS cell death, and highlight potential treatment avenues for restoring brain health. Additionally, as inflammatory processes were identified as key mediators in some of these observations, our findings likely extend to a number of neurodegenerative diseases which are characterized by a neuroinflammatory component. / Biomedical Neuroscience

L’acide rétinoïque (AR) est le ligand des récepteurs nucléaires RAR et RXR qui agissent comme facteurs de transcription ligand-inductibles et médient ses effets biologiques. Il est connu que l’AR a des propriétés prodifférenciatrices et antiprolifératives, notamment sur les cellules de l’épithélium mammaire. Une perte de sensibilité de l’AR a toutefois été mise en évidence dans plusieurs lignées cellulaires mammaires cancéreuses, ce qui pourrait faciliter la croissance des tumeurs. Or jusqu’ici cette perte de sensibilité avait été attribuée à des défauts de la voie de signalisation de l’AR, causée par la perte de l’expression des récepteurs à l’AR dans la tumeur, bien que plusieurs lignées de cellules cancéreuses y soient tout de même très sensibles. Peu d’études se sont intéressées au rôle de la voie de synthèse de l’AR dans la transformation des cellules mammaires. En effet, l’AR est synthétisé à partir de la vitamine A, ou rétinol, son précurseur sanguin provenant de la diète. Les cellules de l’épithélium mammaire normales ont la capacité de synthétiser l’AR à partir du rétinol. Nos rapportons pour la première fois que l’épithélium mammaire est probablement le siège de la synthèse et de la signalisation de l’AR. Cela est dû, au moins en partie, à l’expression d’une enzyme de synthèse de l’AR, RALDH3, dans l’épithélium mammaire normal. Dans cette étude, nous démontrons que les cellules cancéreuses de type luminal, qui ont sensibles à l’AR (et qui expriment le récepteur des estrogènes ER, catégorie qui regroupe 75 % des tumeurs diagnostiquées) n’ont au contraire pas la capacité de sythétiser l’AR, probablement en raison d’une faible expression de RALDH3 dans les tumeurs, sous l’effet des estrogènes. Cela pourrait représenter un nouveau mécanisme favorisant la croissance des tumeurs luminales dont les cellules proliférent en présence du rétinol sanguin. RALDH1, une autre enzyme de la voie de synthèse de l’AR, et qui partage 70 % d’identité de séquence avec RALDH3, est un marqueur de tumeurs plus agressives et de la formation de métastase. Nous montrons au contraire, que RALDH3 est un marqueur d’une moindre probabilité de développer des métastases chez les patientes atteintes d’une tumeur luminale. Cela suggère des rôles different pour ces deux enzymes dans la glande mammaire. Nos résultats indiquent que RALDH1 et 3 ont des propriétés enzymatiques très différentes, ce qui est en accord avec cette dernière hypothèse. Nos données suggèrent aussi que RALDH1 et 3 pourraient être des marqueurs de populations distinctes de cellules dans la glande mammaire normale. Nous proposons d’exploiter les diffèrences entre RALDH1 et 3 afin de mettre au point des méthodes de séparation des différentes population de cellules de l’épithélium mammaire ce qui pourrait aider à comprendre le rôle de la synthèse d’AR dans ce tissu. / Retinoic acid (RA) is ligand of nuclear receptors RARs and RXRs that act as ligand-inducible transcription factors and mediate its biological effects. It was shown that RA has antiproliferative and prodifferenciating properties in mammary cells. A loss of RA sensitivity was associated with increased tumorigenicity in the mammary tissue, potentially facilitating the growth of tumors. It’s believed that is was mainly due to deficiencies in the RA signaling pathway, probably caused by the loss of RAR and RXR expressions. However, some tumorigenic cell lines were still reported to be RA sensitive. The role of RA synthesis in mammary tumorigenesis has been poorly characterized. RA is synthezised in target tissues from vitamin A (retinol) its precursor in blood. It was shown that mammary epithelial cells were able to synthesize RA from retinol in vitro. We show here for the first time that RALDH3, an enzyme involved in RA synthesis, is probably responsible for RA synthesis in normal mammary epithelial cells. Our result suggest that luminal cancer cells (that express ER and represent 75 % of breast tumors) have a very low capacity of RA synthesis, probably due to a low estrogen-mediated RALDH3 expression. It might represent a new mechanism of estrogen-driven tumorigenenesis allowing RA senstive tumors to proliferate in the presence of retinol in the blood. It was suggested that RALDH1, an other enzyme of the RA synthesis pathway that shares 70 % of identity with RALDH3, is a marker of mammary stem cells, of more agressive tumors and higher occurance of metastasis. We shown that unlinke RALDH1, RALDH3 is a marker of a lower occurance of metastasis and probably a marker of differentiation, suggesting different roles in the mammary gland for these 2 enzymes. This is in good agreement with our results showing that they have very different enzymatic properties. All together our data suggest that RALDH1 and 3 might be markers of different populations of cells of in the mammary epithelium. We propose to use the differences between RALDH1 and 3 to rationally develop methods and tools to separate and isolate RALDH1- and 3-expressing cells that would help the understand the role of RA synthesis in the mammary gland.

The human retinoid X receptor alpha (hRXRalpha) is a member of the nuclear receptor super-family of ligand-activated transcription factors. The Doyle laboratory has previously engineered a variety of functional hRXRalpha variants that activate gene expression in response to synthetic ligands (LG335 and γ-oxo-1-pyrenebutyric acid), compounds that are poor activators of wild-type hRXRalpha. The variants generally no longer respond to the wild-type ligand 9-cis retinoic acid. To enable targeting of these engineered receptors to arbitrary DNA sequences, we developed a program, ESPSearch, for identifying short or specific sequences in DNA or protein. ESPSearch enables identification of combinations of known zinc finger motifs to target arbitrary genes, as well having several other applications. The ability to target any DNA sequence means that the engineered receptors can be directed to control any gene. The ligand binding, self-association, coactivator interactions, and unfolding properties of the ligand binding domain of wild-type hRXRalpha were characterized. Our expression and purification protocol improves upon existing methods, providing high purity protein in a single step with more than twice prior yields. A general fluorescence-based method for measuring ligand affinity with hRXRalpha was developed, and used to determine binding constants for the small molecules. The presence of a peptide containing the binding motif from coactivator proteins (LxxLL) differentially increased the affinity of the receptor for the ligands. Assays to determine the self-association give a Kd for the dimer-tetramer equilibrium of 35 µM. hRXRalpha was found to denature irreversibly when heated, but shifts in apparent Tm due to ligands correlates strongly with the ligand binding affinities. Our results clarify disparities in existing reports and provide a benchmark for comparison. Reliable analysis of our data led to the development of a computer program for rigorous, automated data fitting. Nine functional variants of hRXRalpha were characterized to probe correlations between biophysical properties and the observed functional activity of the receptors, which differ significantly from wild-type. Although the correlation between ligand binding affinity and melting temperature was strong for all variants, there was essentially no correlation between ligand binding and activation of the variants. The mutations, which are all contained within the binding pocket, have significant long-range effects on the protein, causing changes in ligand-LxxLL interactions and oligomerization of the variants. Experimental and computational analysis of selected mutations suggests that they are highly coupled, complicating protein design. However, the large variation in properties amongst the variants also suggests that hRXRalpha can be mutated extensively while still retaining function. The long-range impact of binding pocket mutations will need to be taken into account in future engineering projects, as hRXRalpha is a flexible, dynamic protein.

L’acide rétinoïque (AR) est le ligand des récepteurs nucléaires RAR et RXR qui agissent comme facteurs de transcription ligand-inductibles et médient ses effets biologiques. Il est connu que l’AR a des propriétés prodifférenciatrices et antiprolifératives, notamment sur les cellules de l’épithélium mammaire. Une perte de sensibilité de l’AR a toutefois été mise en évidence dans plusieurs lignées cellulaires mammaires cancéreuses, ce qui pourrait faciliter la croissance des tumeurs. Or jusqu’ici cette perte de sensibilité avait été attribuée à des défauts de la voie de signalisation de l’AR, causée par la perte de l’expression des récepteurs à l’AR dans la tumeur, bien que plusieurs lignées de cellules cancéreuses y soient tout de même très sensibles. Peu d’études se sont intéressées au rôle de la voie de synthèse de l’AR dans la transformation des cellules mammaires. En effet, l’AR est synthétisé à partir de la vitamine A, ou rétinol, son précurseur sanguin provenant de la diète. Les cellules de l’épithélium mammaire normales ont la capacité de synthétiser l’AR à partir du rétinol. Nos rapportons pour la première fois que l’épithélium mammaire est probablement le siège de la synthèse et de la signalisation de l’AR. Cela est dû, au moins en partie, à l’expression d’une enzyme de synthèse de l’AR, RALDH3, dans l’épithélium mammaire normal. Dans cette étude, nous démontrons que les cellules cancéreuses de type luminal, qui ont sensibles à l’AR (et qui expriment le récepteur des estrogènes ER, catégorie qui regroupe 75 % des tumeurs diagnostiquées) n’ont au contraire pas la capacité de sythétiser l’AR, probablement en raison d’une faible expression de RALDH3 dans les tumeurs, sous l’effet des estrogènes. Cela pourrait représenter un nouveau mécanisme favorisant la croissance des tumeurs luminales dont les cellules proliférent en présence du rétinol sanguin. RALDH1, une autre enzyme de la voie de synthèse de l’AR, et qui partage 70 % d’identité de séquence avec RALDH3, est un marqueur de tumeurs plus agressives et de la formation de métastase. Nous montrons au contraire, que RALDH3 est un marqueur d’une moindre probabilité de développer des métastases chez les patientes atteintes d’une tumeur luminale. Cela suggère des rôles different pour ces deux enzymes dans la glande mammaire. Nos résultats indiquent que RALDH1 et 3 ont des propriétés enzymatiques très différentes, ce qui est en accord avec cette dernière hypothèse. Nos données suggèrent aussi que RALDH1 et 3 pourraient être des marqueurs de populations distinctes de cellules dans la glande mammaire normale. Nous proposons d’exploiter les diffèrences entre RALDH1 et 3 afin de mettre au point des méthodes de séparation des différentes population de cellules de l’épithélium mammaire ce qui pourrait aider à comprendre le rôle de la synthèse d’AR dans ce tissu. / Retinoic acid (RA) is ligand of nuclear receptors RARs and RXRs that act as ligand-inducible transcription factors and mediate its biological effects. It was shown that RA has antiproliferative and prodifferenciating properties in mammary cells. A loss of RA sensitivity was associated with increased tumorigenicity in the mammary tissue, potentially facilitating the growth of tumors. It’s believed that is was mainly due to deficiencies in the RA signaling pathway, probably caused by the loss of RAR and RXR expressions. However, some tumorigenic cell lines were still reported to be RA sensitive. The role of RA synthesis in mammary tumorigenesis has been poorly characterized. RA is synthezised in target tissues from vitamin A (retinol) its precursor in blood. It was shown that mammary epithelial cells were able to synthesize RA from retinol in vitro. We show here for the first time that RALDH3, an enzyme involved in RA synthesis, is probably responsible for RA synthesis in normal mammary epithelial cells. Our result suggest that luminal cancer cells (that express ER and represent 75 % of breast tumors) have a very low capacity of RA synthesis, probably due to a low estrogen-mediated RALDH3 expression. It might represent a new mechanism of estrogen-driven tumorigenenesis allowing RA senstive tumors to proliferate in the presence of retinol in the blood. It was suggested that RALDH1, an other enzyme of the RA synthesis pathway that shares 70 % of identity with RALDH3, is a marker of mammary stem cells, of more agressive tumors and higher occurance of metastasis. We shown that unlinke RALDH1, RALDH3 is a marker of a lower occurance of metastasis and probably a marker of differentiation, suggesting different roles in the mammary gland for these 2 enzymes. This is in good agreement with our results showing that they have very different enzymatic properties. All together our data suggest that RALDH1 and 3 might be markers of different populations of cells of in the mammary epithelium. We propose to use the differences between RALDH1 and 3 to rationally develop methods and tools to separate and isolate RALDH1- and 3-expressing cells that would help the understand the role of RA synthesis in the mammary gland.

No description available.

This thesis is divided into two parts.Part I describes the organic extraction, separation, and liquid chromatographic-mass spectrometric analysis of chromophores from human and bovine retinal pigment epithelium. Flurorophores in the retinal pigment epithelium have been implicated in age related macular degeneration. In addition, the synthesis and characterization of a number of bis-retinoid type compounds that may potentially be found in such extracts, or that may be used for insight into pyridinium bis-retinoid reactivity, was accomplished.Part II describes a study of pyridinium bis-retinoid-folic acid coupling with respect to linker type, linker length, and nature of the linkage. Folic acid has been used as a targeting compound for a variety of cancer types. Development of HPLC and UV-Vis conditions suitable for the analysis of this new type of macromolecule was performed.

No description available.

The telencephalon is the most highly evolved region of the vertebrate central nervous system (CNS). The major structures of the telencephalon - the cortex and basal ganglia – derive from the dorsally positioned pallium and the ventrally positioned subpallium, respectively. Differences in morphology, gene expression, and connectivity permit a subdivision of the developing telencephalon into domains that give rise to discrete regions of the adult brain. In mammals, the ventral region of the developing telencephalon can be subdivided into the medial (MGE) and lateral (LGE) ganglionic eminences. The dorsal midline cells give rise to the choroid plexus, and cells in the more lateral domain, the dorsal pallium, give rise to the cerebral cortex. Genetic studies have provided evidence that crossregulatory interactions between transcription factors contribute to the regionalization of the telencephalon. Less is known, however, about the secreted signals that induce the initial dorsoventral character of telencephalic cells. Sonic hedgehog (SHH) is required for the specification of ventral character along the entire anteroposterior (AP) extent of the developing CNS, including the telencephalon. We show that WNT activity imposes an early generic dorsal telencephalic character and that Fibroblast Growth Factor (FGF) act sequentially, and in concert with WNT, to specify cells of definitive dorsal telencephalic character. We also show that retinoic acid (RA)-mediated signaling induces intermediate character in telencephalic cells, and that FGFs maintain cells of ventral character by opposing RA activity. The following model emerges from these findings. At gastrula stages, most or all prospective telencephalic cells become specified as ventral cells in response to node-derived SHH signals. At neural fold- and early neural plate stages, cells in the prospective dorsal and intermediate regions of the telencephalon cells are exposed to WNT signaling that induce a generic dorsal character. The head ectoderm adjacent to the telencephalon then starts to express the retinoic acid producing enzyme, Raldh3, thus exposing telencephalic cells to RA signals. At the same time prospective dorsal cells start to express WNT signals. RA signaling appears to promote the generation of intermediate/prestriatal cells, whereas WNT signal suppress the actions of RA on dorsal cells, which therefore maintain their dorsal character. From the neural plate stage, prospective ventral 6 telencephalic cells are exposed to FGF8 derived from the anterior neural ridge, and FGF8 maintains ventral telencephalic character by opposing the influence of RA signals in ventral cells. At early neural tube stages, the domain of Fgf8 expression expands dorsally and FGF signals derived from the dorsal midline region induce definitive dorsal/precortical cells. In the intermediate region of the telencephalon cells evade high levels of WNT and FGF signals, resulting in an environment in which RA signaling is able to induce prestriatal character.

Creating receptors for control of transcription with arbitrary small molecules has widespread applications including gene therapy, biosensors, and enzyme engineering. Using the combination of high throughput docking, codon randomization, and chemical complementation, we have created new receptors to control transcription with small molecules. Chemical complementation, a new method of protein engineering, was used to discover retinoid X receptors (RXR) variants that are activated by compounds that do not activate wild-type RXR. A first library of 32,768 RXR variants was designed for the synthetic retinoid-like compound LG335. The library produced ligand-receptor pairs with LG335 that have a variety of EC50s and efficacies. One engineered variant has essentially the reverse ligand specificity of wild-type RXR and is transcriptionally active at 10 and #64979;fold lower LG335 concentration than wild-type RXR with 9cRA in yeast. The activity of this variant in mammalian cells correlates with its activity in yeast. A second library of 262,144 RXR variants was designed for two purposes: (i) to develop a high-throughput chemical complementation method to select variants that have high efficacies and low EC50s; and (ii) to find variants which are activated by small molecules not known to bind RXR variants. Selection conditions were manipulated to find only variants with high efficacies and low EC50s. This library was also selected for variants that activate transcription specifically in response to gamma-oxo-1-pyrenebutyric acid (OPBA), which is different from any known RXR ligand. OPBA was chosen as a potential ligand using high-throughput docking with the software program FlexX. Two variants are activated by OPBA with an EC50 of 5 mM. This is only ten-fold greater than the EC50 of wild type RXR with its ligand 9cRA (500 nM) in yeast. An improved method synthesizing LG335 and a method for quantifying intracellular ligand concentrations were developed. Although the LG335 synthetic method has an additional step, the overall yield was improved to 8% from 4% in the original publication. Liquid chromatography and mass spectrometry was used to quantify the intracellular concentration of LG335, which was found to be within four fold of the LG335 concentration in the media.