Dissertations / Theses on the topic 'Pregnenolone'

Thesis (M.A.)--Boston University
The central nervous system operates through a tightly balanced relationship of excitatory and inhibitory synaptic transmission. Neurosteroids, synthesized de novo from cholesterol in the human brain as well as converted from precursors circulating in the blood, are proven modulators of this synaptic activity. Pregnenolone sulfate, one of the most abundant endogenous neurosteroids, is a negative modulator of the GABA receptor and positive modulator of NMDA and AMPA receptors, increasing the frequency of excitatory transmission in the brain. Here, we show that low concentrations (50pM) of pregnenolone sulfate increase receptor trafficking in cultured rat hippocampal and cortical cells. Immunocytochemistry data shows that a ten-minute treatment with 50pM PS increases NR2B subunit protein. Preliminary surface biotinylation results highlight a potential increase in NR1 subunit protein. Since NMDA receptors play a pivotal role in learning and memory and have been implicated in neuropsychiatric disorder, an endogenous positive modulator of NMDA receptors is likely to play a major pharmaceutical role in neurodegenerative and memory disorders.

Les mitochondries d'oligodendrocytes de rats ages de 3 semaines transforment le #3h-cholesterol en #3h-pregnenolone(p), en #3h-pregn-5-ene-3 beta, 20 alpha-diol (20-oh-p). Les cultures primaires de cellules gliales de rats nouveaux-nes contiennent 60% d'oligodendrocytes, comme indique par la detection immunocytochimique au galactocerebroside, les oligodendrocytes contiennent aussi un antigene reconnu par des igg anti-p450scc, ces cultures primaires convertissent le #3h-mevolonolactone (mva) en #3h-cholesterol(c), en #3h-p et en #3h-20-oh-p. Les oligodendrocytes subissent un processus de differenciation en culture comme l'indique en l'occurence l'augmentation de la phosphohydrolase de nucleotide 2,3-cyclique et l'activite steroidogenique. L'aminoglutethimide empeche la formation des hormones steroides, le #3h-c s'accumule dans les cellules, une fois les cellules transferees dans un milieu sans #3h-mva et sans aminoglutethimide, elles produisent de la #3h-20-oh-p. La production des steroides est stimulee par le camp et par le dexamethasone. La #3h-pregnenolone formee est ensuite convertie en hormone steroide: la #3h-progesterone et en #3h-3 alpha-hydroxy-5 alpha-pregnane-20-one, un steroide anesthesique. Les resultats obtenus permettent de definir la biosynthese des neurosteroides comme une nouvelle fonction du cerveau, leur role physiologique fera l'objet de recherches ulterieures

Le cannabis sativa est l'une des drogues les plus consommées dans le monde. Le THC, sa principale composante psychoactive, représente un facteur de risque de pathologies mentales, telles que le trouble de la consommation de cannabis, la dépendance et la psychose. Le THC a un effet biphasique, de fortes doses de THC provoquent une hypoactivité et une aversion, tandis que de faibles doses de THC provoquent une hyperactivité et une récompense. Le THC agit sur le récepteur aux cannabinoïdes de type 1 (CB1), dont la signalisation est biaisée avec différents transducteurs pouvant véhiculer une voie spécifique selon différentes conditions. Il a été prouvé que la signalisation biaisée est pertinente dans la découverte de médicaments et la compréhension de la signalisation CB1 est essentielle pour le développement de médicaments à base de cannabinoïdes. Même si il a été montré que différentes doses de THC entraînent des effets comportementaux, cellulaires et moléculaires différents, le lien entre ces phénomènes n'a jamais été étudié.En outre, il a été découvert que la pregnénolone (PREG) est un modulateur allostérique endogène biaisé du CB1, en étant un inhibiteur de signalisation spécifique du récepteur CB1 (CB1-SSi) et capable de bloquer des comportements induits par le THC. Comme la PREG est un précurseur de stéroïde, son administration à forte dose peut induire la production de stéroïdes, avec d'éventuels effets secondaires.Ainsi, dans un but thérapeutique, des composés CB1-SSi analogues à la PREG ont été synthétisés, ayant le même potentiel thérapeutique que la PREG, mais sans être métabolisés en stéroïdes. Le CB1-SSi étudié ici est le composé CB1-SSi leader, l’AEF0117.Le premier objectif de ce travail était de comprendre les voies de signalisation intracellulaires selon des doses faibles, moyennes et élevées de THC, conduisant à respectivement l'hyperlocomotion, l'asociabilité et l'hypolocomotion chez la souris.Le second objectif était de comprendre le mécanisme d'action de l’AEF0117 et sa capacité à bloquer les effets comportementaux et moléculaires du THC à faibles, moyennes et fortes doses.La thèse est divisée en cinq parties. L'introduction sert à préfacer les concepts du système endocannabinoïde, ainsi que l'abus de cannabis chez l'homme et les effets comportementaux du THC chez la souris, comme l'hyperlocomotion, l'asociabilité et l'hypolocomotion. L'état de l'art de la signalisation CB1 impliquant le système CB1 biaisé est décrit avec un accent particulier sur les inhibiteurs spécifiques de la signalisation CB1 (CB1-SSi), en particulier la pregnénolone endogène (PREG), et son analogue synthétique, l’AEF0117.L'article Zanese*, Tomaselli* et al, 2020 (publié dans J. Neurosci. Methods) porte sur la validation de la technique analytique à haut débit (AlphaLISA) de choix dans cette étude pour la détection de la phosphorylation des protéines dans les lysats de tissu cérébral.L'article de Tomaselli et al. (à soumettre) est consacré à l'étude de faible dose de THC qui provoque l'hyperlocomotion chez la souris. Les principales données ont révélé que le THC via CB1 recrute la voie de signalisation β-Arrestin1-PI3K-Akt-GSK3β, dans les zones du cerveau riches en CB1 et pertinentes pour l'activité locomotrice (NAc, Str, CB), qui conduit à l'hyperlocomotion. En outre, PREG et AEF0117 peuvent bloquer l'hyperlocomotion et les modifications de la signalisation CB1 induite par le THC.La troisième partie représente les études sur les effets du THC à des doses moyennes et élevées qui induisent respectivement un comportement asocial et une hypolocomotion. Chaque dose de THC induit des altérations spécifiques des voies de signalisation intracellulaires CB1 et le traitement avec l’AEF0117 réverse les deux comportements.La discussion générale aborde ensuite le rôle des voies CB1 spécifiques dans la dépendance et la psychose induites par le THC, et propose un mécanisme d'action pour les composés CB1-SSi, dont l’AEF0117
Cannabis sativa is among the most abused drugs worldwide. THC, its main psychoactive component, represents a risk factor of several mental pathologies, such as cannabis use disorder, addiction, and psychosis. Being a biphasic drug, high doses of THC cause hypoactivity and aversion, whereas low doses of THC cause hyperactivity and reward. THC acts on the type-1 cannabinoid receptor (CB1), one of the most abundant G-protein coupled-receptors (GPCRs) in the brain, whose signaling is biased, meaning that different transducers can carry specific pathway following different conditions. Biased signaling was proven to be extremely relevant in drug discovery and understanding CB1 signaling in pathologic conditions is essential for cannabinoid-based drug development. It is known that different doses of THC bring along different behavioral, cellular, and molecular outcomes. However, the link between those phenomena has never been investigated.Thus, for a therapeutic purpose PREG-like CB1-SSi compounds have been synthetized that share the same PREG therapeutic potential, but cannot be metabolized in downstream steroids. The CB1-SSi studied in the current work is the CB1-SSi lead compound, AEF0117.The first aim of the current work was to understand the intracellular signaling pathways following low, medium, and high doses of THC, leading to three distinct known behavioral outputs in mice, hyperlocomotion, asociability, and hypolocomotion, respectively.The second aim of the thesis was to understand the mechanism of action of AEF0117, and its capability to block the behavioral and molecular effects of THC at low, medium, and high doses.The doctoral dissertation is divided into five main parts. The introduction serves to preface the concepts of the endocannabinoid system, as well as cannabis abuse in humans and the counterpart behavioral outcomes of THC in mice, including hyperlocomotion, asociability, and hypolocomotion. The state of the art of CB1 signaling involving the biased CB1 system is described with particular emphasis on CB1 Signaling Specific Inhibitors (CB1-SSi), in particular the endogenous pregnenolone (PREG), and its synthetic analogue, the lead CB1-SSi compound, AEF0117.The article Zanese*, Tomaselli* et al., 2020 (published in J. Neurosci. Methods) oversees the validation of the high throughput analytical technique (AlphaLISA) of choice in this study for detection of protein phosphorylation in brain tissue lysates.The article Tomaselli et al. (to be submitted), is devoted to the studies of the low dose of THC that causes hyperlocomotion, with the discovering of its related intracellular CB1 signaling pathway, along with the signaling transducer involved in the CB1-rich brain areas relevant for locomotor activity (NAc, Str, CB). The main data revealed that THC via CB1 recruits the β-Arrestin1-PI3K-Akt-GSK3β signaling pathway that lead to hyperlocomotion. Furthermore, both PREG and AEF0117 were able to block the THC-induced hyperlocomotion and altered signaling in mice.The third part of the data represent studies on the effects of THC at medium and high doses that induce asocial behavior and hypolocomotion, respectively. Each dose of THC induced specific alterations in the CB1intracellular signaling pathways in the most CB1-rich brain areas, and the treatment with AEF0117 rescued both behaviors.The general discussion then addresses conclusions and perspectives, highlighting the role of specific CB1 pathways in THC-induced addiction and psychosis, and proposes a mechanism of action for CB1-SSi compounds, including AEF0117

Thesis (M. S.)--Biology, Georgia Institute of Technology, 2006.
Donald Doyle, Committee Member ; Harish Radhakrishna, Committee Member ; Alfred Merrill, Committee Member ; Marion Sewer, Committee Chair Includes bibliographical references.

Classiquement, il est admis depuis de nombreuses annees que l'ovaire ftal de mammifere ne possede pas d'activite staroidogene alors que, au meme moment, chez le male, une synthese de testosterone apparait. Le but de notre travail a ete de rechercher chez le rat les etapes limitantes de la steroidogenese ovarienne, au cours de la vie ftale. Nous avons etudie les deux etapes conduisant a la production de progesterone. Dans une premiere serie d'experiences, nous avons suivi, par dosage radioimmunologique, la production de pregnenolone et de progesterone d'ovaires ftaux de 18,5 j. P. C. Explantes soit en milieu temoin, soit en presence de dibutyryl ampc (dbampc). Dans une seconde approche experimentale, nous avons evalue l'activite 3-hydroxysteroide deshydrogenase (3-hsd) en mesurant la conversion de pregnenolone #3h en progesterone #3h dabs des homogenats d'ovaires de 18,5 j. P. C. Cette activite est presente dans l'ovaire ftal. De la meme facon, l'activite de clivage de la chaine laterale du cholesterol (activite scc) a ete evaluee dans les mitochondries isolees en mesurant la conversion du cholesterol #3h en pregnenolone #3h. Absente au niveau des ovaires ftaux, cette activite peut etre induite par un pretraitement avec le dbampc. Enfin, l'analyse en immunoblotting nous a permis de mettre en evidence la presence du cytochrome p-450scc (composant du complexe enzymatique de clivage) dans les ovaires ftaux pretraites par le nucleotide, alors qu'il est absent dans les ovaires temoins. En conclusion, la premiere etape de la steroidogenese est une etape limitante pour l'ovaire ftal de rat. Cette etape peut etre induite par le dbampc

Les neurosteroides pregnenolone (preg) et dehydroepiandrosterone (dhea), precurseurs des hormones steroides dans les glandes endocrines classiques, ont ete mis en evidence dans le cerveau de rat a des taux inexpliques par leurs concentrations plasmatiques et les productions gonadiques et surrenaliennes. Leur 7alpha-hydroxylation est importante au niveau des microsomes cerebraux. Elle est observee principalement dans des cultures d'astrocytes de type 1 et d'oligodendrocytes. Son inhibition par l'oxyde de carbone suggere l'implication d'un cytochrome p450, probablement different des 7alpha-hydroxylases hepatiques specifiques du cholesterol ou de la testosterone. Le metabolisme des neurosteroides est influence par la densite cellulaire d'ensemencement. Dans des cultures d'astrocytes a faible densite, une forte activite 3-hydroxy-steroide deshydrogenase-delta#5##4-isomerase conduit a la formation de progesterone a partir de la preg et d'androstenedione a partir de la dhea, et elle est inhibee aux densites elevees. Le mecanisme de cette regulation semble impliquer des contacts intercellulaires. Les cellules gliales et les neurones convertissent la progesterone en 5alpha-pregnane-3,20-dione qui est ensuite transformee en 3alpha et/ou 3beta-hydroxy,5alpha-pregnane-20-one. Au niveau du systeme nerveux peripherique chez le rat, la preg est presente dans le nerf sciatique a des concentrations qui excedent largement celles du plasma, et qui ne sont pas modifiees apres castration et surrenalectomie. Les cellules de schwann synthetisent de la preg a partir du 25-hydroxy-cholesterol. Elles forment aussi les derives 7alpha-hydroxyles de la preg et de la dhea, et reduisent cette derniere en delta5-androstene-3beta,17beta-diol. Le role physiologique des metabolites des neurosteroides et leur mecanisme d'action semblent relever d'un dispositif auto et/ou paracrine. Ces donnees ouvrent ainsi de nouvelles perspectives d'etudes physiologiques et pharmacologiques

Pregnenolona (PREG) e 17-alfa-hidroxipregnenolona (17OHPREG) são dois esteroides produzidos pela glândula adrenal e precursores de vários hormônios esteroidais. A dosagem desses compostos tem aplicações clínicas, como o diagnóstico de doenças relacionadas aos corticoesteroides e mineralocorticóides e especialmente na avaliação da atividade da enzima 3-β-hidroxidesidrogenase que é decisiva no diagnóstico de um dos tipos de hiperplasia da glândula adrenal que causa defeitos severos na síntese de esteroides. Métodos cromatográficos associados à espectrometria de massas superaram a especificidade reduzida dos imunoensaios e tem sido crescentemente utilizados na quantificação de esteroides. Os últimos anos tem sido marcados pela hegemonia da cromatografia líquida acoplada à espectrometria de massas em tandem (LC-MS/MS) em grande parte devido à velocidade e possibilidade da análise direta de vários analitos. Porém, no caso específico dos esteroides de tipo 3-hidroxi-5-eno, que apresentam baixa afinidade protônica e, portanto, baixa eficiência de ionização, são necessárias muitas etapas para a conversão em derivados mais detectáveis. Embora desfavorecida em relação ao LC-MS/MS nos últimos anos, a cromatografia gasosa acoplada à espectrometria de massas (CG-MS) apresenta várias características favoráveis para a análise de esteroides como a eficiência cromatográfica ainda insuperável. Adicionalmente, a incorporação da espectrometria de massas em tandem ao CG (CG-MS/MS) torna a técnica tão seletiva quanto LC-MS/MS. No presente trabalho, foi desenvolvido um novo método que permite a extração e derivatização simultâneas da PREG e 17OHPREG de amostras de soro tornando o método de preparo da amostra tão simples quanto os descritos para LC-MS/MS. O método de detecção desenvolvido baseado em ionização química no modo negativo obteve a sensibilidade necessária para o diagnóstico da deficiência da enzima 3-beta-hidroxidesidrogenase utilizando apenas 250 µ:L de amostra.
Pregnenolone (PREG) and 17α-hydroxypregnenolone (17OHPREG) are two steroid precursors produced by the adrenal gland. The quantification of these compounds is essential for the evaluation of 3-β-hidroxidesidrogenase enzyme activity, which promotes the conversion of PREG in 17OHPREG. The 3-β:-hidroxidesidrogenase deficiency is a rare but severe type of adrenal hyperplasia that causes serious defects in steroid synthesis. Chromatographic methods coupled to mass spectrometry overcame immunoassays limitations such as reduced specificity, and have been widely used for steroids quantification. Recent years have been marked by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) hegemony due to the speed and possibility to analyze directly several analytes. However, in the case of type 3-hydroxy-5-ene steroids, which have low affinity for protons and, therefore, low ionization efficiency, many steps are required for conversion to detectable products. Notwithstanding, gas chromatography coupled to mass spectrometry (GC-MS) has some favorable features for steroid analysis such as unbeatable chromatographic efficiency. In addition, the incorporation of tandem mass spectrometry (GC-MS/MS) makes it as selective as LC-MS/MS. In this study, a new method for simultaneous extraction and derivatization of PREG and 17OHPREG from serum was developed. This procedure makes sample preparation for GC-MS/MS as simples as those described for LC-MS/MS. The detection method based on negative mode chemical ionization achieved the sensitivity required for the diagnosis of 3-β-hidroxidesidrogenase defficiency using only 250 µL of sample.

The first step in the synthesis of steroid hormones occurs in the mitochondria where cholesterol is converted to pregnenolone by cytochrome P450scc (CYP11A1). Cholesterol is insoluble in water and is supplied to the CYP11A1 directly from the inner mitochondrial membrane to which the enzyme is bound. The aim of this study was to characterise the interaction of bovine CYP11A1 with the phospholipid membrane. The effect of osmotic stress provided by glycerol on the spin-state, activity and degree of hydration of CYP11A1 was also investigated. Multiple sequence alignment of mitochondrial P450s revealed that there are 46 absolutely conserved residues, with the highest conservation in the heme-binding region at the C-terminal. The greatest variablility between subfamilies is in the regions believed to be involved in substrate binding (SRSs), as defined for the CYP2B family. The secondary structure prediction for CYP11A1 suggests that there is strong similarity in secondary structure to P450s of known structure. A model structure of CYP11A1 was built from primary sequence alignment to template P450 structures using the SwissModel automated server. From the model and other bioinformatic analyses, the distal face of the P450 which includes the A’ helix, F-G loop and beta sheet 1 regions, were predicted to interact with the membrane. Tryptic digests of CYP11A1 were performed with the aim of identifying membrane bound peptides that may be protected from protease activity. HPLC tryptic maps were similar in profile between soluble and vesicle-bound P450 which suggests that there is not a large region of CYP11A1 protected from protease digestion when the enzyme is attached to a membrane. Mass spectrometric analysis of peptides resulting from tryptic digestion revealed a number of peptides in the soluble digest that were not present in the digest of vesicle-bound P450. These peptides were located at the N-terminal and the J to J’ helix and interestingly, there was an absence of C-terminal peptides for both digests. This C-terminal peptide could be detected in digests of vesicle-bound P450 but not in digests of soluble P450 by tricine SDS polyacrylamide gel electrophoresis, Western transfer and N-terminal sequence analysis. Based upon the bioinfomatic and tryptic digestion data, a set of N- and C-terminal deletion mutants of CYP11A1 were expressed in E. coli and fractionated based on their association with the soluble or membrane fraction of the cells. The N-terminal deletion of the A’ helix resulted in an increase in the proportion of CYP11A1 in the soluble fraction while the C-terminal deletion did not alter membrane localisation. There are eight tryptophan residues in mature CYP11A1. The accessibility of these tryptophans to a water-soluble fluorescence quencher was determined for soluble and vesicle-bound enzyme. When CYP11A1 was associated with the vesicle membrane an average of 2 tryptophan residues were protected from quenching compared to soluble CYP11A1. This suggests that these tryptophan residues become buried within the membrane following association of CYP11A1 with the vesicles and are no longer accessible to quencher. The only free cysteine (C265S) of bovine CYP11A1 was removed by site directed mutagenesis and new cysteine residues introduced at selected sites based upon earlier results and the modelled CYP11A1 structure. The cysteine mutants were expressed, purified and labelled with the environmentally sensitive fluorescent probe, N-(7-nitrobenz-2-oxal-3-diazol-4-yl)ethylenediamine (NBD). There was an increase in the hydrophobicity of the NBD environment following the association of CYP11A1 with vesicles for the labeled mutants V212C and L219C. This indicates that these residues which are in the F-G loop, become localized to a more hydrophobic environment following membrane binding. Labeled cysteine residues introduced into the A’, B’ and G helices and β4-2 did not show major changes in hydrophobicity following membrane integration of CYP11A1. Osmotic stress of CYP11A1 induced by glycerol resulted in a low-spin spectral response and inhibition of activity. The change to low spin correlated with the dissociation of five or six water molecules from CYP11A1 and the inhibition of activity with cholesterol as substrate correlated with the dissociation of two molecules of water. In conclusion, this study shows that CYP11A1 is held to the membrane, at least in part, by the F-G loop region, and that the removal of water from the active site of CYP11A1 by osmotic stress causes a low spin spectral response and inhibition of activity.

Dissertação (mestrado) - Universidade Federal de Santa Catarina, Centro de Ciências Biológicas. Programa de Pós-Graduação em Biotecnologia.
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O receptor pregnano X (PXR) é um receptor nuclear envolvido na regulação transcricional de enzimas envolvidas no metabolismo de fármacos e também de proteínas transportadoras. Em mamíferos, vários xenobióticos induzem a expressão dos genes do citocromo P4503A (CYP3A) e de resistência a multidrogas 1 (MDR1) através da via de sinalização do receptor PXR. Pouca atenção tem sido dada aos estudos sobre a identificação da função biológica dos homólogos do PXR em espécies de não-mamíferos. O peixe-zebra tem se tornado amplamente utilizado e aceito como espécie modelo em estudos toxicológicos e farmacológicos no entendimento dos mecanismos de doenças humanas e na identificação de vias de sinalização conservadas. O objetivo desse estudo foi avaliar a expressão in vivo dos genes PXR, CYP3A e MDR1 no fígado de peixes-zebra tratados com o esteróide sintético pregnenolona 16a-carboninitrilo (PCN), o antimicótico sintético clotrimazol (CTZ) e o composto fármaco nifedipina (NIF). Peixes (n=15 por grupo) foram expostos à PCN, CTZ e NIF (20 mg.Kg-1) por 48 horas. Os fígados foram retirados e utilizados para a extração do RNA total (n=5 por grupo). A partir do cDNA obtido foi realizada a reação de PCR, utilizando diferentes iniciadores para a amplificação de PXR, CYP3A, MDR1 e ß-ACTINA. Fígados de peixes tratados com PCN mostraram uma indução de 1,9 vezes na indução do PXR seguida de 1,8 vezes na indução do CYP3A e de 1,6 vezes na indução do MDR1 nos níveis de mRNA. CTZ e NIF não afetaram estatisticamente a expressão dos genes PXR, CYP3A e MDR1. O padrão similar na expressão do mRNA dos genes PXR, CYP3A e MDR1 encontrado em peixes tratados com diferentes indutores de PXR sugere que a associação intrínseca entre esses três genes é conservada no peixe-zebra.

Les neurostéroïdes sont des composés endogènes qui peuvent moduler la transmission synaptique et avoir un effet neuroprotecteur dans les maladies neurodégénératives. Les systèmes régulant leur biosynthèse ne sont pas connus mais la première étape de celle-ci peut être régulée par la protéine TSPO. Cette protéine mitochondriale facilite le transport du cholestérol vers l’intérieur de la mitochondrie pour y être métabolisé en prégnénolone. Ce stéroïde est le précurseur principal de la biosynthèse des neurostéroïdes et l’utilisation in vitro de ligands du TSPO permet d’augmenter sa sécrétion. Dans ce travail de thèse, nous avons ainsi cherché à développer de nouvelles familles de ligands solubles du TSPO augmentant la sécrétion de prégnénolone. Le développement de ces nouvelles familles a nécessité la réalisation d’une méthodologie de synthèse faisant intervenir une réaction de cyclisation pallado-catalysée de type Buchwald-Hartwig. Une étude de solubilité des composés synthétisés a été effectuée expérimentalement, leur activité a été évaluée par des méthodes fonctionnelles et leur effet neuroprotecteur a été testé sur un modèle cellulaire de la maladie d’Alzheimer
Neurosteroids are endogenous compounds which can alter the synaptic transmission and enhance neuroprotection in neurodegenerative diseases. The systems that regulates their biosynthesis are not described but its first step ca be regulated by the TSPO. This mitochondrial protein facilitates the transport of cholesterol to the mitochondrial matrix to be metabolized in pregnenolone. This steroid is the precursor of neurosteroid biosynthesis and in vitro use of TSPO ligands induces its secretion. For this project, we looked forward to develop new families of soluble TSPO ligands that can increase pregnenolone production. The access to 3-amino-3,4-dihydroquinolin-2-ones required the establishment of a synthesis methodology of a palladium-catalyzed cyclization following Buchwald-Hartwig amination. A solubility study of synthesized compound was performed, their activity was established based on functional assays and their neuroprotective effect was evaluated on a cellular model of Alzheimer disease

Pregnenolone (PREG), the precursor of all neurosteroids, is synthesized in the nervous system from cholesterol and recent clinical studies indicate that reduced cognitive symptoms of schizophrenia correlate with elevated serum levels of pregnenolone sulfate (PregS), its immediate sulfated metabolite. PregS fulfills most of the classical criteria for an endogenous modulator of excitatory synaptic transmission, including: presence in nervous tissue at physiologically relevant concentrations, potentiation of N-methyl-D-aspartate receptor (NMDAR) mediated synaptic activity, and a mechanism for its inactivation. As NMDAR hypoactivity has been implicated in the pathophysiology of schizophrenia, defects in neurosteroid metabolism might play a role in its associated cognitive dysfunction. PregS improves memory performance in rodents and augments long-term potentiation (LTP), an electrophysiological correlate of synaptic plasticity that is stabilized by phosphorylation of the cAMP response element binding protein (CREB). We have previously demonstrated that PregS at low picomolar (pM) concentrations increases intracellular Ca2+ and CREB via synaptic NMDARs. Therefore, we hypothesized that low pM concentrations of PregS might potentiate spontaneous excitatory postsynaptic currents (sEPSCs) and promote molecular events underlying synaptic plasticity. Here, using whole-cell patch clamp recordings, we report that PregS enhances the frequency of sEPSCs of cultured hippocampal neurons by about 2-fold while not altering their amplitude or passive membrane properties. This suggests that PregS acts presynaptically by increasing the frequency of neurotransmitter release or postsynaptically by activating silent synapses. We then investigated the hypothesis that PregS increases α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) and NMDAR subtypes at synapses as a molecular switch for this enhancement. We measured receptor redistribution and phosphorylation using fluorescence imaging and Western blot technology. The results demonstrate that PregS (50pM, 10min): (1) Increases AMPAR (GluA1)/PSD95 colocalization (dependent on L-type voltage-gated Ca+2 channel and synaptic NMDAR activity), and increases phosphorylation of GluA1 at serine-831/845; (2) Increases casein kinase 2 (CK2) dependent surface NMDAR2A (GluN2A) but not GluN1 or GluN2B; and (3) Increases GluN2B serine-1480 phosphorylation. The results show that PregS increases the frequency of excitatory synaptic transmission and increases surface/synaptic AMPARs and surface GluN2A (but not GluN1 or GluN2B) NMDARs, shifting the molecular composition of young glutamatergic synapses toward the adult GluN2A enriched synaptic phenotype.

博士
國立陽明大學
生化暨分子生物研究所
101
Pregnenolone (P5) is a neurosteroid that improves memory and neurological recovery. It is also required for zebrafish embryonic development. However, its mode of action is unclear. This thesis shows that P5 promotes cell migration and microtubule polymerization by binding to a microtubule-plus-end-tracking cytoplasmic linker protein 1 (CLIP-170). CLIP-170 was captured from zebrafish embryonic extract by a P5 photoaffinity probe conjugated to diaminobenzophenone and identified by LC-MS/MS analysis. P5 interacted with CLIP-170 at its coiled-coil domain and changed it into an extended conformation. This increased CLIP-170 interaction with microtubules, dynactin subunit p150Glued, and LIS1; it also promoted CLIP-170-dependent microtubule polymerization. CLIP-170 was essential for P5 to promote microtubule abundance and zebrafish epiboly migration during embryogenesis; overexpression of the P5-binding region of CLIP-170 delayed this migration. P5 also sustained migration directionality of cultured mammalian cells. It shows that P5 activates CLIP-170 to promote microtubule polymerization and cell migration. Besides microtubules, P5 deficient embryos showed abnormal F-actin architectures and decreased endocytotic activity. P5 is required and sufficient for resistance to the F-actin disrupting agent. It shows that P5 stabilizes F-actin. Together, P5 promotes zebrafish embryonic movement by controlling both microtubule and F-actin.

碩士
國立成功大學
生理學研究所
88
The neurosteroid pregnenolone sulfate (PS) modulates neuronal excitability via a non-genomic mechanism. When administered intracerebroventricularly to experimental animals, PS also enhances post-training memory performance. Epilepsy is a disorder of brain function characterized by recurrent seizures that have a sudden onset and are evoked by over-activation of central neurons. During seizure, patients usually generate convulsive movement and sometimes lose consciousness. Memory deficits, particularly deficits of long-term memory, have often been reported in patients with temporal lobe epilepsy. However, whether memory deficits also occur in epileptic animals and whether PS has a memory-enhancing effect on epileptic animals are not clear. In the present study we explore the effect of intracerebroventriclularly injected kainic acid on rat learning processes by using the passive avoidance learning paradigms. In addition, we also study the effects of PS injected intracerebroventricularly on learning performance in normal and epileptic rats. The retention latency in epileptic rats is shorter than that in normal rats, indicating poorer memory performance in epileptic rats. Rotarod treadmills and electric shock sensitivity tests exclude the possibility that the shorter retention latency in epileptic rats is due to their higher motor activity and higher shock sensitivity. Furthermore, administration of high dose (840 pmol) of PS produces a memory-enhancing effect on normal rats but has no significant effect on epileptic rats, suggesting lower sensitivity to PS in epileptic rats.

碩士
臺灣大學
化學研究所
98
The semiconductor nanocrystal quantum dots (QDs) are versatile fluorophores with unique photophysical properties, including narrow and size-dependent luminescence, broad absorption spectra, and high quantum yields. Bioconjugation of surface functionalized QDs is applied to biological imaging and cellular tracking.1 Embryonic cell movement is essential for morphogenesis and the establishment of body shapes. In previous research, we showed that the steroid hormone, pregnenolone, preserves microtubule abundance and promotes cell movement during epiboly.2 To visualize the interaction between pregnenolone and pregnenolone binding proteins (PBPs) on microtubules, the pregnenolone-conjugated QDs are fabricated as a tool to evidence the pregnenolone-related biological events. Hence, we synthesized a series of functionalized water-soluble QDs with heterobifunctional ligands incorporating dihydrolipoic acid (DHLA), a hydrophilic spacer, and a functional (amino) or nonfunctional (hydroxy) terminus. The length of hydrophilic spacer is studied by comparing triethylene glycol (TEG) spacer and poly(ethylene glycol) (PEG) spacer. The covalent conjugation will be finally achieved by amide coupling between functional ligands and pregnenolone derivatives. It is hoped tracking the PBPs in embryonic cells of zebrafish can be achieved with these pregnenolone-conjugate QDs.

Preclinical results support the use of N-methyl D-aspartate receptor (NMDAR) modulators for cognition enhancement therapeutics. Pregnenolone sulfate (PregS) is a neuroactive steroid derived from cholesterol that augments long term potentiation (LTP) in hippocampal slices and improves memory performance in rats and mice. At micromolar concentrations, PregS is a subtype selective positive allosteric modulator of NMDARs at NR2A and NR2B containing receptors, and at concentrations ranging from pM - nM induces NMDAR-dependent dopamine release in the striatum and from striatal synaptosomes. In this report, we observe that micromolar [PregS] induces an increase in levels of neuronal intracellular calcium ([Ca^2+]i) and surface NMDARs in cortical neurons. Moreover, our results show that PregS stimulated upregulation of surface NR1 subunits in cortical neurons is dependent on NMDARs but independent of channel activity. As PregS has been detected in brain at bulk concentrations of 0.1 nM to 5 nM, we asked whether low, picomolar concentrations of PregS might alter [Ca^2+] levels. We report here that PregS increases [Ca^2+]i signal in cortical neurons in a voltage-gated Na^+ channel and NMDAR-NR2B dependent manner with an EC50 of ~2 pM, at least 6 orders of magnitude higher affinity than its rapid potentiating effect upon the NMDAR-mediated ionotropic response, and within the range of PregS detected in bulk brain tissue. Additionally, calcium (Ca^2+) activation of cyclic AMP response element binding protein (CREB) is critical to the protein synthesis-dependent component of LTP and important in associated behavioral measures of learning and memory. Increased [Ca^2+]i levels are known to induce CREB activation and we now show that 50 pM PregS induces a 44 ± 13% increase in the ratio of pCREB to total CREB that is dependent upon ERK signaling and canonical excitatory synaptic transmission: this includes voltage gated Na+ channels, NMDARs, and voltage-gated Ca^2+ channel activation. The results taken together indicate that PregS may be a useful platform for the development of high-affinity positive modulators of NMDAR-signaling that can be used as cognitive enhancers to treat a variety of neurological disorders: such as Alzheimer's disease, Parkinson's disease, and schizophrenia.

博士
國立成功大學
基礎醫學研究所
93
Previous studies have shown that neurosteroids dehydroepiandrosterone (5-androsten-3b-ol-17-one; DHEA) and pregnenolone sulfate (5-pregnen-3b-ol-20-one sulfate; PS) modulate amino acid receptor-mediated responses in brain. Compared to the extensive studies of DHEA and PS effect on amino acid receptors, relatively little is known of interaction of DHEA and PS with the capsaicin receptor. In the present study, we investigated the effects of DHEA and PS on the capsaicin receptor-mediated current in acutely dissociated rat dorsal root ganglion neurons using the whole-cell voltage-clamp recording technique. DHEA and PS rapidly and reversibly inhibited the capsaicin-induced current in a concentration-dependent manner, with EC50 values of 6.7 and 13 mM and maximal inhibition of 100 and 65%, respectively. DHEA increased the capsaicin EC50 with little effect on the capsaicin maximal response, suggesting that the blocking action of DHEA was competitive. Neither the capsaicin response nor inhibition of the capsaicin response by extracellularly applied DHEA was significantly affected by inclusion of a saturating concentration of DHEA in the electrode buffer, arguing that DHEA acted at the extracellular surface of the membrane. Moreover, DHEA did not act through protein phosphatases 1, 2A or 2B to inhibit the capsaicin-induced current. Not all steroids inhibited the capsaicin response. Progesterone did not exert any significant effect on the capsaicin-induced current, suggesting that inhibition by DHEA of the capsaicin response was a specific effect. Furthermore, the stereoisomer of DHEA, 5-androsten-3a-ol-17-one (3a-DHEA), failed to inhibit the capsaicin-induced current, producing instead a potentiating effect on the capsaicin response, demonstrating that interaction of steroids with the capsaicin receptor was stereospecific. Neither the capsaicin response nor inhibition of the capsaicin response by extracellularly applied PS was significantly affected by inclusion of a saturating concentration of PS in the electrode buffer, arguing that PS acted at the extracellular surface of the membrane. Furthermore, PS inhibited the capsaicin maximal response with little effect on the capsaicin EC50, demonstrating that the blocking action of PS was noncompetitive. The fact that antagonism of the capsaicin response by PS was neither voltage- nor agonist-dependent, indicating that PS did not act as an open-channel blocker. In addition, we investigated the effects of intradermal administration of PS on the nociceptive response evoked by intradermal injection of capsaicin into the rat hindpaw. Results revealed that PS dose-dependently inhibited the capsaicin-induced nociceptive response. Moreover, PS did not act through opioid- and cannabinoid-dependent mechanisms to reduce the capsaicin-induced nociception. Furthermore, intradermal injection of capsaicin did not cause the release of endogenous opioids and cannabinoids. Our results suggested that PS depressed the capsaicin-induced nociception via direct inhibition of the capsaicin receptor.

博士
國立臺灣大學
化學研究所
104
The work presented here consists of two parts: Section I describes that pregnenolone (P5) was equipped with benzophenone photoreactive group and biotin tag at C7 position in ether linkage to explore P5-binding proteins in the stage of embryonic development of the zebrafish. Various spacer lengths and orientations of P5-photoaffinity probes had been employed to investigate the influences on the activity of in vitro tubulin polymerization. With the preservation of the biological functions as P5, P5-NBPN was used to label P5-binding proteins from zebrafish embryo lysates and the P5 binding protein (Figure 1), cytoplasmic linker protein 170 (CLIP-170), had ultimately been found by LC-MS/MS identification. The photolabeling experiments of CLIP-170 and/or its various depletion mutants showed that the binding region of P5 on CLIP-170 located in the region between aa 920-970 with remarkable labeling selectivity and specificity. Section II describes that a series of G-quadruplex (G-4)-directing alkylating agents, BMVC-CnM (n = 2, 3, and 6) and BMVC-SW, integrating BMVC with aniline mustard in spacers of various lengths or with longer bridge length to react with different G-4 structures (hybrid-2 type, antiparallel and parallel) (Figure 2). The intact alkylated adducts were elaborately characterized by electrospray ionization mass spectroscopy (ESI-MS), LC-MS, and chemical/enzymatic footprinting to determine precise alkylation sites and plausible binding profiles. These results indicated that alkylation selectivity, specificity, and reactivity are modulated by adjusting linker lengths, whereas intrastrand cross-link efficiency which showed higher cytotoxicity is determined by the distance between two reactive warheads. Our preliminary findings regarding the different distance effects on G-4-specific alkylation provide a structural foundation for the development of G-4-selective bifunctional alkylating agents.

碩士
國防醫學院
生物及解剖學研究所
104
Alzheimer’s disease (AD) is a progress neurodegenerative disorder that associated with the defects of cognition and memory. Recent studies showed that insulin resistant in the brain may play an important role in the etiopathogenesis of the sporadic AD (sAD). In this study, the SD rat with streptozotocin intracerebroventricular (STZ-icv) treatment was used to be a sAD animal model. The open field test (OFT) and radial arm maze (RAM) behavior test were carried out to evaluate the cognitive and memory deficits in STZ-icv rat. Moreover, the [18F]FDG and [18F]T807 coupled with animal positron emission tomography (animal-PET) were also used to evaluate the status of brain glucose metabolism and the expression of paired helical filament (PHF)-tau protein, respectively. Volumes of interest (VOIs) of brain regions were drawn on PET images and compared between control and STZ-icv rats. We also examined the neuroprotective effects of dextromethorphan (DM) and pregnenolone sulfate (PS) in STZ-icv rat. After the last PET scanning, all the rats were sacrificed for the phosphorylated tau and glucose transporter 3 (GLUT3) immunohistochemical (IHC) staining. The results of OFT and RAM tests showed cognitive and memory deficits in STZ-icv induced sAD rat. The PET images showed that [18F]FDG uptakes was decreased and [18F]-T807 uptake was increased in the rat brains after STZ-icv injection. The STZ-icv rats with DM or PS treatments showed significant uptake changes of [18F]FDG and [18F]T807 in rat brains. Previous studies have shown that DM can be improved caused by NMDA receptors adjusting the neurotoxicity hypoxia and neuronal damage. And pregnenolone sulfate is the most abundant and primitive steroid in the rat and human nerve tissue. Early studies have indicated pregnenolone sulfate has anti-inflammatory and inhibit oxidative stress in the central nervous system. In this study the results showed that both drugs have neuroprotective effects against the STZ-induced neurotoxicity. The results of this study may clarify the toxicity of STZ in the rat brains, and the model may provide a platform for in developing novel therapeutic druds for AD.

碩士
國立臺灣大學
化學研究所
100
Affinity-based probes (AfBPs) have been developed recently by several research groups. Both of Cravatt and Yao groups independently reported the first AfBPs for metalloproteases. AfBPs achieves labelling by binding at a specific site of a protein followed by a non-specific covalent bond-forming event. The seminal works of AfBPs research enlighten us to design and synthesize probes for targeting different biomolecules of interest. 　　Embryonic cell movement is essential for morphogenesis and establishment of body shape. In the previous research reported by our group, showed the steroid hormone, pregnenolone, can preserve the abundance of microtubules, and thus effectively promotes the cell movement. In light of this study, we speculate that there might be pregnenolone binding proteins (PBPs), which can bind pregnenolone and further control the formation of microtubules. To verify our assumption, the photoreactive probes (P5C7b-O-NBPN) composed of a pregnenolone as the PBPs binding group, benzophenone as the photo cross-linker and a biotin as the reporter were designed and synthesized. It is hoped that by using this set of probes, PBPs could be identified and further purified prospectively. 　　On the other hand, telomeres are specialized DNA ends providing protection against genomic instability and cell senescence during cell division. The homeostasis of telomere is maintained by telomerase, which is selectively expressed in most tumors, and hence has a crucial role in cellular immortalization.38(?) The G-rich sequence of human telomeric DNA has a strong propensity to form the DNA G-quadruplex secondary structure, which can inhibit the activity of telomerase.53(?) Recently, 3,6-bis(1-methyl-4-vinylpyridium) carbazole diiodide (BMVC) was reported as a G-quartet stabilizer and a fluorescence probe. Preserving the key structural features of BMVC, a series of BMVC-mustard conjugates (BMVC-CnM; n = 2, 3, 6) were designed and synthesized in our group in attempts to develop telomere-directed DNA alkylating agents. The recognition between BMVC-CnM and G-quarduplex were analyzed and confirmed by various spectroscopic tools and DNA-PAGE studies previously. In this thesis, FRET analysis and DNA footprinting were applied to investigate the binding modes of BMVC-CnM with G-quarduplex. Furthermore, the TRAP-assay and cell images experimental results indicate BMVC-CnM have great potentials to be developed as anti-tumor agents.

碩士
國立成功大學
生理學研究所
95
The capsaicin receptor is expressed predominantly by sensory neurons and has been implicated in nociception and inflammatory thermal hyperalgesia. Previous studies from our laboratory have shown that the neurosteroid pregnenolone sulfate (PS) inhibits, but the female sex steroid 17β-estradiol (E2) potentiates, both the capsaicin receptor-mediated current in male rat dorsal root ganglion neurons and the capsaicin receptor-mediated nocifensive response in male rats. However, it is unclear whether PS and E2 also have modulatory effects on capsaicin-induced thermal hyperalegsia. In the present study, we used pharmacological methods and nociceptive behavioral tests to characterize the effects of PS and E2 on capsaicin-induced heat hypersensitivity and nocifensive responses in male rats in vivo. Our results revealed that intraplantar injection of PS dose-dependently attenuated capsaicin-induced thermal hyperalgesia. PS alone had no effect on heat sensitivity, indicating that the effect of PS on capsaicin-induced thermal hyperalgesia was not due to an increase in nociceptive threshold. Moreover, inhibition by PS of capsaicin-induced thermal hyperalgesia was not reduced by FK506, a selective calcineurin inhibitor, suggesting that the effect of PS was not mediated by calcineurin. Furthermore, intraplantar injection of E2 potentiated capsaicin-induced thermal hyperalgesia in male rats. In addition, co-injection of E2 but not 17α-estradiol with a subthreshold dose of capsaicin induced thermal hyperalgesia, indicating that the reduction by E2 of the capsaicin threshold for thermal hyperalgesia was stereospecific. E2 alone did not alter the heat sensitivity. Administration of capsazepine, a capsaicin receptor antagonist, blocked thermal hyperalgesia induced by co-injection of subthreshold doses of E2 and capsaicin, suggesting that thermal hyperalgesia was mediated by the capsaicin receptor. Furthermore, pretreatment of a protein kinase C (PKC) or a PKA inhibitor completely inhibited the synergistic effect of co-injected capsaicin and E2 on thermal hyperalgesia, arguing strongly for the involvement of PKC and PKA. In contrast, administration of PKC or PKA inhibitor did not block the potentiating effect of E2 on capsaicin-induced nocifensive responses. In conclusion, these data demonstrate that PS inhibits capsaicin-induced heat hypersensitivity via a calcineurin-independent mechanism, and that PKC and PKA are involved in the reduction by E2 of the capsaicin threshold for thermal hyperalgesia but not in the potentiating effect of E2 on capsaicin-induced nocifensive responses.

碩士
國立臺灣大學
化學研究所
101
The development of proteomics has opened up new horizons for the research of many diseases and drugs invention. Meanwhile, the related technology of affinity-based probes (AfBPs) provides more direct evidence about the mediation and regulation of protein in physiological condition. The actions of AfBPs are mainly based on molecular recognition, with the introducing of photoreactive groups (PGs) to achieve the specific covalent modification on protein. With the directing of different substrate, allowing scientists for the corresponding binding protein studies to understand the interaction between the protein and disease. This thesis is mainly divided into three parts: The first part is to study the embryonic-cell-movement related proteins. Our previous research indicated pregnenolone (PREG) can preserve the abundance of microtubules and effectively promote the development of embryonic cell, but the corresponding pregnenolone binding proteins (PBPs) is not clear. However, our recent studies found that pregnenolone probe (P5-NBPN) can effectively promote microtubule assembly, increase cell migration rate and specifically target to cytoplasmic linker protein (CLIP1, or CLIP -170). To validate the structural interaction about the binding mode and mechanism between pregnenolone and CLIP-170, we introduced the reactive groups at different positions on pregnenolone. At this point, We designed and synthesized the photoreactive probes (P5C20-NBPN) composed of pregnenolone as recognition unit, benzophenone as the photo cross-linker and a biotin as the reporter. Hope to compare the efficiency among these regio-affinity probes of pregnenolone on photolabeling experiments, and to understand more details about the binding mode, as the basis for drug development in the future. The second part is to study and discuss about G-quadruplex helicase. Many studies have demonstrated that the protein can maintain genomic stability, suppress inappropriate genetic recombination and inhibit of tumor progression, but still do not know about the mechanism of action. We designed and synthesized BMVC-DzN3, directing by the ligand of G-quadruplex. After reactive groups excited by irradiation, helicase is expected to be selectively labeling to obtain the direct binding evidence with G-quadruplex structures and explore its subsequent physiological functions. Although DNA-alkylated agents display excellent ability to inhibit cancer cell growing, but due to low selectivity, resulting in extremely serious side effects on the clinical application. In the third part, we hope to take advantage on molecular recognition and the concept of prodrug. BMVC equipped with DNA-alkylated agents - nitrogen mustard sheltered by phenylboronic ester were synthesized, named BMVC-Ak, to achieve high cytotoxicity toward cancer cells with good selectivity.