The present audiographic study examined ligands for three receptors, chosen on the basis of high abundance of these receptors in the SCN relative to other brain regions([125I)VIP and [125I) angiotensin II) or the ability of pharmacological manipulations to affect the phase and period of circadian rhythms.

Several lines of evidence implicate a dysregulation of tryptophan hydroxylase (TPH)-dependent serotonin (5-HT) synthesis in emotions and stress and point to their potential relevance to the etiology and pathogenesis of various neuropsychiatric disorders. However, the differential expression pattern of the two isoforms TPH1 and TPH2 which encode two forms of the rate-limiting enzyme of 5-HT synthesis is controversial. Here, a comprehensive spatio-temporal analysis clarifies TPH1 and TPH2 expression during pre- and postnatal development of the mouse brain and in adult human brain as well as in peripheral organs including the pineal gland. Four different methods (real time PCR, in situ hybridization, immunohistochemistry and Western blot analysis) were performed to systematically control for tissue-, species- and isoform-specific expression on both the pre- and posttranslational level. TPH2 expression was consistently detected in the raphe nuclei, as well as in fibres in the deep pineal gland and in the gastrointestinal tract. Although TPH1 expression was found in these peripheral tissues, no significant TPH1 expression was detected in the brain, neither during murine development, nor in mouse and human adult brain. Also under conditions like stress and clearing the tissue from blood cells, no changes in expression levels were detectable. Furthermore, the reuptake of 5-HT into the presynaptic neuron by the serotonin transporter (SERT) is the major mechanism terminating the neurotransmitter signal. Thus, mice with a deletion in the Sert gene (Sert KO mice) provide an adequate model for human affective disorders to study lifelong modified 5-HT homeostasis in interaction with stressful life events. To further explore the role of TPH isoforms, Tph1 and Tph2 expression was studied in the raphe nuclei of Sert deficient mice under normal conditions as well as following exposure to acute immobilization stress. Interestingly, no statistically significant changes in expression were detected. Moreover, in comparison to Tph2, no relevant Tph1 expression was detected in the brain independent from genotype, gender and treatment confirming expression in data from native animals. Raphe neurons of a brain-specific Tph2 conditional knockout (cKO) model were completely devoid of Tph2-positive neurons and consequently 5-HT in the brain, with no compensatory activation of Tph1 expression. In addition, a time-specific Tph2 inducible (i) KO mouse provides a brain-specific knockdown model during adult life, resulting in a highly reduced number of Tph2-positive cells and 5-HT in the brain. Intriguingly, expression studies detected no obvious alteration in expression of 5-HT system-associated genes in these brain-specific Tph2 knockout and knockdown models. The findings on the one hand confirm the specificity of Tph2 in brain 5-HT synthesis across the lifespan and on the other hand indicate that neither developmental nor adult Tph2-dependent 5-HT synthesis is required for normal formation of the serotonergic system, although Tph1 does not compensate for the lack of 5-HT in the brain of Tph2 KO models. A further aim of this thesis was to investigate the expression of the neuropeptide oxytocin, which is primarily produced in the hypothalamus and released for instance in response to stimulation of 5-HT and selective serotonin reuptake inhibitors (SSRIs). Oxytocin acts as a neuromodulator within the central nervous system (CNS) and is critically involved in mediating pain modulation, anxiolytic-like effects and decrease of stress response, thereby reducing the risk for emotional disorders. In this study, the expression levels of oxytocin in different brain regions of interest (cortex, hippocampus, amygdala, hypothalamus and raphe nuclei) from female and male wildtype (WT) and Sert KO mice with or without exposure to acute immobilization stress were investigated. Results showed significantly higher expression levels of oxytocin in brain regions which are involved in the regulation of emotional stimuli (amygdala and hippocampus) of stressed male WT mice, whereas male Sert KO as well as female WT and Sert KO mice lack these stress-induced changes. These findings are in accordance with the hypothesis of oxytocin being necessary for protection against stress, depressive mood and anxiety but suggest gender-dependent differences. The lack of altered oxytocin expression in Sert KO mice also indicates a modulation of the oxytocin response by the serotonergic system and provides novel research perspectives with respect to altered response of Sert KO mice to stress and anxiety inducing stimuli. / Durch zahlreiche Untersuchungen ist belegt, dass eine gestörte Tryptophan-Hydroxylase (TPH)-abhängige Serotonin (5-HT)-Synthese an einer veränderten emotionalen Reaktion sowie einer veränderten Stress-Antwort beteiligt ist und damit auch in der Ätiologie und Pathogenese psychischer Erkrankungen eine Rolle spielt. Dennoch werden nach wie vor die unterschiedlichen Expressionsmuster der beiden Isoformen TPH1 und TPH2, die für zwei Formen des Schrittmacherenzyms der 5-HT-Synthese kodieren, kontrovers diskutiert. Zentrales Anliegen dieser Arbeit ist daher eine Klärung der TPH1- und TPH2-Expression während der prä- und postnatalen Entwicklung des murinen Gehirns, sowie im adulten humanen Gehirn und in einigen peripheren Organen und der Zirbeldrüse. Durch die Verwendung von vier verschiedenen Methoden (Real time-PCR, In situ-Hybridisierung, Immunhistochemie und Westernblot-Analysen) wurde systematisch die Gewebs- und Isoform-spezifische Expression in Maus und Mensch auf prä- und posttranslationaler Ebene nachgewiesen. TPH2-Expression wurde Spezies-übergreifend in den Raphe-Kernen des Hirnstamms wie auch in Fasern zur Zirbeldrüse und im Gastrointestinaltrakt detektiert. Auch TPH1 konnte in diesen peripheren Organen (die Zirbeldrüse eingeschlossen) nachgewiesen werden, jedoch fand sich keine signifikante TPH1-Expression im Gehirn, weder während der Entwicklung des Maus-Gehirns noch im humanen und murinen adulten Gehirn. Auch durch veränderte Bedingungen wie der Entfernung von Blutzellen aus dem Gewebe oder der Anwendung von akutem Immobilisierungsstress konnte keine Änderung der Expression gemessen werden. Sert Knockout-Mäuse, stellen ein geeignetes Tiermodell für affektive Erkrankungen dar, insbesondere um eine lebenslang veränderte 5-HT-Homöostase in Verbindung mit belastenden Lebensereignissen zu untersuchen. Um die Bedeutung der TPH-Isoformen und deren korrekte Expression weiter zu untersuchen, wurde die Tph1- und Tph2-Expression in den Raphe-Kernen von Sert Knockout (KO)-Mäusen unter normalen Bedingungen und nach akutem Stress getestet. Interessanterweise konnten keine statistisch signifikanten Expressionsänderungen entdeckt werden. Mehr noch, relativ zu Tph2 konnte unabhängig von Behandlung, Geschlecht oder Genotyp keine relevante Tph1-Expression im Gehirn gemessen werden, was wiederum die Expressionsdaten aus nativen Tieren unterstützt. Die Raphe-Neurone eines Gehirn-spezifischen konditionalen Tph2 KO-Modells zeigten weder Tph2-positive Zellen noch 5-HT, wiesen aber auch keine kompensatorische Aktivierung der Tph1-Expression im Gehirn auf. Zusätzlich repräsentiert eine zeit-spezifische, induzierbare KO-Maus ein Gehirn-spezifisches Tph2 Knockdown-Modell ab dem Erwachsenenalter, das eine stark reduzierte Anzahl an Tph2-positiven Zellen und 5-HT im Gehirn aufweist. Expressionsuntersuchungen zeigten interessanterweise, dass diese Gehirn-spezifischen Tph2 Knockout- und Knockdown-Modelle keine sichtliche Änderung in der Expression von 5-HT-System-assoziierten Genen aufweisen. Diese Ergebnisse bestätigen zum einen, dass die 5-HT-Synthese im murinen Gehirn während der kompletten Lebensspanne ausschließlich durch Tph2 katalysiert wird und weisen außerdem darauf hin, dass eine Tph2-abhängige 5-HT-Synthese weder während der Entwicklung noch im Erwachsenalter für die Ausbildung eines normalen serotonergen Systems benötigt wird, obwohl Tph1 den Verlust des 5-HT-Vorkommens im Gehirn der Tph2 KO-Mäuse nicht kompensiert. Weiterhin beschäftigt sich diese Arbeit mit der Expression von Oxytocin, das hauptsächlich im Hypothalamus produziert. Oxytocin ist maßgeblich bei Angst-lösenden Effekten sowie einer verringerten Stressantwort beteiligt. In dieser Studie wurde die Expression von Oxytocin in verschiedenen Gehirnregionen (Cortex, Hippocampus, Amygdala, Hypothalamus und Raphe Nuclei) von weiblichen und männlichen Wildtyp- (WT) und Sert KO-Mäusen getestet, die entweder unter normalen Bedingungen gehalten wurden oder eine Stunde lang akutem Immobilisierungsstress ausgesetzt waren. Die Ergebnisse zeigten eine signifikant höhere Oxytocin-Expression in Gehirnregionen, die für die emotionale Reizverarbeitung zuständig sind (Amygdala und Hippocampus) in gestressten männlichen WT-Mäusen, während männliche Sert KO-Mäuse sowie weibliche WT- und Sert KO-Mäuse diese Stress-bedingten Unterschiede nicht aufwiesen. Diese Befunde sind im Einklang mit der Hypothese, dass Oxytocin eine schützende Rolle bei Stress, depressiver Stimmung und Angst übernimmt, weisen jedoch auf einen Geschlechterunterschied hin. Ferner legt das Fehlen einer veränderten Oxytocin-Expression in Sert KO-Mäusen eine Modulation der Oxytocin-Expression durch das serotonerge System nahe, was neue Forschungsperspektiven über eine veränderte Reaktion auf Stress und Angst-auslösende Reize in Sert KO-Mäusen eröffnet.

Introdução: A epilepsia é uma doença de grande relevância médica e social, trazendo grande impacto aos pacientes e a sociedade como um todo. A Epilepsia do Lobo Temporal Mesial (ELTM) é a epilepsia refratária mais prevalente, tendo em sua causalidade o impacto do desequilíbrio entre circuitos neuronais excitatórios e inibitórios, necessitando da remoção cirúrgica das estruturas alteradas e da interrupção das suas vias para melhor controle das crises e qualidade de vida dos pacientes. Objetivo: Buscando ampliar o esclarecimento do papel da amígdala junto as modificações intrínsecas nos receptores de neurotransmissores e em suas subunidades nos mecanismos de ictogênese e epileptogênese, possibilitando o aprimoramento das técnicas cirúrgicas atualmente empregadas, além de novas modalidades terapêuticas, o presente estudo analisou as expressões gênicas das subunidades de receptores excitatórios, NMDA (NR2C e NR3A, genes GRIN2C e GRIN3A), Cainato (GluK1 e GluK2, genes GRIK1 e GRIK2), e subunidade de receptor inibitório GABAA (?4 e ?5, genes GABRA4 e GABRA5) e subtipos de receptor de neuropeptídio Y (Y2 e Y5, com genes NPY2R e NPY5R), em núcleos basolaterais de amígdalas humanas de pacientes com ELTM. Material e Métodos: Foram utilizados fragmentos de amígdala de 20 pacientes que fizeram amigdalohipocampectomia junto ao Serviço de Neurocirurgia do HC-FMRP-USP, sendo 10 pacientes com controle efetivo pós-operatório (Engel 1) e 10 pacientes com controle inadequado das crises(Engel 3 e 4), 10 amígdalas obtidas de autópsias (controle), utilizando a qPCR. Resultados: Foram evidenciadas diferenças da expressão nas subunidades NR2C (p=0,006) e ?4 do GABAAr (p=0,008), subtipo de NPYr Y2(p=0.013), com tendência junto a subunidade NR3A(p=0,077). Não evidenciando significância estatísticas nas análises das subunidades GluK1(p=0,147), GluK2(p=0,182) e?5 do GABAAr (p=0,272), para o subtipo NPYr Y1(p=0,242). Conclusão: As análises sugerem diferenças na expressão de receptores de neurotransmissores em pacientes com epilepsia em relação ao controle contendo as subunidadeNR2C e ?4 do GABAAr, com tendências a subunidade NR3A, indicando modificações neuronais amigdalianas possivelmente envolvidas com a zona epileptogênica, possibilitando aprimoramentos terapêuticos junto ao tratamento dasepilepsias refratárias. Também podemos inferir que os mecanismos neuronais envolvendo as subunidades?4 doGABAAr e GRIN2C, e do subtipo Y2 do NPYr na epileptogênese e ictogênese da ELTM podem ser semelhantes entre amígdala e hipocampo, enquanto os envolvendo as subunidades GLUK1 e GLUK2 parecem ser diferenciados; o gene GABRA5 pode ser utilizado como gene de controle endógeno em estudos com amigdala e hipocampo na ELTM. / Introduction: Epilepsy is a disease whith highly medical and social relevance, bringing impact on patients and society as a whole. Mesial Temporal Lobe Epilepsy (MTLE) is the most prevalent refractory epilepsy, in its causality the impact of the imbalance between excitatory neuronal circuits and inhibitory, needing a surgical removal of the altered structures and the interruption of their way to better seizure control and quality of life pacientes. Goal: Searching to increase understanding the role of the amygdala with intrinsic changes in neurotransmitter receptors and their subunits in ictogenesis mechanisms and epileptogenesis, enabling the improvement of surgical techniques currently used, as well as new therapeutic modalities, this study analyzed gene expression on the subunits of excitatory receptors, NMDA (NR2 and NR3A, GRIN2C and GRIN3A genes) and kainate (GluK1 and GluK2, GRIK1 and GRIK2 genes), and inhibitory receptor subunit GABA (?4 and ?5, genes GABRA4 and GABRA5 ), neuropeptide Y receptor subtypes (Y2 and Y5, and NPY5R with NPY2R gene) in the basolateral nucleus of human amygdala of patients with MTLE. Material and Methods: Amygdala fragments were used in 20 patients who made amigdalohipocampectomia with the Service neurosurgery HC-FMRP-USP, 10 patients with postoperative effective control (Engel 1) and 10 patients with inadequate control of seizures (Engel 3:04), and 10 amygdalas obtained from autopsies (control) using qPCR. Results: Were differences evidenced expression in NR2C subunits (p = 0.006) e?4 the GABAAr (p = 0.008), and subtype NPYr Y2 (p = 0.013), along with a tendency of NR3A subunits (p = 0.077). Showing no statistical significance in the analysis of GluK1 subunits (p = 0.147), GluK2 (p = 0.182) e?5 the GABAAr (p = 0.272), and the NPYr Y1 subtype (p = 0.242). Conclusion: The analyzes suggest differences in expression of neurotransmitter receptors in epilepsy patients on control containing the NR2C subunits and ?4 of GABAAr with NR3A subunits trends indicating amygdala neuronal modifications possibly involved in the epileptogenic zone, enabling therapeutic improvements with the refractory epilepsy treatment. As well can infer that the neural mechanisms involving the subunits ?4 GABAAr, GRIN2C and Y2 NPYr subtype in epileptogenesis and ictogenesis of TLE can be similar between the amygdala and hippocampus, while involving GLUK1 and GLUK2 subunits appear to be different; the GABRA5 gene can be used as endogenous control gene in studies of hippocampus and amygdala in TLE.

Acupuncture has been used for thousands of years to treat a wide range of diseases, but the mechanisms involved in the process have remained a mystery. The present study measures EEG responses to stimulation of a specific acupuncture point, GB37 (Guang Ming), with two different types of manual needle stimulation. Previous studies stimulated for a maximum of 2 minutes. The present study reflects the normal acupuncture treatment time of 20 minutes, with EEG recordings during and for 10 minutes prior to and after stimulation. Our results show no changes in the global spatial and temporal properties of EEG during and shortly after acupuncture treatment of acupoint GB37. The second part of this study examines the global protein expression of glutamic acid decarboxylase (GAD) knockout mice. GAD is the rate-limiting enzyme in the synthesis of GABA, the major inhibitory neurotransmitter in the brain. The protein content of wild type, hetero-, and homozygous GAD knockout mice brains were determined using a LC-MS-based gel-free shotgun profiling of complex protein mixtures. The data was analyzed using the Raculovic algorithm to determine the proteins differences. A short list of 32 proteins was determined with four that have been shown to be significant proteins that influence cell survival and excitotoxicity in the brain and have potential relationships with GABA. These proteins include VATPase, Glutamine synthetase, Beta-synuclein, and Micortuble associated protein (MAP). The proteomics results provide a preliminary best guess list of proteins influencing GAD and GABA production. / by Michael L. Marshall. / Thesis (Ph.D.)--Florida Atlantic University, 2011. / Includes bibliography. / Electronic reproduction. Boca Raton, Fla., 2011. Mode of access: World Wide Web.

Introdução: A epilepsia é uma doença de grande relevância médica e social, trazendo grande impacto aos pacientes e a sociedade como um todo. A Epilepsia do Lobo Temporal Mesial (ELTM) é a epilepsia refratária mais prevalente, tendo em sua causalidade o impacto do desequilíbrio entre circuitos neuronais excitatórios e inibitórios, necessitando da remoção cirúrgica das estruturas alteradas e da interrupção das suas vias para melhor controle das crises e qualidade de vida dos pacientes. Objetivo: Buscando ampliar o esclarecimento do papel da amígdala junto as modificações intrínsecas nos receptores de neurotransmissores e em suas subunidades nos mecanismos de ictogênese e epileptogênese, possibilitando o aprimoramento das técnicas cirúrgicas atualmente empregadas, além de novas modalidades terapêuticas, o presente estudo analisou as expressões gênicas das subunidades de receptores excitatórios, NMDA (NR2C e NR3A, genes GRIN2C e GRIN3A), Cainato (GluK1 e GluK2, genes GRIK1 e GRIK2), e subunidade de receptor inibitório GABAA (?4 e ?5, genes GABRA4 e GABRA5) e subtipos de receptor de neuropeptídio Y (Y2 e Y5, com genes NPY2R e NPY5R), em núcleos basolaterais de amígdalas humanas de pacientes com ELTM. Material e Métodos: Foram utilizados fragmentos de amígdala de 20 pacientes que fizeram amigdalohipocampectomia junto ao Serviço de Neurocirurgia do HC-FMRP-USP, sendo 10 pacientes com controle efetivo pós-operatório (Engel 1) e 10 pacientes com controle inadequado das crises(Engel 3 e 4), 10 amígdalas obtidas de autópsias (controle), utilizando a qPCR. Resultados: Foram evidenciadas diferenças da expressão nas subunidades NR2C (p=0,006) e ?4 do GABAAr (p=0,008), subtipo de NPYr Y2(p=0.013), com tendência junto a subunidade NR3A(p=0,077). Não evidenciando significância estatísticas nas análises das subunidades GluK1(p=0,147), GluK2(p=0,182) e?5 do GABAAr (p=0,272), para o subtipo NPYr Y1(p=0,242). Conclusão: As análises sugerem diferenças na expressão de receptores de neurotransmissores em pacientes com epilepsia em relação ao controle contendo as subunidadeNR2C e ?4 do GABAAr, com tendências a subunidade NR3A, indicando modificações neuronais amigdalianas possivelmente envolvidas com a zona epileptogênica, possibilitando aprimoramentos terapêuticos junto ao tratamento dasepilepsias refratárias. Também podemos inferir que os mecanismos neuronais envolvendo as subunidades?4 doGABAAr e GRIN2C, e do subtipo Y2 do NPYr na epileptogênese e ictogênese da ELTM podem ser semelhantes entre amígdala e hipocampo, enquanto os envolvendo as subunidades GLUK1 e GLUK2 parecem ser diferenciados; o gene GABRA5 pode ser utilizado como gene de controle endógeno em estudos com amigdala e hipocampo na ELTM. / Introduction: Epilepsy is a disease whith highly medical and social relevance, bringing impact on patients and society as a whole. Mesial Temporal Lobe Epilepsy (MTLE) is the most prevalent refractory epilepsy, in its causality the impact of the imbalance between excitatory neuronal circuits and inhibitory, needing a surgical removal of the altered structures and the interruption of their way to better seizure control and quality of life pacientes. Goal: Searching to increase understanding the role of the amygdala with intrinsic changes in neurotransmitter receptors and their subunits in ictogenesis mechanisms and epileptogenesis, enabling the improvement of surgical techniques currently used, as well as new therapeutic modalities, this study analyzed gene expression on the subunits of excitatory receptors, NMDA (NR2 and NR3A, GRIN2C and GRIN3A genes) and kainate (GluK1 and GluK2, GRIK1 and GRIK2 genes), and inhibitory receptor subunit GABA (?4 and ?5, genes GABRA4 and GABRA5 ), neuropeptide Y receptor subtypes (Y2 and Y5, and NPY5R with NPY2R gene) in the basolateral nucleus of human amygdala of patients with MTLE. Material and Methods: Amygdala fragments were used in 20 patients who made amigdalohipocampectomia with the Service neurosurgery HC-FMRP-USP, 10 patients with postoperative effective control (Engel 1) and 10 patients with inadequate control of seizures (Engel 3:04), and 10 amygdalas obtained from autopsies (control) using qPCR. Results: Were differences evidenced expression in NR2C subunits (p = 0.006) e?4 the GABAAr (p = 0.008), and subtype NPYr Y2 (p = 0.013), along with a tendency of NR3A subunits (p = 0.077). Showing no statistical significance in the analysis of GluK1 subunits (p = 0.147), GluK2 (p = 0.182) e?5 the GABAAr (p = 0.272), and the NPYr Y1 subtype (p = 0.242). Conclusion: The analyzes suggest differences in expression of neurotransmitter receptors in epilepsy patients on control containing the NR2C subunits and ?4 of GABAAr with NR3A subunits trends indicating amygdala neuronal modifications possibly involved in the epileptogenic zone, enabling therapeutic improvements with the refractory epilepsy treatment. As well can infer that the neural mechanisms involving the subunits ?4 GABAAr, GRIN2C and Y2 NPYr subtype in epileptogenesis and ictogenesis of TLE can be similar between the amygdala and hippocampus, while involving GLUK1 and GLUK2 subunits appear to be different; the GABRA5 gene can be used as endogenous control gene in studies of hippocampus and amygdala in TLE.

γ-Aminobuttersäure-Rezeptoren vom Typ A (GABAARs) und Glyzin-Rezeptoren (GlyRs) sind die wichtigsten Vermittler der schnellen synaptischen Inhibition im zentralen Nervensystem. Von wesentlicher Bedeutung für ihre ordnungsgemäße Funktion in der inhibitorischen Signalübertragung ist ihre präzise Lokalisation und Konzentration innerhalb der neuronalen Oberflächenmembran. Diese Eigenschaften werden durch Gerüstproteine vermittelt, welche direkt an die großen intrazellulären Schleifen der Rezeptoren, sowie an Bausteine des neuronalen Zytoskeletts binden. In meiner Dissertation habe ich die molekularen Details mehrerer zugrunde liegenden Protein-Protein Wechselwirkungen untersucht. Im Speziellen habe ich die Interaktion ausgewählter GABAAR und GlyR Untereinheiten mit den Gerüstproteinen Gephyrin, Radixin und Collybistin analysiert. Ich habe kurze lineare Aminosäuren-Motive innerhalb der großen intrazellulären Schleifen der Rezeptoren identifiziert, welche die direkten und Untereinheit-spezifischen Interaktionen vermitteln. Die Quantifizierung der jeweiligen Bindungsstärke ergab, dass Gephyrins E-Domäne vor allem an die GABAAR α1 (Kd = 17 M) und α3 (Kd = 5 M) -Untereinheiten bindet, wohingegen die SH3-Domäne von Collybistin hauptsächlich mit der GABAAR α2-Untereinheit interagiert (Kd = 1 M). Demgegenüber bindet die FERM-Domäne von Radixin fest an die α5-Untereinheit des GABAAR (Kd = 8 µM). Weiterhin zeigt meine Arbeit, dass diese einfache Beziehung durch (i) fehlende oder (ii) überlappende Bindungsspezifitäten zwischen den Gerüstproteinen und den Rezeptor-Untereinheiten komplex reguliert wird. Ferner beschreibe ich hier, wie im Folgenden ausgeführt, die Möglichkeit einer (iii) negativen Modulation mittels posttranslationaler Modifikation, sowie einer Verstärkung der Bindung durch (iv) Aviditäts-Effekte. (i) Als erstes habe ich mit Hilfe biochemischer Methoden die Radixin-GABAAR α5 Interaktion im Detail untersucht. Meine Strukturanalyse und Kompetitionsstudien legen den Schluss nahe, dass Radixin die betreffende Rezeptor-Untereinheit mittels einer universellen Bindungstasche in der F3 Subdomäne innerhalb seiner FERM Domäne bindet. Diese Bindungsstelle wird durch zwei markante Strukturelemente gebildet: Einer α-Helix, die eine große hydrophobe Tasche bildet, welche eine Vielzahl unterschiedlicher hydrophober Reste in verschiedenen Konformationen akzeptiert, sowie ein β-Strang, der Peptidrückgrat-Interaktionen eingehen kann. Es überrascht nicht, dass eine Vielzahl an Studien die Beteiligung dieser Bindungsseite mit unterschiedlichen Liganden beschrieben hat. Diese Promiskuität unterstreicht die Bedeutung des Aktivierungsmechanismus der zuvor für die Radixin FERM GABAAR α5-Untereinheit beschrieben wurde und impliziert weitere Regulationsmechanismen, die eine koordinierte Interaktion in vivo ermöglichen. (ii) Weiterhin habe ich mich ausführlich der Analyse der Gephyrin-vermittelten GABAAR Clusterbildung gewidmet. Meine röntgenkristallographischen Studien und Bindungsstudien zeigen, dass Gephyrin mit den GABAAR α1, α2 und α3 Untereinheiten über eine universelle Bindungsstelle interagiert, welche auch die Wechselwirkungen mit der β-Untereinheit des GlyR vermittelt. Mittels Struktur-basierter Mutagenesestudien konnte ich die Schlüsselreste innerhalb von Gephyrin und der Rezeptor-Untereinheiten identifizieren, die einen entscheidenden Beitrag zur Gesamt-Bindungsstärke liefern. Insbesondere zwei konservierte aromatische Reste in der N-terminalen Hälfte der Rezeptorbindungsregion gehen entscheidende hydrophobe Wechselwirkungen mit Gephyrin ein. Dementsprechend konnte J. Mukherjee, ein Mitarbeiter in der Gruppe unseres Kooperationspartners Steven J. Moss, zeigen, dass der Austausch dieser Reste innerhalb der α2-Untereinheit des GABAAR ausreicht, um einen deutlichen Rückgang der Rezeptor Cluster-Anzahl und ihrer Größe in primären hippokampalen Neuronen zu verursachen. Die Ausweitung meiner Rezeptor-Interaktions-Studien auf Collybistin (CB) ergab, dass dieses Protein im Vergleich zu Gephyrin eine umgekehrte, aber dennoch überlappende Rezeptor-Untereinheiten-Präferenz aufweist. Die GABAAR α3-Untereinheit bindet ausschließlich an Gephyrin (Kd = 5 µM), während die GABAAR α1-Untereinheit zwar vor allem Gephyrin bindet (Kd = 17 µM), zusätzlich jedoch eine schwache Affinität (Kd ≈ 400 µM) für die SH3-Domäne von CB aufweist. Im Gegensatz dazu bindet die GABAAR α2-Untereinheit hochaffin an die SH3-Domäne von CB (Kd = 1 µM) und zeigt zusätzlich eine schwache Gephyrin Affinität (Kd ≈ 500 µM). Interessanterweise konnte ich Synergieeffekte zwischen der GABAAR α2-Untereinheit, Gephyrins E-Domäne und CBs SH3-Domäne ausschließen und statt dessen zeigen, dass diese Rezeptor-Untereinheit exklusiv entweder Gephyrin oder CB bindet. Diese Ergebnisse lassen vermuten, dass die Rolle von CB in der Rezeptor-Anhäufung allein durch die konkurrierenden Bindungs-Ereignisse seiner konstituierenden Domänen bestimmt wird. Die intramolekulare Assoziation zwischen der PH und der DH-Domäne mit der SH3-Domäne von CB konkurriert mit unterschiedlichen intermolekularen Wechselwirkungen von CB. Und zwar mit der GABAAR α2-Untereinheit-Bindung an die SH3-Domäne, mit der PIP2-Bindung an die PH-Domäne, sowie mit der Gephyrin-Bindung, welche vermutlich von der PH und DH-Domäne von CB vermittelt wird. (iii) Interessanterweise bestätigen frühere Studien, dass die Rezeptor-Motive, die ich hier identifiziert habe und welche direkt mit den Gerüst-Proteinen wechselwirken, in vivo posttranslational modifiziert vorliegen. Insbesondere wurde gezeigt, dass die Gephyrin-Bindemotive der GABAAR α1-Untereinheit und GlyR β-Untereinheiten Ziele des ERK/MAPK und PKC-Phosphorylierungs-Weges sind, während das Radixin-Bindungs-Motiv innerhalb der GABAAR α5-Untereinheit ubiquitiniert vorliegt. In dieser Dissertation habe ich im Besonderen die ERK-Phosphorylierung von Thr348 in der GABAAR α1-Untereinheit untersucht. Tatsächlich konnten meine Bindungs-Assays eine starke Reduktion der direkten Gephyrin Bindungsstärke beim Einbringen eines phosphomimetischen Restes bestätigen. Darüber hinaus konnte J. Mukherjee eine signifikante Reduktion der Cluster-Anzahl und Größe beim Einführen der gleichen Mutation in die α1-Untereinheit beinhaltenden GABAARs in hippokampalen Neuronen beobachten. Der ERK/MAPK-Regulation-Weg ist daher ein aussichtsreicher Kandidat für die Regulation der GABAergen-Signalübertragung. (iv) In vivo bildet Gephyrin vermutlich durch Selbstorganisation seiner G (GephG) und E-Domänen (GephE) ein multivalentes Gerüst. Angesichts der multimeren Natur Gephyrins und der pentameren Rezeptorarchitektur habe ich die Möglichkeit von Aviditäts-Effekten im Prozess der synaptischen Neurotransmitter-Rezeptor-Anhäufung untersucht. Die Kristallstrukturen von GephE im Komplex mit ausgewählten Peptiden zeigen zwei Rezeptor-Bindungsstellen in räumlicher Nähe (15 Å). Auf der Basis dieser Information habe ich bivalente Peptide entworfen, welche beide Rezeptor-Bindungsstellen in Gephyrin simultan besetzen können und, wie erwartet, konnte ich mit Hilfe verschiedener biophysikalischen Methoden eine unübertroffen hohe, durch Avidität potenzierte, Gephyrin-Affinität nachweisen. Mir gelang es diesen Aviditäts-Effekt für einen schwachen Gephyrin Liganden, ein GABAAR-abgeleitetes Peptid, welcher nicht mit herkömmlichen monomeren Liganden untersucht werden konnte, nutzbar zu machen. Darüber hinaus konnte ich zeigen, dass diese Verbindung gezielt die Rezeptor-Bindungsstelle in GephE besetzt und auf diese Weise hemmend auf Gephyrins Rezeptorbindungsaktivität wirkt. Eine weitere Entwicklung dieser Verbindung könnte die Möglichkeit eröffnen, spezifisch die Wirkung der Entkopplung der Gephyrin Rezeptor-Interaktion in der Zellkultur-Experimenten zu analysieren ohne dabei die Anzahl oder die Funktion der Proteine zu beeinträchtigen, was einen Nebeneffekt von konventionellen Methoden wie Gen „knock-out“, RNA-Interferenz oder den Einsatz von Antikörpern darstellt. / γ-Aminobutyric acid type A receptors (GABAARs) and glycine receptors (GlyRs) are the major mediators of fast synaptic inhibition in the central nervous system. For proper synaptic function their precise localization and exact concentration within the neuronal surface membrane is essential. These properties are mediated by scaffolding proteins which directly contact the large intracellular loops of the receptors and tether them to cytoskeletal elements of the neuronal cells. In my thesis I deciphered the molecular details of several underlying protein-protein interactions, namely the interaction of a subset of GABAAR and GlyR subunits with the scaffolding proteins gephyrin, radixin and collybistin. I determined short linear motifs within the large intracellular loops of the receptors that directly engage in subunit specific scaffold protein interactions. My quantitative binding studies revealed that gephyrins E domain primarily recognizes the GABAAR α1 (Kd = 17 M) and α3 (Kd = 5 M) subunits, in contrast, the SH3 domain of collybistin mainly interacts with the GABAAR α2 subunit (Kd = 1 µM), while the FERM domain of radixin tightly binds to the GABAAR α5 subunit (Kd = 8 µM). My work additionally demonstrated that this simple relationship is complicated by (i) missing or (ii) overlapping binding specificities between the scaffold proteins and the receptor subunits. Moreover, this thesis addressed the possibility of (iii) posttranslational negative regulation as well as amplification generated by (iv) avidity effects as summarized below. (i) First, using biochemical methods I mapped the radixin-GABAAR α5 interaction in detail. My structural analysis and competition assays suggest that radixin mediates the receptor subunit binding via a universal binding site within the F3 subdomain of its FERM domain. This binding site is formed by an α-helix that offers a large hydrophobic pocket, which accepts a variety of different hydrophobic residues adopting different conformations, and a β-strand that readily engages in peptide backbone interactions. Not surprisingly, this binding site has been implicated in a wide variety of different scaffold interactions, thus emphasizing the importance of the essential FERM activation mechanism described earlier and suggesting additional pathways to allow tight regulation of this interaction. (ii) Next, I analyzed in detail the process of gephyrin-mediated GABAAR clustering. My X-ray crystallographic studies and binding assays revealed that gephyrin mediates binding of the GABAAR α1, α2 and α3 subunit via a universal binding site that also mediates the interactions with the GlyR β subunit. Using structure-guided mutagenesis I identified key residues within gephyrin and the receptor subunits that act as major contributors to the overall binding strength. Namely, two conserved aromatic residues within the N-terminal half of the receptor binding region engage in crucial hydrophobic interactions with gephyrin. Accordingly, J. Mukherjee from the group of our collaborator Steven J. Moss verified a substantial decrease in GABAAR cluster number and size in primary hippocampal neurons upon exchange of these residues within the GABAAR α2 subunit. Extension of my studies to collybistin (CB) revealed an overlapping but reciprocal subunit preference for this protein in comparison to gephyrin. The GABAAR α3 subunit exclusively binds gephyrin, in contrast the GABAAR α1 subunit mainly targets gephyrin (Kd = 17 µM) but additionally displays a moderate affinity (Kd ≈ 400 µM) towards the SH3 domain of CB. The GABAAR α2 subunit binds tightly to the SH3 domain of CB (Kd = 1 µM) and additionally displays a weak gephyrin affinity (Kd ≈ 500 µM). Notably, I could exclude the possibility of synergistic effects between gephyrins E domain, the SH3 domain of CB and the GABAAR α2 subunit. Instead, I found that the GABAAR α2 subunit binds gephyrin and CB in a mutually exclusive manner. These results suggest that CBs role in receptor clustering is solely determined by competing binding events of its constituting domains. Namely, the intra-molecular association between the PH/DH domain and the SH3 domain within CB competes with different inter-molecular interactions of CB: GABAAR α2 binding to the SH3 domain, PIP2 binding to the PH domain and gephyrin presumably binding to the PH and DH domain of CB. (iii) Interestingly, the receptor motifs, which have been mapped in my thesis to directly interact with the scaffold proteins, were shown in earlier studies to be posttranslationally modified in vivo. In particular, the GABAAR α1 and GlyR β subunits have been implicated as targets of the ERK/MAPK and PKC phosphorylation-pathways, respectively, while the GABAAR α5 subunit motif was shown to be ubiquitinated. In this dissertation, I analyzed Thr348, a possible ERK phosphorylation site within GABAAR α1. My binding assays verified a severe reduction of the direct gephyrin binding strength upon introduction of the respective phosphomimetic residue. The relevance of this in vitro result was highlighted by J. Mukherjee who confirmed a significant reduction in GABAAR cluster number and size upon introduction of the same mutation. The ERK/MAPK pathway is therefore a promising candidate for regulation of GABAergic transmission. (iv) In vivo, gephyrin presumably forms a multivalent scaffold, which is based on the self-association of its G (GephG) and E domains (GephE). Given the multimeric nature of gephyrin and the pentameric receptor architecture, I tested the possibility of avidity in the clustering of inhibitory neurotransmitter receptors. Cocrystallization of selected minimum peptides with GephE and their crystal structure analyses enabled me to define a receptor-derived peptide that offers a maximized gephyrin affinity. The structure of the GephE-GlyR  receptor complex reveals two receptor-binding sites in close spatial vicinity (15 Å). I therefore designed bivalent peptides that enable to target both GephE sites at the same time and, as expected, a variety of biophysical methods verified an avidity-potentiated and unmatched high gephyrin affinity for these bidentate compounds. Notably, I could extend the dimerization approach to low affinity gephyrin ligands, namely short GABAAR-derived peptides that could not be studied using conventional monomeric ligands. Additionally, I verified that this compound specifically targets GephEs receptor binding site, and that it thereby inhibits its receptor binding activity. Further development of this molecule may offer the possibility to specifically analyze the effect of uncoupling the gephyrin-receptor interaction in cell culture-based assays, without altering protein function or expression level that accompanies conventional methods such as protein knock-out, RNA interference or the usage of antibodies.

Part I: Racemic and asymmetric syntheses of a new substance with prospective antidepressant properties were achieved. In vitro assays with synthetic racemates (±)-25 and (±)-26 suggested that the former is a relatively selective inhibitor of serotonin transporter whereas the latter is a more balanced inhibitor of both serotonin and norepinephrine transporter. An initial approach to enantiomers of 25 and 26 via resolution of carboxylic acids 21 and 22 was unsuccessful but a de novo strategy which introduced asymmetry by means of Charette enantioselective cyclopropanation led to (+)-25, (-)-25, (+)-26 and (-)-26. In vitro assays with (+)-26, now known as synosutine synthesis/OSU), indicate that this substance is a highly effective dual inhibitor of serotonin and norepinephrine transporter. With IC₅₀ and K[subscript i] values in the 1-2 nM range, (+)-26 compares favorably with Eli Lilly's duloxetine (Cymbalta®) as a dual reuptake inhibitor of serotonin and norepinephrine and is thus a potential candidate for development as a drug for treatment of clinical depression. Synosutine was also assayed in vivo for its binding to human monoamine transporters. These studies indicate that synosutine, with a K[subscript i] of 1.2 nM for norepinephrine and 2.1 nM for serotonin, is a more balanced inhibitor than duloxetin. Part II: Synthetic studies towards the tetracyclic core structure of neomangicol A (129) led to advanced intermediate 245 which bears rings A and D of the neomangicol nucleus. This bicylic enone carries the correct stereochemical imprint for tetracycle 129 at C5, C6 and C14 and it contains all of carbon atoms needed to assemble the remaining two rings. Synthesis of bicyclic lactone 170, the precursor for ring A, was accomplished from the monoterpene (S)-(+)-carvone via radical cyclization and a series of Baeyer-Villiger oxidations as the key steps. Alkylation of 170 with alkyl iodide 217, obtained from the monoterpene (S)-(-)-citronellol furnished advanced intermediate 218 which was converted into diene 244. Ring closing metathesis of 244 with Grubbs-Hoveyda second generation catalyst afforded 245. Exploratory functionalization of 245 was carried out for the purpose of assembling rings B and C of the complete neomangicol skeleton. / Graduation date: 2012

Zugl.: Giessen, University, Diss., 2009.

Not available / text

The lower esophageal sphincter (LES) is characterized by basal tone and appropriately timed neurogenic relaxation. The physiological mechanisms underlying these crucial LES functions remain poorly understood. The current studies were designed to characterize the electrophysiological properties and neural regulation of LES circular smooth muscle (CSM), and to determine whether interstitial cells of Cajal (ICC) play a role in neurotransmission. Conventional intracellular recordings were performed in CD1, nNOS knock-out, eNOS knock-out and W/Wv mutant mice. Mouse LES consists of “sling” and “clasp” smooth muscle, which were studied separately in CD1 mice. In subsequent studies of mutant mice and respective controls, only the clasp muscle was examined, Immunohistochemical c-Kit staining of ICC was performed in wild-type and W/Wv mutant mice that were first characterized electrophysiologically. The smooth muscle of the LES clasp and sling displayed unitary membrane potentials with a resting membrane potential (RMP) of ~ -43 mV. Spontaneous nifedipine-sensitive action potentials superimposed on the unitary potentials were usually recorded in the LES clasp, but not sling muscle. A monophasic inhibitory junction potential (IJP) was recorded in sling CSM, whereas a biphasic IJP consisting of an initial IJP, followed by long-lasting slow IJP (LSIJP) was recorded in clasp. Further pharmacological studies using control and various knockout mice suggest that: 1. the CSM of the mouse LES is innervated by cholinergic, nitrergic and purinergic nerves; 2. the LSIJP is mediated entirely by nitrergic nerves, whereas purinergic and nitrergic nerves produce the monophasic IJP in the LES sling and initial phase of biphasic IJP in the LES clasp; 3. Ca2+/CaM-kinase II is involved in the regulation of the nitrergic IJPs; 4. TREK-1 K+ channels are not involved in the nitrergic IJP; 5. purinergic and cholinergic neurotransmission is intact in LES CSM of W/Wv mutant mice, whereas nitrergic neurotransmission is impaired in about half of the animals. In animals in which nitrergic neurotransmission was intact, ICC-IM were markedly deficient immunohistologically, suggesting that ICC are not required for nitrergic neurotransmission; 6. impaired nitrergic neurotransmission in W/Wv mutant mice is associated with dysfunction of a Ca2+-dependent signaling cascade primed by spontaneous Ca2+ release from the sarcoplasmic reticulum. / Thesis (Ph.D, Physiology) -- Queen's University, 2008-01-24 15:54:52.175