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Die Familie der Adhesion-G-Protein-gekoppelten Rezeptoren (aGPCR) stellt die zweitgrößte Gruppe der GPCR dar. Ein strukturelles Charakteristikum der aGPCR ist der modular aufgebaute N-Terminus, welcher eine GPCR-proteolytic site (GPS) mit einem konservierten Spaltungsmotiv enthält. Trotz der hohen medizinischen Relevanz dieser Rezeptorgruppe sind für die meisten der 33 humanen Vertreter der aGPCR bis heute weder Funktion noch endogener Agonist bekannt. Um sie jedoch zukünftig als potentielle Angriffspunkte diagnostisch und therapeutisch nutzen zu können, kommt der umfassenden Charakterisierung der aGPCR hinsichtlich ihrer Aktivierungsmechanismen und Signaltransduktion große Bedeutung zu. In dieser Arbeit konnte gezeigt werden, dass eine kurze Peptidsequenz im C-terminalen Bereich des N-Terminus (Stachel-Sequenz genannt) als gebundener Agonist der aGPCR fungiert und G-Protein-vermittelte Signalwege aktiviert. Dazu wurden ortsgerichtete Mutagenesestudien durchgeführt und synthetisierte Peptide analog der Stachel-Sequenz der aGPCR in funktionellen second messenger-Akkumulationsexperimenten getestet. Während die Untersuchungen des Aktivierungsmechanismus an den bereits initial charakterisierten Rezeptoren GPR126 und GPR133 unternommen wurden, konnten 11 weitere aGPCR hinsichtlich ihrer Kopplung an G-Proteine, Expression und ihres autoproteolytischen Spaltungsverhaltens in vitro analysiert werden. Dabei ist herauszustellen, dass alle untersuchten aGPCR an das Gs-Protein koppeln. GPR115, GPR116 und GPR128 zeigten sogar eine multiple Kopplung an Gs-, Gq- und Gi-Proteine. Weiterhin konnte dargelegt werden, dass die Zerstörung hoch konservierter Disulfidbrückenbindungen innerhalb der GPS durch Aminosäuresubstitution in einer konstitutiven Aktivierung des Wildtyp-Rezeptors resultiert.

The ability of organisms to sense – and properly respond to – their environment is crucial to their survival. Higher organisms communicate with conspecifics to ensure the survival of the species. Nematodes, such as the roundworm Caenorhabditis elegans, are ubiquitous across all biomes, and rely on chemical communication to convey information with one another. The small molecules they utilize in this communication are called ascarosides. These modular pheromones are employed by all taxa, ranging from Caenorhabditis to Ascaris. The ascaroside, ascr#8, is release by hermaphroditic C. elegans to attract potential mates. Previous work has shown that a class of male specific neurons are required for sensation of this pheromone. Here, we show that these neurons initiate a neural circuit modulated by the FMRFamide-like neuropeptide, flp-3. This neuropeptide is sensed by a set of G protein-coupled receptors (GPCRs), NPR-10 and FRPR-16. Together, these components determine the behavioral valence of males to ascr#8. Within the male-specific sensory neurons, the CEM, we show that another group of GPCRs sense the ascr#8. Two of these receptors, DMSR-12 and SRW-97, are expressed in the cilia, suggesting their involvement in direct sensation of the cue. As a targeted approach to identifying and confirming receptors for ascr#8, we have developed a bioactive photoaffinity probe. We have also confirmed that the ability of ascr#8 to attract males is conserved across the genus. Together, these studies coalesce to deepen our understanding of sex-specific chemosensation and neuronal processing. These results can be used to better understand the defects that are seen in neurodegenerative diseases – many of which exhibit sex-specific defects in neuronal processing.

Screening compounds with image-based analysis is an important part in the processof drug discovery. It is an efficient way to screen compounds as it gives moreinformation than for example HTS. High-content screening as it is also called, hasreally progressed in recent years, as the field of data science evolves, and with it sodoes the efficiency of how images can be processed into information. Anotherimportant part of the drug discovery field is the family of receptors GPCRs, a largefamily of over 800 different receptors in humans. The reason GPCRs are importantin drug discovery is because of the large number of drugs targeting them. In thisexperiment we wanted to use image-based analysis to challenge drugs orcompounds that were said to be specific and see if they actually are that specific, orif we can see indications of the drug also working somewhere else. While the drugswe tested did not appear to cause any morphological perturbations large enough todistinguish them from the control, some drugs appear to cluster differently. Thismight suggest that they affect multiple targets, but it needs to be followed up upon inorder to draw any substantial conclusions.

The current epidemic of obesity has been linked to an increase in fat intake associated with the Western diet. Nutrient-induced stimulation of enteroendocrine cells in the small intestine leads to the release of hormones that contribute to satiety and the control of food intake. In particular, ingested fat, specifically in the form of free fatty acids, is potent activator of enteroendocrine cells in the proximal small intestine. However, the underlying signaling cascade that free fatty acids initiate in these enteroendocrine cells, which leads to secretion of satiety hormones, is not known. In general, my research is focused on identifying nutrient-responsive pathways in enteroendocrine cells involved with the release of satiety signals and using this information to begin to develop novel drug delivery strategies to reduce food intake. In general, my results revealed that activation of the fatty acid receptor GPR120 was ecessary for the linoleic acid-induced intracellular calcium rise, a necessary precursor for hormone release. Using patch clamp recording, I discovered that linoleic acid activated enteroendocrine cells by inducing membrane depolarization, a process requiring the calcium-activated, monovalent cation permeable channel TRPM5, which is activated downstream of GPR120. To validate the unexpected finding that TRPM5 was involved in fattyacid signaling, I performed experiments using bitter compounds, whose transduction pathway is known to involve TRPM5. Enteroendocrine cells express the bitter taste receptors and release cholecystokinin in response to bitter stimuli, suggesting the probable role of gut in initiation of protective behavior against ingestion of potentially harmful substances. Armed with the data on the specifics of the fatty acid transduction, I performed experiments using nanoparticles to determine their utility for delivering pharmaceuticals specifically to the enteroendocrine cells. I fabricated and characterized PLGA nanoparticles and performed intracellular uptake studies in order to optimally delivery payloads inside cells. Finally, I validated their use by using cell-based assays to determine the effects of internalized PLGA nanoparticles on ion channels and signaling pathways involved in CCK release. Taken together, this dissertation research has identified the signaling pathways (pharmacological targets) involved in fatty acid-mediated satiety hormone release and validated the potential therapeutic use of nanoparticle-mediated drug delivery for the eventual control of food intake.

Indiana University-Purdue University Indianapolis (IUPUI) / Metabolic survival mechanisms that defend body weight and conserve energy are currently at odds with modernized society which has a food supply that is ubiquitous, calorie dense, and highly palatable. Chronic overnutrition leads to a metabolic syndrome of obesity, insulin resistance, inflammation, and cardiovascular diseases that is increasingly prevalent and threatens health on a global scale. The brain is both a victim and culprit of metabolic diseases, and prolonged metabolic dysfunction can exacerbate the pathological mechanisms underlying both metabolic and neurodegenerative diseases. Since neuroendocrine pathways comprise an essential feedback mechanism that detects circulating hormones and nutrients in order to regulate satiety, energy expenditure, and glucose homeostasis, our research goals were to characterize molecular mechanisms within neuroendocrine pathways that could be leveraged for treating obesity, diabetes, and Alzheimer’s disease. First, we identified the expression of a G protein-coupled receptor, Gpr17, in POMC neurons and discovered that it protects aged mice from high-fat diet (HFD)-induced metabolic derangements. We examined the electrophysiological properties of POMC neurons and found Gpr17 deficiency led to increased spontaneous action potentials. Moreover, Pomc-Cre-driven Gpr17 knockout (PGKO) mice, especially female knockouts, had increased POMC-derived alpha-melanocyte stimulating hormone and beta-endorphin despite a comparable level of prohormone POMC in their hypothalamic extracts. Second, we generated a highly insulin resistant mouse model with human GLUT4 promoter-driven insulin receptor knockout (GIRKO) in muscle, adipose, and GLUT4-expressing neuronal subpopulations. This genetic approach recapitulates the primary defect preceding type 2 diabetes (T2D) and revealed additional factors/mechanisms that drive the ultimate progression of overt diabetes. Third, we used 5xFAD mice as a model of Alzheimer’s disease and showed that they were more susceptible to HFD-induced metabolic dysregulation and expression of AD pathological markers in the hippocampus. Our results helped elucidate the molecular and cellular mechanisms responsible for increased AD pathology in high-fat diet-fed 5xFAD mice and suggest that metabolic dysfunctions are a therapeutic target to ameliorate AD pathology. In conclusion, metabolic diseases are pervasive and require nuanced approaches that target the neuroendocrine system in order to restore metabolic homeostasis and protect the brain from neurodegenerative processes that are associated with obesity and diabetes.

京都大学 / 0048 / 新制・課程博士 / 博士(工学) / 甲第20691号 / 工博第4388号 / 新制||工||1682(附属図書館) / 京都大学大学院工学研究科都市環境工学専攻 / (主査)教授 田中 宏明, 教授 高野 裕久, 教授 米田 稔 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DFAM

京都大学 / 0048 / 新制・論文博士 / 博士(農学) / 乙第13146号 / 論農博第2852号 / 新制||農||1056(附属図書館) / 学位論文||H30||N5093(農学部図書室) / (主査)教授 植田 和光, 教授 加納 健司, 教授 三芳 秀人 / 学位規則第4条第2項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(エネルギー科学) / 甲第21193号 / エネ博第367号 / 新制||エネ||72(附属図書館) / 京都大学大学院エネルギー科学研究科エネルギー基礎科学専攻 / (主査)教授 木下 正弘, 教授 森井 孝, 教授 片平 正人 / 学位規則第4条第1項該当 / Doctor of Energy Science / Kyoto University / DFAM

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