Die Familie der G-Protein-gekoppelten Rezeptoren (GPCRs) bildet die größte Gruppe von Membranrezeptoren im menschlichen Organismus. Für viele GPCRs sind bisher die physiologischen Funktionen nicht bekannt. Das biologische Verständnis der Funktionen im menschlichen Organismus dieser sogenannten „orphan“ GPCRs (oGPCRs) hat, aufgrund möglicher kausaler Beteiligung an der Pathogenese von Erkrankungen sowie deren therapeutische Beeinflussbarkeit, hohe medizinische Relevanz. Die GPCRs der P2Y12-ähnliche Rezeptorgruppe besitzen eine große physiologische Bedeutung bei der Thrombozytenaggregation und der Induktion der Migration von immunokompetenten Zellen in Schädigungsgebiete. Der ADP-Rezeptor P2Y12 kann durch verschiedene pharmakologische Wirkstoffe beeinflusst werden, was bereits klinisch-therapeutisch genutzt wird. Diese Gruppe von GPCRs enthält jedoch auch Mitglieder, deren Funktionen völlig unbekannt sind. Einer dieser oGPCRs ist der GPR34. Ziel dieser Arbeit war es, mittels verschiedener in-vitro-Methoden und anhand eines GPR34-defizienten Mausstamms die physiologische Relevanz dieses P2Y12-ähnlichen Rezeptors zu analysieren. Dazu wurde ein GPR34-Knockout-Mausmodell etabliert. Die GPR34-Defizienz hatte keinen wesentlichen Einfluss auf die Entwicklung, Morphologie, das Wachstum oder die Fertilität bei Mäusen. Die Ergebnisse aus Immunisierungs– und Infektionsstudien zeigten jedoch, dass dieser evolutionär hoch konservierte Rezeptor eine wichtige Funktion in der Feinkontrolle der zellulären Immunabwehr ausübt. Neben einer verstärkten Antwort im Delayed-type Hypersensitivity (DTH)-Test war die Abwehr einer Cryptococcus-Infektion in diesem GPR34-defizienten Tiermodell beeinträchtigt. Signifikant erhöhte Zytokinspiegel nach Antigen- bzw. Pathogenexposition deuteten auf eine gestörte Immunregulation in GPR34-defizienten Mäusen hin. Weiterführende Untersuchungen sollten sich der Identifizierung des endogenen Agonisten und der Funktion des GPR34 bei der Koordinierung der zellulären Immunreaktion widmen.

Indiana University-Purdue University Indianapolis (IUPUI) / Regulation of the pyruvate dehydrogenase complex (PDC) is important for glucose homeostasis and control of fuel selection by tissues. Knocking out pyruvate dehydrogenase kinase 4 (PDK4), one of four kinases responsible for regulation of PDC activity, lowers blood glucose levels by limiting the supply of three carbon compounds for gluconeogenesis. Down regulation of PDK4 expression is also important for control of blood glucose by insulin. The primary goal was to determine whether PDK4 should be considered a target for the treatment of diabetes. A major concern is that inhibition of fatty acid oxidation by PDK4 deficiency may promote fat accumulation in tissues and worsen insulin sensitivity. This was examined by feeding wild type and PDK4 knockout mice a diet rich in saturated fat. Fasting blood glucose levels were lower, glucose tolerance was better, insulin sensitivity was greater, and liver fat was reduced in PDK4 knockout mice. The reduction in liver fat is contradictory to the finding that fibrate drugs increase PDK4 expression but ameliorate hepatic steatosis in rodents. To investigate this phenomenon, wild type and PDK4 knockout mice were fed the high saturated fat diet with and without clofibric acid. The beneficial effect of clofibric acid on hepatic steatosis was greater in the PDK4 knockout mice, indicating up regulation of PDK4 is not necessary and likely opposes the effect of clofibric acid on hepatic steatosis. Clofibric acid dramatically lowered the amount of hepatic CD36, a plasma membrane translocase required for fatty acid import, suggesting a novel mechanism for prevention of hepatic steatosis by fibrates. PDK4 deficiency had no effect on CD36 expression but reduced the enzymatic capacity for fatty acid synthesis, suggesting clofibric acid and PDK4 deficiency ameliorate hepatic steatosis by independent mechanisms. Investigation of the mechanism by which insulin regulates PDK4 expression revealed a novel binding site for hepatic nuclear factor 4α (HNF4α) in the PDK4 promoter. The stimulatory effect of HNF4α was sensitive to inhibition by Akt which is activated by insulin. The findings suggest PDK4 is a viable target for the treatment of hepatic steatosis and type 2 diabetes.

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Macrophages belong to the mononuclear phagocyte system and they perform fundamental roles to maintain homeostasis in the organism. Macrophage development, survival, proliferation and functionality depend upon the colony stimulating factor 1 (CSF1) and interleukin-34 (IL-34), which signal through the CSF1 receptor (CSF1R). CSF1R is a type III tyrosine kinase receptor that is present in the plasma membrane of monocytes and macrophages. Mutations in Csf1r in mice produce the loss of many tissue macrophage populations and multiple developmental abnormalities. In humans, abnormal enhancement of CSF1R expression has been correlated to adverse prognosis in a subset of carcinomas; and mutations in the human CSF1R are associated with an autosomal-dominant neurodegenerative disease. CSF1R is encoded by the c-fms proto-oncogene and its expression is partially controlled by the fms-intronic regulatory element (FIRE). The FIRE sequence is highly conserved across species and contains binding motifs for multiple transcription factors, which are relevant for haematopoiesis. Previous results from murine Csf1r transgenes showed that FIRE is essential for driving Csf1r expression, and that interactions between FIRE and multiple myeloid transcription factors contribute to maximal regulatory activity. This project aimed to study the role of FIRE in its normal chromatin context, in vivo. A FIRE knockout (FIRE-/-) mouse model was generated using the CRISPR/Cas9 technology in mouse embryonic stem cells (ESCs) and in mice. In ESCs, the deletion severely compromised the differentiation of macrophages from embryoid bodies generated in vitro. In mice, the frequency of the FIRE- /- genotype in the progeny does not follow a Mendelian distribution and about 5% of the offspring developed hydrocephalus. Unlike Csf1r -/-mice, which die before weaning, most surviving FIRE-/- mice grew normally and were fertile. The impact of the mutation on macrophage populations is selective. FIRE-/- mice are not monocyte deficient (identified as F4/80+ Csf1r+ cells in peripheral blood), although these cells have reduced levels of Csf1r mRNA and do not bind porcine CSF1 Fc fusion protein. The development of peritoneal macrophages and Iba-1+ microglia was abolished, but Adgre1+ (F4/80+) macrophage populations in liver and spleen were unaffected. Csf1r was greatly reduced in bone marrow progenitors, but about 30% of these cells were able to differentiate into macrophages in vitro, upon exposure to recombinant human CSF1 (rhCSF1). This study shows that FIRE is essential for the development of a subset of tissue-resident macrophage populations. In FIRE-/- mice, potential compensation from additional regulatory elements within Csf1r might underlie the development of unaffected tissue-resident macrophages.

Transmembrane signal transduction by ligand-activated G protein-coupled receptors (GPCRs) controls virtually every aspect of mammalian physiology, and this receptor class is the target of 40-50% of currently marketed pharmaceuticals. In addition to the clinical use of direct GPCR agonists and antagonists, it is now believed that GPCR effectors and regulators may also be viable drug targets with improved therapeutic efficacy and specificity. The prototypic role of Regulator of G protein Signaling (RGS) proteins is inhibition of G protein signaling through acceleration of GTP hydrolysis by GΑ, which promotes re-association of GΑ and GΒΓ subunits with the receptor at the cell membrane. In this way, RGS proteins determine the magnitude and duration of the cellular response to GPCR stimulation. Though RGS protein biochemistry has been well elucidated in vitro, the physiological functions of each RGS family member remain largely unexplored. RGS6 belongs to the R7 subfamily of RGS proteins originally identified in brain. Our acquisition of an RGS6-/- mouse allowed us to survey RGS6 expression in all tissues of the body revealing the greatest expression of RGS6 in brain. Despite robust neural RGS6 expression, little is known regarding functional roles of RGS6 in the brain and spinal cord. In addition, we identified several novel, higher molecular weight RGS6 immunoreactive bands specifically present in the nervous system. The plan of this thesis work was multifaceted. We sought to elucidate novel GPCR signaling cascades modulated by RGS6 in brain while simultaneously characterizing the expression patterns and identity of the novel RGS6 species specifically detected in the nervous system. Considering the large diversity of RGS6 isoforms present in brain, the abundance of potential RGS6 binding partners, and the possibility of discovering new mechanisms involved in RGS6 regulation, elucidation of the novel RGS6 molecular species is of paramount importance. Utilizing RGS6-/- mice we identified RGS6 as a critical modulator of two GPCRs in brain. First, by inhibiting the serotonin receptor 1A (5-HT1AR)-adenylyl cyclase (AC) axis, RGS6 functions to promote anxiety- and depression-related behaviors in mice. As a result, RGS6-/- mice exhibit a robust anxiolytic and antidepressant phenotype remarkably similar to that of animals treated chronically with therapeutic doses of selective serotonin reuptake inhibitors (SSRIs). RGS6 also inhibits GABAB receptor (GABABR)-G protein- activated inwardly rectifying potassium (GIRK) channel current in cerebellar granule cells, and loss of RGS6 results in cerebellar ataxia and gait abnormalities reversible by GABABR blockade. Furthermore, evaluation of voluntary alcohol drinking behaviors in WT versus RGS6-/- mice revealed a striking reduction in alcohol intake resulting from RGS6 loss in both acute and chronic alcohol consumption paradigms due, at least in part, to potentiation of GABABR signaling. Thus, RGS6 inhibitors have potential clinical utility in the treatment of mood disorders and alcoholism. We have shown that one novel RGS6 immunoreactive band expressed in the brain and spinal cord is a phospho-protein sensitive to Λ phosphatase-mediated dephosphorylation. Further, new information acquired from PCR amplification of RGS6 mRNA species from human brain cDNA libraries has necessitated substantial revisions to the RGS6 splicing scheme devised by the Fisher laboratory in 2003. To the 36 isoforms generated from two alternate transcription start sites (RGS6L vs. RGS6), the inclusion or exclusion of exons 14 and 17, and variable splicing to one of 7 different 3' terminal exons, we have added the possible insertion of three novel internal exons (A1, A2, A3), a retained intron, and two new 3' terminal exons. As a result, the number of RGS6 mRNAs present in brain could be as many as 248 unique species, an astonishing diversity unprecedented in the RGS protein family.