Bisher wurden sieben verschiedene TRPC-Kanäle (für „classical (oder canonical) transient receptor potential“) beschrieben, die in der Plasmamembran tierischer Zellen lokalisiert sind. Diese Kanäle gehören zu einer von sieben Familien der TRP-Ionenkanäle, deren Mitglieder an einer Vielzahl von physiologischen Funktionen im Körper beteiligt sind. Im Jahr 2005 konnten in Patienten, die an einer autosomal dominant vererbten Form der fokalen segmentalen Glomerulosklerose (FSGS) leiden, Mutationen der TRPC6-Kanäle identifiziert werden, die zu einer Überaktivität dieser Kanäle führen ( sog. “gain-of-function”-Mutationen). Etwas später (2006) wurden aber auch einige FSGS Patienten entdeckt, die keine „gain-of-function“-Mutationen im TRPC6 sondern funktionslose, sog. „loss of function“-Mutationen der Phospholipase Cɛ (PLCɛ) exprimierten. Diese Daten deuten auf eine funktionelle Interaktion zwischen TRPC6 und PLCɛ in Zellen der Niere hin, die bisher noch nicht näher untersucht worden ist. Beide Proteine könnten sich auch als Zielstrukturen für eine Pharmakotherapie der FSGS eignen. Die FSGS äußert sich durch eine Störung des glomerulären Filtrationsprozesses in der Niere, wodurch es unter anderem zu einer Proteinurie kommt. In vielen Fällen führt die FSGS terminal zur ESRD („end stage renal disease“), also zu einem akuten Nierenversagen. Glomeruli bilden die filtrierende Einheit der Niere, wobei der eigentliche Filter, welcher im Inneren des Glomerulus lokalisiert ist, aus Podozyten, Endothelzellen und der dazwischen befindlichen Basalmembran besteht. Da TRPC-Kanäle unter anderem in Podozyten exprimiert werden, liegt die Annahme nahe, dass diese Zellen durch den vermehrten Ca2+-Einstrom mutierter Kanäle bei der FSGS krankhaft verändert sein könnten. Aus diesem Grund wurden in dieser Arbeit Podozyten aus Wildtyp (WT)-Mäusen sowie TRPC6 (TRPC6-/-)- und PLCε (PLCε-/-)-gendefizienten Tieren isoliert und umfangreich durch den Nachweis podozytenspezifischer Markerproteine charakterisiert. Zellfunktionen wie Proliferation, Aktinstressfaserbildung, RhoA- und TRPC6-Aktivität wurden vergleichend in den Zellen der verschiedenen Genotypen analysiert. Es zeigte sich, dass PLCε zwar mit TRPC6 in Zellen des Nierenkortex interagieren kann, aber PLCε-/--Podozyten funktionell in ihrer Angiotensin II-induzierten Aktinstressfiberbildung und GTPγS-induzierten TRPC6-Aktivierung nicht von Wildtyp-Podozyten unterschieden werden konnten, was auf eine redundante Funktion der PLCε-vermittelten TRPC6-Aktivierung hindeutet. Eine Aktivierung von TRPC6 durch PLCε wird wahrscheinlich durch die Stimulation der wesentlich stärker exprimierten anderen PLC-Isoform PLCβ1, zumindest in Podozyten, überdeckt. Eine Expression der klonierten murinen TRPC6-FSGS-Mutanten in primär isolierten Wildtyp- und TRPC6-defizienten Podozyten war für die Zellen lethal, wodurch die Pathogenität eines erhöhten TRPC6-induzierten Ca2+-Einstroms für diese Zellen und damit den gesamten Nierenglomerulus in FSGS-Patienten noch einmal nachgewiesen werden konnte. In Zukunft könnten deswegen spezifische TRPC6-Inhibitoren eine Therapieoption zur Linderung der Symptome bei FSGS-Patienten sein.

Die Alzheimer Demenz (AD) ist eine degenerative zerebrale Erkrankung, die klinisch durch den Verlust vielfältiger kognitiver Fähigkeiten gekennzeichnet ist. Ein geringer Prozentsatz (<5%) der familiären AD kann auf monogenetisch bedingte Mutationen, die autosomal-dominant vererbt werden, zurückgeführt werden. Für den überwiegenden Teil der sporadisch auftretenden AD ist die multifaktorielle Ätiopathogenese unbekannt, wobei eine genetische Vulnerabilität als risikomodulierender Faktor den Ausbruch der Erkrankung begünstigt. Bislang wurden einzelne chromosomale Loci mit moderaten Beiträgen zur phänotypischen Ausprägung der sporadischen AD identifiziert. In der Zelle tritt die RNA typischerweise als Ribonukleoproteinkomplex (RNP) auf, bestehend aus einer oder mehreren RNAs und einer multiplen Anzahl an RNA-bindenden Proteinen. RNA-bindende Proteine (RBP) übernehmen dabei wichtige Aufgaben wie Transport und Lokalisierung der RNA, und sind insbesondere bei der posttranskriptionalen Prozessierung von großer Bedeutung: RBPs regulieren und kontrollieren u.a. Spleißvorgänge, die Polyadenylierung, die mRNA-Stabilisierung und den Translationsvorgang. In der Zelle ist daher das Zusammenspiel einer großen Vielfalt an RNAs und RBPs, sowie die exakte Prozessierung der RNA maßgeblich für eine funktional korrekt verlaufende Genexpression. Dass eine veränderte RNA-Prozessierung zu einer Reihe von Erkrankungen führt, gilt als gesichert. Der Zusammenhang zwischen RBPs und demenziellen Erkrankungen findet gegenwärtig, unterstützt durch neuere Befunde, wissenschaftliche Aufmerksamkeit. In der vorliegenden Fall-Kontroll-Assoziationsstudie wurde der Zusammenhang zwischen 19 Polymorphismen in vier Genen, die für RNA-bindende Proteine codieren (HNRNPA2B1, HNRNPH3, PCBP2 und ELAVL4) und der AD untersucht. Dazu wurden die ausgewählten SNPs an 323 AD-Patienten und 677 gesunden Kontrollprobanden bezüglich ihrer Unterschiede in Allel-, Genotyp-, und Haplotypfrequenz analysiert. Die Diagnose einer "wahrscheinlichen" AD erfolgte nach den NINCDS-ADRDA Kriterien. Eine Unterteilung der Patienten in Early-Onset Alzheimer's Disease (EOAD, Krankheitsausbruch <65 Jahre) bzw. Late-Onset Alzheimer's Disease (LOAD, Krankheitsausbruch >65 Jahre) erfolgte im Sinne einer homogeneren Subgruppierung und sollte eine aussagekräftigere Assoziation der genetischen Vulnerabilitätsmarker ermöglichen. In die Kontrollgruppe wurden ausschließlich gesunde Probanden ohne demenzielle Erkrankungen in der Familie eingeschlossen. Neben einer grundsätzlichen Assoziation mit der AD wurde in einem zweiten Schritt der Zusammenhang zwischen den 19 Polymorphismen und der individuellen Gedächtnisleistung als intermediärer Phänotyp untersucht. Die Gedächtnisleistung wurde mittels standardisierter Tests erhoben. Die 19 SNPs (rs4412638, rs12024093, rs12043872, rs3009113, rs913858, rs12113404, rs12672536, rs4722589, rs10998072, rs10998076, rs1162757, rs16925347, rs1162756, rs1683152, rs784566, rs11170562, rs784563, rs2249381, rs1716966) wurden mittels iPLEX-Verfahren genotypisiert und die massenspezifischen Produkte im MALDI-TOF Massenspektrometer ausgewertet. Signifikante Assoziationen konnten dabei insbesondere zwischen den drei Genen HNRNPA2B1, PCBP2, ELAVL4 und der Alzheimer Demenz, sowie der Gedächtnisleistung festgestellt werden. Auf diesen drei Genen zeigten sich fünf Marker mit dem Phänotyp der AD bzw. insbesondere mit der EOAD assoziierbar: Auf ELAVL4 ließen sich rs3009113 und rs12024093 in der Patienten-Gesamtgruppe, sowie im EOAD-Kollektiv mit der AD assoziieren; auf PCBP2 waren rs784563 und rs2249381 in der Gesamtgruppe der Patienten auffällig; auf HNRNPA2B1 ließ sich rs4722589 mit der EOAD assoziieren. Diese und weitere Polymorphismen der angeführten drei Gene konnten sowohl in der EOAD- als auch in der LOAD-Gruppe mit einer verminderten Gedächtnisleistung assoziiert werden. In der Haplotypanalyse ließen sich diese Befunde grundsätzlich bestätigen. Zusammenfassend lassen die Resultate der vorliegenden Untersuchung einen Zusammenhang zwischen drei der untersuchten RBPs (HNRNPA2B1, PCBP2 und ELAVL4) und dem Phänotyp Alzheimer Demenz, sowie dem intermediären Phänotyp Gedächtnisleistung vermuten. SNPs in intronischen Bereichen können regulatorische Sequenzen wie die Polyadenylierung- oder Spleißfaktorbindungsstellen beeinflussen und zu Veränderungen in der Transkriptionsrate oder im Spleißprozess führen. Zudem kann durch Muationen die natürliche Aggregationsbereitschaft RNA-bindender Proteine, sich in RNA-granules zusammenzulagern, verstärkt werden und damit direkt zellschädigend wirken. Da über Funktion und Struktur der untersuchten RBPs gegenwärtig noch relativ wenig bekannt ist und es bislang keine vergleichbaren Referenzstudien bezüglich Alzheimer gibt, ist weiterführende Forschungsarbeit zur abschließenden Interpretation der Ergebnisse notwendig. Aufgrund der bis dato vorliegenden Ergebnisse stellen RNA-bindende Proteine insgesamt einen interessanten Kandidaten für die Alzheimerforschung dar.

The field of serotonin (5-HT) research continues to expand. A variety of physiological and pathophysiological functions regulated by 5-HT has been identified. Selective serotonin reuptake inhibitors (SSRIs) and 5-HT-3-receptor antagonists are used in medical therapy, and more 5-HT-related medicaments are expected in the future. Circulating 5-HT is stored mainly in the dense granules of platelets. 5-HT stimulates platelets, which has been observed, apart from humans, in various other species. Therefore, the stimulatory effect of exogenous 5-HT in high concentrations (> 0.5 μM) on human platelets in blood is unambiguous. However, results of the platelet-stimulating effect of the endogenous 5-HT, which is stored in dense granules and released upon platelet activation, are contradictory. The aim of this thesis was to investigate the acute effect of SSRIs on human platelet aggregation, the role of the 5-HT2A receptor for platelet function in human blood, and the role of the newly discovered 5-HT4 receptor for platelet function. In some—but not all—clinical studies, SSRIs on the one hand have been suspected to cause bleeding complications, on the other hand they may protect against ischemic cardiovascular diseases. In our studies, the acute addition of the SSRI fluoxetine to blood, alone or in combination with aspirin, indeed inhibited the potentiation of platelet aggregation by exogenous 5-HT (probably by unspecific inhibition of 5-HT2A receptors), the platelet aggregation induced by physiological stimuli was, however, not affected. Therefore, we conclude that the serotonin transporter is not involved in the physiological platelet aggregation process. Yet, SSRIs could cause bleeding complications by reducing the platelet 5-HT content, consequently leading to a reduced 5-HT-mediated vasoconstriction, or contribute to stomach ulcers by disruption of the 5-HT-mediated wound healing. Our results indicate that 5-HT2A receptors mediate the potentiation of aggregation of human platelets in the blood by exogenous 5-HT. However, 5-HT2A receptor antagonists do not inhibit platelet activation by physiological stimuli, shear stress, or flow over atherosclerotic plaque material. Therefore, endogenous 5-HT, which is released upon platelet activation, plays no role in human platelet aggregation. Finally, our studies support the expression of platelet-inhibiting 5-HT4 receptors. However, these played only a minor role in the regulation of human platelet aggregation induced by epinephrine. The question why platelet 5-HT plays a role in platelet aggregation of mice, rats, cats, rabbits, and dogs, but not of men, is intriguing, but has no explanation according to our present knowledge. Species-specific structural differences of the 5-HT2A receptors are suggested. Based on the current knowledge and our experiments, it can be concluded that human platelet function is not influenced by endogenous 5-HT, but platelets are important for transporting 5-HT in the blood to diseased organs and tissues. The absence of a stimulating action of endogenous 5-HT on platelets precludes the use of 5-HT2A receptor antagonists as anti-thrombotic medications.

The process of the production of new neurons is called adult neurogenesis. It occurs in specific regions in the adult mammalian brain. The new neurons are being produced by so called adult neural stem cells (aNSCs). Two niches that harbour these cells are the subgranular zone (SGZ) of the dentate gyrus of the hippocampus and the subependymal zone (SEZ) of the lateral ventricle. aNSCs in the SEZ produce transit-amplifying progenitors (TAPs) which give rise to neuroblasts (NBs) that migrate via the rostral migratory stream (RMS) to the olfactory bulb (OB) where they mature to different interneuron subtypes. The properties of the aNSCs in these regions have been extensively investigated either by in vitro or in vivo population studies. However, little is known about the behaviour of an individual aNSC in the SEZ. In order to overcome this missing knowledge, an analysis method was established in this thesis by using double heterozygous mice for GLASTCreERT2 and R26R-Confetti. This method allows the lineage tracing of aNSCs in the murine brain. By titration of the dose of Tamoxifen that was injected intraperitoneally into these mice, sparse labelling of individual GLAST+ aNSCs was achieved. This method was then used to analyse the behaviour of single aNSCs of the SEZ, as well as their progeny (TAPs, NBs and neurons) over the course of time. These aNSCs are able to produce a large progeny already within 3 and 7 days after induction and show a drive towards neuronal maturation in between 3 and 8 weeks. The amplification step occurs at the level of the progeny. This analysis at the single cell level showed insights into the temporal and spatial profile of aNSCs. Compared to other analyses performed at the population level, the progeny of aNSCs in the SEZ reduces with time. It seems that the continuous neurogenesis is maintained at the population level and therefore reflects a population property. Furthermore, it was found that a single aNSC is capable of producing multiple interneuron subtypes, however the majority of cells produced are the deep granule interneuron subtype.

The intestine is a pivotal organ which is divided into two anatomical parts: the small intestine and the large intestine (colon and rectum). Both parts are made up of single layered epithelium. This epithelium is composed of villi (protrusions) – found only in the small intestine - and crypts (invaginations) leading to an increase of the surface of the intestinal lumen whereby the uptake of nutrients and water is improved. Every five days, the intestinal epithelium is renewed whereby both, crypts and eventually villi, are filled up with new cells. The homeostasis of the crypts/villi relies on adult stem cells (SCs), especially crypt base columnar (CBC) cells, which are located at the base of the crypts. These are regulated by an active Wnt signaling pathway. A deregulation of the Wnt signaling pathway leads to cancer formation found in humans almost exclusively in the colon and rectum. Colorectal cancer (CRC) is worldwide the third most common cause for cancer related deaths. In the majority of CRC, origin and progress are caused by mutations in the adenomatous polyposis coli (APC) gene which encodes an essential component of the β-catenin destruction complex that is the central element of the Wnt signaling pathway. As a consequence of these mutations, the executor of the Wnt signaling pathway, β-catenin, which is in this context a transcription factor, cannot be downregulated any more. As a consequence target genes of β-catenin are expressed in an unregulated manner. These target genes regulate features of stem cell biology which confer cancer stemness, metastasis, EMT (epithelial-mesenchymal transition), chemoresistance and other characteristics to colorectal tumor cells. Interestingly, APC mutations have only an effect when they occur in the adult stem cells. Thus, the descendend tumor cells show characteristics of these cells and have been termed cancer stem cells (CSCs). Like adult stem cells in the normal crypt CSCs are the origin of cancer and are characterized by an activated - here deregulated - Wnt signaling pathway and thus, by the aforementioned features. Clinically, cancer death is caused in most cases by metastasis which is treated by chemotherapy from which most if not all CRCs escape by the development of chemoresistance which is an intrinsic feature of the CSCs. Therefore, CSC specific targeted therapies might be a promising therapeutic tool for a successful treatment of CRCs. One possibility is the interference of CSC sustaining molecules as these molecules are involved in the induction and maintenance of CSCs. Here, a promising molecule is olfactomedin-4 (OLFM4) which was discussed to be a CSC marker. But the role of OLFM4 as a CSC marker and important factor for tumorigenesis has been controversially described. Therefore, I investigated in the first part of my thesis the role of OLFM4 in CRC cells. I demonstrate that OLFM4 was expressed only in two out of 14 CRC cell lines. The assumption that OLFM4 was only expressed in cells with characteristics of CSCs and thus, was not detected in the cell lines as they possess only a small proportion of CSCs, was not confirmed. I found that CSCs showed a reduced OLFM4 expression and thus, OLFM4 was not coexpressed with other SC markers. These results indicate that OLFM4 is not a marker of CSCs in CRC. In order to analyze the functional role of OLFM4 in CRC cells, I overexpressed OLFM4 lentivirally. However, the overexpression of OLFM4 and thus, high OLFM4 protein levels did not influence the expression of CSC, EMT or differentiation marker. Likewise, OLFM4 did not play a functional role for proliferation, stemness and metastatic features. Therefore, this study demonstrates that OLFM4 is not a CSC marker and has no functional role for the driving activity in the process of colorectal carcinogenesis. Additionally, I evaluated in the second part of my thesis the role of the microRNAome (miRNAome) in colorectal carcinogenesis, the influence on CSC features and whether the miRNAome might be a tool for specific CSC targeted therapies. microRNAs (miRNAs) are generally downregulated in tumors whereby the miRNA loss promotes tumorigenesis. As the majority of the CRC cases are driven by an APC mutation in the SC compartment, I used for my investigations a mouse model with a conditional Apc knockout in CBC cells which develops efficiently intestinal adenomas. This mouse model was crossed with another mouse model harboring a conditional knockout of the essential miRNA generator Dicer1 to investigate the role of a loss of the miRNAome in murine Wnt driven intestinal tumors. In this part of my study I demonstrated that hetero- and homozygous deletion of Dicer1 in CBC cells, in combination with an Apc knockout, enhances significantly the number of adenomas. Moreover, deletion of Dicer1 resulted in smaller adenomas caused by reduced proliferation. Further analysis of DICER1 deletion in human CRC cell lines revealed that loss of DICER1 and thus, miRNAs led likewise to a decreased proliferation. Additionally, I showed that loss of miRNAs increased the expression/protein levels of CSC markers and CSC features indicating that loss of DICER1 promotes tumorigenesis. Moreover, I translated these mouse model/cell culture results into human colonic normal and tumor tissue as well as CRC. In a collection of different tissues (normal tissue, adenomas and cancers of stages I to IV), increased DICER1 levels were seen from normal tissue to adenomas followed by decreased levels during carcinoma progression. Increased levels of DICER1 were also found in the murine Wnt driven adenomas. In support with this I provided finally evidence that DICER1 expression is regulated by the Wnt signaling pathway thus already early in the beginning of the colorectal tumorigenesis. Thus, this data showed that DICER1 is a tumor suppressor in intestinal cancer and the loss of DICER1 and hence, of the miRNAome, influences CSC marker expression and marker protein levels as well as proliferation and CSC features. Therefore, the miRNAome might possibly become a therapeutic target for CSC targeted therapy.

Chronic obstructive pulmonary disease (COPD) is projected to be the third leading cause of death by 2020 with cigarette smoke exposure being the main risk factor. Cigarette smoke leads to oxidative stress in the lung, resulting in protein damage and adaptive immune responses. Also, smokers and COPD patients are more susceptible to viral infections often followed by acute exacerbations of COPD pathogenesis. Lungs of COPD patients exhibit increased numbers of innate and adaptive immune cells, among these CD8+ T cells, whose abundance correlates with disease severity. The proteasome degrades more than 90 % of intracellular proteins - including damaged ones - into small peptides and is important to protect the cell from proteotoxic stress. Furthermore, the immunoproteasome, a specialized proteasome subtype which is expressed by default in antigen presenting cells and induced during infection, is involved in shaping adaptive immune responses by enhancing antigen presentation via major histocompatibility complex (MHC) I to cytotoxic CD8+ T cells. The effects of cigarette smoke on (immuno-)proteasome function have not been investigated so far. The first publication included in this thesis (van Rijt et al. 2012) explored the effects of acute cigarette smoke exposure on proteasome expression and activity. We observed that short-term exposure of cells to extracts of cigarette smoke directly impaired proteasome activity, while proteasomal protein expression was not altered. Oxidatively modified and polyubiquitinated proteins accumulated, suggesting augmentation of oxidative stress in cigarette smoke-treated cells. In lungs of mice acutely exposed to cigarette smoke, a similar effect could be observed: one of the three proteasome activities was significantly reduced, and ubiquitinated substrates for the proteasome were found to be accumulated, while proteasome expression levels were not changed. The second publication in this thesis (Keller et al. 2015) shows for the first time the cell-specific expression of immunoproteasomes in the lung and their induction by interferon-γ in vitro and by murid herpesvirus 68 (MHV-68) infection in vivo. Within these experiments, activity-based probes were used to clearly define the kinetics of standard and immunoproteasome subunit incorporation. In human lungs from controls or early-stage COPD patients, immunoproteasome expression was not changed. Immunoproteasomes localized mainly to alveolar macrophages, but not to parenchymal cells in both donors and end-stage COPD. Results from recent experiments were accepted for publication in the meantime (Kammerl et al. 2016): we investigated MHC I antigen presentation in cigarette smoke extract-treated primary immune cells and bronchoalveolar lavage (BAL) cells from mice exposed to cigarette smoke for ten days. In vitro treatment of primary immune cells with cigarette smoke extract led to a decrease in the presentation of an immunoproteasome-dependent “self”-epitope. With the help of activity-based probes, we observed a shift from immuno- to standard proteasome activity in isolated alveolar macrophages from smoke exposed mice. This shift, however, was not sufficient to impact antigen presentation of an immunoproteasome-dependent epitope. The altered ratio of standard and immunoproteasome might be explained by transcriptional downregulation of immuno-, but not standard proteasomes by cigarette smoke in isolated alveolar macrophages of smoke-exposed mice, which was also observed in total BAL cells of early-stage COPD patients. In the lungs of end-stage COPD patients, activities of both standard and immunoproteasome subunits were significantly decreased, while total proteasome protein levels were not changed. Taken together, we show that cigarette smoke directly impairs proteasome function in vitro and in vivo, which may exacerbate oxidative stress resolution in response to cigarette smoke, since the degradation of oxidatively modified and misfolded proteins is impaired. In addition, we observed alterations in immunoproteasome-dependent MHC I antigen presentation, which may contribute to increased susceptibility to virus-induced exacerbations, prolonged infection and possibly result in autoimmune responses.