Emulating the early phases of human tooth development in vitro

Functional in vitro models emulating the physiological processes of human organ formation are invaluable for future research and the development of regenerative therapies. Here, a developmentally inspired approach is pursued to reproduce fundamental steps of human tooth organogenesis in vitro using human dental pulp cells. Similar to the in vivo situation of tooth initiating mesenchymal condensation, a 3D self-organizing culture was pursued resulting in an organoid of the size of a human tooth germ with odontogenic marker expression. Furthermore, the model is capable of epithelial invagination into the condensed mesenchyme, mimicking the reciprocal tissue interactions of human tooth development. Comprehensive transcriptome analysis revealed activation of well-studied as well as rather less investigated signaling pathways implicated in human tooth organogenesis, such as the Notch signaling. Early condensation in vitro revealed a shift to the TGFß signal transduction pathway and a decreased RhoA small GTPase activity, connected to the remodeling of the cytoskeleton and actin-mediated mechanotransduction. Therefore, this in vitro model of tooth development provides a valuable model to study basic human developmental mechanisms.DFG, 414044773, Open Access Publizieren 2019 - 2020 / Technische Universität Berli

3-Iodothyronamine Activates a Set of Membrane Proteins in Murine Hypothalamic Cell Lines

3-Iodothyronamine (3-T1AM) is an endogenous thyroid hormone metabolite. The profound pharmacological effects of 3-T1AM on energy metabolism and thermal homeostasis have raised interest to elucidate its signaling properties in tissues that pertain to metabolic regulation and thermogenesis. Previous studies identified G protein-coupled receptors (GPCRs) and transient receptor potential channels (TRPs) as targets of 3-T1AM in different cell types. These two superfamilies of membrane proteins are largely expressed in tissue which influences energy balance and metabolism. As the first indication that 3-T1AM virtually modulates the function of the neurons in hypothalamus, we observed that intraperitoneal administration of 50 mg/kg bodyweight of 3-T1AM significantly increased the c-FOS activation in the paraventricular nucleus (PVN) of C57BL/6 mice. To elucidate the underlying mechanism behind this 3-T1AM-induced signalosome, we used three different murine hypothalamic cell lines, which are all known to express PVN markers, GT1-7, mHypoE-N39 (N39) and mHypoE-N41 (N41). Various aminergic GPCRs, which are the known targets of 3-T1AM, as well as numerous members of TRP channel superfamily, are expressed in these cell lines. Effects of 3-T1AM on activation of GPCRs were tested for the two major signaling pathways, the action of Gαs/adenylyl cyclase and Gi/o. Here, we demonstrated that this thyroid hormone metabolite has no significant effect on Gi/o signaling and only a minor effect on the Gαs/adenylyl cyclase pathway, despite the expression of known GPCR targets of 3-T1AM. Next, to test for other potential mechanisms involved in 3-T1AM-induced c-FOS activation in PVN, we evaluated the effect of 3-T1AM on the intracellular Ca2+ concentration and whole-cell currents. The fluorescence-optic measurements showed a significant increase of intracellular Ca2+ concentration in the three cell lines in the presence of 10 μM 3-T1AM. Furthermore, this thyroid hormone metabolite led to an increase of whole-cell currents in N41 cells. Interestingly, the TRPM8 selective inhibitor (10 μM AMTB) reduced the 3-T1AM stimulatory effects on cytosolic Ca2+ and whole-cell currents. Our results suggest that the profound pharmacological effects of 3-T1AM on selected brain nuclei of murine hypothalamus, which are known to be involved in energy metabolism and thermoregulation, might be partially attributable to TRP channel activation in hypothalamic cells

3-Iodothyronamin und seine Rolle in der aminergen G-Protein gekoppelten Rezeptor-Signaltransduktion und Neuromodulation

3-Iodothyronamine (T1AM) is an endogenous thyroid hormone metabolite. Its profound pharmacological effects on energy metabolism and thermal homeostasis in rodents have raised interest to elucidate its signaling properties in tissues that pertain to metabolic regulation and thermogenesis. Especially its anapyrexic effect could be utilized in emergency medicine to treat disease conditions like stroke. Although the exact molecular mechanism of T1AM-induced anapyrexia has not been fully enlightened, it is assumed that in rodents its regulation is mediated centrally in the brain. Previous studies identified G protein-coupled receptors (GPCRs) and transient receptor potential channels (TRPs) as targets of T1AM in various cell types. However, known T1AM targets cannot explain the anapyrexic effect. In my dissertation, I investigated whether aminergic receptors that are involved in thermoregulation such as serotonin receptor 1b (5-HT1b) and histamine receptor 1 (HRH1) are new receptor targets for T1AM. 5-HT1b primarily activates the Gai/o-mediated pathway and phospholipase C (PLC) signaling through the Gβγ-subunit of Gi/o, whereas HRH1 is Gq/11 coupled. Since the expression profiles of TAAR1, the first described GPCR target of T1AM, and 5-HT1b coincide, I evaluated heteromerization of these two GPCRs and their signaling properties under co-expression. Next, I aimed at identifying activated brain areas after the intraperitoneal (i.p.) injection of T1AM in mice. I used the proto-oncogene cFOS (FOS) as a marker for neuronal activation. Further, I used murine neuronal cell lines to study their activation through T1AM regarding Gs and Gi/o signaling. In the first part of my dissertation, I showed that T1AM blocked HRH1 activation through its endogenous ligand histamine. Thus T1AM is an antagonist for HRH1. T1AM induced Gai/o signaling through 5-HT1b at a concentration of 10 µM. Strikingly, T1AM only activated the Gai/o mediated reduction of cAMP accumulation at 5-HT1b, but not PLC signaling through the ß?-subunit. Therefore, T1AM is a biased ligand for 5-HT1b. Additionally, I confirmed the heterodimerization between TAAR1 and 5-HT1b using fluorescence resonance energy transfer. When co-expressed, only TAAR1 mediated T1AM-induced signaling, whereas 5-HT1b activation was abrogated. In conclusion, 5-HT1b and HRH1 are new receptor targets for T1AM. Altogether, this indicates a complex interrelation of distinct signaling effects between the investigated GPCRs and respective ligands. In the second part, I observed a significant increase in the amount of FOS expressing neurons in the paraventricular nucleus (PVN) in C57BL/6 mice upon T1AM stimulation. To elucidate the underlying mechanism behind this T1AM signalosome, I used three different murine hypothalamic cell lines (GT1-7, mHypoE-N39 (N39) and mHypoE-N41 (N41)), which are known to express PVN markers. The cell lines expressed various aminergic GPCRs, as well as numerous members of TRP channel superfamily. Effects of T1AM on these cell lines were analyzed for the two GPCR signaling pathways, Gs and Gi/o. Here, I demonstrated that, despite the expression of known GPCR targets of T1AM, this thyroid hormone metabolite had no significant effect on Gi/o signaling and only a minor impact on the Gs pathway in the hypothalamic cell lines N39 and N41. A faint Gs signaling upon T1AM stimulation might have been the reason for FOS-expressing cells in the PVN. Although, the herein identified T1AM targets HRH1 and 5-HT1b are not Gs-coupled GPCRs my findings confirm that the multi-target ligand T1AM can modulate both histamine and serotonin GPCRs.3-Iodothyronamin (T1AM) ist ein endogener Schilddrüsenhormonmetabolit. Seine pharmakologischen Auswirkungen auf den Energiestoffwechsel und die Temperaturregulation in Nagetieren haben Interesse an seiner Signaltransduktion in verschiedensten Organen, welche in den Metabolismus und die Thermogenese involviert sind, geweckt. Vor allem seine anapyrexische Wirkung hat das Potential in der Notfallmedizin zur Behandlung von akuten Krankheitsbildern, wie bei einem Schlaganfall, zur Anwendung zu kommen. Der genaue molekulare Mechanismus von T1AMinduzierter Anapyrexie ist nicht vollständig aufgeklärt, wird aber vermutlich bei Nagetieren zentral im Gehirn reguliert. Frühere Studien identifizierten G-Protein-gekoppelte Rezeptoren (GPCRs) und transiente Rezeptorpotentialkanäle (TRPs) als Interaktionspartner von T1AM. Bereits bekannte T1AM-Interaktionspartner können die ausgelöste Anapyrexia jedoch nicht erklären. In meiner Dissertation untersuchte ich, ob aminerge Rezeptoren, die an der Temperaturregulation beteiligt sind, wie der Serotoninrezeptor 1b (5-HT1b) und der Histaminrezeptor 1 (HRH1), auch Rezeptoren für T1AM sind. 5-HT1b aktiviert in erster Linie den Gai/o Signalweg und Phospholipase C (PLC) durch Gβγ von Gi/o, während HRH1 ein Gq/11-gekoppelter Rezeptor ist. Da TAAR1, der erster beschriebene GPCR von T1AM, und 5-HT1b sich in ihrem Expressionsmuster teilweise überschneiden, untersuchte ich eine mögliche Heterodimerisierung zwischen diesen beiden GPCRs und die Signalwege der Rezeptoren bei Ko-Expression. Darüber hinaus identifizierte ich Gehirnareale, welche durch intraperitoneal (i.p.) injiziertes T1AM aktiviert werden. Dazu verwendete ich das Protooncogen cFOS (FOS) als Marker für stimulierte Neuronen. Um die endogene Gs und Gi/o Signaltransduktion durch T1AM-Stimulation zu untersuchen verwendete ich murine neuronale Zelllinien. Im ersten Teil meiner Dissertation konnte ich zeigen, dass T1AM die Aktivierung von HRH1 durch seinen endogenen Liganden Histamin blockierte, wodurch T1AM als ein Antagonist am HRH1 identifiziert werden konnte. T1AM induzierte Gai/o-Signalisierung am 5-HT1b in einer Konzentration von 10 µM. Allerdings, aktivierte T1AM am 5-HT1b nur die a- und nicht die ßy-Untereinheit. Dies macht T1AM zu einem funktionell selektiven Liganden. Zudem konnte ich zeigen, dass TAAR1 und 5-HT1b dimerisieren können. Bei der Ko-Expression von TAAR1 und HTR1b wurde die T1AM-Wirkung nur über TAAR1 vermittelt und die Aktivierung von 5-HT1b durch T1AM wurde blockiert. In meinem Exprimenten stellten sich 5-HT1b und HRH1 als neue Rezeptoren für T1AM heraus. Insgesamt deutet dies auf einen komplexen Zusammenhang der Signalwirkungen zwischen den untersuchten GPCRs und den jeweiligen Liganden hin. Im zweiten Teil dieser Arbeit konnte ich beobachten, dass die i.p. Injektion von 50 mg/kg Körpergewicht an T1AM die Anzahl an FOS exprimierenden Neuronen im Nucleus paraventricularis (PVN) von C57BL/6-Mäusen signifikant erhöhte. Um den zugrunde liegenden Mechanismus hinter diesem T1AM-induzierten Signalosom aufzuklären, habe ich drei verschiedene murine Hypothalamus-Zelllinien verwendet, da sie verschiedene PVN-Marker exprimieren: GT1-7, mHypoE-N39 (N39) und mHypoE-N41 (N41). In diesen Zelllinien wurden verschiedene aminerge GPCRs sowie TRP-Kanäle exprimiert. Auswirkungen von T1AM auf die hypothalamischen Zelllinien wurden für die zwei GPCR Signalwege Gs und Gi/o untersucht. Ich konnte zeigen, dass der Schilddrüsenhormon-Metabolit keinen signifikanten Effekt auf die Signalisierung von Gi/o und nur gering Gs Signalisierung in den hypothalamischen Zelllinien N39 und N41 aktiviert. Im PVN könnte ein Gs-Signal durch die T1AM Stimulation zu FOS-positiven Neuronen führen.Auch wenn die neu identifizierten T1AM Interaktionspartner HRH1 und 5-HT1b nicht Gs gekoppelte GPCRs sind, zeigen meine Forschungsergebnisse dennoch, dass T1AM ein Multi-Target-Ligand ist und sowohl Histamin-, als auch Serotonin-GPCRs modulieren kann

Single-cell sequencing in translational cancer research and challenges to meet clinical diagnostic needs

The ability to capture alterations in the genome or transcriptome by next-generation sequencing has provided critical insight into molecular changes and programs underlying cancer biology. With the rapid technological development in single-cell sequencing, it has become possible to study individual cells at the transcriptional, genetic, epigenetic, and protein level. Using single-cell analysis, an increased resolution of fundamental processes underlying cancer development is obtained, providing comprehensive insights otherwise lost by sequencing of entire (bulk) samples, in which molecular signatures of individual cells are averaged across the entire cell population. Here, we provide a concise overview on the application of single-cell analysis of different modalities within cancer research by highlighting key articles of their respective fields. We furthermore examine the potential of existing technologies to meet clinical diagnostic needs and discuss current challenges associated with this translation

3-Iodothyronamine Activates a Set of Membrane Proteins in Murine Hypothalamic Cell Lines

3-Iodothyronamine (3-T1AM) is an endogenous thyroid hormone metabolite. The profound pharmacological effects of 3-T1AM on energy metabolism and thermal homeostasis have raised interest to elucidate its signaling properties in tissues that pertain to metabolic regulation and thermogenesis. Previous studies identified G protein-coupled receptors (GPCRs) and transient receptor potential channels (TRPs) as targets of 3-T1AM in different cell types. These two superfamilies of membrane proteins are largely expressed in tissue which influences energy balance and metabolism. As the first indication that 3-T1AM virtually modulates the function of the neurons in hypothalamus, we observed that intraperitoneal administration of 50 mg/kg bodyweight of 3-T1AM significantly increased the c-FOS activation in the paraventricular nucleus (PVN) of C57BL/6 mice. To elucidate the underlying mechanism behind this 3-T1AM-induced signalosome, we used three different murine hypothalamic cell lines, which are all known to express PVN markers, GT1-7, mHypoE-N39 (N39) and mHypoE-N41 (N41). Various aminergic GPCRs, which are the known targets of 3-T1AM, as well as numerous members of TRP channel superfamily, are expressed in these cell lines. Effects of 3-T1AM on activation of GPCRs were tested for the two major signaling pathways, the action of Gαs/adenylyl cyclase and Gi/o. Here, we demonstrated that this thyroid hormone metabolite has no significant effect on Gi/o signaling and only a minor effect on the Gαs/adenylyl cyclase pathway, despite the expression of known GPCR targets of 3-T1AM. Next, to test for other potential mechanisms involved in 3-T1AM-induced c-FOS activation in PVN, we evaluated the effect of 3-T1AM on the intracellular Ca2+ concentration and whole-cell currents. The fluorescence-optic measurements showed a significant increase of intracellular Ca2+ concentration in the three cell lines in the presence of 10 μM 3-T1AM. Furthermore, this thyroid hormone metabolite led to an increase of whole-cell currents in N41 cells. Interestingly, the TRPM8 selective inhibitor (10 μM AMTB) reduced the 3-T1AM stimulatory effects on cytosolic Ca2+ and whole-cell currents. Our results suggest that the profound pharmacological effects of 3-T1AM on selected brain nuclei of murine hypothalamus, which are known to be involved in energy metabolism and thermoregulation, might be partially attributable to TRP channel activation in hypothalamic cells

The Trace Amine-Associated Receptor 1 Agonist 3-Iodothyronamine Induces Biased Signaling at the Serotonin 1b Receptor

Trace amine-associated receptors (TAARs) belong to the class A G-protein-coupled receptors (GPCR) and are evolutionary related to aminergic receptors. TAARs have been identified to mediate effects of trace amines. TAAR1 signaling is mainly mediated via activation of the Gs/adenylyl cyclase pathway. In addition to classical trace amines, TAAR1 can also be activated by the thyroid hormone derivative 3-iodothyronamine (3-T1AM). Pharmacological doses of 3-T1AM induced metabolic and anapyrexic effects, which might be centrally mediated in the hypothalamus in rodents. However, the observed anapyrexic effect of 3-T1AM persists in Taar1 knock-out mice which raises the question whether further GPCRs are potential targets for 3-T1AM and mediate the observed physiological effect. Anapyrexia has been observed to be related to action on aminergic receptors such as the serotonin receptor 1b (5-HT1b). This receptor primarily activates the Gi/o mediated pathway and PLC signaling through the Gβγ of Gi/o. Since the expression profiles of TAAR1 and 5-HT1b overlap, we questioned whether 3-T1AM may activate 5-HT1b. Finally, we also evaluated heteromerization between these two GPCRs and tested signaling under co-expressed conditions. In this study, we showed, that 3-T1AM can induce Gi/o signaling through 5-HT1b in a concentration of 10 μM. Strikingly, at 5-HT1b the ligand 3-T1AM only activates the Gi/o mediated reduction of cAMP accumulation, but not PLC activation. Co-stimulation of 5-HT1b by both ligands did not lead to additive or synergistic signaling effects. In addition, we confirmed the capacity for heteromerization between TAAR1 and 5-HT1b. Under co-expression of TAAR1 and HTR1b, 3-T1AM action is only mediated via TAAR1 and activation of 5-HT1b is abrogated. In conclusion, we found evidence for 5-HT1b as a new receptor target for 3-T1AM, albeit with a different signaling effect than the endogenous ligand. Altogether, this indicates a complex interrelation of signaling effects between the investigated GPCRs and respective ligands