Cilia are small, hair-like structures on the surface of most eukaryotic cells. They are composed of distinct substructures: the basal body, the transition zone, and the axoneme. Proper ciliary function is crucial for human health, and defects can result in a group of disorders known as ciliopathies. Many ciliopathy-associated mutations affect genes encoding proteins of the ciliary transition zone, a key structural and regulatory region at the base of the cilium. Understanding the molecular composition and interactions within subciliary compartments, such as the transition zone, is essential to elucidate their role in ciliary function and disease. Protein interaction studies have played a central role in uncovering the functional landscape of subciliary compartments. In this context, the in situ proximity ligation assay (in situ PLA) has emerged as a valuable tool to investigate whether two proteins are located in close proximity (less than 40 nm) within the cellular environment, implying potential interaction. In situ PLA uses primary antibodies to recognise target proteins, followed by secondary antibodies conjugated with oligonucleotides (PLA probes). When two probes are sufficiently close, added circle-forming oligonucleotides can hybridise and ligate to form a circular DNA strand. This DNA circle serves as a template for rolling circle amplification, which is then detected through hybridisation with fluorescently labelled oligonucleotides. The resulting signals can be visualised using fluorescence microscopy, enabling precise spatial mapping of protein proximities in cells. The in situ PLA technique offers a powerful means of detecting protein proximities in subciliary compartments with high spatial resolution. This method supports the identification of novel protein interactions and contributes to a deeper understanding of subciliary architecture and its disruption in ciliopathies.

Das Jahrbuch der Heinrich-Heine-Universität Düsseldorf versteht sich als Forum für den wissenschaftlichen Dialog der Universität zu Zeitfragen, zu aktuellen Problemlagen und Herausforderungen von Wissenschaft und Gesellschaft, als Brücke der Vermittlung zwischen Forschung und Öffentlichkeit sowie als Gedächtnisort der Innovationen und des Fortschritts in Forschung und Lehre der Universität und als Speicher der wissenschafts- und hochschulpolitischen Entscheidungen für strukturelle Weichenstellungen mit Langzeitwirkung. Zielgruppe ist die an den Arbeitsergebnissen in Forschung und Lehre sowie an wissenschaftlichen Entscheidungen der Heinrich-Heine-Universität interessierte Öffentlichkeit. Diese soll über die Dynamik und das sich wandelnde Profil der Fakultäten kontinuierlich informiert und in die Lage versetzt werden, sich intensiver mit neuen Forschungsfragen und -ergebnissen auseinander zu setzen. Es geht vor allem darum, die Bedeutung der Forschung für die verschiedenen Lebensbereiche und damit auch für unsere gesellschaftliche Entwicklung bewusst zu machen. Die Beiträge vermitteln gleichsam als Momentaufnahme einen Ausschnitt aus dem permanenten Prozess des sich verändernden Profils der Fakultäten. Erst eine Folge von Jahrbüchern eröffnet die Chance, die Tiefe des Gesamtprofils auszuloten und dessen Nachhaltigkeit zu erkennen.

Mastocytosis is a heterogeneous disease characterized by an abnormal accumulation of mast cells (MCs) in 1 or several organs. Although a somatic KIT D816V mutation is detected in similar to 85% of patients, attempts to demonstrate its oncogenic effect alone have repeatedly failed, suggesting that additional pathways are involved in MC transformation. From 3 children presenting with both Greig cephalopolysyndactyly syndrome (GCPS, Mendelian Inheritance in Man [175700]) and congenital mastocytosis, we demonstrated the involvement of the hedgehog (Hh) pathway in mastocytosis. GCPS is an extremely rare syndrome resulting from haploinsufficiency of GLI3, the major repressor of Hh family members. From these familial cases of mastocytosis, we demonstrate that the Hh pathway is barely active in normal primary MCs and is overactive in neoplastic MCs. GLI3 and KIT mutations had a synergistic, tumorigenic effect on the onset of mastocytosis in a GCPS mouse model. Finally, Hh inhibitors suppressed neoplastic MC proliferation in vitro and extend the survival time of mice with aggressive systemic mastocytosis (ASM). This work revealed, for the first time, the involvement of Hh signaling in the pathophysiology of mastocytosis and demonstrated the cooperative effects of the KIT and Hh oncogenic pathways in mice with ASM, leading to the identification of new promising therapeutic targets. (C) 2021 by The American Society of Hematology.

Ventricular septal defects (VSDs) are developmental disorders, characterised by a gap in the septum between the right and the left ventricle, that lead to life-threatening heart defects. At present, the only curative treatment of VSDs is surgical closure. Since these surgeries comprise several severe risks, the development of alternative therapies against VSDs is urgently needed. To develop such therapies, the current knowledge of the molecular factors and mechanisms underlying VSDs has to be increased. Based on our previous data, we analysed the relevance of the HH signalling pathway mediator GLI3 in ventricular septum (VS) formation. GLI3 functions as both a transcriptional activator (GLI3-A) and repressor (GLI3-R). By analysing two different mouse Gli3 mutants, we revealed that the lack of GLI3-A with simultaneous presence of GLI3-R impairs cilia-mediated PDGFRα signalling causing reduced cell proliferation and in consequence the development of VSDs. Moreover, we showed that the rescue of PDGFRα signalling restores cell proliferation. Since VSDs are also appear in humans with comparable gain-of-function mutations in GLI3 , our findings propose activators of PDGFRα signalling as potential agents against the development of VSDs.SUMMARY The article reports how a gain-of-function mutation of Gli3 causes ventricular septal defects and paves the way for therapies tackling these congenital heart defects.

Initially, the function of the fat mass and obesity associated (Fto) gene seemed to be primarily the regulation of the body weight. Here we show that loss of Fto results in a hyperactivation of the hypothalamic-pituitary-adrenal (HPA) axis. In consequence, Fto(-/-) mice display an anxiety-like behavior and impairments in working memory. Furthermore, differentiation of neurons is affected in the hippocampus. As a cause of these impairments we identified a processing defect of the neurotrophin BDNF which is most likely the result of a reduced expression of MMP-9. Therefore, we propose FTO as a possible new target to develop novel approaches for the treatment of diseases associated with hippocampal disorders. In parallel, we also would like to make the point that any anti-obesity therapy via blocking FTO function can have negative effects on the proper function of the hippocampus.

Protein degradation is a pivotal process for eukaryotic development and homeostasis. The majority of proteins are degraded by the ubiquitin–proteasome system and by autophagy. Recent studies describe a crosstalk between these two main eukaryotic degradation systems which allows for establishing a kind of safety mechanism. If one of these degradation systems is hampered, the other compensates for this defect. The mechanism behind this crosstalk is poorly understood. Novel studies suggest that primary cilia, little cellular protrusions, are involved in the regulation of the crosstalk between the two degradation systems. In this review article, we summarise the current knowledge about the association between cilia, the ubiquitin–proteasome system and autophagy.

Since 1967, it is known that the loss of GLI3 causes very severe defects in murine eye development. GLI3 is able to act as a transcriptional activator (GLI3-A) or as a transcriptional repressor (GLI3-R). Soon after the discovery of these GLI3 isoforms, the question arose which of the different isoforms is involved in eye formation – GLI3-A, GLI3-R or even both. For several years, this question remained elusive. By analysing the eye morphogenesis of Gli3XtJ/XtJ mouse embryos that lack GLI3-A and GLI3-R and of Gli3Δ699/Δ699 mouse embryos in which only GLI3-A is missing, we revealed that GLI3-A is dispensable in vertebrate eye formation. Remarkably, our study shows that GLI3-R is sufficient for the creation of morphologically normal eyes although the molecular setup deviates substantially from normality. In depth-investigations elucidated that GLI3-R controls numerous key players in eye development and governs lens and retina development at least partially via regulating WNT/β-CATENIN signalling.

Previously, macroautophagy/autophagy was demonstrated to be regulated inter alia by the primary cilium. Mutations in RPGRIP1L cause ciliary dysfunctions resulting in severe human diseases summarized as ciliopathies. Recently, we showed that RPGRIP1L deficiency leads to a decreased proteasomal activity at the ciliary base in mice. Importantly, the drug-induced restoration of proteasomal activity does not rescue ciliary length alterations in the absence of RPGRIP1L indicating that RPGRIP1L affects ciliary function also via other mechanisms. Based on this knowledge, we analyzed autophagy in Rpgrip1l-negative mouse embryos. In these embryos, autophagic activity was decreased due to an increased activation of the MTOR complex 1 (MTORC1). Application of the MTORC1 inhibitor rapamycin rescued dysregulated MTORC1, autophagic activity and cilia length but not proteasomal activity in Rpgrip1l-deficient mouse embryonic fibroblasts demonstrating that RPGRIP1L seems to regulate autophagic and proteasomal activity independently from each other.

Obesity is one of the major risk factors that can lead to a myocardial infarction and can negatively influence subsequent cardiac remodeling. The onset of obesity is related to different genetic variants of the fat mass and obesity associated gene (Fto). Fto deficient mice were protected from obesity and showed an improved glucose tolerance under a high fat diet. In addition, it is known that Fto acts as an m6A RNA demethylase, whereby it can influence mRNA stability and translation. In a first approach, we used Fto deficient mice to perform an ischemic/reperfusion (I/R) model. At 24 h after reperfusion, Fto deficient mice already had a smaller infarct size compared to their wild type littermates. Over a time period of three weeks, the heart function was investigated by echocardiography. Three weeks after reperfusion, Fto deficient mice showed a preserved heart function and had reduced collagen scar formation compared to wild‐type control mice. Initial molecular studies indicated a reduction of the mTORC1 pathway and consequently a down regulation of S6K phosphorylation. For a first approach of therapeutic intervention, we used the Fto inhibitor rhein, which binds the active center of Fto and thereby inhibits its demethylase activity. Wild type mice were pre‐treated with rhein or the solvent over three weeks before the I/R procedure was performed. The rhein‐treated mice showed a significant reduction in the infarct size compared to the solvent treated littermates. In this study, we demonstrated that Fto deficiency can modulate the outcome of a myocardial infarction by improving left ventricular function and reduction of the infarct size.Support or Funding InformationSupported by the Deutsche Forschung Gesellschaft (DFG) as Part of the SFB 1116 (project number 236177352)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Ciliopathies are life-threatening human diseases caused by defective cilia. They can often be traced back to mutations of genes encoding transition zone (TZ) proteins demonstrating that the understanding of TZ organisation is of paramount importance. The TZ consists of multimeric protein modules that are subject to a stringent assembly hierarchy. Previous reports place Rpgrip1l at the top of the TZ assembly hierarchy in Caenorhabditis elegans. Byperforming quantitative immunofluorescence studies in RPGRIP1L(-/-) mouse embryos and human embryonic cells, we recognise a different situation in vertebrates in which Rpgrip1l deficiency affects TZ assembly in a cell type-specific manner. In cell types in which the loss of Rpgrip1l alone does not affect all modules, additional truncation or removal of vertebrate-specific Rpgrip1 results in an impairment of all modules. Consequently, Rpgrip1l and Rpgrip1 synergistically ensure the TZ composition in several vertebrate cell types, revealing a higher complexity of TZ assembly in vertebrates than in invertebrates.

RPGRIP1L is an evolutionary highly conserved gene encoding a protein that localises at the transition zone of primary cilia. Mutations in RPGRIP1L result in ciliopathies, severe human diseases caused by dysfunctional cilia. Patients with mutations in this gene often suffer from an impaired development of not only one but various organs. To elucidate the function of Rpgrip1l in human development and the mechanisms underlying ciliopathies, different model organisms are used. In this review article, we summarise the findings of these investigations comprising novel functions of Rpgrip1l and the most promising therapeutic approaches.

Genetic variations within the fat mass and obesity associated gene (FTO) are known to correlate with obesity and hypertension in humans, demonstrating a possible key role in vascular health. Therefore, we hypothesized that FTO may be important at the vascular level. Because endothelial cells (EC) are known to have a central role in the development of obesity‐induced metabolic changes and hypertension, we wanted to investigate if murine loss of endothelial Fto may influence obesity‐induced pathophysiological changes. Therefore, EC‐specific Fto deficient mice were generated using a tamoxifen‐inducible Cre system. To analyze obesity‐dependent effects, mice were either fed a normal chow (NC) or a high fat diet (HFD) for 12 weeks. Regardless of the endothelial deletion of Fto, the mice still gained a significant amount of weight and were obese, indicating the Fto gene in endothelium did not inhibit weight gain like global Fto deficiency in mice. Next we determined whether loss of endothelial Fto affected obesity‐induced metabolic changes in HFD‐fed mice. While serum lipid analysis showed no differences in dependence of endothelial Fto, glucose and insulin tolerance was significantly improved in HFD‐fed mice without endothelial Fto. Due to the importance of EC in vascular tone of resistance arteries, we isolated 3rd order mesenteric arteries from NC‐ and HFD‐fed mice with and without endothelial Fto and performed pressure curves to analyze myogenic tone. While loss of endothelial Fto did not influence myogenic tone in resistance arteries of NC‐fed mice, remarkably, endothelial Fto deletion provided protection from HFD‐induced changes on myogenic tone. Subsequently, blood pressure was measured in HFD‐fed mice with and without endothelial Fto using radiotelemetry and it revealed that loss of endothelial Fto significantly reduced blood pressure. Collectively, these data suggest that loss of endothelial Fto was protective against HFD‐induced changes on myogenic tone in mesenteric arteries and subsequently reduced blood pressure in vivo without influencing body weight. Furthermore, glucose homeostasis, but not serum lipid levels, were specifically improved in HFD‐fed mice suggesting that endothelial Fto is not only important in the development of obesity‐induced hypertension, but also in the development of insulin resistance and hyperglycemia.Support or Funding InformationAHA, NHLBI and IRTG1902

Fibrosis, which occurs in various heart diseases like acute myocardial ischemia and pressure overload, is triggered by the differentiation of fibroblasts into myofibroblasts. Dysregulation of this reparative mechanism results in excessive collagen accumulation leading to cardiac stiffness and impaired heart function. The aim of this study was to determine whether the rhubarb anthraquinone Rhein, a drug already used as treatment for chondroarthritis, prevents the transdifferentiation of cardiac fibroblasts. We observed that Rhein pre-treatment ameliorates the cardiac function and reduces adverse remodeling after acute myocardial infarction in mice, in vivo . In primary human cardiac fibroblasts, Rhein incubation dose-dependently inhibited the TGF-β-mediated upregulation of α-SMA, the master marker for myofibrolasts, and prevented the contraction of fibroblast-populated collagen gel lattices upon TGF-β stimulation. Further, Rhein reduced TGFβ-R1 expression in primary human cardiac fibroblast, resulting in decreased SMAD2 phosphorylation and blunting of the fibrogenic response. Furthermore, Rhein stabilized protein levels of SMAD7, a key inhibitor of TGF-β signaling. Collectively, these data show for the first time that Rhein administration prevents cardiac fibrosis in vivo and in vitro by blunting the TGF-β signaling pathway, and identify Rhein as potential therapeutic treatment to prevent excessive fibrosis and adverse remodeling in cardiac pathologies.

Objective Myelosuppression is a life-threatening complication of thiopurine therapy, and the incidence of thiopurine-induced myelosuppression is higher in East Asians than in Europeans. We investigated genetic factors associated with thiopurine-induced leukopenia in patients with IBD. Design A genome-wide association study (GWAS) was conducted in thiopurine-treated patients with IBD, followed by high-throughput sequencing of genes identified as significant in the GWAS or those involved in thiopurine metabolism (n=331). Significant loci associated with thiopurine-induced leukopenia were validated in two additional replication cohorts (n=437 and n=330). Functional consequences of FTO (fat mass and obesity-associated) variant were examined both in vitro and in vivo. Results The GWAS identified two loci associated with thiopurine-induced leukopenia (rs16957920, FTO intron; rs2834826, RUNX1 intergenic). High-throughput targeted sequencing indicated that an FTO coding variant (rs79206939, p.A134T) linked to rs16957920 is associated with thiopurine-induced leukopenia. This result was further validated in two replication cohorts (combined p=1.3×10−8, OR=4.3). The frequency of FTO p.A134T is 5.1% in Koreans but less than 0.1% in Western populations. The p.A134T variation reduced FTO activity by 65% in the nucleotide demethylase assay. In vivo experiments revealed that Fto−/− and Fto+/− mice were more susceptible to thiopurine-induced myelosuppression than wild-type mice. Conclusions The results suggest that the hypomorphic FTO p.A134T variant is associated with thiopurine-induced leukopenia. These results shed light on the novel physiological role of FTO and provide a potential pharmacogenetic biomarker for thiopurine therapy.

Few cell lines have been used to investigate the involvement of c-Fos in differentiation. Furthermore, some data have been derived from organ culture experiments. The potential involvement of c-Fos in the differentiation of embryonal carcinoma (EC) cells was tested by transfection of different c-fos gene constructs into F9 EC cells. The First report about the expression of c-Fos in mice described it as restricted to the extraembryonic tissues and placenta in mouse development and to bone and skin in adult mice. In addition, c-Fos is expressed in the intestine, developing cartilage, and the spinal cord as well as in certain structures in the peripheral nervous system. If c-Fos is a key regulator in development, one would expect its activity to be dominant. The ultimate proof for the function of c-Fos in differentiation and development is the analysis of mice lacking c-Fos. The tissue culture experiments had previously indicated that c-Fos has a distinct function in differentiation.

An incomplete septation of the ventricles in the vertebrate heart that disturbes the strict separation between the contents of the two ventricles is termed a ventricular septal defect (VSD). Together with bicuspid aortic valves, it is the most frequent congenital heart disease in humans. Until now, life-threatening VSDs are usually treated surgically. To avoid surgery and to develop an alternative therapy (e.g., a small molecule therapy), it is necessary to understand the molecular mechanisms underlying ventricular septum (VS) development. Consequently, various studies focus on the investigation of signalling pathways, which play essential roles in the formation of the VS. In the past decade, several reports found evidence for an involvement of Hedgehog (HH) signalling in VS development. In this review article, we will summarise the current knowledge about the association between HH signalling and VS formation and discuss the use of such knowledge to design treatment strategies against the development of VSDs.

The Hedgehog signalling pathway is evolutionarily highly conserved and essential for embryonic development of invertebrates and vertebrates. Consequently, impaired Hedgehog signalling results in very severe human diseases, ranging from holoprosencephaly to Pallister-Hall syndrome. Due to this great importance for human health, the focus of numerous research groups is placed on the investigation of the detailed mechanisms underlying Hedgehog signalling. Today, it is known that tiny cell protrusions, known as primary cilia, are necessary to mediate Hedgehog signalling in vertebrates. Although the Hedgehog pathway is one of the best studied signalling pathways, many questions remain. One of these questions is: How do primary cilia control Hedgehog signalling in vertebrates? Recently, it was shown that primary cilia regulate a special kind of proteasome which is essential for proper Hedgehog signalling. This review article will cover this novel cilia-proteasome association in embryonic Hedgehog signalling and discuss the possibilities provided by future investigations on this topic.

The primary cilium is an essential structure for the mediation of numerous signaling pathways involved in the coordination and regulation of cellular processes essential for the development and maintenance of health. Consequently, ciliary dysfunction results in severe human diseases called ciliopathies. Since many of the cilia-mediated signaling pathways are oncogenic pathways, cilia are linked to cancer. Recent studies demonstrate the existence of a cilia-regulated proteasome and that this proteasome is involved in cancer development via the progression of oncogenic, cilia-mediated signaling. This review article investigates the association between primary cilia and cancer with particular emphasis on the role of the cilia-regulated proteasome.