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Dendritic spines are small membranous protrusions covering the dendritic tree of principal telencephalic neurons, such as the GC or CA2-pc. The CA2-subregion is crucial for social memory. Dendritic spines are a main site of synaptic plasticity, which is a key element of learning and memory. The plasticity-related protein Synaptopodin (SP) is essential to form the spine apparatus (SA), a spine-specific organelle involved in synaptic plasticity. SP stabilizes dendritic spines. This thesis investigated, for the first time, the dendritic SP-distribution and its influence on spine density and spine head size under different conditions in adult mice ex vivo: 1) SP-overexpression (gain-of-function), 2) SP-deficiency (loss-of-function), and 3) wild type-level of SP-expression in male and female mice (sex-differences in dCA2). SP-overexpression in adult male CSPtg-mice led to a ~doubled ratio of SP+ spines in the OML of the DG, while the spine density, the average spine head size and the average SP-puncta size were not affected. Consistently, SP-deficiency in adult male SP-KO animals had no significant effect on average spine head size. Of importance, under SP-overexpression, many small spines and a few large spines become SP+, assumingly assembling a SA. On a functional level, this may indicate an activation of silent synapses. dCA2 showed sex specific differences in spine density and spine morphology in a layer-specific manner: In males, pc-spines of the basal dCA2-compartment showed larger spine heads than females in the diestrus stage of their cycle (females (diestrus), while spine density was not significantly different. In the apical dCA2-compartment (sr), females (diestrus) showed an increased spine density, while spine head size was still shifted towards larger head sizes in males. In addition, dCA2 showed significant layer-specific differences in spine head size, but in a sex-independent manner: In both sexes, average spine head size in the apical sr was significantly smaller than in the basal so. This findings could reflect a yet unknown compartment-specific difference in synaptic plasticity in the basal compartment, which is preferentially targeted by neuromodulatory input from extrahippocampal sources such as the PVN or SUM99,101,170,189-195. In so of dCA2, there was no sex-specific difference in SP-puncta size or in the ratio of SP+ spines, indicating that SP is distributed in a sex-independent manner in dCA2 in adult mice.
Pericytes are capillary-associated mural cells involved in the maintenance and the stability of the vascular network. This thesis aims to investigate the role of pericytes in the heart in the context of ageing and disease. We highlight the malignant effects of the remodelling in the heart and stress the focus on the role of cardiac pericytes in this context. We show that ageing reduces pericyte coverage and that myocardial infarction (MI) causes an activation of these cells. Single-nuclei and single-cell RNA sequencing analysis of murine hearts further revealed that the expression of the Regulator of G-protein signalling 5 (Rgs5) is reduced in cardiac pericytes both in ageing and transiently at day 1 and day 3 after MI. The loss of RGS5 in pericytes drives an entropic state of these mural cells characterized by morphological changes, excessive extracellular deposition and enhanced Gaq mediated GPCR signalling. The deletion of RGS5 in pericytes causes cardiac systolic dysfunction, induces myocardial fibrosis, and drives the activation of cardiac fibroblasts in a TGFb-dependent manner. In conclusion, our results describe the importance of pericytes maintaining cardiac homeostasis, identify RGS5 as a key regulator of this process and propose pericytes as crucial mediators of cardiac fibrosis and possible therapeutic targets to prevent cardiovascular disease.
Characteristics of critical incident reporting systems in primary care: an international survey
(2022)
Aim: The aim of the study was to support the development of future critical incident reporting systems (CIRS) in primary care by collecting information on existing systems. Our focus was on processes used to report and analyse incidents, as well as strategies used to overcome difficulties.
Methods: Based on literature from throughout the world, we identified existing CIRS in primary care. We developed a questionnaire and sent it to operators of a purposeful sample of 17 CIRS in primary care. We used cross-case analysis to compare the answers and pinpoint important similarities and differences in the CIRS in our sample.
Results: Ten CIRS operators filled out the questionnaire, and 9 systems met the inclusion criteria. The sample of CIRS came from 8 different countries and was rather heterogeneous. The reporting systems invited a broad range of professions to report, with some also including reports by patients. In most cases, reporting was voluntary and conducted via an online reporting form. Reports were analysed locally, centrally, or both. The various CIRS used interesting ideas to deal with barriers. Some, for example, used confidential reporting modes as a compromise between anonymity and the need for follow-up investigations, whereas others used smartphone applications and call centres to speed up the reporting process.
Conclusion: We found multiple CIRS that have operated in primary care for many years, have received a high number of reports and were largely developed in accordance with recommendations found in literature. Although primary care in Germany differs from other countries, these CIRS could serve as an inspiration for CIRS in German primary care.
Acute myeloid leukemia (AML) is a neoplastic disease of an early myeloid precursor cell in hematopoiesis. It leads to the accumulation of monoclonal cells in the bone marrow and the peripheral blood, showing a differentiation block and deregulated self-renewal. Frequently, the leukemic cells exhibit genetic aberrations with reciprocal chromosomal translocations. These translocations induce the formation of a fusion protein, that can lead to new cellular functions and a transformation into a leukemic cell. Common chromosomal translocation in AML are t(8;21) or t(15;17), which cause the formation of the fusion proteins AML1/ETO and PML/RARα and determine the leukemic phenotype of the AML.
The translocation t(6;9) leads to the formation of the fusion protein DEK/CAN and is of special interest, because of its association with mostly young patients and a very aggressive course of the disease. The fusion product induces leukemia in a small subset of hematopoietic stem cells, but its mechanism of leukemogenesis is greatly unknown.
The intention of this work was to characterize the DEK/CAN-induced AML on a molecular genetic level to gain a deeper understanding of the disease pathogenesis. Therefore, gene expression analysis with polymerase chain reaction (PCR) and microarray analysis was performed.
To detect DEK/CAN in different cell lines by PCR and real-time quantitative PCR (qPCR), specific primers and probes were designed, and a standardized workflow was established. Emphasis was placed on the optimization of RNA isolation, DNase treatment, cDNA synthesis with following PCR and qPCR, which enabled the detection of the fusion product DEK/CAN in the cell lines 32B, Phoenix and FKH-1. To quantify the fusion product DEK/CAN, the method of qPCR with absolute and relative quantification was used. Absolute quantification enabled the calculation of an exact copy number of the fusion transcript DEK/CAN with a detection limit of 50 copies/µl at a sensitivity of 10-6, which is of importance in determining the minimal residual disease (MRD) of patients with DEK/CAN-positive AML. MRD detection by qPCR is a highly sensitive diagnostic method to identify leukemic cells, even in low cell counts. This enables a thorough evaluation of the treatment response and allows an early detection of changes in the MRD level as part of the remission control.
Additionally, a microarray gene expression analysis was performed to identify alterations in relevant target genes and associated signaling pathways in DEK/CAN-positive cells.
Because of DEK/CAN’s potential to induce leukemia in a subset of hematopoietic stem cells, Sca+/Lin- cells of the bone marrow of C57Bl/6 mice were used and transfected with the gene products DEK/CAN and PML/RARα. Microarray analysis led to the identification of 16 different genes of interest, which demonstrated significant alterations of gene expression in DEK/CAN-positive cells. They were validated and quantified with TaqMan assay assisted qPCR. The elevated expression of the transcription factors TRIM25, HIF1α and ATF2, in DEK/CAN-positive cells, indicated an altered transcription factor activity and interaction with DNA in the nucleus. The localization of DEK/CAN in the nucleus emphasizes this assumption. Also, the upregulated expression of the nuclear export receptor XPO1 suggested changes in nuclear transport processes and impaired export activity in DEK/CAN-positive cells.
Furthermore, the results demonstrated changes of gene expression in genes that are involved in the JAK/STAT signaling pathway. PTPRC, the Protein Tyrosine Phosphatase Receptor Type C, functions as a direct inhibitor of JAKs (Janus Kinases) and STATs (Signal Transducers and Activators of Transcription) and their associated signaling pathway.
It was shown that the gene expression of PTPRC was significantly reduced in DEK/CAN-positive cells. This allowed the assumption, that the reduced expression of PTPRC led to a loss of inhibition and thus a consecutive hyperactivation of the JAK/STAT signaling pathway. This hypothesis was supported by an independent activation of PIM1, a target gene of STAT5 and the activation of LMO2, a direct target gene of JAK2. In addition, the transmembrane receptor CSF1R, which is directly involved in STAT activation, also showed an upregulation in gene expression.
The results of this work show an activation of the JAK/STAT signaling pathway in DEK/CAN-positive cells, which may be a key mechanism in DEK/CAN-induced leukemogenesis.
Considering treatment options in the future, the addition of targeted therapy, such as pan-JAK inhibitors, to the standard therapy, could be a chance to improve the overall survival rate and the prognosis of t(6;9)-positive AML.
BH3 mimetics are novel anticancer therapeutics that induce apoptosis by targeting anti‐apoptotic BCL‐2 proteins. Highly specific inhibitors of the main anti-apoptotic proteins BCL-2, BCL‐XL and MCL‐1 promise new opportunities for the treatment of AML. However, it is currently unclear which of these anti-apoptotic BCL-2 proteins represents the most promising target in AML. Therefore, we investigated the effect of BH3 mimetics targeting either BCL-2 (ABT-199, S55746), BCL-XL (A-1331852) or MCL-1 (S63845) on eleven AML cell lines. Drug sensitivity screening revealed heterogeneous sensitivity towards the different BH3 mimetics, with the best responses observed upon targeting of MCL-1. Selected cell lines that displayed sensitivity towards the specific BH3 mimetics underwent intrinsic apoptosis, which was characterized by loss of mitochondrial membrane potential, exposure of phosphatidylserine and activation of caspases. Furthermore, S63845 turned out to displace BIMS and NOXA from MCL-1 to induce apoptotic cell death. Importantly, the translational relevance of this study was demonstrated by experiments in primary AML blasts, which displayed similar sensitivity towards BH3 mimetics as the cell lines did. Additionally, experiments with nonmalignant cells could confirm the clinical relevance of the MCL-1 inhibitor. There we could show, that S63845 does not cause cytotoxicity on HPCs at efficacious doses.
In conclusion, our findings reveal that the inhibition of BCL-2 proteins, especially MCL-1, by BH3 mimetics can be a promising strategy in AML treatment.
Functional roles of COMP and TSP-4 in articular cartilage and their relevance in osteoarthritis
(2020)
Osteoarthritis (OA) is a slowly progressing disease, resulting in the degradation of cartilage and the loss of joint functionality. The cartilage extracellular matrix (ECM) is degraded and undergoes remodelling in OA progression. Chondrocytes start to express degrading proteases but are also reactivated and synthesise ECM proteins. The spectrum of these newly synthesised proteins and their involvement in OA specific processes and cartilage repair is hardly investigated.
Human articular cartilage obtained from OA patients undergoing knee replacement surgery was evaluated according to the OARSI histopathology grading system. Healthy, non-OA cartilage samples were used as controls. The expression and distribution of thrombospondin-4 (TSP-4) and the closely related COMP were analysed on the gene level by PCR and on the protein level by immunohistology and immunoblot assays. The potential of TSP-4 as a diagnostic marker was evaluated by immunoblot assays, using serum samples from OA patients and healthy individuals. The functional role of both proteins was further investigated in in vitro studies using chondrocytes isolated from femoral condyles of healthy pigs. The effect of COMP and TSP-4 on chondrocyte migration and attachment was investigated via transwell and attachment assays, respectively. Moreover, the potential of COMP and TSP-4 to modulate the chondrocyte phenotype by inducing gene expression, ECM protein synthesis and matrix formation was investigated by immunofluorescence staining and qPCR. The activation of cartilage relevant signalling pathways was investigated by immunoblot assays.
These results showed for the first time the presence of TSP-4 in articular cartilage. Its amount dramatically increased in OA compared to healthy cartilage and correlated positively with OA severity. In healthy cartilage TSP-4 was primarily found in the superficial zone while it was wider distributed in the middle and deeper zones of OA cartilage. The amount of specific TSP-4 fragments was increased in sera of OA patients compared to healthy controls, indicating a potential to serve as an OA biomarker. COMP was ubiquitously expressed in healthy cartilage but degraded in early as well as re-expressed in late-stage OA. The overall protein levels between OA severity grades were comparable. Contrary to TSP-4, COMP was localised primarily in the upper zone of OA cartilage, in particular in areas with severe damage. COMP could attract chondrocytes and facilitated their attachment, while TSP-4 did not affect these processes. COMP and TSP 4 were generally weak inducers of gene expression, although both could induce COL2A1 and TSP-4 additionally COL12A1 and ACAN after 6 h. Correlating data were obtained on the protein level: COMP and TSP-4 promoted the synthesis and matrix formation of collagen II, collagen IX, collagen XII and proteoglycans. In parallel, both proteins suppressed chondrocyte hypertrophy and dedifferentiation by reducing collagen X and collagen I. By analysing the effect of COMP and TSP-4 on intracellular signalling, both proteins induced Erk1/2 phosphorylation and TSP-4 could further promote Smad2/3 signalling induced by TGF-β1. None of the two proteins had a direct or modulatory effect on Smad1/5/9 dependent signalling.
In summary, COMP and TSP-4 contribute to ECM maintenance and repair by inducing the expression of essential ECM proteins and suppressing chondrocyte dedifferentiation. These effects might be mediated by Erk1/2 phosphorylation. The presented data demonstrate an important functional role of COMP and TSP-4 in both healthy and OA cartilage and provide a basis for further studies on their potential in clinical applications for OA diagnosis and treatment.
Role of Orphan G-protein-coupled receptor GPRC5B in smooth muscle contractility and differentiation
(2019)
G protein coupled receptors (GPCRs) are the largest family of cell-surface receptors encoded in the human genome. They mediate the cellular responses to a wide variety of stimuli, ranging from light, odorants, and metabolic cues to hormones, neurotransmitters, and local mediators. Upon ligand binding, the GPCR undergoes conformational changes resulting in the activation of heterotrimeric G-proteins belonging to the families Gs, Gi/o, Gq/11, G12/13, which in turn mediate the downstream signaling. While most of the 360 non-olfactory GPCRs are well studied, approximately 120 GPCRs are still considered "orphan", meaning that their mechanism of activation and biological function is unknown. GPCRs have been functionally described in the regulation of almost all organ systems, and their dysregulation has been implicated in the pathogenesis of a multitude of diseases. In the vascular system, the contractile tone of vessels is crucially regulated by GPCRs. Substances that act through G12/13- and Gq/11-coupled GPCRs are associated with facilitation of contraction, while Gs-coupled GPCRs are usually associated with the induction of relaxation. Furthermore, while Gq/11 pathway activation promotes proliferation and dedifferentiation of vascular smooth muscle cells (VSMC), G12/13 and Gs signaling pathways promote expression of contractile proteins and differentiation.
The functional properties of VSMC depend on the anatomical location, and a recent single-cell expression analysis showed that VSMC from different vascular beds have different patterns of GPCR expression. Interestingly, smooth muscle cells (SMCs) from resistance arteries not only express various GPCRs for known modulators of vascular tone, but also a number of orphan GPCRs. These results suggest a potential role of orphan GPCRs in the modulation of blood pressure. Orphan GPCR GPRC5B was one of the GPCRs enriched in resistance arteries, and this receptor was also upregulated in dedifferentiated aortal SMC. The function of GPRC5B in these types of SMC is currently unknown. In vitro studies suggested that GPRC5B negatively regulates obesity, inflammation, insulin secretion and fibrotic activity, but there are no data available with respect to its function in regulation of vascular tone or other SMC functions.
Our study aimed at the identification of the specific functions of GPRC5B in SMC. To do so, we generated a SMC-specific GPRC5B-deficient mouse line by crossing Gprc5bfl/fl mice with smooth muscle-specific, tamoxifen-inducible Myh11-CreERT2 mice. We found that SMC-specific deletion of GPRC5B did neither affect myogenic tone in pressure myography, nor the response to the contractile agonists in wire myography. In contrast, vessel relaxation in response to prostacyclin analogues cicaprost and iloprost, which act on the prostacyclin receptor IP, were increased. These results suggested a selective improvement of IP receptor signaling. The IP receptor is coupled to Gs protein, it promotes vasorelaxation and acts as a restraint on platelet activation. Using overexpression of IP and GPRC5B in HEK cells, we found that GPRC5B physically interacts with the IP receptor and controls IP trafficking and membrane localization. Furthermore, we found that membrane IP receptor expression was increased in GPRC5B-deficient human aortic SMC and in resistance vessels of SMC-specific GPRC5B. To investigate the importance of increased IP-mediated signaling in SMC in vivo, we measured blood pressure in two mouse models of hypertension. We found that SMC-deletion of Gprc5b resulted in a significant reduction of blood pressure compared with control mice, which suggested that Gprc5b negatively regulated relaxation in hypertensive disease by decreasing IP mediated relaxation. In line with this notion we found that application of the IP antagonist Cay10441 largely abrogated the beneficial effect of GPRC5B inactivation in this hypertension model. Another important function of the IP receptor is the regulation of SMC differentiation, which led us to investigate the differentiation state of GPRC5B-deficient SMC. We found that deletion of GPRC5B enhanced expression of contractile genes and reduced expression of proliferative markers. This improved differentiation was, at least partially, due to increased IP signaling in SMC. Moreover, in a mouse model of atherosclerosis SMC-specific deletion of Gprc5b reduced plaque area and contributed to a more stable fibrous cap by promoting differentiation.
In conclusion, deletion of GPRC5B in SMC significantly improved contractility and differentiation by increasing IP receptor membrane availability and signaling.
Characterization of SPRTN, the first mammalian metalloprotease that repairs DNA-protein-crosslinks
(2019)
DNA is constantly exposed to various endogenous and exogenous sources causing different kinds of DNA damage. To overcome this threat, cells have evolved various repair mechanisms. Impairments of these repair mechanisms result in diverse diseases. Ruijs-Aalfs syndrome is a monogenic disease characterized by accelerated ageing and carcinogenesis, typical features of impaired DNA repair and was shown to be caused by germline mutations of SPRTN, a newly identified and only partially understood protein. A role of SPRTN in DNA damage response was previously shown and an involvement in translesion synthesis (TLS) proposed. However, later discoveries revealed an essential function of SPRTN, being indispensible for embryonic development of vertebrates and cellular survival, whereby this function is independent of SPRTN’s proposed function in TLS. The essential function of SPRTN was proposed to be contained in its protease domain but remained unclear.
In this study we identify SPRTN as the first mammalian metalloprotease that repairs DNA-protein-crosslinks (DPCs). DPCs represent a specific type of DNA-lesions with bulky protein adducts covalently linked to DNA thereby being highly toxic as they potentially stall replication forks and lead to double strand breaks and genomic instability. DPC-repair remains only partially understood despite their frequent appearance and toxicity. With this study we discover and characterize a new mechanism of DPC-repair in mammalian cells - a proteolytic cleavage of the protein adduct by the metalloprotease SPRTN. Accordingly, a proteolytic activity of SPRTN is demonstrated and s SPRTN-recruitment to DNA upon DPC-induction displayed. Furthermore, SPRTN exhibits degradation of different proteins covalently bound to DNA in form of DPCs, but not of unbound fractions of the same protein substrates. Consequently, mutations of SPRTN’s proteolytic core as well as a mislocalization or depletion of SPRTN result in impaired DPC-repair. The importance of SPRTN-mediated DPC-removal is confirmed by a severely compromised response to DPC-inducing agents for cells with impaired SPRTN function. Additionally to the discovery of SPRTN’s essential function this study further provides an explanation of the molecular mechanism underlying Ruijs-Aalfs syndrome (RJALS), the segmental progeroid syndrome resulting from SPRTN mutation. The effects of the identified clinical mutations on the DPC-repair function of SPRTN are explained and a DPC-accumulation in cells carrying clinical SPRTN-mutation displayed. The obtained data provides sufficient evidence that an impaired DPC-repair is the pathophysiologic cause of RJALS-syndrome, confirming the importance of SPRTN’s newly identified function. In conclusion, SPRTN is the first identified mammalian metalloprotease with a DPC-repairing function and the impairment of SPRTN-mediated DPC-removal is the underlying mechanism of RJALS syndrome.
Durch Implementierung eines effizienten Früherkennungsprogramms ist die Inzidenz des Zervixkarzinoms in Industrienationen seit 2005 auf konstant niedrigem Niveau. Ungeachtet dessen ist das Zervixkarzinom mit deutlich höheren Inzidenzraten und weniger als 50% Gesamtüberleben in den nicht industrialisierten Staaten die vierthäufigste Tumorentität der Frau weltweit.
Zur Behandlung des lokal fortgeschrittenen Zervixkarzinoms (FIGO Stadium IIb bis IVa bzw. Ib2/IIa2 mit mehreren histologischen Risikofaktoren) besteht nach aktueller Leitlinie (Stand 2014) und internationalem Konsens Indikation zur platinhaltigen Radiochemotherapie (RCT), subsequent gefolgt von einer (High Dose-Rate) Brachytherapie (HDR-BT). Unter diesen Umständen beträgt die lokale Kontrolle für Patientinnen mit lokal fortgeschrittenem Tumor zwischen 74% und 85%.
Dennoch stagnieren Gesamtüberleben und das spezifische Überleben bezogen auf verschiedene klinische Endpunkte, sodass die Entwicklung neuer Behandlungsstrategien und Therapieoptionen, insbesondere zur Behandlung rezidivierter und metastasierter Erkrankungsstadien, angezeigt ist. Darüber hinaus spielen im Gegensatz zu anderen Tumorentitäten molekulare Marker sowohl als prädiktive als auch als therapeutische Targets bei der Behandlung des Zervixkarzinoms eine bislang untergeordnete Rolle, während molekular-zielgerichtete Therapien in der modernen Krebstherapie einen immer größeren Stellenwert einnehmen.
Ziel der hier vorliegenden Arbeit ist es, neue Biomarker für das Zervixkarzinom und dessen Ansprechen auf simultane Radiochemotherapie und anschließende Brachytherapie zu identifizieren.
Zu diesem Zweck untersuchten wir in einem Patientenkollektiv von 74 Patientinnen mit histologisch gesichertem Zervixkarzinom (FIGO Ib - IVb) prätherapeutisch gewonnenes Biopsiegewebe. Mittels immunhistochemischer Methoden wurde die Expression von Polo-like Kinase 3 (PLK3) und phosphoT273 Caspase-8 erfasst und quantifiziert. Die Ergebnisse wurden anschließend mit klinischen bzw. histo-pathologischen Charakteristika, einschließlich der p16INK4a Expression und den klinischen Endpunkten lokales progressionsfreies- und Fernmetastasen-freies
Überleben bzw. dem tumorspezifischem und dem Gesamtüberleben nach kurativ intendierter Therapie korreliert.
Hierbei konnte zunächst eine signifikante Korrelation zwischen der PLK3 und pT273 Caspase-8 Expression beobachtet werden (p = 0.009). Darüber hinaus war PLK3 signifikant mit dem N-Status (p = 0.046), dem M-Status (0.026) und dem FIGO-Stadium (p = 0.001) assoziiert, wohingegen die pT273 Caspase8-Expression signifikant mit der Tumorgröße (T-Stadium) korreliert war. Bezogen auf univariate Überlebenszeitanalysen war eine erhöhte PLK3-Expression signifikant mit einer geringeren Rate an Fernmetastasen (DMFS p = 0.009) sowie einem signifikant verlängertem tumorspezifischen und Gesamtüberleben assoziiert (CSS p = 0.001, OS p = 0.003). Vergleichbare Ergebnisse konnten auch für die pT273-Caspase 8 Expression mit einer verringerten Metastasierungsrate (p=0.021) und verbessertem tumorspezifischem (p<0,001) sowie Gesamtüberleben (p=<0.001) gezeigt werden. In den multivariaten Analysen verblieb die pT273-Caspase 8-Expression mit einem signifikant verbesserten Gesamtüberleben (p=0.001).
Zusammenfassend belegen diese Daten erstmals eine signifikante Korrelation zwischen einer erhöhten prä-therapeutischen PLK3 und pT273 Caspase 8- Expression und einem zu favorisierenden klinischen Verlauf nach mit Radiochemotherapie behandeltem Zervixkarzinom.