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Aims: We investigated N471D WASH complex subunit strumpellin (Washc5) knock-in and Washc5 knock-out mice as models for hereditary spastic paraplegia type 8 (SPG8). Methods: We generated heterozygous and homozygous N471D Washc5 knock-in mice and subjected them to a comprehensive clinical, morphological and laboratory parameter screen, and gait analyses. Brain tissue was used for proteomic analysis. Furthermore, we generated heterozygous Washc5 knock-out mice. WASH complex subunit strumpellin expression was determined by qPCR and immunoblotting. Results: Homozygous N471D Washc5 knock-in mice showed mild dilated cardiomyopathy, decreased acoustic startle reactivity, thinner eye lenses, increased alkaline phosphatase and potassium levels and increased white blood cell counts. Gait analyses revealed multiple aberrations indicative of locomotor instability. Similarly, the clinical chemistry, haematology and gait parameters of heterozygous mice also deviated from the values expected for healthy animals, albeit to a lesser extent. Proteomic analysis of brain tissue depicted consistent upregulation of BPTF and downregulation of KLHL11 in heterozygous and homozygous knock-in mice. WASHC5-related protein interaction partners and complexes showed no change in abundancies. Heterozygous Washc5 knock-out mice showing normal WASHC5 levels could not be bred to homozygosity. Conclusions: While biallelic ablation of Washc5 was prenatally lethal, expression of N471D mutated WASHC5 led to several mild clinical and laboratory parameter abnormalities, but not to a typical SPG8 phenotype. The consistent upregulation of BPTF and downregulation of KLHL11 suggest mechanistic links between the expression of N471D mutated WASHC5 and the roles of both proteins in neurodegeneration and protein quality control, respectively.
Holocene climate was characterised by variability on multi-centennial to multi-decadal time scales. In central Europe, these fluctuations were most pronounced during winter. Here we present a record of past winter climate variability for the last 10.8 ka based on four speleothems from Bunker Cave, western Germany. Due to its central European location, the cave site is particularly well suited to record changes in precipitation and temperature in response to changes in the North Atlantic realm. We present high-resolution records of δ18O, δ13C values and Mg/Ca ratios. Changes in the Mg/Ca ratio are attributed to past meteoric precipitation variability. The stable C isotope composition of the speleothems most likely reflects changes in vegetation and precipitation, and variations in the δ18O signal are interpreted as variations in meteoric precipitation and temperature. We found cold and dry periods between 8 and 7 ka, 6.5 and 5.5 ka, 4 and 3 ka as well as between 0.7 and 0.2 ka. The proxy signals in the Bunker Cave stalagmites compare well with other isotope records and, thus, seem representative for central European Holocene climate variability. The prominent 8.2 ka event and the Little Ice Age cold events are both recorded in the Bunker Cave record. However, these events show a contrasting relationship between climate and δ18O, which is explained by different causes underlying the two climate anomalies. Whereas the Little Ice Age is attributed to a pronounced negative phase of the North Atlantic Oscillation, the 8.2 ka event was triggered by cooler conditions in the North Atlantic due to a slowdown of the thermohaline circulation.
Holocene climate was characterised by variability on multi-centennial to multi-decadal time scales. In central Europe, these fluctuations were most pronounced during winter. Here we present a new record of past winter climate variability for the last 10.8 ka based on four speleothems from Bunker Cave, Western Germany. Due to its central European location, the cave site is particularly well suited to record changes in precipitation and temperature in response to changes in the North Atlantic realm. We present high resolution records of δ18O, δ13C values and Mg/Ca ratios. We attribute changes in the Mg/Ca ratio to variations in the meteoric precipitation. The stable C isotope composition of the speleothems most likely reflects changes in vegetation and precipitation and variations in the δ18O signal are interpreted as variations in meteoric precipitation and temperature. We found cold and dry periods between 9 and 7 ka, 6.5 and 5.5 ka, 4 and 3 ka as well as between 0.7 to 0.2 ka. The proxy signals in our stalagmites compare well with other isotope records and, thus, seem representative for central European Holocene climate variability. The prominent 8.2 ka event and the Little Ice Age cold events are both recorded in the Bunker cave record. However, these events show a contrasting relationship between climate and δ18O, which is explained by different causes underlying the two climate anomalies. Whereas the Little Ice Age is attributed to a pronounced negative phase of the North Atlantic Oscillation, the 8.2 ka event was triggered by cooler conditions in the North Atlantic due to a slowdown of the Thermohaline Circulation.
Cyclic GMP-dependent protein kinase 1 (PKG1) mediates presynaptic nociceptive long-term potentiation (LTP) in the spinal cord and contributes to inflammatory pain in rodents but the present study revealed opposite effects in the context of neuropathic pain. We used a set of loss-of-function models for in vivo and in vitro studies to address this controversy: peripheral neuron specific deletion (SNS-PKG1-/-), inducible deletion in subsets of neurons (SLICK-PKG1-/-) and redox-dead PKG1 mutants. In contrast to inflammatory pain, SNS-PKG1-/- mice developed stronger neuropathic hyperalgesia associated with an impairment of nerve regeneration, suggesting specific repair functions of PKG1. Although PKG1 accumulated at the site of injury, its activity was lost in the proximal nerve due to a reduction of oxidation-dependent dimerization, which was a consequence of mitochondrial damage in injured axons. In vitro, PKG1 deficiency or its redox-insensitivity resulted in enhanced outgrowth and reduction of growth cone collapse in response to redox signals, which presented as oxidative hotspots in growing cones. At the molecular level, PKG1 deficiency caused a depletion of phosphorylated cofilin, which is essential for growth cone collapse and guidance. Hence, redox-mediated guidance required PKG1 and consequently, its deficiency in vivo resulted in defective repair and enhanced neuropathic pain after nerve injury. PKG1-dependent repair functions will outweigh its signaling functions in spinal nociceptive LTP, so that inhibition of PKG1 is no option for neuropathic pain.
The Masquelet technique for the treatment of large bone defects is a two‐stage procedure based on an induced membrane. The size of a scaffold is reported to be a critical factor for bone healing response. We therefore aimed to investigate the influence of the granule size of a bone graft substitute on bone marrow derived mononuclear cells (BMC) supported bone healing in combination with the induced membrane. We compared three different sizes of Herafill® granules (Heraeus Medical GmbH, Wehrheim) with or without BMC in vivo in a rat femoral critical size defect. A 10 mm defect was made in 126 rats and a membrane induced by a PMMA‐spacer. After 3 weeks, the spacer was taken out and membrane filled with different granule sizes. After 8 weeks femurs were taken for radiological, biomechanical, histological, and immunohistochemical analysis. Further, whole blood of the rat was incubated with granules and expression of 29 peptide mediators was assessed. Smallest granules showed significantly improved bone healing compared to larger granules, which however did not lead to an increased biomechanical stability in the defect zone. Small granules lead to an increased accumulation of macrophages in situ which could be assigned to the inflammatory subtype M1 by majority. Increased release of chemotactic respectively proangiogenic active factors in vitro compared to syngenic bone and beta‐TCP was observed. Granule size of the bone graft substitute Herafill® has significant impact on bone healing of a critical size defect in combination with Masquelet's technique in terms of bone formation and inflammatory.
Measuring NADPH oxidase (Nox)-derived reactive oxygen species (ROS) in living tissues and cells is a constant challenge. All probes available display limitations regarding sensitivity, specificity or demand highly specialized detection techniques. In search for a presumably easy, versatile, sensitive and specific technique, numerous studies have used NADPH-stimulated assays in membrane fractions which have been suggested to reflect Nox activity. However, we previously found an unaltered activity with these assays in triple Nox knockout mouse (Nox1-Nox2-Nox4-/-) tissue and cells compared to wild type. Moreover, the high ROS production of intact cells overexpressing Nox enzymes could not be recapitulated in NADPH-stimulated membrane assays. Thus, the signal obtained in these assays has to derive from a source other than NADPH oxidases. Using a combination of native protein electrophoresis, NADPH-stimulated assays and mass spectrometry, mitochondrial proteins and cytochrome P450 were identified as possible source of the assay signal. Cells lacking functional mitochondrial complexes, however, displayed a normal activity in NADPH-stimulated membrane assays suggesting that mitochondrial oxidoreductases are unlikely sources of the signal. Microsomes overexpressing P450 reductase, cytochromes b5 and P450 generated a NADPH-dependent signal in assays utilizing lucigenin, L-012 and dihydroethidium (DHE). Knockout of the cytochrome P450 reductase by CRISPR/Cas9 technology (POR-/-) in HEK293 cells overexpressing Nox4 or Nox5 did not interfere with ROS production in intact cells. However, POR-/- abolished the signal in NADPH-stimulated assays using membrane fractions from the very same cells. Moreover, membranes of rat smooth muscle cells treated with angiotensin II showed an increased NADPH-dependent signal with lucigenin which was abolished by the knockout of POR but not by knockout of p22phox. In conclusion: the cytochrome P450 system accounts for the majority of the signal of Nox activity chemiluminescence based assays.
Aim: NADPH oxidases are important sources of reactive oxygen species (ROS). Several Nox homologues are present together in the vascular system but whether they exhibit crosstalk at the activity level is unknown. To address this, vessel function of knockout mice for the cytosolic Nox organizer proteins p47phox, NoxO1 and a p47phox-NoxO1-double knockout were studied under normal condition and during streptozotocin-induced diabetes.
Results: In the mouse aorta, mRNA expression for NoxO1 was predominant in smooth muscle and endothelial cells, whereas p47phox was markedly expressed in adventitial cells comprising leukocytes and tissue resident macrophages. Knockout of either NoxO1 or p47phox resulted in lower basal blood pressure. Deletion of any of the two subunits also prevented diabetes-induced vascular dysfunction. mRNA expression analysis by MACE (Massive Analysis of cDNA ends) identified substantial gene expression differences between the mouse lines and in response to diabetes. Deletion of p47phox induced inflammatory activation with increased markers of myeloid cells and cytokine and chemokine induction. In contrast, deletion of NoxO1 resulted in an attenuated interferon gamma signature and reduced expression of genes related to antigen presentation. This aspect was also reflected by a reduced number of circulating lymphocytes in NoxO1-/- mice.
Innovation and conclusion: ROS production stimulated by NoxO1 and p47phox limit endothelium-dependent relaxation and maintain blood pressure in mice. However, NoxO1 and p47phox cannot substitute each other despite their similar effect on vascular function. Deletion of NoxO1 induced an anti-inflammatory phenotype, whereas p47phox deletion rather elicited a hyper-inflammatory response.
Objective: The NADPH oxidase Nox4 is an important source of H2O2. Nox4-derived H2O2 limits vascular inflammation and promotes smooth muscle differentiation. On this basis, the role of Nox4 for restenosis development was determined in the mouse carotid artery injury model. Methods and results: Genetic deletion of Nox4 by a tamoxifen-activated Cre-Lox-system did not impact on neointima formation in the carotid artery wire injury model. To understand this unexpected finding, time-resolved single-cell RNA-sequencing (scRNAseq) from injured carotid arteries of control mice and massive-analysis-of-cDNA-ends (MACE)-RNAseq from the neointima harvested by laser capture microdissection of control and Nox4 knockout mice was performed. This revealed that resting smooth muscle cells (SMCs) and fibroblasts exhibit high Nox4 expression, but that the proliferating de-differentiated SMCs, which give rise to the neointima, have low Nox4 expression. In line with this, the first weeks after injury, gene expression was unchanged between the carotid artery neointimas of control and Nox4 knockout mice. Conclusion: Upon vascular injury, Nox4 expression is transiently lost in the cells which comprise the neointima. NADPH oxidase 4 therefore does not interfere with restenosis development after wire-induced vascular injury.
The KASCADE-Grande experiment has significantly contributed to the current knowledge about the energy spectrum and composition of cosmic rays for energies between the knee and the ankle. Meanwhile, post-LHC versions of the hadronic interaction models are available and used to interpret the entire data set of KASCADE-Grande. In addition, a new, combined analysis of both arrays, KASCADE and Grande, was developed significantly increasing the accuracy of the shower observables. First results of the new analysis with the entire data set of the KASCADE-Grande experiment will be the focus of this contribution.
Reactive oxygen species (ROS) are important mediators of both physiological and pathophysiological signal transduction in the cardiovascular system. The effects of ROS on cellular processes depend on the concentration, localization, and duration of exposure. Cellular stress response mechanisms have evolved to mitigate the negative effects of acute oxidative stress. In this study, we investigate the short-term and long-term metabolic and transcriptomic response of human umbilical vein endothelial cells (HUVEC) to different types and concentrations of ROS. To generate intracellular H2O2, we utilized a lentiviral chemogenetic approach for overexpression of human D-amino acid oxidase (DAO). DAO converts D-amino acids into their corresponding imino acids and H2O2. HUVEC stably overexpressing DAO (DAO-HUVEC) were exposed to D-alanine (3 mM), exogenous H2O2 (10 µM or 300 µM), or menadione (5 µM) for various timepoints and subjected to global untargeted metabolomics (LC-MS/MS) and RNAseq by MACE (Massive analysis of cDNA ends). A total of 300 µM H2O2 led to pronounced changes on both the metabolic and transcriptomic level. In particular, metabolites linked to redox homeostasis, energy-generating pathways, and nucleotide metabolism were significantly altered. Furthermore, 300 µM H2O2 affected genes related to the p53 pathway and cell cycle. In comparison, the effects of menadione and DAO-derived H2O2 mainly occurred at gene expression level. Collectively, all types of ROS led to subtle changes in the expression of ribosomal genes. Our results show that different types and concentration of ROS lead to a different metabolic and transcriptomic response in endothelial cells.