The orphan adapter protein SLY1 as a novel anti-apoptotic protein required for thymocyte development

<p>Abstract</p> <p>Background</p> <p>SH3 containing Lymphocyte Protein (SLY1) is a putative adapter protein exclusively expressed in lymphocytes which is involved in antigen receptor induced activation. We previously have generated SLY1^Δ/Δmice harbouring a partial deletion in the N-terminal region of SLY1 which revealed profound immunological defects in T and B cell functions.</p> <p>Results</p> <p>In this study, T cell development in SLY1^-/-and SLY1^Δ/Δmice was analysed <it>ex vivo </it>and upon cultivation with the bone marrow stromal cell line OP9. SLY1-deficient thymocytes were compromised in inducing nutrient receptor expression and ribosomal protein S6 phosphorylation, indicating a defect in mTOR complex activation. Furthermore, SLY1 was identified as a novel anti-apoptotic protein required for developmental progression of T cell precursors to the CD4⁺CD8⁺double-positive stage by protecting from premature programmed cell death initiation in developing CD4^-CD8^-double-negative thymocytes. In addition, SLY1 phosphorylation was differentially regulated upon Notch ligand-mediated stimulation and expression of the preTCR.</p> <p>Conclusion</p> <p>Thus, our results suggest a non-redundant role for SLY1 in integrating signals from both receptors in early T cell progenitors in the thymus.</p

Platelet G i protein Gα i2 is an essential mediator of thrombo-inflammatory organ damage in mice

Platelets are crucial for hemostasis and thrombosis and exacerbate tissue injury following ischemia and reperfusion. Important regulators of platelet function are G proteins controlled by seven transmembrane receptors. The Gi protein Gα(i2) mediates platelet activation in vitro, but its in vivo role in hemostasis, arterial thrombosis, and postischemic infarct progression remains to be determined. Here we show that mice lacking Gα(i2) exhibit prolonged tail-bleeding times and markedly impaired thrombus formation and stability in different models of arterial thrombosis. We thus generated mice selectively lacking Gα(i2) in megakaryocytes and platelets (Gna(i2)(fl/fl)/PF4-Cre mice) and found bleeding defects comparable to those in global Gα(i2)-deficient mice. To examine the impact of platelet Gα(i2) in postischemic thrombo-inflammatory infarct progression, Gna(i2)(fl/fl)/PF4-Cre mice were subjected to experimental models of cerebral and myocardial ischemia/reperfusion injury. In the model of transient middle cerebral artery occlusion stroke Gna(i2)(fl/fl)/PF4-Cre mice developed significantly smaller brain infarcts and fewer neurological deficits than littermate controls. Following myocardial ischemia, Gna(i2)(fl/fl)/PF4-Cre mice showed dramatically reduced reperfusion injury which correlated with diminished formation of the ADP-dependent platelet neutrophil complex. In conclusion, our data provide definitive evidence that platelet Gα(i2) not only controls hemostatic and thrombotic responses but also is critical for the development of ischemia/reperfusion injury in vivo.Fil: Devanathan, Vasudharani. University of Tübingen; AlemaniaFil: Hagedorn, Ina. University Hospital; AlemaniaFil: Köhler, David. University of Tübingen; AlemaniaFil: Pexa, Katja. Universitat Dusseldorf; AlemaniaFil: Cherpokova, Deya. University Hospital; AlemaniaFil: Kraft, Peter. Universität Würzburg; AlemaniaFil: Singh, Madhurendra. Universitat Dusseldorf; AlemaniaFil: Rosenberger, Peter. University of Tübingen; AlemaniaFil: Stoll, Guido. Universität Würzburg; AlemaniaFil: Birnbaumer, Lutz. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Research Triangle Park; AlemaniaFil: Piekorz, Roland P.. Universitat Dusseldorf; AlemaniaFil: Beer-Hammer, Sandra. University of Tübingen; AlemaniaFil: Nieswandt, Bernhard. University Hospital; AlemaniaFil: Nürnberg, Bernd. University of Tübingen; Alemani

RXFP1 Receptor Activation by Relaxin-2 Induces Vascular Relaxation in Mice via a Gαi2-Protein/PI3Kß/γ/Nitric Oxide-Coupled Pathway

Background: Relaxins are small peptide hormones, which are novel candidate molecules that play important roles in cardiometablic syndrome. Relaxins are structurally related to the insulin hormone superfamily, which provide vasodilatory effects by activation of G-protein-coupled relaxin receptors (RXFPs) and stimulation of endogenous nitric oxide (NO) generation. Recently, relaxin could be demonstrated to activate Gi proteins and phosphoinositide 3-kinase (PI3K) pathways in cultured endothelial cells in vitro. However, the contribution of the Gi-PI3K pathway and their individual components in relaxin-dependent relaxation of intact arteries remains elusive.Methods: We used Gαi2- (Gnai2-/-) and Gαi3-deficient (Gnai3-/-) mice, pharmacological tools and wire myography to study G-protein-coupled signaling pathways involved in relaxation of mouse isolated mesenteric arteries by relaxins. Human relaxin-1, relaxin-2, and relaxin-3 were tested.Results: Relaxin-2 (∼50% relaxation at 10-11 M) was the most potent vasodilatory relaxin in mouse mesenteric arteries, compared to relaxin-1 and relaxin-3. The vasodilatory effects of relaxin-2 were inhibited by removal of the endothelium or treatment of the vessels with N (G)-nitro-L-arginine methyl ester (L-NAME, endothelial nitric oxide synthase (eNOS) inhibitor) or simazine (RXFP1 inhibitor). The vasodilatory effects of relaxin-2 were absent in arteries of mice treated with pertussis toxin (PTX). They were also absent in arteries isolated from Gnai2-/- mice, but not from Gnai3-/- mice. The effects were not affected by FR900359 (Gαq protein inhibitor) or PI-103 (PI3Kα inhibitor), but inhibited by TGX-221 (PI3Kβ inhibitor) or AS-252424 (PI3Kγ inhibitor). Simazine did not influence the anti-contractile effect of perivascular adipose tissue.Conclusion: Our data indicate that relaxin-2 produces endothelium- and NO-dependent relaxation of mouse mesenteric arteries by activation of RXFP1 coupled to Gi2-PI3K-eNOS pathway. Targeting vasodilatory Gi-protein-coupled RXFP1 pathways may provide promising opportunities for drug discovery in endothelial dysfunction and cardiometabolic disease

Dataset on the activation of Muller cells through macrophages upon hypoxia in the retina

The dataset presented in this article complements the article entitled “Myeloid cells contribute indirectly to VEGF expression upon hypoxia via activation of Müller cells” (C. Nürnberg, N. Kociok, C. Brockmann, T. Lischke, S. Crespo-Garcia, N. Reichhart, S. Wolf, R. Baumgrass, S.A. Eming, S. Beer- Hammer, and A.M. Joussen). This complementary dataset provides further insight into the experimental validation of the VEGFfl/fl LysMCre (here named VEGFmcko) knockout model used in the main article through genomic and quantitative Real-Time PCR in various murine tissues as well as additional flow cytometry data and immunohistochemical stainings. By providing these data, we aim to enable researcher to reproduce and critically analyze our data

Intranasal delivery of bone marrow derived mesenchymal stem cells, macrophages, and microglia to the brain in mouse models of Alzheimer's and Parkinson's disease

In view of the rapid preclinical development of cell-based therapies for neurodegenerative disorders, traumatic brain injury, and tumors, the safe and efficient delivery and targeting of therapeutic cells to the central nervous system is critical for maintaining therapeutic efficacy and safety in the respective disease models. Our previous data demonstrated therapeutically efficacious and targeted delivery of mesenchymal stem cells (MSCs) to the brain in the rat 6-hydroxydopamine model of Parkinson’s disease (PD). The present study examined delivery of bone marrow derived MSCs, macrophages, and microglia to the brain in a transgenic model of PD ((Thy1)-h[A30P] αS) and an APP/PS1 model of Alzheimer’s disease (AD) via intranasal application (INA). INA of microglia in naïve BL/6 mice led to targeted and effective delivery of cells to the brain. Quantitative PCR analysis of eGFP DNA showed that the brain contained the highest amount of eGFP-microglia (up to 2.1x104) after INA of 1x106 cells, while the total amount of cells detected in peripheral organs did not exceed 3.4x103. Seven days after INA, MSCs expressing eGFP were detected in the olfactory bulb (OB), cortex, amygdala, striatum, hippocampus, cerebellum, and brainstem of (Thy1)-h[A30P] αS transgenic mice, showing predominant distribution within the OB and brainstem. INA of eGFP-expressing macrophages in 13 month-old APP/PS1 mice led to delivery of cells to the OB, hippocampus, cortex, and cerebellum. Both, MSCs and macrophages contained Iba-1-positive population of small microglia-like cells and Iba-1-negative large rounded cells showing either intracellular Amyloid beta (macrophages in APP/PS1 model) or α-Synuclein (MSCs in (Thy1)-h[A30P] αS model) immunoreactivity. Here we show, for the first time, intranasal delivery of cells to the brain of transgenic PD and AD mouse models. Additional work is needed to determine the optimal dosage (single treatment regimen or repeated administrations) to achieve functional improvement in these mouse models with intranasal microglia/macrophages and MSCs

Intranasal delivery of bone marrow derived mesenchymal stem cells, macrophages, and microglia to the brain in mouse models of Alzheimer's and Parkinson's disease

In view of the rapid preclinical development of cell-based therapies for neurodegenerative disorders, traumatic brain injury, and tumors, the safe and efficient delivery and targeting of therapeutic cells to the central nervous system is critical for maintaining therapeutic efficacy and safety in the respective disease models. Our previous data demonstrated therapeutically efficacious and targeted delivery of mesenchymal stem cells (MSCs) to the brain in the rat 6-hydroxydopamine model of Parkinson’s disease (PD). The present study examined delivery of bone marrow derived MSCs, macrophages, and microglia to the brain in a transgenic model of PD ((Thy1)-h[A30P] αS) and an APP/PS1 model of Alzheimer’s disease (AD) via intranasal application (INA). INA of microglia in naïve BL/6 mice led to targeted and effective delivery of cells to the brain. Quantitative PCR analysis of eGFP DNA showed that the brain contained the highest amount of eGFP-microglia (up to 2.1x104) after INA of 1x106 cells, while the total amount of cells detected in peripheral organs did not exceed 3.4x103. Seven days after INA, MSCs expressing eGFP were detected in the olfactory bulb (OB), cortex, amygdala, striatum, hippocampus, cerebellum, and brainstem of (Thy1)-h[A30P] αS transgenic mice, showing predominant distribution within the OB and brainstem. INA of eGFP-expressing macrophages in 13 month-old APP/PS1 mice led to delivery of cells to the OB, hippocampus, cortex, and cerebellum. Both, MSCs and macrophages contained Iba-1-positive population of small microglia-like cells and Iba-1-negative large rounded cells showing either intracellular Amyloid beta (macrophages in APP/PS1 model) or α-Synuclein (MSCs in (Thy1)-h[A30P] αS model) immunoreactivity. Here we show, for the first time, intranasal delivery of cells to the brain of transgenic PD and AD mouse models. Additional work is needed to determine the optimal dosage (single treatment regimen or repeated administrations) to achieve functional improvement in these mouse models with intranasal microglia/macrophages and MSCs

Gα_i Proteins are Indispensable for Hearing

Background/Aims: From invertebrates to mammals, Gαi proteins act together with their common binding partner Gpsm2 to govern cell polarization and planar organization in virtually any polarized cell. Recently, we demonstrated that Gαi3-deficiency in pre-hearing murine cochleae pointed to a role of Gαi3 for asymmetric migration of the kinocilium as well as the orientation and shape of the stereociliary (“hair”) bundle, a requirement for the progression of mature hearing. We found that the lack of Gαi3 impairs stereociliary elongation and hair bundle shape in high-frequency cochlear regions, linked to elevated hearing thresholds for high-frequency sound. How these morphological defects translate into hearing phenotypes is not clear. Methods: Here, we studied global and conditional Gnai3 and Gnai2 mouse mutants deficient for either one or both Gαi proteins. Comparative analyses of global versus Foxg1-driven conditional mutants that mainly delete in the inner ear and telencephalon in combination with functional tests were applied to dissect essential and redundant functions of different Gαi isoforms and to assign specific defects to outer or inner hair cells, the auditory nerve, satellite cells or central auditory neurons. Results: Here we report that lack of Gαi3 but not of the ubiquitously expressed Gαi2 elevates hearing threshold, accompanied by impaired hair bundle elongation and shape in high-frequency cochlear regions. During the crucial reprogramming of the immature inner hair cell (IHC) synapse into a functional sensory synapse of the mature IHC deficiency for Gαi2 or Gαi3 had no impact. In contrast, double-deficiency for Gαi2 and Gαi3 isoforms results in abnormalities along the entire tonotopic axis including profound deafness associated with stereocilia defects. In these mice, postnatal IHC synapse maturation is also impaired. In addition, the analysis of conditional versus global Gαi3-deficient mice revealed that the amplitude of ABR wave IV was disproportionally elevated in comparison to ABR wave I indicating that Gαi3 is selectively involved in generation of neural gain during auditory processing. Conclusion: We propose a so far unrecognized complexity of isoform-specific and overlapping Gαi protein functions particular during final differentiation processes

Depletion of Dendritic Cells Enhances Innate Anti-Bacterial Host Defense through Modulation of Phagocyte Homeostasis

Dendritic cells (DCs) as professional antigen-presenting cells play an important role in the initiation and modulation of the adaptive immune response. However, their role in the innate immune response against bacterial infections is not completely defined. Here we have analyzed the role of DCs and their impact on the innate anti-bacterial host defense in an experimental infection model of Yersinia enterocolitica (Ye). We used CD11c-diphtheria toxin (DT) mice to deplete DCs prior to severe infection with Ye. DC depletion significantly increased animal survival after Ye infection. The bacterial load in the spleen of DC-depleted mice was significantly lower than that of control mice throughout the infection. DC depletion was accompanied by an increase in the serum levels of CXCL1, G-CSF, IL-1α, and CCL2 and an increase in the numbers of splenic phagocytes. Functionally, splenocytes from DC-depleted mice exhibited an increased bacterial killing capacity compared to splenocytes from control mice. Cellular studies further showed that this was due to an increased production of reactive oxygen species (ROS) by neutrophils. Adoptive transfer of neutrophils from DC-depleted mice into control mice prior to Ye infection reduced the bacterial load to the level of Ye-infected DC-depleted mice, suggesting that the increased number of phagocytes with additional ROS production account for the decreased bacterial load. Furthermore, after incubation with serum from DC-depleted mice splenocytes from control mice increased their bacterial killing capacity, most likely due to enhanced ROS production by neutrophils, indicating that serum factors from DC-depleted mice account for this effect. In summary, we could show that DC depletion triggers phagocyte accumulation in the spleen and enhances their anti-bacterial killing capacity upon bacterial infection