Margitta Elvers

Tumor necrosis factor α triggers proliferation of adult neural stem cells via IKK/NF-κB signaling

BackgroundBrain inflammation has been recognized as a complex phenomenon with numerous related aspects. In addition to the very well-described neurodegenerative effect of inflammation, several studies suggest that inflammatory signals exert a potentially positive influence on neural stem cell proliferation, migration and differentiation. Tumor necrosis factor alpha (TNF-α) is one of the best-characterized mediators of inflammation. To date, conclusions about the action of TNF on neural stem or progenitor cells (NSCs, NPCs) have been conflicting. TNF seems to activate NSC proliferation and to inhibit their differentiation into NPCs. The purpose of the present study was to analyze the molecular signal transduction mechanisms induced by TNF and resulting in NSC proliferation.ResultsHere we describe for the first time the TNF-mediated signal transduction cascade in neural stem cells (NSCs) that results in increased proliferation. Moreover, we demonstrate IKK-α/β-dependent proliferation and markedly up-regulated cyclin D1 expression after TNF treatment. The significant increase in proliferation in TNF-treated cells was indicated by increased neurosphere volume, increased bromodeoxyuridin (BrdU) incorporation and a higher total cell number. Furthermore, TNF strongly activated nuclear factor-kappa B (NF-κB) as measured by reporter gene assays and by an activity-specific antibody. Proliferation of control and TNF-treated NSCs was strongly inhibited by expression of the NF-κB super-repressor IκB-AA1. Pharmacological blockade of IκB ubiquitin ligase activity led to comparable decreases in NF-κB activity and proliferation. In addition, IKK-β gene product knock-down via siRNA led to diminished NF-κB activity, attenuated cyclin D1 expression and finally decreased proliferation. In contrast, TGFβ-activated kinase 1 (TAK-1) is partially dispensable for TNF-mediated and endogenous proliferation. Understanding stem cell proliferation is crucial for future regenerative and anti-tumor medicine.ConclusionTNF-mediated activation of IKK-β resulted in activation of NF-κB and was followed by up-regulation of the bona-fide target gene cyclin D1. Activation of the canonical NF-κB pathway resulted in strongly increased proliferation of NSCs.

The serum- and glucocorticoid-inducible kinase 1 (SGK1) influences platelet calcium signaling and function by regulation of Orai1 expression in megakaryocytes

Platelets are activated on increase of cytosolic Ca2+ activity ([Ca2+](i)), accomplished by store-operated Ca2+ entry (SOCE) involving the pore-forming ion channel subunit Orai1. Here, we show, for the first time, that the serum- and glucocorticoid-inducible kinase 1 (SGK1) is expressed in platelets and megakaryocytes. SOCE and agonist-induced [Ca2+](i) increase are significantly blunted in platelets from SGK1 knockout mice (sgk1(-/-)). Similarly, Ca2+ -dependent degranulation, integrin α(IIb)β3 activation, phosphatidylserine exposure, aggregation, and in vitro thrombus formation were significantly impaired in sgk1(-/-) platelets, whereas tail bleeding time was not significantly enhanced. Platelet and megakaryocyte Orai1 transcript levels and membrane protein abundance were significantly reduced in sgk1(-/-) mice. In human megakaryoblastic cells (MEG-01), transfection with constitutively active (S422D)SGK1 but not with inactive (K127N)SGK1 significantly enhanced Orai1 expression and SOCE, while effects reversed by the SGK1 inhibitor GSK650394 (1μM). Transfection of MEG-01 cells with (S422D)SGK1 significantly increased phosphorylation of IκB kinase α/β and IκBα resulting in nuclear translocation of NF-κB subunit p65. Treatment of (S422D)SGK1-transfected MEG-01 cells with the IκB kinase inhibitor BMS-345541 (10μM) abolished SGK1-induced increase of Orai1 expression and SOCE. The present observations unravel SGK1 as novel regulator of platelet function, effective at least in part by NF-κB-dependent transcriptional up-regulation of Orai1 in megakaryocytes and increasing platelet SOCE.

Multiple alterations of platelet functions dominated by increased secretion in mice lacking Cdc42 in platelets

Platelet activation at sites of vascular injury is crucial for hemostasis, but it may also cause myocardial infarction or stroke. Cytoskeletal reorganization is essential for platelet activation and secretion. The small GTPase Cdc42 has been implicated as an important mediator of filopodia formation and exocytosis in various cell types, but its exact function in platelets is not established. Here, we show that the megakaryocyte/platelet-specific loss of Cdc42 leads to mild thrombocytopenia and a small increase in platelet size in mice. Unexpectedly, Cdc42-deficient platelets were able to form normally shaped filopodia and spread fully on fibrinogen upon activation, whereas filopodia formation upon selective induction of GPIb signaling was reduced compared with wild-type platelets. Furthermore, Cdc42-deficient platelets showed enhanced secretion of alpha granules, a higher adenosine diphosphate (ADP)/adenosine triphosphate (ATP) content, increased aggregation at low agonist concentrations, and enhanced aggregate formation on collagen under flow. In vivo, lack of Cdc42 resulted in faster occlusion of ferric chloride-injured arterioles. The life span of Cdc42-deficient platelets was markedly reduced, suggesting increased clearing of the cells under physiologic conditions. These data point to novel multiple functions of Cdc42 in the regulation of platelet activation, granule organization, degranulation, and a specific role in GPIb signaling.

The Bispecific SDF1-GPVI Fusion Protein Preserves Myocardial Function After Transient Ischemia in Mice

Background CXCR4-positive bone marrow cells (BMCs) are critically involved in cardiac repair mechanisms contributing to preserved cardiac function. Stromal cell–derived factor-1 (SDF-1) is the most prominent BMC homing factor known to augment BMC engraftment, which is a limiting step of stem cell–based therapy. After myocardial infarction, SDF-1 expression is rapidly upregulated and promotes myocardial repair. Methods and Results We have established a bifunctional protein consisting of an SDF-1 domain and a glycoprotein VI (GPVI) domain with high binding affinity to the SDF-1 receptor CXCR4 and extracellular matrix proteins that become exposed after tissue injury. SDF1-GPVI triggers chemotaxis of CXCR4-positive cells, preserves cell survival, enhances endothelial differentiation of BMCs in vitro, and reveals proangiogenic effects in ovo. In a mouse model of myocardial infarction, administration of the bifunctional protein leads to enhanced recruitment of BMCs, increases capillary density, reduces infarct size, and preserves cardiac function. Conclusions These results indicate that administration of SDF1-GPVI may be a promising strategy to treat myocardial infarction to promote myocardial repair and to preserve cardiac function.

Microarray analysis of tumor necrosis factor α induced gene expression in U373 human glioblastoma cells

BackgroundTumor necrosis factor α (TNF) is able to induce a variety of biological responses in the nervous system including inflammation and neuroprotection. Human astrocytoma cells U373 have been widely used as a model for inflammatory cytokine actions in the nervous system. Here we used cDNA microarrays to analyze the time course of the transcriptional response from 1 h up to 12 h post TNF treatment in comparison to untreated U373 cells. TNF activated strongly the NF-κB transcriptional pathway and is linked to other pathways via the NF-κB target genes JUNB and IRF-1. Part of the TNF-induced gene expression could be inhibited by pharmacological inhibition of NF-κB with pyrrolidine-dithiocarbamate (PDTC). NF-κB comprises a family of transcription factors which are involved in the inducible expression of genes regulating neuronal survival, inflammatory response, cancer and innate immunity.ResultsIn this study we show that numerous genes responded to TNF (> 880 from 7500 tested) with a more than two-fold induction rate. Several novel TNF-responsive genes (about 60% of the genes regulated by a factor ≥ 3) were detected. A comparison of our TNF-induced gene expression profiles of U373, with profiles from 3T3 and Hela cells revealed a striking cell-type specificity. SCYA2 (MCP-1, CCL2, MCAF) was induced in U373 cells in a sustained manner and at the highest level of all analyzed genes. MCP-1 protein expression, as monitored with immunofluorescence and ELISA, correlated exactly with microarray data. Based on these data and on evidence from literature we suggest a model for the potential neurodegenerative effect of NF-κB in astroglia: Activation of NF-κB via TNF results in a strongly increased production of MCP-1. This leads to a exacerbation of neurodegeneration in stoke or Multiple Sclerosis, presumably via infiltration of macrophages.ConclusionsThe vast majority of genes regulated more than 3-fold were previously not linked to tumor necrosis factor α as a search in published literature revealed. Striking co-regulation for several functional groups such as proteasome and ribosomal proteins were detected.

Long-term depression activates transcription of immediate early transcription factor genes: involvement of serum response factor/Elk-1.

Long‐term depression (LTD) is one of the paradigms used in vivo or ex vivo for studying memory formation. In order to identify genes with potential relevance for memory formation we used mouse organotypic hippocampal slice cultures in which chemical LTD was induced by applications of 3,5‐dihydroxyphenylglycine (DHPG). The induction of chemical LTD was robust, as monitored electrophysiologically. Gene expression analysis after chemical LTD induction was performed using cDNA microarrays containing >7000 probes. The DHPG‐induced expression of immediate early genes (c‐fos, junB, egr1 and nr4a1) was subsequently verified by TaqMan polymerase chain reaction. Bioinformatic analysis suggested a common regulator element [serum response factor (SRF)/Elk‐1 binding sites] within the promoter region of these genes. Indeed, here we could show a DHPG‐dependent binding of SRF at the SRF response element (SRE) site within the promoter region of c‐fos and junB. However, SRF binding to egr1 promoter sites was constitutive. The phosphorylation of the ternary complex factor Elk‐1 and its localization in the nucleus of hippocampal neurones after DHPG treatment was shown by immunofluorescence using a phosphospecific antibody. We suggest that LTD leads to SRF/Elk‐1‐regulated gene expression of immediate early transcription factors, which could in turn promote a second broader wave of gene expression.

Blood Platelets in the Progression of Alzheimer's Disease

Alzheimer’s disease (AD) is characterized by neurotoxic amyloid-ß plaque formation in brain parenchyma and cerebral blood vessels known as cerebral amyloid angiopathy (CAA). Besides CAA, AD is strongly related to vascular diseases such as stroke and atherosclerosis. Cerebrovascular dysfunction occurs in AD patients leading to alterations in blood flow that might play an important role in AD pathology with neuronal loss and memory deficits. Platelets are the major players in hemostasis and thrombosis, but are also involved in neuroinflammatory diseases like AD. For many years, platelets were accepted as peripheral model to study the pathophysiology of AD because platelets display the enzymatic activities to generate amyloid-ß (Aß) peptides. In addition, platelets are considered to be a biomarker for early diagnosis of AD. Effects of Aß peptides on platelets and the impact of platelets in the progression of AD remained, however, ill-defined. The present study explored the cellular mechanisms triggered by Aß in platelets. Treatment of platelets with Aß led to platelet activation and enhanced generation of reactive oxygen species (ROS) and membrane scrambling, suggesting enhanced platelet apoptosis. More important, platelets modulate soluble Aß into fibrillar structures that were absorbed by apoptotic but not vital platelets. This together with enhanced platelet adhesion under flow ex vivo and in vivo and platelet accumulation at amyloid deposits of cerebral vessels of AD transgenic mice suggested that platelets are major contributors of CAA inducing platelet thrombus formation at vascular amyloid plaques leading to vessel occlusion critical for cerebrovascular events like stroke.

Intracellular cyclophilin A is an important Ca2+ regulator in platelets and critically involved in arterial thrombus formation

Platelet adhesion and aggregation play a critical role in primary hemostasis. Uncontrolled platelet activation leads to pathologic thrombus formation and organ failure. The decisive central step for different processes of platelet activation is the increase in cytosolic Ca(2+) activity ([Ca(2+)](i)). Activation-dependent depletion of intracellular Ca(2+) stores triggers Ca(2+) entry from the extracellular space. Stromal interaction molecule 1 (STIM1) has been identified as a Ca(2+) sensor that regulates store-operated Ca(2+) entry through activation of the pore-forming subunit Orai1, the major store-operated Ca(2+) entry channel in platelets. In the present study, we show for the first time that the chaperone protein cyclophilin A (CyPA) acts as a Ca(2+) modulator in platelets. CyPA deficiency strongly blunted activation-induced Ca(2+) mobilization from intracellular stores and Ca(2+) influx from the extracellular compartment and thus impaired platelet activation substantially. Furthermore, the phosphorylation of the Ca(2+) sensor STIM1 was abrogated upon CyPA deficiency, as shown by immunoprecipitation studies. In a mouse model of arterial thrombosis, CyPA-deficient mice were protected against arterial thrombosis, whereas bleeding time was not affected. The results of the present study identified CyPA as an important Ca(2+) regulator in platelets, a critical mechanism for arterial thrombosis.

The dimeric platelet collagen receptor GPVI-Fc reduces platelet adhesion to activated endothelium and preserves myocardial function after transient ischemia in mice

Platelets play a critical role in the pathophysiology of reperfusion, sepsis, and cardiovascular diseases. In a multiple step process, they adhere to activated endothelium and release proinflammatory cytokines thereby promoting the inflammatory process. Glycoprotein VI (GPVI) is the major collagen receptor on the platelet surface and triggers platelet activation and primary hemostasis. Activation of GPVI leads to stable platelet adhesion and degranulation of platelet granules. However, GPVI is critically involved in platelet adhesion to activated endothelium without exposure of subendothelial matrix. Earlier studies show that the soluble GPVI-Fc binds to collagen and protects mice from atherosclerosis and decreases neointima proliferation after arterial injury. Here, we show for the first time that recombinant GPVI-Fc binds to activated endothelium mainly via vitronectin and prevents platelet/endothelial interaction. Administration of GPVI-Fc reduced infarct size and preserved cardiac function in a mouse model of myocardial infarction. This process was associated with reduced GPVI-induced platelet degranulation and release of proinflammatory cytokines in vitro and in vivo. Taken together, administration of GPVI-Fc offers a novel strategy to control platelet-mediated inflammation and to preserve myocardial function following myocardial infarction.

Pre-activated blood platelets and a pro-thrombotic phenotype in APP23 mice modeling Alzheimer's disease

Platelet activation and thrombus formation play a critical role in primary hemostasis but also represent a pathophysiological mechanism leading to acute thrombotic vascular occlusions. Besides, platelets modulate cellular processes including inflammation, angiogenesis and neurodegeneration. On the other hand, platelet activation and thrombus formation are altered in different diseases leading to either bleeding complications or pathological thrombus formation. For many years platelets have been considered to play a role in neuroinflammatory diseases such as Alzheimers disease (AD). AD is characterized by deposits of amyloid-β (Aβ) and strongly related to vascular diseases with platelets playing a critical role in the progression of AD because exposure of platelets to Aβ induces platelet activation, platelet Aβ release, and enhanced platelet adhesion to collagen in vitro and at the injured carotid artery in vivo. However, the molecular mechanisms and the relation between vascular pathology and amyloid-β plaque formation in the pathogenesis of AD are not fully understood. Compelling evidence is suggestive for altered platelet activity in AD patients. Thus we analyzed platelet activation and thrombus formation in aged AD transgenic mice (APP23) known to develop amyloid-β deposits in the brain parenchyma and cerebral vessels. As a result, platelets are in a pre-activated state in blood of APP23 mice and showed strongly enhanced integrin activation, degranulation and spreading kinetics on fibrinogen surfaces upon stimulation. This enhanced platelet signaling translated into almost unlimited thrombus formation on collagen under flow conditions in vitro and accelerated vessel occlusion in vivo suggesting that these mice are at high risk of arterial thrombosis leading to cerebrovascular and unexpectedly to cardiovascular complications that might be also relevant in AD patients.