Elevation of basal intracellular calcium as a central element in the activation of brain macrophages (microglia): suppression of receptor-evoked calcium signaling and control of release function

Microglia-brain macrophages are immune-competent cells of the CNS and respond to pathologic events. Using bacterial lipopolysaccharide (LPS) as a tool to activate cultured mouse microglia, we studied alterations in the intracellular calcium concentration ([Ca 2+]i) and in the receptor-evoked generation of transient calcium signals. LPS treatment led to a chronic elevation of basal [Ca 2+]i along with a suppression of evoked calcium signaling, as indicated by reduced [Ca 2+]i transients during stimulation with UTP and complement factor 5a. Presence of the calcium chelator BAPTA prevented the activation-associated changes in [Ca 2+]i and restored much of the signaling efficacy. We also evaluated downstream consequences of a basal [Ca 2+]i lifting during microglial activation and found BAPTA to strongly attenuate the LPS-induced release of nitric oxide (NO) and certain cytokines and chemokines. Furthermore, microglial treatment with ionomycin, an ionophore elevating basal [Ca 2+]i, mimicked the activation-induced calcium signal suppression but failed to induce release activity on its own. Our findings suggest that chronic elevation of basal [Ca 2+]i attenuates receptor-triggered calcium signaling. Moreover, increased [Ca 2+]i is required, but by itself is not sufficient, for release of NO and certain cytokines and chemokines. Elevation of basal [Ca 2+]i could thus prove a central element in the regulation of executive functions in activated microglia

TLR2 controls random motility, while TLR7 regulates chemotaxis of microglial cells via distinct pathways

Microglial cells are the pathologic sensor of the brain, and any pathologic event triggers microglial activation, which involves migration of these cells to a lesion site. Employing different migration assays, we show that ligands for toll-like receptor (TLR) 2 stimulate random motility, while TLR7 ligands are chemoattractants. The subtype specificity of the TLR ligands was verified by using different TLR-deficient (TLRKO) mouse lines. PI3K and Rac inhibition impairs both TLR2- and TLR7-stimulated microglial migration. In contrast, Akt phosphorylation is only required for the TLR2-, but not for the TLR7-stimulated pathway. Interestingly, P2Y12 receptor signaling is involved in the TLR2 activation-induced microglial migration but not TLR7. Furthermore, TLR7 mRNA expression is down-regulated by TLR2 and TLR7 activation. We conclude that TLRs control the migratory behavior of microglia in a distinct manner

Mechanisms of C5a and C3a complement fragment-induced [Ca2+]i signaling in mouse microglia

Microglial cells are activated in response to brain insults; the mechanisms of this process are not yet understood. One of the important signaling mechanisms that might be involved in microglia activation is related to changes in the intracellular calcium concentration ([Ca2+]i). Using fluo-3 microfluorimetry, we have found that external application of the complement fragment C5a (4-10 nM) induced [Ca2+]i elevation in microglial cells in situ in corpus callosum slices. Similarly, application of complement fragments C5a (0.1-10.0 nM) or C3a (100 nM) generates biphasic [Ca2+]i transients composed of an initial peak followed by a plateau in cultured microglia. Incubation of microglial cells for 30 min with pertussis toxin (PTX; 1 microgram/ml) inhibited both C5a- and C3a-triggered [Ca2+]i responses, suggesting the involvement of PTX-sensitive G-proteins in the signal transduction chain. Removal of Ca2+ ions from the extracellular solution eliminated the plateau phase and limited the response to the initial peak. The restoration of the extracellular Ca2+ concentration within 30-60 sec after the beginning of the complement fragment-induced [Ca2+]i elevation led to the recovery of the plateau phase. Inhibition of the endoplasmic reticulum Ca2+ pumps with 500 nM thapsigargin transiently increased the [Ca2+]i and blocked the [Ca2+]i signals in response to subsequent complement fragment application. Our data suggest that complement factors induce [Ca2+]i responses by Ca2+ release from internal pools and subsequent activation of Ca2+ entry controlled by the filling state of the intracellular Ca2+ depots

Satellite microglia display spontaneous electrical activity uncorrelated with activity of the attached neuron

Microglia are innate immune cells of the brain. We have studied a subpopulation of microglia, called satellite microglia. This cell type is defined by a close morphological soma-to-soma association with a neuron, indicative of a direct functional interaction. Indeed, ultrastructural analysis revealed closely attached plasma membranes of satellite microglia and neurons. However, we found no apparent morphological specializations of the contact and biocytin injection into satellite microglia showed no dye-coupling with the apposed neuron or any other cell. Likewise, evoked local field potentials or action potentials and postsynaptic potentials of the associated neuron did not lead to any transmembrane currents or non-capacitive changes in the membrane potential of the satellite microglia in the cortex and hippocampus. Both satellite and non-satellite microglia, however, showed spontaneous transient membrane depolarizations which were not correlated with neuronal activity. These events could be divided into fast and slow rising depolarisations, which exhibited different characteristics in satellite and non-satellite microglia. Fast and slow rising potentials differed with regard to voltage dependence. The frequency of these events was not affected by application of TTX, but fast rising event frequency decreased after application of GABA. We conclude that microglial cells show spontaneous electrical activity uncorrelated with the activity of adjacent neurons

Spontaneous Ca(2+) transients in mouse microglia

Microglia are the resident immune cells in the central nervous system and many of their physiological functions are known to be linked to intracellular calcium (Ca2+) signaling. Here we show that isolated and purified mouse microglia-either freshly or cultured-display spontaneous and transient Ca2+ elevations lasting for around ten to twenty seconds and occurring at frequencies of around five to ten events per hour and cell. The events were absent after depletion of internal Ca2+ stores, by phospholipase C (PLC) inhibition or blockade of inositol-1,4,5-trisphosphate receptors (IP3Rs), but not by removal of extracellular Ca2+, indicating that Ca2+ is released from endoplasmic reticulum intracellular stores. We furthermore provide evidence that autocrine ATP release and subsequent activation of purinergic P2Y receptors is not the trigger for these events. Spontaneous Ca2+ transients did also occur after stimulation with Lipopolysaccharide (LPS) and in glioma-associated microglia, but their kinetics differed from control conditions. We hypothesize that spontaneous Ca2+ transients reflect aspects of cellular homeostasis that are linked to regular and patho-physiological functions of microglia

Minocycline rescues decrease in neurogenesis, increase in microglia cytokines and deficits in sensorimotor gating in an animal model of schizophrenia

Adult neurogenesis in the hippocampus is impaired in schizophrenic patients and in an animal model of schizophrenia. Amongst a plethora of regulators, the immune system has been shown repeatedly to strongly modulate neurogenesis under physiological and pathological conditions. It is well accepted, that schizophrenic patients have an aberrant peripheral immune status, which is also reflected in the animal model. The microglia as the intrinsic immune competent cells of the brain have recently come into focus as possible therapeutic targets in schizophrenia. We here used a maternal immune stimulation rodent model of schizophrenia in which polyinosinic-polycytidilic acid (Poly I:C) was injected into pregnant rats to mimic an anti-viral immune response. We identified microglia IL-1{beta} and TNF-{alpha} increase constituting the factors correlating best with decreases in net-neurogenesis and impairment in pre-pulse inhibition of a startle response in the Poly I:C model. Treatment with the antibiotic minocycline (3mg/kg/day) normalized microglial cytokine production in the hippocampus and rescued neurogenesis and behavior. We could also show that enhanced microglial TNF-{alpha} and IL-1{beta} production in the hippocampus was accompanied by a decrease in the pro-proliferative TNFR2 receptor expression on neuronal progenitor cells, which could be attenuated by minocycline. These findings strongly support the idea to use anti-inflammatory drugs to target microglia activation as an adjunctive therapy in schizophrenic patients

Intrathecal heat shock protein 60 mediates neurodegeneration and demyelination in the CNS through a TLR4- and MyD88-dependent pathway

Background Toll-like receptors (TLR) constitute a highly conserved class of receptors through which the innate immune system responds to both pathogen- and host-derived factors. Although TLRs are involved in a wide range of central nervous system (CNS) disorders including neurodegenerative diseases, the molecular events leading from CNS injury to activation of these innate immune receptors remain elusive. The stress protein heat shock protein 60 (HSP60) released from injured cells is considered an endogenous danger signal of the immune system. In this context, the main objective of the present study was to investigate the impact of extracellular HSP60 on the brain in vivo. Results We show here that HSP60 injected intrathecally causes neuronal and oligodendrocyte injury in the CNS in vivo through TLR4-dependent signaling. Intrathecal HSP60 results in neuronal cell death, axonal injury, loss of oligodendrocytes, and demyelination in the cerebral cortex of wild-type mice. In contrast both mice lacking TLR4 and the TLR adaptor molecule MyD88 are protected against deleterious effects induced by HSP60. In contrast to the exogenous TLR4 ligand, lipopolysaccharide, intrathecal HSP60 does not induce such a considerable inflammatory response in the brain. In the CNS, endogenous HSP60 is predominantly expressed in neurons and released during brain injury, since the cerebrospinal fluid (CSF) from animals of a mouse stroke model contains elevated levels of this stress protein compared to the CSF of sham- operated mice. Conclusions Our data show a direct toxic effect of HSP60 towards neurons and oligodendrocytes in the CNS. The fact that these harmful effects involve TLR4 and MyD88 confirms a molecular pathway mediated by the release of endogenous TLR ligands from injured CNS cells common to many forms of brain diseases that bi-directionally links CNS injury and activation of the innate immune system to neurodegeneration and demyelination in vivo

Glioma stem cells but not bulk glioma cells upregulate IL-6 secretion in microglia/brain macrophages via toll-like receptor 4 signaling

Peripheral macrophages and resident microglia constitute the dominant glioma-infiltrating cells. The tumor induces an immunosuppressive and tumor-supportive phenotype in these glioma-associated microglia/brain macrophages (GAMs). A subpopulation of glioma cells acts as glioma stem cells (GSCs). We explored the interaction between GSCs and GAMs. Using CD133 as a marker of stemness, we enriched for or deprived the mouse glioma cell line GL261 of GSCs by fluorescence-activated cell sorting (FACS). Over the same period of time, 100 CD133(+ )GSCs had the capacity to form a tumor of comparable size to the ones formed by 10,000 CD133(-)GL261 cells. In IL-6(-/-)mice, only tumors formed by CD133(+ )cells were smaller compared with wild type. After stimulation of primary cultured microglia with medium from CD133-enriched GL261 glioma cells, we observed an selective upregulation in microglial IL-6 secretion dependent on Toll-like receptor (TLR) 4. Our results show that GSCs, but not the bulk glioma cells, initiate microglial IL-6 secretion via TLR4 signaling and that IL-6 regulates glioma growth by supporting GSCs. Using human glioma tissue, we could confirm the finding that GAMs are the major source of IL-6 in the tumor context

Vascular signal transducer and activator of transcription-3 promotes angiogenesis and neuroplasticity long-term after stroke

BACKGROUND: Poststroke angiogenesis contributes to long-term recovery after stroke. Signal transducer and activator of transcription-3 (Stat3) is a key regulator for various inflammatory signals and angiogenesis. It was the aim of this study to determine its function in poststroke outcome. METHODS AND RESULTS: We generated a tamoxifen-inducible and endothelial-specific Stat3 knockout mouse model by crossbreeding Stat3(floxed/KO) and Tie2-Cre(ERT2) mice. Cerebral ischemia was induced by 30 minutes of middle cerebral artery occlusion. We demonstrated that endothelial Stat3 ablation did not alter lesion size 2 days after ischemia but did worsen functional outcome at 14 days and increase lesion size at 28 days. At this late time point vascular Stat3 expression and phosphorylation were still increased in wild-type mice. Gene array analysis of a CD31-enriched cell population of the neurovascular niche showed that endothelial Stat3 ablation led to a shift toward an antiangiogenic and axon growth-inhibiting micromilieu after stroke, with an increased expression of Adamts9. Remodeling and glycosylation of the extracellular matrix and microglia proliferation were increased, whereas angiogenesis was reduced. CONCLUSIONS: Endothelial Stat3 regulates angiogenesis, axon growth, and extracellular matrix remodeling and is essential for long-term recovery after stroke. It might serve as a potent target for stroke treatment after the acute phase by fostering angiogenesis and neuroregeneration

Copper(II) Complexes with Tetradentate Piperazine-Based Ligands: DNA Cleavage and Cytotoxicity

Five-coordinate Cu(II) complexes, [Cu(Ln)X]ClO4/PF6, where Ln = piperazine ligands bearing two pyridyl arms and X = ClO4− for Ln = L1 (1-ClO4), L2 (2-ClO4), L3 (3-ClO4), and L6 (6-ClO4) as well as [Cu(Ln)Cl]PF6 for Ln = L1 (1-Cl), L4 (4-Cl), and L5 (5-Cl) have been synthesized and characterized by spectroscopic techniques. The molecular structures of the last two complexes were determined by X-ray crystallography. In aqueous acetonitrile solutions, molar conductivity measurements and UV-VIS spectrophotometric titrations of the complexes revealed the hydrolysis of the complexes to [Cu(Ln)(H2O)]2+ species. The biological activity of the Cu(II) complexes with respect to DNA cleavage and cytotoxicity was investigated. At micromolar concentration within 2 h and pH 7.4, DNA cleavage rate decreased in the order: 1-Cl ≈ 1-ClO4 > 3-ClO4 ≥ 2-ClO4 with cleavage enhancements of up to 23 million. Complexes 4-Cl, 5-Cl, and 6-ClO4 were inactive. In order to elucidate the cleavage mechanism, the cleavage of bis(4-nitrophenyl)phosphate (BNPP) and reactive oxygen species (ROS) quenching studies were conducted. The mechanistic pathway of DNA cleavage depends on the ligand’s skeleton: while an oxidative pathway was preferable for 1-Cl/1-ClO4, DNA cleavage by 2-ClO4 and 3-ClO4 predominantly proceeds via a hydrolytic mechanism. Complexes 1-ClO4, 3-ClO4, and 5-Cl were found to be cytotoxic against A2780 cells (IC50 30–40 µM). In fibroblasts, the IC50 value was much higher for 3-ClO4 with no toxic effect