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

Extracellular ATP activates a cation conductance and a K+ conductance in cultured microglial cells from mouse brain

Microglial cells have important functions during regenerative processes after brain injury. It is well established that they rapidly respond to damage to the brain tissue. Stages of activation are associated with changes of cellular properties such as proliferation rate or expression of surface antigens. Yet, nothing is known about signal substances leading to the rapid changes of membrane properties, which may be required to initiate the transition from one cell stage into another. From our present study, using the patch-clamp technique, we report that cultured microglial cells obtained from mouse or rat brain respond to extracellularly applied ATP with the activation of a cation conductance. Additionally, in the majority of cells an outwardly directed K+ conductance was activated with some delay. Since ADP, AMP, and adenosine (in descending order) were less potent or ineffective in inducing the cation conductance, the involvement of a P2 purinergic receptor is proposed. The receptor activation is accompanied by an increase of cytosolic Ca2+ as determined by a fura-2-based Ca(2+)-imaging system. This ATP receptor could enable microglial cells to respond to transmitter release from nerve endings with ATP as a transmitter or cotransmitter or to the death of cells with resulting leakage of ATP

Glioblastoma-induced attraction of endogenous neural precursor cells is associated with improved survival

Neural precursor cells contribute to adult neurogenesis and to limited attempts of brain repair after injury. Here we report that in a murine experimental glioblastoma model, endogenous neural precursors migrate from the subventricular zone toward the tumor and surround it. The association of endogenous precursors with syngenic tumor grafts was observed, after injecting red fluorescent protein-labeled G261 cells into the caudate-putamen of transgenic mice, which express green fluorescent protein under a promoter for nestin (nestin-GFP). Fourteen days after inoculation, the nestin-GFP cells surrounded the tumors in several cell layers and expressed markers of early noncommitted and committed precursors. Nestin-GFP cells were further identified by a characteristic membrane current pattern as recorded in acute brain slices. 5-bromo-2-deoxyuridine labeling and dye tracing experiments revealed that the tumor-associated precursors originated from the subventricular zone. Moreover, in cultured explants from the subventricular zone, the neural precursors showed extensive tropism for glioblastomas. Tumor-induced endogenous precursor cell accumulation decreased with age of the recipient; this correlated with increased tumor size and shorter survival times in aged mice. Coinjection of glioblastoma cells with neural precursors improved the survival time of old mice to a level similar to that in young mice. Coculture experiments showed that neural precursors suppressed the rapid increase in tumor cell number, which is characteristic of glioblastoma, and induced glioblastoma cell apoptosis. Our results indicate that tumor cells attract endogenous precursor cells; the presence of precursor cells is antitumorigenic; and this cellular interaction decreases with aging

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

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

Astrocytes and oligodendrocytes in the thalamus jointly maintain synaptic activity by supplying metabolites

Thalamic astrocytes and oligodendrocytes are coupled via gap junctions and form panglial networks. Here, we show that these networks have a key role in energy supply of neurons. Filling an astrocyte or an oligodendrocyte in acute slices with glucose or lactate is sufficient to rescue the decline of stimulation-induced field post-synaptic potential (fPSP) amplitudes during extracellular glucose deprivation (EGD). In mice lacking oligodendroglial coupling, loading an astrocyte with glucose does not rescue the EGD-mediated loss of fPSPs. Monocarboxylate and glucose transporters are required for rescuing synaptic activity during EGD. In mice deficient in astrocyte coupling, filling of an oligodendrocyte with glucose does not rescue fPSPs during EGD. Our results demonstrate that, in the thalamus, astrocytes and oligodendrocytes are jointly engaged in delivering energy substrates for sustaining neuronal activity and suggest that oligodendrocytes exert their effect mainly by assisting astrocytes in metabolite transfer to the postsynapse

Impact of actin filament stabilization on adult hippocampal and olfactory bulb neurogenesis

Rearrangement of the actin cytoskeleton is essential for dynamic cellular processes. Decreased actin turnover and rigidity of cytoskeletal structures have been associated with aging and cell death. Gelsolin is a Ca(2+)-activated actin-severing protein that is widely expressed throughout the adult mammalian brain. Here, we used gelsolin-deficient (Gsn(-/-)) mice as a model system for actin filament stabilization. In Gsn(-/-) mice, emigration of newly generated cells from the subventricular zone into the olfactory bulb was slowed. In vitro, gelsolin deficiency did not affect proliferation or neuronal differentiation of adult neural progenitors cells (NPCs) but resulted in retarded migration. Surprisingly, hippocampal neurogenesis was robustly induced by gelsolin deficiency. The ability of NPCs to intrinsically sense excitatory activity and thereby implement coupling between network activity and neurogenesis has recently been established. Depolarization-induced [Ca(2+)](i) increases and exocytotic neurotransmitter release were enhanced in Gsn(-/-) synaptosomes. Importantly, treatment of Gsn(-/-) synaptosomes with mycotoxin cytochalasin D, which, like gelsolin, produces actin disassembly, decreased enhanced Ca(2+) influx and subsequent exocytotic norepinephrine release to wild-type levels. Similarly, depolarization-induced glutamate release from Gsn(-/-) brain slices was increased. Furthermore, increased hippocampal neurogenesis in Gsn(-/-) mice was associated with a special microenvironment characterized by enhanced density of perfused vessels, increased regional cerebral blood flow, and increased endothelial nitric oxide synthase (NOS-III) expression in hippocampus. Together, reduced filamentous actin turnover in presynaptic terminals causes increased Ca(2+) influx and, subsequently, elevated exocytotic neurotransmitter release acting on neural progenitors. Increased neurogenesis in Gsn(-/-) hippocampus is associated with a special vascular niche for neurogenesis

Distinct SARS-CoV-2 RNA fragments activate Toll-like receptors 7 and 8 and induce cytokine release from human macrophages and microglia

INTRODUCTION: The pandemic coronavirus disease 19 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and is marked by thromboembolic events and an inflammatory response throughout the body, including the brain. METHODS: Employing the machine learning approach BrainDead we systematically screened for SARS-CoV-2 genome-derived single-stranded (ss) RNA fragments with high potential to activate the viral RNA-sensing innate immune receptors Toll-like receptor (TLR)7 and/or TLR8. Analyzing HEK TLR7/8 reporter cells we tested such RNA fragments with respect to their potential to induce activation of human TLR7 and TLR8 and to activate human macrophages, as well as iPSC-derived human microglia, the resident immune cells in the brain. RESULTS: We experimentally validated several sequence-specific RNA fragment candidates out of the SARS-CoV-2 RNA fragments predicted in silico as activators of human TLR7 and TLR8. Moreover, these SARS-CoV-2 ssRNAs induced cytokine release from human macrophages and iPSC-derived human microglia in a sequence- and species-specific fashion. DISCUSSION: Our findings determine TLR7 and TLR8 as key sensors of SARS-CoV-2-derived ssRNAs and may deepen our understanding of the mechanisms how this virus triggers, but also modulates an inflammatory response through innate immune signaling

Microglia sense neuronal activity via GABA in the early postnatal hippocampus

Microglia, the resident macrophages in the central nervous system, express receptors for classical neurotransmitters, such as γ-aminobutyric acid (GABA) and glutamate, suggesting that they sense synaptic activity. To detect microglial Ca(2+) responses to neuronal activity, we generate transgenic mouse lines expressing the fluorescent Ca(2+) indicator GCaMP6m, specifically in microglia and demonstrate that electrical stimulation of the Schaffer collateral pathway results in microglial Ca(2+) responses in early postnatal but not adult hippocampus. Preceding the microglial responses, we also observe similar Ca(2+) responses in astrocytes, and both are sensitive to tetrodotoxin. Blocking astrocytic glutamate uptake or GABA transport abolishes stimulation-induced microglial responses as well as antagonizing the microglial GABA(B) receptor. Our data, therefore, suggest that the neuronal activity-induced glutamate uptake and the release of GABA by astrocytes trigger the activation of GABA(B) receptors in microglia. This neuron, astrocyte, and microglia communication pathway might modulate microglial activity in developing neuronal networks

Neurofibromatosis 1 - mutant microglia exhibit sexually-dimorphic cyclic AMP-dependent purinergic defects

As critical regulators of brain homeostasis, microglia are influenced by numerous factors, including sex and genetic mutations. To study the impact of these factors on microglia biology, we employed genetically engineered mice that model Neurofibromatosis type 1 (NF1), a disorder characterized by clinically relevant sexually dimorphic differences. While microglia phagocytic activity was reduced in both male and female heterozygous Nf1 mutant (Nf1+/-) mice, purinergic control of phagocytosis was only affected in male Nf1+/- mice. ATP-induced P2Y-mediated membrane currents and P2RY12-dependent laser lesion-induced accumulation of microglial processes were also only impaired in male, but not female Nf1+/-, microglia. These defects resulted from Nf1+/- male-specific defects in cyclic AMP regulation, rather than from changes in purinergic receptor expression. Cyclic AMP elevation by phosphodiesterase blockade restored the male Nf1+/- microglia defects in P2Y-dependent membrane currents and process motility. Taken together, these data establish a sex-by-genotype interaction important to microglia function in the adult mouse brain