Loss of CX3CR1 increases accumulation of inflammatory monocytes and promotes gliomagenesis

The most abundant populations of non-neoplastic cells in the glioblastoma (GBM) microenvironment are resident microglia, macrophages and infiltrating monocytes from the blood circulation. The mechanisms by which monocytes infiltrate into GBM, their fate following infiltration, and their role in GBM growth are not known. Here we tested the hypothesis that loss of the fractalkine receptor CX3CR1 in microglia and monocytes would affect gliomagenesis. Deletion of Cx3cr1 from the microenvironment resulted in increased tumor incidence and shorter survival times in glioma-bearing mice. Loss of Cx3cr1 did not affect accumulation of microglia/macrophages in peri-tumoral areas, but instead indirectly promoted the trafficking of CD11b+CD45hiCX3CR1lowLy-6ChiLy-6G-F4/80-/low circulating inflammatory monocytes into the CNS, resulting in their increased accumulation in the perivascular area. Cx3cr1-deficient microglia/macrophages and monocytes demonstrated upregulation of IL1{beta} expression that was inversely proportional to Cx3cr1 gene dosage. The Proneural subgroup of the TCGA GBM patient dataset with high IL1{beta} expression showed shorter survival compared to patients with low IL1{beta}. IL1{beta} promoted tumor growth and increased the cancer stem cell phenotype in murine and human Proneural glioma stem cells (GSCs). IL1{beta} activated the p38 MAPK signaling pathway and expression of monocyte chemoattractant protein (MCP-1/CCL2) by tumor cells. Loss of Cx3cr1 in microglia in a monocyte-free environment had no impact on tumor growth and did not alter microglial migration. These data suggest that enhancing signaling to CX3CR1 or inhibiting IL1{beta} signaling in intra-tumoral macrophages can be considered as potential strategies to decrease the tumor-promoting effects of monocytes in Proneural GBM

Stimulation of Na⁺/H⁺ Exchanger Isoform 1 Promotes Microglial Migration

Regulation of microglial migration is not well understood. In this study, we proposed that Na+/H+ exchanger isoform 1 (NHE-1) is important in microglial migration. NHE-1 protein was co-localized with cytoskeletal protein ezrin in lamellipodia of microglia and maintained its more alkaline intracellular pH (pHi). Chemoattractant bradykinin (BK) stimulated microglial migration by increasing lamellipodial area and protrusion rate, but reducing lamellipodial persistence time. Interestingly, blocking NHE-1 activity with its potent inhibitor HOE 642 not only acidified microglia, abolished the BK-triggered dynamic changes of lamellipodia, but also reduced microglial motility and microchemotaxis in response to BK. In addition, NHE-1 activation resulted in intracellular Na+ loading as well as intracellular Ca2+ elevation mediated by stimulating reverse mode operation of Na+/Ca2+ exchange (NCXrev). Taken together, our study shows that NHE-1 protein is abundantly expressed in microglial lamellipodia and maintains alkaline pHi in response to BK stimulation. In addition, NHE-1 and NCXrev play a concerted role in BK-induced microglial migration via Na+ and Ca2+ signaling. © 2013 Shi et al

Bradykinin-induced microglial migration mediated by B1-bradykinin receptors depends on Ca2+ influx via reverse-mode activity of the Na+/Ca2+ exchanger

Bradykinin (BK) is produced and acts at the site of injury and inflammation. In the CNS, migration of microglia toward the lesion site plays an important role pathologically. In the present study, we investigated the effect of BK on microglial migration. Increased motility of cultured microglia was mimicked by B1 receptor agonists and markedly inhibited by a B1 antagonist but not by a B2 receptor antagonist. BK induced chemotaxis in microglia isolated from wild-type and B2-knock-out mice but not from B1-knock-out mice. BK-induced motility was not blocked by pertussis toxin but was blocked by chelating intracellular Ca2+ or by low extracellular Ca2+, implying that Ca2+ influx is prerequisite. Blocking the reverse mode of Na+/Ca2+ exchanger (NCX) completely inhibited BK-induced migration. The involvement of NCX was further confirmed by using NCX+/- mice; B1-agonist-induced motility and chemotaxis was decreased compared with that in NCX+/+ mice. Activation of NCX seemed to be dependent on protein kinase C and phosphoinositide 3-kinase, and resultant activation of intermediate-conductance (IK-type) Ca2+-dependent K+ currents (I(K(Ca))) was activated. Despite these effects, BK did not activate microglia, as judged from OX6 staining. Using in vivo lesion models and pharmacological injection to the brain, it was shown that microglial accumulation around the lesion was also dependent on B1 receptors and I(K(Ca)). These observations support the view that BK functions as a chemoattractant by using the distinct signal pathways in the brain and, thus, attracts microglia to the lesion site in vivo

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

Glial cell line‐derived neurotrophic factor increases matrix metallopeptidase 9 and 14 expression in microglia and promotes microglia‐mediated glioma progression

Glial cell line‐derived neurotrophic factor (GDNF) is released by glioma cells and promotes tumor growth. We have previously found that GDNF released from the tumor cells is a chemoattractant for microglial cells, the immune cells of the central nervous system. Here we show that GDNF increases matrix metalloproteinase (MMP) 9 and MMP14 expression in cultured microglial cells from mixed sexes of neonatal mice. The GDNF‐induced microglial MMP9 and MMP14 upregulation is mediated by GDNF family receptor alpha 1 receptors and dependent on p38 mitogen‐activated protein kinase signaling. In organotypic brain slices, GDNF promotes the growth of glioma and this effect depends on the presence of microglia. We also previously found that MMP9 and MMP14 upregulation can be mediated by Toll‐like receptor (TLR) 2 signaling and here we demonstrate that GDNF increases the expression of TLR1 and TLR2. In conclusion, GDNF promotes the pro‐tumorigenic phenotype of microglia

Müller glia provide essential tensile strength to the developing retina.

This is the final version of the article. It first appeared from the Rockefeller University Press via http://dx.doi.org/10.1083/jcb.201503115To investigate the cellular basis of tissue integrity in a vertebrate central nervous system (CNS) tissue, we eliminated Müller glial cells (MG) from the zebrafish retina. For well over a century, glial cells have been ascribed a mechanical role in the support of neural tissues, yet this idea has not been specifically tested in vivo. We report here that retinas devoid of MG rip apart, a defect known as retinoschisis. Using atomic force microscopy, we show that retinas without MG have decreased resistance to tensile stress and are softer than controls. Laser ablation of MG processes showed that these cells are under tension in the tissue. Thus, we propose that MG act like springs that hold the neural retina together, finally confirming an active mechanical role of glial cells in the CNS.This work was funded by a Herchel Smith Postdoctoral Fellowship to R.B.M., the Wellcome Trust programme in Developmental Biology to O.R. and J.O., NIH grants EY14358 (R.O.W.) and EY01730 (Vision Core), MRC Career Development Award and HFSP Young Investigator Grant to K.F., and a Wellcome Trust Investigator Award to W.A.H

The Spider Effect: Morphological and Orienting Classification of Microglia in Response to Stimuli in Vivo

The different morphological stages of microglial activation have not yet been described in detail. We transected the olfactory bulb of rats and examined the activation of the microglial system histologically. Six stages of bidirectional microglial activation (A) and deactivation (R) were observed: from stage 1A to 6A, the cell body size increased, the cell process number decreased, and the cell processes retracted and thickened, orienting toward the direction of the injury site; until stage 6A, when all processes disappeared. In contrast, in deactivation stages 6R to 1R, the microglia returned to the original site exhibiting a stepwise retransformation to the original morphology. Thin highly branched processes re-formed in stage 1R, similar to those in stage 1A. This reverse transformation mirrored the forward transformation except in stages 6R to 1R: cells showed multiple nuclei which were slowly absorbed. Our findings support a morphologically defined stepwise activation and deactivation of microglia cells

High-Throughput Analysis of Calcium Signalling Kinetics in Astrocytes Stimulated with Different Neurotransmitters

Astrocytes express a wide range of receptors for neurotransmitters and hormones that are coupled to increases in intracellular Ca2+ concentration, enabling them to detect activity in both neuronal and vascular networks. There is increasing evidence that astrocytes are able to discriminate between different Ca2+-linked stimuli, as the efficiency of some Ca2+ dependent processes – notably release of gliotransmitters – depends on the stimulus that initiates the Ca2+ signal. The spatiotemporal complexity of Ca2+ signals is substantial, and we here tested the hypothesis that variation in the kinetics of Ca2+ responses could offer a means of selectively engaging downstream targets, if agonists exhibited a “signature shape” in evoked Ca2+ response. To test this, astrocytes were exposed to three different receptor agonists (ATP, glutamate and histamine) and the resultant Ca2+ signals were analysed for systematic differences in kinetics that depended on the initiating stimulus. We found substantial heterogeneity between cells in the time course of Ca2+ responses, but the variation did not correlate with the type or concentration of the stimulus. Using a simple metric to quantify the extent of difference between populations, it was found that the variation between agonists was insufficient to allow signal discrimination. We conclude that the time course of global intracellular Ca2+ signals does not offer the cells a means for distinguishing between different neurotransmitters

Calcium Signals Driven by Single Channel Noise

Usually, the occurrence of random cell behavior is appointed to small copy numbers of molecules involved in the stochastic process. Recently, we demonstrated for a variety of cell types that intracellular Ca2+ oscillations are sequences of random spikes despite the involvement of many molecules in spike generation. This randomness arises from the stochastic state transitions of individual Ca2+ release channels and does not average out due to the existence of steep concentration gradients. The system is hierarchical due to the structural levels channel - channel cluster - cell and a corresponding strength of coupling. Concentration gradients introduce microdomains which couple channels of a cluster strongly. But they couple clusters only weakly; too weak to establish deterministic behavior on cell level. Here, we present a multi-scale modelling concept for stochastic hierarchical systems. It simulates active molecules individually as Markov chains and their coupling by deterministic diffusion. Thus, we are able to follow the consequences of random single molecule state changes up to the signal on cell level. To demonstrate the potential of the method, we simulate a variety of experiments. Comparisons of simulated and experimental data of spontaneous oscillations in astrocytes emphasize the role of spatial concentration gradients in Ca2+ signalling. Analysis of extensive simulations indicates that frequency encoding described by the relation between average and standard deviation of interspike intervals is surprisingly robust. This robustness is a property of the random spiking mechanism and not a result of control

Reactive astrocyte nomenclature, definitions, and future directions

Reactive astrocytes are astrocytes undergoing morphological, molecular, and functional remodeling in response to injury, disease, or infection of the CNS. Although this remodeling was first described over a century ago, uncertainties and controversies remain regarding the contribution of reactive astrocytes to CNS diseases, repair, and aging. It is also unclear whether fixed categories of reactive astrocytes exist and, if so, how to identify them. We point out the shortcomings of binary divisions of reactive astrocytes into good-vs-bad, neurotoxic-vs-neuroprotective or A1-vs-A2. We advocate, instead, that research on reactive astrocytes include assessment of multiple molecular and functional parameters-preferably in vivo-plus multivariate statistics and determination of impact on pathological hallmarks in relevant models. These guidelines may spur the discovery of astrocyte-based biomarkers as well as astrocyte-targeting therapies that abrogate detrimental actions of reactive astrocytes, potentiate their neuro- and glioprotective actions, and restore or augment their homeostatic, modulatory, and defensive functions