Leyton, Integrin-mediated transactivation of P2X7R via hemichannel-dependent ATP release stimulates astrocyte migration, BBA -Molecular Cell Research (2016Research ( ), doi: 10.1016Research ( /j.bbamcr.2016 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT

BackgroundNeuroinflammation involves cytokine release, astrocyte reactivity and migration. Neuronal Thy-1 promotes DITNC1 astrocyte migration by engaging αVβ3 Integrin and Syndecan-4. Primary astrocytes express low levels of these receptors and are unresponsive to Thy-1; thus, inflammation and astrocyte reactivity might be necessary for Thy-1-induced responses.MethodsWild-type rat astrocytes (TNF-activated) or from human SOD1G93A transgenic mice (a neurodegenerative disease model) were used to evaluate cell migration, Thy-1 receptor levels, signaling molecules, and reactivity markers.ResultsThy-1 induced astrocyte migration only after TNF priming. Increased expression of αVβ3 Integrin, Syndecan-4, P2X7R, Pannexin-1, Connexin-43, GFAP, and iNOS were observed in TNF-treated astrocytes. Silencing of β3 Integrin prior to TNF treatment prevented Thy-1-induced migration, while β3 Integrin over-expression was sufficient to induce astrocyte reactivity and allow Thy-1-induced migration. Finally, hSOD1G93A astrocytes behave as TNF-treated astrocytes since they were reactive and responsive to Thy-1.ConclusionsTherefore, inflammation induces expression of αVβ3 Integrin and other proteins, astrocyte reactivity, and Thy-1 responsiveness. Importantly, ectopic control of β3 Integrin levels modulates these responses regardless of inflammation.Electronic supplementary materialThe online version of this article (10.1186/s12974-017-0968-5) contains supplementary material, which is available to authorized users.

Cell adhesion to the extracellular matrix (ECM) proteins occurs through interactions with integrins that bind to Arg-Gly-Asp (RGD) tripeptides, and syndecan-4, which recognizes the heparin-binding domain (HBD) of other proteins. Both receptors trigger signaling pathways, including those that activate RhoGTPases such as RhoA and Rac1. This sequence of events modulates cell adhesion to the ECM and cell migration. Using a neuron-astrocyte model, we have reported that the neuronal protein Thy-1 engages αVβ3 integrin and syndecan-4 to induce RhoA activation and strong astrocyte adhesion to their underlying substrate. Thus, because cell-cell interactions and strong cell attachment to the matrix are considered antagonistic to cell migration, we hypothesized that Thy-1 stimulation of astrocytes should preclude cell migration. Here, we studied the effect of Thy-1 expressing neurons on astrocyte polarization and migration using a wound-healing assay and immunofluorescence analysis. Signaling molecules involved were studied by affinity precipitations, western blots and the usage of specific antibodies. Intriguingly, Thy-1 interaction with its two receptors was found to increase astrocyte polarization and migration. The latter events required interactions of these receptors with both the RGD-like sequence and the HBD of Thy-1. Additionally, prolonged Thy-1-receptor interactions inhibited RhoA activation while activating FAK, PI3K and Rac1. Therefore, sustained engagement of integrin and syndecan-4 with the neuronal surface protein Thy-1 induces astrocyte migration. Interestingly we identify here, a cell-cell interaction that although initially induces strong cell attachment, upon persistant stimulation favors cell migration by engaging the same signaling receptors and molecules as those utilized by ECM proteins to stimulate cell movement.

SummaryThy-1, an abundant mammalian glycoprotein, interacts with avb3 integrin and syndecan-4 in astrocytes and thus triggers signaling events that involve RhoA and its effector p160ROCK, thereby increasing astrocyte adhesion to the extracellular matrix. The signaling cascade includes calcium-dependent activation of protein kinase Ca upstream of Rho; however, what causes the intracellular calcium transients required to promote adhesion remains unclear. Purinergic P2X7 receptors are important for astrocyte function and form large non-selective cation pores upon binding to their ligand, ATP. Thus, we evaluated whether the intracellular calcium required for Thy-1-induced cell adhesion stems from influx mediated by ATP-activated P2X7 receptors. Results show that adhesion induced by the fusion protein Thy-1-Fc was preceded by both ATP release and sustained intracellular calcium elevation. Elimination of extracellular ATP with Apyrase, chelation of extracellular calcium with EGTA, or inhibition of P2X7 with oxidized ATP, all individually blocked intracellular calcium increase and Thy-1-stimulated adhesion. Moreover, Thy-1 mutated in the integrin-binding site did not trigger ATP release, and silencing of P2X7 with specific siRNA blocked Thy-1-induced adhesion. This study is the first to demonstrate a functional link between avb3 integrin and P2X7 receptors, and to reveal an important, hitherto unanticipated, role for P2X7 in calcium-dependent signaling required for Thy-1-stimulated astrocyte adhesion.

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Neuronal electrical activity increases intracellular Ca(2+) concentration and generates reactive oxygen species. Here, we show that high frequency field stimulation of primary hippocampal neurons generated Ca(2+) signals with an early and a late component, and promoted hydrogen peroxide generation via a neuronal NADPH oxidase. Hydrogen peroxide generation required both Ca(2+) entry through N-methyl-D-aspartate receptors and Ca(2+) release mediated by ryanodine receptors (RyR). Field stimulation also enhanced nuclear translocation of the NF-κB p65 protein and NF-κB -dependent transcription, and increased c-fos mRNA and type-2 RyR protein content. Preincubation with inhibitory ryanodine or with the antioxidant N-acetyl L-cysteine abolished the increase in hydrogen peroxide generation and the late Ca(2+) signal component induced by electrical stimulation. Primary cortical cells behaved similarly as primary hippocampal cells. Exogenous hydrogen peroxide also activated NF-κB-dependent transcription in hippocampal neurons; inhibitory ryanodine prevented this effect. Selective inhibition of the NADPH oxidase or N-acetyl L-cysteine also prevented the enhanced translocation of p65 in hippocampal cells, while N-acetyl L-cysteine abolished the increase in RyR2 protein content induced by high frequency stimulation. In conclusion, the present results show that electrical stimulation induced reciprocal activation of ryanodine receptor-mediated Ca(2+) signals and hydrogen peroxide generation, which stimulated jointly NF-κB activity.

Iron is essential for crucial neuronal functions but is also highly toxic in excess. Neurons acquire iron through transferrin receptor-mediated endocytosis and via the divalent metal transporter 1 (DMT1). The N-terminus (1A, 1B) and C-terminus (+IRE, -IRE) splice variants of DMT1 originate four protein isoforms, all of which supply iron to cells. Diverse physiological or pathological conditions induce differential DMT1 variant expression, which are cell-type dependent. Hence, it becomes relevant to ascertain if activation of neuronal plasticity processes that require functional N-methyl D: -aspartate (NMDA) receptors, including in vitro stimulation of NMDA receptor-mediated signaling and spatial memory training, selectively modify DMT1 variant expression. Here, we report for the first time that brief (5 min) exposure of primary hippocampal cultures to NMDA (50 muM) increased 24 h later the expression of DMT1-1B and DMT1+IRE, but not of DMT1-IRE mRNA. In contrast, endogenous DMT1 mRNA levels remained unaffected following 6 h incubation with brain-derived nerve factor. NMDA (25-50 muM) also enhanced DMT1 protein expression 24-48 h later; this enhancement was abolished by the transcription inhibitor actinomycin D and by the NMDA receptor antagonist MK-801, implicating NMDA receptors in de novo DMT1 expression. Additionally, spatial memory training enhanced DMT1-1B and DMT1+IRE expression and increased DMT1 protein content in rat hippocampus, where the exon1A variant was not found. These results suggest that NMDA receptor-dependent plasticity processes stimulate expression of the iron transporter DMT1-1B+IRE isoform, which presumably plays a significant role in hippocampal spatial memory formation.

Astrocytes in the normal brain possess a stellate shape reflecting their non-migratory properties. Alternatively, in neurodegenerative diseases or after injury, astrocytes become "reactive" in a process known as astrocytosis or reactive gliosis, retract their processes, become polarized and acquire front-to-rear asymmetry typical of migratory cells. On the other hand, neuronal migration is a common process during embryonic development, but only few types of neurons can migrate and differentiate during adult life in the central nervous system. Those that do migrate follow tracks made by glial cells and mainly give rise to interneurons. In vitro, molecular mechanisms involved in adhesion of cells to and migration on extracellular matrix proteins have been widely studied; however, signal transduction pathways explaining how particularly neurons and astrocytes, mutually modulate adhesion and migration are less well known. In this review, we describe and discuss how ligand/receptor interactions in astrocytes and neurons trigger signaling events leading to actin and microtubule reorganization, changes in cell morphology, as well as cell adhesion and migration. The biological significance these cell-cell interactions and signaling events might have in the brain are discussed.