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.