We examined the expression and pharmacological modulation of the purinergic receptor P2X7R in a C6 glioma model. Intrastriatal injection of C6 cells induced a time-dependent growth of tumor; at 2 weeks postinjection immunohistochemical analysis demonstrated higher levels of P2X7R in glioma-injected versus control vehicle-injected brains. P2X7R immunoreactivity colocalized with tumor cells and microglia, but not endogenous astrocytes. Intravenous administration of the P2X7R antagonist brilliant blue G (BBG) inhibited tumor growth in a spatially dependent manner from the C6 injection site. Treatment with BBG reduced tumor volume by 52% versus that in controls. Double immunostaining indicated that BBG treatment did not alter microgliosis, astrogliosis, or vasculature vessels in C6-injected animals. In vitro, BBG reduced the expression of P2X7R and glioma chemotaxis induced by the P2X7R ligand, 2',3'-O-(4-benzoyl-benzoyl)adenosine triphosphate (BzATP). Immunohistochemical staining of human glioblastoma tissue samples demonstrated greater expression of P2X7R compared to control nontumor samples. These results suggest that the efficacy of BBG in inhibiting tumor growth is primarily mediated by direct actions of the compound on P2X7R in glioma cells and that pharmacological inhibition of this purinergic receptor might serve as a strategy to slow the progression of brain tumors.

Astrocytes react to brain injury triggering neuroinflammatory processes that determine the degree of neuronal damage. However, the signaling events associated to astrocyte activation remain largely undefined. The nuclear factor of activated T-cells (NFAT) is a transcription factor family implicated in activation of immune cells. We previously characterized the expression of NFAT isoforms in cultured astrocytes, and NFAT activation in response to mechanical lesion. Here we analyze NFATc3 in two mouse models of inflammatory brain damage: hippocampal excitotoxicity induced by intracerebral kainic acid (KA) injection and cortical mechanical lesion. Immunofluorescence results demonstrated that NFATc3 is specifically induced in a subset of reactive astrocytes, and not in microglia or neurons. In KA-treated brains, NFATc3 expression is transient and NFATc3-positive astrocytes concentrate around damaged neurons in areas CA3 and CA1. Complementary Western blot and RT-PCR analysis revealed an NFAT-dependent induction of RCAN1-4 and COX-2 in hippocampus as soon as 6 h after KA exposure, indicating that NFAT activation precedes NFATc3 over-expression. Moreover, activation of NFAT by ATP increased NFATc3 mRNA levels in astrocyte cultures, suggesting that NFATc3 expression is controlled through an auto-regulatory loop. Meanwhile, stab wound enhanced NFATc3 expression specifically in a subclass of reactive astrocytes confined within the proximal layer of the glial scar, and GFAP immunoreactivity was attenuated in NFATc3-expressing astrocytes. In conclusion, our work establishes NFATc3 as a marker of activation for a specific population of astrocytes in response to brain damage, which may have consequences for neuronal survival.

Ca2+-dependent calcineurin is upregulated in reactive astrocytes in neuroinflammatory models. Therefore, the fact that the nuclear factor of activated T cells (NFAT) is activated in response to calcineurin qualifies this family of transcription factors with immune functions as candidates to mediate astrogliosis. Brain trauma induces a neuroinflammatory state in which ATP is released from astrocytes, stimulating calcium signalling. Our goal here is to characterize NFATc1 and NFATc2 in mouse primary astrocyte cultures, also exploring the implication of NFAT in astrocyte activation by mechanical lesion. Quantitative reverse transcriptase-polymerase chain reaction, Western blot analysis and immunofluorescence microscopy identified NFATc1 in astrocytes, but not NFATc2. Moreover, NFATc1 was expressed in the cytosol of resting astrocytes, whereas activation of the Ca2+-calcineurin pathway by ionomycin translocated NFATc1 to the nucleus, which is a requirement for activation. The implication of astrocytic NFAT in brain trauma was analysed using an in vitro scratch lesion model. Mechanical lesion caused a rapid NFATc1 translocation that progressed throughout the culture as a gradient and was maintained for at least 4 h. We also demonstrate that ATP, released by lesion, is a potent inducer of NFATc1 translocation and activation. Moreover, the use of P2Y receptor modulators showed that such ATP action is mediated by stimulation of several G(q)-protein-coupled P2Y purinergic receptors, among which P2Y(1) and P2Y(6) are included. In conclusion, this work provides evidence that newly identified NFATc1 is translocated in astrocytes in response to lesion following a pathway that involves ATP release and activation of metabotropic purinergic receptors.