Recettori P2 coinvolti nella segnalazione tra astrociti e microglia nell'astrogliosi

The astrocytes adopt a reactive phenotype during brain inflammation to counteract the change in neuronal function. The complex response of astrocytes in this pathological context is called astrogliosis, it is characterized by a series of processes, such as an increase in cell proliferation leading to the formation of a glial scar. The glial scar is a histopathological mechanism that acts as a barrier that prevents the extension of the immune response to surrounding healthy tissues but compromises axonal regeneration. Purinergic signalling is involved in the initiation and/or development of this cytotoxic event. It is mediated by extracellular ATP present at the damaged site that activates P2 purinergic receptors. P2 receptors are divided into two macrofamilies, P2Y metabotropic and P2X ionotropic receptors, both subunits are expressed in neuroglia cells. The increase in the concentration of ATP in the injured site acts as a danger signal, making not only the astrocytes reactive but also the microglia that pass from rest M0 to activated M1. The microglial phenotype M1 releases inflammation mediators capable of modulating the action of reactive astrocytes. The paracrine and autocrine communication of ATP between P2X/Y receptors mediates the complicated cross-talk between astrocytes and microglia during astrogliosis. Previous studies have highlighted the role of P2X4, P2X7, P2Y1 and P2Y12 receptors, as they are involved in the early stages of activation of the reactive phenotype of cells: in microglia they stimulate chemotactic movement and phagocytosis, and in astrocytes the proliferation, hypertrophy and overexpression of the GFAP protein. The following study examined the astrocytic proliferation induced by ATPS, a stable ATP analogue to identify which P2 receptors are involved in this marker of astrogliosis and to investigate the influence of microglia in this pathological event. Astrocyte-enriched cultures and co-cultures of astrocytes with microglia were used for the study and tested with the proliferation assay and immunohistochemical assays. Proliferation assay: after cell synchronization (used to block the natural proliferative cycle), the cultures were incubated with drugs for 48 h. The effect of ATPS in proliferation was evaluated with the incorporation of methyl-[3H] -thymidine and quantified with liquid scintillation spectrometry. Our results reveal that in astrocyte cultures, ATPS increased astroglial proliferation by up to 200%, while in co-cultures it was only about 35%. This suggests that microglia release soluble factors that can inhibit astrocytic P2 receptors. The P2Y2 receptor was identified by immunohistochemistry assays with a preference in cultures of astrocytes in accordance with its role in promoting cell division. Treatment with purinergic receptor antagonists indicated that P2Y1/2 receptors are involved in ATPS-induced astrocytic cell proliferation. The simultaneous addition of P2Y1/2 antagonists did not implement the inhibition of proliferation, advancing the hypothesis that these receptors share the same pathway. To clarify the M1 pro-inflammatory action of microglia on ATPS-induced astroglial proliferation, we neutralized the microglia using minocycline. The analysis confirmed the influence of microglia on the proliferation of astrocytes. Finally, the paracrine mechanism by which microglia passes from the M0 to activated M1 state was investigated by examining the P2X4 and P2X7 receptors. The use of P2XR antagonists on both cell cultures has shown that they are not involved in ATPS-induced proliferation of astrocytes but in co-cultures the blockade of this receptors re-establishes the proliferative effect of ATPS to similar levels to those obtained in astrocyte cultures.