Anales RANF

Aida Menéndez Méndez, Juan Ignacio Díaz Hernández, Felipe Ortega, Rosa Gómez Villafuertes, Javier Gualix @Real Academia Nacional de Farmacia. Spain 190 diseases, though the underlying mechanisms are still unclear. Regarding Alzheimer’s disease (AD), this disorder is characterized by complex molecular and cellular alterations, including the development of extracellular amyloid-beta (A β ) deposits, intracellular aggregated phosphorylated tau, dystrophic neurites, loss of synapses and neurons, and a prominent gliosis. The reactive gliosis involves alterations in morphology and function of microglia and astrocytes. It has long been recognized that A β neuritic plaques are surrounded by activated microglial cells that could contribute to A β phagocytosis and/or compaction (2, 3). In this sense, microglial activation could improve the AD pathology reducing the A β levels (4). However, the release of pro- inflamatory mediators, such as cytokines or chemokines, from reactive microglia is thought to contribute to the chronic inflammatory response associated to AD, which has a prominent role in the progression and severity of the disease (5, 6). Nucleotides act as widespread extracellular signaling molecules. The physiological effects of these compounds are mediated through an extended family of purinoceptors that are classified into ionotropic P2X or metabotropic P2Y receptors (7, 8). Microglia express multiple P2 receptors that differentially sense extracellular nucleotides and, therefore, P2 receptor-mediated microglial responses have received much attention (9-12). Among these purinoceptors, the P2X7 receptor has gained prominent recognition as a regulator of inflammatory responses (13). Microglial cells express P2X7 receptors (14, 15) which drive microglial activation and proliferation (16). P2X7 receptors are overexpressed in microglial cells in the neuroinflammatory foci of numerous neurodegenerative conditions (17) and the activation of these P2X7 receptors is coupled to the maturation and secretion of the key pro- inflammatory cytokine IL-1 β (18), thereby triggering or potentiating neuroinflammation. These results lead to propose the use of P2X7 receptor antagonists for the treatment of CNS inflammation (19, 20). Interestingly, the P2X7 receptor seems to be also involved in the regulation of the processing of the amyloid precursor protein (APP) (21) and the in vivo inhibition of this receptor conduced to a reduction of the amyloid plaques in a mouse model of AD (22). Microglia sense ATP or other nucleotides by multiple P2 receptors after which they change in different phenotypes. However, it is necessary to investigate what is the origin of extracellular ATP in the brain under both physiological and pathological conditions. Microglia have been shown to release ATP when the cells are exposed to different stimuli, such as ATP or glutamate (23, 24). The signaling pathways that mediate the ATP release from microglia remain unclear, although Connexin 43 (Cx43) hemichannels have been shown to be involved (25). However, a recent work showed that microglial cells express the nucleotide vesicular transporter (VNUT), which transports cytosolic nucleotides into vesicles using a proton-mediated membrane potential as a driving force, and these cells are able to release ATP by VNUT- dependent exocytotic mechanisms (26). Due to the close relationship that exists among ATP, the P2X7 receptor and microglial cells in inflammatory and neurodegenerative processes, where the extracellular nucleotide can have a vesicular origin, we evaluated whether the expression of VNUT and the P2X7 receptor, as well as the ATP release, could be modified in the reactive microglia. To achieve microglia activation we use the lipopolysaccharide (LPS), one of the main components of gram-negative bacterial cell wall, that induces microglial cells activation through interaction with the Toll-like receptor 4 (TLR4) (27). Additionally, we analyzed the effect of the β -amyloid peptide β 1-42 , which is able to activate and stimulate the phagocytic activity of the microglial cells (28), on the expression of VNUT and the ATP release in the microglia. 2. MATERIALS AND METHODS 2.1. Cell Culture Primary cultures of microglial cells were performed according to the procedure described by (29), with some modifications. Briefly, the whole brain was removed aseptically from postnatal day 5 C57BL/6 mice and submitted to digestion with papain 100 U/ml (Worthington, Lake Wood, NJ) (previously activated in Earle’s Balanced Salt Solution (EBSS) buffer containing 5 mM l-Cys and 2 mM EDTA), in the presence of 100 U/ml of DNAse (Worthington, Lake Wood, NJ), 1 mM CaCl 2 and 1 mM MgCl 2 . Then, cells were resuspended in Dulbecco's Modified Eagle's medium (DMEM) containing 10% (v/v) fetal calf serum, 25 mM glucose, 2 mM glutamine, 100 U/ml penicillin, 100 mg/ml streptomycin and 2.5 µg/ml amphotericin, and they were plated in culture flasks at a density of 70,000 cells/cm 2 . Cells were maintained in culture until they reached confluence (approximately 10–12 days), replacing the medium every 3–4 days. This procedure allows obtaining an astroglial culture, as neurons do not survive under such conditions. To separate astrocytes and microglia, flasks were shaken at 250 rpm for 2 h at 37°C in an orbital shaker, and after that, the supernatant contains microglia whereas astrocytes remains adhered to the culture surface. Culture medium was then centrifuged at 200 x g for 10 min and microglial cells were resuspended and seeded onto culture plates. Treatment with the lipopolysaccharide (LPS) was initiated 48h after seeding and the compound was added at 1µg/ml concentration for 24h (26). In the case of the treatment with β 1-42 , the β -amyloid peptide was prepared according to (30, 31) and added at a final concentration of 5 µM for 18h. Control non-amyloidogenic β 42-1 peptide was prepared and added under the same conditions. 2.2. Reverse Transcription PCR and Quantitative Real- Time PCR Total RNA was obtained from cultured microglia using a SpeedTools Total RNA Extraction kit (Biotools), following the manufacturer’s instructions. After