Anales RANF

Aida Menéndez Méndez, Juan Ignacio Díaz Hernández, Felipe Ortega, Rosa Gómez Villafuertes, Javier Gualix @Real Academia Nacional de Farmacia. Spain 194 Figure 3. Effect of the amyloid peptide β 1-42 in VNUT expression in microglial cells. Release of ATP. Luminometric measure of extracellular ATP (A) and Q-PCR analysis of the VNUT mRNA levels (B) in microglial cells treated with the control peptide β 42-1 or the amyloidogenic peptide β 1-42 for 18h. Values were normalized with respect to those obtained in cells treated with the control peptide and are represented as a percentage. Data are the mean ± SEM of three independent experiments. *p < 0.05 (Student’s t -test). (C) Representative photomicrographs of microglial cells treated with the control peptide β 42-1 (upper panels) or the amyloidogenic peptide β 1- 42 (lower panels) showing immunodetection for VNUT (green) and the microglial cell marker CD11b (red). Nuclei are counterstained with DAPI (blue). Scale bar = 10 µm . 4. DISCUSSION A common characteristic of the CNS diseases that courses with inflammation is that the activation of microglial cells occurs at the initial stages (36, 37). Then, cells migrate to the damaged area where they carry out phagocytic processes and mediate the inflammatory responses (1). Purinergic system plays an important role in the regulation of such processes, ATP being a key molecule in the development of microglial functions. Due to the relevance of the presence of extracellular ATP in the physiopathological context of the microglia, we asked whether such presence could be mediated by a VNUT- dependent mechanism. Our first methodological approach was the activation of the microglia through the use of the lipopolysaccharide LPS. Stimulation with this bacterial endotoxin has been used as a study model for neuroinflammatory processes, which are present in neurodegenerative diseases (38, 39). The conditions of the treatment of the microglia with LPS were based on previous studies carried out in a microglial cell line (26). The effectiveness of this stimulation was confirmed by the morphological change observed, microglial cells acquiring an amoeboid morphology, as well as by the increase in the expression of the P2Y 2 receptor (33). LPS treatment did not modify the mRNA levels of the P2X7 receptor, although a significant reduction in the amount of the protein was observed. These results could reflect a change in the turnover or degradation of the P2X7 protein induced by the lipopolysaccharide. In the case of VNUT, the 24-hour treatment with LPS produced a significant decrease in its * 0 50 100 150 200 250 + β 42‐1 Normalized luminiscence vs Control (%) 0 50 100 150 VNUT mRNA (%) A B VNUT CD11b VNUT CD11b DAPI DAPI DAPI C + β 1‐42 + β 42‐1 + β 1‐42