Anales RANF

Effect of the β -amyloid peptide on microglia activation: ATP release @Real Academia Nacional de Farmacia. Spain 195 expression both at the mRNA and protein levels. These data does not agree to those described in a previous study in which the vesicular transporter was identified in the microglia (26). In this case, although the conditions tested were the same, the authors of the work used a cell line instead of a primary microglial culture. Although it is a widely used study model, it is important to consider the origin of this microglial cell line, obtained from the brain of a transgenic mouse deficient in the p53 gene (40). The absence of this gene, known as the guardian of the genome, enhances the proliferative potential of the cells (41), so that the pattern of gene expression can differ considerably from that of the primary microglia from wild type animals. Likewise, in other microglial cell lines, discrepancies have been observed in the responses to the same treatment, not only with respect to the primary microglial culture, but also in the same cell line used (42). Therefore, this could explain the differences observed regarding the expression of VNUT. On the other hand, LPS caused a significant increase in the amount of ATP released to the extracellular medium in a five-minute interval, which is coincident with previous data obtained by other research groups (35). These results confirm that LPS induces the release of ATP in the microglia, with the initial response being very pronounced. However, the luminescence values recorded in LPS treated cells for 24 hours did not significantly differ from the control values. We cannot define the mechanism involved in LPS-induced ATP release from microglial cells. Despite this, the robust expression of VNUT in both LPS and untreated cells make possible that such release could be mediated at least in part by VNUT-dependent exocytotic mechanism. However, the decrease in transporter expression by treatment of the microglial cells with LPS suggests that another mechanism other than exocytosis, such as release trough connexins or panexins (25, 43), could mediates the ATP release from these cells. Our second methodological approach was based in the use of the amyloid peptide β 1-42 . Again, the microglial cells acquired an amoeboid morphology, similar to observed after LPS treatment and typical of reactive microglia, following stimulation with the β 1-42 peptide. In this case, treatment with the amyloid peptide β 1-42 for 18 hours did not alter the expression of VNUT, but produced a significant increase in the extracellular levels of ATP. β 1- 42 -induced ATP release from microglial cells has been described in a previous work (44). However, in this case acute (less than one hour) stimulation periods were used. Our results, however, showed long-term potentiation of ATP release by β 1-42 peptide treatment in microglial cells. Although it cannot be confirmed that the release of ATP is mediated by a VNUT-dependent exocytotic mechanism, this idea cannot be ruled out, taking into account the expression of the transporter in the microglial cells that, unlike the treatment with LPS, remains unchanged in β 1-42 stimulated cells. It has been previously shown that microglial cells express the amyloid precursor protein (APP) and hence these cells are able to produce and release the β -amyloid peptide (45-47). The ATP released by the microglia in response to β 1-42 peptide stimulation could activate microglial P2 receptors that in turn regulate β -amyloid peptide formation. It has been described that P2X7 and P2Y 2 receptors modulate the α -secretase-dependent processing of the APP protein, these receptors having opposite effects on β -amyloid peptide generation (21). Activation of the P2X7 receptor leads to a reduction of α - secretase activity which enhances the amyloidogenic processing of the APP and the formation of β -amyloid peptide. However, activation of the P2Y 2 receptor increases α -secretase activity, thus precluding the generation of the β -amyloid peptide (21). As activation of the microglial cells lead to reduction in the expression of the amyloidogenic P2X7 receptor and to an increase in the expression of the non-amyloidogenic P2Y 2 receptor, this could represent a negative feedback mechanism by which the β -amyloid peptide limits or reduces its own formation and secretion by the microglia. Additionally, it has been previously described that the nucleotides released by the microglia in response to β 1-42 peptide stimulation enhance cell migration and promote β -amyloid peptide phagocytosis through activation of the P2Y 2 receptor (44). However, to have a complete image of this process it is necessary to take also into account the effect of the microglial ATP release on purinergic receptors expressed in cells other than microglia, such as surrounding neurons and astrocytes. 5. CONCLUSIONS ATP and other nucleotides are important signal molecules that regulate a wide diversity of microglial functions. One of the possible sources of extracellular nucleotides in the CNS is their release from microglial cells. It has been demonstrated that microglia are able to release ATP and other nucleotides in response to a variety of stimuli. Our results demonstrate that activation of microglial cells by LPS or the β -amyloid peptide β 1-42 induces an increase in the ATP release, measured by luminometric techniques. This release could be mediated, at least in part, by a VNUT-dependent exocytotic mechanism, as VNUT appear to be robustly expressed by both resting and reactive microglia. However, in the case of the activation by LPS, the reduction in the expression of the transporter in activated cells, suggests that a different mechanisms, other than exocytosis, could be also involved in the ATP release from these cells. Moreover, activation of microglial cells induces a reduction in the expression of the purinergic P2X7 receptor and an increase in P2Y 2 receptor expression. In the case of the activation by β 1-42 peptide, it can be hypothesized that this could represent a negative feedback mechanism by which the β -amyloid peptide limits their own formation and release by microglial cells. Taken together, our results highlight the relevance of a new element, the nucleotide vesicular transporter (VNUT),