ANALYSIS OF THE DEATH PATHWAYS INDUCED BY ENDOCANNABINOIDS IN RETINAL DEVELOPMENT CELL CULTURE

Background: The cannabinoid system is well described in the retina, which expresses the main components of the cannabinoid system, such as the predominant endocannabinoids, 2-arachidonoylglycerol (2-AG) and anandamide (AEA), the classic cannabinoid metabotropic receptors, and also the enzymes responsible for the degradation and synthesis of these endocannabinoids. We have shown that the inhibition of MAGL, responsible for the hydrolysis of 2-AG, in the retinal development of chicken embryo cultures, causes cell death. In this project the goal is to evaluate the involvement of autophagy and endoplasmic reticulum stress (ERE) in this cell death. Methods:  CEUA number 820. Mixed cell cultures of the retina were made from 7-day-old chick (Gallus gallus)  embryos (E7), maintained in MEM. After 24 hours after cell plating (C1), cells were treated with 0.05% DMSO (control group), URB602 (50 µM), MAGL enzyme inhibitor, and evaluated after 24 hours (C2). Western Blotting was used to analyze the levels of proteins involved in the autophagy pathway, by using anti-Beclin-1 (1:1000, n=6) and anti-LC3A/B (1:1000, n=6), as well as ERE, anti-phospho-eif2α (1:1000, n=5). Anti-α-tubulin (1:100.000) was used as loading control. The data was normalized to control and the statistical analysis used was the One Sample t-test, using GraphPad Prism 8.0.1 considering p < 0.05 and mean ± standard error of mean. Results: After 24 hours of URB602 treatment, cell cultures showed a significant increase in LC3A/B levels (2.504 ± 0.4619, p<0.0225). However, Beclin-1 content was significantly decreased (0.5899 ± 0.1539, p<0.0446). The analysis of the effect of 24 h of URB602 exposure in the p-eif2α showed a tendency to increase in comparison to control (5.032 ± 2.500, p<0.1821). Conclusions: From the observed results, the cell death induced by URB602 could be related to autophagy or/and reticulum stress pathways, but is still to be confirmed.