Cannabinoids Activate Endoplasmic Reticulum Stress Response and Promote the Death of Avian Retinal Müller Cells in Culture.

Background/objectives: Activation of cannabinoid CB1 or CB2 receptors induces the death of glial progenitors from the chick retina in culture. Here, by using an enriched retinal glial cell culture, we characterized some mechanisms underlying glial death promoted by cannabinoids.

Methods and results: Retinal cultures obtained from 8-day-old (E8) chick embryos and maintained for 12-15 days (C12-15) were used. MTT assays revealed that the CB1/CB2 agonist WIN 55,212-2 (WIN) decreased cell viability in the cultures in a time-dependent manner, with a concomitant increase in extracellular LDH activity, suggesting membrane integrity loss. Cell death was also dose-dependently induced by cannabidiol (CBD), Δ⁹-tetrahydrocannabinol (THC), and CP55940, another CB1/CB2 agonist. In contrast to WIN-induced cell death that was not blocked by either antagonist, the deleterious effect of CBD was blocked by the CB2 receptor antagonist SR144528, but not by PF514273, a CB1 receptor antagonist. WIN-treated cultures showed glial cells with large vacuoles in cytoplasm that were absent in cultures incubated with WIN plus 4-phenyl-butyrate (PBA), a chemical chaperone. Since cannabinoids induced the phosphorylation of eukaryotic initiation factor 2-alfa (eIF2α), these results suggest a process of endoplasmic reticulum (ER) swelling and stress. Incubation of the cultures with WIN for 4 h induced a ~five-fold increase in the number of cells labeled with the ROS indicator CM-H2DCFDA. WIN induced the phosphorylation of JNK but not of p38 in the cultures, and also induced an increase in the number of glial cells expressing cleaved-caspase 3 (c-CASP3). The decrease in cell viability and the expression of c-CASP3 was blocked by salubrinal, an inhibitor of eIF2α dephosphorylation.

Conclusions: These data suggest that cannabinoids induce the apoptosis of glial cells in culture by promoting ROS production, ER stress, JNK phosphorylation, and caspase-3 processing. The graphical abstract was created at Biorender.com.