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Published on: October 20, 2014
Cannabinoids Activate Endoplasmic Reticulum Stress Response and Promote the Death of Avian Retinal Müller Cells in
Ana Lúcia Marques Ventura1, Thayane Martins Silva1, Guilherme Rapozeiro França2
1Neuroscience Program, Department of Neurobiology, Federal Fluminense University, Niterói CEP 24210-201, RJ, Brazil.
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.
Insights
Cannabinoids trigger glial cell death in chick retinal cultures by inducing oxidative stress, endoplasmic reticulum stress, and apoptosis. These mechanisms involve reactive oxygen species production and caspase-3 activation, leading to cell death.
Area of Science:
- Neuroscience
- Cell Biology
- Pharmacology
Background:
- Cannabinoid receptor activation (CB1/CB2) is known to induce glial progenitor death in chick retina cultures.
- Understanding the specific mechanisms behind cannabinoid-induced glial cell death is crucial for potential therapeutic applications and understanding neurotoxicity.
Purpose of the Study:
- To elucidate the molecular mechanisms underlying cannabinoid-induced glial cell death in enriched chick retinal cultures.
- To investigate the roles of oxidative stress, endoplasmic reticulum stress, and apoptotic pathways in this process.
Main Methods:
- Utilized enriched glial cell cultures from embryonic chick retinas (E8).
- Assessed cell viability using MTT assays and measured cell membrane integrity via LDH activity.
- Investigated the involvement of CB1 and CB2 receptors using specific antagonists.
- Analyzed endoplasmic reticulum stress markers (eIF2α phosphorylation, vacuole formation), reactive oxygen species (ROS) production (CM-H2DCFDA), and apoptotic markers (JNK phosphorylation, cleaved-caspase 3).
Main Results:
- Cannabinoid agonists (WIN 55,212-2, CBD, THC, CP55940) dose-dependently decreased glial cell viability.
- WIN 55,212-2 induced cell death via mechanisms involving ROS production, ER stress (eIF2α phosphorylation), JNK phosphorylation, and caspase-3 activation.
- CBD-induced cell death was partially mediated by CB2 receptors.
- Chemical chaperones and eIF2α dephosphorylation inhibitors partially blocked cannabinoid-induced cell death, indicating ER stress and eIF2α pathway involvement.
Conclusions:
- Cannabinoids induce glial cell apoptosis in chick retinal cultures through a multi-faceted mechanism.
- Key pathways involved include the generation of reactive oxygen species, endoplasmic reticulum stress, JNK signaling activation, and caspase-3 processing.
- These findings provide insights into the neurotoxic potential of cannabinoids on retinal glial cells.
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