Mechanisms Underlying AMPA-Mediated Excitotoxicity of Retinal Ganglion Cells Under Hypoxic Conditions

Purpose: Excessive AMPA receptor (AMPAR) stimulation has been implicated in producing excitotoxicity in many neurodegenerative diseases such as glaucoma. The purpose of this study was to investigate if AMPAR desensitization attenuates excitotoxicity in purified retinal ganglion cells (RGCs) under normoxic and hypoxic conditions. Methods: Purified RGCs were treated with AMPAR agonists (100µM s-AMPA (desensitizing), 100µM kainic acid (non-desensitizing)), an AMPAR modulator (100µM cyclothiazide), AMPAR antagonist (50µM CFM-2), and kainate receptor antagonist (50µM UBP301) for 72h in RGC defined medium. To determine if excitotoxicity occurs following hypoxic injury, RGCs were subjected to oxygen-glucose deprivation (OGD) for 4h, followed by s-AMPA treatment under OGD for an additional 4h. Live-Dead Assays were carried out to assess cell viability. AMPA receptor mediated calcium influxes in RGCs were determined by imaging with fura-2 AM following 4h either normoxic-glucose-free or OGD treatments. Results: Significantly enhanced viability was found in RGCs treated with 100µM s-AMPA (84 ± 1% viable) compared to vehicle (0.1% DMSO) group (71 ± 4% viable) alone (ps-AMPA in combination with cyclothiazide or kainic acid significantly reduced cell viability to 50 ± 3% and 54 ± 2%, respectively (ps-AMPA (22 ± 5% viable) following OGD injury compared to those under normoxic-glucose-free conditions (67 ± 4% viable). Additionally, no significant decrease in RGC survival was observed when OGD was carried out for 8h in the presence of s-AMPA in the cell culture medium (85 ± 2% viable). Increased calcium influx was observed when RGCs were maintained in OGD (1085 ± 97 nM) compared to control (764 ± 72 nM). However, there was no difference between treatments of normoxic-glucose-free and RGC medium with RGC defined medium. Conclusions: Desensitization of AMPAR is a key determinant of s-AMPA-mediated excitotoxicity, whereby blocking the desensitization of AMPAR induces cell death. Future studies will determine if AMPAR subunits with greater ion current influx possibly mediate increased sensitivity to excitotoxicity following injury.