Involvement of AMPA Receptor and Its Flip and Flop Isoforms in Retinal Ganglion Cell Death Following Oxygen/Glucose Deprivation

• Published in: Investigative ophthalmology & visual science 2016-02, Vol.57 (2), p.508-526
• Main Authors: Park, Yong H, Broyles, Heather V, He, Shaoqing, McGrady, Nolan R, Li, Linya, Yorio, Thomas
• Format: Article
• Language: English
• Subjects: Animals; Animals, Newborn; Caspase 3 - metabolism; Caspase 7 - metabolism; Cell Death; Cells, Cultured; Disease Models, Animal; Glaucoma - metabolism; Glaucoma - pathology; Glucose - metabolism; Oxidative Stress - physiology; Oxygen - metabolism; Protein Isoforms - metabolism; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species - metabolism; Receptors, AMPA - metabolism; Retinal Ganglion Cells - metabolism; Retinal Ganglion Cells - pathology
• ISSN: 1552-5783; 1552-5783
• DOI: 10.1167/iovs.15-18481

The α-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid (AMPA) receptors (AMPAR) subunits can be posttranscriptionally modified by alternative splicing forming flip and flop isoforms. We determined if an ischemia-like insult to retinal ganglion cells (RGCs) increases AMPAR susceptibility to s-AMPA-mediated excitotoxicity through changes in posttranscriptional modified isoforms. Purified neonatal rat RGCs were subjected to either glucose deprivation (GD) or oxygen/glucose deprivation (OGD) conditions followed by treatment with either 100 μM s-AMPA or Kainic acid. A live-dead assay and caspase 3 assay was used to assess cell viability and apoptotic changes, respectively. We used JC-1 dye and dihydroethidium to measure mitochondria depolarization and reactive oxygen species (ROS), respectively. Calcium imaging with fura-2AM was used to determine intracellular calcium, while the fluorescently-labeled probe, Nanoprobe1, was used to detect calcium-permeable AMPARs. Quantitative PCR (qPCR) analysis was done to determine RNA editing sites AMPAR isoforms. Glucose deprivation, as well as an OGD insult followed by AMPAR stimulation, produced a significant increase in RGC death. Retinal ganglion cell death was independent of caspase 3/7 activity, but was accompanied by increased mitochondrial depolarization and increased ROS production. This was associated with an elevated intracellular Ca(2+) and calcium permeable-AMPARs. The mRNA expression of GLUA2 and GLUA3 flop isoform decreased significantly, while no appreciable changes were found in the corresponding flip isoforms. There were no changes in the Q/R editing of GLUA2, while R/G editing of GLUA2 flop declined under these conditions. Following oxidative injury, RGCs become more susceptible to AMPAR-mediated excitotoxicity. RNA editing and changes in alternative spliced flip and flop isoforms of AMPAR subunits may contribute to increased RGC death.