title: Involvement of AMPA Receptor and Its Flip and Flop Isoforms in Retinal Ganglion Cell Death Following Oxygen/Glucose Deprivation.
abstract: OBJECTIVE
  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.
  
  
  METHODS
  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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  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.
authors: University of North Texas Health Science Center, Fort Worth, Texas, United States 2North Texas Eye Research Institute, University of North Texas Health Science Center, Fort Worth, Texas, United States.
authors: Yong H
authors: YH
authors: Park
authors: University of North Texas Health Science Center, Fort Worth, Texas, United States 2North Texas Eye Research Institute, University of North Texas Health Science Center, Fort Worth, Texas, United States 3Texas College of Osteopathic Medicine, University of.
authors: Heather V
authors: HV
authors: Broyles
authors: University of North Texas Health Science Center, Fort Worth, Texas, United States 2North Texas Eye Research Institute, University of North Texas Health Science Center, Fort Worth, Texas, United States.
authors: Shaoqing
authors: S
authors: He
authors: University of North Texas Health Science Center, Fort Worth, Texas, United States 2North Texas Eye Research Institute, University of North Texas Health Science Center, Fort Worth, Texas, United States.
authors: Nolan R
authors: NR
authors: McGrady
authors: University of North Texas Health Science Center, Fort Worth, Texas, United States 2North Texas Eye Research Institute, University of North Texas Health Science Center, Fort Worth, Texas, United States.
authors: Linya
authors: L
authors: Li
authors: University of North Texas Health Science Center, Fort Worth, Texas, United States 2North Texas Eye Research Institute, University of North Texas Health Science Center, Fort Worth, Texas, United States.
authors: Thomas
authors: T
authors: Yorio
mesh_terms: D000818:Animals; D000831:Animals, Newborn; D053148:Caspase 3 / Q000378:metabolism; D053179:Caspase 7 / Q000378:metabolism; D016923:Cell Death; D002478:Cells, Cultured; D004195:Disease Models, Animal; D005901:Glaucoma / Q000378:metabolism / Q000473:pathology*; D005947:Glucose / Q000378:metabolism*; D018384:Oxidative Stress / Q000502:physiology*; D010100:Oxygen / Q000378:metabolism*; D020033:Protein Isoforms / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D017382:Reactive Oxygen Species / Q000378:metabolism; D018091:Receptors, AMPA / Q000378:metabolism*; D012165:Retinal Ganglion Cells / Q000378:metabolism / Q000473:pathology*
chemical_list: D020033:Protein Isoforms; D017382:Reactive Oxygen Species; D018091:Receptors, AMPA; D053148:Caspase 3; D053179:Caspase 7; D005947:Glucose; D010100:Oxygen