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Abstract #66806 Published in IGR 17-4
Involvement of AMPA Receptor and Its Flip and Flop Isoforms in Retinal Ganglion Cell Death Following Oxygen/Glucose Deprivation
Park YH; Broyles HV; He S; McGrady NR; Li L; Yorio TInvestigative Ophthalmology and Visual Science 2016; 57: 508-526
PURPOSE: 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.
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.
Full articleClassification:
3.6 Cellular biology (Part of: 3 Laboratory methods)
3.8 Pharmacology (Part of: 3 Laboratory methods)
11.8 Neuroprotection (Part of: 11 Medical treatment)
