Involvement of ampa receptor and its flip and flop isoforms in retinal ganglion cell death following oxygen/glucose deprivation

Yong H. Park, Heather V. Broyles, Shaoqing He, Nolan R. McGrady, Linya Li, Thomas Yorio

Research output: Contribution to journalArticle

4 Citations (Scopus)

Abstract

PURPOSE. The a-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 lM 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 fluorescentlylabeled 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 Ca2+ 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.

Original languageEnglish
Pages (from-to)508-526
Number of pages19
JournalInvestigative Ophthalmology and Visual Science
Volume57
Issue number2
DOIs
StatePublished - 1 Feb 2016

Fingerprint

AMPA Receptors
Retinal Ganglion Cells
Protein Isoforms
Cell Death
Oxygen
Glucose
RNA Editing
Calcium
Caspase 3
Reactive Oxygen Species
Caspase 7
alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid
Kainic Acid
Alternative Splicing
Cell Survival
Mitochondria
Coloring Agents
Ischemia
Polymerase Chain Reaction
Messenger RNA

Keywords

  • ADAR proteins
  • AMPA
  • Cell death
  • Flip/flop isoforms
  • RGCs

Cite this

@article{4aa89d80056d4885a98ec905fec40357,
title = "Involvement of ampa receptor and its flip and flop isoforms in retinal ganglion cell death following oxygen/glucose deprivation",
abstract = "PURPOSE. The a-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 lM 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 fluorescentlylabeled 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 Ca2+ 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.",
keywords = "ADAR proteins, AMPA, Cell death, Flip/flop isoforms, RGCs",
author = "Park, {Yong H.} and Broyles, {Heather V.} and Shaoqing He and McGrady, {Nolan R.} and Linya Li and Thomas Yorio",
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Involvement of ampa receptor and its flip and flop isoforms in retinal ganglion cell death following oxygen/glucose deprivation. / Park, Yong H.; Broyles, Heather V.; He, Shaoqing; McGrady, Nolan R.; Li, Linya; Yorio, Thomas.

In: Investigative Ophthalmology and Visual Science, Vol. 57, No. 2, 01.02.2016, p. 508-526.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Involvement of ampa receptor and its flip and flop isoforms in retinal ganglion cell death following oxygen/glucose deprivation

AU - Park, Yong H.

AU - Broyles, Heather V.

AU - He, Shaoqing

AU - McGrady, Nolan R.

AU - Li, Linya

AU - Yorio, Thomas

PY - 2016/2/1

Y1 - 2016/2/1

N2 - PURPOSE. The a-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 lM 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 fluorescentlylabeled 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 Ca2+ 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.

AB - PURPOSE. The a-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 lM 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 fluorescentlylabeled 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 Ca2+ 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.

KW - ADAR proteins

KW - AMPA

KW - Cell death

KW - Flip/flop isoforms

KW - RGCs

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