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 Ca 2þ 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.

Identification of Mesencephalic Astrocyte-Derived Neurotrophic Factor as a Novel Neuroprotective Factor for Retinal Ganglion Cells

“…The caspase 3/7 activation in the different groups was evaluated by the Caspase-Glo 3/7 assay (Promega, Milano, Italy), as previously reported Park et al (2016). The luminescence signals from the cell lysate solution were determined with the Tecan Genios microplate reader (Synergy H1, BioTAK).…”

Immunological and molecular basics of the primary open angle glaucoma pathomechanism

“…Ischemia and oxidative stress play key roles in glaucoma [31,38,39]. Oxidative stress induction by glial (mainly microglial) cells activation has been reported [33,36].…”

The role and therapeutic potential of melatonin in age‐related ocular diseases

“…Mitochondrial dysfunction has also been observed in cultured retinal ganglion cells submitted to elevated hydrostatic pressure . Furthermore, high IOP triggers retinal glutamate receptor (mainly N ‐methyl‐ d ‐aspartate, NMDA receptor but also α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazoleproprionic acid, AMPA receptor) activation that permits the rapid influx of calcium ions impairing intracellular calcium homeostasis and at the same time increasing ROS/RNS generation . These effects also evoke mitochondrial dysfunction that finally leads to neuronal death by apoptosis and prompts calcium‐dependent exocytosis of glutamate from injury neurons that induces further excitotoxic damage to adjacent neurons .…”

“…190 Furthermore, high IOP triggers retinal glutamate receptor (mainly N-methyld-aspartate, NMDA receptor but also α-amino-3-hydroxy-5-methyl-4isoxazoleproprionic acid, AMPA receptor) activation that permits the rapid influx of calcium ions impairing intracellular calcium homeostasis and at the same time increasing ROS/RNS generation. [191][192][193] These effects also evoke mitochondrial dysfunction that finally leads to neuronal death by apoptosis and prompts calcium-dependent exocytosis of glutamate from injury neurons that induces further excitotoxic damage to adjacent neurons. 194,195 Additionally, mitochondrial dysfunction also increases the sensitivity of neurons to glutamate excitotoxicity and therefore leads to a lower concentration of glutamate, which is sufficient to induce neuronal death.…”