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

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Involvement of AMPA Receptor and Its Flip and Flop Isoforms in Retinal Ganglion Cell Death Following Oxygen/Glucose Deprivation Physiology and Pharmacology Involvement of AMPA Receptor and Its Flip and Flop Isoforms in Retinal Ganglion Cell Death Following Oxygen/Glucose Deprivation Yong H. Park,1,2 Heather V. Broyles,1–3 Shaoqing He,1,2 Nolan R. McGrady,1,2 Linya Li,1,2 and Thomas Yorio1,2 1University 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 North Texas Health Science Center, Fort Worth, Texas, United States Correspondence: Thomas Yorio, Of- PURPOSE. The a-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid (AMPA) receptors fice of the Provost, UNT Health (AMPAR) subunits can be posttranscriptionally modified by alternative splicing forming flip Science Center, 3500 Camp Bowie and flop isoforms. We determined if an ischemia-like insult to retinal ganglion cells (RGCs) Boulevard, Fort Worth, TX 76107, increases AMPAR susceptibility to s-AMPA–mediated excitotoxicity through changes in USA; posttranscriptional modified isoforms. [email protected]. Submitted: October 22, 2015 METHODS. Purified neonatal rat RGCs were subjected to either glucose deprivation (GD) or Accepted: January 12, 2016 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 Citation: Park YH, Broyles HV, He S, and apoptotic changes, respectively. We used JC-1 dye and dihydroethidium to measure McGrady NR, Li L, Yorio T. Involve- ment of AMPA receptor and its flip mitochondria depolarization and reactive oxygen species (ROS), respectively. Calcium and flop isoforms in retinal ganglion imaging with fura-2AM was used to determine intracellular calcium, while the fluorescently- cell death following oxygen/glucose labeled probe, Nanoprobe1, was used to detect calcium-permeable AMPARs. Quantitative deprivation. Invest Ophthalmol Vis PCR (qPCR) analysis was done to determine RNA editing sites AMPAR isoforms. Sci. 2016;57:508–526. DOI:10.1167/ RESULTS. Glucose deprivation, as well as an OGD insult followed by AMPAR stimulation, iovs.15-18481 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 Ca2þ 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: AMPA, flip/flop isoforms, cell death, RGCs, ADAR proteins laucoma is a heterogeneous group of optic neuropathies ischemia in glaucoma.6 It is thought that tissue modeling at the G associated commonly with elevated IOP that affects optic nerve head, accompanying insufficient blood flow to the approximately 70 million people worldwide.1 It is the second retina, exacerbates cupping of the optic nerve head, thereby leading cause of vision loss, and the number one leading cause compromising the retina’s access to oxygen, nutrients, and the of irreversible blindness.2 Glaucoma is characterized by the ability to remove waste.7,8 In the retina, where metabolic cupping of the optic disc and degeneration of the optic nerve, demand is high, this could lead to depletion of ATP, causing the and is accompanied by slow and progressive death of retinal deregulation of mitochondrial bioenergetics, and provoking the ganglion cells (RGCs), thus leading to the loss of the visual increased production of reactive oxygen species (ROS), causing field.3,4 The etiologic mechanisms underlying the pathogenesis oxidative damage and eventual cell death, in particular of the of glaucoma have yet to be elucidated. RGCs.9,10 Many cellular and molecular mechanisms have been One of the main factors associating retinal ischemia with proposed to account for the death of RGCs in glaucoma. Of RGC death is the excitatory amino acid, glutamate. The these proposed mechanisms, ischemia and excitotoxicity neurotransmitter, glutamate, relays signals in the vertical appear to have a key role in glaucomatous pathogenesis.5 pathway of the retina by the activation of ionotropic Increased immunohistochemical staining of hypoxia-inducible glutamate receptors (iGluRs), allowing the influx of monova- factor-1, a transcription factor induced by hypoxia, was lent and divalent cations, propagating action potentials.11,12 observed in human glaucomatous retinas and optic nerve However, under conditions of retinal ischemia, abnormal heads, providing the supporting evidence for the role of retinal concentrations of glutamate are released into the extracellular iovs.arvojournals.org j ISSN: 1552-5783 508 This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License. Downloaded from iovs.arvojournals.org on 09/26/2021 AMPA Receptor Involvement in RGC Death IOVS j February 2016 j Vol. 57 j No. 2 j 509 2þ milieu of the retina, causing a large influx of [Ca ]through METHODS the activated iGluRs on RGCs, leading to deregulation of calcium-dependent cellular events and, therefore, mediating Purified RGCs Isolation and Culture excitotoxicity in the RGCs.10,13–15 The iGluRs are composed of the N-methyl-D-aspartate receptors (NMDAR), a-amino-3- All animal procedures were performed in compliance with the hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AM- Association for Research in Vision and Ophthalmology (ARVO) PAR), and kainate receptors (KAR).16 All three receptors have policy for the Use of Animals in Ophthalmic and Vision been implicated in glutamate excitotoxicity in RGCs, with Research, and approved by the Institutional Animal Care and NMDAR receptors being the most widely studied recep- Use Committee (IACUC) of the University of North Texas tor.5,17–19 However, recent findings are pinpointing AMPARs Health Science Center. Purified neonatal RGCs were isolated to have an equally large role in mediating excitotoxicity to using a double immunopanning technique as published 30,43 RGCs.20–23 previously. Time-pregnant Sprague-Dawley rats were pur- The AMPARs-mediated excitotoxicity in RGCs is well chased from Charles River (Wilmington, MA, USA), and retinas established in the field evidenced by numerous publications were dissected from euthanized postnatal (days 4–6) rat pups. demonstrating AMPAR-mediated damage to the ganglion cell Collected retinas were dissociated in papain solution (4.5 layer.17,21,23–25 However, many of these studies were per- units/mL, #3125; Worthington, Lakewood, NJ, USA). Dissoci- formed in total retina, in vivo, or mixed culture of retinal ated cell suspension were incubated with rabbit antimacro- cells.17,24,26,27 Additionally, AMPARs-mediated excitotoxicity phage antibody (#CLAD51240; Cedarlane Laboratories, was conducted with either glutamate or kainic acid (KA), Ontario, Canada) and then plated twice to a 150-mm petri neither of which is specific for the AMPARs.24,28,29 These dish coated with goat anti-rabbit IgG (HþL chain) antibody confounding factors make it hard to discern if AMPAR in RGCs (#111-005-003; Jackson ImmunoResearch, West Grove, PA, are contributing to excitotoxicity directly, or as a secondary USA), to remove microglia from the cell suspension. Subse- effect. In our previous studies,30 we demonstrated that quently, nonadherent cells were transferred to a 100-mm petri stimulating AMPAR with s-AMPA (a highly selective agonist) dish coated with Thy1.1 antibody (from hybridoma T11D7; in a purified RGC culture, does not induce excitotoxicity, but American Type Culture Collection, Rockville, MD, USA), a instead promotes RGC survival through the induction of selective RGC marker. Following 1 hour of incubation with cAMP response element-binding protein (CREB) phosphory- intermittent shaking of the plate (every 10 minutes), the 100- lation. We concluded that blocking AMPAR’s desensitization, mm petri dishes were washed with Dulbecco’s phosphate- like using cyclothiazide, induced RGC death and is the buffered saline (DPBS) multiple times (#14287080; Invitrogen, determinant for excitotoxicity. The AMPARs are hetero/homo Carlsbad, CA, USA), removing nonadherent cells and leaving tetrameric structures that are composed of 4 different behind RGCs. The RGCs then were incubated with trypsin subunits, GLUA1-4.31 Each subunit can be posttranscription- (1250 units/mL) (#T9935; Sigma-Aldrich Corp., St. Louis, MO, ally modified by alternative splicing in a region of the USA) in a 378C incubator for 5 minutes and successively extracellular loop between TM3 and TM4, forming flip and mechanically triturated using a pipette, to dissociate the cells flop isoforms.32 Flip and flop isoforms are expressed from the 100-mm petri dish. The RGCs were seeded onto differently during development leading to a high level of plates coated with poly-D-lysine (#P6407; Sigma-Aldrich Corp.) expression of flip isoforms and a low expression of the flop and mouse-laminin-1 (#3400-010-01; Trevigen, Inc., Gaithers- isoforms. However, following development, the flop isoform burg,
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