title: Glutamate receptor activation triggers OPA1 release and induces apoptotic cell death in ischemic rat retina.
abstract: OBJECTIVE
  Glutamate receptor activation-induced excitotoxicity has been hypothesized to cause retinal ganglion cell (RGC) death in glaucoma and to link mitochondrial dysfunction in both acute and chronic neurodegenerative disorders. However, the relationships among elevated intraocular pressure (IOP), glutamate receptor-mediated excitotoxicity, and mitochondrial dysfunction in glaucoma remains unknown. The goal of this study was to determine whether the N- methyl D-aspartate (NMDA) glutamate receptor antagonist MK801 can block optic atrophy 1 (OPA1) release and subsequent apoptotic cell death, as well as whether acute IOP elevation triggers OPA1 release and alters OPA1 gene and protein expression in the rat retina after ischemia.
  
  
  METHODS
  Sprague Dawley rats received injections of MK801 (10 mg/kg) or vehicle and then transient retinal ischemia was induced by acute IOP elevation. Following subcellular fractionation, changes in cytoplasmic and mitochondrial OPA1 were assessed by western blot analysis. Also, the expression of OPA1 mRNA was measured by Taqman qPCR, the distribution of OPA1 protein was assessed by immunohistochemistry, and apoptotic cell death was assessed by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining.
  
  
  RESULTS
  The ~65 and 90 kDa isoforms of OPA1 were increased in the cytosol in the rat retina at 6 h and at 12 h, but only the 90 kDa isoform of OPA1 was decreased at 12 h after ischemia induced by acute IOP elevation. This suggests that ischemic insult induced OPA1 release from the mitochondria in retinas. Pretreatment with MK801 blocked this effect and significantly increased OPA1 immunoreactivity in the inner retinal layers, as well as OPA1 gene expression and total protein expression in retinas at 12 h after ischemia. Further, pretreatment with MK801 prevented apoptotic cell death in retinas at 12 h after ischemia. Following acute IOP elevation, Bcl-2 mRNA expression in retinas was decreased at 3 h and 6 h but increased at 12 h and 24 h. In contrast, Bax mRNA expression in these retinas was increased in the first 12 h and then plateaued. Moreover, pretreatment with MK801 increased Bcl-2 mRNA expression, but did not alter the course of Bax mRNA expression.
  
  
  CONCLUSIONS
  These results indicate that OPA1 release from mitochondria triggered by acute IOP elevation is inhibited by blockade of glutamate receptor activation. Because this effect was accompanied by increases of Bcl-2 expression, no changes of Bax expression, and blockade of apoptosis, these findings indicate that glutamate receptor activation following acute IOP elevation may lead to a distinct mitochondria-mediated cell death pathway in ischemic retina. These results support further studies to determine whether ischemia-induced OPA1 release may be an important component of the biochemical cascade leading to pressure-related ischemic damage in glaucomatous retina.
authors.affiliation: Hamilton Glaucoma Center and Department of Ophthalmology, University of California San Diego, La Jolla, CA 92037-0946, USA. danielju@glaucoma.ucsd.edu
authors.forename: Won-Kyu
authors.initials: WK
authors.lastname: Ju
authors.forename: James D
authors.initials: JD
authors.lastname: Lindsey
authors.forename: Mila
authors.initials: M
authors.lastname: Angert
authors.forename: Ankur
authors.initials: A
authors.lastname: Patel
authors.forename: Robert N
authors.initials: RN
authors.lastname: Weinreb
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D016291:Dizocilpine Maleate / Q000494:pharmacology; D020558:GTP Phosphohydrolases / Q000235:genetics / Q000378:metabolism*; D005786:Gene Expression Regulation / Q000187:drug effects; D007429:Intraocular Pressure / Q000187:drug effects; D007511:Ischemia / Q000378:metabolism* / Q000473:pathology* / Q000503:physiopathology; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D017470:Receptors, Glutamate / Q000378:metabolism*; D012160:Retina / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology*; D051028:bcl-2-Associated X Protein / Q000378:metabolism
chemical_list: D017470:Receptors, Glutamate; D051028:bcl-2-Associated X Protein; D016291:Dizocilpine Maleate; D020558:GTP Phosphohydrolases; C498811:Opa1 protein, rat