title: The mitochondrial complex I inhibitor rotenone induces endoplasmic reticulum stress and activation of GSK-3β in cultured rat retinal cells.
abstract: OBJECTIVE
  Both mitochondrial dysfunction and endoplasmic reticulum (ER) stress have been implicated in the pathogenesis of neurodegenerative disorders. The purpose of the present study was to investigate the mechanisms of cellular damage resulting from mitochondrial dysfunction induced in cultured retinal cells and, in particular, whether ER stress plays a role in this response.
  
  
  METHODS
  Mixed retinal cell cultures containing neurons and glia were prepared from neonatal rats. The complex I inhibitor rotenone was employed to induce mitochondrial dysfunction. Immunocytochemistry, Western blotting, and assays for reactive oxygen species (ROS), adenosine triphosphate (ATP), and TdT-dUTP terminal nick-end labeling (TUNEL) were used as standard to elucidate cellular responses.
  
  
  RESULTS
  Neurons were more rapidly affected by rotenone (1 μM) than glial cells, with significant loss of these cells appearing by 6 hours after application. Glial death was apparent by 24 hours and was associated with positive nuclear labeling by TUNEL. All cell loss was associated with increased ROS production, but neuronal loss was also concurrent with a significant depletion in cellular ATP. Increased expression of the characteristic ER stress components immunoglobulin heavy-chain binding protein (BiP), ATF-4 (activating transcription factor 4), phospho-PERK (pancreatic endoplasmic reticulum kinase/PKR-like endoplasmic reticulum kinase), and growth arrest and DNA damage-inducible protein/C/EBP homologous protein (CHOP) was evident in the rotenone-treated cultures after 6 hours in glial cells. Furthermore, inhibition of glycogen synthase kinase-3β (GSK-3β) with LiCl was able to protect glia cells, whereas inhibition of the calpain with calpain inhibitor III protected only neurons.
  
  
  CONCLUSIONS
  These data together demonstrate that a mitochondrial dysfunction induced in retinal cells can give rise to pathology via a variety of mechanisms including ATP depletion, ROS elevation, ER stress, or activation of GSK-3β or calpain. Such mechanisms predominantly depend upon the concentration and duration of mitochondrial challenge and the type of cell affected.
authors.affiliation: Ophthalmic Research Laboratories, South Australian Institute of Ophthalmology, Hanson Institute Centre for Neurological Diseases, Adelaide, South Australia, Australia Department of Ophthalmology, University of Adelaide, Adelaide, South Australia, Australia.
authors.forename: Guoge
authors.initials: G
authors.lastname: Han
authors.affiliation: Ophthalmic Research Laboratories, South Australian Institute of Ophthalmology, Hanson Institute Centre for Neurological Diseases, Adelaide, South Australia, Australia Department of Ophthalmology, University of Adelaide, Adelaide, South Australia, Australia.
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Casson
authors.affiliation: Ophthalmic Research Laboratories, South Australian Institute of Ophthalmology, Hanson Institute Centre for Neurological Diseases, Adelaide, South Australia, Australia Department of Ophthalmology, University of Adelaide, Adelaide, South Australia, Australia.
authors.forename: Glyn
authors.initials: G
authors.lastname: Chidlow
authors.affiliation: Ophthalmic Research Laboratories, South Australian Institute of Ophthalmology, Hanson Institute Centre for Neurological Diseases, Adelaide, South Australia, Australia Department of Ophthalmology, University of Adelaide, Adelaide, South Australia, Australia.
authors.forename: John P M
authors.initials: JP
authors.lastname: Wood
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000704:Analysis of Variance; D000818:Animals; D015153:Blotting, Western; D002478:Cells, Cultured; D004195:Disease Models, Animal; D059865:Endoplasmic Reticulum Stress / Q000187:drug effects*; D038362:Glycogen Synthase Kinase 3 / Q000378:metabolism*; D000071679:Glycogen Synthase Kinase 3 beta; D007150:Immunohistochemistry; D020287:In Situ Nick-End Labeling; D051381:Rats; D017382:Reactive Oxygen Species / Q000378:metabolism; D055351:Retinal Neurons / Q000187:drug effects* / Q000378:metabolism; D012402:Rotenone / Q000494:pharmacology*; D014475:Uncoupling Agents / Q000494:pharmacology*
chemical_list: D017382:Reactive Oxygen Species; D014475:Uncoupling Agents; D012402:Rotenone; D000255:Adenosine Triphosphate; D000071679:Glycogen Synthase Kinase 3 beta; C000605508:Gsk3b protein, rat; D038362:Glycogen Synthase Kinase 3
keywords: ATP; ER stress; GSK-3β; calpain; rotenone