title: Involvement of autophagy in hypoxic-excitotoxic neuronal death.
abstract: Neuronal autophagy is increased in numerous excitotoxic conditions including neonatal cerebral hypoxia-ischemia (HI). However, the role of this HI-induced autophagy remains unclear. To clarify this role we established an in vitro model of excitotoxicity combining kainate treatment (Ka, 30 µM) with hypoxia (Hx, 6% oxygen) in primary neuron cultures. KaHx rapidly induced excitotoxic death that was completely prevented by MK801 or EGTA. KaHx also stimulated neuronal autophagic flux as shown by a rise in autophagosome number (increased levels of LC3-II and punctate LC3 labeling) accompanied by increases in lysosomal abundance and activity (increased SQSTM1/p62 degradation, and increased LC3-II levels in the presence of lysosomal inhibitors) and fusion (shown using an RFP-GFP-LC3 reporter). To determine the role of the enhanced autophagy we applied either pharmacological autophagy inhibitors (3-methyladenine or pepstatinA/E64) or lentiviral vectors delivering shRNAs targeting Becn1 or Atg7. Both strategies reduced KaHx-induced neuronal death. A prodeath role of autophagy was also confirmed by the enhanced toxicity of KaHx in cultures overexpressing BECN1 or ATG7. Finally, in vivo inhibition of autophagy by intrastriatal injection of a lentiviral vector expressing a Becn1-targeting shRNA increased the volume of intact striatum in a rat model of severe neonatal cerebral HI. These results clearly show a death-mediating role of autophagy in hypoxic-excitotoxic conditions and suggest that inhibition of autophagy should be considered as a neuroprotective strategy in HI brain injuries.
authors.affiliation: Department of Fundamental Neurosciences; Faculty of Biology and Medicine; University of Lausanne; Lausanne, Switzerland.
authors.forename: Vanessa
authors.initials: V
authors.lastname: Ginet
authors.affiliation: Department of Fundamental Neurosciences; Faculty of Biology and Medicine; University of Lausanne; Lausanne, Switzerland.
authors.forename: Amélie
authors.initials: A
authors.lastname: Spiehlmann
authors.affiliation: Department of Fundamental Neurosciences; Faculty of Biology and Medicine; University of Lausanne; Lausanne, Switzerland.
authors.forename: Coralie
authors.initials: C
authors.lastname: Rummel
authors.affiliation: Brain Mind Institute; École Polytechnique Fédérale de Lausanne; Lausanne, Switzerland.
authors.forename: Nikita
authors.initials: N
authors.lastname: Rudinskiy
authors.affiliation: Department of Fundamental Neurosciences; Faculty of Biology and Medicine; University of Lausanne; Lausanne, Switzerland.
authors.forename: Yulia
authors.initials: Y
authors.lastname: Grishchuk
authors.affiliation: Brain Mind Institute; École Polytechnique Fédérale de Lausanne; Lausanne, Switzerland.
authors.forename: Ruth
authors.initials: R
authors.lastname: Luthi-Carter
authors.affiliation: Department of Fundamental Neurosciences; Faculty of Biology and Medicine; University of Lausanne; Lausanne, Switzerland.
authors.forename: Peter G H
authors.initials: PG
authors.lastname: Clarke
authors.affiliation: Clinic of Neonatology; Department of Pediatrics and Pediatric Surgery; Lausanne University Hospital and University of Lausanne; Lausanne, Switzerland.
authors.forename: Anita C
authors.initials: AC
authors.lastname: Truttmann
authors.affiliation: Department of Fundamental Neurosciences; Faculty of Biology and Medicine; University of Lausanne; Lausanne, Switzerland; Clinic of Neonatology; Department of Pediatrics and Pediatric Surgery; Lausanne University Hospital and University of Lausanne; Lausanne, Switzerland.
authors.forename: Julien
authors.initials: J
authors.lastname: Puyal
mesh_terms: D000818:Animals; D000831:Animals, Newborn; D001238:Asphyxia Neonatorum / Q000473:pathology; D001343:Autophagy / Q000502:physiology*; D016923:Cell Death / Q000187:drug effects; D002478:Cells, Cultured; D004195:Disease Models, Animal; D018690:Excitatory Amino Acid Agonists / Q000633:toxicity*; D000860:Hypoxia / Q000378:metabolism; D020925:Hypoxia-Ischemia, Brain / Q000473:pathology; D007608:Kainic Acid / Q000633:toxicity*; D008297:Male; D009474:Neurons / Q000187:drug effects* / Q000502:physiology*; D009498:Neurotoxins / Q000633:toxicity; D051381:Rats; D017207:Rats, Sprague-Dawley
chemical_list: D018690:Excitatory Amino Acid Agonists; D009498:Neurotoxins; D007608:Kainic Acid
keywords: autophagy; excitotoxicity; neonatal hypoxia-ischemia; neuronal death; neuroprotection