1. Involvement of autophagy in hypoxic-excitotoxic neuronal death.
- Author
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Ginet V, Spiehlmann A, Rummel C, Rudinskiy N, Grishchuk Y, Luthi-Carter R, Clarke PG, Truttmann AC, and Puyal J
- Subjects
- Animals, Animals, Newborn, Asphyxia Neonatorum pathology, Cell Death drug effects, Cells, Cultured, Disease Models, Animal, Hypoxia metabolism, Hypoxia-Ischemia, Brain pathology, Male, Neurotoxins toxicity, Rats, Rats, Sprague-Dawley, Autophagy physiology, Excitatory Amino Acid Agonists toxicity, Kainic Acid toxicity, Neurons drug effects, Neurons physiology
- 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.
- Published
- 2014
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