Involvement of autophagy in hypoxic-excitotoxic neuronal death

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 red...