Ginet Vanessa, Spiehlmann Amélie, Rummel Coralie, Rudinskiy Nikita, Grishchuk Yulia, Luthi-Carter Ruth, Clarke Peter G H, Truttmann Anita C, Puyal Julien
Department of Fundamental Neurosciences; Faculty of Biology and Medicine; University of Lausanne; Lausanne, Switzerland.
Brain Mind Institute; École Polytechnique Fédérale de Lausanne; Lausanne, Switzerland.
Autophagy. 2014 May;10(5):846-60. doi: 10.4161/auto.28264. Epub 2014 Mar 11.
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.
在包括新生儿脑缺氧缺血（HI）在内的多种兴奋性毒性条件下，神经元自噬会增加。然而，这种HI诱导的自噬的作用仍不清楚。为了阐明这一作用，我们在原代神经元培养物中建立了一种兴奋性毒性的体外模型，将海藻酸处理（Ka，30 μM）与缺氧（Hx，6%氧气）相结合。KaHx迅速诱导兴奋性毒性死亡，MK801或EGTA可完全阻止这种死亡。KaHx还刺激了神经元自噬流，表现为自噬体数量增加（LC3-II水平升高和点状LC3标记增加），同时溶酶体丰度和活性增加（SQSTM1/p62降解增加，以及在溶酶体抑制剂存在下LC3-II水平增加）和融合（使用RFP-GFP-LC3报告基因显示）。为了确定增强的自噬的作用，我们应用了药理学自噬抑制剂（3-甲基腺嘌呤或胃蛋白酶抑制剂A/E64）或递送靶向Becn1或Atg7的shRNA的慢病毒载体。这两种策略都减少了KaHx诱导的神经元死亡。在过表达BECN1或ATG7的培养物中，KaHx的毒性增强也证实了自噬的促死亡作用。最后，在严重新生儿脑HI大鼠模型中，通过纹状体内注射表达靶向Becn1的shRNA的慢病毒载体对自噬进行体内抑制，增加了完整纹状体的体积。这些结果清楚地表明自噬在缺氧-兴奋性毒性条件下具有死亡介导作用，并表明抑制自噬应被视为HI脑损伤中的一种神经保护策略。
