SUMMARY Aims: To determine the extent to which autophagy contributes to neuronal death in cerebral hypoxia and ischemia. Methods: We performed immunocytochemistry, western blot, cell viability assay, and electron microscopy to analyze autophagy activities in vitro and in vivo.Results: In both primary cortical neurons and SH‐SY5Y cells exposed to oxygen and glucose deprivation (OGD)for 6 h and reperfusion (RP) for 24, 48, and 72 h, respectively, an increase of autophagy was observed as determined by the increased ratio of LC3‐II to LC3‐I and Beclin‐1 (BECN1) expression. Using Fluoro‐Jade C and monodansylcadaverine double‐staining, and electron microscopy we found the increment in autophagy after OGD/RP was accompanied by increased autophagic cell death, and this increased cell death was inhibited by the specific autophagy inhibitor, 3‐methyladenine. The presence of large autolysosomes and numerous autophagosomes in cortical neurons were confirmed by electron microscopy. Autophagy activities were increased dramatically in the ischemic brains 3–7 days postinjury from a rat model of neonatal cerebral hypoxia/ischemia as shown by increased punctate LC3 staining and BECN1 expression. Conclusion: Excessive activation of autophagy contributes to neuronal death in cerebral ischemia.

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AimsTo determine the extent to which autophagy contributes to neuronal death in cerebral hypoxia and ischemia.MethodsWe performed immunocytochemistry, western blot, cell viability assay, and electron microscopy to analyze autophagy activities in vitro and in vivo.ResultsIn both primary cortical neurons and SH‐SY5Y cells exposed to oxygen and glucose deprivation (OGD) for 6 h (hours) and reperfusion (RP) for 24, 48 and 72 h respectively, an increase of autophagy was observed as determined by the increased ratio of LC3‐II to LC3‐I and Beclin‐1 (BECN1) expression. Using Fluoro‐Jade C and monodansylcadaverine (MDC) double‐staining, and electron microscopy we found the increment in autophagy after OGD/RP was accompanied by increased autophagic cell death, and this increased cell death was inhibited by the specific autophagy inhibitor, 3‐Methyladenine (3‐MA). The presence of large autolysosomes and numerous autophagosomes in cortical neurons were confirmed by electron microscopy. Autophagy activities were increased dramatically in the ischemic brains 3 to 7 days post‐injury from a rat model of neonatal cerebral hypoxia/ischemia as shown by increased punctate LC3 staining and Beclin‐1 expression.ConclusionExcessive activation of autophagy contributes to neuronal death in cerebral ischemia. This study was supported by CIHR, MHRC, MICH and Canadian Stroke Network.

Caspase‐independent neuronal death has been shown to occur in neuroexcitotoxicity. Here, we tested the hypothesis that the gene encoding Bcl‐2/E1B‐19K‐interacting protein 3 (BNIP3) mediates caspase‐independent neuronal death in excitotoxicity. BNIP3 was not detectable in neurons under normal condition. BNIP3 expression was increased dramatically in neurons in both in vivo and in vitro models of excitotoxicity. Expression of full‐length BNIP3 in primary hippocampal neurons induced atypical cell death that required protein synthesis but was largely independent of caspase activities. Inhibition of BNIP3 expression by RNA interference protected against glutamate‐induced neuronal cell death. Thus, BNIP3 activation and expression appears to be both necessary and sufficient for neuronal apoptosis in excitotoxicity. These results suggest that BNIP3 may be a new target for neuronal rescue strategies.

Necrostatin‐1 (Nec‐1) has previously been shown to protect neurons from death in traumatic and ischemic brain injuries. This study tests the hypothesis that Nec‐1 protects neural cells against traumatic and ischemic brain injuries through inhibition of the Bcl‐2/adenovirus E1B 19‐kDa interacting protein 3 (BNIP3). We have used biochemical and morphological techniques to determine the inhibition of Nec‐1 on BNIP3‐induced cell death and to identify its mechanism of action in in vivo and in vitro models of neurodegeneration. Here we show that Nec‐1 significantly increased neuronal viability following prolonged exposure to hypoxia in vitro, and attenuated myelin damage and neuronal death in traumatic brain injury and cerebral ischemia in Sprague‐Dawley rats. Nec‐1 alleviated traumatic brain injury‐induced up‐regulation of BNIP3 in mature oligodendrocytes. In isolated mitochondria, Nec‐1 prevented BNIP3 from integrating into mitochondria by modifying its binding sites on the mitochondria. Consequently, Nec‐1 robustly inhibited BNIP3‐induced collapse of mitochondrial membrane potential and reduced the opening probability of mitochondrial permeability transition pores. Nec‐1 also preserved mitochondrial ultrastructure and suppressed BNIP3‐induced nuclear translocation of apoptosis‐inducing factor. In conclusion, Nec‐1 protects neurons and oligodendrocytes against traumatic and ischemic brain injuries by targeting the BNIP3‐induced cell death pathway, and is a novel inhibitor for BNIP3. Cover Image for this issue: https://doi.org/10.1111/jnc.15056.