Abstract
Primary neurons undergo insult-dependent programmed cell death. We examined autophagy as a process contributing to cell death in cortical neurons after treatment with either hydrogen peroxide (H2O2) or staurosporine. Although caspase-9 activation and cleavage of procaspase-3 were significant following staurosporine treatment, neither was observed following H2O2 treatment, indicating a non-apoptotic death. Autophagic activity increased rapidly with H2O2, but slowly with staurosporine, as quantified by processing of endogenous LC3. Autophagic induction by both stressors increased the abundance of fluorescent puncta formed by GFP-LC3, which could be blocked by 3-methyladenine. Significantly, such inhibition of autophagy blocked cell death induced by H2O2 but not staurosporine. Suppression of Atg7 inhibited cell death by H2O2, but not staurosporine, whereas suppression of Beclin 1 prevented cell death by both treatments, suggesting it has a complex role regulating both apoptosis and autophagy. We conclude that autophagic mechanisms are activated in an insult-dependent manner and that H2O2 induces autophagic cell death.
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Abbreviations
- 3-MA:
-
3-Methyladenine
- DIC:
-
Differential interference contrast
- Endo G:
-
Endonuclease G
- GFP-LC3:
-
Green fluorescent protein-tagged microtubule associated protein light chain 3
- H2O2 :
-
Hydrogen peroxide
- LC3:
-
Microtubule associated protein light chain 3
- nRNA:
-
Non-specific RNA
- PCD:
-
Programmed cell death
- PI:
-
Propidium iodide
- RFU:
-
Relative fluorescence units
- siAtg7:
-
siRNA specific for ATG7
- siBeclin1:
-
siRNA specific for BECN1
- STS:
-
Staurosporine
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Acknowledgments
We thank Ms. Sue Ekkel for technical assistance with Western immunoblotting and Dr. Danielle Smith for technical advice. This work was supported by the National Health and Medical Research Council (Australia), of which Philip M. Beart is a Research Fellow. Phillip Nagley and Rodney J. Devenish are also supported by the Australian Research Council.
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Higgins, G.C., Devenish, R.J., Beart, P.M. et al. Autophagic activity in cortical neurons under acute oxidative stress directly contributes to cell death. Cell. Mol. Life Sci. 68, 3725–3740 (2011). https://doi.org/10.1007/s00018-011-0667-9
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DOI: https://doi.org/10.1007/s00018-011-0667-9