Abstract
Cerebral ischemia and excitotoxic injury induce transient or permanent bioenergetic failure, and may result in neuronal apoptosis or necrosis. We have previously shown that ATP depletion and activation of AMP-activated protein kinase (AMPK) during excitotoxic injury induces neuronal apoptosis by transcription of the pro-apoptotic BH3-only protein, Bim. AMPK, however, also exerts pro-survival functions in neurons. The molecular switches that determine these differential outcomes are not well understood. Using an approach combining biochemistry, single-cell imaging and computational modeling, we here demonstrate that excitotoxic injury activated the bim promoter in a FOXO3-dependent manner. The activation of AMPK reduced AKT activation, and led to dephosphorylation and nuclear translocation of FOXO3. Subsequent mutation studies indicated that bim gene activation during excitotoxic injury required direct FOXO3 phosphorylation by AMPK in the nucleus as a second activation step. Inhibition of this phosphorylation prevented Bim expression and protected neurons against excitotoxic and oxygen/glucose deprivation-induced injury. Systems analysis and computational modeling revealed that these two activation steps defined a coherent feed-forward loop; a network motif capable of filtering any effects of short-term AMPK activation on bim gene induction. This may prevent unwanted AMPK-mediated Bim expression and apoptosis during transient or physiological bioenergetic stress.
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Abbreviations
- AKT-CA:
-
constitutively active mutant of AKT
- AMPK-CA:
-
constitutively active mutant of AMPK
- CC:
-
compound C
- CFL:
-
coherent feed-forward loop
- CGNs:
-
cerebellar granule neurons
- 6 × DBE FOXO3 promoter:
-
reporter luciferase plasmid with six copies of the FOXO family protein-binding element
- DN-FOXO3:
-
dominant-negative FOXO3
- FOXO3-6A:
-
construct with FOXO3 sequence mutated at the AMPK phosphorylation sites
- FOXO3-nuclear:
-
construct with FOXO3 sequence mutated at the AKT phosphorylation sites
- GLY:
-
glycine
- GLUT:
-
glutamate
- OGD:
-
oxygen/glucose deprivation
- PI:
-
propidium iodide
- RLUs:
-
relative luciferase counts
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Acknowledgements
This study was supported by Marie Curie IEF (PIEF-GA-2009-237765), Science Foundation Ireland (08/ IN1/ 1949), and the Health Research Board in Ireland (PHD/2007/11). We thank Dr. Hans Georg Koenig for discussions and support, Ina Woods and Sarah Cannon for excellent technical assistance, and Andreas Strasser (WEHI, Melbourne, Australia) for bim-deficient mice.
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Davila, D., Connolly, N., Bonner, H. et al. Two-step activation of FOXO3 by AMPK generates a coherent feed-forward loop determining excitotoxic cell fate. Cell Death Differ 19, 1677–1688 (2012). https://doi.org/10.1038/cdd.2012.49
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DOI: https://doi.org/10.1038/cdd.2012.49
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