Abstract
Mitochondrial dysfunction and release of pro-apoptotic factors such as cytochrome c or apoptosis-inducing factor (AIF) from mitochondria are key features of neuronal cell death. The precise mechanisms of how these proteins are released from mitochondria and their particular role in neuronal cell death signaling are however largely unknown. Here, we demonstrate by fluorescence video microscopy that 8–10 h after induction of glutamate toxicity, AIF rapidly translocates from mitochondria to the nucleus and induces nuclear fragmentation and cell death within only a few minutes. This markedly fast translocation of AIF to the nucleus is preceded by increasing translocation of the pro-apoptotic bcl-2 family member Bid (BH3-interacting domain death agonist) to mitochondria, perinuclear accumulation of Bid-loaded mitochondria, and loss of mitochondrial membrane integrity. A small molecule Bid inhibitor preserved mitochondrial membrane potential, prevented nuclear translocation of AIF, and abrogated glutamate-induced neuronal cell death, as shown by experiments using Bid small interfering RNA (siRNA). Cell death induced by truncated Bid was inhibited by AIF siRNA, indicating that caspase-independent AIF signaling is the main pathway through which Bid mediates cell death. This was further supported by experiments showing that although caspase-3 was activated, specific caspase-3 inhibition did not protect neuronal cells against glutamate toxicity. In conclusion, Bid-mediated mitochondrial release of AIF followed by rapid nuclear translocation is a major mechanism of glutamate-induced neuronal death.
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Abbreviations
- AIF:
-
apoptosis-inducing factor
- ANOVA:
-
analysis of variance
- CCCP:
-
carbonyl cyanide m-chlorophenylhydrazone
- FACS:
-
fluorescent-activated cell sorting
- FITC:
-
fluorescein isothiocyanate
- GFP:
-
green fluorescent protein
- Hq:
-
harlequin
- JC-1:
-
5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolyl-carbocyanine iodide
- MTT:
-
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
- PARP:
-
poly-(ADP-ribose)-polymerase-1
- RT-PCR:
-
reverse transcription-PCR
- siRNA:
-
small interfering RNA
- Smac/Diablo:
-
second mitochondria-derived activator of caspase/direct IAP binding protein with low pI
- STS:
-
staurosporine
- tBid:
-
truncated Bid
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Acknowledgements
The authors gratefully acknowledge outstanding technical support and generation of vectors for transfection experiments by Melinda Kiss as well as additional control experiments by Markus Hofer. This work was supported in part by NIH grant HL082574 to MP.
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Landshamer, S., Hoehn, M., Barth, N. et al. Bid-induced release of AIF from mitochondria causes immediate neuronal cell death. Cell Death Differ 15, 1553–1563 (2008). https://doi.org/10.1038/cdd.2008.78
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DOI: https://doi.org/10.1038/cdd.2008.78
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