Background and aims: Subarachnoid hemorrhage (SAH) results in a high mortality rate, despite sophisticated medical management and neurosurgical techniques. Recent studies have emphasized the importance of acute brain injury after SAH, in which apoptosis is involved as one of the major mechanisms. We have reported the involvement of mitochondrial cytochrome c release in apoptotic cell death after SAH. However, the molecular mechanism of this cytochrome c release remains unresolved. PUMA (p53-upregulated modulator of apoptosis), which is one of the BH3-only proteins, was shown to localize to mitochondria and to function to induce cytochrome c release in several recent in vitro studies. These reports inspired us to investigate the role of PUMA in the mechanisms of acute brain injury after SAH. Methods: To examine the relationship between acute brain injury and the PUMA pathway following SAH, we used a perforation SAH model in rats. One, 6 and 24 hours after SAH, samples were taken from the cerebral cortex and used for Western blot, coimmunoprecipitation or immunohistochemistry. Protein extraction of the mitochondrial fraction was performed using a multiple centrifugation method for Western blot and coimmunoprecipitation. To investigate apoptotic cell death after SAH, DNA fragmentation was analyzed with a commercial enzyme immunoassay. Finally, we intravenously administered an inhibitor against p53, pifithrin-α (PFT), to examine the role of PUMA as a downstream target of p53 and the relationship between PUMA inhibition and acute brain injury. Results: Western blot analysis showed that the expression of PUMA significantly increased in the mitochondrial fraction 6 hours after SAH. Double immunofluorescence for PUMA and COX demonstrated that PUMA colocalized with COX (which was used as a mitochondrial marker) in the cerebral cortex 6 hours after SAH. PUMA expression precipitated by Bcl-XL in the mitochondrial fraction increased time dependently and a significant increase was observed at 1 and 6 hours. Western blot analysis showed a significant increase in cytosolic cytochrome c 24 hours after SAH compared with sham-operated brains. Double immunofluorescence showed that some cortical cells with strong PUMA immunoreactivity colocalized with cytochrome c-positive cells at 24 hours. Western blot analysis showed that mitochondrial localization of PUMA, which was transcriptionally controlled by p53, and cytosolic cytochrome c release were significantly inhibited by PFT 6 hours after SAH. Moreover, DNA fragmentation at 24 hours was significantly decreased in the PFT-treated animals compared with vehicle-treated animals. Conclusions: We found that PUMA is upregulated in mitochondria and directly binds to Bcl-XL after SAH. Mitochondrial localization of PUMA and protein interaction between PUMA and Bcl-XL might induce release of cytochrome c to the cytosol, resulting in acute brain injury following SAH.
|Journal||Journal of Cerebral Blood Flow and Metabolism|
|Issue number||SUPPL. 1|
|Publication status||Published - 2007 Nov 13|
ASJC Scopus subject areas
- Clinical Neurology
- Cardiology and Cardiovascular Medicine