Mitochondrial translocation of P53 following transient global ischemia in rats

Introduction

The mechanism of p53-mediated apoptosis after cellular stress remains unclear. p53 can mediate apoptosis by transcriptional activation of proapoptotic genes like the BH3-only proteins Noxa and Puma, Bax, p53 AIP1, Apaf-1, and PERP. In addition, evidence for transcription-independent p53-mediated apoptosis has been accumulating. Release of cytochrome c from mitochondria to the cytosol is a critical step in apoptotic cell death. Recent studies have shown that the p53 protein can directly induce permeabilization of the outer mitochondrial membrane by forming a complex with the protective Bcl-xL protein, resulting in cytochrome c release. However, the mitochondrial p53 pathway remains unclear in the death of vulnerable hippocampal CA1 neurons after transient cerebral ischemia.

Materials and methods

To examine the p53 mitochondrial pathway following ischemia-reperfusion injury, we used a transient global cerebral ischemia (tGCI) model in rats^{1, 2}. After 1, 4, 8, 24, and 72 h of reperfusion, samples were taken from the hippocampal CA1 subregion to extract protein from the mitochondrial fraction, and used for Western blot and immunoprecipitation analysis. To examine the effects of p53, an inhibitor, 2-(2-lmino-4, 5, 6, 7-tetrahydrobenzothiazol-3-yl)-1-p-tolyethanone hydrobromide (pifithrin á), was administered intravenously.

Results

Western blot analysis of the mitochondrial fraction showed p53 gradually increased in a time-dependent manner after tGCI, which means ischemia-reperfusion injury induces translocation of p53 to mitochondria (Fig. 1A). Pifithrin á had no effect on p53 mitochondrial translocation after ischemia. We did a coimmunoprecipitation study to investigate the change in p53 binding with Bcl-xL or Bcl-2. This analysis for p53 immunoreactivity precipitated by Bcl-xL or Bcl-2 in the mitochondrial fraction revealed that p53/Bcl-xL binding, but not p53/Bcl-2 binding, gradually increased in a time-dependent manner after the tGCI (Fig. 1B), indicating that p53 bound to Bcl-xL in mitochondria in hippocampal CA1 neurons that were subsequently destined to die 3 days later.

Figure 1

Conclusions

In this study, we demonstrated that p53 translocates to mitochondria and binds to Bcl-xL after tGCI. Mitochondrial translocation of p53 and protein interaction between p53 and Bcl-xL might induce release of cytochrome c from mitochondria to the cytosol, resulting in delayed hippocampal CA1 neuronal cell death. We suggest that this is a novel mechanism underlying the convergence of cell death pathways involving both p53 and antiapoptotic proteins in promoting neuronal apoptosis in vulnerable CA1 neurons after cerebral ischemia.