p53, a tumour suppressor, is involved in DNA repair and cell death processes and mediates apoptosis in response to death stimuli by transcriptional activation of pro-apoptotic genes and by transcription-independent mechanisms. In the latter process, p53 induces permeabilization of the outer mitochondrial membrane by forming an inhibitory complex with a protective Bcl-2 family protein, resulting in cytochrome c release in several cell line systems. However, it is unclear how the mitochondrial p53 pathway mediates neuronal apoptosis after cerebral ischaemia. We examined interaction between the mitochondrial p53 pathway and vulnerable hippocampal CA1 neurons using a tGCI (transient global cerebral ischaemia) rat model. We showed mitochondrial translocation of p53 and its binding to Bcl-XL. Mitochondrial p53 translocation, interaction between p53 and Bcl-XL, and cytochrome c release from mitochondria and subsequent CA1 neuronal death were prevented by pifithrin-α, a p53-specific inhibitor. These results suggest that the mitochondrial p53 pathway plays a role in delayed CA1 neuronal death after tGCI.
- cytochrome c
- global cerebral ischaemia
Current evidence indicates that the tumour suppressor gene p53 encodes a sequence-specific transcription factor that controls the expression of genes whose products mediate apoptosis . Furthermore, evidence for transcription-independent p53-mediated apoptosis has been accumulating . In some cell types, a fraction of stabilized p53 rapidly translocates to mitochondria in response to a death stimulus [3–6]. Endogenous mitochondrial p53 forms inhibitory complexes with protective Bcl-XL and Bcl-2 proteins, resulting in cytochrome c release from mitochondria . This suggests that p53 contributes to apoptosis by direct signalling at the mitochondria.
Mitochondria are central integrators and transducers for pro-apoptotic signals in neuronal apoptosis. However, the signalling pathways upstream of the mitochondrial apoptotic mechanism engaged in delayed neuronal death of hippocampal CA1 neurons after tGCI (transient global cerebral ischaemia) are not fully understood. p53 functions as a pro-apoptotic factor [7,8], but its precise mechanism in neuronal injury after stroke remains unknown. The present study was designed to clarify the role of the mitochondrial p53 pathway after tGCI.
p53 immunoreactivity was evident as a band in both the cytosolic and mitochondrial fractions from the hippocampal CA1 subregion of rat brains. Under normal conditions, cytosolic-dominant distribution of p53 was observed (results not shown). Mitochondrial p53 increased as early as 1 h after reperfusion and increased significantly 8 and 24 h after reperfusion (Figure 1A). In contrast, cytosolic p53 was decreased as early as 1 h after reperfusion and significantly decreased 8 h after reperfusion (Figure 1A). Double immunofluorescence for p53 and cytochrome oxidase demonstrated that they co-localized (used as a mitochondrial marker) in the hippocampal CA1 subregion 8 h after tGCI (results not shown). Double immunofluorescence for p53 and neuron-specific nuclear protein demonstrated that p53 expression co-localized with neurons in the hippocampal CA1 subregion (results not shown). These results suggest that mitochondrial localization of p53 occurs in hippocampal CA1 neurons after tGCI.
To investigate the direct interaction between p53 and Bcl-XL or Bcl-2, we performed co-immunoprecipitation. p53 expression precipitated by Bcl-XL in the mitochondrial fraction increased time dependently and a significant increase was observed 8 h after reperfusion (Figure 1B). In contrast, p53 expression precipitated by Bcl-2 showed no significant difference at any time point after reperfusion (results not shown). Double immunofluorescence for p53 and Bcl-XL demonstrated that p53 expression co-localized with Bcl-XL in the hippocampal CA1 subregion 8 h after reperfusion (results not shown). These results suggest that direct binding between p53 and Bcl-XL increased in the hippocampal CA1 subregion after tGCI.
We next addressed whether PFT (pifithrin-α), a p53-specific inhibitor, plays a role in the mitochondrial p53 pathway after tGCI. We intravenously administered 2 mg/kg of PFT immediately after reperfusion. Western-blot analysis showed that mitochondrial p53 expression significantly decreased in the PFT-treated rats compared with vehicle-treated animals 8 h after tGCI (Figure 2A). Moreover, co-immunoprecipitation analysis showed that p53 expression, precipitated by Bcl-XL, significantly decreased in the PFT-treated rats 8 h after tGCI (Figure 2B). To investigate the effect of PFT downstream of the mitochondrial p53 pathway, we examined cytochrome c release and caspase 9 activation. In the vehicle-treated rats, a significant increase in cytosolic cytochrome c was observed 8 and 24 h after reperfusion (Figure 2C). Comparison between treatments with the vehicle and PFT showed that this protein was less abundant 8 and 24 h after ischaemia in the PFT-treated animals. Next, we examined the activated form of caspase 9 as an initiator of the caspase chain reaction. Expression of cleaved caspase 9 showed a significant increase 8 and 24 h after tGCI in the vehicle-treated animals (Figure 2C). Comparison between treatments with the vehicle and PFT showed that this protein was less abundant 8 and 24 h after ischaemia in the PFT-treated animals. These results indicate that PFT administration inhibits mitochondrial p53 translocation and direct binding between p53 and Bcl-XL in mitochondria, resulting in inhibition of cytosolic cytochrome c release and subsequent activation of caspase 9.
To investigate the involvement of the mitochondrial p53 pathway in the delayed death of hippocampal CA1 neurons after tGCI, we did a histological evaluation and a counting study using TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling) in the PFT-treated animals. In the present study, 2 mg/kg of PFT was intravenously administered immediately after reperfusion. In both non-treated and vehicle-treated animals, delayed death of hippocampal CA1 neurons was observed 72 h after ischaemia. Most of the CA1 neurons in both groups had shrunken, triangular-shaped, condensed nuclei in the Cresyl Violet-stained sections. Normal features were preserved, however, in the non-ischaemic and PFT-treated animals (results not shown). Most of the morphologically damaged neurons stained with Cresyl Violet were also positive for TUNEL (results not shown). Neuronal damage in the hippocampal CA1 subregion, which was qualitatively evaluated on the basis of a scoring system , was significantly decreased in the PFT-treated animals (Figure 2D). Furthermore, the counting studies of TUNEL-positive cells showed that they were significantly decreased in the PFT-treated animals (Figure 2D). These results suggest that intravenous PFT administration might be neuroprotective after tGCI through inhibition of the mitochondrial p53 pathway.
The rat tGCI model mimics conditions after transient cardiac arrest and causes selective apoptotic neuronal death in vulnerable regions, such as hippocampal CA1 pyramidal cells [10,11]. Mitochondrial cytochrome c release to the cytosol is considered the critical step towards apoptotic cell death for vulnerable hippocampal CA1 neurons after tGCI . However, upstream signalling of mitochondrial cytochrome c release after tGCI remains unclear. In the present study, we demonstrated that mitochondrial p53 translocation and direct inhibitory binding to Bcl-XL induced mitochondrial release of cytochrome c and subsequent activation of caspase 9, resulting in delayed death of CA1 neurons after ischaemia.
A fraction of induced p53 translocates to the mitochondria at the onset of p53-dependent apoptosis, but not during p53-independent apoptosis or in vitro p53-mediated cell-cycle arrest . Bypassing the nucleus by targeting p53 to mitochondria is still sufficient to launch apoptosis . This evidence suggests the contribution of p53 to apoptosis by direct signalling at the mitochondria in vitro. In the present study, Western-blot analysis revealed increased p53 immunoreactivity in the mitochondrial fraction and decreased p53 immunoreactivity in the cytosolic fraction after tGCI. Moreover, immunofluorescence showed p53 signals co-localized with cytochrome oxidase, which was used as a mitochondrial marker, after tGCI. These results provide evidence of mitochondrial p53 translocation after cerebral ischaemia in vivo. Erster et al.  found that mitochondrial p53 accumulation occurred rapidly in radiosensitive organs like the brain within 2 h after exposure to γ-irradiation in vivo. They proposed that mitochondrial translocation of p53 triggers a rapid pro-apoptotic response that jump-starts and amplifies the slower transcription-dependent response. In our study, Western-blot analysis showed that p53 immunoreactivity in the mitochondrial fraction began to increase 1 h after ischaemia and significantly increased 8 and 24 h after ischaemia. In contrast, nuclear p53 immunoreactivity was first observed 72 h after ischaemia in the immunofluorescence study (results not shown). This result suggests the important role of rapid p53 mitochondrial accumulation in delayed death of CA1 neurons.
The compound PFT can inhibit p53 transcriptional activity in various cell lines and prevents DNA damage-induced apoptosis . PFT protected neurons against oxygen–glucose deprivation in vitro  and reduced ischaemic brain damage in both transient focal cerebral ischaemia [14–16] and tGCI . The mechanisms of PFT neuroprotection against cerebral ischaemia have been reported to include inhibition of p53 translocation into the nucleus and prevention of its DNA-binding activity [15,16], or preservation of nuclear factor κB activity . In the present study, CA1 neuronal death after tGCI was significantly decreased by intravenous administration of PFT. Moreover, PFT inhibited mitochondrial p53 translocation, direct binding between p53 and Bcl-XL in mitochondria, cytochrome c release and subsequent caspase 9 activation. Inhibition of mitochondrial p53 translocation and subsequent apoptotic cell death by PFT were also observed in the renal ischaemia-reperfusion model . Mitochondrial release of cytochrome c is the critical step for delayed neuronal death of hippocampal CA1 neurons in our model of tGCI . The study of PFT administration suggests that mitochondrial p53 translocation and direct interaction between p53 and Bcl-XL might be among the upstream mechanisms of mitochondrial cytochrome c release after cerebral ischaemia. Moreover, a reduction in oxidative stress inhibited cytochrome c release and subsequent neuronal death after tGCI in SOD1 (copper/zinc-superoxide dismutase) transgenic rats . We also observed a reduction in mitochondrial p53 translocation after tGCI in SOD1 transgenic rats (results not shown). This evidence might support the idea that mitochondrial p53 translocation is a key step to cytochrome c release and subsequent neuronal death after tGCI. We have reported that a cytosolic p53 degradation system, such as the MDM2 (murine double minute clone 2 oncoprotein) pathway or the ubiquitin-proteasome system, is inhibited by cerebral ischaemia (Figure 3) . This mechanism might help the mitochondrial p53 pathway to enhance neuronal death after tGCI. In contrast, the role of the transcriptional pathway of p53 needs to be considered. A recent study suggested that the transcriptional p53 pathway has a co-operative relationship with the cytosolic p53 pathway in cell death mechanisms . Thus this co-operative relationship might underlie the delayed neuronal death of hippocampal neurons after tGCI in our study. The relationship between the mitochondrial and nuclear pathways of p53 in neuronal death needs to be investigated in detail in a future study.
In summary, the present study provides evidence of a role for the mitochondrial p53 signalling pathway in the delayed neuronal death of hippocampal CA1 neurons. We found that p53 translocates to mitochondria and directly binds to Bcl-XL after tGCI. Mitochondrial translocation of p53 and protein interaction between p53 and Bcl-XL induce release of cytochrome c from mitochondria to the cytosol, resulting in delayed hippocampal CA1 neuronal death after tGCI.
This work was supported by National Institutes of Health grants P50 NS14543, R01 NS25372, R01 NS36147 and R01 NS38653.
International Symposium on Neurodegeneration and Neuroprotection: Independent Meeting held at University of Münster, Germany, 23–27 July 2006. Organized and Edited by S. Klumpp and J. Krieglstein (Münster, Germany).
Abbreviations: MDM2, murine double minute clone 2 oncoprotein; PFT, pifithrin-α; SOD1, copper/zinc-superoxide dismutase; tGCI, transient global cerebral ischaemia; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling
- © 2006 The Biochemical Society