Redistribution of apoptosis promoters: XAF1, Smac/DIABLO and HtrA2 during transient forebrain ischemia/reperfusion

Abstract

The neuropathology following brain injury after cerebral ischemia includes both, selective neuronal necrosis and apoptosis. Caspases are key regulators of apoptosis and are essential for the execution of cell death. Caspase activity is controlled through a wide range of regulatory mechanisms, including a family of genes encoding for inhibitors of apoptosis proteins (IAPs). Convincing anti-apoptotic activity has been shown for a subset of mammalian IAPs. XIAP, cIAP1, cIAP2. XIAP (X-linked inhibitor of apoptosis protein) is the best characterized mammalian IAP and is also the most potent and versatile regulator of cell death. Regulation of XIAP involves at least three recently discovered proteins: XAF-1 (XIAP-associated factor 1), Smac/Diablo (Second mitochondrial activator of caspase/direct IAP-binding protein with low pI) and HtrA2/Omi (high-temperature requirement serine protease A2 or Omi). XAF-1 is ubiquitously expressed in normal tissues and inhibits XIAP and localized in the nucleus. When co-expressed in cell lines, XAF-1 retains normally cytosolic localized XIAP in the nucleus. To investigate whether IAPs and their inhibitors XAF1, Smac/DIABLO, HtrA2 may play a role in the apoptotic machinery after ischemia, we analyzed dynamics in their expression pattern and in their localisation. Ischemia was performed in Wistar rats by clamping both common carotid arteries and reducing blood pressure to 40 mmHg for 10 min. Animals were decapitated at different times post-ischemia and processed for histology, immunohistochemistry and immunoblotting by using antibodies directed against XIAP-BIR2, Smac/DIABLO, HtrA2 and cleaved caspase-3 (P17). XAF-1 staining and colocalization with XIAP was seen in the selectively vulnerable CA1-subfield of the hippocampus. In damaged neurons, we found that XAF-1 colocalized with XIAP in the nuclei, suggesting that it sequestered XIAP, allowing the pathological apoptosis to proceed. Smac/Diablo is a mitochondrial protein, which binds to XIAP, antagonizing its anti-apoptotic activity. This role of XIAP is supported by our findings, as it colocalized after ischemia very well with Smac, and as these double-labelled cells presented signs of damage. HtrA2 is another mitochondrial protein released upon apoptotic stimuli that binds to XIAP and promotes cell death. The strong HtrA2 immunoreactivity around the nuclei of damaged neurons, that stained also for XIAP and Smac, supports a concerted action of these proteins after ischemia. Increased intensity of Smac and HtrA2 stainings presumably indicates release from mitochondria. Here presented data report to our knowledge for the first time an involvement of XAF-1 and HtrA2 in the apoptotic dynamics following global cerebral ischemia in rats.