Research Report Multiple apoptogenic proteins are involved in the nuclear translocation of Apoptosis Inducing Factor during transient focal cerebral ischemia in rat Ganta Vijay Chaitanya, Phanithi Prakash Babu ⁎ Department of Biotechnology, School of Life Sciences, University of Hyderabad, Hyderabad-50046, India Department of Animal Sciences, School of Life Sciences, University of Hyderabad, Hyderabad-50046, India ARTICLE INFO ABSTRACT Article history: Accepted 19 September 2008 Available online 9 October 2008 Apoptosis Inducing Factor is a mitochondrial protein which upon translocation to nucleus causes large scale DNA fragmentation. The stimulus for the cytosolic release and nuclear translocation for this protein still remains to be understood. The role of calpains, cathepsin- b, Poly ADP (ribose) Polymerase and granzyme-b in the nuclear translocation of AIF has been investigated in the pathology of cerebral ischemia. Calpains, cathepsin-b and PARP-1 which were mostly confined to cytosol, lysosomes and nucleus respectively were found to be elevated in the mitochondrial fraction interacting with AIF in the western blot analysis and double immunofluorescence analysis. Western blot and immunohistochemical analysis revealed elevated levels of granzyme-b secreted by cytotoxic T lymphocytes and natural killer cells in the infarct of ischemic mouse brain. Co-immunoprecipitation revealed and western blot analysis the interaction and break down of Heat Shock Protein-70 an endogenous inhibitor of AIF into signature fragments by granzyme-b facilitating the nuclear translocation of AIF. Break down of HSP-70 correlated with the nuclear translocation of AIF observed in western and immunohistochemical analysis. These results indicate that multiple proteases were involved in the nuclear translocation of AIF during the pathology of cerebral ischemia. © 2008 Elsevier B.V. All rights reserved. Keywords: Cerebral ischemia Apoptosis Inducing Factor Calpain Cathepsin-b Granzyme-b Poly ADP (ribose) Polymerase Heat Shock Protein-70 1. Introduction Cerebral ischemia is one of the devastating neuropathological disorders known to humans. Though several putative mole- cules have been investigated for pharmacological interven- tion, no successful therapy has been achieved so far. The only causal therapy for ischemic stroke is reperfusion. Even if cerebral blood flow is reestablished quickly enough to prevent immediate cell death, a large population of initially surviving neuronal cells will die within the first few hours after reperfusion (Graham and Chen, 2001). The involvement of several suicidal proteases (Chaitanya and Babu, in press), their cross talks (Yamashima, 2000) and interaction with other sub- cellular organelles like mitochondria which harbor apopto- genic proteins like AIF, cyt-c, HtrA2/Omi, Endo-g, Smac/Diablo further amplify the cell death program (Plesnila, 2004), rendering pharmacological intervention inefficient. Apoptotic and necrotic cell deaths were considered to be the crucial partners contributing to the pathology of cerebral ischemia (Charriaut-Marlangue et al., 1996). Though Apoptosis is the BRAIN RESEARCH 1246 (2008) 178 – 190 ⁎ Corresponding author. Fax: +91 40 23010120. E-mail addresses: ppbsl@uohyd.ernet.in, phanithi@yahoo.co.in (P.P. Babu). 0006-8993/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2008.09.075 available at www.sciencedirect.com www.elsevier.com/locate/brainres