A BAX/BAK and Cyclophilin D-Independent Intrinsic Apoptosis Pathway Sebastia´ n Zamorano1,2, Diego Rojas-Rivera1,2, Fernanda Lisbona1,2, Valentina Parra2,3, Felipe A. Court4, Rosario Villegas4, Emily H. Cheng5, Stanley J. Korsmeyer6{, Sergio Lavandero2,3,7, Claudio Hetz1,2,8,9* 1 Biomedical Neuroscience Institute, Faculty of Medicine, University of Chile, Santiago, Chile, 2 Center for Molecular Studies of the Cell, Institute of Biomedical Sciences, University of Chile, Santiago, Chile, 3 Faculty of Chemical & Pharmaceutical Sciences, University of Chile, Santiago, Chile, 4 Millennium Nucleus for Regenerative Biology, Faculty of Biology, P. Catholic University of Chile, Santiago, Chile, 5 Human Oncology and Pathogenesis Program, Memorial Sloan-Kettering Cancer Center, New York, New York, United States of America, 6 Dana–Farber Cancer Institute, and Harvard Medical School, Boston, Massachusetts, United States of America, 7 Cardiology Division, Department of Internal medicine, University of Texas Southwestern Medical Center, Dallas, Texas, United States of America, 8 Harvard School of Public Health, Boston, Massachusetts, United States of America, 9 Neurounion Biomedical Foundation, Santiago, Chile Abstract Most intrinsic death signals converge into the activation of pro-apoptotic BCL-2 family members BAX and BAK at the mitochondria, resulting in the release of cytochrome c and apoptosome activation. Chronic endoplasmic reticulum (ER) stress leads to apoptosis through the upregulation of a subset of pro-apoptotic BH3-only proteins, activating BAX and BAK at the mitochondria. Here we provide evidence indicating that the full resistance of BAX and BAK double deficient (DKO) cells to ER stress is reverted by stimulation in combination with mild serum withdrawal. Cell death under these conditions was characterized by the appearance of classical apoptosis markers, caspase-9 activation, release of cytochrome c, and was inhibited by knocking down caspase-9, but insensitive to BCL-XL overexpression. Similarly, the resistance of BIM and PUMA double deficient cells to ER stress was reverted by mild serum withdrawal. Surprisingly, BAX/BAK-independent cell death did not require Cyclophilin D (CypD) expression, an important regulator of the mitochondrial permeability transition pore. Our results suggest the existence of an alternative intrinsic apoptosis pathway emerging from a cross talk between the ER and the mitochondria. Citation: Zamorano S, Rojas-Rivera D, Lisbona F, Parra V, Court FA, et al. (2012) A BAX/BAK and Cyclophilin D-Independent Intrinsic Apoptosis Pathway. PLoS ONE 7(6): e37782. doi:10.1371/journal.pone.0037782 Editor: Dhyan Chandra, Roswell Park Cancer Institute, United States of America Received November 22, 2011; Accepted April 26, 2012; Published June 12, 2012 Copyright: 2012 Zamorano et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by FONDECYT no. 1100176, Millennium Institute no. P09-015-F, Michael J. Fox Foundation for Parkinson’s Research, Alzheimer’s Disease Foundation, North American Spine Society, Muscular Dystrophy Association, ALS Therapy Allianze (to CH), FONDAP grant no. 15010006 (to CH and SL), Supported by grants from FONDECYT no. 1070377 and Millennium Nucleus no. P-07-011-F (to FC), National Institute of Health R01CA125562 and American Cancer Society RSG-10-030-01-CCG (to EC), and PhD fellowships from CONICYT (to DRR, FL, and VP). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: chetz@med.uchile.cl { Deceased. Introduction Apoptosis is a conserved cell death mechanism essential for normal development and tissue homeostasis in multicellular organisms. Although apoptosis presumably participates in the development of most cell lineages, alterations in the expression of apoptosis-regulatory proteins is implicated in the initiation of a variety of human diseases, including autoimmunity, immunodefi- ciency, cancer, and neurodegenerative diseases, among others [1,2]. The BCL-2 family of proteins is a group of upstream regulators of the caspase cascade, comprised of both pro- and anti- apoptotic components [1,2]. BCL-2 family members are defined by the presence of up to four a-helical conserved BCL-2 homology (BH) domains. Pro-apoptotic BCL-2 family members can be further subdivided into more highly conserved, ‘‘multidomain’’ members displaying homology in the BH1, BH2 and BH3 domains (i.e. BAX and BAK), and the ‘‘BH3-only’’ members which contain a single BH domain critical for activation of apoptosis. Genetic and biochemical studies indicate that BAX and BAK function in concert as a major core of the intrinsic apoptosis pathway at the mitochondria [3,4]. Upstream BH3-only proteins respond to particular apoptotic signals and subsequently trigger the conformational activation of BAX and BAK, inducing their intramembranous homo-oligomerization and resultant mitochon- drial outer membrane permeabilization (MOMP) [5]. MOMP is a key step for the release of cytochrome c and the assembling of the apoptosome [5,6]. Besides, the BH3-only proteins can be functionally separated into two subtypes: (i) activators (i.e. tBID, BIM, and PUMA) that directly engage BAX and BAK to trigger cytochrome c release, but are sequestered by anti-apoptotic BCL-2 molecules; and (ii) sensitizers or inactivators (i.e. BAD and NOXA) that only bind to and antagonize anti-apoptotic BCL-2 members to release activator BH3-only proteins (examples in [7–11]). Alternatively, differential binding to anti-apoptotic proteins may explain the action of activator and sensitizer/inactivator BH3-only proteins [12] or combination of both models [11,13,14]. Under certain conditions, cytochrome c release occurs inde- pendent of BAX and BAK through opening of the mitochondrial permeability transition pore (PTP), a non-specific pore in the inner mitochondrial membrane (see reviews in [15–17]). 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