Cytochrome c oxidase deficiency accelerates mitochondrial apoptosis by activating ceramide synthase 6

OPEN Citation: Cell Death and Disease (2015) 6, e1691; doi:10.1038/cddis.2015.62 & 2015 Macmillan Publishers Limited All rights reserved 2041-4889/15 www.nature.com/cddis Cytochrome c oxidase deficiency accelerates mitochondrial apoptosis by activating ceramide synthase 6 S Schüll1, SD Günther1, S Brodesser2, JM Seeger1, B Tosetti1, K Wiegmann1, C Pongratz1, F Diaz3, A Witt1, M Andree1, K Brinkmann1, M Krönke1,2, RJ Wiesner2,4 and H Kashkar*,1,2 Although numerous pathogenic changes within the mitochondrial respiratory chain (RC) have been associated with an elevated occurrence of apoptosis within the affected tissues, the mechanistic insight into how mitochondrial dysfunction initiates apoptotic cell death is still unknown. In this study, we show that the specific alteration of the cytochrome c oxidase (COX), representing a common defect found in mitochondrial diseases, facilitates mitochondrial apoptosis in response to oxidative stress. Our data identified an increased ceramide synthase 6 (CerS6) activity as an important pro-apoptotic response to COX dysfunction induced either by chemical or genetic approaches. The elevated CerS6 activity resulted in accumulation of the pro-apoptotic C16 : 0 ceramide, which facilitates the mitochondrial apoptosis in response to oxidative stress. Accordingly, inhibition of CerS6 or its specific knockdown diminished the increased susceptibility of COX-deficient cells to oxidative stress. Our results provide new insights into how mitochondrial RC dysfunction mechanistically interferes with the apoptotic machinery. On the basis of its pivotal role in regulating cell death upon COX dysfunction, CerS6 might potentially represent a novel target for therapeutic intervention in mitochondrial diseases caused by COX dysfunction. Cell Death and Disease (2015) 6, e1691; doi:10.1038/cddis.2015.62; published online 12 March 2015 The mitochondrial oxidative phosphorylation (OXPHOS) machinery is composed of four multisubunit respiratory complexes (complex I–IV) creating an electrochemical gradient through the coupled transfer of electrons to oxygen and the transport of protons from the matrix across the inner mitochondrial membrane (IMM) into the intermembrane space (IMS), used by the ATP-synthase to produce ATP.1 The components of the OXPHOS machinery are encoded by nuclear DNA (nDNA) as well as by mitochondrial DNA (mtDNA). Mutations in either nuclear or mitochondrial genes result in mitochondrial dysfunction and precipitate versatile severe degenerative diseases, premature aging phenotypes and mortality.2,3 It is increasingly evident that defects in OXPHOS results in degenerative states based on the accelerated apoptotic death of the damaged cells. However, while numerous pathogenic changes within OXPHOS have been associated with an elevated occurrence of apoptosis, the causal role of mitochondrial dysfunction in the initiation of the apoptotic program and the underlying molecular mechanism linking OXPHOS dysfunction to the cellular apoptotic machinery are incompletely understood.3 Apoptosis is a mode of cell death that is used by multicellular organisms to dispose irreparably damaged cells and is executed by a family of proteases known as caspases.4 Caspase activity can be either initiated extrinsically or 1 intrinsically. Extrinsic apoptosis is triggered by binding of extracellular ligands to tumor necrosis factor receptor family members and results in the activation of the initiator caspase, caspase-8.5 Overwhelming cellular damage may alternatively initiate the death of the cell autonomously by involving mitochondria (intrinsic apoptotic pathway). Accordingly, intrinsic stress cues promote mitochondrial outer membrane permeabilization (MOMP), a process which is tightly regulated by members of the Bcl-2 protein family6 and results in the release of pro-apoptotic factors from IMS including cytochrome c. Cytosolic cytochrome c subsequently initiates the activation of the initiator caspase, caspase-9.7 Once active, these initiator caspases cleave and activate the zymogens of executioner caspases such as caspase-3, which in turn are responsible for the majority of proteolytic events that ultimately result in the apoptotic destruction of the cell.4 Cytochrome c oxidase (COX or complex IV) is a 200-kDa multicomponent enzyme located in the IMM and represents the terminal complex of the electron transport chain. COX is composed of 13 subunits encoded by both the mitochondrial (subunits 1, 2 and 3, which form the catalytic core of the enzyme) and the nuclear genomes.8 Naturally occurring COX dysfunctions are predominantly caused by somatic or inherited mutations in the mtDNA and nDNA. Here we show that COX deficiency, which represents a common defect found in Center for Molecular Medicine Cologne (CMMC) and Institute for Medical Microbiology, Immunology and Hygiene (IMMIH), University of Cologne, Cologne, Germany; Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD) Research Center, University of Cologne, Cologne, Germany; 3 Department of Neurology, University of Miami, Miami, FL, USA and 4Institute for Vegetative Physiology, University of Cologne, Cologne, Germany *Corresponding author: H Kashkar, Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD) Research Center, University of Cologne, Joseph-Stelzmann-Strasse 26, 50931 Cologne, Germany. Tel: +49 221 478-84091; Fax: +49 221 478-84094; E-mail: Abbreviations: RC, respiratory chain; COX, cytochrome c oxidase; CerS6, ceramide synthase 6; OXPHOS, oxidative phosphorylation; IMM, inner mitochondrial membrane; mtDNA, mitochondrial DNA; nDNA, nuclear DNA; ROS, reactive oxygen species; SMase, sphingomyelinase; FB1, fumonisin B1; LAG1, longevity assurance gene 1; LAC1, longevity assurance gene 1 cognate 2 Received 26.12.14; revised 3.2.15; accepted 9.2.15 CerS6 potentiates apoptosis in COX-deficient cells S Schüll et al 2 mitochondrial diseases, increases the mitochondrial apoptotic response to oxidative stress. Our data identify elevated levels of ceramide with an acyl chain of C16 : 0 to be responsible for the increased susceptibility of COX-deficient cells to oxidative stress. Specifically, COX dysfunction enhances ceramide synthase 6 (CerS6) transcription and activity, which in turn results in C16 : 0 ceramide accumulation, cytochrome c release and accelerated apoptosis in response to oxidative stress. Our results provide new insights into how mitochondrial dysfunction mechanistically interferes with the apoptotic machinery and how it induces the apoptotic demise of damaged cells in an affected degenerating tissue. Results Inhibition of COX increases cellular susceptibility to H2O2. We first investigated whether the alteration of mitochondrial respiration in general or the specific inhibition of one of the mitochondrial respiratory chain (RC) complexes directly impacts on cellular viability. Different RC complexes w (...truncated)