Inhibition of the AIF/CypA complex protects against intrinsic death pathways induced by oxidative stress

OPEN Citation: Cell Death and Disease (2014) 5, e993; doi:10.1038/cddis.2013.518 & 2014 Macmillan Publishers Limited All rights reserved 2041-4889/14 www.nature.com/cddis Inhibition of the AIF/CypA complex protects against intrinsic death pathways induced by oxidative stress N Doti*,1,2,3, C Reuther2, PL Scognamiglio1, AM Dolga2, N Plesnila*,3,4, M Ruvo1 and C Culmsee*,2 Delayed neuronal cell death largely contributes to the progressive infarct development and associated functional impairments after cerebral ischemia or brain trauma. Previous studies exposed a key role for the interaction of the mitochondrial protein apoptosis-inducing factor (AIF) and cytosolic cyclophilin A (CypA) in pathways of programmed cell death in neurons in vitro and in vivo. These studies suggested that pro-apoptotic activities of AIF, such as its translocation to the nucleus and subsequent DNA degradation, depend on the physical interaction of AIF with CypA. Hence, this protein complex may represent a new pharmacological target for inhibiting the lethal action of AIF on the brain tissue. In this study, we show that the AIF amino-acid residues 370–394 mediate the protein complex formation of AIF with CypA. The synthetic AIF(370–394) peptide inhibited AIF/CypA complex formation in vitro by binding CypA with a KD of 12 lM. Further, the peptide exerted pronounced neuroprotective effects in a model of glutamate-induced oxidative stress in cultured HT-22 cells. In this model system of AIF-dependent cell death, the AIF(370–394) peptide preserved mitochondrial integrity, as detected by measurements of the mitochondrial membrane potential and quantification of mitochondrial fragmentation. Further, the AIF(370–394) peptide inhibited perinuclear accumulation of fragmented mitochondria, mitochondrial release of AIF to the nucleus and glutamate-induced cell death to a similar extent as CypA-siRNA. These data indicate that the targeting of the AIF-CypA axis is an effective strategy of neuroprotection. Cell Death and Disease (2014) 5, e993; doi:10.1038/cddis.2013.518; published online 16 January 2014 Subject Category: Neuroscience Apoptosis-inducing factor (AIF) is a mitochondrial flavoprotein with several functions regulating cellular survival or death at the mitochondrial level.1 AIF has a role in the maintenance of mitochondrial electron transport chain functions, in the mitochondrial regulation of ROS formation, and in mitochondrial pathways of cell death, particularly in neurons.2,3 AIF deficiency in mouse and human cells hampers oxidative phosphorylation, likely through an effect on the biogenesis and maintenance of respiratory complexes I, III and IV, which ultimately results in decreased respiratory activity.4,5 Harlequin (Hq) mice, exhibiting an 80–90% global reduction in AIF protein expression levels, suffer from slow, progressive neurodegeneration, ataxia, and loss of vision.1,3 These findings propose a vital role for AIF in the maintenance of mitochondrial functions and neuronal survival. In contrast, AIF acquires a detrimental role in release from the mitochondria under conditions of lethal cellular stress. There is evidence for 1 a prominent role of AIF in mediating neuronal cell death in a variety of different rodent models of acute brain injury induced by cerebral hypoxia/ischemia (HI), middle cerebral artery occlusion, cardiac arrest-induced brain damage, epileptic seizures or even brain trauma. In these models AIF translocation from the mitochondria to the nucleus was associated with chromatin condensation and large-scale DNA fragmentation in dying neurons.6–9 Inhibition of mitochondrial AIF release to the nucleus or reduction of AIF expression, such as in Hq mice, is neuroprotective and significantly reduces brain damage in the respective mouse models of acute brain damage.9–12 Despite the findings on the crucial role of AIF in mitochondrial pathways of caspase-independent neuronal death, attempts for direct inhibition of AIF to achieve neuroprotective effects have not been reported and pharmacological inhibitors of AIF are not available. Institute of Biostructures and Bioimaging (IBB)-CNR, CIRPEB, Via Mezzocannone, 16, Naples 80134, Italy; 2Institute of Pharmacology and Clinical Pharmacy, Philipps University of Marburg, Marburg 35032, Germany; 3Department of Neurodegeneration, Royal College of Surgeons in Ireland, Dublin 2, Ireland and 4Institute for Stroke and Dementia Research (ISD), University of Munich Medical School–Campus Grohadern, Max-Lebsche Platz 30, Munich D-81377, Germany *Corresponding author: N Doti, Institute of Biostructures and Bioimaging (IBB)-CNR, CIRPEB, Via Mezzocannone, 16, Naples 80134, Italy. Tel: +39 0 81 2536644; Fax: +39 0 81 2534574; E-mail: or N Plesnila, Institute for Stroke and Dementia Research (ISD), University of Munich Medical School–Campus Grohadern, Max-Lebsche Platz 30, Munich 81377, Germany. Tel: +49 0 89 7095 8357; Fax: +49 0 89 7095 8369; E-mail: or C Culmsee, Institute of Pharmacology and Clinical Pharmacy, Philipps University of Marburg, Karl-von-Frisch-Strae 1, Marburg 35032, Germany. Tel: +49 0 6421 2825963; Fax: +49 0 6421 2825720; E-mail: Keywords: AIF/CypA complex; mitochondria; neuronal death; oxidative stress; peptide inhibitor Abbreviations: AIF, apoptosis-inducing factor; ANOVA, analysis of variance; CCCP, carbonyl cyanide m-chlorophenylhydrazone; CsA, cyclosporin A; CypA and D, cyclophilin A and D; DiOC6(3), 3,30 -dihexyloxacarbocyanine iodide; FACS, fluorescent-activated cell sorting; FITC, fluorescein isothiocyanate; Fmoc, fluorenylmethoxycarbonyl; IMM, inner mitochondrial membrane; JC-1, 5,50 ,6,60 -tetrachloro-1,10 ,3,30 -tetraethylbenzimi-dazolylcarbocyanine-iodide; TMRE, tetramethylrhodamine ethyl ester; MMP, mitochondrial membrane permeabilization; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; OMM, outer mitochondrial membrane; RT-PCR, reverse transcription-PCR; siRNA, small interfering RNA Received 26.6.13; revised 28.10.13; accepted 15.11.13; Edited by D Bano Neuroprotection induced by CypA targeting N Doti et al 2 A direct approach to inhibit AIF-mediated cell death in neurons may be achieved by inhibiting the interaction of AIF with cyclophilin A (CypA). On release from the mitochondria, AIF binding to CypA is supposed to initiate the translocation of both proteins to the nucleus, where they generate a lethal DNA-degrading complex.13,14 CypA exerts peptidyl-prolylisomerase activity in vitro; however, its function is unknown in living cells.15–18 In neurons, CypA is expressed in the cytoplasm and in the nucleus, where it can exert apoptosisrelated DNase activity.17 The molecular model of the AIF/CypA complex13 suggested that the interaction involves the AIF regions 367–399 and one of the AIF helices (aa. 136–146), together with a part of the b-barrel of CypA.13 The predicted interface overlaps neither with the catalytic site of CypA nor with the predicted binding site for Hsp70, nor wit (...truncated)