With the recognition of the central role of mitochondria in apoptosis, there is a need to develop specific tools to manipulate mitochondrial function within cells. Here we report on the development of a novel antioxidant that selectively blocks mitochondrial oxidative damage, enabling the roles of mitochondrial oxidative stress in different types of cell death to be inferred. This antioxidant, named mitoQ, is a ubiquinone derivative targeted to mitochondria by covalent attachment to a lipophilic triphenylphosphonium cation through an aliphatic carbon chain. Due to the large mitochondrial membrane potential, the cation was accumulated within mitochondria inside cells, where the ubiquinone moiety inserted into the lipid bilayer and was reduced by the respiratory chain. The ubiquinol derivative thus formed was an effective antioxidant that prevented lipid peroxidation and protected mitochondria from oxidative damage. After detoxifying a reactive oxygen species, the ubiquinol moiety was regenerated by the respiratory chain enabling its antioxidant activity to be recycled. In cell culture studies, the mitochondrially localized antioxidant protected mammalian cells from hydrogen peroxide-induced apoptosis but not from apoptosis induced by staurosporine or tumor necrosis factor-α. This was compared with untargeted ubiquinone analogs, which were ineffective in preventing apoptosis. These results suggest that mitochondrial oxidative stress may be a critical step in apoptosis induced by hydrogen peroxide but not for apoptosis induced by staurosporine or tumor necrosis factor-α. We have shown that selectively manipulating mitochondrial antioxidant status with targeted and recyclable antioxidants is a feasible approach to investigate the role of mitochondrial oxidative damage in apoptotic cell death. This approach will have further applications in investigating mitochondrial dysfunction in a range of experimental models.

ROS (reactive oxygen species) from mitochondrial and non-mitochondrial sources have been implicated in TNFα (tumour necrosis factor α)-mediated signalling. In the present study, a new class of specific mitochondria-targeted antioxidants were used to explore directly the role of mitochondrial ROS in TNF-induced apoptosis. MitoVit E {[2-(3,4-dihydro-6-hydroxy-2,5,7,8-tetramethyl-2H-1-benzopyran-2-yl)ethyl]triphenylphosphonium bromide} (vitamin E attached to a lipophilic cation that facilitates accumulation of the antioxidant in the mitochondrial matrix) enhanced TNF-induced apoptosis of U937 cells. In time course analyses, cleavage and activation of caspase 8 in response to TNF were not affected by MitoVit E, whereas the activation of caspase 3 was significantly increased. Furthermore, there was an increased cleavage of the proapoptotic Bcl-2 family member Bid and an increased release of cytochrome c from mitochondria, in cells treated with TNF in the presence of MitoVit E. We considered several mechanisms by which MitoVit E might accelerate TNF-induced apoptosis including mitochondrial integrity (ATP/ADP levels and permeability transition), alterations in calcium homoeostasis and transcription factor activation. Of these, only the transcription factor NF-κB (nuclear factor κB) was implicated. TNF caused maximal nuclear translocation of NF-κB within 15 min, compared with 1 h in cells pretreated with MitoVit E. Thus the accumulation of an antioxidant within the mitochondrial matrix enhances TNF-induced apoptosis by decreasing or delaying the expression of the protective antiapoptotic proteins. These results demonstrate that mitochondrial ROS production is a physiologically relevant component of the TNF signal-transduction pathway during apoptosis, and reveal a novel functional role for mitochondrial ROS as a temporal regulator of NF-κB activation and NF-κB-dependent antiapoptotic signalling.

The subcellular localization of TNF-R1 to the Golgi apparatus, initially observed in endothelial cells, has been confirmed using transfection of bovine aortic endothelial cells with a human TNF-R1 expression plasmid. The subcellular interactions of TNF-R1 and the TRADD (TNFR-associated death domain protein) adaptor protein have been analyzed in the human monocyte cell line U937 and the human endothelial cell line ECV304 by confocal immunofluorescence microscopy and by Western blot analysis of fractionated cell extracts. In untreated cells, in which TNF-R1 is found on the cell surface but principally localizes to the trans-Golgi network, TRADD is concentrated in the cis- or medial-Golgi region, but separates from the Golgi during cell fractionation. Coimmunoprecipitation studies have shown that TRADD binds to TNF-R1 within 1 min of TNF treatment in a cell fraction-containing plasma membrane. This association is followed by a gradual dissociation, which is prevented if receptor-mediated endocytosis is inhibited by hypertonic medium. In contrast, no association is detected between TRADD and TNF-R1 in the Golgi in response to exogenous TNF at any time examined. These results suggest that although TNF-R1 is predominantly a Golgi-associated protein and TRADD also localizes to the Golgi region, exogenous TNF causes TRADD to bind to TNF-R1 only at the plasma membrane.