The mitochondria-targeted drugs mitoquinone (Mito-Q) and mitovitamin E (MitoVit-E) are a new class of antioxidants containing the triphenylphosphonium cation moiety that facilitates drug accumulation in mitochondria. In this study, Mito-Q (ubiquinone attached to a triphenylphosphonium cation) and MitoVit-E (vitamin E attached to a triphenylphosphonium cation) were used. The aim of this study was to test the hypothesis that mitochondria-targeted antioxidants inhibit peroxide-induced oxidative stress and apoptosis in bovine aortic endothelial cells (BAEC) through enhanced scavenging of mitochondrial reactive oxygen species, thereby blocking reactive oxygen species-induced transferrin receptor (TfR)-mediated iron uptake into mitochondria. Glucose/glucose oxidase-induced oxidative stress in BAECs was monitored by oxidation of dichlorodihydrofluorescein that was catalyzed by both intracellular H 2 O 2 and transferrin iron transported into cells. Pretreatment of BAECs with Mito-Q (1 M) and MitoVit-E (1 M) but not untargeted antioxidants (e.g. vitamin E) significantly abrogated H 2 O 2 -and lipid peroxide-induced 2 ,7 -dichlorofluorescein fluorescence and protein oxidation. Mitochondria-targeted antioxidants inhibit cytochrome c release, caspase-3 activation, and DNA fragmentation. Mito-Q and MitoVit-E inhibited H 2 O 2 -and lipid peroxide-induced inactivation of complex I and aconitase, TfR overexpression, and mitochondrial uptake of 55 Fe, while restoring the mitochondrial membrane potential and proteasomal activity. We conclude that Mito-Q or MitoVit-E supplementation of endothelial cells mitigates peroxide-mediated oxidant stress and maintains proteasomal function, resulting in the overall inhibition of TfR-dependent iron uptake and apoptosis.

1-Methyl-4-phenylpyridinium (MPP(+)) is a neurotoxin used in cellular models of Parkinson's Disease. Although intracellular iron plays a crucial role in MPP(+)-induced apoptosis, the molecular signalling mechanisms linking iron, reactive oxygen species (ROS) and apoptosis are still unknown. We investigated these aspects using cerebellar granule neurons (CGNs) and human SH-SY5Y neuroblastoma cells. MPP(+) enhanced caspase 3 activity after 24 h with significant increases as early as 12 h after treatment of cells. Pre-treatment of CGNs and neuroblastoma cells with the metalloporphyrin antioxidant enzyme mimic, Fe(III)tetrakis(4-benzoic acid)porphyrin (FeTBAP), completely prevented the MPP(+)-induced caspase 3 activity as did overexpression of glutathione peroxidase (GPx1) and pre-treatment with a lipophilic, cell-permeable iron chelator [N, N '-bis-(2-hydroxybenzyl)ethylenediamine-N, N '-diacetic acid, HBED]. MPP(+) treatment increased the number of TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick-end-labelling)-positive cells which was completely blocked by pre-treatment with FeTBAP. MPP(+) treatment significantly decreased the aconitase and mitochondrial complex I activities; pre-treatment with FeTBAP, HBED and GPx1 overexpression reversed this effect. MPP(+) treatment increased the intracellular oxidative stress by 2-3-fold, as determined by oxidation of dichlorodihydrofluorescein and dihydroethidium (hydroethidine). These effects were reversed by pre-treatment of cells with FeTBAP and HBED and by GPx1 overexpression. MPP(+)-treatment enhanced the cell-surface transferrin receptor (TfR) expression, suggesting a role for TfR-induced iron uptake in MPP(+) toxicity. Treatment of cells with anti-TfR antibody (IgA class) inhibited MPP(+)-induced caspase activation. Inhibition of nitric oxide synthase activity did not affect caspase 3 activity, apoptotic cell death or ROS generation by MPP(+). Overall, these results suggest that MPP(+)-induced cell death in CGNs and neuroblastoma cells proceeds via apoptosis and involves mitochondrial release of ROS and TfR-dependent iron.

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1-Methyl-4-phenylpyridinium (MPP + ), the toxic metabolite of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, induces apoptosis in cerebellar granule neurons (CGNs

In this study, we investigated the molecular mechanisms of toxicity of 1-methyl-4-phenylpyridinium (MPP ؉ Parkinson's disease (PD) 1 is characterized by mitochondrial complex I defects, elevated iron levels in brain tissue, tetrahydrobiopterin (BH 4 ) and dopamine deficiencies, and ␣-synuclein accumulation in Lewy body aggregates (1-4). The exact molecular mechanisms leading to the pathophysiology of PD are not well understood. 1-Methyl-4-phenylpyridinium ion (MPP ϩ ), a mitochondrial complex I inhibitor, produces Parkinson-like symptoms in humans and laboratory animals, and has been used to investigate the mechanism of pathogenesis of PD (5, 6). MPP ϩ is the ultimate metabolite of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), a contaminant found in illicit narcotics (7,8). MPP ϩ is taken up into dopaminergic neurons via the dopamine transporter and accumulates into mitochondria, where it inhibits complex I activity. Although the mechanism of MPP ϩ -induced neurotoxicity is not fully understood, there is increasing evidence supporting the involvement of reactive oxygen species (ROS) and reactive nitrogen species (RNS) (1, 9).)Previous studies using the neuronal nitric-oxide synthase (nNOS) knockout mice implicate nitric oxide ( ⅐ NO) and peroxynitrite (ONOO Ϫ ) in MPP ϩ -induced neurodegeneration (10). However, therapeutic intervention studies with nitric-oxide synthase (NOS) inhibitors in MPTP-treated mice demonstrated the opposite result, i.e. the attenuation of ⅐ NO was more deleterious than protective (11)(12)(13)(14). Decreased formation of ⅐ NOderived metabolites (nitrite and nitrate) in cerebrospinal fluids was observed in PD (15). Furthermore, low levels of tetrahydrobiopterin (BH 4 ) were detected during the onset and progression of PD (2,16,17). BH 4 is a critical cofactor for NOS activity (18,19). Evidence indicates that BH 4 , by acting as a "redox switch," plays a critical role in increasing not only the rate of ⅐ NO generation by NOS, but also in controlling the formation of superoxide and hydrogen peroxide (20 -22). Pharmacological manipulation of BH 4 has been suggested as a therapeutic strategy to modulate ⅐ NO and superoxide generation in neuronal cells and endothelial cells (23)(24)(25)(26)(27). De novo biosynthesis of BH 4 is catalyzed by guanosine 5Ј-triphosphate cyclohydrolase I (GT-PCH I) (28,29). Mammalian cells can also generate BH 4 by