title: 1-Methyl-4-phenylpyridinium (MPP+)-induced apoptosis and mitochondrial oxidant generation: role of transferrin-receptor-dependent iron and hydrogen peroxide.
abstract: 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.
authors.affiliation: Biophysics Research Institute and Free Radical Research Center, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI 53226, USA.
authors.forename: Shasi V
authors.initials: SV
authors.lastname: Kalivendi
authors.forename: Srigiridhar
authors.initials: S
authors.lastname: Kotamraju
authors.forename: Sonya
authors.initials: S
authors.lastname: Cunningham
authors.forename: Tiesong
authors.initials: T
authors.lastname: Shang
authors.forename: Cecilia J
authors.initials: CJ
authors.lastname: Hillard
authors.forename: B
authors.initials: B
authors.lastname: Kalyanaraman
mesh_terms: D015655:1-Methyl-4-phenylpyridinium / Q000494:pharmacology*; D000154:Aconitate Hydratase / Q000187:drug effects / Q000378:metabolism; D000818:Animals; D000975:Antioxidants / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects*; D053148:Caspase 3; D020169:Caspases / Q000187:drug effects / Q000378:metabolism; D002478:Cells, Cultured; D003676:Deferoxamine / Q000494:pharmacology; D004492:Edetic Acid / Q000031:analogs & derivatives* / Q000494:pharmacology; D005260:Female; D005979:Glutathione Peroxidase / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism*; D007501:Iron / Q000378:metabolism*; D007502:Iron Chelating Agents / Q000494:pharmacology; D008297:Male; D008665:Metalloporphyrins / Q000494:pharmacology; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism*; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism; D009474:Neurons / Q000187:drug effects / Q000378:metabolism; D016877:Oxidants / Q000378:metabolism; D018384:Oxidative Stress; D010300:Parkinson Disease / Q000378:metabolism / Q000473:pathology; D017382:Reactive Oxygen Species / Q000378:metabolism; D011990:Receptors, Transferrin / Q000187:drug effects / Q000276:immunology / Q000378:metabolism*; D013194:Staining and Labeling / Q000379:methods; D014168:Transferrin / Q000378:metabolism; D000094765:Glutathione Peroxidase GPX1
chemical_list: D000975:Antioxidants; D007502:Iron Chelating Agents; D008665:Metalloporphyrins; D016877:Oxidants; D017382:Reactive Oxygen Species; D011990:Receptors, Transferrin; D014168:Transferrin; C474019:tetrakis(4-benzoic acid)porphyriniron(III); C037740:N,N'-bis(2-hydroxybenzyl)ethylenediamine-N,N'-diacetic acid; D004492:Edetic Acid; D006861:Hydrogen Peroxide; D007501:Iron; D005979:Glutathione Peroxidase; D009247:NADH, NADPH Oxidoreductases; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases; D000154:Aconitate Hydratase; D003676:Deferoxamine; D015655:1-Methyl-4-phenylpyridinium; D000094765:Glutathione Peroxidase GPX1; C000722067:GPX1 protein, human