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.