1-Methyl-4-phenylpyridinium-induced apoptosis in cerebellar granule neurons is mediated by transferrin receptor iron-dependent depletion of tetrahydrobiopterin and neuronal nitric-oxide synthase-derived superoxide.

In this study, we investigated the molecular mechanisms of toxicity of 1-methyl-4-phenylpyridinium (MPP(+)), an ultimate toxic metabolite of a mitochondrial neurotoxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, that causes Parkinson-like symptoms in experimental animals and humans. We used rat cerebellar granule neurons as a model cell system for investigating MPP(+) toxicity. Results show that MPP(+) treatment resulted in the generation of reactive oxygen species from inhibition of complex I of the mitochondrial respiratory chain, and inactivation of aconitase. This, in turn, stimulated transferrin receptor (TfR)-dependent iron signaling via activation of the iron-regulatory protein/iron-responsive element interaction. MPP(+) caused a time-dependent depletion of tetrahydrobiopterin (BH(4)) that was mediated by H(2)O(2) and transferrin iron. Depletion of BH(4) decreased the active, dimeric form of neuronal nitric-oxide synthase (nNOS). MPP(+)-mediated "uncoupling" of nNOS decreased *NO and increased superoxide formation. Pretreatment of cells with sepiapterin to promote BH(4) biosynthesis or cell-permeable iron chelator and TfR antibody to prevent iron-catalyzed BH(4) decomposition inhibited MPP(+) cytotoxicity. Preincubation of cerebellar granule neurons with nNOS inhibitor exacerbated MPP(+)-induced iron uptake, BH(4) depletion, proteasomal inactivation, and apoptosis. We conclude that MPP(+)-dependent aconitase inactivation, Tf-iron uptake, and oxidant generation result in the depletion of intracellular BH(4), leading to the uncoupling of nNOS activity. This further exacerbates reactive oxygen species-mediated oxidative damage and apoptosis. Implications of these results in unraveling the molecular mechanisms of neurodegenerative diseases (Parkinson's and Alzheimer's disease) are discussed.