Abstract
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.
Highlights
Parkinson’s disease (PD)1 is characterized by mitochondrial complex I defects, elevated iron levels in brain tissue, tetrahydrobiopterin (BH4) and dopamine deficiencies, and ␣-synuclein accumulation in Lewy body aggregates [1,2,3,4]
A major pathway by which neuronal cells acquire iron is via the transferrin receptor (TfR), which facilitates the uptake of iron-loaded transferrin [35]
MPPϩ-induced Iron Signaling in cerebellar granule neurons (CGNs)—Cytosolic aconitase is an indicator of intracellular iron level and oxidant formation and has been shown to play an important role as an iron sensor [47,48,49]
Summary
Materials—1-Methyl-4-phenylpyridinium (MPPϩ), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), deferoxamine, and NG-nitro-L-arginine-methyl ester HCl (L-NAME) were purchased from Sigma. After treatment of CGNs, culture medium was aspirated and cells were washed three times with Hanks’ buffer (110 mM NaCl, 25 mM KCl, 20 mM HEPES, 5.5 mM glucose, 1.25 mM CaCl2, 1 mM MgCl2, pH 7.4). Cells were washed three times with ice-cold PBS and collected in 0.5 mM Tris-HCl, pH 7.4, containing protease inhibitor (Roche Applied Science). TfR antibody (C), TfR mRNA levels were determined by the RT-PCR method as described under “Experimental Procedures” (D), and CGNs were treated with 70 M MPPϩ for different time intervals as indicated and further incubated with 55Fe (0.2 Ci/ml) for 4 h (E). Lysate (40 l) was incubated at 37 °C with the fluorogenic substrate Boc-VLK-AMC (80 M, trypsin-like) or Suc-LLVY-AMC (100 M, chymotrypsin-like) in 500 l of assay buffer (50 mM Tris-HCl, pH 7.4, 2 mM DTT, 5 mM MgCl2, and 2 mM ATP) for 30 and 60 min, respectively. Data Analysis—Statistical significance was obtained using Student’s t test employing the Sigmastat software
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