Peroxynitrite in the pathogenesis of Parkinson's disease and the neuroprotective role of metallothioneins

Parkinson's disease (PD) is characterized by a progressive loss of dopaminergic neurons in the substantia nigra zona compacta and in other subcortical nuclei associated with a widespread occurrence of Lewy bodies. The causes of cell death in Parkinson's disease are still poorly understood, but a defect in mitochondrial oxidative phosphorylation and enhanced oxidative stress has been proposed. We have examined 3-morpholinosydnonimine (SIN-1)-induced apoptosis in control and metallothionein-overexpressing dopaminergic neurons, with a primary objective to determine the neuroprotective potential of metallothionein (MT) against peroxynitrite-induced neurodegeneration in PD. SIN-1 induced lipid peroxidation and triggered plasma membrane blebbing. In addition, it caused DNA fragmentation, alpha-synuclein induction, and intramitochondrial accumulation of metal ions (copper, iron, zinc, and calcium), and it enhanced the synthesis of 8-hydroxy-2-deoxyguanosine. Furthermore, it downregulated the expression of Bcl-2 and poly(adenosine diphosphate-ribose) polymerase, but upregulated the expression of caspase-3 and Bax in dopaminergic (SK-N-SH) neurons. SIN-1 induced apoptosis in aging mitochondrial genome knockout cells, alpha-synuclein-transfected cells, metallothionein double-knockout cells, and caspase-3-overexpressed dopaminergic neurons. SIN-1-induced changes were attenuated with selegiline or in metallothionein-transgenic striatal fetal stem cells. SIN-1-induced oxidation of dopamine (DA) to dihydroxyphenylacetaldehyde (DopaL) was attenuated in metallothionein-transgenic fetal stem cells and in cells transfected with a mitochondrial genome, and was enhanced in aging mitochondrial genome knockout cells, in metallothionein double-knockout cells, and caspase-3 gene-overexpressing dopaminergic neurons. Selegiline, melatonin, ubiquinone, and metallothionein suppressed SIN-1-induced downregulation of a mitochondrial genome and upregulation of caspase-3 as determined by reverse transcription polymerase chain reaction. These studies provide evidence that nitric oxide synthase activation and peroxynitrite ion overproduction may be involved in the etiopathogenesis of PD, and that metallothionein gene induction may provide neuroprotection.