Glutathione redox status in mitochondria and cytoplasm differentially and sequentially activates apoptosis cascade in dopamine-melanin-treated SH-SY5Y cells

Neuromelanin (NM)-containing dopaminergic neurons in the substantia nigra are selectively vulnerable in Parkinson's disease (PD), suggesting the involvement of NM in the pathogenesis. NM is composed of protein, lipid, trace metals and melanin component, a mixture of eumelanin produced from dopamine (DA)-quinone and pheomelanin containing 5-S-cyteinyl-DA-quinone. We reported that NM induces mitochondria-mediated apoptosis in human dopaminergic SH-SY5Y cells, which was suppressed completely by Protease K-treatment, suggesting the essential requirement for the protein component. In this paper, the role of the melanin component in NM-dependent apoptosis was studied using SH-SY5Y cells and synthesized DA-melanin (DAM) and L-Cystemyl-DAM (Cys-DAM). DAM oxidatively decreased glutathione (GSH) and sulfhydryl (SH) content in mitochondria, whereas NM increased GSH by de-S-glutathionylation of complex I. DAM induced mitochondrial permeability transition (mPT), leading to membrane potential collapse and cytochrome c release, whereas Cys-DAM did not. However, the cytotoxicity of DAM itself was rather mild and thiol-targeting reducing reagents, including GSH, dithiothreitol and N-acetyl-cysteine, increased apoptosis significantly. The reducing SH reagents activated caspase 3 and induced apoptosis, but did not affect mPT. On the other hand, NM itself activated mitochondria-initiated apoptotic cascade, which GSH suppressed completely. The results indicate that DAM induces apoptosis through the sequential activation by oxidation of SH status in mitochondria and reduction in cytoplasm. in contrast to the case with NM. The regulation of apoptotic processing by SH redox state is discussed in relation to degeneration of nigra-striatal DA neurons in aging and PD, where oxidative stress is increased with impaired antioxidant capacity. (c) 2009 Elsevier Ireland Ltd. All rights reserved.