Raised calcium and oxidative stress cooperatively promote alpha-synuclein aggregate formation

Cell loss in Parkinson's and Parkinson's-plus diseases is linked to abnormal, aggregated forms of the cytoplasmic protein, alpha-synuclein (alpha-syn). The factors causing alpha-syn aggregation may include oxidative stress, changes in protein turnover and dysregulation of calcium homeostasis, resulting in cytotoxic aggregated alpha-syn species. Recently, we showed that raised calcium can promote alpha-syn aggregation. We have now investigated the effects of raised calcium combined with oxidation/oxidative stress on alpha-syn aggregation both in vitro and in vivo. We treated monomeric alpha-syn with calcium, hydrogen peroxide or calcium plus hydrogen peroxide in vitro and used size exclusion chromatography, fluorescence correlation spectroscopy, atomic force microscopy and scanning electron microscopy to investigate protein aggregation. Our in vitro data is consistent with a cooperative interaction between calcium and oxidation resulting in alpha-syn oligomers. In cell culture experiments, we used thapsigargin or ionophore A23187 to induce transient increases of intracellular free calcium in human 1321N1 cells expressing an alpha-syn-GFP construct both with and without co-treatment with hydrogen peroxide and observed alpha-syn aggregation by fluorescence microscopy. Our in vivo cell culture data shows that either transient increase in intracellular free calcium or hydrogen peroxide treatment individually were able to induce significantly (P = 0.01) increased 1-4 mu m cytoplasmic alpha-syn aggregates after 12 h in cells transiently transfected with alpha-syn-GFP. There was a greater proportion of cells positive for aggregates when both raised calcium and oxidative stress were combined, with a significantly increased proportion (P = 0.001) of cells with multiple (3 or more) discrete alpha-syn focal accumulations per cell in the combined treatment compared to raised calcium only. Our data indicates that calcium and oxidation/oxidative stress can cooperatively promote alpha-syn aggregation both in vitro and in vivo and suggests that oxidative stress may play an important role in the calcium-dependent aggregation mechanism. (C) 2012 Elsevier Ltd. All rights reserved.