Calcium Induces Alpha-Synuclein Aggregates in Solution, on Surfaces and in Cultured Cells

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Pountney, Dean
Nath, S.
Goodwin, Jacob
Engelborghs, Y.
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2010
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Melbourne

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Parkinson's and Parkinson's-plus diseases are associated with abnormal, aggregated forms of the protein, a-synuclein. We have investigated the effects of calcium on a-synuclein aggregation in vitro and in vivo. We treated monomeric a-synuclein with calcium in vitro and used fluorescence imaging, fluorescence correlation and scanning electron microscopy to investigate protein aggregation. Our in vitro data suggests two distinct modes of aggregation: surface-dependent aggregation and aggregation in solution, both of which are accelerated by calcium, but at different concentrations. Incubation of monomeric a-synuclein (24 hrs) at low concentration (10 卩 with calcium resulted in surface aggregates (1.5ᰮ7 孩 saturating at a half-maximum calcium concentration of 80 卬 whilst incubations without calcium showed few protein aggregates. In the presence of calcium, plaques (0.5-1 孩 of a-synuclein aggregates comprising 10-20 nm globular particles were observed by scanning electron microscopy. Incubation of a-synuclein at high concentration (75 卻 6 hrs) resulted in soluble oligomeric aggregates detected by fluorescence correlation in a calcium dependent process, saturating at a half maximum calcium concentration of 180 卮 In cell culture experiments, we used thapsigargin or ionophore A23187 to induce transient increases of intracellular free calcium in human 1321N1 cells expressing an a-synuclein-GFP construct and observed calcium flux and a-synuclein aggregation by fluorescence microscopy. The in vivo data shows that a transient increase in intracellular free calcium significantly increased the proportion of cells bearing cytoplasmic a-synuclein aggregates 12 hrs post-treatment (P, 0.01). Our data indicates that calcium accelerates a-synuclein aggregation in vitro and in vivo and suggests that surface adsorption may play an important role in the calcium-dependent aggregation mechanism.

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OzBio2010 Combined Conference

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© The Author(s) 2010. The attached file is reproduced here in accordance with the copyright policy of the publisher. For information about this Conference please refer to the Conference website or contact the authors.

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Cellular Nervous System

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