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

Abstract

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.