Dopamine promotes formation and secretion of non-fibrillar alpha-synuclein oligomers

Abstract

Parkinson's disease (PD) is characterized by selective and progressive degeneration of dopamine (DA)-producing neurons in the substantia nigra pars compacta (SNpc) and by abnormal aggregation of α-synuclein. Previous studies have suggested that DA can interact with α-synuclein, thus modulating the aggregation process of this protein; this interaction may account for the selective vulnerability of DA neurons in patients with PD. However, the relationship between DA and α-synuclein, and the role in progressive degeneration of DA neurons remains elusive. We have shown that in the presence of DA, recombinant human α-synuclein produces non-fibrillar, SDS-resistant oligomers, while β-sheet-rich fibril formation is inhibited. Pharmacologic elevation of the cytoplasmic DA level increased the formation of SDS-resistant oligomers in DA-producing neuronal cells. DA promoted α-synuclein oligomerization in intracellular vesicles, but not in the cytosol. Furthermore, elevation of DA levels increased secretion of α-synuclein oligomers to the extracellular space, but the secretion of monomers was not changed. DA-induced secretion of α-synuclein oligomers may contribute to the progressive loss of the dopaminergic neuronal population and the pronounced neuroinflammation observed in the SNpc in patients with PD.

Figure 1

DA-induced oligomerization of α-synuclein in vitro. (A) Thioflavin T binding assay. Recombinant α-synuclein was incubated in the presence (red) or absence (green) of DA. (B) CD analysis of α-synuclein monomer (blue), fibrils (green), and oligomer-monomer mixture prepared in the presence of DA (red).

Figure 2

Analysis of DA-induced α-synuclein oligomers Size exclusion chromatography (A) and Western blotting (B) of α-synuclein proteins incubated with DA. (Dotted line: α-synuclein monomer, black line: DA-induced α-synuclein oligomer profiles. △: blue-dextran [2,000 kDa], █: albumin [66,000 Da], ●: cytochrome c [12,400 Da]) (C) Electron microscopy images of control (left) and DA-induced oligomers (right) (Scale bar: 0.2 µm).

Figure 3

DA-induced α-synuclein aggregation and vesicular localization in SH-SY5Y cells. (A) L-dopa treatment increased α-synuclein aggregation in differentiated SH-SY5Y cells in a dose-dependent manner. Differentiated SH-SY5Y neuronal cells overexpressing α-synuclein were treated with L-dopa at final concentrations of 0, 50, 150, and 400 µM for 48 hours. (B) Inhibition of MAO-B by pargyline (0, 1, 5, and 20 µM) further elevated α-synuclein aggregate formation in L-dopa-treated cells (1 mM). The data were obtained from the same blot. (C) Addition of c-dopa (1, 4, and 10 mM) inhibited DA-induced α-synuclein aggregation (L-dopa [1 mM)], pargyline [5 µM]). All the data shown here were originated from the same blot. (D) Quantitation of high MW α-synuclein aggregates in blot (C). β-actin was shown to demonstrate equal loading of proteins on all blots.

Figure 4

Localization of DA-induced α-synuclein aggregates in vesicle fractions. Differentiated SH-SY5Y neuronal cells overexpressing α-synuclein were treated with 1 mM L-dopa and the extract was separated by density gradient centrifugation to obtain vesicle (Ves) or cytosolic (Cyt) fractions. Note the increased α-synuclein aggregates (side bar) in vesicle fraction of L-dopa treated cells.

Figure 5

DA-induced secretion of α-synuclein aggregates from SH-SY5Y cells. Differentiated SH-SY5Y cells were treated with 1 mM L-dopa and 10 µM pargyline for indicated times and the conditioned media were collected and analyzed for α-synuclein release. Note that the monomer levels do not differ but the amount of α-synuclein aggregates increased significantly in L-dopa- and pargyline-treated cells (^***P < 0.001).