Abstract
BackgroundOligomerization and aggregation of α-synuclein molecules play a major role in neuronal dysfunction and loss in Parkinson's disease [1]. However, α-synuclein oligomerization and aggregation have mostly been detected indirectly in cells using detergent extraction methods [2], [3], [4]. A number of in vitro studies showed that dopamine can modulate the aggregation of α-synuclein by inhibiting the formation of or by disaggregating amyloid fibrils [5], [6], [7].Methodology/Principal FindingsHere, we show that α-synuclein adopts a variety of conformations in primary neuronal cultures using fluorescence lifetime imaging microscopy (FLIM). Importantly, we found that dopamine, but not dopamine agonists, induced conformational changes in α-synuclein which could be prevented by blocking dopamine transport into the cell. Dopamine also induced conformational changes in α-synuclein expressed in neuronal cell lines, and these changes were also associated with alterations in oligomeric/aggregated species.Conclusion/SignificanceOur results show, for the first time, a direct effect of dopamine on the conformation of α-synuclein in neurons, which may help explain the increased vulnerability of dopaminergic neurons in Parkinson's disease.
Highlights
Protein misfolding and aggregation, processes involved in several neurodegenerative diseases, are likely preceded by conformational changes in the proteins involved [8]
We have previously shown that conformational changes in aSyn can be monitored in immortalized H4 cells using the sensitive fluorescence resonance energy transfer (FRET) based proximity assay, fluorescence lifetime imaging microscopy (FLIM) [31]
Neurons were transfected at 5–7 days in vitro (DIV) using Lipofectamine 2000 and transfection efficiencies in the order of 5% were achieved
Summary
Processes involved in several neurodegenerative diseases, are likely preceded by conformational changes in the proteins involved [8]. A-Synuclein (aSyn), a small (140 amino acid) neuronal protein of unknown function which is ubiquitously expressed in the brain, displays little secondary structure in vitro and belongs to a group of proteins known as ‘natively unfolded’ [9,10]. In PD, there is substantial loss of dopaminergic neurons in the substantia nigra, with the presence of fibrillar inclusions called Lewy bodies (LBs) comprising aSyn as a major constituent [14]. Oligomerization and aggregation of a-synuclein molecules play a major role in neuronal dysfunction and loss in Parkinson’s disease [1]. A number of in vitro studies showed that dopamine can modulate the aggregation of a-synuclein by inhibiting the formation of or by disaggregating amyloid fibrils [5,6,7]
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