Abstract
Background: Alpha-synuclein is a protein involved in the pathogenesis of Parkinson's disease. In vitro observations have shown that specific brain-enriched polyunsaturated fatty acids, such as arachidonic acid, can give rise to a conformational change in alpha-synuclein and ultimately induce its fibrillation. Arachidonic acid is released by phospholipase A2 activity and clinical observations have shown a link between mutations in PLA2G6, the gene responsible for the production of phospholipase A2, and early-onset types of parkinsonism. It is unknown how phospholipase A2-driven release of arachidonic acid can affect the conformation of alphasynuclein.
Objective: The main objective of this study was to investigate if phospholipase A2-induced release of arachidonic acid can induce changes in conformation and aggregation state of alpha-synuclein.
Methods: Recombinant human alpha-synuclein was expressed and isolated and incubated in the presence of phosphatidylcholine and phosphatidylserine (PC/PS) containing liposomes. The release of free fatty acids from PC/PS liposomes by bee venom phospholipase A2 was measured with the fluorescent probe acrylodated intestinal fatty acid-binding protein (ADIFAB) and radioactive labelling by preparing liposomes in the presence of L- 3-phosphatidylcholine, 1-stearyl-2[1-14C] arachidonoyl. The effect of free fatty acid release on the conformation of alpha-synuclein was assayed by far-UV circular dichroism and resistance against V8 protease-induced limited proteolysis. Aggregation of alpha-synuclein upon exposure to phospholipase A2-induced action on PC/PS liposomes was measured using thioflavin T fluorescence, SDS-PAGE, gel filtration chromatography, and transmission electron microscopy. RAW264.7 cells were transiently transfected with human alpha-synuclein and release of arachidonic acid was quantified using radiolabeling and liquid scintillation counting.
Results: Phospholipase A2 is capable of releasing arachidonic acid from biomimetic phospholipid membranes. Exposure of alpha-synuclein to phospholipase A2-induced release of arachidonic acid from PC/PS liposomes induces a conformational transition of the protein and leads to partial resistance against proteolytic cleavage by V8 protease. Prolonged incubation of alpha-synuclein with arachidonic acid, derived from PC/PS liposomes by phospholipase A2 leads to aggregate formation. In line with this, transiently transfected RAW264.7 cells with alpha-synuclein showed arachidonic acid release and punctate alpha-synuclein staining upon phospholipase A2 activation. The ability of arachidonic acid to drive alpha-synuclein to aggregate was independent of its oxidation state.
Conclusion: We present data that suggest a biological context for the previously reported clinical observation that linked mutations in PLA2G6, the gene responsible for the production of phospholipase A2, and early-onset types of parkinsonism. Release of arachidonic acid, independent of its oxidation state, through activation of phospholipase A2-driven hydrolysis of phospholipid membranes, leads to the structural transition and aggregation of alpha-synuclein.
Keywords: Alpha-synuclein, phospholipase A2, arachidonic acid, oxidation, Parkinson's disease, aggregation.
Graphical Abstract
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