Abstract
The interaction of brain lipids with α-synuclein may play an important role in the pathogenesis of Parkinson disease (PD). Docosahexaenoic acid (DHA) is an abundant fatty acid of neuronal membranes, and it is presents at high levels in brain areas with α-synuclein inclusions of patients with PD. In animal models, an increase of DHA content in the brain induces α-synuclein oligomer formation in vivo. However, it is not clear whether these oligomeric species are the precursors of the larger aggregates found in Lewy bodies of post-mortem PD brains. To characterize these species and to define the role of fatty acids in amyloid formation, we investigated the aggregation process of α-synuclein in the presence of DHA. We found that DHA readily promotes α-synuclein aggregation and that the morphology of these aggregates is dependent on the ratio between the protein and DHA. In the presence of a molar ratio protein/DHA of 1:10, amyloid-like fibrils are formed. These fibrils are morphologically different from those formed by α-synuclein alone and have a less packed structure. At a protein/DHA molar ratio of 1:50, we observe the formation of stable oligomers. Moreover, chemical modifications, methionine oxidations, and protein-lipid adduct formations are induced by increasing concentrations of DHA. The extent of these modifications defines the structure and the stability of aggregates. We also show that α-synuclein oligomers are more toxic if generated in the presence of DHA in dopaminergic neuronal cell lines, suggesting that these species might be important in the neurodegenerative process associated with PD.
Highlights
␣-Synuclein (␣-syn)4 is a 14.5-kDa presynaptic protein, natively unfolded, that plays an important role in the pathogenesis of Parkinson disease (PD) [1]
We show that ␣-synuclein oligomers are more toxic if generated in the presence of docosahexaenoic acids (DHA) in dopaminergic neuronal cell lines, suggesting that these species might be important in the neurodegenerative process associated with PD
In the case of the aggregation conducted using a P/DHA of 1:50, samples do not produce any increase of thioflavin T (ThT) fluorescence, suggesting the absence of ordered amyloid-like structures able to selectively bind the dye
Summary
The spectra were recorded on protein samples in the absence and presence of DHA and after 15 days of incubation. Aliquots (200 l) of samples taken from the aggregation mixture were loaded into the column and eluted at 0.4 ml/min in 20 mM Tris-HCl, 0.15 M NaCl, pH 7.4. The molecular weight value for the ␣-syn samples was estimated using the following standards: blue dextran, 2000 kDa; -amylase, 200 kDa; albumin, 67 kDa; ovalbumin, 45 kDa; carbonic anhydrase, 29 kDa, ␣-lactalbumin, 14.4 kDa and aprotinin, 6.5 kDa. The HPLC analyses were conducted using a Jupiter C4 column (4.6 ϫ 150 mm; Phenomenex, CA) eluted with a gradient of acetonitrile, 0.085% TFA versus water, 0.1% TFA from 5 to 38% in 5 min and from 38 to 43% in 15 min.
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