Unsaturated fatty acids uniquely alter aggregation rate of α-synuclein and insulin and change the secondary structure and toxicity of amyloid aggregates formed in their presence

Abstract

Docosahexaenoic (DHA) and arachidonic acids (ARA) are omega-3 and omega-6 long-chain polyunsaturated fatty acids (LCPUFAs). These molecules constitute a substantial portion of phospholipids in plasma membranes. Therefore, both DHA and ARA are essential diet components. Once consumed, DHA and ARA can interact with a large variety of biomolecules, including proteins such as insulin and α-synuclein (α-Syn). Under pathological conditions known as injection amyloidosis and Parkinson's disease, these proteins aggregate forming amyloid oligomers and fibrils, toxic species that exert high cell toxicity. In this study, we investigate the role of DHA and ARA in the aggregation properties of α-Syn and insulin. We found that the presence of both DHA and ARA at the equimolar concentrations strongly accelerated aggregation rates of α-Syn and insulin. Furthermore, LCPUFAs substantially altered the secondary structure of protein aggregates, whereas no noticeable changes in the fibril morphology were observed. Nanoscale Infrared analysis of α-Syn and insulin fibrils grown in the presence of both DHA and ARA revealed the presence of LCPUFAs in these aggregates. We also found that such LCPUFAs-rich α-Syn and insulin fibrils exerted significantly greater toxicities compared to the aggregates grown in the LCPUFAs-free environment. These findings show that interactions between amyloid-associated proteins and LCPUFAs can be the underlying molecular cause of neurodegenerative diseases.

Keywords: AFM-IR; LDH; ROS; alpha-synuclein; arachidonic acid; docosahexaenoic acid; insulin; unsaturated farry acids.

FIGURE 1

ARA and DHA uniquely alter the rate of α-Syn and insulin aggregation. ThT aggregation kinetics of α-Syn (red) in the lipid-free environment, in the presence of ARA (yellow) and DHA (green); insulin (blue), insulin in the presence of ARA (light blue) and DHA (purple) at 1:1 molar ratio. t_lag was calculated as 10% of ThT intensity maximum, and t_1/2 as 50% of ThT intensity maximum. Data were analyzed using ANOVA, p < 0.05. Tuckey HSD posthoc test was used for further group comparison.

FIGURE 2

Morphologies of α-Syn and insulin aggregates grown in the presence of LCPUFAs, as well as in the LCPUFAs-free environment. AFM images of α-Syn:ARA (A–C), Ins:ARA (D–F), α-Syn:DHA (G–I), Ins:DHA (J–L), α-Syn (M–O), and Ins (P–R) aggregates.

FIGURE 3

IR spectra acquired from α-Syn, α-Syn:ARA and α-Syn:DHA (A,B) and Ins, Ins:ARA and Ins:DHA (C,D) aggregates, as well as from ARA and DHA lipids themselves (A–D).

FIGURE 4

AFM-IR spectra were acquired from α-Syn:ARA (A), α-Syn:DHA (B), and α-Syn (C) fibrils. Histograms of relative amount of anti-parallel β-sheet, unordered protein, and parallel β-sheet in the analyzed fibrils (D).

FIGURE 5

AFM-IR spectra acquired from Ins:ARA (A), Ins:DHA (B), and Ins (C) fibrils. Histograms of relative amount of anti-parallel β-sheet, unordered protein, and parallel β-sheet in the analyzed fibrils (D).

FIGURE 6

α-Syn and insulin aggregates grown in the presence of LCPUFAs exert different cell toxicity compared to the aggregates grown in the LCPUFAs-free environment. Histograms of LDH (A), ROS (B), and JC-1 (C) assays of α-Syn, α-Syn:ARA, α-Syn:DHA, Ins, Ins:ARA and Ins:DHA, as well as ARA and DHA themselves. Black asterisks (*) show significance level of differences between α-Syn and α-Syn:ARA, α-Syn:DHA, ARA, DHA, and control; red asterisks (*) show significance level of differences between Ins, Ins:ARA and Ins:DHA, ARA, DHA, and control; green NS shows insignificance level of differences between ARA, DHA and control. *p ≤ .05; **p ≤ .01; ***p ≤ .001; ****p ≤ .0001; NS, non significant difference.