Large Unilamellar Vesicles of Phosphatidic Acid Reduce the Toxicity of α-Synuclein Fibrils

Abstract

Parkinson's disease (PD) is a severe pathology that is caused by a progressive degeneration of dopaminergic neurons in substantia nigra pars compacta as well as other areas in the brain. These neurodegeneration processes are linked to the abrupt aggregation of α-synuclein (α-syn), a small protein that is abundant at presynaptic nerve termini, where it regulates cell vesicle trafficking. Due to the direct interactions of α-syn with cell membranes, a substantial amount of work was done over the past decade to understand the role of lipids in α-syn aggregation. However, the role of phosphatidic acid (PA), a negatively charged phospholipid with a small polar head, remains unclear. In this study, we examined the effect of PA large unilamellar vesicles (LUVs) on α-syn aggregation. We found that PA LUVs with 16:0, 18:0, and 18:1 FAs drastically reduced the toxicity of α-syn fibrils if were present in a 1:1 molar ratio with the protein. Our results also showed that the presence of these vehicles changed the rate of α-syn aggregation and altered the morphology and secondary structure of α-syn fibrils. These results indicate that PA LUVs can be used as a potential therapeutic strategy to reduce the toxicity of α-syn fibrils formed upon PD.

Keywords: AFM-IR; LDH; fibrils; phosphatidic acid; α-synuclein.

Figure 1

ThT aggregation kinetics (A) with corresponding t_lag and t_1/2 (B) of α-syn aggregation in the lipid-free environment (α-syn), as well as in the presence of PA-C_18:0 (α-syn:PA-C_18:0), PA-C_18:1 (α-syn:PA-C_18:1), and PA-C_16:0 (α-syn:PA-C_16:0) at 37 °C. According to one-way ANOVA, * P < 0.05; *** P < 0.001; **** P < 0.0001. NS, nonsignificant differences. Standard deviations of three individual repeats are shown in gray.

Figure 2

IR (top) and CD (bottom) spectra acquired from α-syn, α-syn:PA-C_18:0, α-syn:PA-C_18:1, and α-syn:PA-C_16:0.

Figure 3

Averaged AAFM-IR spectra (top) acquired from α-syn, α-syn:PA-C_18:0, α-syn:PA-C_18:1, and α-syn:PA-C_16:0. A bar graph (bottom) summarizes the distribution of the protein secondary structure in the protein aggregates according to the fitting of the amide I band. Parallel β-sheet (1624 cm^–1) in blue, α-helix and random coil (1660 cm^–1) in orange, β-turn (1675 cm^–1) in gray, and antiparallel β-sheet (1695 cm^–1) in yellow.

Figure 4

Length and saturation of FAs in PAs alter the morphology of the α-syn aggregates. AFM images (top) and height histograms (bottom) of α-syn fibrillar aggregates formed in the lipid-free environment (α-syn), as well as in the presence of PA-C_18:0 (α-syn:PA-C_18:0), PA-C_18:1 (α-syn:PA-C_18:1), and PA-C_16:0 (α-syn:PA-C_16:0) formed at 37 °C. Scale bars are 500 nm.

Figure 5

Histograms of LDH assays reveal cell toxicity of (A) α-syn, α-syn:PA-C_18:0, α-syn:PA-C_18:1, and α-syn:PA-C_16:0 and (B) the lipid themselves. Black asterisks (*) show significance level of differences between protein aggregates and the control; blue asterisks show significance level of difference between α-syn and α-syn:PA-C_18:1, α-syn:PA-C_16:0, and α-syn:PA-C_18:0; according to one-way ANOVA, * P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. NS shows the absence of statistical significance between α-syn and α-syn fibrils formed in the presence of LUVs with different PAs. The results of the LDH assay show no cell toxicity for PA-C_18:0 and PA-C_18:1, whereas PA-C_16:0 was only slightly toxic to N27 rat dopaminergic cells (B). Black asterisks show significant levels of differences between LUVs and the control.