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. 2024 May 25;11(6):nwae182. doi: 10.1093/nsr/nwae182

Figure 5.

Graphs and TEM images labelled a to g. a includes the graphs of ThT kinetic curves (left) and the analysis of soluble α-syn (right), and TEM images of fibril (middle) in low LPC/ α-syn ratios. b includes the graphs of ThT kinetic curves (left) and the analysis of soluble α-syn (right), and TEM images of fibril (middle) in high LPC/ α-syn ratios. c is the schematic illustration of the cell experiments. d is the graphs of western blot image and analysis for α-syn aggregates in cell adding LPC. e is graph comparing total LPL intensity from control vs. BEL-treated cells. f is the heatmap of lipidomic profiling of BEL-treated cells. g is graph showing western blot image and analysis of α-syn aggregates in cell treating with BEL.

LPC inhibits α-syn aggregation in vitro and in cells. (a, b) The ThT kinetic assay (graph on left) and the analysis of soluble α-syn (graph on right) shows the inhibition effect of LPC on α-syn aggregation at relatively low LPC/α-syn ratios (a) and high LPC/α-syn ratios (b). α-Syn aggregation was enhanced by the addition of 0.05% (v/v) preformed α-syn fibril seeds in (b). Transmission electron microscopy (TEM) images in the middle were taken at the end of the ThT assay for each sample. Scale bar = 200 nm. Data represent the mean ± SD (n = 3). **, P-value < 0.01; ***, P-value < 0.001; Student's t-test. (c) Schematic illustration of the cellular experiments. Chemical inhibitor BEL was added to inhibit the phospholipase that converts PLs to LPLs, to inhibit the production of LPLs. Reduced LPL production may impair the protection of LPLs against α-syn aggregation. (d) Western blot and the analysis of α-syn in-cell pellets show a dose-dependent inhibition of intracellular α-syn aggregation by treating cells with LPC. Preformed α-syn fibril seeds were used to promote α-syn aggregation. Data represent the mean ± SD (n = 4). *, P-value < 0.05; ***, P-value < 0.001; Student's t-test. (e) Normalized intensity of total LPL amounts in the control cells and BEL-treated cells. The total amounts of LPLs were calculated using the sum of Z-score-normalized (x/standard deviation) intensity for each LPL. ***, P-value < 0.001; Student's t-test. (f) Heat map of the lipidomic profiling of BEL-treated cells. LPLs with a significant change in amount (q-value < 0.05) are presented and colored by Z-scores. The lipid species within each cluster are shown in the right column. pLPE is a 1Z-alkenyl ether (plasmalogen) substituent of LPE. aLPC is an alkyl ether substituent of LPC. Four biological replicates for each sample were measured. Student's t-test followed by FDR correction. (g) Western blot and the analysis of α-syn in-cell pellets show that in the presence of BEL, insoluble α-syn aggregates dramatically increased compared to those in the control. Data are shown as means ± SD (n = 4). **, P-value < 0.01; Student's t-test.