Fig. 3. Synthetic FABP7 ligands reduce αSyn oligomerization and aggregation.

a Confocal microscopy of immunofluorescence staining of αSyn (red) and FABP7 (green) in U251 cells. The αSyn-overexpressed U251 cells were incubated with the vehicle, AA (100 µM), and H₂O₂ (100 µM), for 48 h. The FABP7-positive and αSyn-positive aggregates in AA- and H₂O₂-treated cells were increased. In addition, ligand 6 disrupted the FABP7/αSyn interaction and decreased the number of FABP7-/αSyn-positive aggregates in each cell. b–e Quantification of FABP7- and αSyn-positive aggregates in U251 cells, based on immunofluorescent staining, is illustrated. Small aggregates (<2 µm) were significantly increased in the AA-treated cells (n = 48) and H₂O₂-treated cells (n = 43), and normal sized aggregates (2–4 µm) and large aggregates (>4 µm) are significantly increased only in the AA-treated cells (n = 48). Ligand 6 treatment significantly decreased FABP7-/αSyn-positive aggregates in each size (n = 44). The data are shown as mean ± standard error of the mean and were obtained using one-way analysis of variance. The scale bar represents 50 µm. *P < 0.05; **P < 0.01; ^##P < 0.01. f Western blot (WB) analysis of αSyn (left) and FABP7 (right) reveals that 48 h incubation with FABP7 ligand 6 and ligand 8 significantly reduced the formation of αSyn oligomers weighing 70–140 kDa. g, h Quantification of αSyn (h) and FABP7 (g) oligomers in SDS-soluble fractions, based on WB analysis, is illustrated. FABP7 ligand 6 (n = 10) and ligand 8 (n = 10) significantly reduced αSyn oligomerization, and ligand 1 (n = 4) fails to inhibit αSyn oligomerization (left). Ligand 6 (n = 4) also disrupts FABP7-αSyn interaction and significantly reduced FABP7 oligomerization (right). i Schematic draft of FABP7 ligands inhibition of αSyn oligomerization. The data are shown as mean ± standard error of the mean and were obtained using one-way analysis of variance. *P < 0.05; **P < 0.01; ^#P < 0.05; ^##P < 0.01. αSyn alpha-synuclein, AA arachidonic acid, FABP7 fatty acid-binding protein 7, H₂0₂ hydrogen peroxide, NS not statistically significant.