Figure 8. Fbxo7 small molecule inhibitor elevates PINK1 and protects against MPP+ toxicity in human cells and primary neurons.

(A) Human SH-SY5Y cells stably expressing PINK1-FLAG were treated with the indicated amounts of BC1464 for 3 hours, followed by incubation for 1 hour in the presence of CHX (10 μg/mL) before immunoblotting analysis. (B) Densitometry analysis revealed an IC₅₀ of ~5.2 μg/mL by nonlinear regression (n = 6; interpolated mean with 95% CI bands; Prism v 8.2.1, 2019, sigmoidal 4PL; 32 Degrees of Freedom, R² = 0.81; Hill coefficient = 2.9). (C and D) Duolink Proximity Ligation Assay of Fbxo7 and PINK1-FLAG in SH-SY5Y cells treated with BC1464 titration. EC₅₀ ~1.4 μM by nonlinear regression (mean ± SEM; **P < 0.01; ***P < 0.001; ****P < 0.0001 vs. vehicle by 1-way ANOVA with Dunnett’s post hoc test). PLA is detected with red fluorescence, and nuclei counterstained using Hoescht 33342. Scale bar: 20 μm. (E–G) SH-SY5Y cells were treated with vehicle or the indicated compound (5 μg/mL) for 16 hours and then lysed for Western blot for ubiquitin phosphorylated at S65 (E) and phosphorylated PKAc (F). (G) Densitometric analysis of the indicated phospho-epitopes. (mean ± SD, n = 3 wells, representative of 2 independent experiments; 1-way ANOVA with Bonferroni’s post hoc test). (H) SH-SY5Y cells were treated with the indicated concentrations of MPP+ or vehicle control in the presence of 5 μg/mL BC1465 or BC1464 for 24 hours. Cell viability was measured using AlamarBlue fluorescence intensity. (I) Mouse E16 primary cortical neurons were treated with the indicated concentrations of MPP+ in the presence of either 5 μg/mL BC1465 or BC1464 for 24 hours, and cell numbers were measured as in H. Data in H and I are shown as mean ± SD; n = 4 independent experiments; 1-way ANOVA with Bonferroni-corrected 2-tailed t test.