Fig. 6
Tyrphostin A9 decreases αSyn protein expression. (A) Anti-αSyn Western blotting of mDA neuron lysates. Before lysis > D50 mDA neurons were treated with 0.3 or 2.5 µM Tyrphostin A9 for 72 h. Three independent neuronal differentiations were performed. Gel images are cropped. Full-length blots/gels are presented in Figure S8. (B) Caspase and Chemtrypsin activities were determined in approximately D30 mDA neurons treated with 1 or 2.5 µM Tyrphostin A9 or the proteasome inhibitor MG132 for 72 h. Three independent experiments with at least three technical replicates each were performed. The boxplots show the median and the 2nd and 3rd quartiles of the data. Whiskers indicate 1.5X of the interquartile range. (C) SNCA-4x mDA neurons were treated with 2.5 µM Tyrphostin A9 for 72 h and Western blotting was performed using antibodies against p-AMPK and β-actin. (D) SNCA-4x mDA neurons were treated with 2.5 µM Tyrphostin A9 for 72 h and Western blotting was performed using antibodies against p62, LC3B, and β-actin. Bar plots indicate the means of two or three independent differentiations. Welch’s unequal variances t-test was performed using the (technical) replicates of all independent experiments. ns = not significant, * = p < 0.05, ** = p < 0.01, *** = p < 0.001. Uncropped Western blot images are shown in Figure S8 and Figure S11. (E) Scheme depicting the use of morphological profiling to identify compounds that produce a healthy control-like (SNCA-corr) profile in mutant (SNCA-4x) mDA neurons. The identified compound Tyrphostin A9 increases mitochondrial uncoupling and decreases ROS and αSyn levels which can explain the rescued morphological profile.
