Figure 3

Cellular activities of anti-aggregation compound 576755. (a) 576755 reduces αSyn oligomerization in H4 cells as measured by complementation of αSyn proteins with split luciferase tags executed as described in methods. Trace firefly luciferase co-transfected provides a normalization measure for transfection efficiency. 0.3% DMSO is present in all samples. Data are plotted as means +/− SD. Shown are representative results from 3 independent experiments. A reduction in cellular oligomerization by 576755 was determined by one-way ANOVA with Dunnett’s post-test. (b) 576755 does not reduce on αSyn levels in H4 cells as determined by Western analyses (trend towards small increase seen). (Right) representative Western from one experiment of a merged image detecting actin and αSyn from H4 cells transiently transfected with vector control or αSyn containing N or C terminal split luciferase tags (SynGN + SynGC) and treated with 0.3 percent DMSO or 150 µM 576755. The two αSyn bands correspond to different tags. Entire length of blot shown. Outline of full blot shown by black lines. (Left) αSyn was quantitated in 3 separate biological replicates. Samples were normalized to cells transfected with αSyn without drug treatment to allow comparisons between blots. There is no significant difference in αSyn levels in 576755 treated vs. untreated cells (100) as determined by one sample t test. Data are plotted as mean ± SD. (c) 576755 is not toxic and (d) alleviates loss of dopaminergic neurons induced by the A53T mutant of αSyn. Primary rat embryonic midbrain cultures were non-transduced or transduced with adenovirus encoding A53T αSyn (+Ad SynA53T), in the absence or presence of 576755. The cells were then stained immunocytochemically for MAP2 and TH. Preferential dopaminergic cell death was assessed by evaluating the percentage of MAP2-positive cells that also stained positive for TH. Data are plotted as the mean ± SEM. n = 2–3 for the neuron toxicity analysis and n = 5 for the reversal of αSyn toxicity. Shown are representative results from 5 independent experiments. Statistics used a one-way ANOVA with Tukey post-test after square root transformation of the data. *p < 0.05 where shown. ***p < 0.001. **** p < 0.0001. ns is not significant.