Figure 2

FK506 restores polyQ-induced alteration of BDNF-vesicular transport in rat cortical neurons. A) Rat cortical neurons were electropored to silence endogenous huntingtin and samples were analyzed by Western blotting for huntingtin expression. (B) Immunostaining of electroporated cortical neurons silenced or not for endogenous huntingtin and ectopically expressing the first 480 amino acids of huntingtin containing 17Q repeats (480-17Q, normal) or 68Q repeats (480-68Q, mutant). Whereas huntingtin silencing depletes efficiently endogenous huntingtin in individual neurons, it does not impair the re-expression of the various huntingtin constructs nor the expression of BDNF-mCherry. (C and D) FK506 treatment at 1 μM concentration restores both anterograde and retrograde transport of BDNF vesicles to wild type values (anterograde ***p < 0.0001 and retrograde ***p < 0.0001) but has no significant effect on the wild-type huntingtin fragment (anterograde velocity p = 0.10; NS and retrograde velocity p = 0.78; NS). (E) Pausing time altered in 480-68Q condition is reduced by FK506 treatment (***p < 0.0001) without having any effect in wild-type condition (p = 0.38; NS). (F) Total traveled distance of BDNF-Cherry tagged vesicles are significantly decreased in 480-68Q expressing neurons (***p < 0.001) and restored by FK506 treatment (***p < 0.001). Data are from three independent experiments: 6850 tracks, 24 neurons for 480-17Q with DMSO, 1383 tracks, 8 neurons for 480-17Q with FK506 1 μM, 3934 tracks, 15 neurons for 480-68Q with DMSO, 4910 tracks, 19 cells for 480-68Q with FK506 1 μM. Scale bar corresponds to 10 μm.