Fig. 6.

HDAC inhibition limited the reduction of histone acetylation in TDP-43^G348C-expressing motor neurons. After microinjection of plasmid encoding TDP-43^G348C or mCherry as control, cultures were treated with vehicle (DMSO), 4 µM arimoclomol, 4 µM SAHA, 1 µM RGFP963, alone and in combination for 3 days. Injected neurons were identified by double-labeling with rabbit anti-flag M2 to visualize TDP-43^G348C or by mCherry epifluorescence. (a) Acetylation of histone 3 at lysine residues 9 and 14 (H3K9/K14Ac), evaluated by immunocytochemistry using an acetylation-specific antibody was categorized as: strong (H3K9/K14Ac+/+), weak (H3K9/K14Ac+/−) or absent (H3K9/K14Ac−/−). (b) Reduction in H3K9K14ac in motor neurons expressing TDP-43^G348C was prevented by the HDAC inhibitors (c) SAHA or (d) RGFP963 demonstrating target engagement, whereas arimoclomol had no impact. (e) No alteration of histone acetylation by wild-type TDP-43. Data are presented as mean ± SD. Each data point on the graph represents the mean % of neurons in each of 6–13 cultures (10–59 neurons/culture). Statistical significance was evaluated by one-way ANOVA followed by Bonferroni post hoc analysis. *P < 0.05, **P < 0.01, ***P < 0.001. Scale bar = 20 µm. Abbreviations used: HDAC, histone deacetylase; SAHA, suberoylanilide hydroxamic acid; SD, standard deviation; TDP-43, TAR DNA binding protein 43 kDa. 43 kDa; DMSO dimethyl sulfoxide.