Fig. 4.

Ethosuximide induces FOXO target gene expression and reduces polyglutamine protein aggregation in mammalian neurons. a mRNA levels of classical FOXO target genes were analysed by qRT-PCR in differentiated mouse N2A neuroblastoma cells treated with the indicated concentrations of ethosuximide. Data are shown as mean ± SEM (n = 3; *p ≤ 0.05, **p ≤ 0.01). (b-c) Ethosuximide reduces polyglutamine protein aggregation. b Visualisation of EGFP-tagged non-pathological (polyQ25) and pathological (polyQ97) polyglutamine tracts in N2A cells 72 h post-transfection. Phase contrast, GFP (green) and SYTOX orange staining (red, to identify dead cells) confocal images are shown to illustrate that aggregates are specific to polyQ97 and that ethosuximide redistributed polyQ97-EGFP away from aggregates into the cytoplasm and neuronal processes C) Quantification of polyQ aggregation. The number of polyQ-EGFP transfected N2A cells bearing fluorescent aggregates as a percentage of the total number of viable transfected (green) cells was counted at the indicated post-transfection times. Cell/aggregate counting was performed manually and confirmed using ImageJ software if cells were sufficiently sparse to allow this. Data are shown as mean ± SEM (n = 3, counting ~100 cells in each experiment; *p < 0.05)