Fig. 7

SAG attenuates neurotoxicity induced by FXN-deficient reactive astrocytes. A Schematic representation of ACM collection and treatment of mouse cortical neurons. For these experiments, cortical neurons were cultured for 5 days (120 h) in ACM from HAs cultured under standard conditions (untreated) or transduced with either the LV-scrambled or LV-shRNA37 and treated daily or not with SAG at 1 μM. 48 h prior to ACM collection, the medium was shifted to complete neuronal medium. B Quantification of the cellular metabolic activity (n = 3) and total cell number estimated by DAPI counts (n = 4) in all experimental groups of mouse cortical neurons after ACM treatment. C The left panels show representative confocal images of mouse cortical neurons cultured in ACM from the indicated experimental conditions, that were stained with specific antibodies against MAP2 to stain dendrites and SMI-31 to stain axons (n = 4). Scale bars: 50 μm. The graphs on the right show axonal and dendritic lengths calculated by measuring the total length per field corrected by the number of cells per field on each acquired image. D The left panels show representative confocal images of mouse cortical neurons cultured in the different ACM and stained with the presynaptic marker synaptophysin and the postsynaptic marker PSD-95 (n = 4). Synapse number was estimated by analyzing synaptophysin and PSD95 co-localization using the plugin JACoP (ImageJ). Insets are threefold enlargements of the boxed region. Scale bars: 25 μm (expanded fields) and 5 μm (insets). All values were normalized to the group cultured in ACM from untreated astrocytes (data not plotted). Data in all graphs represent mean values ± S.E.M. Statistical significance in A–C was determined using one-way ANOVA followed by Tukey’s post hoc test