Figure 3

Figure 3

ALS-linked mutations in FUS lead to post synaptic alterations. Representative confocal images of DIV21 rat primary neurons transduced with eGFP, HA-FUS^WT, HA-FUS^R514G and HA-FUS^ΔNLS. Cells were stained for HA (green), PSD-95 (red) and MAP2 (merge). HA staining shows cytoplasmic mislocalisation of the mutant FUS. Regions of interest show magnified dendrites used to quantify the PSD-95. Nuclei were counterstained with DAPI. Scale bar = 10 μm. (B) Representative confocal images show HA-FUS expression (left) and dendritic branching shown by MAP2 (greyscale) staining (right). Scale bar = 100 μm. (C) Quantitative analysis comparing the average number of PSD-95 puncta per dendrite after transduction of WT and mutant FUS synaptic alterations. There was a significant increase in the number of PSD-95 puncta in the neurons expressing HA-FUS^R514G compared to HA-FUS^WT (P < 0.05) and a significant decrease in puncta for those cells expressing HA-FUS^ΔNLS compared to HA-FUS^WT (P < 0.001). Statistical analysis was performed using a One-Way ANOVA with a post-hoc Tukey’s multiple comparisons test; error bars are ± SEM (n = three neurites from five cells per condition, three independent replicates). Data represent mean PSD-95 puncta on each dendrite per 10 μm ± SEM. Scale bar = 100 μm. (D,E) Quantitative analysis of dendritic branching after transduction with WT and mutant FUS. Sholl analysis revealed no overall significant change when assessing how dendritic branching was affected by WT or mutant FUS (n = ten transfected cells from three individual replicates). Statistical analysis was performed using a two-Way ANOVA with a multiple comparisons test which compared the simple effects within each row; error bars are ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 ****p < 0.0001.