Fig. 6: Reduced EHMT activity leads to aberrant neuronal activity.
A Calcium imaging of hiPSCs-derived neurons. Differentiated neurons were analysed for spontaneous calcium events using Cal-520tm AM staining. Neurons were treated with UNC0638 for two weeks and calcium imaging was performed around day 50 of differentiation to compare the number of calcium event frequencies between control neurons and those treated with UNC0638. Representative images and traces of calcium influx of neuronal cultures in the presence and absence of UNC0638 (250 nM) are shown. Vertical scale bar shows 0.025 (ΔF/F); horizontal bar shows 25 s. N = 300 ROIs for all cell lines across 2 imaging regions over 3 coverslips/line. The percentage of responsive cells and average calcium events/ROIs for control untreated hiPSCs and cells treated with UNC0638. B qRT-PCR analysis to examine changes in the expression of GluN1, GRIA1 and GRIN2A in EHMT1−/+-derived neurons relative to their expression in isogenic control neurons. The expression of GRIN1 was significantly elevated in EHMT1−/+ cells, while those of GRIA1 and GRIN2A were unchanged, n ≥ 3. Error bars represent SEM, all data points shown, *P < 0.05. C Equivalent stage neurons derived from KS-patient iPSC and control hiPSC were stained for neuronal marker MAP2 and the synaptic marker Synaptophysin positive puncta normalised to the dendritic area stained positive for MAP2. No significant difference in synaptic density, measured by the number of synaptophysin-stained puncta per unit area of MAP2 stained neurite, was observed. Representative images of Synaptophysin (SYN, green) positive puncta in MAP2 (red) positive control, KS1 and KS2 neurons. Scale bar = 50 µm. n ≥ 3 independent experiments. Data shown as Mean±SEM and analysed by student’s t-test or One-way ANOVA with post hoc comparisons using Dunnett’s multiple comparisons test comparing to control samples.
