Fig. 6: Tideglusib treatment reversed the action potential anomalies in MPS II neurons.

A Tideglusib treatment (10 μM, 1 day) reversed the abnormal downregulation of the genes associated with calcium signaling pathways (PLCD3, MYLK2, and PDE1B) and genes encoding sodium or potassium channels (SCN9A, KCNMB, and KCND2), and reversed the abnormal upregulation of the phospholipase-encoding PLCB2 gene in 15-week MPS II neurons (N = 6 clones from 4 patients). B Tideglusib mitigated the intracellular calcium increase caused by TG treatment in MPS II neurons. The intracellular calcium changes were quantified by assessing the fluorescence intensity of the Fura-2 dye at 340 nm and 380 nm wavelengths using fluorescence microscopy in 15-week MPS II neurons and categorized as vehicle control (N = 9 differentiations from 6 clones), Tideglusib pretreatment (N = 9 differentiations from 6 clones), and Tideglusib intra-treatment (N = 9 differentiations from 6 clones). To assess the effect of treatment, we subtracted the baseline 340/380 ratios from the post-treatment fluorescence intensity ratios, calculating the change, namely Δ (340/380). The Tideglusib pretreatment and Tideglusib intra-treatment groups both showed reduced ∆ (340/380) values compared to the Vehicle group. The area under the curve (AUC) of the ∆ (340/380) over a span of 10 min was computed, providing a quantitative depiction of intracellular calcium concentration changes. The arrow indicates the time when TG alone or TG combined with Tideglusib was added. Vehicle pretreatment: MPS II neurons pre-treated with the vehicle prior to TG treatment; Tideglusib pretreatment: MPS II neurons pre-treated with Tideglusib for 24 h prior to TG addition; Vehicle intra-treatment: MPS II neurons treated with TG and vehicle; Tideglusib intra-treatment: MPS II neurons treated simultaneously with both Tideglusib and TG. C The rescuing effect of Tideglusib on the electrophysiology of MPS II neurons. The 15-week MPS II 1-2 neurons were treated with 10 μM Tideglusib or vehicle (DMSO) for 2 weeks. The APs in MPS II neurons, as mentioned above, were measured using current-clamp recordings in response to increasing current steps (−50 to 150 pA). Tideglusib treatment trended toward an increased recordable AP percentage in MPS II neurons (left upper panel). N = 16 cells from 3 independent differentiations of 1 clone for vehicle group; N = 14 cells from 5 independent differentiations of 1 clone for Tideglusib group. p = 0.2, analyzed by chi-square test. Representative change of the AP morphology of MPS II neurons treated with Tideglusib compared to those treated with vehicle (right upper panel). Scale bars: 10 ms (x-axis) and 10 mV (y-axis). Histograms show the differences of AP parameters between vehicle- and Tideglusib-treated MPS II neurons, including AP threshold, AP spike height, AP half-width, after-hyperpolarization (AHP), the slope of 10–90% rise, and the slope of 90–10% decay. N = 12 cells from 3 independent differentiations for vehicle group; N = 12 cells from 5 independent differentiations for Tideglusib group. D The rescuing effect of Tideglusib on the neurite morphology of MPS II neurons. The 15-week MPS II neurons were treated with 10 μM Tideglusib or vehicle (DMSO) for 2 weeks. Neurite morphometrics were measured using Neurolucida Neuron Tracing software. Representative images of the 15-week MPS II neurons transduced with AAV9-CaMKII α-EYFP. Neuron morphology through fluorescence microscopy is displayed in the upper panels, while their reconstructed images by Neurolucida are shown in the lower panels. Scale bar, 10 μm. The morphometric parameters of neurites included soma size, segments, bifurcation nodes, terminal ends, and total branch lengths. N = 4 clones from 2 MPS II patients for each group. Data represent mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.005, analyzed by Wilcoxon signed-rank test for A, B, and D, Mann–Whitney U test for C. Cell clone details are in Supplementary Table 1.