Plasma membrane remodeling in GM2 gangliosidoses drives synaptic dysfunction
Fig 4
Increased spontaneous and stimulated neuronal network activity in ΔHEXA neurons compared with SCRM neurons.
A. Calcium signalling was monitored over 30 dpi for SCRM, ΔHEXA and ΔHEXB cell lines. Calcium signalling of cells starts to become synchronous (measured as correlation of bursting objects) from about 15 dpi with increasing signal correlation over time. Four technical repeats, n = 4 of N = 2 independent experiments are shown with data from all cell lines (mean of SCRM, ΔHEXA and ΔHEXB ± SEM) combined to demonstrate that within an experiment, all cell lines are well correlated but between experiments, time to 100% correlation can differ until about 25 dpi. B. Quantification of gangliosides GM2 and GT1b from whole cell (left) and PM-enriched (right) samples of neurons at 14 and 28 dpi for N = 3 biological replicates of SCRM and ΔHEXA cells. Significance was determined by two-way ANOVA, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001. C. Representative activity traces (raster plots) of spontaneous neural activity for SCRM (left) and ΔHEXA (right) neurons. Activity is recorded over time on each of the 15 electrodes with spikes (black), bursts (blue) and network bursts (pink boxes) shown over the same 180 s time period. The intensity of network activity is represented as a peak (above). D. Analysis of neuronal activity including network burst (NWB) frequency, number of spikes per NWB and NWB duration for data collected from N = 3 biological replicates across n = 52 wells over a time window from 38 to 45 dpi. Mean is displayed for SCRM (grey line) and HEXA (red line) and significance was determined with an unpaired t test, **p ≤ 0.01. E. Representative activity traces (raster plots) for SCRM (upper) and ΔHEXA (lower) neurons in response to repetitive field stimulation (white triangles). Activity over time is recorded on each of the 15 electrodes. Network bursts (red) are shown over the same 120 s time period. Underlying data used to generate these figures are available in S1 Data at https://doi.org/10.17863/CAM.118836.